bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vitro Protection of Human Endothelial Cells from Adenovirus Vector Toxicity by Activated Protein C.
Authors: Lozier Jay Nelson , D’Agnillo Felice
Published: April 2020

High dose adenovirus vector administration in vivo has been associated with toxicity toward many cell types, including endothelial cells. Some of the prominent pathological features of an adenovirus vector death in a gene therapy trial included capillary leak syndrome and disseminated intravascular coagulation (DIC). We investigated the hypothesis that activated protein C (APC) might have a protective effect on primary human microvascular endothelial cells exposed to a first-generation adenovirus vector. We exposed primary human endothelial cells to a first-generation (E1, E3 deleted) adenovirus vector, AVC3FIX5 at concentrations ranging from 103 to 105 vector particles per cell and showed dose-dependent cell death as early as 6 hours (40% cell death at the highest dose). Phase contrast and immunofluorescence microscopy revealed that some cells died rapidly by primary necrosis while others died by apoptosis over a longer time course. By 40 hours, only 40% of the cells were viable. We then tested the effect of pretreatment of endothelial cells with APC concentrations ranging from 1 nM to 100 nM. Dose-dependent protection was seen in which cell death was reduced to 9 and 2 % at APC concentrations of 50 and 100 nM, respectively. We also tested the effect of timing of the APC treatment and showed that 1 hour pre-treatment or concurrent APC treatment were protective, but APC administered one hour after the adenovirus exposure was substantially less protective. This suggested that APC exerts its protective actions on endothelial cells either by interfering with early steps in the interaction of the vector with the cells, (e.g., vector entry) or by modulating death signaling pathways. It has been proposed that APC protects against cell damage in sepsis by interaction with the endothelial cell protein C receptor (EPCR) and protease activated receptor 1 (PAR1) on the endothelial cell surface to induce MCP-1 and other immunomodulatory genes by proteolytic signaling (Riewald et al., Science296:1880–1882, 2002). Other investigators have shown protective effects of APC for endothelial cells subjected to hypoxia through normalization of levels of p53, Bax, and Bcl-2 gene expression (Cheng et al., Nat Med9:338–342, 2003). The APC concentrations in our experiments that were maximally protective (50–100 nM) were of the same order of magnitude as was shown to be protective in vitro by these investigators. If APC can be shown to have a protective effect against adenovirus-induced endothelial cell toxicity and DIC in vivo, this may be a useful therapeutic strategy to explore as treatement of gene therapy vector toxicity.

Cell Viability vs. APC Concentration Cell Viability vs. APC Concentration
γ-Carboxylation Is Not Required for gas6-Axl Binding but Is Necessary for gas6-Dependent Endothelial Cell Survival.
Authors: Blostein Mark D. , Rajotte Isabelle , Hasanbasic Ines
Published: April 2020

Gas6 is a novel member of the vitamin K-dependent family of γ-carboxylated proteins and is a ligand for the receptor tyrosine kinase Axl. Gas6-Axl interactions have been shown to mediate cell survival in vascular endothelium. Although the receptor-binding portion of gas6 lies in the C-terminus, the significance of the N-terminal γ-carboxylated residues (Gla domain) is not clear. To address this question, we recombinantly produced both carboxylated and decarboxylated gas6 and confirmed that the latter is not γ-carboxylated by both Western blotting and fluorescence spectroscopy. Using DAPI staining and flow cytometry, we show that carboxylated gas6 mediates protection of endothelial cells from serum starvation-induced apoptosis whereas decarboxylated gas6 does not. Furthermore, carboxylated gas6, but not decarboxylated gas6, activates Axl and phosphorylates Akt during gas6-Axl-mediated protection of endothelial cells. Interestingly, although decarboxylated gas6 cannot activate Axl and rescue endothelium from serum starvation-induced apoptosis, it can inhibit the survival effect of carboxylated gas6. To further explore the properties of Gla domain of gas6, the binding each form of gas6 to endothelial cells was determined. Both carboxylated and decarboxylated gas6 bind to endothelial cells with an equal affinity of 50 nM. The binding of both forms of gas6 to endothelium is inhibited by an antibody to the extracellular domain of Axl thereby demonstrating that the Gla domain is not required for the direct binding of the C-terminus of gas6 to Axl. These findings support the conclusion that inhibition of gas6-mediated survival by decarboxylated gas6 is not mediated through inhibition of binding of gas6 to its receptor Axl. Rather, the Gla domain of gas6 has a key role by impacting Axl activation via heretofore unknown mechanism(s). Taken together, the results of this study suggest a novel role for γ-carboxylation in gas6 function.
RBC Histogram Predictive of HE.
Authors: Chakmakjian Carl , Rappaport Ed
Published: April 2020

Hereditary elliptocytosis (HE) encompasses a family of inherited erythrocyte disorders characterized by red blood cells with an oval, elongated shape. Diagnosis is most commonly made upon peripheral smear examination. Although the disorder is usually clinically silent, these patients may have some degree of chronic hemolysis that may necessitate splenectomy on occasion. We report a consistent finding in HE common to most electronic cell counters. The red blood cell histogram reliably has a two to three millimeter elevation above baseline at the extreme left of the graph. This “foot” on the histogram is predictive of possible red blood cell elliptocytes. Please see example.

Figure Figure
Long-Term Follow up of Patients with Cold Agglutinin Disease.
Authors: Go Ronald S. , Serck Stephanie L. , Davis Jeffrey J. , Bottner Wayne A. , Cole Craig E. , Farnen John P. , Jumonville Alcee J.
Published: April 2020

Background: Data regarding long-term follow up of patients (pts) with cold agglutinin disease (CAD) are limited.

Methods: We reviewed our institutional database of pts with CAD from 1981–2003. The diagnosis of CAD was confirmed by the following criteria: hemolysis and/or cold induced agglutination symptoms, cold agglutinin titre of ≥ 1:40, and typical direct anti-globulin test findings. We retrospectively collected pertinent clinical data including: demographics, dates of diagnosis and last follow up, associated hematologic diseases, baseline laboratory test results, treatment, clinical course, and subsequent development of lymphoproliferative diseases.

Results: We identified 18 pts with CAD. Most (15) were females. The median age at diagnosis was 71.5 years (range, 22–92). While all pts presented with hemolysis, only 2 had cold-induced acral signs or symptoms. The median presenting hemoglobin level was 8.4 gm/dl (range, 4.5–13.5). The cause was considered idiopathic in 10 pts, while 8 pts had concomitant lymphoproliferative disorders at diagnosis: chronic lymphocytic leukemia, 1 pt; non-Hodgkin lymphoma (NHL), 3 pts; and monoclonal gammopathy of undetermined significance (MGUS), 5 pts. A serum protein electrophoresis was obtained in 16 pts. Five pts had monoclonal proteins, all IgM (3 κ, 2 λ). Thirteen (72%) pts required treatment for CAD or associated lymphoid malignancies at some point in the course of their disease. The most common treatments administered for CAD were prednisone (9 pts) and cyclophosphamide (3 pts). Prednisone produced an overall response rate of 78%. The responses were durable with a median of 95 months (range, 15–414). No one responded to cyclophosphamide. After a median follow up of 55.5 months (mean, 99.5; range, 12–449), 8 pts were alive, 5 in complete remission (2 maintained on prednisone), and 3 in partial remission (all off therapy). Of those who died (10 pts), only 1 died of CAD due to complications from severe anemia. Two pts subsequently developed lymphoproliferative diseases, MGUS (IgMk) and low grade NHL, 125 and 175 months after diagnosis, respectively. The clinical course of CAD did not seem to be any worse in pts with lymphoid malignancies.

Conclusions: In our series, pts with CAD are frequently associated (44%) with lymphoproliferative disease at diagnosis. They have a relatively benign clinical course with a good likelihood of long-term disease remission. Unlike previous reports, prednisone is quite effective. Among those pts considered to have idiopathic CAD, a lymphoproliferative disease may develop later during the course of their disease.
Initial Screening Transferrin Saturation Values, Serum Ferritin Concentrations, and HFE Genotypes in Native Americans and Whites in the Hemochromatosis and Iron Overload Screening (HEIRS) Study.
Authors: Barton James C. , Acton Ronald T. , Lovato Laura , Speechley Mark R. , McLaren Christine E. , Harris Emily L. , Reboussin David M. , Adams Paul C. , Dawkins Fitzroy W. , Gordeuk Victor R. , Walker Ann P.
Published: April 2020

There are few reports of transferrin saturation (TfSat) and serum ferritin (SF) phenotypes and HFE C282Y and H63D genotypes in native Americans. We compared initial screening data of 645 native American and 43,453 white HEIRS Study participants who reported a single race/ethnicity and who did not report a previous diagnosis of hemochromatosis or iron overload. Each underwent TfSat and SF measurements without regard to fasting, and HFE C282Y and H63D genotyping. Elevated measurements were defined as: TfSat >50% (men), >45% (women); and SF >300 ng/mL (men), >200 ng/mL (women). Mean TfSat was lower in native American men than in white men (31% vs. 32%, respectively; p = 0.0337), and lower in native American women than in white women (25% vs. 27%, respectively; p <0.0001). Mean SF was similar in native American and white men (153 μg/L vs. 151 μg/L; p = 0.8256); mean SF was lower in native American women than in white women (55 μg/L vs. 63 μg/L, respectively; p = 0.0015). The respective percentages of native American men and women with elevated TfSat or elevated SF were similar to those of white men and women. The respective mean TfSat and SF values of native American and white participants with genotype HFE wt/wt were similar. The C282Y allele frequency was 0.0340 in native Americans and 0.0683 in whites (p <0.0001). The H63D allele frequency was 0.1150 in native Americans and 0.1532 in whites (p = 0.0001). We conclude that the screening TfSat and SF phenotypes of native Americans do not differ greatly from those of whites. The respective allele frequencies of HFE C282Y and H63D are significantly lower in native Americans than in whites.
Comparison of Liver Iron Concentration Measured by the SQUID Biosusceptometers at Hamburg, Torino and Oakland in Patients with Thalassemia and Sickle Cell Disease.
Authors: Fung Ellen B. , Longo Filomena , Fischer Roland , Engelhardt Rainer , Pakbaz Zahra , Optiz Herbert , Fagaly Robert L. , Nick Hanspeter , Nielsen Peter , Vichinsky Elliott , Piga Antonio , Harmatz Paul
Published: April 2020

Assessing iron chelator efficacy in clinical trials requires standardization of liver iron concentration measurements. In a blinded round-robin test, liver iron concentration (LIC) was measured within 1 month in 18 patients with thalassemia or sickle cell disease and in 10 normal subjects at the 3 SQUID biosusceptometer systems located in Hamburg (UKE), Torino (TOR), and Oakland (CHO). Mean LIC values (range: normal up to 8000 μg/g-liver) were determined from 5 separate vertical scans. The observed intrasite precision (SD of Altman-Bland differences from duplicate measurements with repositioning) was found in the expected range of ±130 (UKE), ±200 (TOR), and 220 μg/g-liver (with 3 operators involved at CHO). Prediction of LIC at TOR and CHO in comparison with UKE was very good, with coefficients of determination between sites of R2 = 0.97, resulting in intersite standard deviations (SD) of 247 and 326 μg/g-liver, respectively. Differences of 24, 20, and 5% were noted for the comparisons UKE-TOR, CHO-TOR, and UKE-CHO, respectively (see Fig. 1). This suggests the need for further standardization of analysis methods. In conclusion, we found that intra-site precision was within an acceptable range for repeat measurements in the majority of iron overloaded subjects. Prediction of liver iron concentration at the three centers was highly correlated.

Figure 1. Agreement (Passing-Bablok regression) between liver iron measurements (mean LIC from 2 positions) by the SQUID biosusceptometer system in Hamburg (LIC-UKE) and the systems in Torino (LIC-TOR) and Oakland (LIC-CHO). Figure 1. Agreement (Passing-Bablok regression) between liver iron measurements (mean LIC from 2 positions) by the SQUID biosusceptometer system in Hamburg (LIC-UKE) and the systems in Torino (LIC-TOR) and Oakland (LIC-CHO).
Three Kinships with ALAS2 P520L Mutations, Two in Association with Severe Iron Overload, and One with Sideroblastic Anemia and Severe Iron Overload.
Authors: Lee Pauline L. , Barton James C. , Rao Sreenivas V. , Acton Ronald T. , Adler Brian K. , Beutler Ernest
Published: April 2020

Aminolevulinate synthase 2 (ALAS2) is an erythroid-expressed gene located on chromosome Xp11.21. Mutations in ALAS2 have been shown to be associated with sideroblastic anemia. We have identified a novel mutation in ALAS2, P520L, in three kinships. The P520L mutation was not found in 316 white male control subjects. The proline in this position is highly conserved across species from humans to zebrafish.

In the C kinship, the P520L mutation was first identified in a white man who presented with severe iron overload at an early age and was a HFE C282Y homozygote. Genetic analyses of members of the C kinship suggest that the presence of P520L alone in hemizygous males, or simple heterozygosity in females, is not associated with anemia or iron overload. Females heterozygous for both HFE C282Y and ALAS2 P520L also had normal ferritin levels. Only subjects homozygous for HFE C282Y and hemizygous or heterozygous for ALAS2 P520L had severe iron overload. Sequencing studies revealed that the propositus did not have missense mutations in HAMP, HJV, FPN1, ABC7, IL6, or RAG1.

In the H kinship, the propositus was a white woman with severe iron overload who was heterozygous for ALAS2 P520L; she had a wildtype HFE genotype. The pedigree of the H kinship identified 5 additional females who were heterozygous for the P520L ALAS2 mutation. Only the propositus had iron overload and none of the subjects with P520L had sideroblastic anemia. The propositus did not have missense mutations in FPN1, HAMP, HJV, TFR2, B2M, IRP2, ABC7, or SFT. We speculate that the propositus in the H kinship has a mutation in a currently unknown gene that contributes to her severe iron overload. In the S kinship, the P520L mutation was identified in a white man with sideroblastic anemia and severe iron overload. The patient had a second mutation in ALAS2, R560H, a mutation previously described in two brothers with sideroblastic anemia of variable penetrance (Blood100:4236–4238, 2002). It is not possible to determine the effect of the P520L mutation on the penetrance of the R560H-associated sideroblastic anemia and iron overload.

We conclude that the ALAS2 P520L occurs at a very low allele frequency in the white population. The present observations also suggest that there is no distinctive phenotype associated with the P520L mutation alone, but that P520L may act as a modifier of iron overload in the presence of HFE homozygosity, other missense mutations of ALAS2, or mutations of uncharacterized iron regulatory genes.
Defective Recycling and Exocytosis of Transferrin in Reticulocytes of hbd (Hemoglobin Deficit) Mice.
Authors: Garrick Michael D. , Gniecko Kathleen , Garrick Laura M.
Published: April 2020

Mice with hemoglobin deficit (hbd) have a hypochromic, microcytic anemia inherited as an autosomal recessive. The mice also exhibit reticulocytosis, hyperferremia and elevated free protoporphyrin in red cells (Bannerman et al 1986 Proc Soc Exp Biol Med 182:52). The spontaneous mutation has been mapped to chromosome 19 (Bloom et al 1998 Mamm Genome 9:666), making it ripe for identification of the gene, given the power of mouse genetics. Until the actual locus is determined, however, some mice must be identified as +/? as dominant inheritance obscures the second allele. Prior studies established that hbd/hbd reticulocytes take up 125I-labelled transferrin as well as +/? controls, but are less effective at incorporating 59Fe from transferrin (Garrick et al 1987 Exp Hemato 15:671). Scatchard analysis showed the affinity of transferrin receptors for transferrin was similar in hbd/hbd versus +/? reticulocytes; slightly more receptors were present, however, on the surface of hbd/hbd reticulocytes.

We now report on aspects of transferrin-dependent iron metabolism in hbd/hbd reticulocytes to resolve the nature of the alterations in the transferrin cycle in these rodent mutants. Improved time resolution for 59Fe uptake from transferrin by hbd/hbd versus +/? reticulocytes reveals that uptake in the first 2.5 min is indistinguishable; but that, by 5 min, incorporation by the mutant cells lags behind. Similarly, endocytosis of 125I-labelled transferrin by mutant versus control reticulocytes occurs at essentially the same rate; while exocytosis is about 4-fold slower for hbd/hbd reticulocytes compared to +/? reticulocytes. We conclude that recycling and exocytosis of transferrin in reticulocytes of hbd/hbd mice is strikingly impaired. The mutation in these mice should be in a gene that causes this impairment.
The Course of Plasmodium.yoelii Infection in Hereditary Persistence of Fetal Hemoglobin.
Authors: Nishino Tamon , Yaegashi Junko , Ohashi Toya , Matsumoto Yoshitsugu , Yoshikatsu Eto
Published: April 2020

Malaria, caused by plasmodium species has great impact on human generations. Each year 300–500 million of people are diagnosed and 1 million die of the disease. Malaria probably become endemic about 10,000 years ago and it has affected human genome by strong selective pressure. Several human genetic diseases are thought to be the outcome of this pressure. Our current research is focused on understanding natural host defense mechanism of this disease, especially effect to human fetal hemoglobin expression to plasmodium species.

Other investigator indicated growth of Plasmodium falciparum is retarded in the cell containing human fetal hemoglobin in vivo (Nature, 270:171), and transgenic mouse (transgenic of human fetal hemoglobin and knockout of mouse beta-major gene) infected with Plasmodium has longer survival compare to control.(Blood, 92:2520). Here we use transgenic mouse express human fetal hemoglobin (human Gamma globin) which simulate human hereditary persistence of fetal hemoglobin (PNAS86:7033) to investigate fetal hemoglobin itself has a protective effect on Plasmodium infection in vivo.

We injected 1,000 Plasmodium.yoelii infected RBCs to transgenic mouse expressing human fetal hemoglobin (n=5) and C57BL6J mouse (n=8). Blood smears obtained daily and smears were stained with Giemsa. Parasitemia is calculated from about 1000 RBCs. 5 days after injection, infected RBCs was 26.4 ±4.7% for C57BL6J and 32.6± 17.3% for human fetal hemoglobin transgenic mouse. We couldn’t show significant difference between these groups. And 60% of C57BL6J mice (3 of 5) were recovered from illness. In contrast, 20% of transgenic mouse (1of 5) was recovered from illness.

These results demonstrates only human fetal hemoglobin expression is not enough to contribute defense mechanism against plasmodium species.
Nitrite Ion Fluxes in Human Erythocytes.
Authors: d’Agostino Chiara , Dejam Andre , Pelletier Mildred , Sibmooh Nathawut , Rizzatti Fabiola , Schechter Alan N.
Published: April 2020

Our recent studies suggest that nitrite ions serve as a storage pool of nitric oxide (NO•) bioactivity, with reduction of nitrite to NO• within red blood cells (RBC), thus potentially explaining endocrine effects of NO•. We have investigated nitrite uptake and decay in human RBC using recently described methodologies based on assaying after stabilization of nitrite to reaction with hemoglobin by treatment with ferricyanide and quantificating by reductive chemiluminescence. Uptake of nitrite added to whole blood is very rapid (sec) and independent of temperature (from 4°C to 37°C) and state of oxygenation of the cells, or treatment with carbon monoxide. We obtain intracellular values as high as 711 ± 45.44 nM/l with an extracellular value of 10,935.089 ± 897.370 nM/l. Decay of nitrite levels within the RBC is, however, strongly temperature and ligand dependent. At 37°C only 22% of the original maximal value remains at 30 minutes (half life: 5.24 min), while there is no apparent decay at 4°C. Carbon monoxide also prevents nitrite destruction. If the nitrite-containing RBC are suspended in nitrite-free plasma, decay of nitrite levels is faster (half life 2.1min) suggesting export of nitrite as well as reaction with hemoglobin. We have also observed whole blood and RBC artery-to-vein gradients of nitrite levels in human forearm circulation suggesting rapid loss during RBC transit of the microcirculation. These results suggest rapid fluxes of nitrite ions into and out of RBC, as well as controlled intra-erythrocytic metabolism, which could contribute to local, rapid modulation of blood flow by mechanisms involving nitrite activation to NO• by intra-erythrocytic hemoglobin.
Maintenance Dosing with Epoetin alfa Every Two Weeks (Q2W) in Anemic Patients with Cancer Receiving Chemotherapy Every One or Four Weeks: Final Results.
Authors: Gregory Stephanie A. , Xie John , Woodman Richard C.
Published: April 2020

Epoetin alfa administered weekly is a proven treatment for chemotherapy (CT)-induced anemia. To increase patient convenience we examined a dosing regimen that would allow patients to receive epoetin alfa 60,000 U Q2W during maintenance therapy. This open-label, multi-center study had two phases: Initiation Phase (IP) of 60,000 U once weekly (QW) to achieve a target hemoglobin (Hb) of 12 g/dL (maximum duration 12 weeks), followed by a Maintenance Phase (MP) of 60,000 U Q2W to maintain Hb between 11.0 and 12.5 g/dL. Only those patients achieving Hb ≥ 12 g/dL in the IP entered the MP. Patients were withdrawn from the MP if Hb decreased to < 11 g/dL. Doses were held for Hb > 13 g/dL and reduced for rate of rise of > 1.3 g/dL/2weeks. Maximum study duration (IP + MP) was 24 weeks. Patients ≥ 18 years of age with non-myeloid malignancy, baseline Hb ≤ 11 g/dL, and CT planned QW or every 4 weeks for at least 16 weeks were enrolled. Endpoints included proportion of patients achieving hematopoietic response in the IP (≥ 2 g/dL Hb increase from baseline or Hb ≥ 12 g/dL) and proportion maintaining mean Hb between 11.0 and 12.5 g/dL in the MP. A total of 129 patients were enrolled and received at least one dose of epoetin alfa; mean age was 62.6 ± 13.0 years, 65.9% were female, mean baseline Hb was 10.0 ± 0.9 g/dL, and 92.2% had solid tumors. During the IP, 84 of 129 (65.1%) patients achieved hematopoietic response (≥ 2 g/dL Hb increase from baseline or Hb ≥ 12 g/dL). The eighty-four patients who achieved Hb ≥ 12 g/dL entered the MP after a median of 4.0 weeks in IP; treatment duration in MP was 13.2 weeks on average. Fifty-one of 84 (60.7%) maintained an average Hb between 11.0 and 12.5 g/dL over the course of the MP. Sixteen (19.0%) patients were withdrawn from the MP because Hb decreased to < 11 g/dL. Of 129 patients dosed in IP, 14 (10.9%) had doses reduced and 8 (6.2%) had doses held; of 84 patients dosed in MP, 47 (36.4%) had doses reduced and 53 (41.1%) had doses held. Sixty-five (50.4%) patients experienced serious adverse events. Fifteen (11.6%) patients experienced clinically relevant thrombotic vascular events. Three and 5 patients discontinued due to adverse events in the IP and MP, respectively. Fifteen (11.6%) patients died during the study or within 30 days after last study dose. Although 60,000 U QW was used during the IP to achieve a HR, 40,000 U QW is the recommended initiation dose of epoetin alfa. This study of patients receiving CT every 1 or 4 weeks, demonstrated that the majority of those who achieved a HR are able to maintain an acceptable Hb level with epoetin alfa 60,000 U every two weeks.
Hydroxyurea Therapy Reduces the Gene Expression of Adhesion Molecules VLA-4 and CD36 in Sickle Erythrocytes, with a Concomitant Decrease in Adhesion to Fibronectin.
Authors: Gambero Sheley , Conran Nicola , Albuquerque Dulcineia M. , Santos Camila O. , Saad Sara T.O. , Costa Fernando F.
Published: April 2020

Adhesive interactions between sickle red cells, leukocytes and the endothelium are thought to initiate vaso-oclusive events in the microcirculation. An increased expression of adhesion molecules such as CD 36 (thrombospondin receptor) and VLA-4 integrin (α4β1, CD49d/CD29) has been observed on the surface of sickle reticulocytes and the increased adhesive properties of sickle erythrocytes are well documented. Gene expression of adhesion molecules and their modulation by drugs such as hydroxyurea (HU) have yet to be fully explored. We compared the expression of the CD36 gene and the VLA-4 integrin subunit genes, CD29 and CD49d, in the reticulocytes of steady-state SCD patients and patients on HU therapy (20–30 mg/Kg/day) using Real Time PCR and also compared the basal adhesion of erythrocytes from these subjects using static adhesion assays. Basal adhesion of sickle erythrocytes (2x108 cells/ml) to fibronectin (20mg/ml) - coated plates was significantly higher than normal erythrocyte adhesion (15.4 ± 2.8% compared to 5.9 ± 1.1%, n=9, p=0.008). In contrast, the adhesion of erythrocytes from SS patients on HU therapy to fibronectin was significantly lower than that of SS erythrocytes of patients not on HU (5.8 ± 0.7%, n=9, p=0.006). Pre-treatment of cells, in vitro, with varying concentrations of HU (0.1 → 250μM, 30 minutes, 37oC), had no significant effect on either normal or sickle cell erythrocyte adhesion under our experimental conditions, suggesting that HU may not reduce sickle cell adhesion by altering adhesion molecule function due to affinity changes. We next used real time PCR to investigate the expression of the mRNA of the CD36 gene and the genes for the VLA-4 subunits, CD49d and CD29, in the reticulocytes of steady-state SCD patients and patients on HU therapy. CD 36 gene expression was reduced by 62.5% ± 10.4% in the reticulocytes of patients on HU compared to the reticulocytes of SCD patients not on HU (n=8, p < 0.001). The β1 (CD29) integrin subunit expression was reduced by 87.9 ± 8.7% (n=8, p < 0.001) in patients on HU compared to SS reticulocytes; likewise α4 integrin (CD49d), expression was reduced by 50.0% ± 2.6% (n=8, p <0.001). In addition, reticulocyte mRNA was collected from one steady-state SCD patient before and 3 months after initiation of HU therapy and decreases of 99.5%, 98.0% and 98.0% were observed in the expressions of CD36, CD29 and CD49d mRNA, respectively, in the reticulocytes of this patient following HU therapy. We speculate that the decrease in adhesive properties observed in the erythrocytes of sickle cell patients during HU therapy may be the consequence of a down-regulation of the gene expressions of adhesion molecules and, consequently, protein expression by sickle cells rather than as a result of alterations in adhesion molecule function.
Adverse Events in Sickle Cell Patients with Nocturnal Hypoxia.
Authors: Campbell Andrew D. , Okumura Megumi , Unaka Ndidi , Hutchinson Sally , Onyekwere Onyinye
Published: April 2020

The relationship between hemoglobin dexoygenation and sickling is well known. However, the relationship between hypoxia and severity of disease in sickle cell patients has not been well established. Recently, nocturnal hypoxemia has been associated with higher incidence of CNS events including strokes, and elevated TCDs. We present our case series on 13 patients(12 SS, 1 SC) with sickle cell disease (SCD) who have nocturnal hypoxia. Approximately 75 patients were screened at the University of Michigan Sickle Cell clinic for nocturnal hypoxia either by symptoms of obstructive sleep apnea or by longitudinal baseline clinic 02 saturations (02 Sat <92%). Of the 13 hypoxic pts, median baseline O2 Sat 90%(n=13, mean 90) and the median nocturnal O2sat (Nctnl 02 sat) 84%(n=13, mean 80%) with 10/13 with moderate-severe nocturnal hypoxia (O2sats<85%)based on sleep studies. Multiple adverse events noted in the cohort were pulmonary hypertension (PHTN TRJV>2.5, n=9, median 2.74/mean 2.74,) frequent pain episodes(>3visits to ER or hospitalizations/year, n=7, with 5 pts >5/year ), recurrent acute chest syndrome( ≥ 3 episodes, n= 10), CNS events (n=3 silent infarcts, vascular stenosis), priapism( (n=4, among 6 males ). Also reported were possible causes of the underlying hypoxia including obstructive sleep apnea(OSA)(n=7 of 11 pts), asthma(n= 10 of 13 pts), and chronic lung disease( n=8). In conclusion, the persistence of nocturnal hypoxia in pediatric sickle cell disease could possibly contribute to the development of severe complications of sickle cell disease. Treatment of underlying hypoxia (ie nighttime oxygen, maximize asthma treatment, T&A for OSA)may help prevent complications and lead to the improvement clinical symptoms. Further, chronic nocturnal hypoxia may complicate pulmonary disease and accelerated the development of PHTN. More studies are needed to clarify the mechanism of hypoxia in SCD.

Table I. Clinical &Demographic Data of 13 SCD Patients with Nocturnal Hypoxia. Age:(6–22y/o, mean 15) Sex: M=6 F=7 Clinical: Total Mild Mod Sev. Genotype: SS=12, SC=1 Mean Range Nctnl Hypoxia(<%) 13 3 5(<85) 5(<80) Baseline O2 Sat(%) 90 +3.0 86–97 Obstr Sleep Apnea 7 3 3 1 Nctnl 02 sat (%) 80+8.4 59–87 Pulm HTN 9 4 4 1 Total #Apneic Events(11) 65.6+80 6–256 Rest. Lung Ds. 8 2 5 1 Obstr. Apneic Events(7) 27+68.5 0–221 # of Episodes <3 3–4 >4 Hypopneic Events(9) 32.5+38 0–132 ACS 2 5 5 TRJet Velocity 2.74+.42 2–3.5 Severe Pain Crises/yr 1 2 5
Mortality Trends in People with Sickle Cell Disease in North Carolina.
Authors: Redding-Lallinger Rupa
Published: April 2020

Background. Using data from the Cooperative Study of Sickle Cell Disease which ran from 1978 -19**, published figures for median life expectancy for men with sickle cell disease is 42 years and for women 46 years. Recent data from Texas show a declining childhood mortality rate, compared to the CSSCD data, in a cohort who have been followed from infancy. However, despite the strides made in reducing childhood mortality and improving length and quality of life in some adults, there is still very significant morbidity and mortality from cumulative damage to organs, especially the lungs and the kidneys.

Aims. The purpose of this investigation is to analyze the numbers and causes of death in individuals with sickle cell disease over the past several years in North Carolina. The information gathered may suggest areas in which health services to people with sickle cell disease may be improved.

Methods. The North Carolina State Center for Health Statistics provided the following data for the years 1997–2003. Total number of SCD deaths for each of those years, and the age, county of residence, place of death, the ICD codes for the underlying and contributing cause of death as listed on the death certificates of the decedents.

Results. There were 277 deaths over the 7 year period. Fifty-three percent of these deaths occurred during the earlier half of this period. Overall, 49% of the deaths occurred in people from age 30 to age 49, and this proportion remained quite stable during the 7 years. Deaths of people 20 years and younger made up 14% of total deaths; there were fewer in the second half of the time period than in the first half—40% of the total deaths in this age group occurred from late 2000 through 2003 vs 60% from 1997 through early 2000. Deaths in the youngest age group (0–5) were only 4.3% of the total deaths for the period. For the individuals who died between birth and 20 years of age, the underlying cause of death from the death certificates were grouped as “sickle cell disease with or without complications” in 50%, congenital heart disease in 11%, bacterial infection in 17%, gastroenteritis in 9.5%, UTI in 6% and other in 4%. For those who died between 30 and 40 years of age the underlying cause of death as listed on the death certificates were grouped as: sickle cell anemia, with and without crisis—58%, pulmonary complications—12%, cardiovascular problems—10%, CNS problems—5%, hepatic disorders and infections—each <5%, and other—10%.

Conclusions. Mortality in the youngest children appears to have lessened in comparison with historical data, and this age group no longer leads in mortality rates. Deaths in the young adult age range of 30–49 years have showed no downward trend over 7 years. The cause of death from death certificates do not match well with the clinical problems typically seen in people in this age group. The lack of precise diagnoses on death certificates may reflect a lack of understanding of sickle cell complications among doctors in the community.
Sickled Erythrocyte Morphology in the Bone Marrow of Patients with Sickle Cell Disease: Previously Unrecognized Forms of Sickled Erythrocytes May Have a Pathogenetic Role in Bone Pain of Painful Crises.
Authors: Roberts George T.
Published: April 2020

Background: The painful crises of sickle cell disease (SCD) are characteristically localized to the skeleton. Various lines of evidence suggest that bone and bone marrow (BM) infarction contribute to the pathophysiology of such pain in which sickled red blood cells (SRBC) are believed to play a part. However, there is little information about the morphological characteristics and role of SRBC in BM during the crises or indeed, in the “steady state”. Occasional light microscopic observations in the BM of SCD patients show numerous markedly distorted cells, presumed to be SRBC, types of which have not been previously reported from this site or the peripheral blood (PB). We therefore studied a series of BM biopsies from SCD patients to determine whether such changes are consistently present.

Methods: We microscopically reviewed archival BM biopsies from SCD patients for abnormally shaped RBC and compared the findings with SRBC on Wright-stained smears of PB collected simultaneously with the biopsies. The material consisted of Wright-Giemsa stained smears of aspirated BM and hematoxylin-eosin stained histological sections of clotted aspirate and / or trephine biopsies. We also examined fresh BM RBC by scanning and transmission electron microscopy (EM). We determined the relationship between the percentage of SRBC in BM and PB on the one hand, and fetal hemoglobin (HbF) levels on the other.

Results: We studied the BM of 43 SCD patients. By LM, we observed markedly distorted RBC in the BM of the majority of patients who also had BM SRBC that were more typical forms. The distorted forms included vastly elongated erythrocytes, measuring up to 40 microns in length and less than 1 micron in width, some fixed into rod like structures, but others as sinuous, snake-like forms that have not been previously described. Scanning EM appearances also confirmed the features observed by LM. By contrast, none of PB samples showed similarly distorted SRBC. Although the percentage of typical SRBC in BM was concordant with that in the PB (p<0.001) SRBC numbers in both PB and BM were inversely proportional to the level of HbF.(Table 1)

Conclusion: We conclude that the proportions of markedly distorted SRBC present in the BM of SCD patients are inversely proportional to HbF level and may thus play a pathogenetic role in the localization of severe pain to the skeleton during painful crises.

Table 1. Correlations between sickled RBC estimates and blood fetal hemoglobin levels MSRBC MARROW MBMSRBC MSRBCLOT+TREPHINE MSRBC PB MSRBC= Mean SRBC marrow and peripheral blood together. MBMSRBC = Mean SRBC in bone marrow aspirates only. MSRBCLOT+TREPHINE = Mean SRBC, clotted marrow aspirate and trephine together. MSRBCPB = Mean SRBC in peripheral blood. MHbF = Mean HbF level in blood. r = correlation coefficient. NS = Not significant. MHbF MHbF MHbF MHbF r −0.3806 −0.3293 −0.2685 −0.384 p < 0.05 < 0.05 NS < 0.05
Increased Chromosomal Breaks in Sickle Cell Disease as Evidenced by the Presence of Micronuclei in Erythrocytes.
Authors: Lal Ashutosh , Bhagat Amrita , Atamna Wafa , Offer Tal , Vichinsky Elliott P. , Kuypers Frans A. , Ames Bruce N.
Published: April 2020

Double-stranded DNA breaks are serious lesions that contribute to the pathogenesis of cancer and chronic illnesses. Free radical injury is an important mechanism for the production of DNA modifications that eventually lead to chromosomal breaks. The generation of free radicals is increased in sickle cell disease (SCD) owing to the presence of the relatively unstable HbS and recurrent ischemia-reperfusion events. In the present study, a sensitive reticulocyte micronucleus assay was used to determine the frequency of chromosomal breaks in patients with SCD (Offer et al. FASEB J. 2005;19:485). A micronucleus is a piece of a chromosome left behind in a reticulocyte as a consequence of DNA double-stranded break after the nucleus is extruded. Blood samples were obtained from patients with SCD (n=33) and healthy volunteers (n=23). After immunomagnetic enrichment of CD71+ reticulocytes (Trf-Ret), RNA was removed with RNAse, and the cells were stained for miconuclei using the DNA dye 7-aminoactinomycin D (7-AAD) followed by flow cytometric evaluation. The appearance of reactive oxygen species in cytosol and membrane lipid oxidation in RBCs were measured by flow cytometry using the fluorescent markers 2, 7-dichlorofluorescin diacetate (DCF) and C11-BODIPY, respectively. The frequency of micronucleated Trf-Ret was significantly higher (p<.0001) in patients with SCD (mean 3.061/10,000 Trf-Ret, range 0.256 to 9.779) as compared with normal controls (mean 0.750/10,000 Trf-Ret, range 0.043 to 2.659). Mean DCF (125.1%) and C11-BODIPY (117.6%) fluorescence intensities were significantly greater than the mean control value (100%, p 0.0001). Our data indicate that patients with sickle cell disease have increased double stranded DNA-breaks compared with healthy controls, and suggest that elevated production of oxidants could contribute to the development of genetic damage in these patients.
Results of Arthroplasty for Avascular Necrosis of the Humeral Head in Sickle Cell Patients.
Authors: Lau Michelle W. , Galatz Leesa M. , Williams Kimberly , Blinder Morey A.
Published: April 2020

Avascular necrosis (AVN) of the humeral head is a debilitating complication of sickle cell disease (SCD) estimated to occur in ~5% of patients for which the optimal therapy is not well defined. Although shoulder replacement is often used for AVN in other settings, little information is available about the outcome of shoulder arthroplasty in this population. In this study, medical records, radiographs, and pre-operative assessment scores of eight adult patients with sickle cell disease were reviewed. Post-operative assessment scores and radiographs were prospectively collected by an independent observer at follow-up appointments. All eight patients had >2 years of follow up. The mean age of the patients at the time of surgery was 37 years old and there were 4 males and 4 females. Six of the patients had Hgb SS, one had Hgb SC and one had Hgb Sβ+ thalassemia. The average duration of symptoms prior to surgery was 26 months. Based on pre-operative shoulder radiographs, 5 of 7 evaluable patients had grade 3 or greater disease. Seven of eight patients had a hemiarthroplasty and one patient had a total arthroplasty. The mean follow up was 51 months. Complications included two episodes of sickle cell crises in the immediate post-operative period, and one intraoperative rotator cuff tear. One patient developed stiffness that required arthroscopic capsular release 22 months after his initial surgery. No infections were seen in the operated shoulder and no shoulder revisions were performed. At the most recent follow-up, the average American Shoulder and Elbow Society (ASES) score improved 31 points, indicating substantial functional improvement (J Shoulder Elbow Surg. 3(6):347–352, 1994). However, only 25% of the patients reported improvement in pain as assessed by a visual analog scale. While two patients had dramatic improvements in all aspects of our outcome measures, two other patients had decreased functional capacity with no improvement in pain. Radiographs at the most recent follow up revealed slight loosening of the prosthetic stem in one patient but no other radiographic complications. In conclusion, shoulder arthroplasty provides improvements in range of motion and joint function in the majority of patients. However, pain relief is less predictable, suggesting that other causes of sickle cell related pain are occuring. Nevertheless, given the limited options available for the treatment of AVN in sickle cell patients who have failed conservative approaches, shoulder arthroplasty is a reasonable treatment option.
Mean Platelet Volume in Children with Sickle Cell Disease on Chronic Transfusion Therapy.
Authors: Lakshminarayanan Sonali , Aygun Banu
Published: April 2020

The symptoms in sickle cell disease are related to ischemic/vaso-occlusive episodes involving the bones, spleen, brain and lungs. One of the possible mechanisms for vaso-occlusion is platelet activation and increased platelet reactivity which is demonstrated by increased platelet volume resulting in an elevated platelet distribution width. We hypothesized that chronic transfusion therapy given to patients with recurrent severe ischemic episodes and/or stroke may lower mean platelet volume (MPV) in addition to decreasing hemoglobin S. We performed a retrospective chart analysis of all patients with sickle cell disease who were on a chronic transfusion regimen at Schneider Children’s Hospital between January 1995 and March 2005. Out of 60 eligible patients, only 42 were eligible for this study. The remaining patients were excluded for less than 3 transfusions in total or incomplete records. The data retrieved included age and sex of the patient, type of sickle cell disease, indication for chronic transfusion therapy, duration of transfusions and blood count variables during the three phases, i.e. prior to, during and after transfusion therapy. The variables included hemoglobin, mean cellular volume, platelet count, reticulocyte count and MPV for each patient with a minimum of one value in each phase. As many values were recorded based on the availability in the records, with at least an interval of 3 months between each blood count and at least 3 months apart from a transfusion.

Results: Sixty-four percent were males and 95% had HbSS genotype. The major indication for transfusion therapy (85%) was stroke. Mean values of each variable and their ranges were compared across the 3 periods using the Kruskal Wallis test. Although the platelet count decreased significantly during transfusions compared to pre-transfusion period, the mean value of MPV did not differ across the 3 periods with a range between 5.9 and 11.3 in all the 3 periods. Furthermore, using the Anacova method proposed by Bland and Altman, there was an overall significant negative correlation between MPV and platelet count, while no correlation at all was present between MPV, reticulocyte count and hemoglobin in any of the 3 periods. Our findings suggest that multiple transfusions do not alter platelet size or volume, which indirectly may indicate that there is no change in platelet function or activity in these patients on chronic transfusion therapy. The changes in MPV values seen in this study are real, as they follow the natural thrombokinetics by maintaining a negative correlation with the platelet count during all 3 periods.
Magnetic Ressonance Imaging (MRI) in the Evaluation of Iron Overload in Patients with Beta Thalassaemia. The Brazilian National Program Preliminary Results.
Authors: Hamerschlak Nelson , Rosemberg Laercio , Parma Alexandre , Assir Fernanda F. , Moreira Frederico R. , Parga Jose R. , Amaro Edson , Samira Morrhy , Edgar Lira , Claudio Fisher , Sandra Loggeto , Monica Verissimo , Aderson Araujo , Giorgio Baldanzi , Antonio Fabron , Claudia Velloso , Merula Steagall A. , Fernando Tricta , Alexandre Cury , Marcelo Vieira , Marcelo Funari G. , Fernanda Magao S. , Joice Nogueira C.
Published: April 2020

Magnetic Ressonance Imaging (MRI) using T2 star (T2*) tecnique appears to be a very useful method for monitoring iron overload and iron chelation therapy in thalassaemia. In Brazil, we have around 400 thalassaemic major patients all over the country. They were treated with hipertransfusion protocols and desferroxamine and/or deferiprone chelation. We developed a cooperative program with the Brazilian Thalassaemic Patients Association (ABRASTA) in order to developT2* tecnique in Brazil to submit brazilian patients to an annual iron overload monitoring process with MRI.. We performed the magnetic ressonance T2* using GE equipment (GE, Milwaukee USA), with validation to chemical estimation of iron in patients undergoing liver biopsy. Until now, 60 patients were scanned, median age=23,2 (12–54); gender: 18 male (30%) and 42 female (70%). The median ferritin levels were 2030 ng/ml (Q1=1466; Q3=3296). As other authors described before, there was a curvilinear inverse correlation between iron concentration by biopsy, liver T2*(r=0,92) and also there were a correlation with ferritin levels. We also correlated myocardial iron measured by T2* with ventricular function.. As miocardial iron increased, there was a progressive decline in ejection fraction and no significant correlation was found between miocardial T2* and the ferritin levels.

Liver iron content can be predicted by ferritin levels. On the other hand, cardiac disfunction is the most important cause of mortality among thalassaemic patients. Since Miocardio iron content cannot be predicted from serum ferritin or liver iron, and ventricular function can only detect those with advance disease, intensification and combination of chelation therapy, guided by T2* MRI tecnique should reduce mortality from the reversible cardiomyopathy among thalassaemic patients.