Hemolysis Of Red Cells Research Articles

Modification of the effects of aflatoxin B1 on the glutathione system and its regulatory genes by zeolite.

The purpose of the present study was to use oxidative stress markers for investigating the effect of zeolite (315 mg/kg of complete feed) in the case of aflatoxin B1 contamination (92 μg/kg complete feed). In a 21-day feeding trial with broiler chickens, oxidative stress parameters such as conjugated dienes, conjugated trienes, malondialdehyde, reduced glutathione content and glutathione peroxidase activity were not changed significantly by supplementation with this mycotoxin absorbent. The relative gene expression of transcription factors KEAP1 and NRF2 was not modified by the absorbent either. Still, the expression of GSS, GSR and GPX4 genes increased significantly due to the aluminosilicate supplementation. The results suggest that zeolite reduced lipid peroxidation in the blood plasma but not in the red blood cell haemolysate or the kidney. The relative expression of the genes encoding the glutathione redox system also changed as a result of zeolite supplementation, but these changes were not found at the protein level.

Pseudohyperkalaemia in essential thrombocytosis: an important clinical reminder.

SummaryAn 82-year-old female was admitted to a general hospital due to progressive bilateral lower limb weakness. A T8–T9 extramedullary meningioma was diagnosed by MRI, and the patient was referred for excision of the tumour. During the patient’s admission, she was noted to have persistent hyperkalaemia which was refractory to treatment. Following a review by an endocrinology team, a diagnosis of pseudohyperkalaemia secondary to thrombocytosis was made. This case demonstrates the importance of promptly identifying patients who are susceptible to pseudohyperkalaemia, in order to prevent its potentially serious consequences.Learning pointsPseudohyperkalaemia should be considered in patients with unexplained or asymptomatic hyperkalaemia. It should also be considered in those patients who are resistant to the classical treatment of hyperkalaemia.A diagnosis of pseudohyperkalaemia is considered when there is a difference of >0.4 mmol/L of potassium between serum and plasma potassium in the absence of symptoms and ECG changes.In patients who are presenting with consistently elevated serum potassium levels, it may be beneficial to take venous blood gas and/ or plasma potassium levels to rule out pseudohyperkalaemia.Pseudohyperkalaemia may subject patients to iatrogenic hypokalaemia which can be potentially fatal.Pseudohyperkalaemia can occur secondary to thrombocytosis, red cell haemolysis due to improper blood letting techniques, leukaemia and lymphoma.

Observation of targeted gold nanoparticles in nasopharyngeal tumour nude mice model through dual-energy computed tomography.

This study was performed to specify the efficiency of imaging nanoparticle concentration as contrast media in dual‐energy computed tomography (DECT). Gold nanoparticles (AuNPs) and gold nanoparticles‐conjugated folic acid through cysteamine (FA‐Cya‐AuNPs) were both considered as contrast agents. Characterization of NPs was performed using Dynamic Light Scattering (DLS) and zeta potential. The hemocompatibility of NPs was confirmed by different blood parameters such as white blood cell, red cell distribution width, hemoglobin, lymphocytes counts and haemolysis assay. DECT algorithm was confirmed using calibration phantom at different concentrations of NPs and tube potentials (80 and 140 kVp). Then, DECT was used to quantify the concentration of both AuNPs and FA‐Cys‐AuNPs in human nasopharyngeal cancer cells. Mice were injected with non‐targeted AuNPs and targeted AuNps at a concentration of 3 × 103 μg/ml. Then, they were scanned with different tube potentials. The concentration of nanoparticles in the various organs of nude mice was measured through DECT imaging and inductively coupled plasma mass spectrometry (ICP‐MS) analysis. The results of DECT images were compared with ICP‐MS analysis and indicated that they were approximately similar. In sum, FA‐Cys‐AuNPs can be a proper candidate for targeted contrast media in DECT molecular scanning of human nasopharyngeal tumours.

Pre-analytical variables in haemostasis: Findings from the United Kingdom National External Quality Assessment scheme for Blood Coagulation (UK NEQAS BC) haemolysis exercise.

Haemolysis is considered one of the major contributors of nonconformities and sample rejection in coagulation testing. Two lyophilized plasmas were distributed to 800 centres registered for prothrombin time (PT), activated partial thromboplastin time (APTT) and either Clauss fibrinogen or thrombin time (TT) in the UK NEQAS BC programme. The same pool of normal plasma was used to prepare both samples, to one of which red blood cell haemolysate was added to mimic haemolysis at 3g/L haemoglobin concentration. Participants were asked to complete a questionnaire about their laboratory approach to dealing with haemolysed samples, including strategies used to deal with different levels of haemolysis. Results for tests performed did not show great differences between the two samples. It should be noted that artificially constructed haemolysed samples may not behave in the same way as patient samples (ie, may not be commutable). However, the possibility of carrying out a large multicentre study for detection of haemolysis was demonstrated. Inconsistency in practice was observed with 226/551 (41%) of centres indicated they reject haemolysed samples solely on visual checks, and 163 (30%) using initial visual checks with further sample rejection evaluation by analyser flags. Furthermore, 333 (72%) of centres indicated that the level of haemolysis affects sample rejection decisions, while 132 (28%) stated it did not. Variability of responses for dealing with haemolysed samples reflects a lack of clear consistency in the pre-analytical area of sample processing.

Visual hemolysis Index for routine clinical chemistry laboratory parameters

Introduction: Interference substances can be significance source of error in the clinical laboratory measurements. Such errors may, in some cases represents hazards in patients. Hemolysis interferes with the Clinical Chemistry Analysis, which influence results of analytes and its leads to erroneous clinical interpretations. Hemolysis of Red cells is inevitable during the collection and handling of blood samples. In usual practice it is very difficult task to perform the rigorous analysis to evaluate interference in routine laboratories. Our objective was to assess effect of Hemolysis on routine biochemistry analysis Materials and Methods: The study followed the CLSI EP7-A2 protocol, sample were collected from 5 male subjects, aged 25-35 years, [0% (normal pool serum), serial dilution of different hemolysis grades [25% (1+coulter analyzer. The data was analyzed and ±5% difference from interferent free sample was taken as acceptable criteria.) ,50%(2+), 75%(3+) and 100%(4+)] of sample were prepared by spiking pooled serum with red cell lysate. The interference was studied up to concentrations of 0.5gm/dl for hemoglobin. Hemolysis indices graded on the basis of the appearance of the sample qualitatively ranked from “no visible hemolysis” to “4+ hemolysis. Aliquots were analyzed in triplicates using Beckman Results: The experimental design for laboratory method validation for hemolysis showed that Hemolysed samples starting from 1+ of hemolysis grade will be rejected for laboratory analysis for clinical chemistry parameters like Total bilirubin, Aspartate amino transferase, Alkaline Phosphatase, Gamaglutamyle transferase, Uric acid and serum potassium. While serum Creatinine hemolysed sample with 4+ hemolysis will not be accepted. Conclusions:Study results are useful for rejecting / to comment about the potentially misleading results in clinical chemistry parameters of clinical laboratory Keywords: Interferences, Hemolysis, Clinical laboratory analysis, Clini

Real World Clinical Experience with Oxbryta Therapy in Individuals with Sickle Cell Disease

Background: Oxbryta is a recently approved drug for patients with Sickle Cell Disease (SCD), which decreases HbS polymerization, thus reducing red cell hemolysis and increasing hemoglobin concentration. It was first available for Expanded Access May 9, 2019 and received fast track approval for commercial use by the FDA on November 25, 2019. Objective: This analysis aims to gain insights to the real word experience for patients with SCD who are prescribed Oxbryta, identify barriers to its use, and measure health outcomes. Methods: Electronic medical records of 54 patients with SCD who were prescribed Oxbryta from January until June 2020 were reviewed and evaluated for data regarding demographics, initiation of treatment, hematological response, side effects, dosage modifications and terminal discontinuations. Results: A total of 54 patients were prescribed Oxbryta in the study period who were 96% (52/54) HbSS, and 4% HbSβ0 (2/54); 46% (25/54) male and 54% (29/54) female, and a mean age of 41 years (range 21-70). At time of initiation, 52% (28/54) of patients were taking hydroxyurea (HU). Indication for initiation of therapy was anemia and hemolysis. In this cohort, 65% of patients (35/54) were able to access drug with a mean time to obtain drug of 45 days (range 0-118 days). The most common barriers to obtaining drug were: requiring a prior authorization or appeal 9% (5/54), incomplete patient follow up 7% (4/54), missing documentation for application 6% (3/54) and high patient co-pay 6% (3/54). Of the 35 patients who started Oxbryta, 46% (16/35) experienced at least 1 side effect with 31% (11/35) reporting multiple side effects. The most common side effects reported were gastrointestinal distress (abdominal pain, nausea, vomiting and/or diarrhea) in 23% (8/35), dermatologic effects (itching and/or rash) in 17% (6/35), and experiencing an acute VOC or increase in daily pain in 11% (4/35). Twenty nine percent (10/35) of patients required a dose modification to address side effects (half who successfully continued the drug on a modified dosage), an additional 23% (8/35) of patients terminated usage of Oxbryta in response to side effects. After two to four weeks of taking Oxbryta 63% of patients had a hemoglobin increase of at least 1g/dl or greater. Conclusion: Oxbryta produces a substantial rise in hemoglobin for persons living with sickle cell and high baseline levels of hemolysis in about 60 % of patients, similarly to the results from the HOPE trial. However, there are significant barriers to access that require close follow up in the immediate period following prescription of the drug in order to minimize delays in access. Furthermore, in our cohort we observed a higher incidence of side effects than the 23% (900mg) -26% (1500mg) reported in the pivotal trial that led to its approval. While some individuals were able to be continue therapy after dose modifications, most discontinued therapy because of side effects. Figure Disclosures Minniti: CLS Behring: Consultancy; Roche: Consultancy; Emmaus: Consultancy; Forma: Consultancy; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Teutona: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Other; Sodium Nitrate for SCD leg ulcers: Patents & Royalties.

No evidence of hemoglobin damage by SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) disease (COVID-19) has affected over 22 million patients worldwide as of August 2020. As the medical community seeks better understanding of the underlying pathophysiology of COVID-19, several theories have been proposed. One widely shared theory suggests that SARS-CoV-2 proteins directly interact with human hemoglobin (Hb) and facilitate removal of iron from the heme prosthetic group, leading to the loss of functional hemoglobin and accumulation of iron. Herein, we refute this theory. We compared clinical data from 21 critically ill COVID-19 patients to 21 non-COVID-19 acute respiratory distress syndrome (ARDS) patient controls, generating hemoglobin-oxygen dissociation curves from venous blood gases. This curve generated from the COVID-19 cohort matched the idealized oxygen-hemoglobin dissociation curve well (Pearson correlation R2=0.97, P<0.0001; a coefficient of variation of the root-mean-square deviation [CV(RMSD)] =7.3%). We further analyzed hemoglobin, total bilirubin, lactate dehydrogenase, iron, ferritin, and haptoglobin levels. For all analyzed parameters, patients with COVID-19 had similar levels compared to patients with ARDS without COVID-19. These results indicate that patients with COVID-19 do not exhibit any hemolytic anemia or a shift in the normal hemoglobin-oxygen dissociation curve. We therefore conclude that COVID-19 does not impact oxygen delivery through a mechanism involving red cell hemolysis and subsequent removal of iron from the heme prosthetic group in hemoglobin.

PLA membranes loaded with dba analogs. Controlled release study during hydrolitic degradation

In this research two dibenzylideneacetone (DBA) analogs compounds: (1E,4E)-1-(4-(dimethylamino)phenyl)-5-(4-methoxyphenyl)penta-1,4-dien-3-one (DBA-1) and (1E,4E)-1-(4-methoxyphenyl)-5-(4-nitrophenyl)penta-1,4-dien-3-one (DBA-2) were encapsulated in poly(lactic acid) (PLA) membranes. These DBA analogs can have several applications such as in the development of controlled drug release systems and tissue engineering. The membranes were elaborated by solvent casting. It was found that these fluorescent compounds have a small percentage of hemolysis in human blood red cells at concentrations between 200-500 µg/mL. Therefore, they can be considered not-toxic at these concentrations. The hydrolytic degradation of PLA membranes loaded with the DBA analogs was studied at a temperature of 37 °C under solutions at acid, neutral, and basic pH conditions for a maximum time of six weeks. The hydrolysis was monitored by measuring the loss of mass of the membranes, changes in pH environments, variations in the molecular weight of PLA matrix, and changes in surface morphology observed through Scanning the Electron Microscopy (SEM) technique. Applying UV-visible spectrophotometry, the amount released from the DBA analogs in the PLA membranes was determined during the degradation time, and finally, the release profile was obtained. It was observed employing SEM that the membranes presented a major degradation under basic pH conditions, with a higher percentage of release in an acid medium for both analogues of DBA studied

Surface Antigen 1 Is a Crucial Secreted Protein That Mediates Babesia microti Invasion Into Host Cells.

Babesia microti, a tick-borne intraerythrocytic zoonotic protozoan, causes most of human babesiosis in the world, and patients usually experience intermittent fever, fatigue, and chills, followed by a combination of additional symptoms and even death in severe cases. Unfortunately, there is no curable drug or effective vaccine available, and the mechanism of related virulence factors in invasion to host cells during the merozoite stage is unclear. Here, we evaluated a secreted protein annotated as B. microti surface antigen 1 (BmSA1) and identified from in vitro culture supernatant by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). BmSA1 fragment was expressed in Escherichia coli to prepare polyclonal antiserum. Western blot analysis revealed the existence of BmSA1 in the lysate of the parasites and the hemolysate of infected red blood cells (iRBCs). Laser confocal microscopy confirmed BmSA1 as a secreted protein with diffuse distribution around the parasites in red blood cells (RBCs). The adhesion capacity of BmSA1 against the host RBCs was tested by RBC binding assays using the recombinant BmSA1 protein (rBmSA1), which was shown to specifically bind to host RBCs. Further in vitro antiserum-neutralization test demonstrated that the growth of parasites could be significantly inhibited by the anti-BmSA1 antiserum. These results indicate that BmSA1 is a crucial factor for B. microti invasion into host RBCs with an important role in host-parasite interactions during the merozoite stage and has the potential use as a vaccine candidate due to its high secretion amount.

Prevalence of A2and A2B subgroups along with anti-A1antibody in patients and donor population and its clinical significance

BACKGROUND: The clinical significance of anti-A1antibody among A2and A2B subgroups is controversial. Few case reports have shown clinically significant anti-A1antibody at 37°C, causing hemolysis of donor red cells. The present study aims to find the prevalence of A2and A2B and the clinical significance of anti-A1antibody in these subgroups of A. MATERIALS AND METHODS: This prospective observational study was done in a tertiary care center in the eastern part of India. Gold standard test tube technique was used to determine blood group and distinguish A1from A2based on agglutination reaction with anti-A1lectin. Serum of A2and A2B subgroups was tested with A1red cells to confirm the presence of anti-A1antibodies. Further testing of anti-A1antibody was also performed to determine whether the antibody is IgM or IgG and of any clinical significance. RESULTS: A total of 2874 blood samples of both patients and donors were analyzed. Among A blood group, 98.4% were A1 and, whereas 1.6% were A2, whereas among AB blood group, A1B and A2B were 93.9% and 6.1%, respectively. Only 3 out of 13, A2B patients (23.07%) showed the presence of anti-A1antibody but without any clinical significance. CONCLUSIONS: Our study could not find any clinically significant anti-A1antibodies. Thus, we insinuate any requirement of mandatory testing for the determination of A1, A2,and anti-A1antibodies. Subtyping will help in the use of A2donor for renal transplant in O and B group patients and the use of A2platelets also in O and B group patients.

Quality of red blood cell and platelet concentrates after transportation by a pneumatic tube system

Background and objectivesPneumatic tube systems (PTSs) are used in some hospitals to transport patients’ specimens. We investigated the feasibility of applying PTS for blood products for transfusion in our hospital.Materials and methodsWe transported red blood cells (RBCs) and platelet concentrates (PCs) by the PTS and measured biomarkers such as red cell haemolysis and platelet dysfunction before and after one‐way transportation, in addition, after one‐way transportation for three consecutive times by PTS.ResultsUsing 25 RBCs, K, free Hb, Hb, LD, AST and pH were compared before and after transportation. There were no significant differences. Haemolysis rates ranging between 0·13% and 0·35% were acceptable under the guideline. Using 6 PCs, platelet concentration, aggregation and activation function were examined. There were no significant differences before and after transportation. Positive rates of CD62P ranging between 3·1% and 11·4% showed no remarkable changes after the transportation. After the third consecutive transportation, LD and AST were slightly increased. However, haemolysis rate changed from 0·16% to 0·21%, again stayed in guideline level. As for platelet products, platelet aggregation activities to collagen (approximately 80%) did not change significantly.ConclusionOur study demonstrated that PTS is feasible for transporting them since one‐ and three‐time transportation by PTS does not appear to influence the important quality of RBCs and PCs. In our hospital, we have applied PTS for the transportation of blood products without major troubles such as loss of products or transfusion‐related adverse events.

Assessment of Blood Storage Effect Using CPDA-1 on Packed Cell Volume, Oxyhaemoglobin and Methaemoglobin in Different ABO/Rhesus Blood Types

Aims: The aim of this study is to determine the effect of blood storage using CPDA-1 on packed cell volume, methaemoglobin and oxyhaemoglobin in different ABO/Rhesus blood types donated by some residents of Port Harcourt, Rivers State, Nigeria.&#x0D; Study Design: This is a comparative study aimed at evaluating the effect of storage on the levels of methaemoglobin, oxyhaemoglobin and packed cell volume using CPDA-1. A total of eight donors were recruited with each sample obtained from the eight (8) known blood groups A+,B+,O+,AB+, A-,B-,O-,AB- and analysis of samples were in triplicate. The donors were adult males with age ranging from 35-45 years and they were apparently healthy and free from transfusion transmissible infections. The different blood group samples were stored for 30 days and samples for analysis were collected at 5 days interval.&#x0D; Place and Duration of Study: The study was conducted in Port Harcourt, Rivers State, Nigeria. All blood donors were residents of Port Harcourt. Blood donated was stored at Military Hospital Blood Bank, Port Harcourt, in a blood bag of 450 ml containing 63 ml of citrate phosphate dextrose adenine-1 (CPDA-1). The analysis was carried out at Rivers State University, Post Graduate Laboratory within March 1st to May 27th, 2019.&#x0D; Methodology: A total of eight (8) different ABO/Rhesus blood types (A+,B+,AB+,O+,A-,B-,AB- and O-) were collected and stored using a blood bank refrigerator at temperature of 4°C. Day 0 was taken to be control and 5 days intervals in-between to day 30 acted as the test. Packed cell volume was estimated using micro-haematocrit method while oxyhaemoglobin and methaemoglobin levels were estimated spectrophotometrically as described by Evelyn and Malloy.&#x0D; Results: The result showed a significant decrease in mean packed cell volume, oxyhaemoglobin and methaemoglobin levels compared to a higher mean of these parameters in the control; and these differences were statistically significant (p&lt;0.05) across all blood groups under study. The decrease in values were as a result of haemolysis that occurs during storage.&#x0D; Conclusion: Storage of blood irrespective of the blood group type using CPDA-1 for 30 days indicates that there are “storage lesions”. This is attributed to red cell haemolysis and ageing of red blood cells. In general, all blood types showed no significant difference in their haematological (packed cell volume, methaemoglobin, oxyhaemoglobin) characteristic deterioration or storage lesion based on blood type differences. It is therefore necessary to state that storage lesion characteristics are the same irrespective of the blood type, and that fresh blood be transfused, and if blood is stored, prolonged storage beyond 10 days should be avoided.

Prevalence and specificity of clinically significant red cell alloantibodies in pregnant women - a study from a tertiary care hospital in Southeast Michigan.

BackgroundMaternal red cell IgG antibodies can cross the placenta and cause hemolysis of fetal red cells in case of antigenic differences between maternal and fetal RBCs, leading to hemolytic disease of the fetus and newborn (HDFN). Although the incidence of anti-D associated HDFN has drastically reduced with Rh immune globulin prophylaxis, HDFN due to other maternal red cell alloantibodies still remains a concern. Prevalence and specificities of clinically significant red cell alloantibodies in pregnant females have rarely been reported in the USA.MethodsA retrospective chart review was conducted to determine the prevalence and specificity of clinically significant red cell alloantibodies in pregnant females who delivered at Beaumont Hospital Royal Oak between May 1, 2017 and December 31, 2017. A total of 4548 pregnant females were screened using electronic medical records. One female above 50 years age and two females with invalid ABO type were excluded from the study per IRB approved protocol. The remaining 4545 pregnant females with a valid ABO/RhD type and valid red cell antibody screen were included.ResultsOut of the 4545 included females, 440 had a positive red cell antibody screen. Of these 440 females, 34 had clinically significant alloantibodies, giving an overall prevalence of 0.74%. Anti-E was the most frequently identified significant alloantibody followed by anti-K. The most prevalent significant alloantibodies in RhD positive and RhD negative females were anti-E and anti-K, respectively. Significant association (p-value <0.001) was found between RhD type and the presence of clinically significant alloantibodies amongst females with positive antibody screen.ConclusionOur study aims to reiterate the importance of maternal red cell antibody screening during early pregnancy to help identify and manage high-risk pregnancies. Minimizing the exposure of childbearing age females to incompatible red cell antigens through unnecessary transfusions can help reduce the incidence of red cell alloimmunization and the risk of HDFN.

Effect of Sterigmatocystin or Aflatoxin Contaminated Feed on Lipid Peroxidation and Glutathione Redox System and Expression of Glutathione Redox System Regulatory Genes in Broiler Chicken.

Authors studied the effect of sterigmatocystin from infected corn (STC), purified sterigmatocystin (PSTC), and aflatoxin B1 from infected corn (AFB1) on lipid peroxidation and glutathione redox parameters, including the expression of their encoding genes in a sub-chronic (14 days) trial. A total of 144 three-week-old cockerels was divided into four experimental groups (n = 36 in each). Control feed was contaminated with STC or PSTC (1590 µg STC/kg or 1570.5 µg STC/kg feed), or with AFB1 (149.1 µg AFB1/kg feed). Six birds from each group were sampled at day 1, 2, 3, 7 and 14 of mycotoxin exposure. As parameters of lipid peroxidation, conjugated dienes (CD) and trienes (CT) were measured in the liver, while malondialdehyde (MDA) concentration was determined in blood plasma, red blood cell hemolysate and liver. Reduced glutathione (GSH) concentration and glutathione peroxidase (GPx) activity were determined in the same samples, and expression of glutathione peroxidase 4 (GPX4), glutathione synthetase (GSS) and glutathione reductase (GSR) genes was measured by RT-PCR in the liver. STC, PSTC or AFB1 caused a slight, but not significant, increase in CD and CT levels; however, in the case of MDA, no increase was found in the liver. Glutathione redox system was activated in the liver by AFB1, but less markedly by STC/PSTC. PSTC and AFB1 resulted in a higher expression of GPX4, while GSS expression was down-regulated by AFB1 on day 1, but up-regulated by STC on day 2 and by both mycotoxins on day 7. However, on day 14, GSS expression was down-regulated by PSTC. Expression of GSR was low on day 1 in AFB1 and PSTC groups, but later it was up-regulated by AFB1. The observed changes regarding gene expression strengthen the hypothesis that the mild oxidative stress, caused by the applied STC doses, activates the glutathione redox system of broiler chickens.

Inhibition of Sickle Hemoglobin Polymerization in Patients with Sickle Cell Anemia

Red blood cell sickling and hemolysis due to polymerization of sickle hemoglobin upon deoxygenation is a hallmark abnormality in those with sickle cell

Long-Term Effects of Ochratoxin A on the Glutathione Redox System and Its Regulation in Chicken.

The purpose of this study was to evaluate the effect of three-weeks ochratoxin A (OTA) exposure on some lipid peroxidation parameters, reduced glutathione concentration and glutathione-peroxidase activity, as well as expression of oxidative stress response-related (KEAP1, NRF2) and glutathione system (GPX3, GPX4, GSS, GSR) genes in chickens. Three levels of exposure (106, 654 and 1126 μg/kg feed) were applied. The results showed that OTA initiated free radical formation, which was suggested by the increase in the malondialdehyde content in the liver and kidney, which was more marked in the liver, depending on the length of exposure and dose. Reduced glutathione concentration increased as an effect of the highest OTA dose in blood plasma and in liver, but not in red blood cell hemolysates and the kidney. Glutathione peroxidase activity did not change in the blood and showed increasing tendency in the liver, and significant increase in the kidney. Expression of KEAP1 gene showed up-regulation in the liver, and down-regulation in the kidney, but overexpression of NRF2 gene was found in the liver and kidney at the highest dose. However, down-regulation of Nrf2 dependent genes, GPX3, GPX4, GSS and GSR, suggested an improper antioxidant response at the protein level, thus oxidative stress occurred, even at the dose of the EU regulatory limit for poultry diets.

A phase 1/2 ascending dose study and open-label extension study of voxelotor in patients with sickle cell disease

AbstractNew treatments directly targeting polymerization of sickle hemoglobin (HbS), the proximate event in the pathophysiology of sickle cell disease (SCD), are needed to address the severe morbidity and early mortality associated with the disease. Voxelotor (GBT440) is a first-in-class oral therapy specifically developed to treat SCD by modulating the affinity of hemoglobin (Hb) for oxygen, thus inhibiting HbS polymerization and downstream adverse effects of hemolytic anemia and vaso-occlusion. GBT440-001 was a phase 1/2 randomized, double-blind, placebo-controlled, single and multiple ascending dose study of voxelotor in adult healthy volunteers and patients with SCD, followed by a single-arm, open-label extension study. This report describes results of voxelotor (500-1000 mg per day) in patients with sickle cell anemia. The study evaluated the safety, tolerability, pharmacokinetic, and pharmacodynamic properties of voxelotor and established proof of concept by improving clinical measures of anemia, hemolysis, and sickling. Thirty-eight patients with SCD received 28 days of voxelotor 500, 700, or 1000 mg per day or placebo; 16 patients received 90 days of voxelotor 700 or 900 mg per day or placebo. Four patients from the 90-day cohort were subsequently enrolled in an extension study and treated with voxelotor 900 mg per day for 6 months. All patients who received multiple doses of voxelotor for ≥28 days experienced hematologic improvements including increased Hb and reduction in hemolysis and percentage of sickled red cells, supporting the potential of voxelotor to serve as a disease-modifying therapy for SCD. Voxelotor was well tolerated with no treatment-related serious adverse events and no evidence of tissue hypoxia. These trials were registered at www.clinicaltrials.gov as #NCT02285088 and #NCT03041909.

Fe-based alloy for stent application inhibits platelet activation

Bioresorbable polymer stents have been used to provide a transient scaffold after coronary angioplasty. However, an increase in stent thrombosis has been observed. A current challenge is thus to develop new bioresorbable stents combining optimized mechanical and biodegradation properties together with limited thrombogenicity. In this context Fe-based alloys are amongst the good candidates for stent manufacture. We aimed to explore in vitro blood compatibility of a new Fe-based alloy. Human whole blood was incubated for 60 min with either the Fe-based alloy, pure iron, cobalt-chromium (CoCr) alloy (composing the bare metal stent used as a reference) or magnesium (Mg) alloy (composing a bioresorbable stent newly on the market). After centrifugation, haemolysis was calculated based on the optical density (540 nm) of the supernatant. For platelet activation assays, human washed platelets were incubated for 60 min with each metal before measuring their reactivity to a platelet agonist by flow cytometry, using CD62P and PAC-1 antibodies. In addition, phosphorylation of PKC substrates was evaluated by western blot. None of the alloys induced significant red cells haemolysis. In addition, Co-Cr did not affect CD62P exposure and PAC-1 presence at platelet surface upon thrombin (0.03 to 0.3 U/ml) stimulation. In contrast, Fe-based alloy and pure iron decreased significantly platelet response to agonist, as well as Mg but to a lesser extent. A drastic inhibition of the phosphorylation state of PKC substrates was also observed with the Fe-based alloy after activation with thrombin. Since similar inhibitory effects were obtained when using a conditioned-medium (incubated for 60 min with the alloy), we postulated that its bio-corrosion might release components counteracting platelet activation. The Fe-based alloy seems to be a promising platform for next-generation stent technologies.

Blood-compatible, stable micelles of sodium alginate – Curcumin bioconjugate for anti-cancer applications

The bioconjugate of alginate and curcumin (AA-CUR) was synthesized in a simple, one-step process and used to prepare the stable calcium cross-linked spherical micelles serving as a delivery vehicle for curcumin. Above its critical micelle concentration (0.6 mg/ml) AA-CUR forms colloidally stable micelles of ca. 200 nm. Prolonged, well controlled release of curcumin was observed from the AA-CUR micelles cross-linked with calcium ions for 5 h under the physiological conditions. To assess the safety of applying the bioconjugate into the bloodstream for possible anti-cancer applications, the interaction between AA-CUR and cells isolated from human blood was analyzed. No red cells aggregation or hemolysis was observed. AA-CUR was also shown to have no significant cytotoxicity to the human Peripheral Blood Mononuclear Cells (PBMC) isolated from peripheral blood of healthy donors and to the mouse primary brain endothelial cells. To evaluate the efficiency of the obtained bioconjugate in the anti-cancer therapy, the micellar solution of AA-CUR was tested against various cancer cells lines: mammary carcinoma 4T1, melanoma B16F10 and colon carcinoma CT26-CEA and MC38-CEA. The bioconjugate at the concentration of 0.7 mg/ml decreased the viability of cancer cells by ca. 80%. The cellular uptake of AA-CUR was rapid and the highest micelles accumulation was detected within 1 h after treatment. The AA-CUR micellar system can serve as an effective and safe delivery vehicle for curcumin.

The role of redox-dependent mechanisms in heme release from hemoglobin and erythrocyte hemolysates

Toxicity mediated by free heme has emerged as an important element of end organ injuries and adverse outcomes in critically ill disease states. Free heme is thought to be derived from oxidative denaturation of free hemoglobin, secondary to red cell hemolysis. In this study, we evaluated the ability of oxidants (H2O2, nitrite, peroxynitrite and hypochlorous acid) formed during inflammation to cause heme release from purified hemoglobin and hemolysates, at pH 7.4 and 6.8. Supraphysiological concentrations of nitrite, peroxynitrite or hypochlorous acid were required to cause appreciable heme release from either free hemoglobin or hemolysates. However, H2O2 administered as a bolus or generated in situ, was more potent at promoting free heme release with free hemoglobin. With hemolysates, only in situ H2O2 formation resulted in significant free heme release. In all cases, free heme release was higher at lower pH and required oxidation of ferrous heme, but was not dependent on ferrylHb formation. Moreover, ligating ferric heme with cyanide or blocking the β93Cys did not prevent, but in fact increased free heme release. The salient observations from this study are that free heme release is likely mediated by continuous generation of H2O2 versus other heme oxidants, and facilitated at low pH.