bluuebunny/crossref_metadata_2025 huggingface dataset:
Mu Opioid Receptor (OPRM-1) Polymorphisms among Patients with Sickle Cell Disease.
Authors: Mehta Sharmila , Campbell Nicholas , Bailey Lakiea , Kutlar Ferdane , Elam Dedrey , Clair Betsy , Kutlar Abdullah
Published: April 2020

Opioid analgesics form an important component of the management of acute and chronic pain in patients with sickle cell disease (SCD). Chronic opioid use, sometimes associated with dependence and addiction in a subset of patients, may pose difficult management problems. This adds to the sometime antagonistic relationship that can develop between providers and patients with SCD. Pain management can become a sociological and psychological issue in the management of SCD. The Mu opioid receptor (OPRM-1) is the primary site of action of endogenous opioid peptides (enkephalins and endorphins) and of opioid analgesics, such as morphine, methadone, fentanyl, heroin, and related compounds. Rapid activation of the mu receptor results in a euphoric effect, conferring the reinforcing or rewarding effects of opioids and thus contributing to the development of addiction. It has been known that there is variation between individuals in sensitivity to opioids suggesting potential variation in the receptor protein and the gene. Some recent data have shown that polymorphisms in the OPRM-1 gene affect pain threshold and tolerance as well as opioid requirements for optimal pain control. In an effort to unravel the complex issues surrounding pain frequency, pain tolerance, opioid usage, and opioid addiction in this patient population, we conducted a study of our adult sickle cell patients for the frequency of the two common cSNPs and another SNP in the IVSII (G691C) of the OPRM-1 gene. DNA samples from randomly selected patients were used in this study. The OPRM-1 gene was PCR amplified and subjected to cycle sequencing on an ABI Prism automated sequencer. The results showed that of the 97 adult SCD patients screened for the C17T polymorphism (GenBank accession #AY292291 and 292290), 67 (69.7%) had the wild type (CC), 28 (29.1%) were heterozygous (CT), and 11 (11.4%) were homozygous (TT) for the SNP. This represents an allele frequency for the T of 0.26. Of the 39 sequenced samples for the A1189G, all (100%) showed the wild type (AA). These results closely resemble those reported by Bond et al (PNAS, 95;9608,1998) for the frequency of the two cSNPs in the African-American population (allele frequency of 0.21 for the C17T and 0.016 for the A118G). Of the 16 samples screened for IVS II G691C polymorphism (GenBank accession #AY299483), 6 (37.5%) had the wild type (GG), 9 (56.3%) were heterozygous (GC), and 1 (6.2%) was homozygous (CC). Our results among SCD patients show a high frequency of C17T polymorphism in the OPRM-1 gene. Detailed studies in higher numbers of SCD patients and clinical correlations with pain frequency and threshold, opioid usage, opioid abuse and drug seeking behavior will be undertaken. It is expected that these studies will clarify the role of OPRM-1 polymorphisms as a genetic modifier associated with pain frequency, with tolerance as well as opioid use and abuse.
Haptoglobin Genotypes among Pediatric and Adult Sickle Cell Patients: An Emerging Genetic Modifier?.
Authors: Hughes Heather Y. , Kutlar Ferdane , Clair Betsy , Elam Dedrey , McKie Kathleen M. , Kutlar Abdullah
Published: April 2020

The role of hemolysis and its end product, cell free hemoglobin, as a Nitric Oxide (NO) scavenger in the pathogenesis of some complications of sickle cell disease (SCD) such as pulmonary hypertension has recently been recognized. Hemoglobin (Hb) released from red blood cells as a result of hemolysis complexes with haptoglobin (Hp). Hb-Hp complexes are removed from the circulation by tissue macrophages in the liver and spleen through CD163, the newly identified hemoglobin scavenger receptor expressed on the monocytes and macrophages. Hp is a polymorphic protein with two well-known alleles, Hp-1 and Hp-2 with worldwide distribution. Although the worldwide frequencies of these two allelles are approximately equal, there is significant ethnic and geographic variation in the distribution of Hp-1 and Hp-2 among different populations. Hp locus is on chromosome 16q22. each Hp monomer consists of one α-chain and one β-chain. An intragenic duplication event during evolution resulted in an elongated α-chain thus generating the Hp-2 allele. Thus individuals homozygous for the long α-2 chain express large multimeric molecules (Hp 2-2). Over the past 5 years, Hp-2 allele has emerged as a major susceptibility gene for development of vascular complications such as coronary artery restenosis and risk of cardiovascular disease, particularly in diabetic patients. It has been reported that the Hb-Hp-2 complexes have a 10-fold greater affinity for the CD163 receptor compared to Hb-Hp-1, and the interaction of Hb-Hp-2 complexes with CD163 on macrophages generates a more powerful inflammatory response with the release of pro-inflammatory cytokines such as IL-1β, IL-6, and GM-CSF. These observations led us to examine the role of Hp polymorphisms in sickle cell disease. We performed genotyping of the Hp-alleles with PCR in 21 pediatric patients (age 4 mo-10 years) and 41 adults patients (age 16–60). The results of this genotyping are shown in the table below. The Hp-1 allele frequency was 0.24 among pediatric patients and 0.52 among adults; while that of Hp-2 was 0.76 in the pediatric age group and 0.48 in adults. This difference was significant (p=0.0023). The significantly higher allele frequency of Hp-2 in pediatric patients compared to adults may indicate a selection against this genotype and suggests that Hp-1 allele may be associated with a survival advantage. This is in keeping with the deleterious effects of Hp-2 allele reported in the literature and suggests yet another mechanism whereby hemolysis may lead to adverse outcomes associated with Hp polymorphisms, in addition to the NO scavenging effects of cell free hemoglobin. Detailed studies are currently being undertaken to elucidate the mechanism whereby Hp polymorphisms may impact on the pathophysiology of SCD.

PERCENTAGE OF HAPTOGLOBIN GENOTYPES BETWEEN ADULT AND PEDIATRIC SICKLE CELL PATIENTS ADULT PEDS sHp 1–1 41.4% 9.5% Hp 1–2 22.5% 28.6% Hp 2–2 36.6% 62.0%
K-Cl Cotransport and MCHC in HbE Transgenic Mice and Human HbE.
Authors: Romero Jose R. , Rivera Alicia , Etzion Zipora , Suzuka Sandra M. , Vichinsky Elliott , Nagel Ronald L. , Fabry Mary E.
Published: April 2020

In HbE, HbC, and HbOArab a glutamic acid is mutated to a lysine resulting in a cumulative increase in positive charge of +4 units per tetramer when compared to HbA. These mutations occur at β−26, β−6, and β−121, respectively. We have reported an increase in both K-Cl cotransport and in red cell density (that is directly proportional to MCHC) for HbC and HbOArab. In the case of HbC, we demonstrated that transgenic mice expressing HbC also have these properties. We also previously reported that human homozyous HbE red blood cells (hRBC) have normal red cell density; that is, they have the same MCHC as HbA hRBC. We now report that RBC from mice (mRBC) expressing HbE have elevated K-Cl cotransport and normal MCHC and that these properties have been found in HbE hRBC as well. In mice expressing exclusively human α and βE, K-Cl cotransport in mRBC with HbE versus C57Bl was 10±0.9 versus 2.0±0.9 mmol/L cells x hr (FU), respectively. Mice expressing exclusively HbE have mRBC that are microcytic vs C57 (MCV=32.9±1.5 vs 47.5±2.0 fl, N=5, p<0.0003), have low MCH vs C57 (MCH=9.8±0.1 vs 14.7±0.8 pg, N=5, p<0.0004), but have normal MCHC vs C57 (CHCM=29.7±1.1 vs 30.9±1.9 g/dl, p=NS). In a human patient with HbE β0 thalassemia, we found elevated K-Cl cotransport (10±0.9 versus 2.0±0.9 FU) and red cell indices vs HbA that were analogous to those seen in HbE transgenic mice: (MCV=63.8 vs 88.1±2.3 fl), (MCH=20.9 vs 30.9±1.0 pg), but a normal MCHC vs HbA (CHCM=33.0 vs 34.9±1.2 g/dl). Because this patient suffers from severe thalassemia and hence has an elevated reticulocyte count (17%), the elevation in K-Cl cotransport may be due, in part or totally, to the presence of reticulocytes. However, reticulocytes have a lower MCHC than mature RBC, and blood with a high percent of reticulocytes generally has a low average MCHC; however, we observed a normal MCHC for this patient and others in whom K-Cl was not measured. We attribute the normal MCHC observed in both human patients with HbE and in our transgenic mouse model to the effect of K-Cl cotransport on a red cell with a thalassemic background. In conclusion, the low MCHC anticipated from the well-known thalassemic nature of the HbE mutation is not realized due to enhanced K-Cl cotransport. This demonstrates that the mouse model reproduces the hematologic features of the human disease and will provide a suitable model for gene therapy and other therapeutic approaches for HbE hemoglobinopathies.
Ten Year-Old Male with Hemoglobin Lufkin/Beta-Zero Thalassemia Compound Heterozygote Who Exhibited Beta-Thalassemia Major Characteristics.
Authors: Blutreich Ahna M. , Shu Peihong , Bragdon Jeremy S. , Kurtin Paul J. , Hoyer James D. , Karayalcin Gungor
Published: April 2020

Hemoglobin Lufkin is a rare and mildly unstable hemoglobin variant with increased oxygen affinity. Since 1977, two cases of hemoglobin Lufkin trait and one hemoglobin Lufkin/hemoglobin S have been described. This report is the first case of hemoglobin Lufkin/beta-zero thalassemia in a 10 year-old Caucasian male of Irish/Italian/German background. The patient presented with jaundice, splenomegaly and thalassemia major facies. On blood smear examination, RBC morphology showed hypochromia, microcytosis, many target cells, some spherocytes and basophilic stippling of RBC’s. On both alkaline hemoglobin (Hb) electrophoresis and isoelectric focusing (IEF) there was an absence of Hb A with a predominant band slightly anodal to the Hb A position. Hb A2 (4.8%)and Hb F (5.3%) were elevated as measured by high performance liquid chromatography. The heat unstable Hb test was abnormal. DNA sequencing of the beta globin gene confirmed a GCC to GAC mutation at codon 29 (gly to asp) consistent with Hb Lufkin. DNA sequence analysis also revealed a beta-zero thalassemia mutation, IVS-1-1, (G to A). The mother’s sample also showed the same beta-thalassemia mutation. Neither hemoglobin Lufkin nor beta-thalassemia were identified in the father; further studies are being done.
Effect of Zoledronic Acid on Bone Mineral Density in Thalassemia-Induced Osteoporosis: Results of a Phase II Clinical Trial.
Authors: Taher Ali , Azar Sami , Shamseddeen Wael , Habr Dany , Inati Adlette , Koussa Suzane , Mahfouz Rami , Otrock Zaher
Published: April 2020

Background: Osteoporosis is an important cause of morbidity in beta-thalassemia patients. Bisphosphonates are potent inhibitors of osteoclast activity and have been recently used for the treatment of osteoporosis in beta-thalassemia. The aim of this study is to assess the efficacy and safety of zoledronic acid in Lebanese thalassemics with osteoporosis.

Methods: Eighteen thalassemic patients (13 thalassemia major and 5 intermedia) with osteoporosis defined as Z-score <−2.5 were given zoledronic acid 4 mg i.v. every 3 months over a period of 12 months (Total of 4 doses administered). The efficacy of treatment was assessed by measuring Bone Mineral Density (BMD) at the lumbar spine, femoral neck and hip at baseline, 6 and 12 months. Other efficacy measurements included markers of bone formation and resorption (bone alkaline phosphatase (BAP), osteocalcin (OC), and urinary deoxypyridinoline (Dpd)), assessment of pain score, analgesic score, and performance score measured at baseline and at 3-month intervals. Safety assessment included regular physical exams, standard hematology and clinical chemistry tests, and adverse events recording. All patients were on Ca/Vitamin D supplementation prior to and during the study. Ten thalassemic osteoporotic patients were followed up only with serial BMDs as controls.

Results: The characteristics of all patients are shown in Table 1. Both groups had no significant difference with respect to age, gender and baseline BMD. Patients taking zoledronic acid had a significant increase in their spine, femoral neck, trochanter and total hip BMD measurements over the 12-month period (all p-values<0.05). Patients in the control group, on the other hand, did not have any significant change except in the spine BMD. The BMD values are presented in Table 2. There was a significant change in the levels of the OC and BAP over the 12-month follow-up in the treatment group (p=0.00 for both). Dpd levels did not significantly change overall (p=0.06) although they decreased throughout the study. Reported adverse events included joint pain in 9 patients (50%) after the 1st dose and in 2 (11.1%) after the 2nd dose and responding very well to oral analgesics. Two patients (11.1%) had perioral numbness and 3 (16.7%) had low grade fever after the 1st dose. No treatment-related adverse events were reported after the 3rd and 4th doses. No patients withdrew from the study.

Conclusions: Treatment of Lebanese thalassemic osteoporotic patients with zoledronic acid 4 mg every 3 months is effective in increasing BMD at the lumbar spine and hip and is well-tolerated. Well-controlled studies with longer follow-up are needed to determine the fracture-reduction benefits and the most optimal zoledronic acid treatment dose and frequency in this patient population.

Table 1: Main characteristics of the studied groups Table 2: BMD values of the treatment and control groups
Thalassemia Major, Intermedia & Sickle-Thal Patients in Lebanon.
Authors: Taher Ali , Shamseddeen Wael , Arnaout Samir , Gharzuddin Walid , Zeineh Nabil , Alam Samir , Isma’eel Hussein
Published: April 2020

Introduction: Cardiac dysfunction in thalassemia remains of primary concern. The prevalence of these abnormalities varies in different phenotypic/genotypic groups of thalassemia.

Objective: To describe the difference in cardiac function among Thalassemia Major ™, Thalassemia Intermedia (TI), and Sickle-Thalassemia (ST) patients.

Methods: 66 TM, 41 TI and 17 ST patients were randomly selected from a population of 450 TM,100 TI and 26 ST patients respectively. The patients were interviewed and their charts reviewed and all had an echocardiography. Pulmonary Hypertension (PHT) was defined as Pulmonary Artery Systolic Pressure> 30mmHg.

Results: 58.8%, 50% and 41.5% of ST,TM and TI patients respectively were males. Mean age was 24.4 ± 11.6, 19.4 ± 7.6, 19.5 ± 10.9 year old for TI, TM and ST. Tricuspid Regurgitant jet was detected in 47%, 30% and 82% of TI, TM & ST patients respectively. PHT was found in 83%, 18% and 10% of TI, ST, TM patients. Deceleration Time (DT) was significantly longer in TI than in TM and ST, with no significant difference between TM and ST. End diastolic dimension (EDD) was significantly larger in TI than TM and ST (for more details about cardiac parameters refer to tables 2a and 2b). Thrombotic events occurred in 6% of TI patients and in 0 TM and ST patients.

Discussion: PHT is primarily prevalent in TI patients which further supports the notion that a different pathophysiological process leads to this than in TM and ST patients. Moreover these results stress the need for better method of assessment of PHT, because of the limited number of patients in whom a TR jet could be detected. A prolonged DT is one of the earliest sign of diastolic dysfunction in thalassemics. TI patients have revealed themselves to have more impaired diastology by both prolonged DT and Isovolumic relaxation time (IVRT). Splenectomy was performed more in Lebanese TI than that reported in the literature (87% vs 65%), because most of our TI patients were managed as TM. This was before the currently recognized association of splenectomy and PHT and thrombosis. In addition chamber sizes (EDD) was significantly more in TI than both TM and ST patients. This can be attributed to the more profound chronic dilutional volume overload secondary to uncorrected anemia. Finally as is evident TI patients are developing more complications than both TM and ST. Transfusion/ chelation therapy to avoid PHT, diastolic dysfunction and thrombosis in TI patients has been proposed as a solution. If the pathophysiology of these complications is iron-mediated then prophylactic transfusion/ chelation therapy would be more advisable than waiting for them to develop. Especially in view of the morbidity associated with thrombosis, and limitation of exercise tolerance secondary to PHT.

Table 1: Clinical characteristics of patients

Table 2a: Significant differences between TI and ST patients Table 2b: Significant differences between TM and ST patients
Rapid Identification of the β-Thalassemia Mutations by Applying CEL I Nuclease Mutation Detection System and Capillary Electrophoresis.
Authors: Hung Chia-Cheng , Lin Win-Li , Jou Shiann-Tarng , Su Yi-Ning
Published: April 2020

Background: Beta-thalassemia is a common autosomal recessive hereditary disease in the Meditertanean, Asia and Africa area. It is a single gene inheritable disease resulting from one or more of a total of more than 200 different mutations in the beta-globin gene (HBB). For the clinical practice, the detection of beta-globin mutations were mainly depends on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequence technique. A more sensitive, efficient and reliable mutation-screening method is warrant and essential in order to establish appropriate prevention programs for at-risk populations based upon a molecular diagnosis.

Methods: To further enhance the detection efficiency and accuracy, the purpose of this study is to develop a novel genotyping method for common mutations in beta-globin gene. We have developed a rapid and highly-specific mutation screening test for the diagnosis of beta-thalassemia by CEL I nuclease mutation analysis based on the denaturing high performance liquid chromatography (DHPLC) system and ABI PRISM 310 Genetic Analyzer. To illustrate the efficacy of this approach, the exons of the beta-globin gene were amplified by PCR using primers 5′-labeled with fluorescent dyes of two colors. This study will demonstrate the usefulness of CEL I Nuclease as a method to genotype mutations in beta-globin gene for applying in daily clinical practice.

Results: Assays conditions were established based on the analysis of 50 DNA samples from heterozygotes or compound heterozygotes for the mutations in beta-globin gene. Homozygous wild-type DNA samples produced electropherograms containing only single peak, whereas samples heterozygous for a specific mutation displayed two peaks for that mutation site. In the double-blind validation analysis, all 50 DNA samples were showed 100% assay specificity.

Conclusions: Compared to classic approaches of mutation screening, when coupling with the previous established heteroduplex and primer-extension analysis, this method allows a rapid, highly sensitive, cost effective and semi-automated mutational screening of a large number of samples. Therefore, mutational analysis by CEL I Nuclease Mutation Detection System will be helpful as the first-line clinical screening and diagnosis tool of beta-thalassemia.
Antineutrophil Antibodies Lead to Mistaken Identity in Severe Congenital Neutropenia.
Authors: Boxer Laurence A. , Bolyard Audrey Anna , Schwinzer Beate , Glaser Darryl , Dougan Kenneth , Bonilla Mary Ann , Alter Blanche P. , Cham Bonnie , Kinsey Sally E. , Zeidler Connie , Welte Karl , Dale David C.
Published: April 2020

Autoimmune Neutropenia of Infancy (ANI) is generally regarded as a benign condition diagnosed by finding neutropenia and a positive test for antineutrophil antibodies. To determine whether the presence of antineutrophil antibodies in infancy always predicts a benign disease, data were reviewed for young children with positive antineutrophil tests who are enrolled in the Severe Chronic Neutropenia International Registry (SCNIR). A set of identical twins presented with severe neutropenia at age 5 months. Twin A1 had a preceding history of aphthous ulcers, cellulitis, recurrent otitis media, pneumonitis, an episode of bacteremia and a positive test for antineutrophil antibodies. Twin A2 had a preceding history of cellulitis, otitis media, and pneumonitis. The median absolute neutrophil count (ANC) of twin A1 and twin A2 was 0.280 x 109/L and 0.116 x 109/L, respectfully and bone marrow examination for twin A1 showed maturation arrest at the myelocyte-metamyelocyte level. With the diagnosis of ANI in one twin, both twins were placed on granulocyte-colony stimulating factor (G-CSF), because of frequent fever and recurrent infections. Fifty-one months after the diagnosis of immune neutropenia, twin A1 developed acute myelogenous leukemia. Further studies then revealed that both twins had a mutation in the neutrophil elastase gene (ELA2) at exon 5 resulting in a nucleotide substitution from TGC to TGA, which indicated a diagnosis of severe congenital neutropenia. Another set of fraternal twins were identified to have ANI at six months because of recurrent abscesses, mouth ulcers, otitis media and positive tests for antineutrophil antibodies. The ANC was 0.067 x 109/L in twin B1 and 0.166 x 109/L in twin B2. At age 31 months twin B1 developed a severe Clostridial infection, which led to the loss of his right leg and part of the anterior abdominal wall. Subsequently both twins were placed on G-CSF. Prior to G-CSF treatment, bone marrow studies revealed an arrest at the promyelocyte stage and an ELA2 mutation in exon 4 leading to a TCG to TTG substitution in both twins thereby indicating a diagnosis of severe congenital neutropenia. Upon further review of the SCNIR, eight other congenital patients were identified to have antineutrophil antibodies and bone marrow patterns consistent with severe congenital neutropenia. In conclusion, it is important to be aware that positive antineutrophil antibodies can occur associated with severe congenital neutropenia. Infants with severe neutropenia and repeated or severe bacterial infections should have a bone marrow examination and ELA2 evaluation to establish correctly the underlying pathogenesis of the neutropenia.
Left Ventricular Cardiac Function during the Course of a One Year Multicenter Trial of the Safety and Efficacy with ICL670 5–40mg/kg/day and Deferoxamine 20–60 mg/kg/day in Beta-Thalassemia Patients with Transfusional Hemosideosis.
Authors: Kleinert Dorothy A. , Jones Erica , Sison Cristina P. , Marks Peter , Giardina Patricia J.
Published: April 2020

Historically regularly transfused patients (pts) with Thalassemia Major (TM) have received prolonged infusions of subcutaneous deferroxamine (sc DFO) to prevent the lethal cardiac complications of transfusional hemosiderosis. Cardiac function is monitored with annual electrocardiograms (EKG) and 2 Dimensonial Doppler Echocardiogram (Echo). ICL670 (Exjade) is a newly identified oral iron chelator that has completed a one-year worldwide multicenter phase III clinical study of its safety and efficacy in comparison to sc DFO in TM. 17 TM pts in our institution participated in the Novartis ICL670-107 and 108 trials. They ranged in age from 2–36 years (yrs), the youngest being naive to chelation, five pts were previously noncompliant with the use of sc DFO, one of which developed acute heart failure more than 15 yrs prior to the study which resolved with vigorous DFO therapy and in whom all cardiac medications were discontinued 10 yrs prior to study entry. 10 TM pts ranging in age from 2–36 yrs received ICL670 at 10–30mg/kg/day and 7 TM pts ranging in age from 7–33 yrs received sc DFO at 40–60 mg/kg over 8 hour infusions 5 days per week. Each pt underwent a 2 dimensional Doppler Echo as part of their routine care prior to entry and after one year of study as well as regular EKGs before and during the study. Pre and post Liver Iron Concentration (LIC) was performed by biopsy in 15 adolescent/adult patients and 2 children had SQUID LIC. Laboratory analysis of blood counts, serum chemistries and ferritin levels were performed by a central laboratory. No clinically significant EKG changes were observed during the course of the study. Normal Ejection Fractions (EF) = 0.55 ± 0.75 ml/m2 and Shortening Fractions (SF) = >26%. One of the ten pts on ICL670 developed an abnormal EF = 47 and a SF = 24% compared to 0 of 7 pts on sc DFO. None of the pts experienced clinical cardiac decomensation. In conclusion regularly transfused TM pts receiving oral ICL670 for one year had no statistically significant mean changes or relative mean changes in EF or SF on 2 dimensional Dopler Echo in comparison to those pts receiving sc DFO.

Echo Results Mean Pre EF Mean Post EF Mean Change Mean Relative Change ICL670 60.3 ± 5.5 58.6 ± 5.4 −1.7 ± 6.9 −2% ± 12% DFO 63.7 ± 4.6 67.1 ± 4.2 3.4 ± 6.3 6% ± 11% Mean Pre SF Mean Post SF Mean Change Mean Relative Change ICL670 32.4 ± 3.4 31.2 ± 3.3 −1.2 ± 4.3 −3% ± 13% DFO 35.0 ± 3.1 37.4 ± 3.3 2.4 ± 4.6 8% ± 14%
Transforming Growth Factor-β1 Inhibits the Maturation of Dendritic Cells Via Toll-Like Receptor 4 Signaling Pathway.
Authors: Mou Haibo , Lin Maofang , Huang He
Published: April 2020

We have demonstrated that transforming growth factor-β1(TGF-β1) inhibits the maturation of mouse bone marrow derived dendritic cells (DCs). TGF-β1 treated DCs (TGFβ-DCs) are resistant to maturation stimulus -lipopolysaccharide (LPS) and might have some correlation with the down-modulation of Toll-like receptor 4 (TLR4) expression. It was known TLR4 binds LPS from Gram-negative bacteria, triggering signaling pathways that lead to the activation of NF-κB, ERK1/2 and p38 MAPK and ensuing gene expression of proinflammatory factors. In the current study, we further estimated the activities of NF-κB, ERK1/2 and p38 proteins involved in TLR4 signaling pathway. Using EMSA method, we found the NF-κB DNA binding activity in immature DCs (imDCs) was significantly increased in response to LPS, but addition of TGF-β1 to DCs inhibited NF-κB binding. Moreover, TGF-β1 was effective in suppressing LPS-induced activation of ERK1/2 and p38 kinase, the level of phosphorylation of ERK1/2 and p38 kinase were lower than imDCs measured by Western Blot. After treatment of imDCs and TGFβ-DCs with LPS for 24 h, the production of IL-12p70 of TGFβ-DCs was significantly less than that of imDCs(115.4±15.2 pg/ml vs 517.0±29.7 pg/ml, P<0.01), but the level of Th2 cytokine-IL-10 was elevated(132.1±17.5 pg/ml vs 75.1±16.6 pg/ml, P<0.05), indicating that exposure to TGF-β1 impaired the capability of DCs to produce high amounts of bioactive IL-12p70. According to the semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), the expressions of chemokines MIP-1α mRNA on TGFβ-DCs after LPS stimulation were lower than imDCs at each time point. The lower expressions of MCP-1 and IP-10 on TGFβ-DCs at irregular pattern after LPS treatment, whereas the expressions RANTES were no different. Hence, the results suggested TGF-β1 maybe directly inhibit TLR4 expression on DCs, and then interfere with the activity of downstream key proteins, such as NF-κB, ERK1/2 and p38. Ultimately, TGF-β1 treated DCs were resistance to LPS, down-regulated the expression of costimulatory molecular on DCs and decreased the secretion of inflammatory cytokines.
Identification of Chitotriosidase Isoforms in Plasma of Gaucher Disease Patients by Two Dimensional Gel Electrophoresis.
Authors: Quintana Lucia , Monasterio Alberto , Escuredo Kepa , Amo Jokin del , Alfonso Pilar , Elortza Félix , Santacruz Simon , Simon Laureano , Martinez Antonio , Pocovi Miguel , Castrillo Jose L. , Giraldo Pilar
Published: April 2020

Introduction: Chitotriosidase protein (CT) is the most important biochemical marker described for Gaucher disease (GD). CT activity is increased several hundred-fold in plasma of GD patients and shows a strong positive correlation with the severity of the disease. However, a recessively inherited enzyme deficiency, with an incidence of about 6% in the Caucasian population, means that not all patients with GD can be monitored by measuring CT activity.

Materials and Methods: This study was performed with plasma samples from 22 type 1 GD patients and 23 healthy subjects. All patients are from Spanish GD Registry. Applying two dimensional gel electrophoresis (2-DE) we compared the plasma proteome profile of GD patients with that of healthy subjects. Differential proteins were excised and prepared for MALDI-TOF analysis. Results were verify with 2-DE western blot.

Results: In this study we have described five CT isoforms in a 2-DE gel from GD plasma samples. We show and compare the 2-DE images of each CT spot in plasma protein samples from healthy controls, GD patients with wild-type CT protein, and GD patients either homozygous or heterozygous for the 24 bp duplication. We propose a connection between the quantitative image analysis of each CT spot and the enzymatic activity of CT in each patient. Lastly, our results verifies that post-translational glycosylation explains at least some of differences between CT isoforms.

Conclusion: The possibilities of proteomic technology in the investigation of biological biomarkers of GD and the nature of the CT protein are demonstrated, as well as in detecting new biochemical markers useful for the diagnosis of the disease and for monitoring patient response to therapy.
Characteristics of Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis (HLH) in European Children.
Authors: Wagner Hans-Joachim S. , Beutel Katrin , Schneider Marion E. , Janka Gritta E.
Published: April 2020

Hemophagocytic lymphohistiocytosis (HLH) represents a severe sepsis-like disease with massive hypercytokinemia and with proliferating and organ-infiltrating phagocytes leading to cytopenia of different hematopoietic lineages. Besides primary familial cases of HLH, infections can cause secondary forms of HLH, among which Epstein-Barr virus (EBV) infections are the most frequent trigger. Between 2003 and 2005, 10 cases of EBV-associated HLH were analysed at a local subcenter of the international HLH study group. Serial blood samples from these 4 boys and 6 girls (age range: 2–14 years, median of age: 4 years) were examined for EBV load in peripheral blood mononuclear cells (PBMC) and plasma by real-time PCR analysis. EBV loads in both PBMC and plasma were significantly elevated in all HLH patients as compared with healthy controls and also with immunocompetent patients suffering from primary EBV infections. In one patient with excessively elevated EBV load in peripheral blood, analysis of EBV-DNA in separated T and B cells by real-time PCR showed an EBV infection of both cell types. During the course of disease, EBV load in HLH patients reflected the activity of disease. Regarding the clinical course and biochemical markers of disease such as LDH, ferritin or soluble IL2-receptor levels in plasma, patients with EBV-associated HLH did not differ from other patients with HLH. Conclusion: Measurement of EBV-load by real-time PCR facilitates detection of EBV-associated cases of HLH and monitoring of affected patients during the course of disease. EBV load analysis should become part of the initial routine examinations of newly diagnosed HLH patients in order to identify the frequency of EBV-associated forms of HLH. Without molecular diagnosis by means of EBV load measurement in peripheral blood, EBV-associated cases of HLH may be underdiagnosed in non-Asian populations.
Low Molecular Weight and Unfractionated Heparins Induce a down Regulation of Inflammation: Decreased Levels of Pro-Inflammatory Cytokines and Nuclear Factor-kB in LPS-Stimulated Human Monocytes.
Authors: Hochart Helene M. , Jenkins Vincent P. , White Barry D.
Published: April 2020

Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are highly sulphated proteoglycans widely applied as anticoagulant drugs. They represent pivotal agents for the prevention and treatment of thromboembolic disorders, pulmonary embolism, disseminated intravascular coagulation and unstable angina. Several different studies have suggested that aside from their anticoagulant capacity, heparins possess anti-inflammatory properties, however reports have been conflicting. UFH and sulfated heparin derivatives have been shown to inhibit pro-inflammatory cytokine gene expression in lipopolysaccharide (LPS)-stimulated human mononuclear cells and to inhibit the nuclear factor-kB (NF-κB) activation in a tumor necrosis factor alpha (TNF-α) stimulated human endothelial cell line. However, other studies indicate that heparin in fact amplifies the inflammatory cytokine release in monocytes. Nevertheless, heparin appeared in several clinical trials to have the potential to treat inflammatory bowel disease, arthritis, rhinitis, and human asthma, with an anti-inflammatory effect dissociable from its anticoagulant activity. In the present study, we assessed the effect of LPS stimulation on heparin pre-treated and untreated pure human monocytes. Cells were isolated from whole blood and resuspended in RPMI supplemented with 10% platelet free autologous serum, before addition of unfractionated and low molecular weight heparins (1IU=10μg/ml and 0.1IU=1μg/ml per million cells) 15 minutes before LPS stimulation (1ng per million cells). Pro-inflammatory cytokine levels produced were measured by ELISA and NF-κB translocation using the TransAM Chemi Transcription Factor Assay. The production of TNF-α, interleukin (IL)-8, IL-6 and IL-1β was upregulated by LPS, and their levels significantly reduced when the cells were exposed to heparin, up to 3 fold with 0.1IU UFH. LPS-induced NF-κB translocation from the cytoplasm to the nucleus was also significantly reduced in heparin pre-treated cells; even at low heparin doses, as shown by a 1.5-fold reduction induced with 0.1IU UFH. NF-κB activation, a critical phenomenon in host inflammatory response, is implicated in a wide range of inflammatory diseases and therefore represents an ideal and novel molecular target. Here we report that both unfractionated and low molecular weight heparins possess the equal ability to significantly reduce the monocytic inflammatory reaction through inhibition of NF-κB activation. This indicates a potential mechanism responsible, in part, for the protective effect of the drug in inflammatory disorders. In clinical practice, heparin use as an anti-inflammatory agent is restricted by its potential to induce bleeding complications. Further investigations are required to elucidate the precise mechanism of action of the drug at the cellular level, and optimise the development of non-anticoagulant variants.
Involvement of Fps/Fes and Fer Kinases in c-Kit Signalling in Mast Cells and Mast Cell Leukemias.
Authors: Samayawardhena Lionel , Craig Andrew W.B.
Published: April 2020

The c-Kit receptor protein-tyrosine kinase (PTK) is required for mast cell differentiation and function. Signalling by c-Kit normally depends on binding its ligand, Stem Cell Factor (SCF). However, several constitutively activating mutations in c-Kit have been described in myeloid leukemias (including mast cell leukemias (MCLs)) and in solid tumors. The objective of this study is to define the involvement of two non-receptor PTKs called Fps/Fes (hereafter referred to as Fps) and Fer in c-Kit signaling in mast cells and MCLs. Recent studies using transgenic mouse strains bearing kinase-inactivating or null mutations in Fer and Fps have revealed roles in hematopoiesis, and in limiting the innate immune response. Recently, we showed that Fer and Fps are activated downstream of the high affinity IgE receptor, FcεRI, in bone marrow-derived mast cells (BMMCs), and that Fer kinase is required for sustained p38 Mitogen-activated protein kinase (MAPK) activation and chemotaxis upon activation of either FcεRI or c-Kit.

In this study, we focus on the roles of Fer and Fps in c-Kit signaling in BMMCs. SCF-induced c-Kit activation led to rapid association of both kinases with c-Kit, and increased tyrosine phosphorylation of Fer and Fps. Interestingly, kinase-dead Fps protein was also phosphorylated upon c-Kit activation, suggesting the involvement of an upstream kinase. Pretreatment of BMMCs with the Src family kinase inhibitor SU6656, caused reduced phosphorylation of Fer and Fps upon SCF treatment. This suggests an involvement of one or more Src family kinases (SFKs) in phosphorylating Fps and Fer upon c-Kit activation. We next examined the potential involvement of the Fyn SFK, since it is known to interact with a juxtamembrane site in activated c-Kit and has been linked to Fer activation in other cell types. SCF treatment of BMMCs from Fyn knock-out mice (fyn-null) revealed normal activation of c-Kit, Erk MAPK, but reduced phosphorylation of Fer kinase. Furthermore, we have observed reduced phosphorylation of Shp2 phosphatase, and the p38 and Jnk MAPKs in Fyn-null BMMCs compared to control. We are currently attempting to restore Fyn expression by retroviral transduction to attempt to rescue these signalling defects, and also evaluate potential requirements for Fyn in chemotaxis and cytokine production. Using MCL cell lines harbouring constitutively actived c-Kit (RBL-2H3 and HMC-1), we have observed that phosphorylation of Fer and Fps kinases are inhibited by treating cells with either a c-Kit inhibitor (SU11652) or SFK inhibitor (SU6656).

In conclusion, Fer and Fps kinases participate in c-Kit signaling in normal and leukemic mast cells, and Src family kinases likely contribute to their activation. Using mast cells from Fer/Fps-deficient mice, we will identify their downstream targets and determine if they are required for leukemogenesis by an activated c-Kit transgene.
Modulation of DNA Binding Properties of CCAAT/Enhancer Binding Protein Epsilon by Heterodimer Formation and Interactions with NFkappaB Pathway.
Authors: Chumakov Alexey M. , Silla Agness , Williamson Elizabeth A. , Koeffler Phillip H.
Published: April 2020

Specific regulation of gene expression is achieved through interaction of multiple transcriptional activators and repressors with their cognate DNA sites. This activity is subject to several levels of control, including post-translational modification by phosphorylation, acetylation, selective degradation and interaction with co-activators and co-repressors.

Recent studies directed at discovering C/EBP- responsive genes resulted in identification of a diverse and degenerate collection of putative C/EBP binding sites. Identification of binding sites for each individual member of the C/EBP family is complicated by common co-expression of several of these proteins in the same cell and their ability to form hetero-dimeric complexes with each other and structurally-related ATF/CREB proteins. Activity of C/EBP proteins is regulated by phosphorylation mediated through several major kinase pathways, that affect DNA-binding, subcellular localization in the nucleus, and interaction with cell cycle proteins.

Our studies of C/EBP-e phosphorylation identified several sites, including one for p38 MAPK within the transactivation domain of C/EBP-e. As a result of phosphorylation at Threonine-75 (T75), C/EBP-e DNA binding and specific transcriptional activity was significantly enhanced.

In this study, we identified consensus binding sequences for C/EBP-e and for heterodimers of C/EBP-e with its most common dimerization partner ATF4. Furthermore, we find that C/EBP-e- DNA interaction is highly upregulated by interaction with activated NFkappaB pathway protein p65-RelA. This interaction depends on the T75 phosphorylation status of C/EBP-e and can be enhanced by introduction of phosphomimetic Thr to Asp mutation. By using selective removal of p65-RelA and siRNA mediated p65 knock-down, we demonstrate that activated NFkappaB pathway is required for high level expression of genes with C/EBP-e responsive promoter. This novel interaction may play an important role in regulation of a C/EBP-e - dependent genes in myeloid cells, for example at sites of infection or other inflammations.
Feline Immunodeficiency Virus Depletes Naïve (CD45RA+) CD4+ Lymphocytes from the Blood and Lymph Nodes of Domestic Cats during Acute Pediatric Infection.
Authors: Johnson Calvin M. , Mergia Ayalew , Novak Janelle , Gengozian Nazareth
Published: April 2020

Feline immunodeficiency virus (FIV) is an immunosuppressive lentivirus of domestic cats that serves as an animal model for the pathogenesis of CD4+ lymphopenia and thymus dysfunction in HIV infected humans. During most cases of adult and pediatric HIV infection, naïve CD4+ T lymphocytes recognized by the expression of the RA isoform of the leukocyte common antigen (CD45RA) are infected at a lower level than memory CD4+ T− lymphocytes; however, children with rapidly progressive disease due to thymic insufficiency harbor high levels of HIV within the CD45RA+ subpopulation. In FIV infected cats, the fate of naïve CD4 lymphocytes is unknown due to the lack of specific markers. Recently, a mAb (755) was reported to recognize the feline homologue to CD45RA, allowing the enumeration of naïve CD4 and CD8 lymphocytes in cats. The purpose of this study was to characterize the fate of CD4+CD45RA+ blood cells eight weeks after FIV infection. One-day-old kittens (n=6) were infected with virions either from a wild type clone (JSY3) or mutant ORF-A clone at equivalent reverse transcriptase units and compared to historical control data. Eight weeks after inoculation, the percentages of CD4+ and CD8+ cells belonging to the CD45RA+ subpopulation were measured by two-color flow cytometry. Both FIV inocula were associated with a reduction in total CD4+ lymphocytes from a median of 13% in controls to 8% in infected cats (P=0.004), contributing to a reduction in the CD4:CD8 ratio from 2.45 in controls to 0.76 in infected cats (P=0.007). The decline in CD4+ lymphocytes was attributable to a disproportionate loss of CD4+CD45RA+ cells: 69% of CD4+ cells were CD45RA+ in controls, as compared to 7% in FIV infected cats (P=0.004). In contrast, naïve CD8+ lymphocytes did not change significantly with FIV infection (67% of CD8+ cells were CD45RA+ in FIV infected cats as compared to 80% in controls). The distribution of CD45RA+ cells in the lymph nodes of FIV infected cats mirrored those in the blood. Together, these data suggest that acute FIV infection results in a rapid depletion of naïve CD4 lymphocytes throughout the blood and secondary lymphoid tissues, while proportions of naïve CD8 lymphocytes remain unchanged. CD4+CD45RA+ cells may be depleted during pediatric FIV infection through lytic infection or a transition to a memory phenotype lacking CD45RA.
Tolerability of Pegylated Liposomal Doxorubicin 20 mg/m2 Every 2 Weeks in the Management of Kaposi’s Sarcoma and Hematologic Malignancies.
Authors: Henry David H. , Kilcoyne Susan , Latham Jonathan N. , Staddon Arthur P.
Published: April 2020

Pegylated liposomal doxorubicin (PLD, Doxil) was developed to decrease the risk of cumulative-dose cardiotoxicity associated with conventional doxorubicin. However, hand-foot syndrome (HFS) and stomatitis were associated with PLD in early clinical trials and were more common when patients with ovarian cancer received 50 mg/m2 q4w (HFS, 37.4%; stomatitis, 37.4%) than when patients with AIDS-related Kaposi’s sarcoma (KS) received 20 mg/m2 q3w (HFS, 3.4%; stomatitis, 6.8%). PLD is now commonly used with a dose intensity of 10 mg/m2 per week (20 mg/m2 q2w, 30 mg/m2 q3w, or 40 mg/m2 q4w). Recent reports with this reduced dose intensity have shown a marked decrease in the rate and grade of HFS and stomatitis. This retrospective chart review was performed to examine the tolerability and effectiveness of PLD 20 mg/m2 q2w in patients with KS and hematologic malignancies. Patient charts from a community oncology clinic from January 2000 through December 2004 were reviewed. Data abstracted included patient demographics, PLD dosing, treatment response, and tolerability for all PLD recipients. Data were censored for patients with solid tumors or an initial PLD dose other than 20 mg/m2 q2w. Of the 157 patients receiving PLD, 65 received 1206 cycles (range, 1–116; median, 17) of PLD at an initial dose of 20 mg/m2 every 2 weeks as a component of care for KS (n=55), multiple myeloma (n=4), non-Hodgkin’s lymphoma (NHL, n=4), Hodgkin’s disease (n=1), or chronic lymphocytic leukemia (n=1). Most patients with KS also received highly active antiretroviral therapy (HAART). No signs (eg, decreased ejection fraction) or treatment-related symptoms (eg, shortness of breath) of cardiotoxicity were reported in any patient. Four patients (6.2%) had documented mild HFS; 3 patients with KS and 1 patient with a hematologic malignancy One case of HFS (1.5%) led to discontinuation of PLD. Three patients (4.6%) had documented symptoms of mucositis; all 3 patients had KS (5.5%). PLD was effective in most patients at this dose intensity; clinical response (complete or partial response) was seen in 55 patients (84.6%), stable disease in 1 patient (1.5%), and progression in 3 patients (4.6%, all with NHL), and response could not be determined in 5 patients (7.7%). The results of this retrospective review suggest that PLD at an initial dose of 20 mg/m2 q2w is active and well tolerated, and that HFS and stomatitis occur in a low percentage of patients and rarely result in discontinuation. Moreover, the rates of HFS seen in patients with KS and hematologic malignancies seem similar. Prospective studies in larger populations are required to confirm the efficacy and safety of PLD 20 mg/m2 q2w as a component of care for hematologic malignancies.

Incidences of Targeted Adverse Events During Treatment with PLD 20 mg/m2 KS (n = 55) Hematologic Malignancies (n = 10) Cardiotoxicity 0 (0.0%) 0 (0.0%) Mucositis 3 (5.5%) 0 (0.0%) HFS 3 (5.5%) 1 (10.0%)