bluuebunny/crossref_metadata_2025 huggingface dataset:
The Acute-Phase Protein Serum Amyloid A Induces G-CSF Expression.
Authors: He Rong , Zhou Jian , Ye Richard D.
Published: October 2019

Host response to injury and infection is accompanied by a rapid rise of acute-phase proteins in the blood. Serum amyloid A (SAA) is a major acute-phase protein, and has been used as a marker for inflammatory diseases. However, its precise role in inflammation has not been defined. In our previous study, we found that SAA can induce IL-8 and TNF-alpha secretion from peripheral blood neutrophils, which indicates that SAA has cytokine-like functions. We have also identified SAA as an endogenous ligand that induces the expression of interleukin-23 (IL-23), a hetero-dimeric cytokine that promotes autoimmune inflammation. In this study, we reported that SAA stimulates monocytes to secret granulocyte colony-stimulated factor (G-CSF), a cytokine and a major hematopoietic growth factor. Injection of SAA into mouse peritoneum significantly increases the number of neutrophils in both peritoneal cavity and blood circulation. These results suggest that SAA can induce granulocytosis during infection and inflammation. We hypothesize that during the inflammation, locally or systematically produced SAA can increase the number of neutrophils in blood circulation and recruits them to injured or infected sites through induction of G-CSF.
Evaluation of Ethnic Differences in Mannose-Binding Lectin (MBL) Level or MBL Genotype.
Authors: Buchbinder Aby , Basile Thomas , Ding Cliff , Hester Jeff , Yu Jing , Eckhardt Thomas , Conover Charles , Horak Ivan
Published: October 2019

Background: MBL is a natural human plasma protein that plays an important role in the humoral innate immune defense. MBL specifically recognizes a broad range of microorganisms, including bacteria, fungi, viruses, and parasites, by binding to common carbohydrate structures located on their surfaces and activating the lectin pathway of complement. The concentration of MBL in plasma is largely genetically determined and ranges from 5 to 10,000 ng/mL. Deficiency is caused by mutations within the mbl2 gene or its promoter region. Individuals heterozygous (A/O) and homozygous (O/O) for these mutations display a reduced level of MBL in plasma. Low levels of MBL have been associated with increased risk of serious infection in situations in which other components of the immune system are compromised. Studies in patients with hematologic malignancies undergoing chemotherapy suggest that low MBL levels or variant MBL genotypes (A/O or O/O) are correlated with infectious complications of chemotherapy and/or duration of febrile neutropenia. The effect of ethnicity on MBL level and genotype was examined as part of the development of recombinant human MBL (rhMBL), a novel molecule that is entering clinical trials.

Methods: The plasma MBL level (protein concentration) and MBL genotype of 80 healthy volunteers were determined by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR), respectively.

Results: No differences in MBL level or genotype were observed among the 3 ethnic groups (see table below). Overall, the plasma MBL level was <100 ng/mL for 10% of the volunteers and <300 ng/mL for 16%. Regarding MBL genotype, 4% had an O/O genotype and 34% had an A/O genotype.

Conclusions: The MBL level and genotype data from 80 healthy volunteers of different ethnic backgrounds were observed to be generally concordant. Further analyses in individuals with other ethnicities are ongoing.

MBL Level and MBL Genotype by Ethnicity White Black Hispanic Total (N=50) (N=15) (N=15) (N=80) n (%) n (%) n (%) n (%) MBL Level (ng/mL) <100 5 (10) 1 (7) 2 (13) 8 (10) 100 to <300 3 (6) 2 (13) 0 5 (6) ≥300 42 (84) 12 (80) 13 (87) 67 (84) MBL Genotype A/A 32 (64) 9 (60) 9 (60) 50 (63) A/O 16 (34) 6 (40) 5 (33) 27 (34) O/O 2 (4) 0 1 (7) 3 (4)
Effects of Protease Inhibiton and Proteasome, Soluble Interleukin-2-Receptor (sIL-2R) in Biclonal Gammopathy-Human Immunodeficiency Virus Patients (BG-HIV).
Authors: McPherson Eugene , Hazel E. , Tassy P.
Published: October 2019

Human immunodeficiency virus-1 (HIV-1) encodes for a protease required for the cleavage of the viral gag-pol polyprotein. HIV-1 protease inhibition (PI) has revolutionized HIV therapy. Recently the 20S proteasome has been shown to cleave the same sites as PI, therefore proteasome inhibition may contribute to some of the effects of PI independent of virus inhibition. Tumor cells of multiple myeloma have been shown to be heavily dependent proteasome regulated proteins for their growth and interaction with stromal cells. Nuclear factor-kb (NF-kb) a transcription factor is an important promoter of myeloma cell growth within bone marrow microenvironment by upregulating IL-6, CRP, VEGF and cell adhesion. We observe a 60+ yo female with HIV-myeloma (biclonal Gammopathy IgG- kappa, IgM-lambda with hypercoagulable state, hyperviscosity syndrome with epistaxis, CHF, fatiqueness, and HIV-1, CD4-728/cumm (22%) but a viral load > 100,000 copies/ml, M-protein IgG of 6.9g/L, IgM of >2 g/L, kappa and lambda (biclonality) respectively. C-reactive protein of 40 mg/dl. With ritonavir-based HAART over 12 weeks lead to over a 50% M-protein reduction and normalization of sIL-2R.

CONCLUSION: HIV-1 protease inhibitors-Ritonavir based therapy-HAART may modulate proteasome activity and cytokines in HIV-myeloma (biclonal gammopathy) patients independent of virus inhibition and reduce tumor mass burden with increase survival and QOL.
A TACI Mutation in a Patient with Autoimmune Lymphoproliferative Syndrome.
Authors: Campagnoli Maria Francesca , Garelli Emanuela , Baravalle Valentina , Carando Adriana , Alliaudi Carla , Quarello Paola , Ramenghi Ugo
Published: October 2019

The autoimmune lymphoproliferative syndrome (ALPS) is an inherited disorder of lymphocyte apoptosis caused by defects in the Fas apoptotic pathway. It is characterized by the association of chronic lymphoid accumulation and autoimmune manifestations. In a subgroup of patients the disease progresses through antibody deficiency and its clinical and laboratory features overlap those of common variable immunodeficiency (CVID). Some CVID cases are associated with mutations in gene TNFRSF13B, encoding TACI. Aim of this study was to determine whether TNFRSF13B mutations are also associated with ALPS. Methods. Genomic DNA from 31 ALPS patients was extracted from PBMCs after informed consent. Exons and intron-exon boundaries of TNFRSF13B gene were amplified by PCR and sequenced on an ABI PRISM 310 Genetic Analyzer. Results. A T>A transition at nt 275, that determined the non-conservative substitution of Isoleucine with Asparagine (I92N), was found in exon 3 from one patient. The mutation involved the second extracellular cysteine rich domain, which is critical for BAFF binding. It was not found in 100 control chromosomes. The patient showed a classical ALPS phenotype with lymphadenopathy, massive splenomegaly that required splenectomy, hepatomegaly and immune cytopenias (neutropenia and immune thrombocytopenic purpura); he had alopecia and high ANA levels. Serum immunoglobulins were normal (IgG titers at the lower level of the range); lymphocytes showed defective Fas-induced apoptosis. Discussion. TACI mutations might contribute to the ALPS phenotype in some cases. Our data confirm the relationships between ALPS and CVID and provides a possible molecular explanation for overlapping phenotypes.
Adult Langerhans’ Cell Histiocitosis (LCH). A Single Institution Experience.
Authors: Fayad Luis , Cannata Jimena
Published: October 2019

Langerhans’ cell histiocytosis (LCH) is a rare clonal disorder that affects mainly children. Clinical presentation as well as behaviour are heterogeneous and can range from solitary, relatively benign lesions to a fatal disseminated disease. Although the features have been well described in children, they remain poorly defined in adults. We report the clinical characteristics and outcome of 30 adults from a single institution.

Clinical presentation: From 1957 to 2003, 30 patients were diagnosed with LCH at MDA. Median age at presentation was 37 years-old, with 60% of being male. Depending on the organs involved and according to the pediatric classification, 17 cases (57%) had a limited form of LCH (single-system) and 13 cases (43%) had disseminated disease (multisystemic). Patients with single-system LCH had mainly bone (76%) and skin (24%) involvement, which was localized to a single site in 10 cases (58%) and multifocal in the remaining. Patients with limited disease were younger (median age 30 yo). Patients with multisystemic LCH were subdivided into a “high risk” category (5 patients) if lung, liver, spleen, or bone marrow was involved, or into a “low risk” category (8 cases), if none of the risk organs was affected. Six patients had pulmonary involvement, typically as bilateral small nodules although diffuse progressive infiltrates were also present in 2 cases.

Clinical course: Patients with limited disease were mainly treated with steroids, radiation, or immunomodulatory agents while those with multifocal or multi-systemic disease had a more agressive behaviour and were treated with systemic therapy. Patients with pulmonary nodules usually responded to systemic steroids and stop of smoking habit. Median of 3 treatments were given (range 1 to 7). Sixty-one percent of patients had a 30-year overall survival (OS). Single-system LCH had a more indolent course (79% OS at 30 years) than multi-systemic LCH (median OS of 16 years). Risk organ involvement was the most important adverse prognostic factor (median OS of 2.2 years vs. 65% OS at 30 years, p: 0.007).

Figure 1. Global overall survival on LCH patients Figure 1. Global overall survival on LCH patients Figure 2. Overall survival according to the extent of the disease Figure 2. Overall survival according to the extent of the disease
Extended TNF Genotyping Identifies TNF -857T as a Risk Factor for Acute Graft-Versus-Host Disease Following Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Mullighan Charles , Heatley Sue L. , Villalta Natalie R. , Doherty Kathleen V. , Danner Silke , Hahn Uwe , Bradstock Kenneth , Schwarer Anthony P. , Szer Jeffrey , Bardy Peter G.
Published: October 2019

Extensive evidence supports a role for non-HLA immunogenetic polymorphisms in determining risk and severity of acute GVHD following allogeneic hematopoietic stem cell transplantation (HCT). Tumor necrosis factor (TNF) polymorphisms have been frequently associated with GVHD risk. However, TNF is highly polymorphic and the TNF polymorphism(s) most strongly associated with GVHD remain to be determined. We previously studied three TNF single nucleotide polymorphisms (SNPs; −308, −238 and 488) in a retrospective study of 160 myeloablative HLA-matched related allo-HCTs and found a highly significant association between the intronic TNF 488A allele and risk and severity of acute GVHD and early mortality (Transplantation 2004;27:587). It was unclear if this association was primarily with TNF 488A or secondary to other functionally important TNF alleles in linkage disequilibrium. To clarify the importance of TNF variants in GVHD, we performed extended genotyping of TNF promoter SNPs in the previously described cohort, and in a prospective cohort of myeloablative (n=78) and non-myeloablative (n=56) sibling HLA-matched allo-HCT. Six TNF polymorphisms (−1030, −863, −857, −308, −238, 488) were genotyped in donors and recipients by PCR-SSP. TNF −308/−238/488 haplotypes were directly amplified by PCR, and full TNF haplotypes inferred using Haploview. The incidence of acute GVHD was 53% and 45.8% for myeloablative and non-myeloablative transplants, respectively. Nine TNF haplotypes were identified, and the TNF 488A allele was in strong, but not complete linkage disequilibrium with TNF −857T. TNF −857T was more strongly associated with acute GVHD (OR 11.1) and grade II–IV GVHD (OR 12.3) than 488A (acute GVHD OR 8.6 and grade II–IV GVHD OR 8.9) in the first cohort. These alleles were also associated with acute GVHD in the second cohort, but only in recipients receiving myeloablative conditioning (all acute GVHD, TNF 488A OR 2.9, −857T OR 3.4). Non-significant trends were observed with grade II–IV GVHD, possibly reflecting the reduced frequency of this complication in the later cohort (29% v. 48%). No association or trends were seen in non-myeloablative transplants. Moreover, no associations were identified with the extensively studied −308 or −238 SNPs in either cohort. Together, these data confirm the association between TNF polymorphisms and GVHD in myeloablative transplants, and suggest that −857T, which lies in closer proximity to TNF regulatory regions than 488A, may be the most useful marker for risk prediction. Our results also suggest that the immunogenetic determinants of GVHD vary according to the intensity of conditioning. Further work examining non-myeloablative transplants is required.
Regulation of Phosphatidylserine Exposure by AMP-Dependent Kinase in Human Platelets.
Authors: Verhoeven Arthur J. , Dekkers Dave W.C. , De Cuyper Iris , Gouwerok Eric , De Korte Dirk
Published: October 2019

Introduction: One of the properties of platelets is the ability, upon activation, to expose phosphatidylserine (PS), providing a negative surface necessary for the docking of the blood coagulation complexes. During storage, PS exposure should be kept to a minimum to achieve optimal transfusion recoveries. Many events underlying PS exposure are still unknown despite intensive research over the last 20 years. The involvement of a phosphorylation event has been suggested and we investigated the involvement of AMP-dependent kinase (AMPK) as a candidate involved in the cascade leading to PS exposure.

Methods: Platelets were washed free of plasma by three consecutive wash steps in substrate-free medium, preventing aggregation during the first two steps by acidification of the platelets with acid citrate dextrose (ACD). Platelets were subsequently incubated with different metabolic substrates for 18 h. The nucleotide levels in the cytosolic fractions of the platelets were analyzed by HPLC, PS exposure via flow cytometry using AnnexinV-FITC as label and phosphorylated AMPK (reflecting activation of AMPK) was determined by immunoblotting using antibodies against phosphorylated AMPK (P-AMPK).

Results: When glucose was present, hardly any phosphorylation of AMPK was observed. Under substrate-deprived conditions, AMPK becomes highly phosphorylated. In the presence of mitochondrial substrates (pyruvate, acetate) AMPK phosphorylation was clearly higher than with glucose. The analysis of the nucleotide profiles showed a good correlation between phosphorylated AMPK and AMP/ATP ratios. Interestingly, PS exposure showed a strong correlation with AMP/ATP ratios and the phosphorylation of AMPK. Inhibition of LKB1, the upstream kinase of AMPK, with 5-iodotubericidin partially suppressed AMPK phosphorylation and PS exposure in response to substrate deprivation.

Conclusion: In human platelets, activation of AMPK induced by metabolic variation strongly correlates with PS exposure, suggesting a role for AMPK in the induction of PS exposure.
Definition of the Apoptotic Pathway Induced in Proliferating Endothelial Cells by Cleaved High Molecular Weight Kininogen.
Authors: Sun Danyu , McCrae Keith R.
Published: October 2019

Previous studies from our laboratory and others have demonstrated that cleaved high molecular weight kininogen (HKa) induces selective apoptosis of proliferating endothelial cells and inhibits angiogenesis. Recently we reported that the induction of endothelial cell apoptosis by HKa was matrix dependent and requires the generation of reactive oxygen species. To further define the mechanisms of HKa-induced endothelial cell apoptosis, we have investigated the pathway through which apoptosis occurs. HKa-induced endothelial cell apoptosis was caspase-dependent, as demonstrated by the cleavage of pro-caspase 3 and the inhibition of apoptosis by the broad spectrum caspase inhibitor z-VAD-fmk. Activation of the intrinsic pathway of apoptosis was also confirmed by demonstrating cleavage of pro-caspase 9 as well as the release of cytochrome C from mitochondria. These results are consistent with our previous work, as apoptosis induced through oxidative stress is a well known activator of the intrinsic apoptotic pathway. Given the resistance to HKa-induced endothelial cell apoptosis conferred by culture of endothelial cells on collagen I or IV, we questioned whether MMPs may be involved in this process. Interestingly, we observed that the broad spectrum MMP inhibitor GM6001 completely protected endothelial cells from HKa-induced apoptosis. Moreover, we observed that HKa-induced apoptosis was associated with PECAM-1 cleavage within 1 hour of exposure, and that inhibition of apoptosis by either GM6001 or z-VAD-fmk also inhibited the cleavage of PECAM-1. These findings are likely to be relevant to the apoptotic pathway, as intact PECAM-1 has been previously reported to inhibit cytochrome c release after exposure of cells to cytotoxic stimuli, while a cleaved cytoplasmic domain of PECAM-1 enhances the susceptibility of cells to apoptosis. In summary, our studies demonstrate that the antiangiogenic activity of HKa may be critically dependent on MMP activation, which in turn leads to ROS generation and caspase activation. The subsequent cleavage of PECAM-1, presumably by caspases, may be another important event in HKa-induced apoptosis.
Analysis of Causes and Outcome of Thrombocytopenia as a Presenting Symptom in a Single Institution.
Authors: Kraemer Doris M. , Bauer Barbara , Rath Joerg C. , Einsele Hermann
Published: October 2019

Patients suffering from thrombocytopenia admitted to the emergency room during nights and weekends might be a problem, depending from the availability of a haematologist on duty. We evaluated retrospectively the medical history of all patients entering our department receiving a bone marrow biopsy due to severe thrombocytopenia in the years from 2000–2003. We analyzed 441 patients admitted due to thrombocytopenia. 146 patients had a platelet count < 50,000/μl. 69 of these patients suffered from severe thrombocytopenia < 20,000/μl.

All patients entering the ER during daytime received a bone marrow biopsy during the first three hours post admission. If patients were admitted during the night, they received a biopsy the following morning. Bone marrow smears were stained according to Pappenheim and evaluated as soon as they were dry. Diagnoses were as the following: acute leukaemia 54; immune thrombocytopenia 31; aplastic anaemia/toxic bone marrow failure/myelodysplastic syndrome 36; Non Hodgkin’s Lymphoma 7; carcinosis of bone marrow 8; myeloproliferative syndrome 3; viral infection (Hanta, HIV) 2; sepsis 1; hypersplenism 2; thrombotic thrombocytopenic purpura 1; heparin induced thrombocytopenia type II 1.

During their time in the hospital only one patient of the 146 patients developed severe bleeding with the need of transfusion of blood packs. The 73 y old male patient with primary diagnosis of rectal carcinoma and 23,000 platelets at admission was diagnosed with secondary acute myeloid leukaemia. He showed lower gastrointestinal bleeding since rectal carcinoma could not be treated by surgery due to the leukaemia.

In summary 1 out of 146 patients with severe thrombocytopenia showed clinical bleedings (<1%), indicating this as a very rare event. Due to the fact that only 31 out of 146 patients (or 20 of 69 patients with platelets < 20,000/μl) with severe thrombocytopenia suffered from immune mediated thrombocytopenia immediate consultation of the haematologist is essential to allow rapid diagnosis and adequate treatment strategies in patients presenting with thrombocytopenia in the emergency department.
Three Novel ADAMTS-13 Gene Mutations in Thrombotic Thrombocytopenic Purpura (TTP).
Authors: Kim Nam Keun , Jang Moon Ju , Kim Sun , Lee Sun Ju , Park Hye Won , Oh Doyeun , Kang Myung Seo
Published: October 2019

ADAMTS-13 is an enzyme which cleave von Willebrand factor (VWF) to prevent excessive platelet thrombus formation. Mutation of ADAMTS-13 is associated with congenital thrombotic thrombocytopenic purpura (TTP). In this study, we report three novel missense mutations of ADAMTS-13 gene in TTP families. Genetic analysis of ADAMTS-13 gene was performed in 6 TTP families. Three novel ADAMTS-13 gene mutations (2708C>T [S903L], 3650T>C [I1217T] and 3941C>T [S1314L]) detected in 3 families. They were all found with heterozygous genotype in exon 21, 26 and 28, respectively. The 3650T>C mutation was found at exon 26 in the patient and his mother. A heterozygous guanine to adenine substitution was found at 5′ splicing site of intron 3(IVS+ 1) in the patient, his brother and his father. The plasma ADAMTS-13 activities of the patient, father, mother, and brother were less than 3%, 56%, 55% and 62% respectively. ADAMTS-13 inhibitors were not detected in all family members. 3941C>T was found at exon 28 in the patient and her father. The plasma ADAMTS-13 activities of the patient, father, and mother were 96%, 83%, and 83% respectively.
Anti-D Immunglobulin in the Treatment of Childhood Chronic Idiopathic Thrombocytopenic Purpura.
Authors: Turkkan Emine , Pekun Fugen
Published: October 2019

Chronic idiopathic (immune) thrombocytopenic purpura (ITP) develops in approximately 20% of children with acute ITP. The therapies for chronic refractory ITP include splenectomy, steroids, intravenous immunoglobulin (Ig), anti-D Ig, monoclonal antibodies, danazol, chemotherapy, plasma exchange, and others. We report five 5 RhD-positive, pediatric,, non-splenectomized patients with chronic immune thrombocytopenic purpura (ITP), treated with anti-D. Patients were refractory to steroids and high dose intravenous immunoglobulin (IVIG). The mean age of the patients was 10.5 years, duration of thrombocytopenia was more than 6 months, and the median platelet count was 6x 10(9)/l. Patients received a single intravenous dose of 75 microg/kg anti-D, and were monitored for response to treatment and adverse events. Complete blood cell and renal function tests were assessed at baseline and 1, 7, 14 and 28 days after infusion. By 24 hours after treatment two of patients had achieved a platelet count>20 x 10(9)/l. Mild headache, fever, or chills occurred in one patients and ameliorated with prednisone and acetaminophen premedication. None of the patients had more than 0.5 gm/dl fall in the hemoglobin level and renal dysfunction following anti-D infusion by day 1,7, 14, 28. A single 75 microg/kg dose of anti-D may increase the platelet count in some children with chronic resistant immune thrombocytopenic purpura with an acceptable safety profile. It is a valuable, safe, well tolerated, and cheap alternative to conventional chronic ITP treatments in selected non-splenectomized patients.
Treatment of Thrombotic Thrombocytopenic Purpura with Heparin, Vincristine and Dexamethasone.
Authors: Wang Jinghua , Liu Na , Liu Fang , Ruan Changgeng , Liu Juan , Cen Xi , Li Xiaoyun , Dai Haibin , Guo Bo , Su Yanhua , Zhang Yanqing , Zhang Jian , Guan Yue , Wang Hongmei
Published: October 2019

Thrombotic thrombocytopenic purpura(TTP) is a serious, low morbidity and high mortality disease, which can simultaneously affect multiple systems in the patients’ body. In the event that the patients cannot be treated by plasma exchange(PE), mortality will be 95–100%(1). Between September, 2000 and May, 2003, thirteen patients with TTP were treated mainly by heparin, vincristine, dexamethasone, six of whom have acceptted one or two PE. The results were excellent. Twelve of the thirteen patients survived. One patient was dead. The ADAMTS13 activity was measured in 10 patients using a Residual-Collagen Binding Assay(R-CBA).
Bortezomib-Induced Down-Regulation of KIT Is Mediated by Inhibition of Sp1 and NF-kB in AML1/ETO-Positive Cells.
Authors: Liu Shujun , Pang Jiuxia , Yu Jianhua , Liu Zhongfa , Huynh Lenguyen , Kearney Jessica , Paschka Peter , Klisovic Rebecca B. , Baiocchi Robert , Blum William , Perrotti Danilo , Marcucci Guido
Published: October 2019

Activating mutations of KIT, encoding a type III receptor tyrosine kinase, are frequently detected in core binding factor AML (i.e., AML1/ETO and CBFB/MYH11 AML), promote cell survival and proliferation of leukemic cells and predict poor outcome. Kinase inhibitors (e.g., imatinib or PKC-412) have been shown to block constitutively activated KIT. However, novel therapeutic approaches that target mutated KIT are necessary, since resistance to these agents can be predicted in a substantial proportion of patients in these subgroups of AML. We observed that expression levels of KIT in AML1/ETO-positive Kasumi-1 cells are more than 25 fold higher than in AML1/ETO-negative AML cell lines (i.e., THP-1, K562, and MV4–11). We also found that in Kasumi-1 cells, bortezomib (Velcade), a proteasome inhibitor already used in the clinic, induces time- (60nM for 0, 1, 3, 6 12 and 24hr) and dose- (0, 1, 6, 20, 60 and 100nM for 24hr) dependent down-regulation (>90%) of total KIT expression. Noticeably, dephosphorylation of KIT occurred 6hr before reduction of the total KIT level after bortezomib exposure. We also found >50% down-regulation of KIT expression in patients’ primary blasts treated with 60nM bortezomib for 24hr and in Kasumi-1 cells treated with the proteasome inhibitor MG132 (300nM for 24hr). Down-regulation of KIT appeared to be associated with inhibition of NF-kB and Sp1, which is necessary for regulation of the KIT promoter activity by the SCL complex. In fact, treatment with bortezomib inactivated NF-kB and decreased transcription of Sp1 in Kasumi-1 cells. Furthermore, exposure to the NF-kB or Sp1 inhibitors parthenolide (30μM for 24hr) and mithramycin (100ng/ml for 24hr), respectively, resulted in a dose-dependent decrease in KIT expression in Kasumi-1 cells. When cells were treated with bortezomib (20nM) in combination with mithramycin (30ng/ml for 24hr), we observed synergy in down-regulation of KIT RNA and KIT protein. This correlated with growth arrest and increased cell death. Interestingly, the magnitude of these effects was higher when Kasumi-1 cells were pretreated with mithramycin for 24hr before being exposed to bortezomib. Taken all together, our data suggest that bortezomib downregulates KIT expression and might also inhibit KIT phosphorylation and should be considered in future therapeutic strategies targeting AML subgroups harboring mutated KIT.
Cell Penetrating Activity of Bax Inhibiting Peptides (BIPs) and Its Utilization for Protein Transduction.
Authors: Gomez Jose A. , Gama Vivian , Distelhorst Clark , Matsuyama Shigemi
Published: October 2019

The cellular membrane has a strict regulation for molecular trafficking, and there is a strong need for the development of new technology for drug delivery into cells with minimum toxicity. Previously, we developed the cell permeable penta-peptides designed from Bax binding domain of Ku70 that are called “Bax Inhibiting Peptides (BIPs)” (i.e. VPMLK, VPTLK, VPALR). Among BIPs, VPTLK showed the best cell penetrating activity. We also found that one of the scrambled control mutant peptides (i.e. KLPVM) shows a strong cell permeable activity without an activity to bind and inhibit Bax-mediated cell death. We named these cell permeable peptides: BIP-derived cell permeable penta-peptides (BCPs). In this study, we determined the mechanism of membrane penetration by these penta-peptides. Human umbilical endothelial cells (HUVEC), Dami cells (human megakaryocytic cell line), and HeLa cells were used for the examination of cell entry of BCPs. Fluorescent dye (Fluorescein) was conjugated to the N-terminus of BCPs, and the cell entry of BCPs was monitored by FACS and confocal micropscope. BCP entered the cells in a dose dependent manner (10 uM-1.6 mM range was tested), and it did not show any significant toxicity even at 1.6 mM. The entry of BCP into the cells was detected within 15 minutes at 37C, and it reaches maximum approximately at 6 hrs of incubation. We found that BCPs entered the cells even at 0 or 4 C, suggesting that energy-independent mechanism is involved in the cell penetration of BCPs. We also tested whether BCPs has an ability to deliver a cargo protein (protein transduction activity) such as 30 kDa Green Fluorescent Protein across the cellular membrane. We used VPTLK and KLPVM for the examination of GFP delivery from the medium into the cell. Recombinant GFP proteins with and without VPTLK or KLPVM on the C-teminus were generated. The delivery of GFP was detected at a concentration as low as 20 nM within 6 hours of incubation at 37C. GFP without BCP was not imported into the cell, indicating that BCP has a cargo delivery activity (protein transduction). There are several bioactive proteins that can become effective therapeutics if they can be delivered into the cell. To be noted, BCPs are less toxic than the previously examined CPPs that had protein transduction activity, such as TAT peptide designed from the human immunodeficiency virus (HIV). BCPs did not show significant toxicity to bone marrow stem cells and endothelial cells, indicating that BCPs are effective tools for drug delivery in stem cells, and vasculature cells. BCPs may become a useful tool to deliver bioactive molecules into the cells, to protect cells from damage, to regulate cellular activity, and to control differentiation.
Hematopoietic Microenvironment Regeneration Using Adipose-Derived Stem Cells.
Authors: Ogawa Rei , Fujimura Juri , Mizuno Hiroshi , Hyakusoku Hiko , Shimada Takashi
Published: October 2019

Backgrounds: We have studied hematopoietic microenvironment regeneration using adipose-derived stem cells (ASCs) harvested from mice. We showed that not only bone matrix but also bone marrow could be regenerated subcutaneously, in vivo. Transplanted ASCs proliferated and differentiated into osteoblasts in micropores on the surface of scaffolds made of hydroxyapatites (HA). We could regenerate a subcutaneous microenvironment in which hematopoietic cells survived, which may lead to new treatments for fibrotic bone marrow diseases, such as idiopathic myelofibrosis and osteopetrosis.

Objective: In this report, we examined the optimal shapes of scaffolds for bone and hematopoietic microenvironment regeneration using ASCs.

Methods: Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, ASCs were isolated from the inguinal fat pads of GFP transgenic mice. ASCs were cultured in vitro and after three passages were seeded into the small pores of scaffolds of various shapes made of HA. The ASC-containing scaffolds were subsequently implanted into isogenic mice subcutaneously. Two months later, the scaffolds were extirpated for histological, immunohistochemical and flow cytometry analyses.

Results: Histological examination showed that the pores were filled with bone matrix and that the bone marrow was composed of adipocytes, hematopoietic cells, and vasculatures. Bone marrow regeneration on small, thin scaffolds of high porosity was better than on other scaffolds. Immunohistochemical analysis confirmed that the GFP+ ASCs that had differentiated into osteoblasts were composed of bone matrix. Flow cytometry analyses showed that the bone marrow was composed of blood cells, including populations of monocytes, lymphocytes and granulocytes.

Conclusions: Hematopoietic microenvironment engineering has great potential for hematopoietic disease therapy. The regeneration of the hematopoietic system using ASCs might be useful in the future for treating hematopoietic stem cell diseases such as leukemia as well as fibrotic bone marrow diseases.
Clinical and Experimental Study in 21 Cases of Acute Leukemia with Trisomy 4.
Authors: Pan Jin L. , Xue Yong Q. , Qiu Hui Y. , Zhu Yong J. , Wu Ya F. , Wang Yong , Shen Juan
Published: October 2019

To explore the clinical and experimental features of acute leukemia(AL) with trisomy 4, a retrospective study on the clinical and laboratory data in 21 cases of AL with trisomy 4 was performed. Chromosomes were prepared using direct method and/or short-term(24h) cultures of bone marrow cells. Karyotypic analysis was carried out using R- banding techique. 14 cases were studied by interphase FISH using a chromosome 4-specific α-satellite DNA probe labeled by spectrum Green to ascertain the presence of a clone with trisomy 4. 5 cases with t(8;21) revealed by karyotypic analysis were detected by dual-color FISH using t(8;21) translocaton probe to confirm the AML1/ETO rearrangement. The patients with AL and trisomy 4 had a median age of 50 years and a median survival of 10 months. They all had de novo AL expect two cases with secondary AL. M2 was the most frequent FAB subtype in this series (9/21 cases). The initial leukocyte count more than 10×109/L was seen in 16 cases. An enlargement of liver, spleen and/or lymph nodes in varying degrees was found in 15 cases. Among 15 cases received immunophenotypic analysis, 11 cases showed CD34 positivity and 6 cases co-expressed myeloid and lymphocytic antigens. Karyotypic analysis disclosed clonal trisomy 4 in 18 cases and one cell with +4 in 3 cases. Isolated trisomy 4 was found in 7 cases, while 14 cases had other abnormalities besides trisomy 4 including numerical abnormality (+6) in one case, and structural abnormalities in 13 cases among which t(8;21) was found in 8 cases. Interphase FISH confirmed that all 13 cases including 3 cases having one cell with +4 on karyotypic analysis had clonal trisomy 4. Dual-color FISH confirmed that all 5 cases with t(8;21) had AML1/ETO rearrangment. So we concluded that AL patients with trisomy 4 had unique clinical features and a poor prognosis.
Arsenic Trioxide Induces Mitochondrial DNA Mutation on D-Loop Region in Patients with Acute Myeloid Leukemia.
Authors: Zhou Jin , Meng Ran
Published: October 2019

Objective:To investigate the effect of arsenic trioxide (As2O3) on mitochondrial DNA (mtDNA) in acute myeloid leukemia cells.

Methods: NB4 cell line and primary leucocytes, isolated from peripheral blood of eight cases with newly diagnosed acute myeloid leukemia (the ratio of the blast cell was more than 80%), were incubated in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, and treated with 0.1, 1.0, 2.0 micromol/L of As2O3 for 48hrs respectively in vitro. Blood samples in eight cases above were collected before and 14 days after As2O3 treatment with the dosage of 0.16mg/kg.d in vivo. Genome DNA was isolated from the cells and blood samples by salt fractionation, and the D-loop fragment of mtDNA was isolated from the genome DNA and amplified by polymerase chain reaction, and the sequencing techniques were applied to identify positive clones. MTT assay, electrophoresis of genomic DNA, and protein/DNA dual parameter flow cytometry were used to examine the effect of As2O3 on cell proliferation and apoptosis.

Results: mtDNA mutations were found on D-loop fragment of mtDNA in both NB4 cells and primary leucocytes, the number of single nucleotide polymorphism (SNP) or mutation were increased remarkably after 1.0, 2.0 micromol/L of As2O3 treatment both in vitro and in vivo than non-As2O3 treated baseline. The types of mutation were included the base transversion, transition, depletion and insertion. The 1.0 and 2.0 micromol/L of As2O3-treated samples revealed apoptosis, reduced proliferation and mtDNA mutation, while in the 0.1 micromol/L of As2O3-treated samples, showed reduced proliferation, differentiation and mtDNA mutation, but no apoptosis was appeared.

Conclusions: As2O3 promoted leucocytes’ mtDNA mutation on D-loop fragment both in NB4 cell line and in primary leucocytes isolated from patients with acute myeloid leukemia. Mitochondrial DNA might be one of the targets of As2O3 act on leukemia cells. Mitochondrial DNA mutation as well as nuclear DNA might participate the process of As2O3 inducing differentiation and reducing proliferation.
Clinical Relevance of Multidrug Resistance Proteins Expression in Patients with De Novo Acute Myeloid Leukaemia.
Authors: Podolak-Dawidziak Maria , Dus Danuta , Kielbinski Marek , Paprocka Maria , Haus Olga , Kuliszkiewicz-Janus Malgorzata , Kuliczkowski Kazimierz
Published: October 2019

Background. Early occurrence of the multidrug resistance (MDR) in de novo acute myeloid leukaemia (AML) may contribute to the treatment failure.The aim of this study was to investigate prospectively the clinical significance of the MDR proteins overexpression and their functional augmenting, in the context of other AML prognostic factors, such as age, immunophenotype and cytogenetic profile. We examined expression of MDR proteins: MDR1, MRP1, MDR3, BCRP, LRP and GSTπ and performed drug resistance functional assay in peripheral blood blasts of 25 patients with de novo AML at diagnosis and after the first chemotherapy cycle consisting of a 3 + 7 combination of DNR/Ara-C. MDR proteins presence and their functional activity (measured by fluorescent Rh123 dye efflux) were estimated by flow cytometry.

Results: Thirteen out of the 25 AML patients (52%) attained a CR with induction treatment, one had PR and eleven did not achieve remission. Out of 10 patients without CR, 2 died in aplasia and 9 were classified as an early death due to disease progression. All patients who achieved remission were younger than 55 years. Among 11 patients without remission, 9 expressed CD34; 6 of them had intermediate and 5 unfovorable cytogenetic profiles. In 10 out of 25 patients (40%) overexpression od MDR1 was shown at diagnosis, and was irreversible in 9 of them, those with poor clinical outcome (6 did not achieve CR, 1 had PR and one who obtained CR relapsed), whereas one patient who reversed achieved CR. The functional MDR assay showed the increased Rh123 efflux, both at diagnosis and after the first chemotherapy cycle in 12 patients (48%) and it influenced patients’ outcome in similar manner as MDR1 expression. At diagnosis other MDR proteins were also elevated in some AML patients:GSTπin 19 (76%), LRP in 9 (36%), MRP in 4 (16%) and MDR3 in 2 (8%). Seven AML patients (20%), both at diagnosis and after the first chemotherapy cycle co-expressed MDR1 with other multidrug resistance proteins and had enhanced Rh123 efflux. This co-expression resultes in 6 of them in chemotherapy resistance; the remaining one who achieved remission was young and had favorable cytogenetic profile. The results obtained demonstrate that the advanced age, unfavorable cytogenetic profile, overexpression of MDR1 at diagnosis and co-expression of other MDR proteins together with their functional activity contribute to the treatment failure in de novo AML.
Prevention of Homoharringtonine Induced Apoptosis by Antioxidants in K562 Cells.
Authors: Mai Wenyuan , Lin Maofang
Published: October 2019

Homoharringtonine (HHT) is a cephalotaxine ester derived from an evergreen tree found wildely throughout southern China, which has antileukemic activities against a variety of acute myeloid leukemic cells. HHT was recently shown by us to have differentiative and apoptotic effects on human leukemic cell lines. Recently, apoptosis has been associated with oxidative stress in a number of cell systems. Here we assessed the inhibitory capacity of antioxidant on HHT-induced apoptosis in the human leukemic cell line, K562. The apoptosis was analyzed by agarose gel electrophoresis of DNA, flow cytometry and terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick labeling. K562 cells were treated with HHT at doses of 0ug/ml, 25ug/ml for 24 hours. Vitamin C (0.2 ug/ml) were added to medium 16 hour before addition of HHT. Results showed vitamin C inhibited oxidative stress and K562 cells apoptosis in HHT-treated groups (P < 0.05). Our data indicated that HHT treatment primarily induces K562 cells apoptosis at concentration up to 100 ug/ml. The underlying mechanism appears to involve mitochondrial damage via an increase in oxidative stress.
Downregulation of Survivin by Arsenic Trioxide in Acute Myeloid Leukemia Cell Line.
Authors: Silva Karina Lani , de Freitas Prazeres Martina , Maia Raquel Ciuvalschi
Published: October 2019

Caspases are proteins that play a central role in apoptosis. Therefore, triggering apoptosis through chemotherapeutical caspases inductor drugs is the major path in cancer treatment. However, hindering apoptosis by inhibitor of apoptosis proteins (IAPs) overexpression, have been described in many cancer types including leukemia and, is frequently related to drug resistance. Survivin, a member of IAPs family, is expressed in most human cancers but undetectable in the majority of normal adult tissues. In acute myeloid leukemia (AML), Survivin expression has been correlated with poor prognosis and chemotherapy resistance. These characteristics make Survivin eligible for a promising target for AML treatment. To explore the relationship between Survivin and drug resistance we investigated the alteration of Survivin expression in two AML cell lines HL60 (AML-M2) and U937 (AML-M5) and one chronic myeloid leukemia cell line in blast crisis for M6 (K562) treated with two chemotherapeutic drugs used in leukemia treatment: arsenic trioxide (As2O3) and doxorubicin (Dox). MTT assay was performed to determine the dose of drugs capable to induce cell death in 50% of treated cells (DL50). To verify the percentage of apoptosis induced by As2O3 and Dox at DL50 concentrations determined by MTT, the annexin V/propidium iodide-staining assay was performed and analyzed by flow cytometer. Western blot was used to analyze Survivin expression before and after drugs treatment at DL50 concentrations. Among the cell lines studied, HL60 was the most sensitive for both drugs tested. The DL50 concentrations obtained for As2O3 were 2, 4 and 5 μM at 24 hours for HL60, U937 and K562, respectively. Dox DL50 concentrations were 10 μM at 24 hours for HL60, 5 μM at 48 hours for K562 and 1 μM at 72 hours for U937. The annexin-V/PI staining showed that these drugs were capable to induce apoptosis in all cell lines tested. The percentages of apoptosis induction for As2O3 were 50% for HL60, 21,84% for U937 and 32,7% for K562 in comparison with control cells, while for Dox, the index of apoptosis were 86,8%, 35,7% and 2,2% for HL60, U937 and K562, respectively. Interestingly, at DL50 concentrations As2O3 and Dox inhibited Survivin expression in 72,7% and 69,2%, respectively. No significant alteration in Survivin expression was observed in U937 and K562 cell lines. Thus, HL60 cell line was the most sensible cell line studied and it was correlated with downregulation of Survivin expression. It suggests that Survivin could be considered a therapeutic target for AML-M2 and that As2O3 and Dox are capable to decrease drug resistance. However, the mechanism of action of As2O3 and Dox in Survivin expression seems to be cell type specific.
Trisomy 22 as the Sole Abnormality Is an Important Marker for Inversion 16 in Acute Myeloid Leukemia.
Authors: Li Jianyong , Zhou Huifen , Chen Lijuan , Pan Jinlan , Qiu Hairong , Xu Wei , Xue Yongquan , Lu Hua
Published: October 2019

Inv(16) has been reported in 10%~12% of acute myeloid leukemia (AML), mostly being associated with the M4Eo subtype, and is associated with a relatively favorable outcome. However, it is a cryptic rearrangement and often difficult to recognize in conventional cytogenetics (CC). Trisomy 22 is an uncommon karyotypic aberration in AML and is often associated with inv(16)(p13q22). In order to explore the value of trisomy 22 in the diagnosis of AML with inv(16), dual-color interphase fluorescence in situ hybridization (FISH) was performed in 19 AML cases with trisomy 22 abnormality. The probe was two-color break apart probe for CBFb with SpectrumRed on the centromeric side and SpectrumGreen on the telomeric side. And the results were compared with that of R-banding CC. CC did not reveal inv(16) in any of the 19 AML with trisomy 22, but FISH analysis showed inv(16) in 11 cases and del(16)(q22) in one case. Among 11 cases with inv(16), 9 were trisomy 22 as the sole abnormality, one was complicated with trisomy 8, and one was del(16)(q22). Four AML patients with trisomy 22 and inv(16) were analyzed by multiplex FISH (M-FISH) which revealed trisomy 22 only. This study further confirmed that trisomy 22 as the sole abnormality can be regarded as an important marker for the inv(16) in AML.
Fluorescence In Situ Hybridization Studies in 22 Cases of Plasma Cell Leukemia.
Authors: Li Jianyong , Xu Wei , Chen Lijuan , Zhu Yu , Pan Jinlan , Qiu Hairong , Xue Yongquan , Lu Hua
Published: October 2019

Background Plasma cell leukemia (PCL) is a rare malignant plasma cell disorder that usually carries an aggressive course with a rapidly fatal outcome. Cytogenetic studies performed on plasma cell disorder are scarce and difficult because of the low proliferation rate of plasma cells. Fluorescence in situ hybridization (FISH) analysis is an attractive alternative for evaluation of numerical and structural chromosomal changes in PCL.

Methods Interphase FISH (I-FISH) and two different specific probes for the regions containing 13q14.3 (D13S319) or 14q32 (IGHC/IGHV) were used to detect 13q14 deletion [del(13q14)] or IgH rearrangement in 22 PCL patients. For patients with IgH rearrangement, probes for IgH(JH) and 11q13 (CCND1) or 4p16 (FGFR3) were used to detect t(11;14)(q13;q32) or t(4;14)(p16;q32).

Results Molecular cytogenetic aberrations were found in 19 of 22 (86.4%) PCL patients. Del(13q14) was detected in 13 cases (59.1%), and IgH rearrangement in 17 (77.3%) patients including 7 with t(11;14) and 3 with t(4;14). 14q32 rearrangement and 13q14 deletion were found concurrently in 11 cases (50%).

Conclusions Chromosomal abnormalities are frequent in PCL. The most frequent aberrations among the cases was the 14q32 rearrangement and 13q14 deletion. I-FISH technique is useful to detect molecular cytogenetic aberrations and should be used in the routine evaluation of PCL.