Hemolysis of Human Red Blood Cells by Riboflavin-Cu(II) System: Enhancement by Azide

Photoactivated riboflavin in the presence of Cu(II) generates reactive oxygen species (ROS) which can hemolyze human red blood cells (RBC). In the present work we examined the effect of sodium azide (NaN3) on RBC in the presence of riboflavin and Cu(II). The addition of NaN3 to the riboflavin-Cu(II) system enhanced K+ loss and hemolysis. The extent of K+ loss and hemolysis were time and concentration dependent. Bathocuproine, a Cu(I)-sequestering agent, inhibited the hemolysis completely. Among various free radical scavengers used to identify the major ROS involved in the reaction, thiourea was found to be the most effective scavenger. Thiourea caused almost 85% inhibition of hemolysis suggesting that *OH is the major ROS involved in the reaction. Using spectral studies and other observations, we propose that when NaN3 is added to the riboflavin-Cu(II) system, it inhibits the photodegradation of riboflavin resulting in increased *OH generation. Also, the possibility of azide radical formation and its involvement in the reaction could not be ruled out.     

Losartan inhibits cellular uptake of oxidized LDL by monocyte-macrophages from hypercholesterolemic patients

Angiotensin II (Ang II) and oxidized LDL (Ox-LDL) are risk factors for atherosclerosis, and both of them contribute to macrophage cholesterol accumulation, the hallmark of early atherosclerosis. As Ang II was shown to increase macrophage uptake of Ox-LDL, we investigated the effect of losartan, an Ang II receptor antagonist with antiatherogenic properties, on the cellular uptake of Ox-LDL by human monocyte-derived macrophages (HMDM) from hypercholesterolemic patients. Eight normotensive hypercholesterolemic patients were treated with losartan (50 mg/day) for a period of 4 weeks. Losartan therapy did not significantly affect the degradation of native LDL by the patients' HMDM. However, losartan therapy significantly reduced HMDM uptake of Ox-LDL as shown by a 78% reduction in Ox-LDL cell-association and a 21% reduction in Ox-LDL degradation. CD36 (an Ox-LDL receptor) mRNA expression in HMDM obtained after losartan treatment was decreased by 54% compared to HMDM obtained before treatment. The ability of losartan to inhibit HMDM CD36 mRNA expression and, hence, Ox-LDL uptake and macrophage foam cell formation is probably related to the blockage of Ang II binding to the cell surface and thus to the prevention of Ang II atherogenic effects.     

Magneto‐optical characteristics of human sperms: normal and deformed

In this study we report on magnetic orientation of human sperms. Samples were taken from 17 donors. Normal human sperms became oriented with their long axis perpendicular to the magnetic field (1 T maximum). Total orientation was achieved with magnetic field of about 1 T, while for abnormal sperms the magnetic behavior was different. The dependence of the measured degree of orientation on the intensity of the magnetic field was in good agreement with the theoretical equation for the magnetic orientation of diamagnetic substances. As a result of a numerical analysis based on the equation, the anisotropic diamagnetic susceptibility of normal sperm was found to be Δ_χ = 8 × 10^–20 J/T². The degree of orientation was influenced by the alterations in the shape of the head, body or the tail. It has been suggested that the DNA in the sperm head retain the strong magnetic anisotropy to counterbalance the magnetic anisotropy retained by flagellum microtubules. Recent studies demonstrated a well‐defined nuclear architecture in human sperm nucleus, where the head morphology has significant correlation with sperm chromatin structure assay SCSA. Then, as the methods to evaluate SCSA can be difficult and expensive our simple magnetic orientation technique can be an alternative to diagnose alteration in DNA. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Magnetic behavior of human erythrocytes at different hemoglobin states

The effect of a static magnetic field on human erythrocytes at different hemoglobin states (normal, oxidized and reduced hemoglobin) was investigated. Three different blood samples, normal, iron deficiency anemic and beta thalassemia minor, were studied. Measurements of the magnetization curves of the erythrocytes for all blood samples in all states showed diamagnetic behavior; however, oxidation was found to enhance this behavior. These measurements have also shown that the normal and iron deficiency samples in the reduced states exhibit a less diamagnetic response in comparison with the normal state. This result indicates that the reduction process gave rise to a paramagnetic component of the magnetization. Analysis of the measured paramagnetic behavior, using a Brillouin function, gave an effective magnetic moment of 8 muB per reduced hemoglobin molecule for both normal and anemic samples. This result shows that both anemic and normal blood have similar magnetic behavior and the only difference is the number of hemoglobin molecules per erythrocyte. For the beta thalassemia minor blood sample, magnetic measurements showed that both the normal and reduced states have almost the same diamagnetic behavior. However, this diamagnetic response is less than that for the normal state of the iron deficiency anemic sample. This result may indicate a low oxygen intake for the blood in the normal state for the beta thalassemia minor blood. All magnetic measurements were made using a vibrating sample magnetometer using field steps of 0.001 T from 1 T to -1 T.     

In vitro and in silico assessment of the developability of a designed monoclonal antibody library

Despite major advances in antibody discovery technologies, the successful development of monoclonal antibodies (mAbs) into effective therapeutic and diagnostic agents can often be impeded by developability liabilities, such as poor expression, low solubility, high viscosity and aggregation. Therefore, strategies to predict at the early phases of antibody development the risk of late-stage failure of antibody candidates are highly valuable. In this work, we employ the in silico solubility predictor CamSol to design a library of 17 variants of a humanized mAb predicted to span a broad range of solubility values, and we examine their developability potential with a battery of commonly used in vitro and in silico assays. Our results demonstrate the ability of CamSol to rationally enhance mAb developability, and provide a quantitative comparison of in vitro developability measurements with each other and with more resource-intensive solubility measurements, as well as with in silico predictors that offer a potentially faster and cheaper alternative. We observed a strong correlation between predicted and experimentally determined solubility values, as well as with measurements obtained using a panel of in vitro developability assays that probe non-specific interactions. These results indicate that computational methods have the potential to reduce or eliminate the need of carrying out laborious in vitro quality controls for large numbers of lead candidates. Overall, our study provides support to the emerging view that the implementation of in silico tools in antibody discovery campaigns can ensure rapid and early selection of antibodies with optimal developability potential.     

Optimizing selectivity of anion hydrophobic multimodal chromatography for purification of a single‐chain variable fragment

Single‐chain variable fragments (scFv) are widely used in several fields. However, they can be challenging to purify unless using expensive Protein L‐based affinity adsorbents or affinity tags. In this work, a purification process for a scFv using mixed‐mode (MM) chromatography was developed by design of experiments (DoE) and proteomics for host cell protein (HCP) quantification. Capture of scFv from human embryonic kidney 293 (HEK293) cell feedstocks was performed by hydrophobic charge induction chromatography (MEP HyperCel?), whereafter polishing was performed by anion hydrophobic MM chromatography (Capto Adhere?). The DoE designs of the polishing step included both binding and flow‐through modes, the latter being the standard mode for HCP removal. Chromatography with Capto Adhere? in binding‐mode with elution by linear salt gradient at pH 7.5 resulted in optimal yield, purity and HCP reduction factor of 98.9 > 98.5%, and 14, respectively. Totally, 258 different HCPs were removed, corresponding to 84% of identified HCPs. The optimized conditions enabled binding of the scFv to Capto Adhere? below its theoretical pI, while the majority of HCPs were in the flow‐through. Surface property maps indicated the presence of hydrophobic patches in close proximity to negatively charged patches that could potentially play a role in this unique selectivity.     

Transfusion regimen in beta thalassemia major using magnetic measurement techniques

The magnetic behaviour of erythrocytes from patients suffering from beta thalassemia major was investigated. All samples exhibited diamagnetic behaviour. Measurements were made before and after blood transfusion treatment. We suggest that the amount of blood given to the patient should be controlled in a way that the diamagnetic susceptibility after blood transfusion should be equal to that for the normal erythrocytes. In this way a specific range of haemoglobin level (Hgb) should be maintained when the patient is subjected to long-term hypertransfusional treatment.