bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Myeloma-Specific Markers in Bone Marrow Spicules Using a Novel Immunohistochemical Technique
Authors: Santra Madhumita , Shaughnessy John , Williams David R. , Barlogie Bart , Bellamy William
Published: April 2020

In addition to its role in diagnosis and assessing the prognosis of numerous malignancies, immunohistochemistry (IHC) is an important tool for the analysis of protein biomarker expression in tissue sections. The combination of bone marrow (BM) biopsy and IHC is frequently used to study histopathological alterations in diseases such as multiple myeloma (MM). However, due to the harsh decalcification process generally used for processing of bone marrow biopsies, protein epitopes are occasionally rendered unsuitable for IHC detection. We have developed a novel technique for processing BM spicule samples into a fibrin-clot matrix that allows for IHC detection of MM protein markers. This method does not require decalcification and results in a consistent, reliable assay. Briefly, bone marrow aspirates drawn from MM patients were subjected to a brief processing step to isolate the marrow spicules. Once isolated, the spicules were admixed with thromboplastin and pooled normal human plasma to create a clot. The clot was allowed to harden at 37°C after which it was placed into an embedding cassette for fixation and histological processing. The result was a formalin-fixed, paraffin-embedded clot suitable for IHC studies, including the construction of TMAs. Using patient paired spicule-clot and core biopsies from patients diagnosed with myeloma, we studied several antibodies including, but not limited to, kappa and lambda immunoglobulin light chains, CD138 and Cyr61, a member of the CCN family of extracellular matrix-associated signaling proteins. Results demonstrated that IHC staining for all antibodies was comparable in both the spicules and in the cores. While BM core biopsies had areas of non specific staining for several antibodies, this was not generally observed in the BM spicule samples with the exception of immunoglobulin light chains which displayed an element of non-specific staining with both samples. Moreover, we observed a consistently superior morphology in the BM spicule samples than in matched bone marrow core biopsies. In conclusion, our study demonstrates that BM spicule samples processed from MM patients using this novel fibrin-clot matrix technique were suitable for IHC and had generally lower background and non-specific staining coupled with better morphology when compared to matched core biopsies.
Effects of CK2 Inhibition on Multiple Signaling Pathways in Myeloma Cells
Authors: Piazza Francesco Alessandro , Montini Barbara , Arcidiacono Diletta , Gurrieri Carmela , Manni Sabrina , Colpo Anna , Tubi Laura Quotti , Zambello Renato , Trentin Livio , Semenzato Gianpietro
Published: April 2020

CK2 serine-threonine kinase plays a key role in promoting cell survival of multiple myeloma (MM) cells through modulation of extrinsic and intrinsic apoptotis and by activating pro-survival signaling such as the NF-κB and STAT3 pathways (Piazza FA et al. 2006, Blood 105:1698–1707). Here we deepened our previous results and placed the CK2 action inside critical pathways for MM cell growth, like the MAPK, STAT3 and Wnt regulated cascades.

We employed MM cell lines, primary cells from MM patients and human cervical carcinoma cell line HeLa. Inhibition of CK2 was obtained with well characterized chemical compounds (TBB, K27 and K17) or by RNA interference.

CK2 inhibition led to an increased p38 MAPK activation and to a decreased basal and growth factor stimulated-ERK1,2 phosphorylation. These changes were associated to increased activation of apoptotis and cell death. Inhibition of PP1 and PP2A phosphatases led to a decreased p38 MAPK phosphorylation upon CK2 inhibition, suggesting that CK2 may control p38 activity through the modulation of these molecules. Activation of upstream JAK tyrosine kinase upon cytokine or growth factor stimulation are a prerequisite for STAT3 activation. Following inhibition of CK2 activity, Tyk2 phosphorylation and JAK2 activation were found impaired and these effects were associated to a decreased STAT3 phosphorylation in tyr705 and ser727. In turn, activated-STAT3 nuclear migration and STAT3-dependent transcription of target genes, like the pro-survival bcl-2 family member mcl-1 and the cell-cycle promoter cyclin-D1, were significantly reduced.

Finally, Wnt activation was also affected by CK2 manipulation. Indeed, β-catenin activation and transcriptional activity were impaired by the treatment of MM cells with CK2 inhibitors; these effects were associated with reduced cell proliferation and cell death.

Altogether, these data demonstrate that CK2 is a key player placed at the cross-road of important signaling pathways for MM cell growth and reinforce the possibility that it may represent an ideal target molecule for therapeutic purposes.
Combination of DNA Methylation Inhibitor 5-Azacytidine and Arsenic Trioxide Has Synergistic Activity in Multiple Myeloma
Authors: Chen Guanghua , Lin Fengru , Depei Wu , Wang Yi , Huang Haiwen , Sun Aining , Chang Huirong , Feng Yufeng
Published: April 2020

This study was undertaken to study the effect of 5-azacytidine on XAF1 expression in myeloma cells and efficacy of 5-azacytidine and arsenic trioxide combination treatment in myeloma in vivo and in vitro. XAF1 expression was analyzed by semi-quantitative PCR and Western blotting. Methylation specific PCR (MSP) was used to detect methylation status of XAF1 promoter CpG islands. RPMI 8226 and XG-7 cells were treated with various concentrations of arsenic trioxide and 5-azacytidine. Expression of XAF1 mRNA variants were confirmed by gel electrophoresis and sequencing. Untreated RPMI 8226 cell expresses two variants of XAF1 mRNA. Untreated XG-7 cell has no expression of XAF1. Hypermethylation of XAF1 promoter CpG islands was detected in both cell lines. Both cell lines express full-length XAF1 mRNA transcript after treated with 2.5 μmol/L 5-azacytidine for 72 h. Our studies demonstrated that 5-azacytidine exhibits antimyeloma synergy with arsenic trioxide. 5-azacytidine treatment reversed XAF1 promoter region hypermethylation status, leading to expression of full-length XAF1 transcript. In addition, combination of 5-azacytidine and arsenic trioxide was effective in slowing myeloma growth and prolonging survival of myeloma-loaded nude mice. The findings suggested that 5-azacytidine and arsenic trioxide may be an effective combination in the therapy of myeloma patients.
Alternative Treatment for POEMS Syndrome: A Lazarus Response to Lenalidomide
Authors: Patel Tejal , Aspitia Alvaro Moreno
Published: April 2020

Introduction: POEMS syndrome is characterized by chronic progressive polyneuropathy (P) with constellation of other symptoms including organomegaly (O), endocrinopathy (E), serum monoclonal protein (M), and skin changes (S). Other features not included in the acronym include sclerotic bone lesions, Castleman disease, papilledema, thrombocytosis, peripheral edema, ascites, fatigue and clubbing. With limited available data, autologous peripheral blood stem cell transplant (APBSCT) is probably the mainstay of therapy in good performance status patients.

Case Description: A 37 year old male presented to the Mayo clinic with a three year history of progressive severe peripheral neuropathy leaving him wheelchair bound, as well as lymphadenopathy, hepatosplenomegaly, diabetes, proteinuria, monoclonal gammopathy and hyperpigmentation. Patient also had significant pain from diffuse sclerotic lesions, blurry vision due to papilledema and generalized edema. Based on the above findings, a diagnosis of POEMS was suspected. The diagnosis was confirmed by a biopsy of a left axillary node showing plasma cell Castleman’s disease. Patient’s bone marrow was negative for multiple myeloma and amyloidosis. Patient underwent APBSCT and had a remarkable clinical improvement that persisted over three years. Patient was able to live a normal, active life during those years.

After three years, patient developed ascites, massive splenomegaly and pancytopenia. Patient was referred again to our institution for possible splenectomy. On further evaluation, he was found to have significant portal hypertension and underwent TIPS (transjugular intrahepatic portosystemic shunt) procedure with some improvement in his symptoms. Patient remained in observation for another 18 months but returned oxygen dependent with worsening ascites, peripheral neuropathy, and encephelopathy. Further evaluation confirmed worsening POEMS. Patient’s ECOG performance status was poor therefore repeat APBSCT was not considered an option. Lenalidomide was started at 10mg/day for 21 days of a 28 day cycle with once weekly dexamethasone 40mg. The dose of lenalidomide was gradually increased to standard 25mg dose which the patient tolerated well.

By the second month patient was on therapy, his performance status improved to ECOG 1, his ascites and encephalopathy resolved, the splenomegaly improved and he no longer required oxygen. Patient has currently completed a total of 10 cycles and feels as good as 10 months post-APBSCT. The only side effect experienced by the patient is weight gain due to chronic dexamethasone therapy. Dexamethasone was weaned and discontinued after which lenalidomide continued as a single agent.

Conclusion:: Lenalidomide is a less toxic but effective alternative treatment for POEMS syndrome. The Lazarus response to the use of lenalidomide supports the principle that POEMS is a cytokine-mediated syndrome
The Incidence of Deep Vein Thrombosis Is Lower in Japanese Patients with Multiple Myeloma Treated with Thalidomide and Corticosteroids or Chemotherapy
Authors: Takei Naoko , Kami Masahiro , Tsubokura Masaharu , Miura Yuji , Issiki Takehiro , Satou Yuji , Matsue Kosei , Komatsu Tsunehiko
Published: April 2020

Background: Deep vein thrombosis (DVT) is an important complication of thalidomide therapy especially when it is combined with corticosteroids or chemotherapy. Japanese patients with multiple myeloma (MM) are currently prescribed thromboprophylaxis after ASCO guideline, although there are limited data for asian population. We examined the incidence of DVT in Japanese patients with MM.

Methods: We reviewed the records of 145 patients receiving thalidomide for newly diagnosed or relapsed/refractory MM at Kameda Medical Center and Tsukuba Memorial Hospital during the 10-year period 1998–2008. Patients were considered to have developed DVT if they presented with suggestive symptoms of leg swelling and had a Doppler ultra-sound study confirming acute thrombosis.

Results: 95 of the 145 patients received thalidomide in combination with a corticosteroid or other chemotherapeutic agents. 70 of 95 patients were prescribed thromboprophylaxis; of these, 53 patients received warfarin, 17 patients received aspirin during combination treatment. 38 of 70 patients were newly diagnosed. Only 1 of 70 patients (1.4%) developed DVT. None of the patients who were not received any prophylaxis developed DVT during combination treatment.

Conclusion: The incidence of deep vein thrombosis is lower in Japanese patients with MM treated with thalidomide and corticosteroids or chemotherapy than previous report from western countries.
Long Period of Complete Haematological and Molecular Remission of Chronic Eosinophilic Leukaemia after 6months Treatment with Imatinib
Authors: O’Mara Stephen K , Iland Harry
Published: April 2020

We report a 25 year old male patient who was diagnosed with chronic eosinophilic leukaemia based on clinical features of Skin rash, peripheral neuropathy of right hand, malaise, and hepatosplenamegaly detectable by clinical examination His blood contained 30 × 109/L Eosinophils.. A bone marrow was heavily replaced by eosinophils and eosinophil precursors. ANCA was negartive and normal Ig E levels. PCR for the PDGFR-FIP1L1 fusion gene was positive. He was initially treated with a combination of corticosteroids and alpha Interferon-2alpha and anticoagulated. At 5 months of therapy complete haematological remission had not been achieved. He was started on Imatinib mesylate 400mg per day and rapidly achieved haematological and molecular remission. Imatanib was stopped at 6 months for liver dysfunction. He was rechallenged with 100mg of imatanib after four weeks without treatment. He again developed hepatic dysfunction and imatanib was ceased completely. He remains in complete haematological and molecular remission 2 years after ceasing imatanib. He has normal performance status. This demonstrates that patients with chronic eosinophilic leukaemia who are treated with a 400mg dose of imatanib for six months may be cured or have substantial remissions. It is unclear whether the use of corticosteroids and interferon prior to the introduction of imatanib influenced the outcome. Previous published data showed that using lower doses of Imatanib does not lead to sustained remission of chronic eosinophilic leukaemia after completing drug therapy.
A Quantitive Assay and Clinical Significance for JAK2V617F Mutation in Chinese Patients with Essential Thrombocytosis
Authors: Zhang Ri , Chao Hongying , Shen Yimin , Fan Zhen , Zhang Xuhui
Published: April 2020

Essential thrombocytosis (ET) is a subtype of Bcr-Abl negative the chronic myeloproliferative disorders and the mutated JAK2V617F has been identified and correlated with ET. The aim of this study was to analyze the mutational status and quantitative expression of JAK2V617F mRNA transcripts in cohort of 98 Chinese patients with ET and to determine the disease features. The cohort consisted of 46 men and 52 women with median age at diagnosis was 50 years (range 17–87), granulocyte cDNA was enriched from blood samples obtained at the time of clinical assessment. A quantitative JAK2V617F measurements were made using amplification-refractory mutation sequencing polymerase chain reaction (ARMS-PCR) and capillary electrophoresis assay. Fifty-nine(60.2%) patients harboring the JAK2 V617F mutation were found, including 17 homozygous and 42 heterozygous changes. The quantitative assay by capillary electrophoresis showed that the mutated mRNA ratio was (89.9±6.7)% in the JAK2V617F homozygote and was (57.1±6.7)% in the JAK2V617F heterozygote patients, the difference was significant(P<0.005). JAK2V617F-positive ET patients were older than patients without the mutation (55 versus 38 years, p<0.001), and the patients whose age ≥ 60 years showed significantly higher JAK2 mutated RNA levels as compared to patients whose age ≤60 years(68.2% versus 80.2%, P =0.0056). Males and females had similar JAK2V617F prevalence (63.0% versus 57.7%) and frequency of homozygosity (17.4% versus 17.3%). As compared to heterozygous ET patients, higher leukocyte count was observed in homozygous ET patients (27.9×109/L versus 15.2×109/L, P©‚0.05), JAK2V617F-positive patients also had a higher median white cell count than wild-type patients (19.4×109/L versus 9.7×109/L, P©‚0.05), but there were no significant differences in hemoglobin concentration or platelet count. During follow-up, 30 patients suffered from documented thrombotic events, with 24 having JAK2V617F mutations. The rate of thrombotic complications in JAK2V617F-positive and homozygous patients was higher than in JAK2 wild-type and heterozygous patients, respectively. (52.2% versus 17.6%, 75% versus 32.8%, respectively ). Compared with patients without thrombotic events, there were higher levels of mutated JAK2 mRNA in patients with thrombotic events(76.9% versus 58.4%, P©‚0.05), thrombotic events were also significantly correlated with leukocytosis and older age. Our results suggest that there are different clinical features between JAK2V617F-positive and JAK2V617F-negative, homozygote and heterozygote, higher and lower levels of mutated JAK2 mRNA, and correlated with leukocytosis and thrombosis. Therefore, detection of the JAK2V617F mutational status and mutated JAK2 mRNA can help not only identify the disease state and progression, but also affect the management of ET patients.
ABC-Me (ABCB10) Is Required for Erythroid Development in the Mouse Embryo and Is Protective against Mitochondrial Oxidative Stress
Authors: Hyde Brigham B , Elorza Alvaro A , Mikkola Hanna K , Schlaeger Thorsten M , Shirihai Orian S
Published: April 2020

We previously described that mitochondrial ATP-Binding Cassette transporter, ABCme (ABCB10) is induced by GATA-1 in erythroid cells and that its over-expression leads to increased heme production. However, the role of ABCme in vivo and the hematologic phenotype of its deficiency have not been described thus far. Here we report for the first time that ABCme is essential for embryonic blood development in vivo. ABCme deficient mouse was generated on 129SvEvBrd/C57BL6J background and backcrossed to n = 4 on the C57BL6J. The homozygous KO mouse is embryonic lethal. ABCme KO dies at 12.5 days of gestation and displayed aplastic embryonic anemia by 10.5 days of gestation, manifested as improper blood development as well as erythroid progenitor apoptosis (Figure 1). Ex vivo analysis of maturation of isolated yolk sac blood determined that ABCme KO erythroid cells fail to differentiate beyond the stage of basophilic erythroblast and exhibit 55% of apoptosis in CD71 positive cells in comparison with 29.25% in ABCme HET, and 17.5% in WT. Erythroid colony assay indicated 50% reduction in the formation of CFU-E and an 80% reduction in BFU-E in comparison with WT. Disruption of heme synthesis may result in accumulation of heme intermediates that are pro-oxidants. To test the hypothesis that apoptosis is induced by oxidative damage we tested both mitochondrial oxidation and the rescue effects of mitochondrial antioxidants in the ABCme KO. Isolated mitochondria of ABCme KO erythroid cells demonstrated significantly elevated levels (5 fold) of oxidized protein in comparison to both WT and HET cells. This elevated level of superoxide were confirmed and identified to be mitochondrial in nature by FACS analysis of superoxide sensitive dye MitoSox. Using antioxidants we tested the patho-physiological role of the elevated ROS in the hematologic phenotype. Treatment of ABCme KO cells with the SOD2 mimetic TBAP resulted in partial and complete rescue of the apoptotic phenotypes of the KO and the HET respectively. Our findings suggest that ABCme is required for erythroid development in vivo and that its deficiency results in impaired erythroid maturation, hemoglobinzation, and increased mitochondrial oxidative damage.

Figure SEQ Figure \* ARABIC 1: Impaired Blood Development of ABCme deficient embryos Figure. SEQ Figure \* ARABIC 1: Impaired Blood Development of ABCme deficient embryos

Light microscopy of Day 10.5 p.c. Embryo and Yolk Sac (Top). At this stage KO embryos are still alive as denoted by heartbeat as well as normal development of other organ systems. However, lack of red cell development is apparent. H/E staining of day 10.5 p.c. yolk sac blood islands (Bottom). KO blood islands demonstrate reduced number, increased nuclear fragmentation associated with apoptosis, and a lack of developed erythroid progenitors. (WT= Wildtype mice, KO= ABCme −/− mice.)
The Predictive Role of Germinal Center or Non-Germinal Center Phenotypes in Diffuse Large B-Cell Lymphoma Patients Receiving CHOP or R-CHOP
Authors: Lee Sun-ah , Ryoo Hun-Mo , Bae Sung Hwa , Jung Hyun Young , Jang Saet Byul , Keum Yoon Seop
Published: April 2020

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin’s lymphoma. It can be divided into the germinal center (GC) and non-GC subtypes by immunohistochemistry based on the expression of CD10, Bcl-6 and MUM-1. The GC Subtype had a significantly better survival rate than the non-GC subtype. These subtypes are predictors of outcome in DLBCL patients receiving CHOP (cyclophosphamide, adriamycin, vincristine and prednisone). Recently a significant improvement of outcome has been obtained by combining a monoclonal anti-CD20 antibody, Rituximab, with chemotherapy. We conducted the retrospective study to evaluate the predictive role of GC or non-GC subtypes in DLBCL patients receiving CHOP or rituximab-CHOP (R-CHOP). We searched the DLBCL patients diagnosed at Daegu Catholic University Hospital between 2000 and 2007. Rituximab was introduced into the treatment since 2003. Both the immunohistochemical and clinical data could be analyzed in forty six patients. According to the expression of CD10, Bcl-6 and MUM-1, we divided DLBCLs into the GC or non-GC subgroups (Blood. 2004;103:275-282). The median follow-up duration was 22.5 months. 21 and 25 patients were received CHOP and R-CHOP, respectively. 12 and 34 patients were classified into the GC and non-GC subgroups. The GC subtype showed a better overall survival (OS) than the non-GC subtype (p=0.046), but not progression-free survival (PFS) (p=0.633). The GC subtype did not predict the outcome in CHOP and R-CHOP group, respectively. In R-CHOP group, the expression of Bcl-6 was associated with a better OS (p=0.033). The GC subtype showed a better OS in low IPI (IPI 0–2) with borderline significance (p=0.054), but not in high IPI group (IPI 3–5) (p=0.429). In conclusion, the GC subtype showed a better OS in whole patients group, but didn’t show the superior outcome than the non-GC subtype in CHOP subgroup or R-CHOP subgroup, respectively, in this study. It may be due to the small size of the GC subtype. The prospective, large scale study is recommended.
Clinical and Biological Characteristics of Plasmacytoid Dendritic Cell Lymphoma in Children
Authors: Hama Asahito , Yagasaki Hiroshi , Muramatsu Hideki , Nishio Nobuhiro , Abe Akihiro , Takahashi Yoshiyuki , Kojima Seiji
Published: April 2020

Plasmacytoid dendritic cell (pDC) lymphoma is a disease entity in the WHO/EORTC cutaneous lymphoma classification. The pDC lymphoma co-expresses CD4 and CD56 without any other lineage-specific markers and has been identified as arising from pDC. In addition, pDC lymphoma is rare and characterized by a rapid aggressive course, with notable skin involvement and a frequent evolution towards overt leukemia. While this disease mainly affects the elderly, a few pediatric cases have been reported. In children, there are two distinct types that exist. These include an aggressive type that involves the skin similar to that seen in the adult type of the disease, along with a second type that is less aggressive and does not involve the skin or behave like acute leukemia. We describe the clinical and biological features of three pDC lymphoma children (Table 1). Morphologically, we observed a peculiar arrangement of the vacuoles along the cytoplasmic outline, the so-called pearl necklace appearance. The blast cells expressed CD4, CD56, HLA-DR, CD123 in three of the patients while BDCA-2 was noted in two patients (Table 2). After the initial AML-oriented therapy, two patients developed an early relapse and subsequently died; they were considered as aggressive type of pDC lymphoma. In our hospital, 3 of 22 patients with malignant lymphoma were diagnosed as pDC lymphoma for five years. In conclusion, the CD4+, CD56+, CD3−, CD13−, CD19−, CD33− phenotype is highly suggestive of pDC lymphoma. In the current cases, additional analyses using CD123 and BDCA-2 antibodies helped to confirm our initial diagnosis. Since an optimal therapy for pDC lymphoma in children is as of yet still unknown, a cooperative study needs to be undertaken to further investigate potential effective therapies for this disease.

Table 1. Clinical presentation of the patients with plasmacytoid dendritic cell lymphoma

Patient 1 Patient 2 Patient 3 AraC, cytosine arabinoside; PSL, prednisone; VCR, vincristine; MTX, methotrexate Year/Sex 5/M 9/M 6/F Skin lesion + + + Lymphadenopathy − − + WB C (×109/L) 5.0 7.4 6.4 Hemoglobin (g/dL) 11.4 12.6 13.2 Platelets (×109/L) 284 294 264 BM blast cells (%) 13 0 0 Initial therapy AraC, etoposide AraC, etoposide PSL, VCR, MTX Complete remission + + + Relapse + + − Survival (month) 22 36 2 < Outcome Death Death Alive

Table 2. Immunophenotypes of the blasts with plasmacytoid dendritic cell lymphoma

Surface marker Patient 1 Patient 2 Patient 3 NA, not available CD3 − − − CD4 + + + CD7 + − − CD13 − − − CD19 − − − CD33 − − + CD34 − − − CD56 + + + CD123 + + + HLA-DR + + + BDCA-2 + NA +
Pediatric Acute Lymphoblastic Leukemia Outcome Predictor Genes OPAL1 and RANTES Modulate Transformation by the V-Abl Oncogene
Authors: Rogers Jason H. , Owens Kristin S. , Vargas Jake M. , Willman Cheryl L. , Hromas Robert , Dahl Richard
Published: April 2020

B-precursor acute lymphoblastic leukemia (ALL) is the most common cancer of childhood. While it represents a highly curable malignancy, a significant number of children still relapse or present with disease that is resistant to therapeutic intensification. With the increasing intensity of curative treatment, there is also an increased incidence of late effects that adversely affect the quality of life of survivors. Previously, a large scale microarray gene expression analysis was undertaken in order to identify genes predictive of outcome that could enhance risk classification, thereby identifying children who might be cured with less intensive therapy or those who fail current regimens and require novel therapies for cure. From analysis of 254 pediatric ALL samples registered to various COG clinical trials, gene expression classifiers were identified that are highly predictive of poor and favorable outcome in precursor B-cell ALL. The goal of this project is to study the biologic function and role in hematopoiesis and leukemogenesis of the genes predictive of outcome and to determine whether any of these genes may serve as novel drug targets. To this end, we have developed a system to examine the effect of these genes on the cancer-promoting activity of the v-Abl oncogene. We have established pre-B cell lines by infecting primary mouse bone marrow cells with a virus expressing v-Abl. These lines were then engineered to express two of the genes we identified that were associated with poor or good treatment outcomes, RANTES (CCL5) and OPAL1 respectively. Consistent with the association of RANTES expression with poor outcome, v-Abl/RANTES pre-B cells are more proliferative than v-Abl cells, and form larger colonies in methylcellulose cultures without added cytokines. OPAL1 had the opposite effect on pre-B cell growth. V-Abl pre-B cells expressing OPAL1 were less proliferative and generated fewer colonies in methylcellulose cultures. To determine whether RANTES made the v-Abl pre-B cells more leukemic, we transplanted v-Abl or v-Abl/RANTES pre-B cells into syngeneic mice. Mice were noticeably ill 30 days post-transplant. Mice injected with v-Abl/RANTES cells had enlarged lymph nodes and increased numbers of white cells in their peripheral blood. The percentage of pre-B cells in the bone marrow, lymph nodes and spleen was higher in v-Abl/RANTES transplanted mice compared to v-Abl transplanted mice. Currently our data demonstrates that the outcome genes are playing a mechanistic role in the susceptibility of leukemic cells to therapy, and are not merely epiphenomenon. This suggests that the products of these genes and their associated pathways may be novel drug targets. Such targeted therapy has the potential of being less toxic than current nonspecific treatment regimens.
Risk Determinants of Nonarteritic Ischemic Optic Neuropathy (NAION)
Authors: Zotz Rainer B. , Finger Clara , Gerhardt Andrea , Scharf Rudiger E.
Published: April 2020

Background: The aim of our present study was to examine the risk factors for atherosclerosis as well as thrombophilic risk factors for their role in patients with NAION in comparison to healthy controls.

Methods: Prospective case-control-design with 109 Patients and 109 age-and sex-matched healthy controls.

Results: Elevated levels of fibrinogen, FVIII:C, FIX:C, FXI:C, plasminogen activity, von Willebrand antigen (vWF:Ag) and activity, triglycerides, elevated erythrocyte sedimentation rate and decreased levels of high density lipoprotein (HDL) proved to be significant risk factors associated with NAION (OR 1.9 to 5.4; p<0.05). The combination of these risk determinants further increased the risk in a multiplicative or supra-multiplicative way (up to OR 16.5; p<0.0001). None of the examined genetic risk factors (factor V Leiden mutation, Prothrombin mutation, platelet polymorphisms KOZAK and VNTR of glycoprotein 1bα, Hpa-1 polymorphism of GPIIb-IIIa, C807T polymorphism of GPIa/IIa, 4G/5G polymorphism of PAI-1 and C667T mutation of MTHFR gene) was significantly associated with the disease. However, when the HPA-1b allele, GPIa807T allele or the PAI-1 polymorphism were combined with increased levels of fibrinogen or vWF:Ag, a significant interaction with a supra-multiplicative increase in risk could be demonstrated.

Conclusions: In this first comprehensive analysis of a wide range of risk determinants of atherosclerosis and thrombophilia in NAION patients, several risk factors could be identified and quantified. Risk factors of arterial thrombosis appear to interact with risk factors of inflammatory atherosclerosis triggering NAION.
Combined Jugular and Subclavian Vein Thrombosis Following Assisted Reproductive Technology: The Mystery Is out
Authors: Salomon Ophira , Schiby Gina , Heiman Zehava , Avivi Kamila , Dor Joshua , Itzchak Yacov , Levran David , Sigal Carol
Published: April 2020

In assisted reproductive technology (ART) high doses of human menopausal gonadotropins are used to induce controlled ovulation. This technology puts women at high risk of developing the ovarian hyperstimulation syndrome and thombotic events.

Objective: To discern the reason for predilection of combined jugular and subclavian following ART.

Methods: Women who developed combined jugular and subclavian vein thrombosis following assisted reproductive technology were included in the study. The thrombotic events were demonstrated by ultrasound Doppler or computerized tomography angiography. All women were interviewed and data obtained from outpatient clinic and hospital medical charts. Magnetic resonance imaging and complete thrombophilic profile work-up was performed in each woman. Open biopsy from the lesions was taken from one of the women for histological sections and immunohistochemical stains.

Results: Five women developed combined jugular and subclavian vein thrombosis following ART. They were found to harbor clusters of rudimentary branchial cysts filled with fluid at the time of ovarian hyperstimulation syndrome, which compressed the jugular and subclavian veins at their junction at the base of the neck. Four patients (80%) were found to be carriers of factor V Leiden.

Conclusion: The predilection of combined jugular and subclavian vein thrombosis early in pregnancy is the result of mechanical compression mediated by rudimentary branchial cysts filled with fluid during ovarian hyperstimulation syndrome, particularly in subjects who are carriers of factor V Leiden.
Increased Expressions of Toll-Like Receptor 4 on Platelet Are Related to Type of Bacteria and Disease Severity in Patients with Sepsis
Authors: Ma Liping , Wang Xiu-Ju , Nie Da-Nian , Li Yi-Qing , Xie Shuang-Fen , Wu Yu-Dan , Zhang Cheng , Song-Mei Yin
Published: April 2020

Early diagnosis and treatment of patients with sepsis remain a common and severe problem, especially in leukopenic patients.. Toll-like receptor-4 (TLR4) plays a crucial role in immunity as the first defenses system against microbial infection through binding gram-negative bacterial LPS. Blood platelets are not only involved in hemostasis, they also have many features of classic inflammatory cells. The expression of TLR4 on platelet in patients with sepsis, including gram-positive bacteria and gram-negative bacteria, is not known. Studying the differences between them, we investigated whether the expressions of TLR4 on platelet were associated with platelet activation, serum TNF-a and endotoxin levels in patients with sepsis, 8 patients of them with gram-positive bacteria and 15 patients of them with gram-negative bacteria. The number of platelet and function of coagulation were normal before infecting. Comparing with the heath subjects, the expressions of TLR4 and P-selectin on platelets, the levels of serum TNF-a and endotoxin were higher (P<0.05),and there was a positive correlation was observed between TLR4 and P-selectin, TNF-a, endotoxin respectively, among the gram-negative bacterial subjects. The phenomena were not found among the gram-positive bacterial subjects. In addition, among the gram-negative bacterial subjects, the expression of TLR4 was higher in patients with decreased number of platelets than with normal number of platelets. These results suggest that increased TLR4 on platelet might be an important early sign of gram-negative bacteria in patient with sepsis, it contributes to platelet activating, the inflammatory process and disease activity in infecting host. Above has be doing in more patients with positive blood bacteria culture in our Lab.
IL-34, in Synergy with RANK Ligand, Promotes Osteoclast Development through the CSF-1 Receptor
Authors: Huang Min Mei , Leo Cindy , Wei Suwen , Stanley E. Richard , Doberstein Stephen , Lin Haishan
Published: April 2020

IL-34 is a newly identified cytokine that binds to the CSF-1 receptor (CSF-1R), promotes monocyte viability, and stimulates macrophage colony formation (Lin et al., 2008). Here we demonstrate that human IL-34 induced tyrosine phosphorylation of the CSF-1R in cells of a mouse MacCsf1r−/−.huCSF-1R macrophage line. MacCsf1r−/−.huCSF-1R cells were obtained by retroviral transduction of the human CSF-1R into cells of the MacCsf1r−/− macrophage line (Yu et al., 2008) derived from bone marrow macrophages isolated from a CSF-1R-deficient mouse. In addition, IL-34 stimulated the downstream signaling of ERK1/2 and the IL-34-induced phosphorylation of ERK1/2 was blocked by a CSF-1R-specific inhibitor. IL-34, in synergy with RANK ligand, promoted formation of tartrate resistant acid phosphatase (TRAP)-positive and multinucleated osteoclasts from monocytes in dose-dependent manner. The activity of IL-34 on osteoclast differentiation was inhibited by the soluble CSF-1R extracellular domain. Therefore, IL-34 functions as a new ligand of CSF-1R and participates in the regulation of osteoclast development.
Leukemia-Associated Primary Granule Proteins (PGPs) Elastase-2 and Proteinase-3 Are Aberrantly Expressed in Solid Tumors: A Potential Therapeutic Target for PR1-Directed Immunotherapy
Authors: Alatrash Gheath , Vence Luis M , Woodward Wendy , Ueno Naoto T , Molldrem Jeffrey J
Published: April 2020

Neutrophil elastase (ELA2) and proteinase-3 (PRTN3) are the source proteins for the nonomeric HLA-A2-restricted peptide PR1 (VLQELNVTV), a recognized leukemia-associated antigen (LAA). In myeloid leukemia, high expression of ELA2 and PRTN3 has been correlated with improved clinical outcomes following hematopoietic stem cell transplant (HSCT). Vaccination with PR-1 peptide showed efficacy in myeloid leukemia and myelodysplastic syndrome, and was associated with immunologic response (≥ 2 fold increase in PR1-specific cytotoxic T lymphocytes (PR1-CTL)) and long-lasting clinical remissions. PR1-CTLwere shown to mediate immunity elicited by PR-1 vaccine, as they preferentially recognize and kill myeloid leukemia cells. Since ELA2 and PRTN3 have been reported in breast cancer biopsies, we hypothesized that ELA2 and PRTN3 may be mislocalized in solid tumors, leading to MHC-I presentation, therefore providing a therapeutic target for PR1 vaccine. To study the subcellular localization of ELA2 and PRTN3, the cytoplasmic, nuclear, membrane, and golgi/ER fractions were obtained from inflammatory breast cancer (IBC) (MDA-IBC1 and SUM149) and melanoma (526, 624, 888 and 926) cell lines. ELA2 was preferentially expressed in the cytoplasmic fraction in the IBC cell line SUM149, while PRTN3 was expressed in the membrane and cytoskeletal fractions of the IBC cell line MDA-IBC1. In the melanoma cell lines, ELA2 was expressed in the cytoplasmic fractions, while PRTN3 was noted in the nuclear, cytoplasmic and cytoskeletal fractions. Furthermore, the HLA-A2+ melanoma cell line 888 was susceptible to lysis by PR1-CTL (approximately 30% lysis; Effector:Target ratio=1:2). Together, these results demonstrate that ELA2 and PRTN3 are aberrantly localized in non-hematopoietic cell lines, and that this can be associated with susceptibility to PR1- CTL lysis. In particular, PR1 is a potential immunotherapeutic target for patients with melanoma and breast cancer.
VG5Q Inhibition Suppresses Proliferation of Endothelial and Cancer Cell Lines
Authors: Bodempudi Vi dya , Dudek Olivia Anna , Terai Kaoru , Dudek Arkadiusz Zbigniew
Published: April 2020

Background: Angiogenesis is a process involved in the formation of new vessels, and is essential for tumor growth and metastases. Angiogenic factors are critical for the initiation of angiogenesis and maintenance of the vascular network. VG5Q is a newly discovered protein that acts as a potent angiogenic factor in promoting angiogenesis and has also been implicated in the vascular pathophysiology of Klippel-Trenaunay syndrome. However, little is known about its role in cancer progression. Method: In this study, VG5Q expression was screened using RT-PCR and Western blotting in the following cancer cell lines: breast (MCF-7), renal (Caki, ACHN), mesothelioma (H2373, H2596), ovarian (OVCAR5), lung (H226, A549) and pancreatic (Panc-1, MiaPaCa). Human umbilical vein endothelial cells (HUVEC) and blood outgrowth endothelial cells (BOEC) were also screened. Results: All cancer cell lines expressed high levels of VG5Q that were comparable to the levels expressed in endothelial cells. The only exception was MiaPaCa cells. In order to understand the biology of VG5Q in cancer cells, we used siRNA knockdown of VG5Q expression in the mesothelioma cell lines H2373 and H2596. Gene-specific silencing of VG5Q resulted in markedly decreased proliferation, invasion, and soft-agar colony formation in these cells. Furthermore, VG5Q silencing increased apoptosis as seen by an increase in annexin levels. Conclusion: VG5Q silencing has profound effects on cancer cell biology, suggesting the hypothesis that the role of VG5Q may not be limited to angiogenic signaling in endothelial cells.
A Reliable Marker of Endothelial Cell Dysfunction and Vascular Damage: Legend or Reality?
Authors: Corinaldesi Giorgio , Corinaldesi Christian
Published: April 2020

Endothelial dysfunction is considered to be a pre-clinical stage of atherosclerosis and represents an initial step of plaque progression. The aim of this study is to investigate whether the overproduction of biochemical markers is related to vascular damage. We studied 72 patients split into three groups:

20 patients had no vessels significant disease; 18 patients had multi-vessels disease; 34 patients had multi-vessels disease and stenosis >50%.

Patients with known coronary artery disease, stroke, hypertension, diabetes and/or dyslipidemia were excluded from the study.

We measured plasma levels of vWF (a marker of endothelial stress), VEGF (a marker of neointimal proliferation), TF and E-selectin (a marker of endothelial activation), all were measured by ELISA, and we investigated if their increased levels are related to the endothelial damage (table 1).

Table 1.

No disease Multi vessels Multi vessels disease+stenosis vWF (IU/dl) 84–102 128–154 146–182 VEGF (pg/ml) 28–124 120–180 160–240 TF (pg/ml) 16–98 80–220 230–370 E-Selectin (ng/ml) 30–74 72–96 94–128

There is a significant relation between vWF, VEGF, TF and E-Selectin and vessels disease severity; patients with no vessels disease had an average thickness of 0.48 mm, (IMT: 0.6–1.18mm), patients with multi vessels disease had a median atheroma score of 1.12, (IMT:1.18–2.42mm), patients with multi vessels disease and stenosis had a mediam atheroma score of 2.40, (IMT:>2.42mm), indeed we did not find any significant relation between increased arterial stiffness and laminar shear-stress. Healthy endothelium exerts a favourable and athero-protective effects by a numbers of factors.

Table 2. Favorable and Atheroprotective Effects of the Healthy Endothelium

Promotion of vasodilation Antioxidant effects Anti-inflammatory effects Inhibition of leukocyte adhesion and migration Inhibition of smooth muscle cell proliferation and migration Inhibition of platelet aggregation and adhesion Anticoagulant effects Profibrinolytic effects

Table 3. Risk factors and predictors of endothelium dysfunction

Recruiment of inflammatory cells SMC proliferation Neointimal formation Oxidative stress Activation of procoagulant activities Activation of plateolet adhesion and aggregation Stasis of blood flow

In conclusion, we demonstrated that high levels of vWF, VEGF, TF, E-Selectin may play a critical role as for the development and the progression of atherosclerosis, even if further and wider studies are needed to confirm this statement.
Peripartum Haemostatic Changes in Women in Labour
Authors: Lefkou Eleftheria , Parmar Kiran , Mamopoulos Apostolos , Masouridou Sevasti , Karagiannis Vassilios , Hunt Beverley J
Published: April 2020

Introduction: Obstetric haemorrhage (OH) is a major cause of maternal mortality and morbidity worldwide. Appropriate management requires understanding of normal peripartum haemostatic changes during labour, and yet there are few studies.

Aim: In this pilot study we aimed to evaluate peripartum haemostatic changes in healthy women.

Methods: We collected blood samples from 20 women peripartum, into 0.102 M trisodium citrate tubes (ratio9:1) (Becton Dickinson) from the antecubital fossa using flawless venepuncture at the following time-points:

antepartum; after delivery of the placenta; 24hrs post-partum and 48hrs post-partum.

Plasma was aliquoted and stored in −70°C until the following ELIZA assays were performed: D-Dimer levels (Dade Behring, Sysmex UK), prothrombin fragments 1+ 2 (PF 1+2) (Dade Behring, Sysmex, UK) and tissue plasminogen activator antigen (t-PA:Ag) (Biopool, Trinity Biotech, UK). Prothrombin time (PT) (PT-Fib HS), activated partial thromboplastin time (APTT) (APTT micronized silica) and clauss fibrinogen (Fib-C), reagents were from Instrumentation Laboratories Ltd UK. Statistical analysis was performed using XLSTAT 2008.

Results: The median (range) age was 28 (19–36) years and the body mass index (BMI) 28.9 (21.7– 35.3).

Table 1: To show the median (range) for the haemostatic assays

Assay (normal ranges) D-dimers (4–52ug/l) PF1+2 (0.2–1.2 pmol/l) t-PA:Ag: 3–11 ng/ml) PT (12–16s) APTT (26–38s) Fibrinogen (1.52–4.12 g/l) * p<0.05 using the Mann Whitney U test between the 1st and each of the other three samples Antepartum 231 (122–900) 570 (352–1533 12 (7.2–23) 12 (10–14) 31 (24–36) 5.2 (3.1–8.7) Post Labour 482 (117–1044) 968 (480–1858)* 18 (8.6–40.6)* 13 (10–15) 30 (24–36) 5.3 (2.2–9.8) 24h 309 (65–931) 568 (264–1080)* 7 (4–23)* 13 (10–16)* 33 (27–41)* 5.3 (4–11) 48hrs 235 (85–614) 648 (371–2287) 7 (3–18)* 12 (10–16) 31 (23–43) 5.7 (3.5–11)

Conclusion: These findings demonstrate that peripartum haemostasis is prothrombotic with shortened PT, APTT and prolonged fibrinogen. There is increased thrombin generation and fibrinolytic activity. t-PA antigen is most notably elevated immediately post delivery.
Inactivation of PUMA Protects Hematopoietic Stem Cells and Confers Long Term Survival in Response to High Dose γ-Irradiation
Authors: Yu Hui , Shen Hongmei , Xu Feng , Hu Xiaoxia , Li Yanxin , Ling Yun , Yu Jian , Zhang Lin , Zambetti Gerard P. , Cheng Tao
Published: April 2020

Radiation injury remains a significant health problem. New medical intervention to prevent or manage radiation damage is highly dependent on a deeper understanding of how radiation-induced cell death is accomplished in the irradiated tissue cells such as stem and progenitor cells. To date, relatively specific or untainted molecular mediators in apoptosis of tissue stem and progenitor cells upon radiation injury have not been clearly defined. The p53 pathway is known as a major molecular mechanism for cell apoptosis, upon the exposure of lethal radiation. Targeting p53 confers a radioprotective effect, but may increase tumorigenesis due to impaired cell cycle arrest for DNA repair. In our current study, we have examined the specific role of PUMA (p53 up-regulated mediator of apoptosis) in the radiosensitivity of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). By quantitative RT PCR, we found that the level of PUMA mRNA was relatively low in the most primitive long-term repopulating hematopoietic stem cells (LT-HSC, isolated based on the immnunophenotype “CD34−LKS”) as compared to other hematopoietic cell populations from mice, but it was significantly elevated in response to γ-irradiation. In the mice lacking PUMA, while neither HSC number nor HSC function was altered under homeostatic conditions, the PUMA−/− HSCs appeared to be resistant to radiation damage in vivo as retrospectively quantified in a competitive HSC transplant model. Our further direct measurement with a single cell culture system for HSC growth in vitro, demonstrated that PUMA, but not p21 (the chief mediator of p53 in cell cycle arrest), is primarily responsible for the radiosensitivity of HSC in the p53 pathway (200 LT-HSCs analyzed for each cell type). Together, these data provide definitive evidence for PUMA as an essential mediator in radiation-induced apoptosis of tissue stem cells. We finally focused on the beneficial effects of targeting PUMA in HSCs and HPCs on the animal survival upon the exposure of lethal irradiation. Strikingly, the wild-type mice reconstituted with PUMA−/− hematopoietic cells exhibited a significant survival advantage after two rounds of 9-Gy γ-irradiation (18 Gy in total) as compared to the mice reconstituted with PUMA+/+ hematopoietic cells (95 % vs. 0 % survival in 20 days, n=21/each group; 50% vs. 0 % survival in 180 days, n=20 or 11/each group, respectively) as shown in the figure below. Moreover, unlike the p53−/− mice, those PUMA−/− reconstituted mice did not have an increased incidence of hematopoietic malignancies (n=20) within 180 days. Therefore, our current study establishes PUMA as an attractive molecular target for the development of therapeutic agents for the prevention and treatment of radiation injury.
An Ultrastructural Investigation of the Fibrin Clot Utilizing Ferritin-Labelled Anti-Human Fibrinogen Antibody
Authors: WHITE JAMES G. , KRIVIT WILLIAM , VERNIER ROBERT
Published: April 2020

A study of fibrin ultrastrtucture utilizing ferritin-conjugated antihuman fibrinogen antibody is reported. The structure of fibrin strands in thin section, and the periodic banding of the fibrin by ferritin conjugated antibody permits conclusions regarding the structural formation of the clot. The observations are in accord with the model of fibrin strand formation suggested by Hall and Slayter. Further areas of investigation utilizing this technic are suggested.