bluuebunny/crossref_metadata_2025 huggingface dataset:
CD34+ Marrow Cells from MDS Patients Express Homing-Associated Cell Surface Molecules but Impaired In Vitro Migration.
Authors: Thanopoulou Eleni , Kouraklis-Symeonidis Alexandra , Symeonidis Argiris , Tzouvara Evangelia , Pherson Lorna , Zoumbos Nicholas
Published: October 2019

Homing and retention of hematopoietic stem cells in the marrow microenvironment are multi-step processes mainly controlled by interactions between chemokines, adhesion receptors and proteases. In the case of MDS, whether these interactions are perturbed and how they are implicated in the pathogenesis of the disease or progression to AML is not known. In this study we evaluated the cell surface expression of homing-associated adhesion molecules, CD11a, CD26, CD45, CD49d, CD49e and CXCR4 by flow cytometric analysis in BM mononuclear (MN) and CD34+ cells from 19 MDS patients [7 RA, 1RARS, 11RAEB(t)] and age-matched healthy individuals (n=9). CD34+ and MN cells from RAEB(t) samples expressed higher levels of CD11a and CD49e (2–3 fold increase) and 1,5–3 fold lower levels of CD45 compared with the cells from the healthy control group, as measured by the mean fluorescent intensity. None of the remaining molecules was found to have altered expression in the RAEB(t) subtype. A similar expression pattern of all homing-associated molecules was seen in the cells from the RA/RARS and normal control samples. However, when CD34+ cells from MDS patients were allowed to migrate in vitro toward a gradient of SDF-1α (125ng/ml), they displayed an impaired chemotactic response [mean values of % input: 9% (0,5–30%)], whereas those from healthy individuals actively migrated [mean values of % input: 20% (10–50%)]. Both migrated and non migrated CD34+ cells from MDS patients displayed little hematopoietic activity in vitro (CFC and LTC-IC). The addition of neutralizing monoclonal antibody anti-CXCR4 decreased in vitro chemotactic response of normal and MDS derived CD34+ cells by 90% and 80% respectively indicating that migration was dependent on SDF1/CXCR4 interactions. To further investigate the differences observed in response to SDF1a, immunofluorescence detection of intracellular expression of CXCR4 was performed. MDS derived CD34+ and MN cells from all the subtypes studied significantly expressed high levels of intracellular CXCR4 and were induced to cell surface CXCR4 expression after 48 hours of SCF (100ng/ml) and IL-6 (20ng/ml) stimulation, as was seen with normal cells.

In conclusion marrow CD34+ cells from MDS patients showed reduced capacity to migrate in vitro, although no significant perturbations of adhesion molecules expression were detected. We are currently investigating the signaling events and pathways, which control homing and adhesion of hematopoietic cells in the marrow.
Overcoming TNF-α Induced Suppression of Erythroid Differentiation of Human Cord Blood CD34+ Cells with Arsenic Trioxide.
Authors: Won Jong-Ho , Cheong Hee-Jeong , Kim Sook-Ja , Bae Sang-Byung , Kim Chan-Kyu , Lee Nam-Su , Lee Kyu-Taeg , Park Sung-Kyu , Hong Dae-Sik , Park Hee-Sook
Published: October 2019

The anemia of chronic disease-which encompasses inflammation, infection, tissue injury, and conditions associated with the release of proinflammatory cytokines (such as cancer)- is one of the most common forms of anemia seen clinically. Symptomatic anemia requires treatment. The two major forms of treatment are transfusions and erythropoietin. Arsenic trioxide (As2O3) used to treat human diseases for centuries in traditional Chinese medicine. Our recent studies suggest that low dose of As2O3 induces erythroid differentiation of K562 human leukemic cells and high dose of As2O3 induce apoptosis. In this study, we have investigated in vitro effect of As2O3 on the erythroid differentiation and it could inhibit TNF-α induced suppression of erythroid differentiation of human cord blood CD34+ cells. Expression of glycophorin A was 35.94 ± 7.94% after 7 days culture of human cord blood CD34+ cells and was decreased to 17.63 ± 7.33% when culture of human cord blood CD34+ cells with 100ng/mL of TNF-α. Expression of glycophorin A was increased in dose dependent manner after 7 days treatment with As2O3 and As2O3 increased percentage of glycophorin A in culture with TNF-α compared to TNF-α alone. The results of colony assay of CFU-MIX and BFU-E after culture with various conditions revealed similar patterns with expression of glycophorin A. These results suggest that As2O3 induces erythroid differentiation of human cord blood CD34+ cells and can reverse TNF-α induced suppression of erythroid differentiation of human cord blood CD34+ cells.

The BFU-E colony assay of the human cord blood CD34+ cells after culture with TNF-α or/and Arsenic trioxide. The BFU-E colony assay of the human cord blood CD34+ cells after culture with TNF-α or/and Arsenic trioxide.
Large Deletion of Chromosome 11 Causes Acute Intermittent Porphyria in an Italian Family.
Authors: Di Pierro Elena , Besana Valeria , Moriondo Valeria , Tavazzi Dario , Casalgrande Giovanna , Rocchi Emilio , Cappellini Maria D.
Published: October 2019

Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by a partial deficiency of hydroxymethylbilane synthase (HMBS), the third enzyme of the heme biosynthetic pathway. The clinical features of AIP are intermittent attacks of neurological dysfunction, including abdominal pain and neuropsychiatric symptoms. Most patients remain asymptomatic throughout their life but 10–20% have severe acute attacks. AIP is normally diagnosed on the basis of clinical symptoms and urinary overproduction of porphyrin precursors, d-aminolevulinic acid (ALA) and porphobilinogen (PBG). The measurement of erythrocyte HMBS activity can confirm the diagnosis and it is also used to detect asymptomatic relatives. So far, more than 290 different mutations in the HMBS gene have been identified among AIP patients. Most of the reported mutations have been detected only in single families. In an Italian family with typical clinical and biochemical signs of AIP, no mutation was found by direct sequencing of 5′ UTR, 3′ UTR, erythroid promoter and the entire coding region of HMBS gene. All sympomatic patients showed apparent homozigosity for eleven common polymorphisms along the HMBS gene. Further family studies established the absence of Mendelian segregation. Excluding interference of polymorphisms in the primer sites, we assumed the presence of a complete deletion of the HMBS gene. In order to verify this hypothesis we performed a semiquantitative RT-PCR analysis on fresh RNA and different long PCR semiquantitative analysis on DNA. All analyses revealed the presence of a normal length fragment which was less abundant compared to controls. In order to identify the size of the deletion, SNPs analysis was extended to flanking genes, H2A Histone Family member X (H2AX) and Dolichyl-Phosphate N-Acetylglucosamine Phosphotransferase 1 (DPAGT1), downstream and Vacuolar protein sorting 11 (VPS11), upstream. We found heterozygotic polymorphisms in VPS11 and DPAGT1 genes. We performed XL-PCR of 19317bp with primers situated in regions outside the heterozygotic polymorphisms that showed a shorter band of 5434bp. Sequence analysis of the abnormal fragment revealed a loss of 77 intergenic base pairs upstream of the HMBS and H2AX genes and 2500bp of DPAGT1 gene. Simultaneously, an inverted complementary sequence of 223bp of intron 9 of HMBS gene was incorporated. Use of ElDorado-Genomatix software revealed the presence of different Alu sequences near breakpoints, that could cause this deletion. The H2AX gene is a member of a classical gene family and its loss has no adverse effects on phenotype. Defects in DPAGT1 are the cause of a congenital disorder of glycosylation type Ij, characterized by severe mental and psychomotor retardation. Our patients had no other symptoms than AIP, supporting the suggestion that DPAGT1 inheritance is probably recessive.
The Complex Mcl-1/Bim Regulates the Sensitivity of Human Myeloma Cells to Melphalan.
Authors: Gomez-Bougie Patricia , Oliver Lisa , Menoret Emmanuelle , Le Gouill Steven , Bataille Régis , Amiot Martine
Published: October 2019

Multiple myeloma is a fatal plasma-cell malignancy that evolves mainly in the bone marrow. Melphalan is widely used to treat patients with Multiple Myeloma, however its mechanism of action remains poorly documented. Thus, we studied the effect of this drug on the molecules of the Bcl-2 family. We showed that melphalan induces a drastic down-regulation of the anti-apoptotic proteins Mcl-1L and Bcl-xL and the pro-apoptotic BimEL in human melphalan-sensitive myeloma cells while the most potent pro-apoptotic Bim isoforms, L and S, are less affected. Interestingly, the disappearance of Mcl-1L and BimEL is associated with the generation of the respective cleaved forms, described as pro-apoptotic. We determined that the cleaved forms were generated by caspase cleavage since the addition of zVADfmk inhibited their formation. Indeed, we observed that caspase 3 activation occurred as early as Mcl-1 cleavage. Recently, we demonstrated that Mcl-1, the major anti-apoptotic molecule for myeloma cells, neutralizes the pro-apoptotic Bim molecule through endogenous complex formation and therefore, preventing the activation of death effectors. In this study, we demonstrate that melphalan disrupts the Mcl-1/Bim complex while the Bcl-2/Bim complex is not affected. In turn, the disappearance of full length Mcl-1 allows the release of Bim isoforms, particularly L and S, which can exert their pro-apoptotic function. In addition, we demonstrate both Bax activation and cytochrome c release, supporting the role of free Bim as a major initiator of the mitochondrial apoptotic cascade. Thus, we propose that in myeloma cells melphalan induces the disruption of Mcl-1/Bim complex resulting in the release of Bim from Mcl-1 sequestration and therefore leading to the initiation of the apoptotic process. Moreover, the cleaved 26kDa pro-apoptotic Mcl-1 and the 19kDa and 12kDa of Bim, generated during melphalan treatment could contribute to the amplification loop of apoptosis. MM remains a fatal disease which develops multi-drug resistance, including melphalan. A potential therapeutic approach could be the disruption of the Mcl-1/Bim complexes by the BH3 small molecules that might target those complexes leading to the release of sequestered Bim.
Mitochondrial Reactive Oxygen Species (ROS) Are Involved in Coated Platelet Formation and Platelet Procoagulant Activity.
Authors: Jobe Shawn , Lentz Steven R. , Di Paola Jorge
Published: October 2019

Increased vascular oxidative stress has been demonstrated in many pathological conditions associated with an increased predisposition for thrombotic events, including atherosclerosis, diabetes mellitus, and hypertension. We hypothesized that oxidative stress may promote thrombosis through increases in platelet procoagulant activity and coated platelet formation. Upon activation with convulxin (an agonist for the collagen receptor GPVI) and thrombin, approximately 70% of murine platelets acquired the coated platelet phenotype, characterized by increased alpha granule protein retention, decreased binding of the α2bβ3 activation-dependent antibody JON/A, and increased binding of annexin V. Dual stimulation with convulxin and thrombin also resulted in a 4-fold increase in platelet procoagulant activity (measured in a prothrombinase assay) together with rapid oxidation of the redox-sensitive dye, H2DCF. Only small increases in H2DCF oxidation were observed in platelets stimulated with thrombin or convulxin alone demonstrating a unique effect of dual agonist stimulation on platelet oxidant production. Formation of coated platelets and platelet procoagulant activity following stimulation with convulxin and thrombin were inhibited with the non-specific antioxidants, Tiron or Trolox. No inhibitory effect was observed using the NADPH oxidase inhibitor diphenylene iodonium or the NO synthase inhibitor L-NNA. The mitochondrial electron transport inhibitor, rotenone, effectively inhibited ROS production, coated platelet formation, and platelet procoagulant activity (53 ± 7%) (p<0.01) following dual agonist stimulation. We also found that hydrogen peroxide (H2O2) could effectively substitute for convulxin in mediating these dual-stimulated activities. Stimulation with H2O2 (100 mM) in the presence of thrombin (0.5 U/mL) resulted in a 4-fold increase in coated platelets and annexin V binding and a 3-fold increase in platelet procoagulant activity relative to platelets stimulated with thrombin alone (p<0.05). This response was dose-dependent, and no effect was observed with H2O2 in the absence of thrombin. Together these results suggest a central role for oxidant signaling and mitochondrial ROS production in platelet procoagulant activity and coated platelet production and point to a possible mechanism for thrombosis in pathological conditions associated with increased oxidative stress.
Improved Myocardial T2* in Transfusion Dependent Anemias Receiving ICL670 (Deferasirox).
Authors: Porter John B. , Tanner Mark A. , Pennell Dudley J. , Eleftheriou Perla
Published: October 2019

ICL670 (deferasirox) is an orally active iron chelator, intended as once daily mono-therapy for the treatment of transfusional iron overload, which has undergone extensive multi-centre Phase II and Phase III trials. Data from such studies suggest that 20mg/kg as a single daily dose will achieve iron balance in the majority of transfusionally dependent patients, whereas 30mg/kg/day typically achieves negative iron balance (Cappellini, Abstract 3619, ASH 2004) (Porter, Abstract 3193, ASH 2004). Patients with transfusion dependent anaemia and iron overload who were entered into these multi-centre studies at UCL Hospitals had myocardial T2* CMR performed at the Royal Brompton Hospital, as part of their routine monitoring, in line with previous clinical management at our centre. Study 108, a non-randomized study, included poorly chelated patients with -thalassemia and other iron overload conditions receiving regular blood transfusion: all patients received 10-30mg/kg/day of ICL670. Study 107 consisted exclusively of transfusion dependent -thalassemia patients, randomized to receive either 8–10h sc DFO (30–50mg/kg/day) or ICL670 (10–30mg/kg/day) with doses stratified for baseline liver iron concentration (LIC). We report here changes in cardiac T2*, LIC and serum ferritin, as well as LVEF in all patients on these two studies treated at UCLH for a mean of 13.1± 0.78 months, in whom CMR was obtained. In a total of 23 patients treated with ICL670, mean age 24.6y (range 9–50y), 18 with transfusion dependent -thalassaemia and 6 other iron overload conditions (2 Pyruvate kinase deficiency, 2 sideroblastic anemia, 2 Diamond Blackfan Anemia), myocardial T2* improved significantly from a pre-treatment geometric mean of 18.0ms to 23.1ms (p = 0.013, paired student t test). In the same patients, serum ferritin fell significantly from 3173 ±410 μg/L to 2451 ±242μg/L (p= 0.023, paired student t test) and LIC fell significantly from 18.3 ±2.2 mg/g dry wt to 10.0 ±1.49 (p= 0.0002, paired student t test). There was no significant change in LVEF before or after treatment over the same period. Patients treated in the DFO arm of study 107 (n=8) also showed a small non-significant increase in myocardial T2* from 18.1 ms to 21.1 ms (p= 0.11), These studies suggest that once daily mono-therapy with ICL670 will be effective at improving myocardial T2* and by inference myocardial iron loading in a wide range of patients with transfusional iron overload. Prospective randomised controlled studies in larger patient numbers are now indicated.
A Population of Small CXCR4+ SSEA-1+ Oct-4+ Embryonic-Like Stem Cells Identified in Adult Bone Marrow.
Authors: Kucia Magda , Reca Ryan , Ratajczak Janina , Ratajczak Mariusz Z.
Published: October 2019

We demonstrated that bone marrow (BM) stem cells are heterogenous and in addition to hematopoietic stem cells (HSC) BM also contains non-hematopoietic tissue committed stem cells (TCSC) for skeletal muscle, heart, neural tissue, epidermis and liver (Leukemia2004:18;29–40). In our follow up studies by employing multiparameter sorting we identified in murine BM a homogenous population of rare (~0.02% of BMMNC) Sca-1+ lin− CD45− cells that express by RQ-PCR and immunhistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1 and highly express Rif-1 telomerase protein. More important the direct electronmicroscopical analysis revealed that these cells display several features typical for primary embryonic stem cells such as i) small size (~3 μm in diameter), ii) poses large nuclei surrounded by a narrow rim of cytoplasm, and iii) contain open-type chromatin (euchromatin) that is typical for embryonic stem cells. Their number is highest in BM from young (1–2 month-old) mice and decreases with age. It is also significantly diminished in short living DBA/2J mice as compared to long living B6 mice. These cells in vitro respond strongly to several motomorphogens such as SDF-1, HGF and LIF and co-express the corresponding receptors such as CXCR4, c-met and LIF-R respectively on their surface. Interestingly, they adhere to fibronectin, and undergo emperipolesis in fibroblasts, thus they may be co-isolated with BM adherent cells. Furthermore, they are mobilized into peripheral blood during tissue/organ injuries (e.g., heart infarct, stroke). In in vitro cultures they differentiate into cells from different germ-layers (e.g., form neurospheres, grow cardiomyocytes). Thus, these findings support the theory of BM containing a reserve population of embryonic-like/pluripotent stem cells and it is also possible that several of the recently described BM-derived CD45− stem cell populations (e.g., MAPC, USSC or MIAMI cells) could in fact overlap with these rare non-hematopoietic CD45− stem cells identified by us, but due to the differences in the experimental approaches employed for their isolation and identification, were assigned different names. We postulate that this population of CD45− embryonic-like cells expressing pluripotent and tissue committed markers identified by us is an ideal source of cells for regeneration.
Specific Activation of Human beta-Globin Gene Expression by the Transcription Factor Ikaros.
Authors: Perkins Andrew C. , Papathanasiou Peter , Goodnow Christopher C. , Keys Janelle R.
Published: October 2019

The zinc finger transcription factor Ikaros is recognized as a key regulator of lymphocyte differentiation. Recently generated dominant negative mutants have hinted at a broader role in haematopoietic stem cell generation. Most recently, a mouse strain, IkarosPlastic, with a point mutation in Ikaros that disrupts DNA binding but preserves efficient assembly of Ikaros protein complexes, is embryonically lethal due to severe defects in erythrocyte differentiation (Papathanasiou P, et al,. Immunity, 2003). (1). These mice display normal murine globin gene expression in the fetal liver. However in humans the globin locus is under alternative regulatory control, particularly with respect to the fetal-to-adult globin switch. Thus, to determine if Ikaros plays a role in human globin switching we crossed the IkarosPlastic mice with mice transgenic for a YAC containing the entire human b-globin locus, which show human fetal to adult globin gene switching from E12 to E17. Embryos were harvested from E12.5 to E15.5 and globin expression was determined in the fetal liver by real-time PCR (relative to actin). At all time points human gamma-globin gene expression was not significantly altered by the presence of the IkarosPlastic mutatation (relative expression Ikaroswt/wt 1±0.11, IkarosPlastic/Plastic 0.82±0.12). In contrast, human beta-globin gene expression was significantly down-regulated in IkarosPlastic fetal livers (relative expression Ikaroswt/wt 1±0.14, IkarosPlastic/Plastic 0.18±0.07). Interestingly, neither murine a- or b-globin gene expression was significantly different to wild type mice, which suggests that the transcription factor Ikaros plays a specific role in the transcriptional activation of the human b-globin gene during development. The mechanism by which this occurs remains to be elucidated, however it is intriguing to consider that Ikaros may act as a potentiator of transcription for erythroid specific transcription factors such as EKLF. Experiments to address this will be presented.
Recombinant Human Activated Protein C Suppresses Endotoxin Induced Hypotension in Mice Deficient of the Endothelial Protein C Receptor (EPCR).
Authors: Joyce David E. , Donahue Deborah L. , Liaw Patricia C. , Castellino Francis J.
Published: October 2019

In Clinical sepsis and human endotoxin studies, administration of the recombinant human form of the anticoagulant activated protein C (rhaPC) demonstrates benefit in parameters of coagulation, inflammation, shock, and sepsis survival. Utilizing transgenic mice deficient in the endothelial protein C receptor, (EPCR), we investigated whether provision of rhaPC improved the hypotensive response and survival subsequent to administration of a lethal dose of endotoxin (LPS). A novel central venous infusion protocol for rhaPC was developed using low expressing EPCR mice (EPCRδ/δ surface expression <10% of WT EPCR) which were generated in our center. WT C57Bl/6 (>F8) mice were used as controls. An aortic arch continuous blood pressure (B.P.) monitor and right internal jugular vein catheter were placed. The continuous infusion dosing of rhaPC (Eli Lilly) was approximately 1.8 μg/g/hr for 20 hr through the central venous catheter (N=3 per group). The control group received saline. Bolus LPS (10 μg/g i.p.) was administered concomitant to rhaPC. Continuous B.P. measurements were obtained until death or for up to 72 hr. RhaPC kinetic parameters (N=4 per group) collected from WT citrate-benzamidine plasma samples were determined by monoclonal antibody capture assays. In initial kinetic studies plasma levels of rhaPC in WT mice given LPS achieved steady state concentrations (90 ng/mL at 4 hr) similar to that found in human studies (45 ng/mL at 2hr). RhaPC dosing produced no allergic reaction or overt bleeding. The subsequent 72 hour B.P. study achieved the goal of suppressing endotoxin-induced hypotension in both WT and EPCRδ/δ mice. The two LPS/rhaPC-treated groups were able to overcome the hypotensive stress and survive longer than the two LPS-only treated groups. RhaPC treated EPCRδ/δ mice were able to recover blood pressure to the same or greater degree than the rhaPC treated WT mice, suggesting either enhanced cell signaling potential by less cell surface EPCR or an alternate aPC receptor. The administered volume of 30 μL/hr (0.6 mL over 20 hr) did not appear to counter the onset of hypotension in EPCRδ/δ mice, and the rhaPC B.P. effect persisted beyond the 20 hr infusion. The expected sequence of LPS-induced mortalitv, viz., EPCRδ/δ > WT > WT/rhaPC > EPCRδ/δ/rhaPC was demonstrated in this study. These transgenic murine studies implicate the protein C pathway as linking coagulation, inflammation and hypotension. By reducing the burden of hypotension in WT and EPCR deficient mice with rhaPC treatment in endotoxin-induced sepsis, this infusion model provides a valuable approach to study effects of aPC in vascular inflammation and hypotension.
New Reference Ranges in Haematology for Healthy Adults Using the Modern Sysmex XE-2100 Automated Analyser.
Authors: Wakeman Lisa , Munro Roger , Russell Chris , Benton Ann , Hartnell Sally , Al-Ismail Saad
Published: October 2019

Establishing clearly defined, accurate reference ranges facilitates good interpretation and effective discrimination between health and disease. These can be used to obviate the need for unnecessary follow-up medical examinations thereby reducing costs. Our data represent findings from one of the most comprehensive studies ever undertaken with the XE-2100 to establish reference ranges (RRs) in healthy adults. Early morning venous samples were collected into Greiner EDTA Vacuettes (Ref: 454286) from 221 healthy laboratory personnel (F= 159;M = 62) aged 20–63 yrs for both gender. Age groups were equally represented. Samples were processed on a Sysmex XE-2100 analyser within 1 hour of collection. NCCLS guidelines (C28-A and H3-A4) were followed throughout. Outliers were excluded, data examined for normal distribution from histograms, Q-Q normality plots, skewness and kurtosis and significance levels calculated from the Kolmogorov-Smirnov and Shapiro-Wilk tests of normality. RRs for near normally distributed parameters were calculated using means ± 2SDs. RRs for non-normally distributed parameters were calculated using the log natural transformation and the antilog of the 2.5- and 97.5- percentiles. Bold parameters shown below have near-normal distribution. Non emboldened values are non-normally distributed. P values are derived from Mann-Whitney U test for differences between males and females.

New Limits Historical Limits Test of M&F diff. (P value) *=sig. diff. Haemoglobin (g/dL) M 13.7–17.2 13.0–17.5 <0.05* F 12.0–15.2 11.7–15.7 RBC (x1012/L) M 4.5–5.6 4.5–5.9 <0.05* F 3.9–5.1 3.8–5.9 Hct (L/L) M 0.40–0.50 0.40–0.52 <0.05* F 0.37–0.46 0.37–0.47 MCV (fL) M 83–98 80–100 0.090 F 85–98 80–100 MCH (pg) M 28–33 27–32 0.391 F 28–33 26–31 MCHC (g/dL) M 32–36 30–36 <0.05* F 32–35 30–36 RDW (%) M 11.6–14.1 11.0–15.0 0.067 F 12.0–14.7 11.0–15.0 Reticulocytes (x109/L) M 27–93 25–85 0.138 F 22–76 25–85 Platelets ( (x109/L) M 140–320 140–450 <0.05* F 180–380 140–450 MPV (fL) M 9.4–12.2 6.3–10.1 0.426 F 9.2–12.9 6.3–10.1 Leucocytes (x109/L) 3.6–9.2 4.0–11.0 0.854 Neutrophils (x109/L) 1.7–6.2 2.0–7.5 0.760 Lymphoctes (x109/L) 1.0–3.4 1.0–4.0 0.854 Monocytes(x109/L) 0.2–0.8 0.2–0.8 0.073 Eosinophils(x109/L) 0.00–0.4 0.04–0.4 0.847 Basophils(x109/L) 0.00–0.1 0.00–0.1 0.279

Reference limits determined for total leucocytes and neutrophils are significantly lower than historical ranges. However, leucocyte counts are at their lowest in the early morning. Our findings are in general agreement with previously published data from more limited trials undertaken in other countries.
Hematological Damage in Systemic Lupus Erythematosus (SLE) and It’s Relationship with Immunological Paramerters.
Authors: Zhao Xiaoying , Zhang Ping , Huang Liansheng
Published: October 2019

Objective To investigate the incidence of hematological damage in systemic lupus erythematosus (SLE) and it’s relationship with disease state of SLE and immunological paramerters.

Methods 155 inpatients with SLE were enrolled in this retrospective study in our hospital from Jan. 2002 to May 2005. All patients fulfiled the criteria of SLE which drawn up by American rheumatism association (ARA) in 1982. Fasting blood samples were obtained and routine of blood, liver and renal function, immunological paramerters (ANA, ENA, Anti -dsDNA,Anti-RNP,Anti-Sm,Anti-SSA,Anti-SSB and C3,C4 constituents of complement)were determined. Acording to SLE Disease activity Index(SLEDAI),disease stata were evaluated in 155 patients and their correlotion with hematological damage and immunological paramerters were analyzed.

Results 67.74% of patients existed hematological disorder. Single cell line damage were in 32.90% of patients while 34.84% of patients were involved several cell lines. The detectable rate of Anti-Sm (43.81%) and Anti-RNP(63.81%) in patients with hematological disorder. were were higher than those in patients without hematological disorder, Anti-Sm in 26.00%, Anti-RNP in 46.00%.The positive rate of Anti-RNP were 68.97% in patients with red cell line damage while only 46.00% in patient without hematological disorder(p<0.05). There were no significant difference in Anti-RNP between patients without hematological disorder and patients with leucocytopenia or thrombocytopenia. 49 patients were in progressive stage and 106 were in stationary stage among 155 patients. hematological damage occurred in approximately 75.51% of patients in progressive stage while it occurred in 64.15% of patients in stationary stage. The prevalence of Anti-dsDNA,Anti-RNP,Anti-Sm were significantly higher in patients with progressive stage than those in patient with stationary stage. single cell line damage and renal damage were early sign of progressive stage of SLE.

Conclusions Hematological system were easily damaged in patients with SLE and Which was related to disease state. Anti-RNP could reflected red cell line damage.
Case Report of Priapism in a Patient with Thalassemia Minor.
Authors: Mankan Nagender , Samavedi Venkata , Mazumder Sudipta , Razaq Mohammad , Gloria Fernandez , Maini Archana
Published: October 2019

Background - Priapism is a urologic emergency caused by hematological problems in about 35% of cases. Its occurrence in sickle cell anemia, leukemia /other malignancies and primary thrombocythemias is well established. A very few case reports of priapism occurring in Thalassemia Intermedia especially after splenectomy have been documented in literature. Veno-occlusive priapism (low flow priapism) beyond 4hours is a compartment syndrome requiring emergent medical intervention. The most important late complication of priapism is fibrosis and impotence and their incidence is related to the duration and aggressiveness of treatment of priapism. We report a case of priapism in a patient of Thalassemia minor.

Case report- A 42 year Asian male was referred from urology service for a hematologic work-up for recurrent episodes of priapism. He was admitted with a 24 hours-long episode of priapism. The history of priapism dates back to 1998, a few months after marriage, when he was admitted for the first episode of priapism and had a decompressive procedure. Since then, for 6 years, he had intermittent, 15 minutes to 1 hour episodes of penile erection. These episodes usually occur after midnight and were unrelated to sexual activity. He did not have any other medical problems and was not on any medications. He was not on any pro-erectile medications. He denied any history of masturbation. He denied any history of alcohol or illicit drug use. He was unaware of any family history of anemia. On physical examination, he did not have pallor, icterus, lymphadenopathy, skin ulcers or hepatosplenomegaly. The penis was circumcised without any trauma or necrosis. His hemoglobin was 10.5 g/dl, hematocrit 34%, MCV 65fl, RDW14.5 %, reticulocyte count 1.3%, platelets 274k/μl, and WBC 6.9k/μl. Peripheral smear showed poikilocytosis, hypochromia and microcytosis without any sickle cells or nucleated RBCs. Hemoglobin electrophoresis showed Hb A of 94%, Hb A2 of 5.6% and Hb F of 0.4%. His clinical picture, blood count, peripheral smear and electrophoresis were consistent with Thalassemia minor. He was managed by removal of inspissated blood and injection of phenylephrine into the corpora cavernosa. This was repeated a few times and the priapism was relieved and patient discharged.

Conclusion- This is the first reported case of priapism in a patient with Thalassemia minor.
Calibration of Myocardial T2* Values in Post-Mortem Hearts.
Authors: Pennell Dudley J. , Anderson Lisa J. , Paul Kirk , Forni Gian Luca , Ellis Gareth , Walker J. M. , Porter John B.
Published: October 2019

Introduction:. Myocardial iron can now be rapidly and reproducibly assessed using a validated cardiovascular magnetic resonance (CMR) T2* technique. However, the calibration of T2* value to absolute myocardial iron concentration is currently unknown in humans.

Purpose: To calibrate myocardial T2* values in post-mortem hearts.

Methods: Two patients’ hearts have been examined. Each was scanned at 37 Celsius using optimised T2* MR sequences. Iron assays were performed with 100 samples per heart, from 5 short axis slices of the left ventricle. For each slice, 6 segments were cut and a sample taken from the endocardium, mid-wall and epicardium, and 2 from the right ventricular free wall (20 samples per slice). This trial was supported by NIH grant R01 DK66084-01.

Results: Heart 1 from Genova Italy, was from a patient with beta-thalassemia major who had been transfused from age 2 to his death at age 47 from a stroke. The patient had heart failure aged 21 years, from which he had recovered. At age 46, the patient had bradycardia and arrhythmia, and his long-term deferoxamine treatment was augmented with deferiprone for 6 months, and then switched to deferiprone alone at 69mg/kg/day for 7 days per week, starting 1 year prior to death. Ex vivo mid-ventricle full thickness septum T2* was 45ms.

Heart 2 from London was from a transfusion dependent patient with sideroblastic anemia with poor compliance with deferoxamine. The patient died in heart failure. Further clinical details are awaited. Pre-mortem mid-ventricle septum T2* was 5.2ms, and the T2* value in the same region post-mortem was identical at 5.2ms. The results of the ex-vivo comparison of segmental myocardial T2* and matching segmental absolute iron level are shown in figure 1. Calibration is shown on a log-log scale in accord with previous liver sample evidence showing this allows fitting with a linear function (Anderson LJ. Eur Heart J2001; 22: 2171.9).

Conclusion: These are the first human data calibrating human myocardial T2* against absolute iron levels. Myocardial T2* clearly decreases as myocardial iron increases. Of interest was the identical myocardial T2* result between the in-vivo and post-mortem scans in patient 2. Further hearts are now needed to complete the calibration and to determine the consistency of calibration between hearts.

Figure Figure
Ambulatory Blood Pressure Monitoring (ABPM) in Children with Thalassemia Major (TM).
Authors: Kuskonmaz Baris , Unal Sule , Duzova Ali , Bilginer Yelda Yilmaz , Bakkaloglu Aysin , Gumruk Fatma
Published: October 2019

In TM chronic anemia, blood transfusions and iron over load result in impairments in many organs. Little is known about the changes in blood pressure (BP) patterns of children with TM. To evaluate ABPM changes and associated laboratory and clinical features in children with TM. ABPM was performed in 20 TM patients with no cardiac or renal dysfunction (12M, 8F; 11.3+/−3.8 years), and 20 healthy children (11M, 9F; 11.6+/−2.4 years). Blood and timed urine samples were collected for hematological and biochemical tests. [Dipping: a fall in the mean systolic (diastolic) BP during nighttime of more than 10% of the mean daytime systolic (diastolic) BP]. Mean daytime (8AM–8PM) and nighttime (midnight to 6AM) systolic (SBP) and diastolic (DBP) blood pressures were comparable. No patient in TM and control group had mean 24-h, day time, nighttime SBP or DBP above 90th percentile. SBP and DBP- loads were always less than 20% in control group. In TM group: SBP-load was more than 20% in 1, and 1 patient during daytime and nighttime, respectively; and DBP- load was more than 20% in 1, and 2 patients during daytime and nighttime, respectively. The percentage of systolic (60% vs 35%; p:0.102) and diastolic (30% vs 20%; p:0.118) non-dippers among TM patients were higher, compared to control group. In TM group: the percentage of systolic (80% vs 40%; p:0.063) and diastolic (50% vs 10%; p:0.044) non-dippers among older (12–16 years) patients were higher, compared to younger (5–11 years) patients. There was no association between non-dipping pattern and hemoglobin, ferritin or albuminuria level. Our preliminary results suggest that BP anomalies in TM occur before the development of cardiac or renal dysfunction. There is an association between non-dipping and the age of the patient. Long-term follow-up will make clear whether abnormalities in ABPM patterns have a predictive value in the development of renal and cardiac dysfunction in TM patients.
Differential Activation of Stress Kinases Pathways and Elaboration of Inflammatory Cytokines MCP-1 and RANTES by Major and Minor Group Rhinovirus.
Authors: Wilson Megan , Nakahara Shashima , Benson Laurel , Hall David
Published: October 2019

Given that virus-induced asthma has been resistant to therapy, knowledge concerning the cellular and physiological processes that are critical in the development of virus-induced asthma is likely to lead to the generation of new forms of intervention. In this regard, previous studies have implicated the macrophage as playing an important role in the elaboration of the inflammatory environment observed in rhinovirus-induced exacerbation of asthma. However, little is known about the cell signaling events that are initiated upon macrophage infection with rhinovirus. Major and minor group rhinovirus are genetically similar although they bind to two different cellular receptors. To determine if the attachment of major (HRV16) and minor group (HRV1a) rhinovirus to the human monocyte-derived macrophages elicits different kinetics of activation for ERK5 and p38, macrophages cells were exposed to HRV16 or HRV1a at an MOI of 10 for 15,30,60, 90 and 120 min. Cell lysates were generated, separated by SDS-PAGE and immunoblotted with anti-phospho-ERK5 or p38. For MAP kinase p38, major and minor group rhinovirus stimulated phosphorylation occurred after 15 min and sustained phosphorylation up to 60 min. Interestingly at the 90 and 120 min time points, there was no p38 phosphorylation for the major group rhinovirus and sustained phosphorylation stimulated by the minor group rhinovirus. A similar kinetics profile holds true for the MAP kinase ERK5. Furthermore, activation of these two MAP kinases and the small molecular weight G-protein RAC are important in the elaboration of the inflammatory mediators MCP-1 and RANTES.
Potent Phosphorodiester-Based DNA Immunomodulators (dSLIM) Exhibit Enhanced Safety Compared to Phosphorothioate-Based Oligonucleotides.
Authors: Schmidt Manuel , de Christobal Javier , Hagner Nicole , Sander Astrid , Marco Alberto , Koenig-Merediz Sven A. , Wittig Burghardt
Published: October 2019

Linear, single-stranded oligonucleotides (ODN) with non-methylated cytosine-guanine (CpG) motifs are immunomodulatory since they resemble bacterial DNA and serve as “danger signals”. These CpG-ODNs promote predominately a TH1-response, with secretion of IL-12 and IFN-γ, and B-cell, natural killer (NK)-cell and dendritic cell (DC) activation and have a broad potential as therapeutic agents, i.e. for cancer gene therapy and for the treatment of allergic diseases. Distinct groups of CpG-ODNs were characterized differing in structure and function: one group promotes B-cell proliferation, monocyte stimulation and secretion of IgM and IL-6; another activates plasmacytoid DC to produce IFN-α/-β and thus γδT-cells and NK-cells to express CD69 and secrete IFN-γ. And a third group exhibits combined properties of stimulating IL-6 and IgM secretion from B cells as well as IFN-α production from pDCs. Phosphorothioate (PS) modifications, usually introduced to enhance stability, result in several side-effects, like prolongation of the blood clotting time, non-specific binding to various proteins and acute toxicities in primates. In addition, a recent publication showed severe side-effect like significant organ enlargements and morphological changes in mice [Heikenwalder et al., Nat Med. 10:187, 2004]. We generated short covalently-closed dumbbell-like structures (dSLIM) to stabilize the DNA without the otherwise necessary PS-modification. Moreover, the covalently closed constructs do not signal for apoptosis, as high intracellular concentrations of open DNA ends would do. These dSLIM molecules are stable in serum and during long-term storage regarding both DNA integrity and biological function. Their broad activity, like increasing surface expression of CD80/B7.1, CD40, HLA-DR/MHC-II and CD54/ICAM-1 and enhancing production of a wide range of cytokines (IL-6, IFN-α, IFN-γ, IL-12, IL-2), was strictly dependent on molecule structure and size. Increasing or decreasing of stem size lead to reduced potency of the dumbbell-shaped dSLIM molecule. This was observed for a decreased size of the loops as well. But the most intriguing result was the significantly reduced toxicity of dSLIM compared to PS-ODN: After repeated injection of dSLIM or PS-ODN, respectively, into mice the mice receiving PS-ODN developed enlargement of liver, spleen and lymph nodes whereas dSLIM did not induce such changes. In addition, damage of liver and spleen - such as necrotic hepatocytes or hyperplasia - was observed in PS-ODN treated but not in dSLIM treated mice. Nevertheless, both dSLIM and PS-ODN induced a comparable IL-12 production in these mice in vivo. The significant differences of side-effect were true for PS-ODN and dSLIM molecules with various nucleotide sequences. In conclusion, we present the new class of potent immunomodulators (dSLIM) with significantly reduced side-effects.
Enhancement of Lymphokine-Activated Killer Cell Cytotoxicity by Mistletoe Plant Extracts.
Authors: Seifert Georg , Lode Holger N. , Lobitz Stephan , Seeger Karl , Laengler Alfred , Henze Guenter
Published: October 2019

Anthroposophical cancer therapies focus on the use of mistletoe (MT) preparations and extracts from European mistletoe (Viscum album l.) belong to the complementary therapeutic regimens. Although widely used in Europe as an additional therapy in the treatment of patients suffering from different malignancies, these cancer therapies have remained relatively unknown in North America. To date, mistletoe is the most commonly used complementary cancer drug in Germany, applied as monotherapy or in combination with conventional therapies. ABNOBAviscum fraxini is a standardized extract from all parts of fresh mistle, obtained from the ash tree. This study aims to investigate the effects of MT on the activity of lymphokine-activated killer (LAK) cell cytotoxicity in vivo.

Here, we demonstrate that in vivo treatment of mice with a standardized MT plant extract results in activation and function of LAK cells. We show an enhanced cytotoxicity against the NK-sensitive target cell line YAC-1 by flow cytometry. Our data show that in vivo application of MT can double the cytotoxic activity against malignant disease and suggest that one mechasim of action of MT is the induction of LAK-cell mediated immune response.
Alterations in the Umbilical Cord Blood NK Cells Related to the Mode and the Time of Delivery.
Authors: Królak-Olejnik Barbara , Olejnik Igor
Published: October 2019

Natural killer (NK) cells take part in the early immunological response to infection. Their lower cytotoxic activity in the neonates, especially premature ones, compared to children and adults, is assumed to be one of the factors responsible for high susceptibility to infections. Moreover, alterations in every components of immune response during anesthesia and surgery have been suggested. The numbers of natural killer cells are decreased postoperatively. The aim of the study was to estimate the influence of the mode and time of delivery on the number of leukocytes, number and percentage of lymphocytes and natural killer (NK) cells. The NK cells were examined by the three-color flow cytometry with the use of monoclonal antibodies of Becton Dickinson in the following study groups: (1) full-term neonates born by normal spontaneous vaginal delivery (n=19); (2) preterm neonates born by normal spontaneous vaginal delivery (n=15); (3) full-term neonates born by elective cesarean section under epidural anesthesia (n=23); (4) preterm neonates born by cesarean section under epidural anesthesia (n=22). The number of leukocytes was similar in all examined neonates. The numbers of leukocytes were lower albeit not significantly in preterm neonates born by cesarean section. The numbers of lymphocytes were also similar in all examined neonates but the percentage of lymphocytes was higher in the preterm neonates than in the full-term ones (p<0,05). The number and percentage of natural killer (NK) cells were higher in the neonates born by normal spontaneous vaginal delivery both full-term and preterm ones. The significant lowest value of NK cells was in the preterm neonates born by cesarean section under epidural anesthesia. These results suggest that either mode of delivery or time of delivery might influence the NK cell numbers in the umbilical cord blood of the neonates.
Comparison of Cationic Propyl Gallate and Adenosine Diphosphate for the Meassurement of Aspirin Effectivity with Optical Aggregometry.
Authors: Motovska Zuzana , Sujanova Zdenka , Wimmerova Sona , Ardo Jan , Skrakova Marcela , Widimsky Petr
Published: October 2019

Background. The goal of our interest is to bring attention to standardisation of the aspirin effectiveness examination in patients profiting from (life)long antithrombotic terapy. Optical platelet aggregation tests have been most widely used. Examination with endogenous inductors of platelet aggregation - thrombin, colagen, arachidonic acid, adenosine diphosphate (ADP) has a low specificity and reproducibility. Aim. To compare a newer inductor of platelets aggregation cationic propyl gallate (CPG) with ADP for the examination of aspirin (ASA) efffectivity with optical aggregometry.

Methods. We prospectively enrolled 116 consecutive patiens with a stable cardiovascular disease on ASA 100mg/day for ≥ 1 month. The controll group was formed from 62 healthy volunteers, without antithrombotic treatment. Analysis. We investigated platelet aggregation by optical aggregometry (aggregometer LASER 4x, BIOART). CPG and ADP were added as aggregating agents. The measured parameter were: CPG-slope (this parameter expresses steepness of the aggregation curve and also the speed of aggregation (%/minutes)) and ADP-max (the maximum percent change in light transmittance from baseline). Results. Definition. First, we determined the CPG-slope cut-off value for the definition of ASA-non-effective treated patient. We established it on the ground of CPG-slope values in the control group. The values from controll group followed a normal distribution (test Shapiro - Wilk). We calculated the cut-off value using the 95% - confidence interval. The CPG-slope cut-off value was 79 %/min for ASA-effective treated patient. We marked the patients as ASA-non efffective treated when the CPG-slope was ≥ 79%/min. The same way we used to define the cut-off value for ADP-max. We identified the aspirin treatment as uneffective when the value of ADP-max had been > 62%. Comparison. The values of CPG-slope and ADP-max were in a close correlation in the group of patients treated with aspirin. The correlation index (r = 0,671) was highly significant. By CPG-induced optical aggregation were 33,6% ASA-non-effective treated patients. While using both inductors (CPG, ADP) were the proportion of ASA-non-effective treated patients 25%. By both tests (CPG, ADP) were equally divided 71,6% patients. ASA-non-effective treated patients were more commonly obese (46,2%) with hypertension (94,9%) and hypercholesterolemia (73,7%) and were less commonly treated with statins (30,8%) as the aspirin effective treated patients (42,%, 88,2%, 59,2% resp.42,1%).

Conclusion. Meassurement of a platelet aggregation by CPG is reproducible with high sensitivity and specificity. The study has also brought attention to the importance of control the cardiovascular risk factors.

Figure. Statistical parameters Figure. Statistical parameters