bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytoprotective Effect of ICL670 (Deferasirox) on At20 Endocrine Cells Susceptible to Iron-Evoked Oxidative Damage.
Authors: Glickstein Hava , Nick Hanspeter , Cabantchik Zvi Loav
Published: October 2019

A major cause of biological damage associated with tissue iron accumulation is the cellular acquisition of circulating labile plasma iron (LPI) by endocytic mechanisms and ensuing metal-catalyzed oxidations. Endocrine cells are presumed to have endocytic abilities and/or high susceptibility to the formation of reactive oxygen intermediates (ROIs), particularly those generated in the presence of labile iron. As these properties might be implicated in the early onset of endocrine dysfunctions observed in transfusional hemosiderosis, a major goal of iron chelation therapy is to prevent iron ingress into endocrine cells and ensuing toxicity and/or removal of cell-accumulated iron. Such properties of chelators have been afforded in cardiac cells (Glickstein et al. 2006, Blood In press) by treating LPI-carrying plasma with pharmacologically attainable concentrations of orally active iron chelators in clinical practice. The latter also evoked neutralization of metal-catalyzed ROS formation, by iron chelation and extrusion from cells and thereby supported cardiac functional restoration. We explored here whether addition of ICL670 (deferasirox) could rescue acutely iron-overloaded At20 pituitary cells from iron-toxicity, using fluorescence-plate reader and microscopy-imaging for live monitoring of cell functions and assessing toxicity in terms of:cell integrity (calcein cell retention assay);metabolic activities (mitochondrial Alamar-Blue assay); andintracellular (ACTH) hormone levels. Our studies indicate that in cell culture conditions and at pharmacological chelator concentrations, ICL670 can preserve both At20 endocrine cell viability and functional properties that were compromised by iron-catalyzed formation of ROIs.
Chance Detection of CD16 Deficiency on Polymorphonuclear Neutrophils in Iron Deficiency Anemia.
Authors: Kamat Anil V. , Ezekwesili Raphael
Published: October 2019

Introduction CD16 (FcgRIII) is a low affinity receptor for immunoglobulin G(IgG)constitutively expressed on polymorphonuclear neutrophils, monocytes and NK-cells with various effector functions including phagocytosis of opsonized particles or of immune complexes and in antibody-dependent cellular cytotoxicity (ADCC). Congenital defect of neutrophil CD16 expression has been reported with variable phenotypic associations such as anaemia, neutropenia, autoimmune thyroiditis, recurrent infections, acute myeloid leukaemia, systemic lupus erythematosis and in mothers whose newborns have developed transient neonatal alloimmune neutropenia.

CD 16 deficiency on neutrophils -A 46 year old lady presented with extreme tiredness with her full blood count showing Hb 7.6 g/L MCV 76.0 fl MCH 23.8 pg WBC 5.9 X 10^9/L Neutrophil count 3.5 X 10^9/L Platelet count 290 X 10^9/L.Her serum ferritin level was 2.2 mcg/l with normal B12 & folate levels. Hemoglobinopathy screen was normal. Blood film was consistent with iron deficiency. She denied any history of blood loss. In view of positive faecal occult blood, she underwent upper & lower gastrointestinal tract endoscopies which failed to reveal any bleeding lesion. Red cell scan was considered but not arranged as clinically she was not bleeding at a rate more than 1 ml per minute. Capsule enteroscopy was not available. Coeliac screen was negative. Due to intolerance of oral iron supplementation, she needed parenteral iron infusions, which improved her hemoglobin.

However, the response was not sustained which prompted further work up. She underwent bone marrow aspirate & trephine biopsy which was suggestive of mild dyserythropoeisis. Cytogenetics was normal. Hemolytic screen was negative. On immunophenotyping as part of Paroxysmal Nocturnal Hemoglobinuria (PNH) screen, neutrophils showed normal expression of CD 59 (89%)& CD 66b (100%)but completely lacked CD 16 (02%). Urinary haemosiderin was negative. She had no history of increased frequency or of exceptionally severe or persistent infections. Family studies are pending. She remains iron replete on parenteral supplementation.

Conclusion- Polymorphonuclear neutrophils totally deficient in CD16 were detected by chance. CD16 deficiency has not compromised the host defence in this patient. We have not assessed CD16 specific mRNA or functional parameters such as phagocytosis of opsonized bacteria and superoxide production since the patient had no history of increased infections. The other receptors for IgG apparently can compensate for the lack of CD16. Congenital deficiencies of CD16 are very rare. The clinical relevance of congenital defect of neutrophil CD16 expression remains unclear. This case substantiates the inferences of other sporadic reports of there being no compromise of host defence resulting from isolated CD16 neutrophil expression defect.
High Levels of Soluble P-Selectin Are Associated with the Risk of Venous Thromboembolism and the P-Selectin Thr715 Variant.
Authors: Ay Cihan , Jungbauer Lea V. , Sailer Thomas , Tengler Theres , Koder Silvia , Kaider Alexandra , Panzer Simon , Quehenberger Peter , Mannhalter Christine , Pabinger Ingrid
Published: October 2019

The cell adhesion molecule P-selectin, which is found in the alpha granula of platelets and in Weibel-Palade bodies of endothelial cells, has been shown to play an important role in the pathophysiology of thrombosis. However, the meaning of soluble P-selectin (sP-selectin) in venous thromboembolism (VTE) is still uncertain because clinical data are limited. The purpose of this study was to investigate whether sP-selectin is associated with the risk for VTE and to elucidate the association of sP-selectin and the P-selectin gene variation (SELP) Thr715Pro in high risk patients with recurrent VTE. We recruited cases and control individuals between January 2005 and November 2005 for an analysis of sP-selectin plasma levels and the SELP Thr715Pro. Patients with a history of objectively confirmed recurrent VTE and at least one event of an unprovoked deep venous thrombosis or pulmonary embolism were enrolled. Plasma was obtained at least 3 months after the most recent event of VTE. Age and sex-matched healthy individuals served as controls. sP-selectin levels were measured with a high sensitive ELISA. The variant for the P-selectin gene was determined by a mutagenically separated PCR followed by high resolution gel-electrophoresis. Hundred-sixteen patients (53 female / 63 male; mean age +/−SD: 56 +/−12 yrs) and 129 controls (66 female / 63 male; mean age +/−SD: 53 +/−11 yrs) were enrolled. Mean concentration (+/−SD) of sP-selectin (ng/mL) was significantly higher in patients than in controls (47.28 +/−15.00 vs. 36.77 +/−11.00, p<0.001). The unadjusted odds ratio (OR) of elevated sP-selectin (cut-off level 55.10 ng/mL representing the 95th percentile of the controls) was 8.5 (95% confidence interval (CI): [3.7–23.3], p<0.001) and increased after adjustment for factor V Leiden, prothrombin G20210A variant, elevated factor VIII level, hyperhomocysteinemia and BMI (OR=11.1, 95% CI [4.3–33.0], p<0.001). Carriers of the SELP Pro715 were more prevalent among controls than patients (21.7% versus 14.7%), however, the difference was statistically not significant (p=0.19). The subgroup of carriers of the SELP Pro715 (n=45) had significantly lower sP-selectin levels than non-carriers (31.31+/−7.94 vs. 44.10+/−14.08, p<0.001). In conclusion, our study shows a highly significant association between elevated sP-selectin levels and VTE. Furthermore, sP-selectin levels correlate with genotype status and individuals carrying the SELP Pro715 have lower levels of sP-selectin.
Detection of HTLV1 in Patients with B and T-Cell Malignancies Which Are Different to Adult T-Cell Leukemia-Lymphoma: A Prospective Study.
Authors: Marquez Jose J.M. , Ortiz Natalia N.O. , Cisterna Ramon R.C. , de Heredia Jose Maria J.B. Beltran
Published: October 2019

Human T-lymphotropic virus type I (HTLV-I), is the pathogenic agent of adult T-cell leukemia-lymphoma (ATLL), which always shows monoclonal HTLV-I provirus DNA integration. HTLV-I is rarely associated with B-cell disorders. The gen Tax of a human T-cell leukemia virus type I (HTLV-I) induces the expression of several cellular genes that are involved in T cell activation and proliferation. In the current study was to analyze prospectively HTLV-I proviral DNA presence in peripheral blood mononuclear cells (PBMCs) in patients with leukemia or lymphoma which are different to ATLL. Following this pattern we have studied in 61 patients with T and B-cell malignancies the presence of the virus HTLV-I on the during our service between April 2004 and May 2006.

A real time PCR assay using SYBR Green intercalation was established. Primer and hybridization probes targeting tax region were standardized against MT2 cell line DNA for HTLV-I. The assay reliably detected a single copy of HTLV-I proviral genome in DNA from 1X106 PBMCs. Also included for each sample the HLA-DQ alpha gene (a measure of genomic DNA), We have detected the presence of HTLV-I in five patients: 2 patients with B-CLL (lymphocytes count: 1.3 x 109/L and 16,8 respectly), one patient with T-ALL CD4 and CD8 positive; other with NHL and one with B-ALL Ph+. As an interesting data, one of the patients with B-CLL presented as complication a immune thrombocytopenia after Fludarabine therapy. Two patients were died and the two cases with B-CLL show stable sickness. Only the patient with T-ALL achieved complete remission after chemotherapy. In conclusion, we have detected on our series, in a unsuspected way, the presence of HTLV-I on some of the patients with lymphoid malignancies, which are different to the ATLL, showing in these cases an atypical clinic course. After knowing the repercussion of HTLV-I over the immune competition of the infected patients it seems suggestive speculate on the possible implication of the virus in a deterioration of the immunity favouring the progress of neoplasia and/or a greater propensity to infections and immunological upheavals, like the observed ones in our series.
The Effects of Glycoprotein IIb/IIIa Antagonist RGDS on Platelet Aggregation and Release Reaction In Vitro.
Authors: Xie Shuangfeng , Yin Songmei , Nie Danian , Li Yiqing , Wang Xiuju , Ma Liping , Yang Guolei
Published: October 2019

Platelet activation, including platelet adhesion, platelet aggregation and platelet release reaction, played an important role in thrombogenesis. We all knew that Platelet glycoprotein IIb/IIIa antagonist was the most effective drug for anti-aggregation, while we don’t know clearly its effect on platelet release reaction and the relations between its effects on platelet aggregation and release reaction. Platelet release reactions included α-granules and dense granules releasing. When α-granules were released, its membrane glycoprotein CD62p was expressed in the platelet membrane. We used the CD62p expression as the index of platelet release reaction. In the current study, the 4-peptides RGDS (Arg-Gly-Asp-Ser) was used as glycoprotein IIb/IIIa antagonist. We detected the effects of RGDS on platelet aggregation and CD62p expression induced by adenosine diphosphate (ADP) (finial concentration, 5μmol/L) in vitro. 50, 100, 200, 400 and 800μmol/L RGDS were used separately in the test. RGDS of each concentration could significantly inhibited maximal platelet aggregation (PAG(M)) induced by ADP, the 50% inhibiting concentration was approximately 200μmol/L. 800μmol/L RGDS could inhibited PAG(M) by 80.48±8.18%. Only ≥200μmol/L RGDS could significantly inhibited platelet CD62p expression. 800μmol/L RGDS could inhibit platelet CD62p expression by 27.31±9.74%. The inhibiting effect of RGDS on PAG(M) and platelet CD62p expression had significantly correlation (r =0.976, P<0.05). These results indicated that RGDS in low concentration (<200μmol) had little negative effect on platelet release reaction induced by ATP, while in relatively high concentration (≥200μmol) RGDS could inhibit platelet release reaction. When RGDS concentrations were same its effect on platelet release reaction was much less than that on platelet aggregation, which indicated that platelet glycoprotein IIb/IIIa compound could only partly participated in the platelet release reaction but fully participated in platelet aggregation induced by ADP.
Use of CSGE, TspRI- RFLP and Western Blot in Carrier Detection in an Indian Family with Type I Glanzmann Thrombasthenia.
Authors: Kannan Meganathan , Ahmad Firdos , Kumar Rajive , Choudhry Ved P. , Saxena Renu
Published: October 2019

Glanzmann Thrombasthenia (GT) is an inherited, autosomal recessive, bleeding disorder which is characterized by absent/reduced platelet Glycoprotein IIb/IIIa. The sub classification of GT into Types I, II and III is based on the levels of GPIIb/IIIa by flow cytometry. Type I is the most severe form of GT and is found to be most common in north Indian population. Since not much study is available on carrier detection based on western blot analysis, it is suggested to confirm the defect in carriers by molecular diagnosis. Here we present a carrier status using TspRI in a family with Glanzmann Thrombasthenia patient. Glanzmann Thrombasthenia was diagnosed in a patient with bleeding manifestations accompanied by absent platelet aggregation, secondary to ADP, ADR, Arachidonic acid and collagen. The patient was sub typed as Type I based on flow cytometry analysis as he had absent GPIIb/IIIa. Patient’s DNA was analyzed for mutation in both the gpIIb and gpIIIa genes by CSGE, followed by sequencing. The patient was found to have mutation, CTG>CCG at exon 12 of GPIIb gene. The mutation caused amino acid change from Leu to Pro in the GPIIb protein. The same mutation was looked for in all the family members (Both parents and two siblings) using CSGE and by TspRI- RFLP analysis. Both the parents and siblings were heterozygous for this mutation, where as patient was homozygous (Fig 1). As this mutation is not present in the normal individuals and is not reported earlier, this considers being a novel mutation. Presence of abnormal protein in the family members was revealed by western blot analysis for GPIIb (Fig 2). The same mutation is being looked for in more number of patients with Glanzmann Thrombasthenia using TspRI- RFLP. So far, a total of two out of 25 GT patients found to carry this mutation. It is possible that abnormal GPIIb protein by western blot in family members may reflect their carrier status. It is also postulated that western blot and CSGE of GPIIb and IIIa in parents/siblings may detect carrier status in Glanzmann Thrombasthenia.

Fig 1: Carrier detection by restriction digestion using TspRI Fig 1:. Carrier detection by restriction digestion using TspRI Fig 2: Immunoblot followed by chemiluminescent detection shows absent/reduced protein in patient and abnormal band pattern in the family members Fig 2:. Immunoblot followed by chemiluminescent detection shows absent/reduced protein in patient and abnormal band pattern in the family members
Rituximab in Children with Autoimmune Thrombocytopenia Complicating Underlying Congenital or Acquired Immunodeficiency State.
Authors: Junagade Pritesh S. , Height Susan , Ruiz Gary , Hadzic Dino , Ball Colin , Rees David
Published: October 2019

Rituximab (chimeric anti-CD20 monoclonal antibody) has been reported to be effective in various autoimmune conditions, especially in autoimmune cytopenias, but there are few reports of its use in paediatric population with underlying immunodeficiency states. We report three cases of autoimmune cytopenias (Evan’s syndrome in DiGeorge syndrome, autoimmune thrombocytopenia in a HIV positive patient and lastly autoimmune thrombocytopenia in a post liver transplant patient) where rituximab was effective in reversing the cytopenias. First patient (11yrs) had DiGeorge syndrome which was complicated by Evan’s syndrome. It was treated with multiple courses of steroids and immunosuppressive agents, with resultant recurrent systemic bacterial and fungal infections. She had rituximab as a last resort, which resulted in sustained improvements in her Haemoglobin and platelet count. Second patient (12yrs) was diagnosed with HIV in 1993. She had sudden onset Intra-cranial bleed as a result of immune thrombocytopenia. She was treated with Intravenous immunoglobulin (IVIg) and subsequently with prednisolone with no response. She however responded to rituximab and then went on to have rituximab for a further 2 courses, each 12 months apart. The third patient (14yrs) had auxiliary liver transplant in 2002. He developed immune thrombocytopenia and neutropenia in which remission was sustained only for a maximum of 6 weeks following IVIg. As he was already on immunosuppression, he was given rituximab to which he has had a sustained remission. Side effects: The patient with DiGeorge syndrome had recurrent chest infection which was thought to be due to aspiration as a result of her hiatus hernia. The other patients did not have major side-effects. Immunoglobulin levels in all three patients were in normal range. Thus, in our experience rituximab is a useful adjunct in management of autoimmune cytopenias with an underlying immunodeficiency state and should be considered early in steroid refractory cases, as it leads to sustained remissions in the blood counts. It is well tolerated without major infections in patients who are already immunosuppressed due to their immunodeficiency state and the fear of immuneparesis is not seen.
A Case Control Study of Deep Venous Thrombosis in Relation to Factor V G1691A (Leiden) and A4070G (HR2 Haplotype) Polymorphisms.
Authors: Almawi Wassim Y. , Borgi-Bouaziz Lobna , Nathalie Hezard , Philipe Nguyen , Touhami Mahjoub
Published: October 2019

Activated protein C resistance (APCR) is a significant risk factor for venous thromboembolism (VTE), and the factor V (FV) gene mutations G1691A (FV-Leiden) account for the majority of inherited APCR cases. An additional FV gene mutation, the A4074G (FV-HR2), was recently reported to induce a risk of VTE by some but not all groups. The aim of this study was to determine the prevalence of single and combined SNPs in 126 patients with documented deep venous thrombosis (DVT), and 197 control Tunisian subjects. The frequencies of FV-Leiden A allele (p <0.001; OR = 5.031), and HR2 G allele (p = 0.014; OR = 2.463) were significantly higher among DVT patients. Genotype differences were found between FV-Leiden G/A (p <0.001; OR = 3.936) and A/A (p = 0.013; OR = 11.529), but not HR2 A/G genotypes (p = 0.862; OR = 1.166), between patients and controls. While no linkage disequilibrium was noted between the FV 1691A and 4070G or A alleles, higher prevalence of the 1691G/4070G (p = 0.002; OR = 5.189) and the 1691A/4070A (p = 0.007; OR = 3.670) were noted among DVT patients than in control subjects. Collectively, this indicates that FV-Leiden, and to a lower extent HR2 haplotype, are important independent risk factors for DVT, and that their coinheritance does not increase significantly the DVT risk imparted by either.

Factor V-Leiden and HR2 Haplotype Allele and Genotype Analysis Cases Controls P OR Factor V-Leiden G 0.8492 0.9670 0.004 0.087 A 0.1508 0.0609 <0.001 5.031 G/G 0.754 0.939 <0.001 0.199 G/A 0.190 0.056 <0.001 3.936 A/A 0.056 0.005 0.013 11.529 HR2 Haplotype A 0.8913 0.9492 1.000 0.352 G 0.1087 0.0508 0.014 2.463 A/A 0.855 0.873 0.862 0.858 A/G 0.145 0.127 0.862 1.166 G/G 0.000 0.000
The Residual Risk of Transfusion-Transmitted HIV Infection in a Public Brazilian Blood Center.
Authors: Pinto Ana Cristina S. , Valente Vanderléia B. , Ubiali Eugênia Maria A. , Angulo Ivan L. , Covas Dimas T.
Published: October 2019

Estimating risks of transfusion-transmitted infections is essential for monitoring blood safety and for helping physicians and patients decide about the risks/benefits of an allogeneic transfusion. In 2000, the residual risk of HIV in our institution was estimated in 1/325,000 units transfused and the prevalence of HIV among all blood donors was 0.06%. The goal of this study was to calculate the current residual risk of transfusion-transmitted HIV infection in the Regional Blood Center of Ribeirão Preto, University of São Paulo, Brazil. We evaluated 292,587 repeated donations of a total of 403,568 donations between january/2002 and december/2005. During this period, we identified 43 seroconversions among the repeated donors. The serological tests performed were 2 different enzyme immunoassays (EIA): HIV 1/2 and a conjugated p24 antigen/antibody as screening tests and Western Blot as a confirmatory test for all positive samples. In order to calculate the residual risk, we used the incidence rate/window period (IR/WP) model, developed in parallel by investigators from the National Heart, Lung and Blood Institute-sponsored Retrovirus Epidemiology Donor Study (REDS), the CDC and the American Red Cross. The IR takes into account the number of incident cases (numerator), the total number of repeated donations and the mean interdonation interval length (multiplication of the last two entities provides the number of person-years at risk or denominator). The mean interdonation interval length can be substituted for the mean seroconversion interval length for all incident cases if the first one can not be accurately measured, so we used this alternative. Once the IR is known, we calculated the residual risk dividing the IR by 365.25 days and then multiplying by the WP of the test used (p24=15 days). Our results are: IR= 43 cases/292,587 repeated donations × 3.2 years = 4.59×10−5. The current residual risk is (IR/365.25 days) × 15 days= 1.5/million units transfused or 1/667,000 units. The prevalence of HIV among all blood donors in 2005 was 0.035%. Although we know that the correlation between prevalence and residual risk is not straightforward, they have a certain degree of correlation and they both have declined since 2000. During these last 5 years, we have made some modifications in our donor selection towards a more rigorous donor screening and since january/2002 we have introduced the p24 antigen/antibody EIA as part of the screening serological tests. We concluded that the more stringent donor risk factor screening and the introduction of the p24 antigen test have substantially reduced the residual risk of transfusion-transmitted HIV infection in our institution.
Signaling from the Oncogenic Fusion Protein BCR/ABL1 Leads to Expression of Wilms’ Tumor Gene 1 Protein (WT1), Which Induces Transcriptional Repression of Interferon Consensus Sequence Binding Protein (ICSBP) in Human Cells.
Authors: Vidovic Karina , Svensson Emelie , Borg Ake , Vallon-Christersson Johan , Lassen Carin , Hakansson Petra , Richter Johan , Olofsson Tor , Fioretos Thoas , Gullberg Urban
Published: October 2019

The Wilms’ tumor gene 1 (WT1) encodes a transcription factor highly expressed in most leukemias. Overexpression of WT1 and the fusion protein AML1-ETO in transgenic mice rapidly induces acute myeloid leukemia (AML), indicating an oncogenic effect by WT1. However, mechanisms behind expression of WT1 in leukemic cells, as well as mechanisms downstream of WT1, are largely unknown. Here, we report that the fusion protein BCR/ABL1, associated with chronic myeloid leukemia (CML), increases expression of WT1 mRNA and protein, which induces transcriptional represssion of ICSBP. Inhibition of BCR/ABL1 signaling by imatinib mesylate reduced WT1 mRNA levels in five different human CML cell lines, confirming previous reports (Cilloni D et al. Cancer 101:979, 2004). In extended investigations, we show that signaling via the phosphatidylinositol-3 kinase (PI3K)-Akt pathway is critical for BCR/ABL1 induced WT1 expression, while independent of JAK-STAT signaling. BCR/ABL1 signaling did not affect half-life of WT1 mRNA, but inhibition of BCR/ABL1 or PI3K strongly suppressed the transcriptional activity of WT1 promoter/enhancer reporters, indicating that BCR/ABL1 signaling increases transcription of the WT1 gene. Downstream of WT1, we report the interferon consensus sequence binding protein (ICSBP) gene as a new target gene of WT1. Increased levels of WT1 reduced the amount of ICSBP mRNA in both normal progenitors and in U937 leukemic cells. Moreover, WT1 repressed transcription from the ICSBP-promoter. Finally, we report that forced expression of BCR/ABL1 in human CD34+ bone marrow progenitor cells resulted in increased expression of WT1 mRNA and protein and repressed levels of ICSBP mRNA and protein, indicating that WT1 can repress expression of ICSBP. In contrast to the almost ubiquitously high expression of WT1 in leukemia, expression of ICSBP is very low or absent in most leukemias, consistent with the notion of WT1 as a repressor of ICSBP in vivo. Moreover, several reports point out ICSBP as an important suppressor gene in CML. Our results provide a mechanistic explanation for BCR/ABL1 induced repression of ICSBP via induction of WT1, and a general mechanism by which high expression of WT1 could contribute to repression of ICSBP also in other leukemias.
Enhancement of Imatinib-Induced Apoptosis through Catalase Activity Modulation in Imatinib-Resistant Chronic Myelogenous Leukemia Blastic Phase Cells.
Authors: Kwee Jolie Kiemlian , Ferreira Ana Carolina dos Santos , Luque Diogo Gomes , da Cunha Vasconcelos Flavia , Silva Karina Lani , Klumb Claudete Esteves , Maia Raquel Ciuvalschi
Published: October 2019

Therapeutic selectivity and drug resistance are two major issues in cancer chemotherapy. Strategies to improve therapeutic selectivity largely rely on our understanding of the biological difference between cancer and normal cells, and on the availability of therapeutic agents that target biological events critical for cancer cells but not essential for normal cells. Imatinib is a selective inhibitor of the BCR-ABL tyrosine kinase activity in chronic myelogenous leukemia (CML). Resistance to imatinib is a major problem in CML patients especially in blast phase (BP). Recently, it was also described that reactive oxygen species (ROS) is another mechanism through which imatinib induces apoptosis. To evaluate the possible role of catalase (a well known ROS enzyme scavenger) in imatinib resistant cells, we studied through apoptosis/necrosis analysis, K562 cells in presence or absence of 3-amino-1,2,4-triazole (AT), an inhibitor of catalase activity. Cell apoptosis index was tested in cells labeled with Annexin V-FITC/PI and analysed by flow cytometry. In order to examine the efficacy of imatinib apoptosis induction we evaluated firstly the apoptosis index in K562 cells after 24 hours of incubation with imatinib at the less cytotoxic concentrations of 0.5, 1.0 and 5.0 μM. Induced imatinib early apoptosis, was significantly higher, (25%, p < 0.05) than control in all concentrations tested. After that, we conducted the same assay in K562 cells previously treated with aminotriazole (AT-K562) and, to address the question of whether the resistance to imatinib could also be counteracted by glutathione modulation, another ROS scavenger, K562 cells previously treated with buthionine S,R-sulphoximine (BSO) a specific inhibitor of g-glutamylcysteine synthetase the rate limiting enzyme in glutathione synthesis, were also analysed. Untreated K562 cells and those previously treated with AT or BSO were incubated with imatinib (0.5, 1.0 and 5.0 μM) and hydrogen peroxide (H2O2) 1.0 μM, a well known apoptosis inductor inhibited by catalase. Only AT-K562 cells had their apoptosis significantly enhanced when compared with non treated cells (p < 0.05). Indeed, we showed that inhibition of catalase activity in K562 cells overcame imatinib resistance in 35% of these cells in the final concentration of 0.5 μM which is equivalent to the half mean plasma concentration used in clinical practice. The present findings suggest that combination of catalase inhibitors with imatinib in lower dosage represent an emerging therapeutic concept for preventing or overcoming resistance formation in CML. Therefore, further studies with drugs clinically available that inhibit catalase activity should be explored.
Ferrocenyl Nucleoside Analogs, a New Class of Cytostatic Drugs, Overcome Multiple Drug Resistance in Acute Lymphoplastic Leukemia Ex Vivo.
Authors: Prokop Aram , Eissmann Moritz , Jesse Patrick , Henze Guenter , James Philippe , Neudoerfl Joerg , Schmalz Hans-Guenther
Published: October 2019

In spite of the relatively good prognosis for de novo acute lymphoplastic leukemia (ALL), the survival rate for relapsed ALL is only 40%. The therapy of relapsed ALL and secondary leukemias with the common cytostatic drugs often show a poor response. Therefore new substances are required to lead multiple drug resistant leukemia cells into cell death. Nucleoside analogs play a very important role in anti-leukemic therapy. Here we present a new, own-synthesized class of Ferrocenyl Nucleosides, a type of organometallic sandwich compounds, where two cyclopentadenyl rings bind to a central Fe2+ ion. We could show anti-leukemic and anti-cancer activity in different leukemia and tumor cell lines and primary cells from children with primary ALL and AML. The apoptosis induction was efficient in low micro molar concentrations (LC50 = 10–20μM). The DNA fragmentation has been extraordinary high (LC90 = 30–50μM). Ex vivo experiments showed that some Ferrocenyl Nucleoside Analogs, like PJ184, are able to overcome resistance against anthracyclines like Doxorubicin (p<0,001), Daunorubicin (p<0,001) and Idarubicin (p<0,001) or alkaloids like Vincristin (p<0,001). Furthermore Ferrocenyl Cytosin Analog (PJ184) mediated apoptosis induction is significant higher than apoptosis induced by the clinical used nucleoside analogs like Fludarabin (p<0,001), Cytarabine (p<0,001) and Cladribine (p<0,001; all p-values by ANOVA test). These results have been achieved by testing primary lymphoblasts of seven primary ALL patients after incubation for 60h with LC50 determined in leukemic cell lines. The testing of one AML revealed similar results. Apoptosis induction has been investigated by flowcytometric measurement of DNA-fragmentation, mitochondrial membrane potential reduction and phosphatedylserin-staining on cell membrane surface. The cell death by necrosis could be excluded by a lactatdehydrogenase-release assay. In order to characterize the Ferrocenyl Nucleoside apoptosis pathway we used cellular model systems with deficiencies in different molecular parts of the apoptosis cascade. Apoptosis reveals to be independent of CD95 receptor and the pro-apoptotic factor SMAC, but shows a dependency of the apoptosis inhibitor Bcl-2. We also investigated a loss of mitochondrial membrane potential, which indicates the involvement of the mitochondrial apoptosis machinery. Taken together, these new class of Ferrocenyl Nucleoside Analogs revealed very promising in vitro and ex vivo results and may lead to a new approach in antileukemic therapy. In vivo experiments will be performed soon.
Potential Application of Differential Glycosylation in Childhood Acute Lymphoblastic Leukemia.
Authors: Chowdhury Suchandra , Mandal Chandan , Chandra Sarmila , Mandal Chitra
Published: October 2019

Acute lymphoblastic leukemia (ALL) is a malignant transformation of lymphoblasts representing the commonest type of cancer in pediatric population. We have earlier demonstrated the differential expression of 9-O-acetylated sialoglycoproteins (9-OAcGP) [Pal et.al.2004,] as disease-associated molecules on these lymphoblasts. These are functionally active signaling molecules [Ghosh et.al.2005] capable of inducing disease-specific anti-9-OAcGP antibodies [6–8]. Subclass switching of anti-9-OAcGP from IgG1 to IgG2, alteration in their glycosylation profile along with impairment of effector functions hint toward imbalanced homeostasis thereby circumventing host defense [Bandyopadhyay et.al 2005]. Aim of the present study was to explore the expression of 9-OAcGP during chemotherapy (MCP841 protocol) and establish it as a universal marker for minimal residual disease (MRD) detection. Along with B/T- lineage-specific CD antigens, the status of 9-OAcGP have been investigated by flow cytometry in paired-samples of bone marrow (BM, 11-times from each patient) and peripheral blood (PB, 18-times from each patient) during the treatment protocol in a longitudinal follow-up study. 89 patients were monitored throughout the study period, of which the number of B-ALL and T-ALL are n =75 and n= 14 respectively. The special templates used for MRD detection, 9-OAcGP+CD10+CD19+/9-OAcGP+CD34+CD19+/9-OAcGP+CD7+CD3+ increased the sensitivity to 0.01% (i.e. one cancer cell could be detected in 10,000 normal cells during clinical remission)while those with high MRD followed by relapse. The number of patients having a clinical relapse during the two years treatment were n = 2. The MRD in BM correlated well with PB both in B and T- ALL. We propose that close monitoring of patients, irrespective of lineage, even in PB, may provide an efficient and reliable index for the MRD detection, enabling more long-term management of these children.
Therapy-Related Acute Lymphoblastic Leukemia (t-ALL).
Authors: Escobar Carolina , McLarty Jerry , Ballester Oscar
Published: October 2019

Introduction: Myelodysplasia and Acute Myeloid Leukemia are recognized and well characterized complications of cancer therapy. Much less is known about t-ALL, but an increasing number of cases are being reported.

Materials and Methods: We conducted a MEDLINE search to identify patients with ALL diagnosed after exposure to chemotherapy and/or radiotherapy. Data extracted included: latency period, radiation and type of chemotherapy drug exposure, phenotype, cytogenetics and survival.

Results: 74 patients were identified with a median age at the time of t-ALL of 33 years, (range 2 to 90; 16 cases age ≤ 10 and 9 cases age ≥60). History of a prior malignancy, including solid tumors in 45 (sarcomas 12, breast cancer 11, others 22) and hematological malignancies in 25 (Hodgkin’s lymphoma 17, others 8), was documeted in all patients with the exception of 4, who received chemotherapy or radiation for a non-malignant disorder. Exposure to radiotherapy was documented in 65% of patients (4 cases had exposure only to radiotherapy), 64% had recieved topoisomerase II inhibitors and 84% other chemotherapy drugs. Median time to t-ALL was 38 months. Follow up data was available in 56/74 (75.6%), 20 were reported alive (27%), 3 to 132 months from t-ALL diagnosis. Twelve patients received stem cell transplants, 7 of them were survivors (8 to 79 months). Based on phenotype and/or cytogenetics, 5 subsets of patients were identified. Group A: 32 patients (43%) with 11q23 translocations and/or MLL rearrangements: 23 of these patients (72%) presented with pro-B cell phenotype (CD19+, CD10−)(2 pre-B cell, 2 T cell, 5 unknown); 90% had history of exposure to topoisomerase II inhibitors; median time to t-ALL was 21 months in Group A, significanlty shorter (p<.0001) when compared to all other patients (median 60 months). Median overall survival was 8 months, 5 year survival 27%. Group B: 11 patients (14.8%), with L3/Burkitt’s leukemia; all with t(8;14), t(8;22) or t(2;8) abnormalities. None of them are long term survivors. Group C: 4 patients with t(9;22) (5.4%). Group D: 4 patients with T cell phenotype (5.4%). Group E: the remaining 23 patients (31%) with primarily a pre-B cell phenotype (CD19+, CD10+) with normal (21.7%), complex (21.7%) or unknown cytogenetics (56%). Of the 18 patients who received induction therapy in Group E, 9 (50%) are survivors (median 60 months from diagnosis).

Conclusions: Various subsets of t-ALL can be recognized, resembling the pattern seen in patients with de novo ALL. However, high-risk subtypes such as 11q23/MLL + cases are overrepresented. Patients with t-ALL not identified by 11q23 or t(9;22) abnormalities, L3/Burkitt’s or T cell phenotype (Group E) appear to have a better prognosis: survival at 5 years 45% compared to 24% for all other patients.
Non-Hodgkin Lymphoma Involving the Bone (NHLB): Report of Ten Cases.
Authors: Vigliotti Maria Luigia , Fazioli Flavio , De Chiara Anna , D’Arena Giovanni , Villani Oreste , Pinto Antonio
Published: October 2019

Background: NHLB is an unusual entity. Primary NHL of the bone comprising approximately 5–12 % of all bone cancer and less than 12% of all extra-nodal lymphomas; surgery resection does not have a therapeutic role and diagnosis is usually established by bone biopsy.

Aim of the study: To demonstrate the clinical features of bone lesion in order to optimize diagnostic approaches and to evaluate prognostic factors and treatment.

Patients and Methods: Ten pts with NHLB were diagnosed with surgical bone biopsy in the last 5 years. Pain and soft tissue swelling were the commonest symptoms. Two pts showed a unique bone lesion which was classified as primary lymphoma of the bone (PLB). In 8 cases, dissemination of the disease with multiple bone and/or visceral involvement was apparent (dNHL). The characteristics of pts were: median age 58,4 years (range 39–82 yrs), male: 70%; female: 30%; CS I, 20%, and CS IV, 80%, BM+ vs BM− (20% vs 80%); IPI was: low, 0–2, in 50%, vs high, 3–5 in 50%; bulky disease in 20%. All pts had B-phenotype; DLBCL in 8 pts, lymphoplasmocitoid lymphoma in 1 pt and follicular lymphoma, grading 2, in 1 pt, according to REAL-classification. Nine pts received CT with CHOP- like regimens (6 courses) to be repeated every 2 or 3 weeks. Only one pt received HDT with PBSCT. Three pts received CT + local irradiation (30 Gy).

Results: CR was achieved in 80% of cases; all these pts were alive and disease free. Only the pt with unique bone lesion and lymphoplasmocitoid features, received only surgery therapy and is alive and disease free. Two pts died for PD at +1 and +6 months at initial sites of disease. Median OS was 19.6 months (range 1–62) and DFS was 20 months (range 2–58).

Conclusions: The long-term survival of these pts is favourable but dependent on histology and clinical stage and appears to parallel that of pts with lymphoma of similar histology involving other sites.
Vinblastine Based Short-Pulse B-Non-Hodgkin’s Lymphoma Type Chemotherapy (CT) with Maintenance Therapy Is Highly Efficacious Treatment for Anaplastic Large Cell Lymphoma (ALCL).
Authors: Banavali Shripad D. , Goyal Lovenish , Bhagwat Roshni V. , Narayan Prasad , Mandhaniya Sushil , Arora Brijesh , Vora Amish , Pai Suresh K. , Gujral Sumeet , Kurkure Purna A. , Adde Melissa , Magrath Ian , Parikh Purvish M.
Published: October 2019

OBJECTIVE: ALCL is a rare but biologically well characterized disorder. Though ALCLs are highly chemo-sensitive, 20% to 40% of patients develop recurrent disease. No standard therapeutic regimen exists for the treatment of ALCL. The objective of the present study is to characterize the clinical features and treatment response of children and young adults with ALCL in India

Methods: 30 patients of ALCL who were previously untreated were enrolled between Jan 1991 and July 2006. Treatment consisted of eight alternating cycles of two regimens A and B. Regimen A included Cyclophosphamide, Adriamycin, Vincristine and Cytosine-arabinoside. Regimen B included Etoposide, Vincristine, Methotrexate (standard dose), and Ifosfamide with Mesna. Intrathecal Methotrexate and Cytosine Arabinoside were administered in the first 4 cycles (MCP 842. Ann. Oncol.1997; 8: 893). No high dose chemotherapy or radiotherapy was given. Since Jan. 2004, Vincristine was replaced with Vinblastine and patients received 6 months (Stg. I and II) or 12 months (Stg. III and IV), of oral maintenance therapy with 6-MP and Methotrexate (11 patients).

Results: The median age was 14 years (range 2.6 to 31 years). The male to female ratio was 2.8:1. Common predominant disease sites included: Abdomen-27%; Nodes-33%; Bones-17%, Head and Neck-10%; and Mediastinum-13%. Four patients (13%) presented with stage I disease, 7 (23%) with stage II, 14 (47%) with stage III and 5 (17%) with stage IV. The response rate was 93% for the 29 evaluable patients (1 still on second cycle), with complete response in 86%. There were 6 relapses (20.7%) with majority (83%) of relapses showing newer sites of involvement. For the whole group, the projected 10 year EFS was 52.7% and OS was 73.3%. The EFS analyzed stage wise was 66.7% and 44.1% for localized Stages (I and II) and advanced Stages (III and IV) respectively. For the 20 post-2000 patients, the probability of 5 yr. OS is 100% (Stg I and II) and 84.4% (Stg III and IV) (3 relapsed patients have received Vinblastine based chemotherapy and are in subsequent remission). Most importantly, the EFS so far is 100% for all 11 patients treated with Vinblastine and maintenance therapy.

Conclusions: Most Indian patients with ALCL present in advanced stages with abdomen and nodes as the dominant primary sites of disease. Although, in the past, initial treatment responses were good, long term survival was suboptimal due to relapses. Simple addition of Vinblastine and oral maintenance may improve EFS in ALCL without the need of adding high dose or more aggressive CT. This protocol would be especially useful to treat both children and adults with ALCL in countries with limited resources & supportive care.
b2a2 Type bcr-abl Transcript Chronic Myelogenous Leukemia (CML) Patients in Chronic Phase (CP) on Glivec Achieve Complete Cytogenetic Remission (CCyR) Earlier Than Those with b3a2.
Authors: Lomaia Elza G. , Martinkevich Irina S. , Ogorodnikova Yulia S. , Lyschov Anton A. , Bogdanov Konstantin V. , Zaritskey Andrey Yu
Published: October 2019

There are few data on different biological significance of b2a2 and b3a2 transcripts. The main one is thrombocytosis in b3a2 CML patients (R.A.Perego e.a.,2000), probably associated with coexpression of m-bcr-abl (T.Liu e.a.,2006). Noteworthy that the level of transcripts in primary CML patients did not differ(Rodrigues J. e.a.,2005). That was the reason for evaluating Glivec efficacy in CP CML patients with bcr-abl trabscripts- b2a2 and b3a2.

Patients and Methods. 97 Ph+, CP CML patients were studied. They received Glivec either as front line therapy or after resistance to preceeding therapy. Analysis of b3a2 and b2a2 transcripts was performed in 27 of them. Cytogenetics was performed routine technology with G-banding. Statitics: Kaplan-Meier survival analysis and Cox regression model were used.

Results. The median time to CCyR in whole cohort of patients was 320 days. Of 27 bcr-abl studied patients, 9 had b2a2 and 18 b3a3 transcripts. They did not differ in age, duration of the disease and Sokal Score. Median time to CCyR in b2a2 patients was 172 days and 362 in b3a2 patients(p=.0001). Time to CCyR was shorter in b2a2 patients(p=.003) and insignificantly longer in b3a2 patients when compared to the whole cohort of studied patients. Cox analysis revealed that type of transcripts is independant prognostic factor, including platelet level and spleen size. Difference was more pronounced in patients with front line Glivec therapy.

Conclusion. b2a2 transcript is independent prognostic marker for early achievement of CCyR on Glivec therapy.
Evaluation of the Clinical Significance of Immunophenotyping in Myelodysplastic Syndrome.
Authors: Liu Dan D. , Jiao Xue L. , Geng Mei J. , Zhu Ming Q. , Gong Jing X. , Wu Wei L. , Ma Qin F. , Xue Yong Q. , Chen Zi X.
Published: October 2019

The myelodysplastic syndrome (MDS) is a clonal hematologic disorder with extremely heterogeneous cell component. Diagnosis is currently depending on the dysplastic morphology of bone marrow cells and the presence of specific cytogenetic abnormalities. Since no specific marker could be identified in MDS, it is sometimes hard to distinguish from other anemias, such as aplastic anemia (AA). The cell immunotype may be helpful in differential diagnosis and predicting the disease progress. To evaluate the clinical usage of immunotyping in MDS patients, we have compared the immunotypes between 36 MDS patients, 18 patients with AA and 11 healthy controls by using flow cytometry. Moreover, in combination with karyotype analysis and prognosis indicator IPSS, the value of immunotyping was further analyzed and discussed.

Our results demonstrated:

In MDS-RA patients, the distribution of surface antigen on BM cells had no preferential difference between lymphoid and myeloid lineages. In RAEB patients, expressions of CD13, CD33 and CD34 were prevailing, significantly higher than others(P<0.05). In contrast, in AA patients, expressions of CD2, CD7, CD19, CD20 were significantly higher than other surface antigens(P<0.05). In high risk MDS (RAEB/RAEB-t) patients, the cells expressing B cell lineage antigens (CD19,CD20) are markedly less than that in healthy people(P<0.05)while percentage of myeloid lineage antigen (CD13,CD33) are much higher than that in health control(P<0.05), However, in low risk MDS patients (RA) the frequency of expression of all myeloid, T and B lymphoid lineage antigens were not different from that in health controls. The distribution pattern of these three lineage antigens in high risk MDS patients was remarkably different with those of AA patients(P<0.05). Comparing with low-risk MDS, high-risk MDS expressed more myeloid lineage antigens (P<0.05)while expressed less B lineage antigens(P<0.05). In MDS patients, expression of CD14 was correlated with special chromosomal abnormalities defined by IPSS

Our results suggested:

The pattern of immunotype distribution in MDS cells are highly heterogenous. Expressions of myeloid antigens was predominant in MDS patients. Immunotyping can be helpful for discriminating MDS from AA and for monitoring the disease progression from low risk to high risk MDS. Expressions of certain immunotype are correlated with prognosis of MDS.
The Potentials of Constitutive NF-κB Activation in U266 Cell Lines.
Authors: Amin Jakia , Otsuyama Ken-ichiro , Uchida Emi , Abroun Saeid , Shamsasenjan Karim , Islam Khademul , Kawano Michio M.
Published: October 2019

NF-κB has been described as a family of ubiquitously expressed transcription factors, composed of a dimer of distinct members of the Rel protein family (RelA/p65, cRel, RelB, p50, p52), and after stimulation, NF-κB dimer translocate into the nucleus to activate the transcription of various target genes. In the present study, we aimed to elucidate the mechanism of constitutive activation of NF-κB in human myeloma cells. For this purpose here, we used a human myeloma cell line, U266 cells, and performed gel shift assay and also examined the expression of different target genes of NF-κB by RT-PCR and determined the up-regulation of some of the anti-apoptopic genes (IAP-1, IAP-2, Survivin, Bcl-2, Bfl-1) and cell cycle regulators (Cox-2, Cyclin-D1, Cyclin-D2). This NF-κB-mediated gene expression provided an approach for investigating the protein expression of NF-κB family in U266. Our data revealed that the expression of p65, p50, RelB and cRel was strongly detected in the whole lysate of U266, while weak expression of p52 was found in U266. Furthermore, in the nuclear fractions of U266 cells, we strongly detected the expression of p65, RelB and p50, but not the cRel, for cRel was present in the cytoplasm. It has been recognized that dimerization is essential for NF-κB activity. Then, we performed immunoprecipitation-immunoblot analysis to demonstrate the dimer that might present in the U266, and we detected the possible dimerization such as p65-RelB in the nucleus. Therefore, these results suggest that U266 shows high constitutive activation of NF-kB, and possible hetero- and homodimer of NF-kB except for p52 may be responsible for the constitutive activation of NF-kB in U266 cells.