Development and validation of an isoform-independent monoclonal antibody–based ELISA for measurement of lipoprotein(a)

The study aims were to develop a new isoform-independent enzyme-linked immunoassay (ELISA) for the measurement of lipoprotein(a) [Lp(a)], validate its performance characteristics, and demonstrate its accuracy by comparison with the gold-standard ELISA method and an LC-MS/MS candidate reference method, both developed at the University of Washington. The principle of the new assay is the capture of Lp(a) with monoclonal antibody LPA4 primarily directed to an epitope in apolipoprotein(a) KIV₂ and its detection with monoclonal antibody LPA-KIV9 directed to a single antigenic site present on KIV₉. Validation studies were performed following the guidelines of the Clinical Laboratory Improvement Amendments and the College of American Pathologists. The analytical measuring range of the LPA4/LPA-KIV9 ELISA is 0.27–1,402 nmol/L, and the method meets stringent criteria for precision, linearity, spike and recovery, dilutability, comparison of plasma versus serum, and accuracy. Method comparison with both the gold-standard ELISA and the LC-MS/MS method performed in 64 samples with known apolipoprotein(a) isoforms resulted in excellent correlation with both methods (r=0.987 and r=0.976, respectively). Additionally, the variation in apolipoprotein(a) size accounted for only 0.2% and 2.2% of the bias variation, respectively, indicating that the LPA4/LPA-KIV9 ELISA is not affected by apolipoprotein(a) size polymorphism. Peptide mapping and competition experiments demonstrated that the measuring monoclonal antibodies used in the gold-standard ELISA (a-40) and in the newly developed ELISA (LPA-KIV9) are directed to the same epitope, ⁴⁰⁷⁶LETPTVV⁴⁰⁸², on KIV₉. In conclusion, no statistically or clinically significant bias was observed between Lp(a) measurements obtained by the LPA4/LPA-KIV9 ELISA and those obtained by the gold-standard ELISA or LC-MS/MS, and therefore, the methods are considered equivalent.     

Encapsulation of Alpha-1 antitrypsin in PLGA nanoparticles: In Vitro characterization as an effective aerosol formulation in pulmonary diseases

ABSTRACT: BACKGROUND: Alpha 1- antitrypsin (alpha1AT) belongs to the superfamily of serpins and inhibits different proteases. alpha1AT protects the lung from cellular inflammatory enzymes. In the absence of alpha1AT, the degradation of lung tissue results to pulmonary complications. The pulmonary route is a potent noninvasive route for systemic and local delivery. The aerosolized alpha1AT not only affects locally its main site of action but also avoids remaining in circulation for a long period of time in peripheral blood. Poly (D, L lactide-co glycolide) (PLGA) is a biodegradable and biocompatible polymer approved for sustained controlled release of peptides and proteins. The aim of this work was to prepare a wide range of particle size as a carrier of protein-loaded nanoparticles to deposit in different parts of the respiratory system especially in the deep lung. Various lactide to glycolide ratio of the copolymer was used to obtain different release profile of the drug which covers extended and rapid drug release in one formulation. RESULTS: Nonaqueous and double emulsion techniques were applied for the synthesis of nanoparticles. Nanoparticles were characterized in terms of surface morphology, size distribution, powder X-ray diffraction (XRD), encapsulation efficiency, in vitro drug release, FTIR spectroscopy and differential scanning calorimetry (DSC). To evaluate the nanoparticles cytotoxicity, cell cytotoxicity test was carried out on the Cor L105 human epithelial lung cancer cell line. Nanoparticles were spherical with an average size in the range of 100 nm to 1mu. The encapsulation efficiency was found to be higher when the double emulsion technique was applied. XRD and DSC results indicated that alpha1AT encapsulated in the nanoparticles existed in an amorphous or disordered-crystalline status in the polymer matrix. The lactic acid to glycolic acid ratio affects the release profile of alpha1AT. Hence, PLGA with a 50:50 ratios exhibited the ability to release %60 of the drug within 8, but the polymer with a ratio of 75:25 had a continuous and longer release profile. Cytotoxicity studies showed that nanoparticles do not affect cell growth and were not toxic to cells. CONCLUSION: In summary, alpha1AT-loaded nanoparticles may be considered as a novel formulation for efficient treatment of many pulmonary diseases.     

How is the relationship between TWIST-1 and BCR-ABL1 gene expressions in chronic myeloid leukaemia patients?

Background: The activation and increased expression of BCR-ABL1 lead to malignant chronic myelogenous leukaemia (CML) cells, as well as the resistance to antitumour agents and apoptosis inducers. Moreover, TWIST-1 protein is a prognostic factor of leukemogenesis, and its level is raised in CML patients with cytogenetic resistance to imatinib. So, there is a likely relationship between BCR-ABL1 and TWIST-1 genes.

Objective: The aim of the study was to assess the relationship between TWIST-1 and BCR-ABL1 expressions.

Methods: Peripheral blood samples were obtained from 44 CML patients under treatment and also from ten healthy subjects as normal controls. The expression of TWIST-1 and BCR-ABL1 genes was measured using real-time PCR, and ABL1 was used as the reference gene. The gene expression was evaluated by REST software.

Results: The expression levels of TWIST-1 and BCR-ABL1 genes in CML patients was changed 40.23?±?177.75-fold and 6?±?18-fold, respectively.

Discussion: No significant relationship was observed between the expressions of TWIST-1 and BCR-ABL1 genes. All patients with TWIST-1 expression levels?≥100-fold had failure of response to treatment.

Conclusion: The probability of the relationship between BCR-ABL1 and TWIST-1 is still debatable, and the average of TWIST-1 expression has been higher in patients without response to treatment. Definitive conclusion needs further investigations.     

A two-scale approach for CFD modeling of endovascular Chemofilter device

Two-scale CFD modeling is used to design and optimize a novel endovascular filtration device for removing toxins from flowing blood. The Chemofilter is temporarily deployed in the venous side of a tumor during the intra-arterial chemotherapy in order to filter excessive chemotherapy drugs such as Doxorubicin from the blood stream. The device chemically binds selective drugs to its surface thus filtering them from blood, after they have had the effect on the tumor and before they reach the heart and other organs. The Chemofilter consists of a porous membrane made of microscale architected materials and is installed on a structure similar to an embolic protection device. Simulations resolving the microscale structure of the device were carried out to determine the permeability of the microcell membrane. The resulting permeability coefficients were then used for macroscale simulations of the flow through the device modeled as a porous material. The microscale simulations indicate that greater number of microcell layers and smaller microcell size result in increased pressure drop across the membrane, while providing larger surface area for drug binding. In the macroscale simulations, the study of idealized prototypes show that the pressure drop can be reduced by increasing the membrane’s tip angle and by decreasing the number of membrane’s sectors. Such design, however, can conversely affect the overall drug binding. By decreasing the concentration of toxins in the cardiovascular system, the drug dosage can be increased while side effects are reduced, thus improving the effectiveness of treatment.     

Theoretical study of chemisorption of cyanuric fluoride and S-triazine on the surface of Al-doped graphene

We studied the adsorption of cyanuric fluoride (CF) and s-triazine (ST) molecules on the surface of pristine as well as Al-doped graphenes using density functional theory calculations. Our results reveal low adsorption on the surface of pristine graphene; but by modification of surface using aluminium, resulted Al-doped graphene becomes more reactive towards both CF and ST molecules. We aimed to focus on the adsorption energy, electronic structure, charge analysis, density of state and global indices of each system upon adsorption of CF and ST molecules on the above-mentioned surfaces. Our calculated adsorption energies for the most stable position configurations of CF and ST on Al-doped graphene were ?76.53 kJ mol^?1 (?57.45 kJ mol^?1 BSSE corrected energy) and ?115.55 kJ mol^?1 (?86.87 kJ mol^?1 BSSE corrected energy), respectively, which point to the chemisorption process. For each CF and ST molecule, upon adsorption on the surface of Al-doped graphene, the band gap of HOMO-LUMO was reduced considerably and it becomes a p-type semiconductor, whereas there is no hybridisation between the above-mentioned molecules and pristine graphene.     

Rheumatoid Arthritis Susceptibility Is Associated with the KIR2DS4-Full of Killer-Cell Immunoglobulin-Like Receptor Genes in the Lur Population of Iran

Background:The pathophysiology underlying the progression and development of autoimmune conditions, such as Rheumatoid Arthritis (RA), is a result of dysregulations of the immune system. Research has explored the genetic alterations present in RA; however, limited studies have examined the role of Killer cell Immunoglobulin-like Receptors (KIR) and Human Leukocyte Antigen (HLA) molecules in RA. Therefore, the aim of this study was to examine KIR genes, their HLA ligands, and KIR-HLA compounds in patients with RA.Methods:In this case-control study, a total of 50 patients with RA and 100 healthy individuals were enrolled. DNA samples were evaluated using PCR with sequence specific Primers (PCR-SSP). Odds ratio (OR) with a 95% confidence interval (CI) were reported.Results:Among the KIR genes examined, KIR2DLA (p= 0.0255, OR= 0.389, 95% CI= 0.210-0.722) and KIR2DS4-full (p< 0.0001, OR= 6.163, 95% CI= 3.174-11.968) were observed to have a statistically significant correlation with disease susceptibility to RA. As an inhibitory gene, KIR2DLA was observed to have a protective effect against RA while KIR2DS4-full as an activating gene, was found to increase risk for RA. No significant associations were found between any of the other KIR genotypes, HLA ligands, or KIR-HLA compounds examined in this study to RA susceptibility. Conclusion:In this study of RA in the Lur population of Iran, KIR2DS4-full was observed to increase susceptibility to RA, while KIR2DL5A was found to act as a protecting factor based on both the cross Table and regression analyses. Further research should focus on repeating this study in additional populations.Key Words: HLA, KIR, NK cells, Rheumatoid Arthritis     

Metformin treatment has no beneficial effect in a dose-response survival study in the SOD1(G93A) mouse model of ALS and is harmful in female mice

Background

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurological disorder characterized by selective degeneration of upper and lower motor neurons. The primary triggers for motor neuron degeneration are unknown but inflammation, oxidative stress and mitochondrial defects have been identified as potential contributing factors. Metformin is an anti-type II diabetes drug that has anti-inflammatory and anti-oxidant properties, can bring about mitochondrial biogenesis and has been shown to attenuate pathology in mouse models of Huntington''s disease and multiple sclerosis. We therefore hypothesized that it might increase survival in the SOD1^G93A murine model of ALS.

Methodology/Principal Findings

Treatment of male and female SOD1^G93A mice (n = ≥6 per sex) with 2 mg/ml metformin in the drinking water from 35 days, resulted in a significant increase in motor unit survival, as measured by in vivo electrophysiology at 100 days, in male EDL muscles (24+/−2 vs. 14+/−2 motor units, p<0.005) and female TA muscles (21+/−1 vs. 15+/−2 motor units, P = 0.0134). We therefore continued to test the effect of 0.5, 2 and 5 mg/ml metformin in the drinking water from 35 days on disease onset and progression (identified by twice weekly determination of weight and neurological score) as well as survival in male and female SOD1^G93A mice (n = ≥14 per sex). Results for all groups were compared using Kaplan-Meier time to event analyses. In this survival study, metformin was unable to reduce pathology at any dose and had an unexpected dose-dependent negative effect on the onset of neurological symptoms (P = 0.0236) and on disease progression (P = 0.0362) in female mice.

Conclusions/Significance

This study suggests that metformin is a poor candidate for clinical trial in ALS patients and that the possibility of harmful effects of metformin in female ALS patients with type II diabetes should be investigated.     

Lymphopenia in COVID‐19: Therapeutic opportunities

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is uncontrollably spread all over the world. The host immune responses strongly try to confront it with all the potential cells and cytokines. With chronically condition of SARS‐CoV‐2, natural killer cells and T cells become exhausted and decreasing their count leads to lymphopenia. Inability to eradicate the infected organ makes hyperinitiation of the immune system, which releases the excessive inflammatory cytokines to compensate the exhausted one as well as the low lymphocytes counts; it consequently leads to the cytokine storm syndrome. These mechanisms and the potential therapeutic targeting are discussed in this paper.     

Computational modeling of drug transport and mixing in the chemofilter device: enhancing the removal of chemotherapeutics from circulation

Biomechanics and Modeling in Mechanobiology - Intra-arterial chemotherapy (IAC) is the preferred treatment for non-resectable hepatocellular carcinoma. A large fraction of IAC drugs, e.g.,...