bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Wnt/β-Catenin Pathway Activation by Tobacco Smoke Modulates Cyclooxygenase-2 Expression in Cardiac Microvascular Endothelial Cells.
Authors: Barbieri Silvia S. , Ip Dawn , Howe Louise R. , Weksler Babette B.
Published: April 2020

While cigarette smoking is a major risk factor for atherosclerosis and for acute cardiovascular events, the toxic components of tobacco smoke (TS) and mechanisms producing cardiovascular dysfunction are unknown. Exposure to TS increases inflammation, thrombosis, and oxidation of LDL cholesterol and we have previously found that TS exposure activates several inflammatory genes in vascular cells. To address the mechanisms of TS-induced proatherosclerotic effects, we derived an immortalized line of mouse cardiac microvascular endothelial (MMCEC) cells by transfection of primary cells with lentiviral vectors encoding genes for telomerase and SV40 T antigen. These MMCEC grow indefinitely while retaining a normal endothelial phenotype, show contact inhibition, display EC markers such as PECAM, VE-cadherin, and von Willebrand factor, and develop an inflammatory phenotype when stimulated with cytokines. In response to interleukin-1β (IL-1 β), MMCEC induce cyclooxygenase-2 (COX-2) and produce prostaglandins; these effects are enhanced by a saline extract of TS in a dose- and time-dependent manner. Increased COX-2 expression accompanies atherosclerosis, while its prostaglandin products favor cell proliferation and decrease apoptosis. Moreover, β-catenin, a key component of cell-cell interactions, and a coactivator of the TCF/LEF (T-cell transcription factor/lymphoid enhancer binding factor) family of transcription factors, is also increased in atherosclerotic plaques. We therefore studied how TS exposure affected the Wnt/β-catenin pathway. IL-1 β and TS each stimulated β-catenin translocation from the plasma membrane to the nucleus of MMCEC within 1–2 hr, but addition of TS to cells exposed to IL-1 β stimulated translocation within minutes shown by confocal microscopy and western blotting. The enhanced β-catenin translocation induced by IL-1 β/TS was linked with rapid phosphorylation of Akt and glycogen synthase kinase 3β, which in turn decreased phosphorylation of β-catenin, preventing its proteosomal degradation. Conversely, when GSK3β, which stabilizes β-catenin, was blocked, COX-2 was induced. If β-catenin expression in MMCEC was directly inhibited by transfection of cells with short interfering RNA (siRNA) induction of COX-2 expression and prostaglandin production by IL-1 β/TS was markedly diminished. In addition, LY294002, an inhibitor of phosphatidylinositol-3-kinase, and AG1478, an EGFR inhibitor, also blocked IL-1 β/TS induced Akt phosphorylation, β-catenin translocation and COX-2 expression, as did selective inhibitors of p38 MAPK and MEK-1. Our data, using a novel endothelial cell model, demonstrate that the PI3K/GSK-3β regulated β-catenin pathway is important for COX-2-related proinflammatory and proatherosclerotic effects of tobacco smoke on vascular cells and indicate the interrelated signaling pathways involved. TS-related effects may similarly modulate other proatherosclerotic genes.
Relationships of Serum Ferritin, Transferrin Saturation, and HFE Mutations and Self-Reported Diabetes Mellitus in the Hemochromatosis and Iron Overload Screening (HEIRS) Study.
Authors: Acton Ronald T. , Barton James C. , Passmore Leah , Adams Paul C. , Speechley Mark R. , Dawkins Fitzroy W. , Sholinsky Phyliss , Reboussin David M. , McLaren Gordon D. , Harris Emily L. , Bent Thomas C. , Vogt Thomas , Castro Oswaldo
Published: April 2020

Mean serum ferritin concentration (SF) has been positively associated with diabetes mellitus in some racial/ethnic groups. We evaluated the associations of self-reported diabetes mellitus with SF, serum transferrin saturation (TfSat), and HFE C282Y and H63D mutations in six racial/ethnic groups recruited at five Field Centers in the multi-center, primary care-based HEIRS Study. Analyses were conducted on the 97,470 participants for whom pertinent data were available. Participants who reported that they had been previously diagnosed with diabetes mellitus and/or hemochromatosis or iron overload were compared to participants who did not report a previous diagnosis. The overall prevalence of diabetes mellitus was 13.8%; the highest prevalence was observed in Native Hawaiian/Pacific Islanders (20.1%). 2.0% of all participants with diabetes mellitus and 2.2% of whites with diabetes mellitus reported that they also had hemochromatosis or iron overload. Mean SF was elevated in women with diabetes mellitus in all racial/ethnic groups and in American Indian/Alaska Native men with diabetes mellitus. Mean SF was significantly lower in Asian men with diabetes mellitus. Mean TfSat was lower (or not increased) in participants with diabetes mellitus than in those without diabetes mellitus. There was no significant association of diabetes mellitus with HFE genotype. Mean SF was greater (p <0.0001) in women with diabetes mellitus than in those without diabetes mellitus for HFE genotypes except C282Y/C282Y and C282Y/H63D. There was a significant positive association of log SF and diabetes mellitus in a logistic regression analysis after adjusting for age, gender, racial/ethnic group, HFE genotype, and Field Center. We conclude that SF is significantly associated with diabetes mellitus, even at levels of SF below those typically associated with hemochromatosis or iron overload.
Ferrochelatase Mutation Screening by Denaturing High Performance Liquid Chomatography in Idiopathic Acquired Sideroblastic Anemia (Refractory Anemia with Ringed Sideroblasts) and Erythrocytic Protoporphyria.
Authors: Hecksel Kathleen A. , Dewald Gordon W. , Steensma David P.
Published: April 2020

BACKGROUND: The molecular etiology of idiopathic acquired sideroblastic anemia (IASA), now considered a form of myelodysplastic syndrome, is currently unknown. Romslo et al (Blood 1982) reported a patient with IASA who had moderately elevated free erythrocytic protoporphyrin (FEP); this observation is now frequently made in IASA, helping distinguish IASA (high FEP) from inherited sideroblastosis (low FEP). In addition, rare patients with erythropoietic protoporphyria (EPP)—defined by cutaneous photosensitivity, dramatically elevated FEP levels, and germline point mutations in the ferrochelatase (FECH) gene at 18q21.3—have ringed sideroblasts in their marrow. We hypothesized that patients with IASA might have acquired somatic FECH mutations.

METHODS AND RESULTS: We designed a denaturing high performance liquid chromatography (DHPLC) assay to explore this possibility and to avoid problems related to mutation screening in the setting of mixed clonality. To validate the DHPLC assay, the coding region of the FECH gene from 2 molecularly undiagnosed EPP patients without liver disease was analyzed. In one patient, both a heterozygous 69delG mutation (GenBank Accession NM_000140) and heterozygosity for the FECH expression-modulating IVS3-48C/T polymorphism were detected. In the other patient, no FECH coding mutations or polymorphisms were detected, either by DHPLC or conventional dye sequencing. FEP measurements were then obtained on 2 IASA patients and were elevated (65 and 115 mcg/dL; normal 1–10 mcg/dL) with normal urine and fecal porphyrins. Genomic DNA obtained from these 2 patients as well as archival DNA from 30 other patients with IASA was amplified and tested for FECH mutations. No coding region mutations were detected. Synonymous polymorphisms in exon 7 (rs536765) and 9 (rs536560) were found in 6/32 (19%; normal heterozygosity 0.398) and 14/32 (44%; normal heterozygosity 0.402) samples, respectively. The IVS3-48C/T polymorphism (rs2272783) was found in 3/32 (9%) (prevalence in the general population is 11%, Gouya Nat Genet 2002). In addition, 3 intronic polymorphisms not in the refSNP database were detected: IVS8+34 C/T (2/32), IVS8-61delG (5/32), and IVS9-59delA (2/32).

CONCLUSION: IASA is not associated with coding mutations in FECH. Elevation of FEP in ASA must instead be due to the lack of mitochondrial iron in the proper form for incorporation into the porphyrin ring; attention should instead focus on factors responsible for maintaining the appropriate redox state and compartmentalization of iron. DHPLC can detect FECH mutations and polymorphisms, but because of the high frequency of the latter and the consequent need to sequence multiple exons, it is not a practical screening tool for studying undiagnosed EPP patients.
Gene Expression Profiling Reveals the Profound Up-Regulation of Hypoxia-Responsive Genes in Primary Human Astrocytes.
Authors: Mense Sarah M. , Zhou Mei , Sengupta Amitabha , Lan Changgui , Bentsman Galina , Volsky David J. , Zhang Li
Published: April 2020

Oxygen is vital for the development and survival of mammals. Lack of oxygen and oxygen sensing mechanisms have been implicated in the pathology of many CNS disorders, including stroke, head trauma, neural developmental problems associated with preterm birth, neoplasia and neurodegenerative diseases. In response to hypoxia, the brain initiates numerous adaptive responses at the organ level as well as at the molecular and cellular level, including the alteration of gene expression. Astrocytes play critical roles in the proper functioning of the brain. They are important for maintaining ion and pH homeostasis, for the synthesis and removal of neurotransmitters, such as GABA and glutamate, and for providing glucose supply to the brain. Recent evidence indicates that astrocytes regulate synaptic activity, synaptogenesis, and neurogenesis. Thus, the manner in which astrocytes respond to hypoxia is likely important in determining the outcome of brain hypoxia. Astrocytes are generally more resistant to energy failure than neurons. Astrocytes account for at least 15% of oxidative metabolism in the brain, and their respiratory rates are maintained at 85% of the basal level until oxygen is virtually exhausted. In this report, to better understand the molecular mechanisms governing astrocytic response to hypoxia, we used microarray gene expression profiling and data analysis algorithms to identify and analyze hypoxia-responsive genes in primary human astrocytes. We also compared the gene expression patterns in astrocytes with those of human HeLa cells and pulmonary artery endothelial cells (ECs). Remarkably, in astrocytes, 5 times as many genes were up-regulated as down-regulated, whereas in HeLa and pulmonary ECs, as many or more genes were down-regulated as up-regulated. More genes encoding hypoxia-inducible functions, such as glycolytic enzymes and angiogenic growth factors, were strongly induced in astrocytes compared to HeLa cells. The extent of induction was also greater than in HeLa cells. Further, gene ontology and computational algorithms revealed that many target genes of the EGF and insulin signaling pathways and the transcriptional regulators Myc, Jun and p53 were selectively altered by hypoxia in astrocytes and in HeLa cells to a lesser degree.

Figure Figure Figure Figure

In the case of astrocytes, the overwhelming induction of target genes of the EGF and insulin pathways, which are pro-survival, and other genes encoding growth factors and functions involved in carbohydrate metabolism may underlie their relative strong resistance to ischemic/hypoxic insults. These results provide a global view of the signaling and regulatory network mediating oxygen regulation in astrocytes, in comparison with that in HeLa cells.
Immature Reticulocyte Fraction after Iron Therapy for Iron Deficiency Anemia.
Authors: Yuh Young Jin , Kim Sung Rok , Han Tae Hee
Published: April 2020

Background: Automated counting of reticulocyte introduced new reticulocyte parameters such as immature reticulocyte fraction(IRF). IRF is thought to be more sensitive than absolute or corrected reticulocyte count for detecting recovery of bone marrow function. The aim of this study is to assess the role of IRF for predicting the response of therapy after treatment of iron deficiency anemia.

Methods: Patients with previously untreated iron deficiency anemia (blood hemoglobin < 10.0g/dL and serum ferritin < 10ng/mL) were enrolled into this study. On the 8th day of iron therapy, Complete blood count(CBC) with reticulocyte count and IRF was assessed by RAM-1 reticulocyte autoanalyzer(Sysmex. Tokyo, Japan). IRF was defined as sum of the reticulocyte fraction of high-fluorescence intensity regions plus the fraction of middle fluorescence intensity regions. After 1 month of iron therapy, CBC was reassessed to evaluate the response of iron therapy. The correlation of the 8th day IRF and the change of hemoglobin level after 4 weeks of therapy was evaluated. The 8th day corrected reticulcyte count was also evaluated.

Results: From Jan. 2001 to May 2003, 27 patients with iron deficiency anemia (3 men and 24 women) entered into this study. We evaluated 21 patients, excluding 6 patients who were lost to follow-up or refused blood sampling after 1 month of iron therapy. The range of pretreatment hemoglobin level was 3.3–9.7g/dL (median 7.5/dL). The 8th day IRF was 0.06–0.39(median 0.15). Ten patients had the low IRF (IRF ≤ 0.15). Four of them (40%) had their hemoglobin level increased by more than 3g/dL. On the other side, 11 patients had high IRF (IRF > 0.15) and 10 of them (91%) had their hemoglobin level increased by more than 3 g/dL. This difference was significant (P=0.024). However, the 8th day IRF was not correlated with the change of hemoglobin as strongly as the 8th day corrected reticulocyte count (Pearson coefficient 0.420 vs 0.693).

Conclusion: For the patients with iron deficiency anemia, the 8th day of treatment IRF correlates with the response of iron treatment, but the 8th day of treatment corrected reticulocyte count has a stronger correlation.
The Prevalence of Anemia among Healthy Women in Japan.
Authors: Kusumi Eiji , Kami Masahiro , Shoji Midori , Endou Shigeyuki , Kishi Yukiko , Shibata Taro , Murashige Naoko , Hamaki Tamae , Matsumura Tomoko , Yuji Koichiro , Yoneyama Akiko
Published: April 2020

Introduction

Anemia is common among young women. National Health and Nutrition Examination Survey (NHENES) revealed that the insufficient iron intake is one of the leading causes of anemia in the US. In Europe and the US, iron fortification of flour increased oral iron intake and decreased anemia prevalence from 30% to 10%. The National Nutrition Survey in Japan by the Ministry of Health, Labour and Welfare in 2002 revealed the estimated anemia prevalence of 19 %. Currently, no nationwide preventive policy aims at iron deficiency anemia. However, the prevalence of 19% was estimated based on 3,156 persons in the Survey.

Objective

The endpoint of this study was to estimate anemia prevalence among healthy Japanese woman based on a larger sample size.

Method

We collected data from consecutive check-up examination records of apparently healthy women in different age groups in Toranomon Hopital (between January 2002 and March 2005, n = 8,264) and Yuai Memorial Hospital (between February 1998 and February 2005, n = 5,153). We defined hemoglobin lower than 12 g/dL as anemia, hemoglobin lower than 10 g/dL as severe anemia, and mean corpuscular volume lower than 80 fl as microcytic anemia.

Results

The median age was 47 years (range, 11–87 years). Of the 13,147 persons, anemia was identified in 2,331 (17.3 %), and severe and microcytic anemia in 438 (3.3 %) and 700 (5.2 %), respectively. The prevalence of anemia and median hemoglobin values by age groups are shown in Figure. The median hemoglobin values tended to be lower in women of the age groups with high anemia prevalence.

Conclusion

The prevalence of anemia among women of child-bearing age and of working age is high in Japan. As the Japanese population is rapidly decreasing and aging, some action needs to be considered to improve quality of life during pregnancy and working efficiency.

Figure Figure.
Treatment of Severe Aplastic Anemia with Combined Immunosuppression: Antithymocyte Globulin (ATG), Cyclosporine A (CSA), and Mycophenolate Mofetil (MMF).
Authors: Scheinberg Phillip , Nunez Olga , Wu Colin O. , Young Neal S.
Published: April 2020

Severe aplastic anemia can be successfully treated with immunosuppressive therapies (IST) or hematopoietic stem cell transplantation (HSCT). Response rates with horse anti-thymocyte globulin (ATG) plus cyclosporine (CsA) are about 60–70%, and robust responders have excellent long term survival (Rosenfeld, Follmann et al. 2003). Relapse require reinstitution of IST and does not negatively affect survival. We introduced a third immunosuppressive agent to standard horse ATG/CsA, mycophenolate mofetil, in an attempt to improve response rate, survival and to decrease the relapse rate and clonal evolutions to myelodysplasia or leukemia. MMF blocks the proliferation of active lymphocytes by the inhibition of the enzyme inosine monophosphate dehydrogenase, an important step in purine and therefore DNA synthesis. MMF immunosuppression is due to direct inhibition of T cell proliferation and therefore, would be anticipated to favor the induction of tolerance by selective depletion of active lymphocytes. MMF has been employed as a “kidney-sparing” adjunct to CSA in renal and other solid organ transplantations, post-HSCT, and in certain autoimmune diseases with a favorable toxicity profile. A total of 104 consecutive patients with SAA were treated with horse ATG/CsA/MMF from May 1999 to June 2003 at the Warren Grant Magnuson Clinical Center at the National Institutes of Health in Bethesda, MD. Response was defined by blood counts no longer satisfying criteria for severe AA. The overall response rate at 6 months was 62%, with 24 (37% of responders) patients relapsing at a median of 389 days from ATG. Fifteen relapses occurred after CsA was discontinued at 6 months but before 18 months, while patients were still on MMF. Nine patients showed evidence of clonal evolution following ATG, five responders and 4 nonresponders. The median survival among responders was not reached and among nonresponders was 1,759 days. There was no difference in overall survival among patients who relapsed and those who did not relapse. Over half of the relapses occurred during MMF administration (15 of 24), suggesting that this drug is not active in preventing relapses among responders. Evolution to clonal disorders did not change with the intensification of immunosuppression with MMF. Despite a strong theoretical rationale for its use, MMF did not result in improvement in response or relapse rates, and it cannot be routinely recommended as a CsA-sparing agent in responders with poor tolerability to CsA.
Hematopoietic Response to Extended Dosing of Epoetin alfa 60,000 U Every 2 Weeks in Anemic Cancer Patients Not Receiving Therapy: Final Results.
Authors: Shasha Daniel , Henry David H.
Published: April 2020

Epoetin alfa administered weekly raises hemoglobin (Hb) in anemic cancer patients not receiving therapy. For optimal management of this group of patients more information is needed about extended dosing intervals. The objective of this open-label, single arm, multicenter study was to evaluate the hematopoietic response (HR) when anemic cancer patients not receiving chemotherapy (CT) or radiotherapy (RT) were treated with 60,000 U of epoetin alfa every two weeks (Q2W). Patients with non-myeloid malignancy, no CT within 8 weeks or RT within 4 weeks prior to study entry, and baseline Hb ≤ 11 g/dL were enrolled. Epoetin alfa was administered at 60,000 U SC Q2W for 12 weeks. If Hb increased < 1 g/dL after 4 weeks, epoetin alfa was increased to 80,000 U SC Q2W. Epoetin alfa was held for Hb > 13 g/dL. Patients were to receive iron supplementation daily at physician’s discretion. The primary efficacy endpoint was the proportion of patients achieving HR (defined as a ≥ 2 g/dL increase in Hb from baseline and/or Hb ≥ 12 g/dL during the treatment period, independent of transfusion within 28 days). Fifty-seven patients were enrolled and 56 met the criteria for efficacy analyses (received ≥ 1 dose of epoetin alfa and had ≥ 1 post-baseline Hb or transfusion evaluation). Mean age was 71.7 ± 9.3 years, 35.7% of patients were male, and mean baseline Hb was 10.2 ± 0.7 g/dL. HR was achieved in 43/56 (76.8%) patients. Mean change in Hb from baseline was 1.5 ± 0.8 g/dL (n=51) after 4 weeks, and 1.8 ± 1.0 g/dL (n=49) after 8 weeks on study. Nine (15.8%) patients had a dose increase and 22 (38.6%) had a dose held. Three (5.4%) patients received RBC transfusion and 37 (64.9%) received iron supplementation. The most common adverse events were peripheral edema, dizziness, nausea, and asthenia. Two (3.5%) patients experienced a clinically relevant thrombovascular event (TVE). No deaths were reported. This study suggests that cancer patients with anemia not receiving CT or RT can be treated effectively and safely with 60,000 U epoetin alfa every two weeks.
Vascular Growth Factors in Patients with Sickle Cell Disease.
Authors: Duits Ashley J. , Schnog John B.
Published: April 2020

Angiogenesis plays a central role in neovascular processes. In sickle cell patients neovascularization is considered a common feature that is associated with complications such as retinopathy and cerebrovascular disease. Several studies have reported increased vascular growth factor levels such as vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) in small groups of patients. Recently the angiopoietin/Tie2 system has been shown to be of major importance in vessel maturation. In order to thoroughly characterize vascular growth factor profiles in sickle cell patients we analyzed serum levels of VEGF, PlGF, Angiopoietin-1, Angiopoietin-2, Tie-2 and erythropoietin (EPO) during the asymptomatic phase as well as during painful crises in HbSS patients (n=42) and compared these to levels in HbAA controls (n=30). Also, levels were compared between patients with and without a history of acute vaso-occlusive complications the year prior to sample collection. Serum Angiopoietin-2, Tie-2 and EPO levels were significantly higher in sickle cell patients as compared to controls (p<0.01, p=0.03 and p<0.01 respectively), whereas PlGF and VEGF serum levels were similar between patients and controls (p= 0.21 and p=0.5). Only Angiopoietin-2 and EPO serum levels were significantly increased in sickle cell patients during acute vaso-occlusive crises as compared to levels in asymptomatic patients (p<0.01 and p=0.03 respectively). No differences were detected in measured parameters between patients with a history of acute vaso-occlusive complications as compared to patients without acute vaso-occlusive complications in the preceding year. Our results show a specific pattern of angiopoietins in sickle cell patients. Considering the importance of endothelial cell activation in sickle cell disease and the regulation of endothelial cell survival and blood vessel maturation by the angiopoietin/Tie-2 system, further analysis of angiogenesis in sickle cell disease is warranted.
Inside-Out Signaling through ADAP Is Required for VWF-GPIb-IX-V Mediated Integrin AIibb3 Activation.
Authors: Kasirer-Friede Ana , Moran Barry P. , Nagrampa-Orje Jennifer , Swanson Ken , Schraven Burkhart , Neel5 Ben , Ruggeri Zaverio M. , Shattil Sanford J.
Published: April 2020

The interaction between von Willebrand factor (VWF) and platelet GPIb-IX-V is a key biological mechanism in hemostasis. In a previous study of signal transduction between GPIb-IX-V and integrin α Iibβ 3, we observed that platelet adhesion to a dimeric VWF A1 domain fragment via GP Ib-IX-V induced the tyrosine phosphorylation of the adapter protein, ADAP (Slap-130, FYB). While this protein has been implicated in cell proliferation and integrin clustering in leukocytes, its role in platelet function has not been determined. Here we used ADAP knockout mice to address this issue. Initiation of signaling in platelets adherent to murine dimeric VWF A1 domain caused α Iibβ 3 activation in wild-type, ADAP+/+ platelets, as determined by the binding of FITC-fibrinogen or POW-2, an anti-α Iibβ 3 antibody Fab specific for high-affinity α Iibβ 3. However, α Iibβ 3 activation through GP Ib-IX-V was diminished by 70–80 % in ADAP−/− platelets (P <0.01), despite normal platelet adhesion under these static conditions. α Iibβ 3 activation by other agonist pathways, such as those stimulated through ADP or Par4 thrombin receptors was also decreased in ADAP−/− platelets (P < 0.05 and 0.01 respectively). In addition, following adhesion of ADAP−/− platelets to VWF A1, tyrosine phosphorylation of numerous proteins, including the ADAP binding partner, SLP-76, was markedly reduced. Surprisingly, deletion of ADAP also affected the expression of another ADAP binding partner, Skap-HOM, and transient transfection of CHO cells confirmed that ADAP expression regulated Skap-HOM protein levels. Platelets from Skap-HOM−/− mice, which have normal ADAP levels, showed normal α Iibβ 3 activation, indicating that the inside-out signaling defect in ADAP−/− platelets was specifically due to deficiency of ADAP and not Skap-HOM. ADAP−/− mice displayed more re-bleeding from tail wounds compared to ADAP+/+ mice (64% vs. 16%, p < 0.01). Under shear flow conditions over a combined matrix of VWF A1A2 and fibronectin to test interactions involving GP Ib-IX-V and α Iibβ 3, respectively, ADAP−/− platelets displayed reduced α IIbβ 3-dependent adhesion. These studies establish that ADAP is a necessary component of the inside-out signaling pathways downstream of GP Ib-IX-V and other receptors in platelets that regulate α IIbβ 3 affinity.
Hypertensive Encephalopathy in Children with Sickle Cell Disease.
Authors: Minniti Caterina P. , Weinstein Steven L. , Khademian Zarir
Published: April 2020

Cerebrovascular (CVA) accidents are a serious complication of Sickle Cell Disease (SCD). Acute cerebral infarction occurs in approximately 10% of children with SCD under the age of 16 years, with silent infarcts identified in an additional 22%. Children however can present with acute neurologic deterioration mimicking a CVA but demonstrate a picture consistent with hypertensive encephalopathy (HTNE). The incidence of HTNE in children with SCD is unknown with few annedoctal reports available in the literature. We retrospectively identified 83 children with SCD, who received their care at Children’s National Medical Center in Washington, DC, from 1992 to 2005, who presented with neurologic complaints that prompted Magnetic Resonance Imaging (MRI). There were 37 females and 46 males, age 13 months to 17 years, with mean age of 5 years and 8 months. Clinical and neuro-imaging data identified 8 children (7 females and 1 male) with clinical picture compatible with HTNE (BP > 2 std deviation for age), prior to neuroimaging. At the time of the hypertensive episode, the ages ranged from 6 to 16 years and 8 months, with an average of 14 years and 2 months. All patients had Hb SS. Neurologic complaints included seizure, sudden onset headache, confusion, loss of consciousness, and urinary retention. Elevated blood pressures were aggressively treated and all patient received an exchange transfusion. No specific etiology for the hypertension was determined. Initial MRI, obtained within 24–48 hours of presentation, did not show acute or prior infarction in this group. However, there was absence of diffusion restriction on T1 and T2 weighted images, and presence of cerebral cortical edema, which differentiate HTNE from acute infarction. The magnetic resonance angiogram (MRA) did not demonstrate evidence of arteriopathy. The hallmark of HTNE is a complete resolution of abnormalities of both the neurologic exam and the imaging studies at follow up. There was complete resolution of cerebral edema in all patients and mild interval prominence of the cerebral sulci, which can indicate cerebral volume loss, in three out of the 8 patients, in follow up studies obtained 10 days to 4 months later. No recurrences of the symptoms have been reported, with follow up ranging from 5 months to 8 years. None of the patients with HTNE has received chronic transfusions. We conclude that HTNE should be considered in the differential diagnosis of an acute neurologic event in a child with SCD. Accurate recording of vital signs and prompt correction of hypertension is indicated. MRI can accurately distinguish between HTNE and acute infarction, even when clinical symptoms are similar. This distinction helps physicians to establish proper treatment such as chronic transfusion following infarction.
Neonatal Cyanosis Due to a Novel Fetal M-Hemoglobin: Hemoglobin F-M Circleville (Gγ63 His→Leu).
Authors: Kutlar Ferdane , Shell Richard , Atkin Joan , Elam Dedrey , Holley Leslie , Davis Deborah , Harbin Jeanette , Kutlar Abdullah
Published: April 2020

Neonatal cyanosis can result from a multitude of acquired and inherited causes. Cyanosis resulting from fetal M-Hemoglobin variants is a rare cause of this condition. In fact, to date, only two Gγ variants causing methemoglobinemia and cyanosis in the newborn have been reported. These are Hb F-M Osaka (Gγ63 His→Tyr) and Hb F-M Fort Ripley (Gγ92 His→Tyr). We report a novel fetal M-hemoglobin presenting with neonatal cyanosis. The propositus was a 1-day old Caucasian male admitted to the hospital because of cyanotic episodes. The patient had an O2 saturation of 85% on room air and required supplemental O2 during his 10-day hospitalization. The family history was significant for a 4-year old sister having a 5-day hospitalization after delivery for cyanosis. The sister required supplemental O2 for 4–5 months to maintain a satisfactory O2 saturation. Both parents were healthy and had no recollection of neonatal cyanosis or O2 requirement. The newborn screening for hemoglobinopathies reportedly showed Hb F, A, and a variant migrating between Hb S and Hb F. Cation exchange HPLC performed at 1-month of age revealed 68.4% Hb F; 17.5% Hb A, and 14.0% Hb X, eluting between Hb F and Hb A. Reverse phase HPLC showed only βA, αa, Gγ, and Aγ chains without any detectable abnormal chains. O2 affinity showed a P50 of 24.2mmHg (control 23.4mmHg). Sequencing of the PCR amplified Gγ gene revealed heterozygosity for a CAT→CTT mutation (His→Leu) at codon 63. (GenBank Acession # AY662983). This novel variant was named Hb F-Circleville [GγE7(63)His→Leu]. The M-hemoglobins arise from mutations in the conserved proximal (F8) or distal (E7) histidine residues, which are important heme contacts. Substitution of these residues lead to methemoglobinemia and cyanosis. Fetal Hb variants causing methemoglobinemia are rare causes of neonatal cyanosis. Hb F-Circleville is only the third fetal M-Hb reported to date. The prognosis of cyanosis is excellent in these cases, as Hb F declines post natally and is replaced by normal Hb A. Supplemental O2 may be required for several months during the postnatal period as was the case with this patient. Some fetal M-Hbs have been shown to arise as de novo mutations. In our case, an older sister and the mother were both proven to carry the Gγ63 His→Leu mutation consistent with autosomal dominant inheritance.
Signalling by the Platelet C-Type Lectin Receptor CLEC-2 Is Mediated by a Novel Mechanism Involving Syk and a Single YxxL Motif.
Authors: Pearce Andrew C. , Fuller Gemma L.J. , Suzuki-Inoue Katsue , Tomlinson Michael G. , Watson Steve P.
Published: April 2020

We have recently identified the C-type lectin-like receptor CLEC-2 as a novel receptor for the snake toxin rhodocytin on platelets. CLEC-2 is a 32 kDa type II transmembrane protein with a single cytoplasmic tyrosine residue in a YxxL consensus sequence. It is the first C-type lectin receptor that has been shown to activate platelets. The aim of the present study was to investigate the mechanism of signalling by CLEC-2 using model cell lines and to establish the role of the YxxL motif in this process.

We have expressed CLEC-2 in wild type DT40 B cells and Jurkat T cells and in mutant derivatives of these cell lines that lack key signalling proteins. We have assayed PLCγ activation using an NFAT-luciferase reporter assay. Site directed mutagenesis of the cytoplasmic tyrosine residue of CLEC-2 has been used to assess its role in CLEC-2 signalling. We have compared the signalling pathway of CLEC-2 with that of the ITAM-coupled collagen receptor GPVI by expressing GPVI in the mutant cell lines and monitoring responses to the GPVI specific agonist convulxin as above.

Cells transfected with CLEC-2 respond to rhodocytin. Mutation of the CLEC-2 YxxL motif to FxxL abrogates CLEC-2 signalling. CLEC-2 signalling is abrogated in cells deficient in Src or Syk family kinases or PLCγ . CLEC-2 signalling is partially dependent on Tec family kinases and the adaptors LAT and SLP-76/BLNK. Interestingly, GPVI signalling and CLEC-2 signalling show a differential requirement for the SLP-76 family adaptor proteins SLP-76 or Blnk; GPVI signalling is entirely dependent on SLP-76/Blnk, whereas CLEC-2 signalling is only partially dependent on these proteins.

These observations are consistent with a model whereby CLEC-2 recruits Syk via its YxxL motif and initiates a signalling cascade similar to the major platelet glycoproteins GPVI and integrin α IIbβ 3. CLEC-2 is the first platelet receptor shown to regulate Syk through a single YxxL motif. Importantly, despite the similarity in the signalling proteins involved in signalling by the ITAM receptor, the integrin receptor and the C-type lectin receptor, the signalling pathways are organised and initiated differently.
Pharmacological Inhibition of K-Cl Cotransport Alters In Vitro Maturation of Normal and β-Thalassemic Human Erythroid Progenitors.
Authors: De Franceschi Lucia , Ronzoni Luisa , Iolascon Achille , Cimmino Francesca , Alper Seth L. , Servedio Valeria , Turrini Franco , Cappellini Maria D.
Published: April 2020

The K-Cl cotransporter family (KCC) plays a crucial role in cell volume regulation, and KCC1 and KCC3 have been reported to participate in cell growth events (Shen MR, PNAS98, 2001; Shen MR JBC278, 2003). Expression of KCC1, KCC3 and KCC4 has been reported in erythroid cells. In β-thalassemic red blood cells (RBCs), K-Cl cotransport activity is abnormally activated and contributes to red cell loss of water and K. This study evaluated the gene expression of two KCC gene products and the effects of the KCC inhibitor [(dihydroindoenyl)oxy]alkanoic acid (DIOA) on in vitro liquid-culture expansion of human normal and β thalassemic (β thal) erythroid precursors from peripheral blood CD34+ cells. Cells from normal subjects and from β thalassemia major patients (cod39cod39) were cultured for 7 days (to the pro-normobast stage) and 14 days (to the eythroblast stage) in the presence or absence of 10 mM DIOA, At each time point the following parameters were evaluated;

cells counts; cytospins stained with Wright-Giemsa to assess differential cell counts and morphology, cell cycle stage by fluorescence-activated cell sorting after propidium iodide staining; KCC protein expression by Western-blot analysis with antibody to the shared KCC carboxy-terminus; mRNA by real time-PCR analysis.

KCC protein expression increased during erythropoiesis in both normal and β thal cells, and was higher in β thal cells than in normal controls. KCC1 mRNA level was increased only in β thal cells at day 14, whereas KCC3 mRNA level was increased at day 14 in both normal and β thal cells. DIOA significantly reduced the number of both normal and β thal cells, parallelled by increases in the percentage of polychromatophilic normoblasts among normal progenitors and of basophilic normoblasts among b thal progenitors. We further investigated the inhibitory effects of DIOA on cell growth by FACS evaluation of cell cycle distributions and by determination ofCycD, p21, Casp3 and Casp8 gene expression. At day 14 DIOA exposure was associated with:

significant reduction in the percentage of β thal cells in S-phase compared to either untreated cells or DIOA-treated normal controls; up-regulation of CycD gene expression in both normal and β thal cells; down-regulation of p21 in β thal cells; up-regulation of casp3 and casp8 in both normal and β thal cells. These data suggest that KCC is involved in the late phase of erythropoiesis mainly in β thal cells, and support a novel role of KCC in erythroid cell growth.
Negative Hematopoietic Scaffold Lnk Upregulates Integrin Outside-In Signaling in Platelets.
Authors: Eto Koji , Takizawa Hitoshi , Nishikii Hidekazu , Seita Jun , Oda Atsushi , Tamura Noriko , Goto Shinya , Takatsu Kiyoshi , Takaki Satoshi , Nakauchi Hiromitsu
Published: April 2020

The adaptor molecule Lnk negatively regulates signaling downstream of cytokine receptors in self-renewal of hematopoietic stem cells, proliferation of B precursors and differentiation/maturation of megakaryocytes. The circulating number of platelets in Lnk-deficient mice is increased by 5-folds to control, presumably owing to its negative effects on TPO-elicited signaling. Lnk expression is maintained in platelets, however, the precise functions in platelets remain unknown. Here we report that Lnk deficiency results in the prolonged bleeding time (2-folds, p<0.01) due to, at least in part, defective the outside-in signaling from α IIbβ 3 integrin. Lnk-null platelets exhibited less activation of PAK kinase, a Rac effector in fibrinogen-adherent platelets. These were concomitant with the defect of lamellipodial formation upon adhesion to fibrinogen in the absence of Lnk, compared to control platelets (p<0.01). Defective Lnk-null platelet functions were also demonstrated by the reduced three-dimensional growth (height) of platelet thrombi on the collagen surface under whole blood flow condition with the adjusted same platelet number at high wall shear rate of 1,500 s-1 (8.1±1.4 μ m in Lnk-deficient vs. 12.1±2.2 μ m in control, p<0.001), while the two-dimensional surface coverage was not impaired. On the other hand, there was no difference between Lnk-null and control platelets in aggregation and α IIbβ 3 integrin activation in response to stimulation with thrombin or collagen. Taken together, implication of Lnk to sites of adhesion may explain the phenotype of Lnk-deficient platelet function. The findings indicate that Lnk is a unique “dual-functioning” scaffolding molecule, which upregulates thrombus formation, while suppressing cytokine-mediated signaling.
Anxiety, Self-Esteem and Social Functioning in Adolescents with Thalassemia Major.
Authors: Sachdeva Anupam , Yadav Satya , Gujral Satinder Kaur , Gupta Ashum , Khanna Varinder Kumar
Published: April 2020

Whereas it has been established that it is important to provide a therapeutic strategy in Thalassemia major that includes not only medical but also psychological and social interventions from childhood to adulthood, the modalities and instruments of intervention and methods of evaluation and verification still have to be defined. In the present study an attempt has been made to obtain compare thalassemic adolescents to that of healthy adolescents in the areas of psychological factors - anxiety, self-esteem and social functioning. In this study we prospectively studies 40 adolescents (age 13–19 years) of which 20 had thalassemia major and 20 were normal healthy adolescents. In order to compare the two groups on anxiety and self-esteem the t-test was used. A semi-structured interview was used to assess psychological factors. In order to find significant difference between two groups on this semi-structured interview, chi square test was used. The measure of anxiety in the present study is that of trait anxiety stable personality measure which is resistant to situational fluctuation. There was no difference found between two groups in the present study on standardized measure of trait anxiety (t-value 0.96). Most theoretical formulations have stressed that thalassemia can have effect on self-image. Specially, body changes and disruption of activities are seen as lowering the patient self worth however, no significant difference was found between two groups in present study on standardized measure of self-esteem. (T-value 0.70) In the social functioning the chi square test found a significant difference in the extent to which the two groups liked the school (p value <0.05) as 35% of thalassemics like the school a lot against only 10% of healthy. In leisure activity thalassemic adolescents were more sedentary type (p value <0.25). In daily functioning no significant difference was noted among two groups. In illness issues the only question which revealed significant difference were that “ are you able to run as much as others”(P value < 0.01) Thalassemic adolescents responded to be less able to run and falling more often ill than the healthy adolescents. In regards to future 50% thalassemics responded to be cured in future whereas 45% of healthy adolescents had responded to having friends as the future hope. In the views on employment prospects significant difference was found (p value <0.01) where 30 % of thalassemics were pessimistic and rest 70% optimistic whereas 100% of healthy adolescents were optimistic. In the field of marriage and parenthood majority of adolescents in each group anticipated marriage and parenthood. No significant difference was noted. In conclusion this study shows that, in general, the self-concepts of thalassemics were normal.
Kinetic of Iron Absorption and Expression of Iron Related Genes in Beta-Thalassemia.
Authors: Breda Laura , Gardenghi Sara , Guy Ella , Amariglio Ninette , Adamsky Konstantin , Weizer-Stern Orly , Rachmilewitz Eliezer , Rechavi Gideon , Giardina Patricia J. , Grady Robert W. , Rivella Stefano
Published: April 2020

We generated the first transplantable adult mouse models of beta-thalassemia intermedia and major by infusing mouse hematopoietic-fetal-liver cells (HFLC) heterozygous or homozygous for a deletion of the beta-globin gene (respectively with th3/+ and th3/th3 cells) into lethally irradiated congenic C57BL/6 mice. Six to 8 weeks post transplantation, mice transplanted with th3/+ HFLCs show 7 to 9 g/dL of hemoglobin levels, splenomegaly, abnormal red cells and increased iron overload. Mice transplanted with th3/th3 HFLCs, unless blood transfused, die 8 to 10 weeks after engraftment showing profound anemia, massive splenomegaly and very rapid and dramatic iron overload. For this reason, we began a systematic study to compare iron content and the expression level of iron related genes in normal and thalassemic mice of varying ages and sex in different organs (liver, duodenum, spleen, kidney and heart). In liver, we observed that iron content increases proportionally with the level of anemia, age and if the blood transfusion is included. We are currently analyzing the other organs. The expression of hepcidin, ferroportin, Hfe, ferritin, transferrin, transferrin-receptor 1 and 2, ceruloplasmin, divalent metal transporter 1 and hemojuvelin are being tested also in all these organs. In particular, we observed that hepcidin is dramatically downregulated in liver of beta-thalassemic animals. Our hypothesis is that low expression of this gene leads to high iron content in these animals. We intend to demonstrate that administration or increasing hepcidin levels of this peptide can prevent iron absorption in beta-thalassemia. We developed two alternative strategies to test our hypothesis. In the first one, we synthesized the active form of the mouse hepcidin peptide that will be administered intraperitoneally to mice affected by beta-thalassemia. In the second, lentiviral vectors have been generated in order to constitutively secrete hepcidin in the bloodstream of animals affected by beta-thalassemia. These vectors were introduced into hematopoietic stem cells derived from mouse embryos of normal and mice affected by beta-thalassemia and engrafted in myeolablated normal mice. The engrafted mice express hepcidin 6 weeks post transplantation by RT PCR. These animals, along with the animals in which hepcidin will be administrated intraperitoneally, will be analyzed at the endpoint of the experiment (> 4 months) for their hematological values and iron content to see if the use of hepcidin can be used to prevent excessive iron absorption in beta-thalassemia.
Elevated Levels of Homocysteine Compromise Blood-Brain Barrier Integrity in Mice.
Authors: Chauhan Anil K. , Kamath Atul F. , Kisucka Janka , Dole Vandana S. , Loscalzo Joseph , Handy Diane E. , Wagner Denisa D.
Published: April 2020

Elevated levels of plasma homocysteine (Hcy) correlate with increased risks of cardiovascular and Alzheimer’s diseases. We studied the effect of elevated Hcy on the blood-brain barrier (BBB) to explore the possibility of a vascular link between the two diseases. On a methionine-enriched diet, cystathionine beta-synthase (CBS) heterozygous mice develop hyperhomocysteinemia (HHcy). To evaluate the extent of HHcy induced by an 8-week methionine-enriched diet (Diet), we determined plasma Hcy concentration. Concentrations were 23.5 ± 5 μM for wild type (WT) and 98.4 ± 22 μM for CBS+/− on Diet and 4.1 ± 0.20 μM for WT on normal chow. Inflammation can lead to BBB breakdown, therefore we first determined whether we could detect systemic endothelial activation/increased leukocyte adherence in the CBS+/− mice as an indicator of inflammation. We observed mesenteric venules (200–300 μm) in WT and CBS +/− mice fed Diet and compared them with WT on chow. Leukocyte rolling velocity was slower in CBS+/− than in WT mice indicating an increase in the density of adhesion molecules on the endothelium. In CBS+/− mice, 60% of leukocytes rolled at a velocity of < 10 μm/s as compared with only 8% in WT mice on Diet (P<0.002) and 3% WT on chow. The slower leukocyte velocity was due at least in part to higher expression of P-selectin on the endothelium. Infusion of fluorescent beads coated with antibody to P-selectin showed a several fold increase in their binding to venules of CBS +/− mice with HHcy as compared with WT on Diet (P<0.05). We also observed increased leukocyte adherence in CBS +/− mice (P<0.05). We then compared BBB function in mice after the 8-week Diet. We injected Evans blue (EB) dye intraperitoneally. EB binds to albumin and does not readily cross the BBB. The CBS+/− mice exhibited 25% greater EB in cortex compared with WT. EB leakage in WT mice was 3.19 ± 0.09, while CBS+/− leakage was 3.99 ± 0.29 (P < 0.03). WT mice on Diet showed slightly higher plasma Hcy levels than on normal chow; but this increase was not sufficient to break the BBB, as the BBB leakage in the WT mice on Diet was similar to that of WT mice on normal chow. Our study suggests an important toxic effect of elevated Hcy on brain microvessels and implicates Hcy in the disruption of the BBB.