bluuebunny/crossref_metadata_2025 huggingface dataset:
HMGB1 Derived from Macrophages Acts as an Angiogenic Factor.
Authors: Maruyama Ikuro , Kawahara Ko-ichi , Ito Takashi , Oyama Yoko , Takenouchi Kazunori , Shimizu Toshiaki , Kikuchi Kiyoshi , Hashiguchi Teruto
Published: October 2019

High Mobility Group Box-1 (HMGB1) protein, released from the most of necrotic cells and activated macrophages, has been identified as a novel cytokine through the receptor for advanced glycation endproducts (RAGE) and Toll-like receptor(TLR)-2 and -4. The HMGB1-RAGE and TLRs-2, -4 interactions contribute to cellular migration and the production of proinflammatory cytokines, and participate in pathomechanisms in tumor growth and invasion, in which an angiogenesis development is an important aspect.

We here show that HMGB1 stimulates the expression of Vascular Endothelial Growth Factor (VEGF), the most potent angiogenic factor in tumors, through the HMGB1-RAGE, but neither TLR-2 nor TLR-4 in macrophage-lineage cells in vitro. The mechanism of VEGF production is mediated through the Akt pathway, which is linked to tumor growth and invasion. Furthermore, HMGB1 induced angiogenesis was also observed in an in vivo rabbit corneal assay (Fig). These results suggest that HMGB1, released from the tumor-associated macrophages, may act as a key cytokine in the development of angiogenesis by producing VEGF in tumor growth and invasion. Thus the tumor-associated HMGB1/RAGE system may contribute to our understanding of the mechanism of cancer cell escape from macrophage-associated acceleration of inflammation.
The Auto-Antibodies on Bone Marrow Cells in the Patients with Systemic Lupus Erythematosus.
Authors: Fu Rong , Deng Jizi , Yuan Shang , Gong Lu , Sun Jun , Shao Zonghong
Published: October 2019

Objective:To explore the pathogenesis of cytopenia in the patients with systemic lupus erythematosus (SLE), the auto-antibodies on bone marrow mononuclear cells (BMMBC) in the patients with SLE were determined.

Methods:Twenty one patients with SLE and ten healthy controls were enrolled in this study. BMMNC Coombs test was used to determine the aotoantibodies. The correlation between the types of auto-antibodies on BMMNC, the types of serum auto-antibodies and the counts of blood cells in the patients with SLE were also investigated.

Results:Positive results of BMMBC-Coombs test were seen in 12 patients with SLE (57.1%), among them, 10 with hemocytopenia (58.82%), and 2 without hemocytopenia (50%). The positive rate of BMMNC Coombs test was higher in the patients with SLE than that in healthy controls, and was higher in SLE patients with hemocytopenia than that in healthy controls. There were no significant difference of BMMNC-Coombs positive rate between the SLE patients without hemocytopenia and healthy controls, and there were also no significant differences between the SLE patients without hemocytopenia and SLE patients with hemocytopenia. In the 12 SLE patients with positive BMMBC-Coombs tests, IgM auto-antibody accounted for 75.0%, and C3 50.0%, IgG 8.33%, IgG+IgM 8.33%, C3+IgM 16.67%, IgG+IgM+C3 16.67%. In the SLE patients without hemocytopenia, IgG+IgM accounted for 8.33%, C3 8.33%, but IgA autoantibody were not seen in any case. There was a significant positive correlation between the auto-antibodies on BMMNC and peripheral anti-SSA, but there was no significant correlation between the results of BMMBC Coombs tests and peripheral blood cell counts.

Conclusion:There were auto-antibodies on BMMNC in the patients with SLE. The hemocytopenia in the patients with SLE maybe resulted from the destructions of bone marrow hematopoietic cells by the autoantibodies.
Analyses of Prognostic Factors for Survival in East African Patients with AIDS-Related Non-Hodgkin’s Lymphoma (AR-NHL) Treated with Dose Modified Oral Chemotherapy.
Authors: Subbiah Vivek , Mwanda O.W. , Orem Jackson , Fu P. , Banura C. , Kakembo J. , Ness A. , Johnson J. , Bako J. , Katongole-Mbidde E. , Remick Scot
Published: October 2019

Background: The results of our feasibility trial of dose-modified oral chemotherapy for AR-NHL in East Africa are encouraging to frame / sustain alternative non-myelotoxic and pragmatic therapeutic strategies suitable for evaluation in the resource-constrained setting. To define a group of patients who may have a better outcome univariate and multivariate analyses of prognostic factors were performed.

Methods: Forty nine patients with AR-NHL received dose-modified oral chemotherapy. The following baseline variables were included as potential predictors of survival: age (≤60 vs. >60 yrs.); sex; stage (≤2 vs. >2); ECOG performance status (0/1 vs. 2/3); LDH (≤270 vs. >270 U/L); extranodal sites of involvement (≤1 vs. >1 site); prior thrush; prior AIDS [wasting or opportunistic infection(s)]; A/B symptoms; m-CHOP salvage chemotherapy (n=10); antiretroviral (ARV) therapy (n=18); and baseline albumin. International Prognostic Index (IPI) excluding LDH since not available for all patients (N Engl J Med1993;329:987) and ACTG 142 prognostic variables (J Clin Oncol1998;16:3601) were also analyzed.

Results: By univariate analysis, only LDH (p=0.0013) was significantly related to overall survival (OS). Hypoalbuminemia and LDH affected event free survival (EFS). By multiple regression, LDH and IPI were significantly related to OS (p=0.012). Similarly, the hazard ratio of patients without and with ARV therapy was 17.45 (p=0.0007); and the hazard ratio of patients IPI 2 comparing to those with IPI 0 was 6.62 (p = 0.028). LDH, ARV status and IPI were significantly related to EFS. The complete response rate was 56% and objective response rate was 77%. Median OS and EFS were 12.3 and 7.9 months respectively.

Conclusions: For patients with AR-NHL in East Africa and treated with dose-modified oral chemotherapy IPI and not ACTG 142 prognostic covariates best predicts survival. Albumin (perhaps substituting for LDH since more readily tested in East Africa) and ARV status provide further independent prognostic information. These factors may further guide therapeutic options in future trials in resource poor settings.
Identification and Characterization of a Novel Lymphotoxin Alpha Mutant Recovered from an ENU Mutagenesis Screen.
Authors: Wang Hongsheng , Feng Jianxun , Qi Chenfeng , Morse Herbert
Published: October 2019

N-ethyl-N-nitrosourea (ENU)-induced mutagenesis in mice has been an important approach in identifying genes that are involved in immune regulation and diseases. We have characterized the features of a new mutant C57BL/6J strain, HLB382, derived from an ENU mutagenesis screen and identified initially as having hereditary leukocytosis. Grossly, the mutant mice had no peripheral or mesenteric lymph nodes or Peyer’s patches. Histological analysis showed that there were no marginal zone B cells or germinal centers. The development of T cells and myeloid cells was normal, as assessed by flow cytometry (FACS), the development of NK cells was impaired. Although this phenotype is strikingly similar to that of mice with genetically engineered knockouts of the Lta gene, we found that the numbers of peritoneal B1b cells was significantly increased, a feature not reported for the knockouts. Since FACS analyses did not detect LTA protein on splenic B cells, we sequenced cloned Lta genomic DNA from the mutant mice and identified a single base insertion that caused a frame shift and a premature stop codon, consistent with the lack of detectable LTa proteins in mutant mice. This new mutant strain on a clean C57BL/6 background provides a useful model to study the functions of LTa in the immune system development and in various disease conditions.
The Role of Calcium/Calmodulin-Dependent Protein Kinase Cascade on MIP-1α Gene Expression in ATL Cells.
Authors: Matsumoto Kensuke , Murao Koji , Imachi Hitomi , Nishiuchi Takamasa , Ohnishi Hiroaki , Ishida Toshihiko
Published: October 2019

Adult T-cell leukemia (ATL) is a mature CD4+ T-cell malignancy caused by infection with human T-lymphotrophic virus (HTLV-1) and is associated with a marked hypercalcemia on many patients. Recently, it is proposed that Macrophage inflammatory protein-1α (MIP-1α) is the clinical hallmark of hypercalcemia in ATL, but the regulation of MIP-1α secretion has not been clarified yet. In this study, we examined the effect of calcium on the MIP-1α secretion and cell proliferation of ATL cells, and also the role of the Ca2+/calmodulin (CaM)-dependent protein kinase (CaM-K) cascade in transcriptional activation of MIP-1α. The addition of calcium nitrate to the medium enhanced the secretion of MIP-1α by ATL cell lines (ATN, MT-2, OKM-2T) and the proliferation in a dose dependent manner. The maximum response of MIP-1α secretion was induced at 10.42mM calcium in OKM-2T cells. CaM-KK selective inhibitor STO-609 inhibited the calcium dependent secretion of MIP-1α and proliferation of ATL cell lines. We investigated the effects of CaM-KK/CaM-KIV signaling pathway on MIP-1α promoter activity in OKM-2T. The transfection of CaM-KIV stimulated the MIP-1α promoter activity and the upstream kinase, CaM-KK enhanced the stimulatory effect of CaM-KIV on its activity. Furthermore, mutation of the cAMP response element (CRE) within the MIP-1α promoter significantly reduced the effect of CaM-KIV and calcium, and it wasn’t less enhanced by the addition of calcium nitrate to the medium than the wild type.

Our studies have indicated that hypercalcemia enhances MIP-1α secretion and the cell growth in ATL cells, and these mechanisms require the CaM-KK/CaM-KIV cascade. These findings raise the possibility that the inhibitory of CaM-KK/CaM-KIV cascade may be of therapeutic value for ATL.
High Activity of Peripheral Blood γδ T Cells in Term and Preterm Neonates.
Authors: Haque Syeda F.Y. , Gibbons Deena L. , Hamilton Katherine , Carr Robert , Hayday Adrian C.
Published: October 2019

The peripheral blood white cell compartment is well characterised in adults but relatively little is known in neonates. T cell function has been reported to be very different between neonates and adults. This is significant because the newborn faces the most precipitous encounter with environmental challenges. To further understand the neonatal T cell compartment, we have focussed on γδ T cells which are known to make essential contributions to the immunoprotection of very young mice. We compared γδ cells and αβ T cells from human cord and neonatal blood from term and preterm babies, and compared them with adults. We investigated T cell cultures ex vivo and derived clones. Several findings were evident. First, we confirmed that fresh neonatal αβ cells and clones are profoundly deficient in IFNγ production. Second, we showed that this is not so pronounced for γδ cells and we note that IFNγ production was higher in preterm babies than in term neonates, perhaps reflective of stress in utero. Neonatal γδ clones also made adult quantities of GM-CSF and TRAIL. We note thirdly that neonatal γδ clones make IL-4 and IL-5 (Th2 cytokines) and the immunosuppressive cytokine IL-10, which is not produced at all by adult Vγ9+ clones. Fourth, these pleiotropic activities of human neonatal γδ clones appears to be determined by the type of T cell receptor expressed.

Vδ1-expressing clones have broad functional potentials whereas Vγ9-expressing clones polarise to either a Th1 or Th2 like profile. Fifth, particularly high Th1 cytokine production is observed in Vγ9-Vδ1 (DP) cells which are more common and therefore more easily cloned from neonatal versus adult blood. We conclude that the neonatal γδ cells are highly active and broader in their cytokine production than either their adult γδ cell or their neonatal αβ T cell counterparts. Thus, γδ cells should be better understood vis-à-vis perinatal vaccination regimens and the development of childhood allergies.
Functions of Glycosylation Cooled Rabbit Platelets In Vivo and In Vitro.
Authors: Chen Bao-An , Huang Cheng-Yin , Pei Xiao-Ping , Gao Chong , Ding Jia-Hua , Sun Yun-Yu , Wang Jun , Cheng Jiang , Shi Guang-Yao , Chen Ning-Na , Fei Fei
Published: October 2019

This study was aimed to investigate functions of glycosylation cooled rabbit platelets in vivo and in vitro and the method to store cold platelets with UDP-gal. We collected rabbit heart blood, prepared concentrated platelet suspensions in a normal way to which we added UDP-Gal, and then stored them for ten days in 4° refrigerator. Thereafter platelet counts, mean platelet volume, platelet distributing width, platelet aggregation function, the activity to urge coagulation including PF3aT and APCT and apoptosis were determine- d. Meanwhile, survival time in vivo was tested after cold-stored rabbit platelets labeled with Cr51 were transfused into rabbits. Rabbit ear bleeding time and percentage plate recovery(PPR) were determined 1 hour and 24 hour after they were transfused into rabbit thrombocytopenia model. Results show that there was not significant difference in PLT counts, MPV, PDW, PF3aT and APCT between UDP-Gal cold-stored platelet group and fresh platelet group(p>0.05). On the contrary, platelet counts decreased significantly, MPV, PDW jumped and PF3aT and APCT went down in cold control group compared to fresh platelet group(p<0.01). Apoptosis increase in UDP-Gal cold-stored platelet group compared with fresh platelet group(p<0.05), but was significantly lower than that in cold control group(p<0.01). Although PagT(inducing reagent: C-PG) decreased, it could still be above 50% of fresh platelets. Survival time in rabbit vivo was close between UDP-Gal cold-stored platelet group and fresh platelet group(p<0.05). Survival rate seventy-two hours after transfusion in fresh platelet group, UDP-Gal cold-stored platelet group and cold control group was 57.5%±7.2%,50.3%±6.3% and 0.1%±0.1% respectively. Rabbit ear bleeding time was significantly shortened after transfusion of galactosylation cooled rabbit platelet (p<0.01), In contrast, it had less change in cold control group(p>0.05). PPR was 66.1%±0.5%,47.8%±0.6%;60.9%±0.3%,41.6%±0.4%;47.7%±0.5%,9.4%±0.5% respectively in fresh platelet group, UDP-Gal cold-stored platelet group and cold control group. PPR after transfusion of galactosylation cooled rabbit platelet had no statistical difference compared with that of fresh platelet group(p>0.05), and they in both groups were much higher than that in cold control group(p<0.01).

Conclusion: Galactosylation can improve functions of cooled rabbit platelets in vivo and in vitro. and prolong the storage time of them.
Bruton’s Tyrosine Kinase Is Essential for Botrocetin/vWf-Induced Signaling and GPIb-Dependent Thrombus Formation In Vivo.
Authors: Liu Junling , Fitzgerald Malinda , Berndt Michael C. , Jackson Carl W. , Gartner T. Kent
Published: October 2019

Botrocetin (bt) facilitated binding of von Willebrand factor (vWf) to the platelet membrane glycoprotein (GP) Ib-IX-V complex on platelets in suspension initiates a signaling cascade that causes αIIbβ3 activation and platelet aggregation. Previous work has demonstrated that bt/vWf-mediated agglutination activates αIIbβ3 and elicits ATP secretion in a thromboxane A2 (TxA2)-dependent manner. The signaling that results in TxA2 production is initiated by Lyn, enhanced by Src and propagated through Syk, SLP-76, PI3K, PLCγ2 and PKC. Here, we demonstrate that the signaling elicited by GPIb-mediated agglutination that results in TxA2 production is dependent on Bruton’s tyrosine kinase (Btk). The results demonstrate that Btk is downstream of Lyn, Syk, SLP-76 and PI3K, upstream of ERK1/2, PLCγ2 and PKC, and greatly enhances Akt phosphorylation. The relationship(s), if any between ERK1/2, PLCγ2 and PKC were not elucidated. The requirement for Btk and TxA2 receptor function in GPIb-dependent arterial thrombosis was confirmed in vivo by characterizing blood flow in ferric chloride treated mouse carotid arteries. These results demonstrate that the Btk family kinase, Tec, cannot provide the function(s) missing because of the absence of normal Btk function, and that Btk is essential for both bt/vWf-mediated agglutination-induced TxA2 production and GPIb-dependent stable arterial thrombus formation in vivo. Finally, consistent with the TxA2 receptor requirement for stable arterial thrombus formation in vivo, aspirin (at the appropriate concentration) prevented GPIb-dependent stable thrombus formation in vivo on ferric chloride damaged arterial endothelium.
The Endocannabinoid 2-Arachidonoylglycerol Regulates Platelet Function.
Authors: Baldassarri Samantha , Bertoni Alessandra , Lova Paolo , Reineri Stefania , Sarasso Chiara , Zanfa Manuela , Sanna Elena , Maccarrone Mauro , Torti Mauro , Sinigaglia Fabiola
Published: October 2019

2-Arachidonoylglycerol (2-AG) is a naturally occurring monoglyceride that activates cannabinoid receptors and meets several key requisites of an endogenous cannabinoid substance. It is present in the brain and hematopoietic cells, including macrophages, lymphocytes and platelets. 2-AG is released from cells in a stimulus-dependent manner and is rapidly eliminated by uptake into cells and enzymatic hydrolysis in arachidonic acid and glycerol. 2-AG might exert a very fine control on platelet function either through mechanisms intertwining with the signal transduction pathways used by platelet agonists or through mechanisms modulating specific receptors. The aim of this study was to define the role of 2-AG in human platelets and characterize the mechanisms by which it performs its action. Platelets from healthy donors were isolated from plasma by differential centrifugations and gel-filtration on Sepharose 2B. The samples were incubated with 2-AG (10–100 μM) under constant stirring in the presence or absence of various inhibitors. Platelet aggregation was measured by Born technique. We have found that stimulation of human platelets with 2-AG induced irreversible aggregation, which was significantly enhanced by co-stimulation with ADP (1–10 μM). Furthermore, 2-AG-dependent platelet aggregation was completely inhibited by ADP scavengers, aspirin, and Rho kinase inhibitor, as well as by antagonists of the 2-AG receptor (CB2), of the ADP P2Y12 receptor, and of the thromboxane A2 receptor. We further investigated the role of endocannabinoids on calcium mobilization. Intracellular [Ca2+] was measured using FURA-2-loaded platelets prewarmed at 37°C under gentle stirring in a spectrofluorimeter. 2-AG induced rapid increase of cytosolic [Ca2+] in a dose-dependent manner. This effect was partially blocked by ADP scavengers and CB2 receptor antagonists. Furthermore, 2-AG-induced [Ca2+] mobilization was totally suppressed by aspirin or the thromboxane A2 receptor antagonist. These results suggest that 2-AG is able to trigger platelet activation, and that this action is partially mediated by CB2 receptor and ADP. Furthmore, 2-AG-dependent platelet activation is totally dependent on thromboxane A2 generation.
Platelet Proinflammatory and Hemostatic Function Is Differentially Regulated in Cystic Fibrosis.
Authors: Sturm Alexander , Hebestreit Helge , Grossmann Ralf
Published: October 2019

Introduction: Platelet function and mechanisms of platelet-leukocyte interactions have been investigated in several vascular und inflammatory disorders. In most studies, platelet activation and an increase of platelet-leukocyte-aggregates (PLA) could be observed. We investigated platelet function in clinically stable patients with cystic fibrosis (CF).

Methods: In addition to routine markers of inflammation (e. g. CRP, IgG, ESR) parameters of platelet function were measured in 54 clinically stable CF patients and 55 healthy controls (age range 3 to 41 years): The percentage of P-selectin (CD62P) and PAC-1 (activated integrin αIIbβ3) positive resting and activated platelets (in-vitro activation with the thrombin receptor activating peptide 6) and the number of PLA were determined by flow cytometry. The plasma markers of platelet activation soluble P-selectin (sCD62P) and soluble CD40 ligand (sCD40L) were measured by ELISA. Furthermore, 15 CF patients and 14 healthy controls were investigated to determine CD41a-expression (integrin αIIbβ3) on resting and activated platelets as well as leukocyte expression of P-selectin-glycoprotein ligand 1 (PSGL-1, receptor of CD62P) and integrin αMβ2.

Results: Chronic inflammation leads to a decrease of PAC-1-binding to resting and activated platelets. The effects were stronger in patients with higher markers of inflammation. CD41a expression was reduced on in-vitro-activated CF platelets. In contrast, proinflammatory platelet functions remained unchanged (CD62P-expression on resting and activated platelets) or increased (sCD62P, sCD40L, PLA). Leukocyte integrin αMβ2 expression was increased and PSGL-1 expression remained unchanged in CF.

Discussion: Platelet function in clinically stable patients with CF is differentially regulated: Chronic inflammation leads to an upregulation of platelet proinflammatory function. In the presence of several procoagulatory mechanisms in inflammation there is a compensatory loss of platelet hemostatic function, shown by the decreased activation and exocytosis of the platelet major integrin αIIbβ3.
Anti-Platelet Glycoprotein Antibodies in Thrombocytosis Are Frequently Detected and Are Associated with Platelet Activation and Thrombosis.
Authors: Bidot Carlos J. , Jy Wenche , Bidot Loreta , Horstman Lawrence L. , Fontana Vincezo , Gonzalez Marta , Dudkyewicz Pamela , Ahn Yeon S.
Published: October 2019

Introduction: Thrombosis is the major cause of morbidity and mortality in patients with thrombocytosis (TS) associated with myeloproliferative disorders (MPD), but predicting which patients are at risk has been challenging. We recently reported that antiphospholipid antibodies (APLA) and platelet activation are risk factors in TS [C Bidot et al; Hematology, 10:451, 2005]. To shed further light on the cause of platelet activation in TS, we here report on measuring anti-platelet antibodies in a group of these patients.

Material and Methods: Thirty-three TS patients, 15 with clinical thrombosis and 18 without, were evaluated using PAICA method of L. Macchi et al [Thromb Haemost76:1020,1996]. We assayed anti-platelet antibodies against 3 target antigens, GPIIb/IIIa (CD41b), GP Ib/IX (CD42b) and GP IV (Cd36) for IgG and IgM. We also measured platelet activation marker CD62P, and microparticles from platelets (PMP, defined by CD31+/CD42+) and endothelial cells (EMP, defined by CD31+/CD42−) by flow cytometry.

Results: At least one anti-platelet antibody was detected in 19/33 (57.5%) of TS patients. Considering first IgG in the19 who were anti-platelet antibodies positive, 14/19 (73.6%) were positive for CD36, 12/19 (63%) for CD41b and 8/19 (42.1%) for CD42b. For IgM in these 19 patients, incidences were 58.8% for CD41b, 38.8% for CD36 and 35% for CD42b. When we compared the anti-platelet antibody positive group with the negative group, we observed that activation marker CD62P was significantly higher in the former (p= 0.04), as were EMP levels (p= 0.02), but PMP levels were not different. Next, we compared results with incidence of thrombosis. None of the IgG results discriminated thrombosis from non-thrombosis. However, all three IgM results were significant in this regard: CD36 (p< 0.05), CD42b (p< 0.02), and most notably, CD41b (p< 0.0003).

Conclusions: These results show that anti-platelet antibodies of IgM class, especially anti-CD41b, are associated with platelet activation, endothelial activation, and clinical thrombosis in TS. These findings parallel our previous report on APLA in TS. We suggest that IgM-CD41b may activate platelets, predisposing to hypercoagulable state in this disorder.
The Relationship between Circulating Microparticles and Complications of Pregnancy.
Authors: Salomon Ophira , Dardik Rima , Katz Ben-Zion , Steinberg David M. , Achiron Reuven , Seligsohn Uri
Published: October 2019

Uteroplacental thrombosis has been implicated in the pathogenesis of fetal loss, intrauterine growth restriction (IUGR) and preeclampsia. The role of inherited and acquired thrombophilias in such pregnancy complications is controversial. Recently, increased levels of circulating procoagulant microparticles (MPs) were related to fetal loss and preeclampsia (Laude et al. Thromb Haemost2001;85:18, Gonzalez-Quintero et al. Am J Obstet Gynecol2004;191:1418)). In this study, we measured the level of MPs derived from endothelial cells, platelets, and monocytes by flow cytometry using monoclonal antibodies against CD31, CD41 and CD14, respectively in 262 healthy unrelated primipara with a spontaneous singleton pregnancy. Expression of tissue factor by a monoclonal antibody and phosphatidyl serine by annexin 5 on MPs were also measured. All women were followed from the 24th week of gestation until delivery and IUGR, small for gestational age (SGA) at birth, pregnancy induced hypertension (PIH) or preeclampsia (PE) or none of the above were recorded. Blood sampling was carried out at 24th week of gestation. The levels of CD31 and CD41- positive MPs in women with and without the above pregnancy complications are presented in the table. Not shown are values of CD14, annexin 5 or tissue factor on MPs since their median levels were negligible in all study groups. There was no statistically significant difference in CD31 and CD41- positive MPs between women who developed or not the indicated pregnancy complications. Also, there was no significant difference in levels of these MPs between women with and without the common inherited thrombophilias, factor V Leiden and or prothrombin mutation (28 patients) and lupus anticoagulant (10 patients). These data indicate that measurement of the level of MP at the 24th week of gestation has no predictive value for development of PIH, PE, IUGR or SGA.

CD31 CD41 Number Median % p Median % p PIH or PE No 226 1.50 0.22 1.80 PIH Yes 13 1.57 3.07 0.53 PE Yes 22 0.62 1.87 IUGR No 207 1.53 0.13 1.95 0.08 IUGR Yes 32 1.30 1.71 SGA No 232 1.48 0.64 1.85 0.81 SGA Yes 26 1.27 2.24
Acceptibility of Patient Self-Testing of Oral Anticoagulation in New Patients.
Authors: Pescott Marie A. , Longair Ian , Gardiner Chris , Austin Steven K. , Machin Samuel J.
Published: October 2019

Monitoring oral anticoagulation therapy (OAT) remains a significant burden on health care resources. Patient self-testing (PST) of OAT using point of care devices may alleviate these demands and improve the quality of treatment. Previous studies have shown limited benefit, as recruitment amongst patients previously established on OAT has proven to be difficult with only around 10–15 % of those invited agreeing to participate, suggesting that PST may only be suitable for a minority of patients. We aimed to assess whether PST is more acceptable to patients if offered at the commencement of treatment. 216 consecutive adults referred to anticoagulation clinics were assessed for their suitability for PST. Exclusion criteria included: short therapeutic duration (e.g. pre-cardioversion); known drug/alcohol abuse; atypical INR target values; language barriers; physical/intellectual impairment. Suitable patients were offered the opportunity to perform PST with 2 training sessions and thereafter monitor their INR every 1–2 weeks using CoaguChek S (Roche Diagnostics). It was noted during recruitment of the first 100 patients, that patients awaiting cardioversion accounted for a large proportion of the patients excluded (15/54, 28%), but many patients awaiting cardioversion received OAT in excess of six months. We therefore modified initial entry criteria, to include those with atrial fibrillation awaiting cardioversion. Of the 116 patients subsequently approached 37 (32%) were then recruited to PST (compared with only 17 cardiac patients from the first 100 enrolled into the study). 98 (45%) patients fulfilled our entry criteria for PST, of whom 54 (25%) consented to PST. Of the 118 ineligible patients: 23 had previously received OAT; 15 were awaiting cardioversion; the expected duration of OAT was less than 6 months in 13 patients; and PST was logistically impractical in 32 other patients. A further 35 were excluded for a wide variety of reasons including non-standard INR ranges, language barriers, discontinuation of OAT and planned surgical procedures. Only two of the 54 patients who started PST elected to return to hospital testing. Overall 53 % (52/98) of eligible patients have continued to self-test. The INR values obtained from these patients showed that their oral anticoagulant control remained stable (mean time in therapeutic range of 71%), and compared favourably to those patients electing to attend our routine anticoagulant clinic. Our data show that patients offered PST as an alternative to routine clinic management during induction of anticoagulation were more likely to participate in PST than established patients. Furthermore, the vast majority of patients (96%) continued to self-test, a higher figure than previous studies involving established patients.
Genomic Distribution of Transcripts and DNA Associated Proteins in One Percent of the Genome of Erythroid and Myeloid Cells.
Authors: Lian Jin , Lian Zheng , Karpikov Alexander , Mahajan Milind , Gerstein Mark , Snyde Michael , Weissman Sherman
Published: October 2019

The NIHGRI has sponsored a multi-group enterprise to use a variety of approaches to map DNA elements in one per cent of the human genome-the ENCODE Project. As part of this effort we have mapped sites of active transcription (transcriptionally active regions or TARS) for myeloid cells including the undifferentiated and retinoic acid treated promyelocytic NB4 cell line and normal neutrophils, as well as the erythroid cell line K562. The results show a large number of intergenic and intronic transcripts, particularly in neutrophils. One feature of note is the amount of cell type specific intergenic transcription in the HoxA cluster. In addition we have been developing simple methods to map the sites of polyadenylation of RNAs and applied this to several cell types. This method also indicates the presence of polyadenylated transcripts derived from the multiple introns of known genes as well as intergenic regions.

In parallel with the transcript mapping, we have been performing microarray based chromatin immunoprecipitation studies (Chip-chip) on several factors, analyzing them on oligonucleotide arrays tiling the ENCODE selected genomic regions. These include various phosphorylated forms of RNA polymerase, various methylated forms of histone H3, certain DNA sequence specific transcription factors, and some other factors either broadly involved in chromatin remodeling or specifically found to be associated with certain sequences in the beta globin cluster. Among other things, our results as well as those of other members of the ENCODE consortium, provide a signature motif commonly associated with promoters.
The Expression and Clinical Significance of FLT3/ITD Gene Mutation in Different Hematologic Malignancy.
Authors: Xu Bing , Tang Jia-hong , Li Lin
Published: October 2019

Objective To analyze Fms-like tyrosine kinase 3 (FLT3) gene internal- tandem duplications(ITD) mutation in various kinds of hematologic malignancy. Methods Polymerase chain reaction(PCR)was used to detect FLT3/ITD gene mutation in 103 AML cases, 63 ALL cases, 53 CML in chronic phase(CML-CP), 34 CML cases in blast crisis(CML-BC), 11 CLL cases, 36 MDS cases, 9 MM cases and 13 NHL cases with marrow infiltration. Results The mutation of FLT3/ITD gene was detected in 22.3% AML cases, in 6.5% CML -BC cases, in 5.6% MDS cases and in 2.6% ALL cases, This two ALL cases with FLT3/ITD mutation were both with myeloid antigen expression and were diagnosed as acute mixed-lineage leukemia after immunology examination. FLT3/ITD gene mutation were not detected in CML-CP, MM, NHL and CLL cases. FLT3/ITD+ AML shew high white blood cell count and high percentage of bone marrow blast cells and had a unfavourable cumulative relapse rates. Conclusion FLT3/ITD gene mutation was majority in AML cases and FLT3/ITD+ AML had a poor prognosis. Detection of FLT3/ITD gene mutation may be valuable in genotyping of leukemia, differential diagnosis between AML and ALL and estimate the prognosis of leukemia.
E6-Associated Protein (E6-AP): A Potential Tumor Suppressor Protein for Non-Hodgkin Lymphoma.
Authors: Zafar Shazia , Srinivasan Sathish , Nawaz Zafar
Published: October 2019

Over the past decade considerable progress has been made in cloning and characterization of potential tumor suppressor genes. Tumor suppressors have a repressive effect on the regulation of the cell cycle or promote apoptosis and sometimes do both. The function of tumor suppressor proteins fall into several categories, tumor suppressor genes are presumed to encode negative regulator of proliferation and inhibit mitotic activity. Loss of tumor suppressor protein or function of a tumor suppressor protein has been shown to be associated with the cancer formation. Continued investigation into the biochemical and cell biological functions of the tumor suppressor is critical to elucidate the mechanisms by which they normally inhibit proliferation/tumor development and to provide a molecular explanation for their frequent inactivation in cancer. Our laboratory has previously shown that the expression of E6-associated protein (E6-AP), which is an E3 ubiquitin-protein ligase and a coactivator of nuclear hormone receptors, is significantly reduced in human cancers having epithelial cell origin such as breast cancer. In this prospective study, we want to extend our observation to the cancers originating from lymphoid tissue. Non-Hodgkin lymphoma is a cancer of lymphoid tissue. The main cell type found in lymphoid tissue is the lymphocyte. The 2 main types of lymphocytes are B-lymphocytes (B-cells) and T-lymphocytes (T-cells). B-cell lymphomas are much more common than T-cell lymphomas. In the U. S., 85% of all cases of non-Hodgkin lymphoma come from B lymphocytes (B-cell) and 15% from T lymphocytes (T-cell). We performed immunohistochemistry analysis to investigate the expression pattern of E6-AP in normal lymph nodes and lymphoid tumors. Tissue micro arrays representing samples from 60 different patients were analyzed in this study. Our analysis suggest that on an average there was about 55 % reduction in E6-AP protein levels in B-cell lymphomas (P =0.0001) and 98.5 % reduction in E6-AP levels in T-cell lymphomas (P =0.0002) compared to normal lymph node. Based on our previous studies in breast and prostate tumors and considering our current finding of reduced/loss of E6-AP in lymphoid tumors, we propose that E6-AP may act as a potential tumor suppressor protein. This proposed idea is consistent with our in vivo data generated from E6-AP null mice which shows that the number of B- and T-cells are significantly increased in spleen compared to normal wild-type animals. Taken together our data establish the role of E6-AP as a potential growth and tumor suppressor protein.
The Four and a Half LIM Domain Protein 2 (FHL2) Interacts with CALM and Is Highly Expressed in AML with Complex Aberrant Karyotpes.
Authors: Pasalic Zlatana , Tizazu Belay , Archangelo Leticia , Krause Alexandre , Philipp Greif , Bohlander Stefan K.
Published: October 2019

The balanced chromosomal translocation t(10;11)(p13;q14) results in the CALM/AF10 fusion gene. This translocation is found in acute myeloid leukemia (AML), T-cell acute lymphoblastic leukaemia (T-ALL) and malignant lymphoma. The CALM/AF10 fusion gene has recently been shown to cause an aggressive biphenotypic leukemia in a murine bone marrow transplant model. The CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene) gene product is a clathrin assembly protein which plays a role in clathrin mediated endocytosis and trans Golgi network trafficking. AF10 is a putative transcription factor most likely involved in processes related to chromatin organization and has polycomb group gene like properties.

To learn more about the function of the CALM/AF10 fusion protein, we searched for protein interaction partners of CALM. In a yeast two hybrid screen the four and a half LIM domain protein FHL2 was identified as putative CALM interacting partner. The CALM FHL2 interaction was confirmed by co-transformation assay in yeast and by GST-pulldown experiments. The FHL2 interaction domain of CALM was mapped to amino acids 294 to 335 of CALM using the yeast two hybrid assay. In co-localization studies with transiently expressed fluorescent protein tagged CALM and FHL2, both proteins showed cytoplasmatic localization. Expression analysis (Affymetrix based) in different AML subtypes showed a significantly higher expression of FHL2 in AML with complex aberrant karyotypes compared to AML with normal karyotypes or balanced chromosomal translocations like the t(8;21), inv(16) or t(15;17).

FHL2, which is also known as DRAL (downregulated in rhabdomyosarcoma LIM protein), is a TP53 responsive gene known to interact with numerous proteins in both the nucleus and the cytoplasm and can function as a transcriptional cofactor. Known FHL2 interactors include TP53, BRCA1, PLZF (promyelocytic leukemia zinc finger protein), the proto-oncogene SKI1 and beta-catenin. High expression of FHL2 in breast cancer has recently been shown to be associated with an adverse prognosis. CALM has been shown to shuttle between the nucleus and the cytoplasm because inhibition of CREM-mediated nuclear export by leptomycin B leads to the accumulation of CALM in the nucleus. Reporter gene assays using a GAL4-DNA binding domain CALM fusion protein and a GAL4 responsive luciferase reporter were able to demonstrate a transcriptional activation function of CALM. We are currently investigation the effect of FHL2 co-expression on this aspect of the CALM function.

It is thus conceivable that FHL2 is playing an important role in CALM/AF10-mediated leukemogenesis by tethering the CALM/AF10 fusion protein to various nuclear transcription factor complexes.
Identification of Peptide Epoxy-Ketone Proteasome Inhibitors with Differential Active Site Selectivity.
Authors: Demo Susan D. , Aujay Monette A. , Goldstein Eileen , Laidig Guy J. , Parlati Francesco , Lu Yan , Shenk Kevin D. , Sun Congcong M. , Sylvain Catherine , Woo Tina M. , Zhou Hanjie , Bennett Mark K.
Published: October 2019

The proteasome is a multi-subunit structure responsible for degrading the majority of proteins in the cell. The three catalytic subunits in the 20S core of the proteasome are defined by their preference for amino acid side chains at the P1 position: the beta5 subunit (chymotrypsin-like), the beta2 subunit (trypsin-like), and the beta1 subunit (caspase-like) hydrolyze substrates after hydrophobic, basic, and acidic residues, respectively. Bortezomib (Velcade®), currently an approved drug for the treatment of multiple myeloma, is a potent inhibitor of the beta5 subunit but also has activity against the beta1 subunit (9-fold less active) and the beta2 subunit (250-fold less active). PR-171, a peptide epoxy-ketone proteasome inhibitor currently in Phase I clinical trials in multiple myeloma and NHL patients, has a distinct profile of inhibition. Similar to bortezomib, PR-171 is most potent against the beta5 subunit, but differs in its activity against the beta2 and beta1 subunits (45-fold less active and 140-fold less active, respectively). Although both bortezomib and PR-171 are cytotoxic to a variety of tumor cell types, the connection between inhibition of specific proteasomal subunits and cellular consequences, including induction of apoptosis, is currently unclear. To investigate this relationship, we synthesized a series of PR-171 analogs and profiled them in our proteasome-specific active site ELISA binding assay to identify compounds with differential binding profiles for the three catalytic subunits. Using this approach, we have identified compounds that bind all three catalytic sites with approximately equivalent activity as well as compounds that have increased specificity for the beta5 subunit. We have extended this approach to search for compounds with differential binding profiles for the immunoproteasome catalytic subunits. The immunoproteasome is the predominant form of the proteasome expressed in cells of hematopoetic origin and in other cell types after interferon-g stimulation. In the 20S core of the immunoproteasome the beta5, beta2 and beta1 subunits of the “constitutive” proteasome are replaced by the homologous subunits LMP7, MECL1, and LMP2. We have successfully identified inhibitors with distinct active site profiles for the immunoproteasome as well as the constitutive proteasome. The impact of differential active site inhibition on tumor cell viability is under investigation in a variety of solid and hematological tumor cell lines expressing the constitutive proteasome or the immunoproteasome.
Immunogenicity of Influenza Vaccination in Patients with Non-Hodgkin Lymphoma: Final Report after Two Epidemic Seasons.
Authors: Centkowski Piotr , Brydak Lidia B. , Machala Magdalena , Kalinka Ewa , Blasinska-Morawiec Maria , Federowicz Irena , Walewski Jan , Wegrzyn Joanna , Wolowiec Dariusz , Maranda Ewa , Sawczuk-Chabin Joanna , Bilinski Przemyslaw , Warzocha Krzysztof
Published: October 2019

Vaccination against influenza is recommended for immunocompromised individuals. However, there is little information concerning the efficacy of vaccination in patients (pts) with non-Hodgkin lymphoma (NHL). The purpose of this study was to assess humoral response to standard intramuscular trivalent subunit influenza vaccine in pts with NHL as compared to healthy subjects. In two consecutive epidemic seasons, 2003/2004 and 2004/2005, 163 pts and 92 healthy controls were vaccinated. Antibody titers to hemagglutinin (HA) and neuraminidase (NA) were measured in serum samples collected before vaccination, and 1 and 6 months apart. Changes in the hemagglutination inhibition (HAI) and neuraminidase inhibition (NII) antibody titers were assessed by comparing geometric mean titers and mean fold increases to baseline values and by comparing changes in the HA seroconversion and seroprotection rates. Pts who received influenza vaccine during 2003/2004 season had after one month increases in the geometric mean titers by a factor of 8,64–26,60 for HI and 6,93–12,66 for NI, as compared with respective increases by a factor of 9,12–24,41 and 4,83–10,31 for the healthy controls. At one month after vaccination seroprotection and seroresponse rates were similar in the two groups, ranging from 68,42 to 84,21 % and 71,93 to 94,74 % in NHL and 66,67–82,22 % and 62,22–86,67 % in controls, respectively. After six months, seroprotection and seroresponse rates had decreased in NHL group to 31,91–38,30% and 46,81–72,34%, respectively. Pts who received influenza vaccine during 2004/2005 season had after 1 month increases in the geometric mean titers by a factor of 38,76–41,49 for HI and 26,59–30,31 for NI, as compared with respective increases by a factor of 81,19–104,32 and 52,16–54,52 for the healthy controls. Seroprotection and seroresponse rates were lower in the former group, ranging from 62,11 to 65,26 % and 74,47 to 77,66 %, respectively. After six months, these parameters had decreased to 24,72–31,46% and 57,30–59,55%, respectively. In both studied seasons, pts achieved titres of functional antibodies greater than the protective threshold, irrespective of the previous chemotherapy administration. The results of this study indicate that standard influenza vaccination induces sufficient immune responses in pts with NHL. Previous chemotherapy adminstration seems to have no impact on the efficacy of vaccination.
RNA Interference Against Bcl-2 mRNA Increases Radiosensitivity of Raji Cells.
Authors: He Dongmei , Zhang Yuan , Liu Gexiu
Published: October 2019

Bcl-2 is the prominent member of a family of proteins responsible for dysregulation of apoptosis, and resistance to chemotherapy. It has been shown that reduction in Bcl-2 protein levels could ultimately induce a lower apoptotic threshold and restore chemosensitivity in a variety of malignancies. Short interfering RNA (siRNA) has been evaluated as an attractive and effective tool for suppressing a target protein by specifically digesting its mRNA. In our lab, we have identified a siRNA targeting against Bcl-2 could effectively down-regulate Bcl-2 protein. In this study, we investigated the effect of gamma radiation combined with the siRNA targeting Bcl-2 on proliferation and apoptosis in B-lymphoma Raji cells. The siRNA was introduced into cells using Oligofectamine transfection. Cells were treated with Bcl-2 siRNA alone or with 2–8 Gy dose of (60)Co gamma rays. Expression of Bcl-2 mRNA and protein was assayed by quantitative reverse transcriptase-polymerase chain reaction and Western blotting analysis. Radiosensitivity was determined by clonogenic cell survival assay. Apoptosis was determined by Giemsa staining, Annexin-V binding assay and flow cytomertry. Furthermore caspase-3 activity and poly (ADP-ribose) polymerase (PARP) cleavage were evaluated. Transfection of Raji cells with 100 nmol/L siRNA targeted against Bcl-2 resulted in reduction of Bcl-2 mRNA by 75% compared with control-siRNA treated group and the vehicle control group(p<0.05). The levels of Bcl-2 protein were significantly reduced by 70% compared with the two control groups (p<0.05). There was significant difference in the radiosensitivity of Raji cells in which Bcl-2 was silenced compared with the cells transfected vehicle or control siRNA.Apoptosis index of the Raji cells treated with Bcl-2 siRNA combined with radiation was significantly increased (p<0.05), compared with either control siRNA / radiation combination or radiation-treatment cells alone, or Bcl-2 siRNA-treatment cells alone.Raji cells treated with Bcl-2 siRNA combined with radiation revealed enhanced caspase-3 and PARP cleavage as compared to Bcl-2 siRNA treated cells alone or only irradiated cells. These findings show that Bcl-2 siRNA synergistically enhances radiation-induced apoptosis through the expression of proteins involved in the programmed cell death.
Diffuse Large B-Cell Lymphoma: Relevance of bcl-2 Expression at a National Hospital from Lima Peru 2000–2002.
Authors: Egoavil R. Cecilia M. , Delgado G. Victor M. , La Serna Jesus
Published: October 2019

Diffuse large B-cell lymphoma (DLCL) exhibits heterogeneous clinical features and varies in response to treatment and prognosis. Establishment of parameters that can predict outcome could help to identify patients who may benefit from risk-adjusted therapies. Because apoptosis-related proteins may play an important role in predicting the prognosis of DLCL, the current study investigated the significance of bcl-2 expression in relation to clinical characteristics in this patients. We studied 125 patients (74/51 men/women; median age 64.17 years) consecutively diagnosed with de novo DLBCL in a single institution from Peru (HNGAI) during 3-year period (2000–2002).Morphology, and clinical characteristics were analyzed according to the primary site of the lymphoma and edge. Paraffin-embedded specimens from 88 were analyzed immunohistochemically for bcl-2 protein expression. Cases with a positive immunohistological stain in more than 20% of the tumor cells were considered positive expression. Results: Sites of the disease were: lymph node, 78 cases (62.4%); Waldeyer’s ring, 18 (14.4%); and extranodal sites, 45 (36%), including GI tract in 33 cases. 5-year overall survival (OS) was 19.7%. Log-rank analyses of survival showed significant differences between ≥60 years old and younger patients (P=0.003).bcl-2 expression was identified in 34 patients (38.6%), There was no significant difference in the OS (P=0.453) between the bcl-2+ (n=34) and bcl-2− (n=54) groups. In conclusion, bcl-2 expression appeared to be non predictive of a good OS in patients with DLCL, however, there was still a clear trend that the bcl-2+ patients died sooner than the patients with tumors that contained bcl-2− lymphoma cells. The current study show all patients as were staged or confined in a group with high or high intermediate IPI scores and could be candidates for alternative therapeutic approaches.