bluuebunny/crossref_metadata_2025 huggingface dataset:
A Contribution to the Cytogenetic Study of Plasmacytoid Dendritic Cell Acute Leukemia.
Authors: Papadhimitriou Stefanos I. , Paterakis Georgios S. , Kritikou-Griva Elpiniki , Zomas Athanassios , Davea Aggeliki , Goumakou Eleni , Tsakiridou Anastasia A. , Gligori Ioanna , Michalis Eurydike , Anagnostopoulos Nikolaos I. , Georgiakaki Maria
Published: October 2019

BACKGROUND: Plasmacytoid dendritic cell (DC2) acute leukemia is a recently described hematological malignancy with distinct clinical and phenotypic features. Yet, with fewer than 40 cases studied so far, the cytogenetic profile of this entity is still elusive. Previous studies (Leroux et al, 2002) have revealed recurrent chromosomal aberrations affecting certain genomic targets and the analysis of additional cases is expected to clarify whether these aberrations - alone or in combination - may constitute specific markers for the disease.

METHODS: Seven patients with a well-established diagnosis of DC2AL (based on clinical, morphological and phenotypic features) were included in the study. A conventional cytogenetic analysis (CCA) was performed on unstimulated bone marrow cultures. Additionally, the diagnostic marrow smears were studied with interphase fluorescent in-situ hybridization (FISH), using commercially available probes for 5q31, 6q21, 7q31, 9p21 (p16 gene), 9q34, 11q23 (MLL gene), 12p13 (TEL gene), 13q14, 17p13 (p53 gene) and 21q22 (AML1 gene) regions. In case of any abnormal finding, further study was performed with the use of centromeric or other appropriate arm/chromosome enumeration probe to distinguish between region deletion or overrepresentation and numerical aberrations of the carrier arm/chromosome.

RESULTS: An abnormal karyotype (by either CCA or FISH) was documented in all seven cases studied, with most lesions indicating the loss of genomic material. The most common finding was a rearrangement of the 21q22 region (involving the AML1 gene). Deletions of 6q21, 9q34, 11q23, 12p13 and 13q14, trisomy 12 and trisomy of 13q were detected in only one case each. Interestingly, some aberrations, including the three cases of AML1 rearrangement, were missed on CCA, suggesting a submicroscopic abnormality.

CONCLUSIONS: The study failed to define recurrent chromosomal lesions in DC2AL, other than 21q22 rearrangements. Our results point to a possible pathogenetic role of the AML1 gene. Since AML1 is involved in both myeloid and lymphoid malignancies, it makes a reasonable candidate for leukemogenesis also in the case of DC2AL. Additional investigation at the cytogenetic and molecular level is needed to verify this assumption.
AP2 Overexpression: Correlation to a Poor Prognosis in CLL.
Authors: Silva Karina Lani , Vasconcellos Deborah Vidal , de Paula Castro Eric Delfraro , Moellman Arthur Coelho , Dobbin Jane , Maia Raquel Ciuvalschi
Published: October 2019

Inhibitors of apoptosis proteins (IAPs) are a group of structurally related proteins that block apoptosis either by binding and inhibiting caspases or through caspases-independent mechanisms. Overexpression of IAPs has been detected in several types of cancers, including hematological malignancies, and it is correlated with chemotherapeutic resistance. B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of mature lymphocytes due to defective apoptosis. During disease progression, many patients acquire resistance to chemotherapeutic drugs, perhaps attributable, in large part, to the defects in programmed cell death. To verify whether IAPs expression could be responsible for chemotherapeutic resistance or disease progression we analyzed, by immunocitochemistry, the expression of c-IAP1, c-IAP2 and XIAP in 30 peripheral blood samples from B-CLL patients (16 samples from untreated patients) and compared it to well-known prognosis factors such as clinical stage (Rai and Binet), lymphocyte doubling time, gender, age and previous chemotherapeutic treatment or not. High levels of c-IAP1 and XIAP expression were not associated with any prognosis factor. On the other hand, interestingly, high levels of c-IAP2 expression were correlated with advanced clinical stage (p = 0,021 and p= 0,031 for Rai and Binet respectively) and previous chemotherapeutic treatment (p = 0,043). Our results suggest that the analysis of c-IAP2 expression in B-CLL cells might be considered a powerful tool as a marker for poor prognostic and disease progression.
Varicella Vaccine (VarilRix) in Children with Acute Lymphoblastic Leukemia.
Authors: Ass Teresa Jackowska , Wasilewski Robert , Górska Elzbieta , Wasik Maria , Loch Teresa
Published: October 2019

Background: To assess the effectiveness of vaccination against varicella in children with acute lymphoblastic leukemia (ALL).

Methods: 105 children without a history of varicella, were qualified for immunization against varicella with VARILRIX (Oka-strain varicella vaccine). 48 children had ALL and 57 were healthy. 25 of the children with ALL were receiving maintenance therapy, 23 children were after chemotherapy.

Results: White blood cells (WBC), lymphocytes, and sub-populations of T- and B-lymphocytes were compared in the healthy and leukemic children before and after vaccination. The ALL children had significantly lower counts of WBC and lymphocytes before vaccination. After vaccination there were no significant differences in the counts of WBC in the healthy and leukemic children. However the ALL children had significantly lower mean counts of lymphocytes. Before vaccination the leukemic children showed a significantly lowered percentage of T-lymphocytes with decreased CD4+ and increased CD8+, what resulted in a lowered CD4 to CD8 ratio. After vaccination, only increased numbers of T CD8+ lymphocytes and a lowered CD4 to CD8 ratio were present while there was no significant difference in CD4. In the healthy and leukemic children alike there was no statistically significant difference between B-lymphocytes (CD 19+) and NK cells.

In 10 children (20%), out of the 48 ALL vaccines, varicelliform rash occurred ~1 month after immunization. No adverse effects we observed in healthy children. Seroconversion to varicella-zoster virus was higher in healthy children and ALL children who had skin rash after vaccination. Two ALL children and three healthy ones had varicella one-two years after the vaccination. Those children received only single vaccine doses (double vaccine doses received children above 12 years).

Conclusion: Varicella vaccine was safe and immunogenic in leukemic children during maintenance and after chemotherapy.
Natriuretic Peptides in Children with Acute Lymphoblastic Leukaemia*.
Authors: Jackowska Teresa , Wasilewski Robert , Golabek Malgorzata , Syczewska Malgorzata
Published: October 2019

Background: Antracyclines (ANTR) are powerful antineoplastic agents and play an important role in treatment of acute lymphoblastic leukaemia (ALL). However, a potential heart damage caused by ANTR in patients who have been cured of cancer may lessen the success of their cure. Dexrazoxane (ICRF-187), a chelator of intracellular iron, has been shown to offer protection against the cardiomyopathic properties of antracyclines. Atrial natriuretic peptide (ANP) levels, especially receptors for proANP (1–98) and plasma brain natriuretic peptide (BNP) levels are markedly increased in cardiac impairment.

Objectives of the study: We aimed to measure the immunoreactive proANP (1–98) and N-terminal proBNP (8–29) (Nt-proBNP) in children with ALL after completion of treatment with ANTR under the protective cover of ICRF-187.

Methods used: Concentration of plasma N-terminal part proANP and NT-proBNP was measured in children by enzyme immunoassay (Biomedica). Median age at diagnosis was 5.5 years.

Results: Mean proANP plasma levels were within the normal range both in girls (1061.2 fmol/ml) and boys (778 fmol/ml). However there was a statistically significant difference between the levels in girls and boys. Mean Nt-proBNP plasma levels were normal - 273.3 fmol/ml in all examined children. Out of the 81 patients 5 (6.2%) had slightly elevated Nt-proBNP levels in comparison with healthy subjects. Nt-proBNP plasma levels were not significantly different in boys (261.0 fmol/ml) in comparison with girls (285.4 fmol/ml).

Conclusion: Our preliminary results suggest that measurement of proANP and Nt-proBNP plasma levels is useful in the detection of subclinical left ventricular dysfunction in children receiving antracycline therapy. Cardioprotection with Dexrazoxane in ALL patients treated with 240mg/m² of ANTR was justified and effective. Further, long term follow-up of the studied patients would be necessary to determine the clinical significance of Nt-proBNP increase.
Identification of B-Cell Translocation Gene 1 as a Biomarker for Monitoring the Remission of Acute Myeloid Leukemia.
Authors: Kim Hyeoung-Joon , Cho Jae-We , Kim Jeong Jung , Park Sung Goo , Lee Do Hee , Park Byoung Chul , Cho Sayeon
Published: October 2019

Acute myeloid leukemia (AML) is a biologically heterogeneous disease of the hematopoietic system characterized by a clonal accumulation of immature blast cells in the bone marrow. In this study, we used a proteomic approach based on two-dimensional electrophoresis and mass spectrometry to search for biomarkers related to the complete remission (CR) state of AML patients. We detected one AML-related protein spot using two-dimensional electrophoresis analysis, which is identified as the B-cell translocation gene 1 (BTC1) protein that belongs to anti-proliferative protein family. In the CR state of AML-m2 and M3 patients, BTG1 protein was upregulated in the bone marrow mononuclear cells. In addition strong BTG1 protein spot was detected in the normal bone marrow mononuclear cells while BTG1 levels in samples from AML-M2 patients in the non-remission state after therapy were not increased compared to those prior to therapy. Overexpression of BTG1 mRNA was also observed in the CR state of all-trans retinoic acid (ATRA)-treated AML-M3 patients and ATRA-treated HL-60 cells. Taken together, these results suggest that BTG1 may play a role in the differentiation process of myeloid cells and thus be a potential treatment-related biomarker for monitoring the remission status of AML-M2 and M3 patients following appropriate therapy.
Adult Patients with Acute Lymphoblastic Leukemia (ALL) and Central Nervous System (CNS) Leukemia at Diagnosis May Attain Durable Complete Remissions (CR). Results from the International ALL Trial (MRC UKALL-XII/ECOG E2993).
Authors: Lazarus Hillard M. , Richards Sue M. , Chopra Raj , Litzow Mark R. , Burnett Alan K. , Wiernik Peter H. , Franklin Ian M. , Tallman Martin S. , Buck G. , Durrant Jill , Rowe Jacob M. , Goldstone Anthony H.
Published: October 2019

In 1993, we initiated an international trial in newly diagnosed adult (age 15–60 years) ALL patients. 1637 patients have been entered and data analyzed as of June 2004. All patients receive two induction phases of therapy and CNS prophylaxis (or therapy if CNS involved) as assessed by lumbar puncture for cytospin. Patients age ≤ 50 yr in CR1 who have a matched-related donor (MRD) undergo an allograft, and the remainder in CR1 are randomized either to an autograft or 4 cycles intensive consolidation and maintenance chemotherapy. Matched unrelated donor (MUD) allograft is an option in Philadelphia chromosome (Ph) positive patients. 46 (3%) patients had CNS leukemia at presentation. Median (range) age was 29 (16–56) years, and 5 were Ph+. Five patients never attained CR1 (3 died early during induction on days 4, 25 and 36). 41/46 (89%) patients attained CR1. 23 patients underwent transplant, 17 in CR1 (10 MRD, 2 MUD, and 5 autografts), while 3 MUD and 3 MRD allografts were performed for > CR1. 31/46 patients died, 12 of leukemia, 8 of infection, 3 of GVHD and 8 other causes. 12 patients died in remission due to infection (5), bleeding (2), GVHD (3), and visceral organ failure (2). 15 patients remain alive, one Ph+ in relapse at 58 mo and 14 (30%) Ph- remain in continuous CR at a median 67 (range: 2–133) mo after diagnosis. Treatments for these 15 long-term survivors were: MRD allograft (6 @ 45,53,63,67,98,120 mo), MUD allograft (1 @ 67 mo; performed in relapse), chemotherapy only (6 @ 2,55,87,98,125,133 mo) and autograft (1 @ 31 mo). In this study we noted a low incidence of CNS leukemia but we cannot exclude avoidance of enrollment due to suspected CNS involvement. Adult Ph- ALL patients with CNS leukemia at diagnosis can attain long-term disease-free survivorship using transplantation as well as conventional chemotherapy.

Therapy (and status) No. Pts Survivors in CR (mo) Induction death 3 Died: no remission (resistant disease) 2 MUD without remission 1 67 MUD in CR1 2 MUD post-relapse 2 MRD in CR1 10 45,53,63,67,98,120 MRD >CR1 3 58 (in relapse) Auto (CR1) 5 31 Chemotherapy (CR1) 18 2,55,87,98,125,133 Total 46
Autoimmune Disease Is a Risk Factor for the Development of Non-Hodgkin’s Lymphoma and May Account in Part for Its Increase in Women.
Authors: Cuttner Janet , Spiera Harry , Troy Kevin , Wallenstein Sylvan
Published: October 2019

The incidence of non-Hodgkin’s lymphoma (NHL) has increased dramatically over the last several decades especially in people over 60 years of age. The reasons for this are likely to be multifactorial and may include a diminution of immune surveillance which occurs with aging. Patients with autoimmune disease may be at increased risk of developing NHL. Women have a higher incidence of autoimmune disease (AIMD). The use of immunosuppressive drugs to control autoimmune diseases decreases immune surveillance and may therefore increase the likelihood of developing lymphoma. We sought to determine if prior autoimmune disease increases the risk of developing NHL.

Methods: All patients seen by JC & KT from 1993 to 2002 form the basis of this report. There were 278 patients with NHL and these were compared with 317 patients with other hematological disorders (controls) seen at the same time period.

Findings: Thirty-six (13%) of NHL patients had an autoimmune disease prior to or at diagnosis of NHL compared to 5% of controls (p=.001). Sixty-nine percent of NHL patients with a prior AIMD were female compared to 43% without a prior AIMD and this was similar in control patients (69% and 48%) respectively. Twenty percent of all women with NHL had a prior history of autoimmune disease compared to 7% of women in the control group (p=.001). A logistic regression was performed to evaluate simultaneously the effects of gender, age and AIMD on disease status. Even adjusting for age and gender, autoimmune disease was significantly more likely to be associated with NHL, with an odds ratio of 2.6 and 95% confidence intervals of 1.4–4.9. Thus for patients of the same age and gender, patients with AIMD are 2.6 times more likely to have NHL than the control patients. The median time from AIMD to the diagnosis of NHL was 10 years. It was 14 years in the control group. The two most common autoimmune diseases in the NHL patients were rheumatoid arthritis and autoimmune hypothyroidism. In the control group it was autoimmune hypothyroidism and inflammatory bowel disease.

Conclusion:

Autoimmune disease may account in part for the increase in NHL especially in women.
Evaluation of Early Stage Hodgkin’s Lymphoma Treatment: Two Decades of Progress.
Authors: Alsaeed Eyad F. , Samant Rajiv , Victor Gallant , Huebsch Lother , Kendal Wayne
Published: October 2019

Introduction

The treatment of early stage Hodgkin’s lymphoma is controversial. Radiotherapy alone, chemotherapy alone or a combination of radiotherapy and chemotherapy are all considered effective options.

Purpose

The purpose of this study is to review the treatment approaches and outcomes used for early stage Hodgkin’s lymphoma over the past two decades at ORCC

Methods

Retrospective chart review of all patients with stage IA /IIA treated from 1984–2002 was performed. Patients were separated into three groups according to initial treatment modality: radiation alone (Rads), chemotherapy alone (Chemo), or combined modality (Combined). Disease-free survival and overall survival were estimated using the Kaplan-Meier analysis.

Result

Between May 1984 and January 2003, 172 patients with newly diagnosed Hodgkin’s lymphoma (28% stage 1A, 72% stage 2A) were seen at our centre. Treatment was as follows: 49% Rads, 13% Chemo and 38% Combined. The median age was 33.7 years (range: 17 – 82 years) and the median follow-up of 73 months (range: 3 – 204 months). The 5-year disease free and overall survival rates for the entire group were 90% and 96 % respectively. The 5-year disease-free and overall survival by treatment modality was: Rads 87% and 93 %; Chemo 80 % and 100 %; Combined 97 % and 98%. In the Combined group, there was no difference in outcome between patients receiving involved-field radiation and those receiving extended-field radiotherapy. The relapse rate in a patient who received abbreviated chemotherapy and greater than four cycles was 4.3% (1 out of 23) and 2.4% (1 out of 41) respectively. The incidence of acute Grade 3 and 4 toxicities were 9 % and 0.5% respectively.

Conclusions

Our data confirms the excellent prognosis of early stage Hodgkin’s lymphoma with all the approaches used at our centre. At present, we favour combined modality treatment with involved-field radiotherapy and our results support its continued use.
Study on Specific Anti-Leukemia Function of Dendritic Cells (DC) Vaccine Induced from Chronic Myeloid Leukemia (CML).
Authors: Qiao ZhenHua , Zheng Z.Z. , Zhu L.
Published: October 2019

Objective In this article, we explore the feasibility of dendritic cells (DCs) induced from chronic myeloid leukemia (CML) with different cytokine cocktails and the mechanisms of immunological responses by specific T lymphocytes primed by this kind of DC vaccine.

Methods Monocytes (MNCs) for the generation of DC were collected from patients with high numbers of circulating peripheral blood CML cells, then cultured with different cytokine cocktails in different cultural medium (Fetal calf serum or Human serum albumin medium) to induce DCs. DCs were studied for morphology and immunofluorescent staining. RT-PCR was used to analyze the specific fusion genes and chromosome banding technique were used to detect Philadelphia chromosome (ph’). In vitro, DCs were used to induce auto-lymphocytes to be activated CTL, killing rates and T subgroups were assayed, IL-12 contents in the supernents were assayed also.

Results After induction, all these CML-derived MNCs developed typical DC morphology and the DC-associated surface molecules upregulated. The contents of IL-12 in the co-culture system of CML-DC and lymphocytes increased and the activated lymphocyte skewed from Th1 to Th2. At and effector: target ratio of 20:1, 80.67±6.43%, 62.33±12.01%, 23.92±5.62% cytotoxicity was noted with CML-DC/AL against autologous leukemia cells, K562, HL60, exhibiting more killing activities to auto-CML cells than K562 and HL60 cell.

Conclusion The results of this research will supply new approach for specific immunotherapy of CML and supply the application value for the CML derived DC vaccine against CML.
Imatinib Does Not Inhibit Neutrophil Oxidative Burst Function in Patients with Chronic Myelogenous Leukemia (CML).
Authors: Eksioglu-Demiralp Emel , Bizargity Peyman , Adiguzel Cafer , Ozilhan Gozen , Bayik Mahmut
Published: October 2019

Imatinib mesylate is a competitive Bcr-Abl tyrosine kinase inhibitor which has been used in treatment of chronic myelogenous leukemia (CML). In addition to the inhibition of the Bcr-Abl protein tyrosine kinases, it also shows activity against other protein kinases such as platelet-derived growth factor receptor (PDGF-R), c-Kit and protein kinase C (PKC) in higher doses (Manley et al., 2002). Myelosuppression has been reported up to 60% of patients with CML under imatinib therapy (Deininger et al., 2003). Microbicidial activity of the neutrophils is mainly generated by the enzyme NADPH oxidase which produces superoxide anion. Several protein kinases including protein kinase C (PKC) that phosphorylate components of the NADPH oxidase and regulate oxidase activity have been identified. Little has been known about the effect of imatinib on neutrophil function, so far. Therefore, we aimed to evaluate the neutrophil respiratory burst activity using Phorbol Myristate acetate (PMA), a PKC activator, in vitro, in patients with CML achieving imatinib. Heparinized peripheral blood samples were collected from 14 patients with CML under imatinib therapy (400 mg daily) for 9.4 ± 4 months and 13 voluntary healthy controls. Oxidative burst was evaluated using a standard method defined previously by flow cytometry (Robinson et al., 1993) Oxidative burst stimulation index was calculated as the ratio of mean value of fluorescence intensity of DCFH-DA labelled neutrophils before and following PMA stimulation. It has been found that the stimulation index of neutrophils in patient achieving imatinib was 14.5±5 which was comparable to healthy control group 14±6.6. Furthermore, no serious infection disease was clinically detected during 8 month follow up period. So this study suggest Imatinib which has ability to inhibit bcr-abl fusion protein selectively and also can inhibit some others kinases, however has no inhibitory effect on the neutrophil function in patients with CML.
Effect of 5-Aza-2′-Deoxycytidine in High-Risk Myelodysplasia Cells in Vitro.
Authors: Tong Hongyan , Lin Maofang , Jin Jie
Published: October 2019

5-Aza-2′-deoxycytidine (5-Aza-CdR; Decitabine) is an active antineoplastic agent in patients with leukemia. Due to its unique mechanism of action of demethylating DNA, 5-Aza-CdR has the potential to activate tumor suppressor and differentiation genes that have been accidentally silenced by DNA methylation in leukemic cells. Since5-Aza-CdR can induce clinical remissions in patients with high-risk myelodysplastic syndromes (MDS), we therefore attempt to investigate the mechanisms of the effect of 5-Aza-CdR by using MUTZ-1 cells in vitro, which are confirmed as a high-risk myelodysplasia Cell line that derived from a MDS patient (FAB subtype refractory anemia with excess of blasts). Our results indicated that 5-Aza-CdR showed inhibition of the growth of MUTZ-1 cells. The IC50 values of 24 hours, 48 hours and 72 hours were 6.75mmol/L, 2.82mmol/L and 5.45mol/L respectively. Characteristic changes of apoptosis emerged in MUTZ-1 cells after being exposed to 5-Aza-CdR in the different concentration from 0.8 mmol/L to 3.2 mmol/L, and the positive cells of Annexin VFITC on the membrane of MUTZ-1 cells were analyzed by flow cytometry. The percentage of apoptosis cells treated by 5-Aza-CdR at a dose of 3.2mmol/L for 48 hours was higher than that in untreated cells (16.7%±5.3% vs 2.9%±1.2%, p<0.05). The CpG islands in the promoter regions of p15INK4B gene were found to be hypermethylated in MUTZ-1 cells, resulting in a significant reduced expression of p15INK4B gene in the cells. However, the expression of p15INK4B gene was recovered and the methylation level of p15INK4B gene was also decreased (densitometry readings: 253.994±26.536 vs 369.641±28.915, p=0.003), after the cells exposed to 5-Aza-CdR at a dose of 3.2 mmol/L for 48 hours. Furthermore, 5-Aza-CdR could significantly down-regulate the expressions of DNA methyltransferase genes DNMT3A, which encodes a deno methyltransferase, at mRNA level in a dose dependent manner(r=−0.879,p=0.03). However, it had no effects on DNMT3B gene and DNMT1 gene that codes a maintenance methylase. The expression level of DNMT3A mRNA in MUTZ-1 cells treated with 5-Aza-CdR at a concentration 3.2 mmol/L for 48 hours was lower than that in untreated cells (densitometry readings:0. 385±0.086 vs 0.654±0.074 p<0.05). These observations suggest that 5-Aza-CdR can inhibit the growth and induce the apoptosis of MUTZ-1 cells within the range of concentration from 0.8 mmol/L to 3.2 mmol/L, which might be one of the mechanisms of antitumor effects of 5-Aza-CdR. The drug can activate the expression of p15INK4B gene in MUTZ-1 cells by demethylation of the p15INK4B gene through inhibiting the expression of DNMT3A gene. That might be the mechanisms of 5-Aza-CdR in the treatments of MDS.
Increased Platelet Turnover and Platelet P-Selectin in Patients with Essential Thrombocythemia and Thrombosis.
Authors: Arellano-Rodrigo Eduardo , Alvarez-Larran Alberto , Cervantes Francisco , Reverter Juan-Carlos , Villamor Neus , Montserrat Emili
Published: October 2019

Essential thrombocythemia (ET) is a myeloproliferative disorder associated with an increased tendency to thrombosis, the pathogenesis of which is not well known. The aim of the present study was to analyze the relationship between platelet turnover and activation and a history of thrombosis in patients with ET. The expression of platelet P-selectin (measured both in unstimulated platelets and after agonist-stimulation with ADP, thrombin, arachidonic acid (AA), collagen or epinephrine) and the percentage of reticulated platelets (RP) were measured in 53 ET patients with (n=26) or without (n=27) previous thrombosis and in 26 healthy controls. Both variable were assessed using quantitative whole blood cytometry and the results were expressed as percentages and in MESF (molecules of equivalent soluble fluorochrome) units. The results were as follows: 1) patients with thrombosis showed a significantly higher basal platelet P-selectin expression (6.1%, 95% CI: 4.6–6.8) than those without thrombosis (4.6%, 95% CI: 3.7–5.5)) and the healthy controls (3%, 95% CI: 2–4.2) (p= 0.0001 for all comparisons); 2) following thrombin activation, ET patients had a higher mean percentage of platelet P-selectin expression than the controls (61% versus 46%, p = 0.01), but no difference was seen between the two groups of patients; 3) after AA activation, patients with ET and thrombosis also showed higher platelet P-selectin values (78%, 95% CI: 69–86) than those without thrombosis (63%, 95% CI: 55–76) and the controls (46%, 95% CI: 33–58) (p=0.001 for all comparisons); 4) ET patients with thrombosis had a significantly higher mean percentage of RP (18%, 95% CI: 13–20) than patients without thrombosis (13%, 95% CI: 11–18) and the controls (8%, 95% CI: 5–10) (p = 0.0001 for all comparisons). No differences were observed when the patients were separated on the basis of the treatment that they received at the time of study. The above results point at a possible role of AA-induced platelet acitvation in the development of thrombosis in ET and give conceptual support to the prohylactic use of aspirin in such patients.
Comparison of Cytokine Efficacy on Ex Vivo Expansion of Megakaryocyte (MK) Progenitor Cells between Essential Thrombocythemia (ET) and Normal Person.
Authors: Do Young-Rok , Kwon Ki Young , Song Hong Suk , Kim Heung Sik , Hyun Myung Soo
Published: October 2019

Essential thrombocythemia (ET) is a chronic myeloproliferative disorder (MPD) of insidious onset and characterized by regulatory defect in stem cells. It is possible that ET can induce autonomous megakaryocyte (MK) growth. The physiology of megakaryocytopoiesis was not well defined, and, as a consequence, the pathophysiology of ET characterized by excessive platelet production was poorly understood. Secondary thrombocytopenia, especially post-transplantation, could be fatal so there were many trials to overcome this problem. This experiment was designed to test the effect of cytokines on ex vivo expansion of MK progenitor cells from ET. The MK colony forming unit (CFU-MK) expansion degree according to single or combination cytokines were analyzed in normal person and ET patients. Mononuclear cells were isolated from bone marrow aspiration and then cultured in serum-free medium (MegaCult™) supplemented with SCF, G-CSF, TPO, IL-11 and 3 types of cytokine combination. After 14 days of ex vivo expansion and then the CFU-MK were counted by microscope. In the absence of cytokines, CFU-MK of ET significantly expanded than normal (p<0.05), which represented autonomous CFU-MK formation. There were significantly increased mixed CFU-MK of ET in SCF, G-CSF and TPO (SGT) combined cytokine added well (p<0.05) but pure and total CFU-MK of SGT added well did not show significant expansion. When the effect of cytokines were compared between normal and ET, all of the cytokines significantly expand CFU-MK in ET (p<0.05) than normal and especially in pure CFU-MK. In summary, autonomous MK growth was shown in ET and SGT can be relatively effective cytokine combinations in ex vivo CFU-MK expansion. Further investigation will be needed to find out the most effective culture system and cytokine combination for ex vivo MK progenitor expansion.
Comparison of Antigen-Loading Strategies for DC-Based Immunotherapy of B-CLL.
Authors: Mellstedt Hakan , Kokhaei Parviz , Osterborg Anders , Choudhury Aniruddha
Published: October 2019

The slow indolent nature of B-CLL makes it eminently suitable for immune-based treatment strategies. Currently, there exist very few CLL-associated, defined antigen which can be used in vaccination approaches. The use of whole tumor preparations in conjunction with dendritic cells (DC) as cellular adjuvants presents an alternative method for generating therapeutic T-cell responses in CLL patients. We have examined various strategies of loading DC with whole tumor antigens; viz. tumor-DC hybrids, endocytosed apoptotic bodies, RNA or lysate from B-CLL cells. Dendritic cells were generated in vitro, using GM-CSF and IL-4 from immunomagnetically purified, CD14+ monocyte precursors obtained from the peripheral blood of B-CLL patients. The immature DC were loaded with whole tumor antigen using the above methods and matured using TNFα. The tumor antigen-loaded DC were compared for their ability to stimulate autologous T-cell responses. Among all the antigen-loading methods tested, DC that had endocytosed apoptotic bodies (Apo-DC) consistently generated the greatest numbers and magnitude of reactive T-cells as quantified in proliferation and ELISPOT assays. RT-PCR analysis for cytokine mRNA revealed that T-cells stimulated by Apo-DC resulted in an immune response that was almost entirely of the TH1 type as manifested by the production of mRNA for IFN-γ and TNFα. In contrast, other methods of loading antigen resulted in a mixed TH1-TH2 population with varying amounts of TH2 cytokines like IL-4 and IL-10.

In a separate series of experiments we compared the T-cell stimulatory capacities of cryopreserved and thawed, with fresh antigen-loaded DC. The results of these studies are essential for the development of a protocol for clinical therapy of B-CLL patients using Apo-DC. Our results indicated that cryopreserved DC could be recovered with high viability. A comparison of functional abilities demonstrated that no significant differences in T-cell stimulatory capacity between cryopreserved and fresh Apo-DC could be noted over a wide variety of immunological assays. Cumulatively, our results suggest that Apo-DC may be a suitable vaccine candidate for immunotherapy of B-CLL patients.
A Randomised Controlled Trail of Plasma Exchange in Rapidly Progressive Renal Failure of Myeloma.
Authors: Clark William F. , Stewart A. Keith , Rock Gail A. , Sternbach Marion , Sutton David M. , Barrett Brendan J. , Heidenheim Paul A. , Churchill David N. ,   members of the Canadian Apheresis Group
Published: October 2019

In myeloma, plasma exchange (PE) has been suggested to prevent rapidly progressive kidney failure by reducing exposure to nephrotoxic light chains. We carried out a randomized controlled multi-centre trial comparing PE or no PE in 104 patients of whom 101 met the inclusion, exclusion criteria and 4 were lost to follow-up. We compared baseline characteristics as well as renal outcomes and performed a futility analysis to determine the sample size necessary for potential statistical significance for the changes noted. Thirty-nine patients were randomized to the control group and 58 to the PE group with a 6-month follow-up. The baseline characteristics of these 2 groups were similar including serum creatinine, dialysis dependence, age, gender, serum calcium, serum albumin, 24 -hour urine for protein levels and Durie-Salmon myeloma staging. Thirteen (33.3%) of the control group and 19 (33.3%) of the PE group died within 6 months of follow up. Ten patients (31%) in the control and 10 patients (21%) in the PE arm were dialysis dependent at 6 months. Seven patients (47%) came off dialysis in the control and 13 patients (59%) in the PE arm with the mean number of dialysis days from 0–6 months being 45.7±67.6 in the control versus 29.2±56.1 in the PE arm at 6 months. The mean serum creatinine in the control group was 314.6±256.1 μmol/L versus 215.4±215.3 μmol/L in the PE group and the composite end point of death, dialysis or serum creatinine >254 μmol/L occurred in 12 (30.8%) in the control and 11 (19.3%) in the PE arm. The futility analysis to indicate the per group sample size necessary to achieve statistical significance at 6 months for the difference we observed was infinite for cumulative mortality, 805 for dialysis dependence, 2418 for coming off dialysis, 321 for number of dialysis days, 132 for creatinine difference of 100 μmol/L and for the composite outcome of death, dialysis or creatinine>354 μmol/L, 737. We did not observe a statistically significant difference in mortality or renal morbidity for PE versus no PE in patients with myeloma and rapidly progressive kidney failure.
Fetal-Maternal Microchimerism: Basis for a Potential New Therapy for Malignancy?.
Authors: Gilmore Gary L. , Holm Melissa M. , Lister John , Shadduck Richard K.
Published: October 2019

Fetal-maternal microchimerism [MC] occurs during gestation or delivery, as cells traffic from the fetal circulation to the maternal circulation (and vice versa) via the placenta, and persist for decades post-partum. The reported incidence of MC is 33% in normal parous women. To explore the incidence and extent of MC among normal females and cancer patients for potential therapy, we obtained blood samples from 162 normal parous women, and 21 parous cancer patients. 30 non-parous women served as controls. The presence of male DNA was detected using two rounds of PCR with nested sets of primers specific for the Y chromosome. The sensitivity of the assay was validated at 1 male cell per 1 million female cells using DNA prepared from serial dilutions of (male) KG-1 cells in (female) K562 cells. The observed frequency of MC was dependent on the amount of DNA assayed: with 1 ug DNA [~300,000 genomes] per test, the frequency of MC was 18%. At 5 ug DNA [1.5 million genomes], this rose to 46%, and at ≥ 25 ug DNA [7.5 million genomes], 66% of normal parous women had MC. Among parous cancer patients, MC was present at 20% with 5 ug DNA, rose to 76% when ≥ 25 ug DNA or more was tested. The lower frequency of MC among cancer patients at 5 ug DNA may reflect the effects of prior chemotherapy. 2 out the 30 normal non-parous donors were chimeric; this number did not increase when more DNA was assayed. The reason for the MC among non-parous subjects is unclear. Our results indicate the MC is much more frequent among parous women than previously reported. Provided that MC confers long-lasting tolerance of fetal tissue antigens, this suggests that son-to-mother cellular therapy may be feasible for treatment of malignancy.
The Arrest of Cell Cycle Progression in Mixed Lymphocyte Reaction by Cultured Mouse Mesenchymal Stem Cell.
Authors: Kim Jeong-A. , Hong Sungyoul
Published: October 2019

Mesenchymal stem cells (MSCs) possess in vitro immunosuppressive properties that appear not to be major histocompatibility complex (MHC)restricted. This study evaluated the immune suppressive effect of mouse MSCs on mixed lymphocyte reaction (MLR), and the mechanisms were investigated. MSCs were obtained from BALB/c bone marrow and cultured in low-glucose DMEM media. The expression of surface antigens and cell cycle were analyzed by flow cytometry. The MSC-induced suppression was assessed by MLR and transwell culture. The BALB/c MSCs constitutively expressed MHC class I and CD54 (ICAM-1) antigens but were negative for MHC class II, CD40, CD80 (B7–1) and CD86 (B7–2) antigens. MSCs suppressed allogeneic C57BL/6 T lymphocytes proliferation by adding them to MLR in which C3H spleen cells were used as a stimulator. This inhibition was dependent on the dose of BALB/c MSCs but independent of MHC. C57BL/6 T lymphocytes proliferation was still inhibited when BALB/c MSCs were added in culture 3 days after starting of MLR. When MSCs were separated from C57BL/6 T cells by using the transwell membrane, the suppression of immune response wasn’t observed, which suggested that the suppressive effect was dependent on cell-cell contact between BALB/c MSCs and C57BL/6 T cells. When C57BL/6 T lymphocytes were cultured with MSCs, the percentage of C57BL/6 T cells in G0 phase increased from 51.8±7.66% to 77.2±7.39% compared with the case that only C57BL/6 T cells were cultured. When the C57BL/6 T cells were cultured with C3H spleen cells, most of C57BL/6 T cells were in G2/M (96.38±3.33%). But by the addition of MSC to MLR, the percentage of T cells in G2/M decreased to 33.0±9.66% while that of T cells in G0 increased from 2.0±0.71% to 66.2±7.46%. We concluded that the cell cycle of responder T lymphocytes in MLR is arrested at G0 phase by MSCs.

Figure Figure
Glucocerebroside Alleviates Chronic Graft-Versus-Host-Disease in a Murine Model.
Authors: Nagler Arnon , Margalit Maya , Ohana Meir , Alper Ruslana , Pappo Orit , Rabbani Elazar , Engelhart Dean , Ilan Yaron
Published: October 2019

Background: Chronic graft-versus-host-disease (cGVHD) is a major obstacle to successful bone marrow transplantation. NKT lymphocytes may have a role in regulation of the immune attack in cGVHD. Glucocerebroside (GC) is a naturally occurring glycolipid that plays a role in the immune modulation of NKT lymphocytes.

Objectives: To determine the immune modulatory effect of GC in a murine model of cGVHD.

Methods: cGVHD was generated by infusion of 2x107 splenocytes from B10.D2 donor mice into Balb/c recipient mice, which received 7Gy 60Co total body irradiation (TBI) prior to transplantation. Recipient mice were followed for 55 days. Histological parameters of cGVHD-associated cutaneous, liver and bowel injury were assessed. To determine the mechanism of GC-mediated immune modulation, intrahepatic and intrasplenic lymphocytes were isolated and analyzed by FACS for CD4+, CD8+,CD56+ and NKT subpopulations. Serum cytokine levels were determined.

Results: Treatment with GC significantly alleviated cGVHD. GC-treated mice gained weight and manifested a significant decrease in skin cGVHD clinical score (0.91 vs. 1.66 in GC treated vs. control mice, respectively). The beneficial effect of GC was associated with a 58% and a 31% reduction of the intrasplenic and the intrahepatic NKT cell number, respectively, accompanied by a 27% decrease of intrasplenic CD3+CD4+ T cells. Intrasplenic (but not intrahepatic) decrease in NK cell number was also observed in GC treated animals (48% vs. 7%). Administration of GC led to increased serum IFNγ and TNFa levels in GC treated compared to untreated controls (46 vs. 27, and 92 vs. 52 pg/ml, respectively), while IL10 levels decreased (61 vs. 83 pg/ml, respectively).

Conclusions: Administration of GC led to significant amelioration of chronic GVHD. This effect was associated with an altered NKT lymphocyte distribution, decreased numbers of effector T lymphocytes and a shift towards a Th1-type cytokine profile. Glucocerebroside may serve as a new therapeutic measure for amelioration of cGVHD.
Role of IL-4-Producing CD8+ T Cells in Patients with Chronic Graft-Versus-Host Disease (cGVHD) and in Normal Healthy Volunteer.
Authors: Nakamura Kengo , Takebayashi Masashi , Tajima Kenichirou , Tanijiri Tsutomu , Yokoi Takashi , Son Yonsu , Matsumoto Noriaki , Zen Katsuhiro , Kishimoto Yuji , Amakawa Ryuichi , Fukuhara Shirou
Published: October 2019

The pathophysiology of cGVHD still remains unclear. To gain more insight into the immunological mechanism of cGVHD, we examined surface marker and cytokine production of peripheral blood T cells from 19 patients in the chronic phase of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The median age of patients was 31 years (range 13–47 years). The median days from allo-HSCT to examination was 43 months (range 17–94 months). Ten patients in whom 6 have treated with immunosuppressive agents and 4 without them developed chronic GVHD, whereas 9 patients in whom 5 have treated with immunosuppressive agents and 4 without them did not develop chronic GVHD. Peripheral blood mononuclear cells were extracted from patients and 10 healthy volunteers, followed by isolation into CD4+ T cells and CD8+ T cells. The analysis of surface marker and intracellular cytokine production of these cells was performed using fluorescence activated cell sorter. The percentage of IFN-g-producing CD8+ T cells among CD8+ T cells was significantly higher in patients with or without cGVHD than in healthy volunteers (p<0.001). Since all 19 patients were in remission, the IFN-g-producing CD8+ T cells might be related to graft-versus-leukemia effect. On the other hand, the percentage of IL-4-producing CD8+ T cells among CD8+ T cells was significantly higher in patients with cGVHD (mean 3.3%, range 1.3–8.2%) than in patients without cGVHD (mean 1.2%, range 0.8–1.7%) and healthy volunteers (mean 1.1%, range 0.1–1.6%) (both p<0.001). By contrast, the percentage of IL-4-producing CD4+ T cells was not different among patients with and without cGVHD and healthy volunteers. Then, we minutely explored the immunological role of IL-4-producing CD8+ T cells in healthy volunteers. These cells expressed the surface marker of CD25, CCR4 and CTLA-4. In the analysis of cytokine production in these cells, we identified the type 2 cytokine, such as IL-4, IL-5, IL-10, and IL-13, although we could not find IFN-g. In conclusion, these findings suggest that IL-4-producing CD8+ T cells may be an immunological marker of cGVHD in which these cells may play a crucial role as regulatory T cells.
Protective Effect of Curcumin in an F1-Hybrid Model of Acute Graft-vs.-Host Disease.
Authors: Ellison Cindy A. , Natuik Shannon A. , Fischer Jacqie M. , Gartner John G.
Published: October 2019

Curcumin (diferuloylmethane), a pigment from the rhizomes of Curcuma Longa, has anti-oxidant, anti-tumor and anti-inflammatory properties. It has been shown that curcumin can inhibit the development of experimental allergic encephalomyelitis, a Th1-mediated, autoimmune, demyelinating disease that affects the central nervous system in mice. Because acute GVHD involves the development of a Th1-mediated immune response, and is characterized by the development of a potent, systemic, inflammatory response, we wished to determine whether curcumin might also protect mice from developing acute GVHD in the C57BL/6→(C57BL/6 x DBA/2)F1-hybrid model. We found that ip injections of curcumin (100 micrograms dissolved in 25 microliters of DMSO), given once a day, every 2 days, protected recipients from developing acute, lethal GVHD. Two thirds of the treated group survived well past day 100 post-transplantation. Control GVH mice treated with DMSO alone became moribund within three weeks of transplantation, the time at which mice with acute GVHD typically succumb. To ensure that the protective effects of curcumin were not due to abortion of the graft, we analyzed T cell engraftment in some of the long-term survivors. In the surviving mice tested on days 135 and 275 post-transplantation, we were unable to detect any cells of recipient origin in the spleen. Furthermore, the percentage of donor-derived CD4+ cells ranged from 32–47% and the percentage of CD8+ T cells ranged from 25–42% in the non-adherent spleen cell fraction. Data from these experiments suggest that curcumin can protect mice from developing acute, lethal GVHD.
Genetic Susceptibility Loci for Lethal Graft-Versus-Host Disease in MHC-Identical Mice: Initial Results from a Genome-Wide Scan.
Authors: Cao Thai M. , Pang Wendy W. , Grumet F. Carl , Lazzeroni Laura C. , Shizuru Judith A.
Published: October 2019

Minor histocompatibility antigens (mHAg) are T-cell peptide antigens derived from polymorphic endogenous gene products encoded throughout the genome. Despite extensive human genetic diversity, recent experimental evidence suggests that a mHAg set may be limited in size because alloimmune responses directed against them can be dominated by only a few specific peptides. To extend upon these observations, we performed a genome-wide scan to identify susceptibility loci for graft-versus-host disease (GVHD) in a murine model. We previously identified B10.BR (H2k) mice to be resistant, and BALB.K (H2k) mice susceptible, to lethal GVHD when transplanted with hematopoietic cells from MHC-matched AKR/J (H2k) donors (Cao et al. PNAS2003:11571). In the present study B10.BR, BALB.K, or (B10.BR x BALB.K)F1 mice were lethally irradiated and injected with AKR/J bone marrow at radioprotective doses plus 2.0 or 3.0 x 106 purified splenic T-cells. Confirming our prior observations, B10.BR mice survived to day 60 whereas all BALB.K mice died prior to day 10 from GVHD. All (B10.BR x BALB.K)F1 mice also died after a median of 10 days, indicating that GVHD susceptibility in BALB.K mice is a dominant trait. (B10.BR x BALB.K)F1 mice were then backcrossed to the GVHD-resistant B10.BR parent. Resulting [B10.BR x (B10.BR x BALB.K)]BC mice (n = 180) were genotyped and similarly irradiated and injected with AKR/J bone marrow plus 3.0 x 106 splenic T-cells. Forty (22%) BC mice died with signs of GVHD prior to day 100. Surprisingly, a 90-marker genome-wide scan for this phenotype, lethal GVHD prior to day 100, revealed highly significant linkage (P <.001) only to chromosome 16. Linkage analysis was performed with MapManager QTX 0.29 using Kosambi’s map function. As shown in Figure 1, interval mapping for chromosome 16 with additional markers revealed a broad and complex linkage pattern encompassing 2 markers, D16Mit4 (27.3 cM) and D16Mit189 (55.2 cM), that had peak LOD scores of 8.8 and 8.0 respectively. Another genetic locus on chromosome 13 flanked by markers D13Mit248 and D13Mit149 with a peak LOD score of 2.3 by interval mapping was suggestively linked. While an evaluation of candidate genes within the support intervals is in progress, these results thus far suggest that the critical gene dose required for inducing lethal GVHD may be remarkably small. If validated as encoding for immunodominant mHAg, these results furthermore suggest that a functionally relevant mHAg set may be definable for clinical genotyping.