scholarly journals P1101A BIFUNCTIONAL ADSORBER PARTICLE FOR THE REMOVAL OF HYDROPHOBIC UREMIC TOXINS FROM WHOLE BLOOD OF RENAL FAILURE PATIENTS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Marieke Sternkopf ◽  
Thimoteus Speer ◽  
Claudia Göttsch ◽  
Vera Jankowski ◽  
Joachim Jankowski ◽  
...  
Keyword(s):  
Adsorption Capacity ◽  
Binding Affinity ◽  
Whole Blood ◽  
Uremic Toxins ◽  
In Vivo Studies ◽  
Uremic Toxin ◽  
High Binding ◽  
High Binding Affinity

Abstract Background and Aims Hydrophobic uremic toxins accumulate in patients with chronic kidney disease, contributing to a highly increased cardiovascular risk. The clearance of these uremic toxins using current hemodialysis techniques is limited due to their hydrophobicity and their high binding affinity to plasma proteins. Adsorber techniques may be an appropriate alternative to increase hydrophobic uremic toxin removal. Method We developed an extracorporeal, whole-blood bifunctional adsorber particle consisting of a porous, activated charcoal core with a hydrophilic polyvinylpyrrolidone surface coating. The adsorption capacity was quantified using analytical chromatography after perfusion of the particles with an albumin solution or blood, each containing mixtures of hydrophobic uremic toxins. Results A time-dependent increase in hydrophobic uremic toxin adsorption was depicted and all toxins showed a high binding affinity to the adsorber particles. Further, the particle showed a sufficient hemocompatibility without significant effects on complement component 5a, thrombin-antithrombin III complex, or thrombocyte concentration in blood in vitro, although leukocyte counts were slightly reduced. Conclusion In conclusion, the bifunctional adsorber particle with cross-linked polyvinylpyrrolidone coating showed a high adsorption capacity without adverse effects on hemocompatibility in vitro. Thus, it may be an interesting candidate for further in vivo studies with the aim to increase the efficiency of conventional dialysis techniques.

scholarly journals A Bifunctional Adsorber Particle for the Removal of Hydrophobic Uremic Toxins from Whole Blood of Renal Failure Patients

Toxins ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 389 ◽  
Author(s):  
Marieke Sternkopf ◽  
Sven Thoröe-Boveleth ◽  
Tobias Beck ◽  
Kirsten Oleschko ◽  
Ansgar Erlenkötter ◽  
...  
Keyword(s):  
Adsorption Capacity ◽  
Binding Affinity ◽  
Whole Blood ◽  
Uremic Toxins ◽  
In Vivo Studies ◽  
Uremic Toxin ◽  
High Binding ◽  
High Binding Affinity

Hydrophobic uremic toxins accumulate in patients with chronic kidney disease, contributing to a highly increased cardiovascular risk. The clearance of these uremic toxins using current hemodialysis techniques is limited due to their hydrophobicity and their high binding affinity to plasma proteins. Adsorber techniques may be an appropriate alternative to increase hydrophobic uremic toxin removal. We developed an extracorporeal, whole-blood bifunctional adsorber particle consisting of a porous, activated charcoal core with a hydrophilic polyvinylpyrrolidone surface coating. The adsorption capacity was quantified using analytical chromatography after perfusion of the particles with an albumin solution or blood, each containing mixtures of hydrophobic uremic toxins. A time-dependent increase in hydrophobic uremic toxin adsorption was depicted and all toxins showed a high binding affinity to the adsorber particles. Further, the particle showed a sufficient hemocompatibility without significant effects on complement component 5a, thrombin-antithrombin III complex, or thrombocyte concentration in blood in vitro, although leukocyte counts were slightly reduced. In conclusion, the bifunctional adsorber particle with cross-linked polyvinylpyrrolidone coating showed a high adsorption capacity without adverse effects on hemocompatibility in vitro. Thus, it may be an interesting candidate for further in vivo studies with the aim to increase the efficiency of conventional dialysis techniques.

scholarly journals An Omalizumab Biobetter Antibody With Improved Stability and Efficacy for the Treatment of Allergic Diseases

2020 ◽  
Vol 11 ◽  
Author(s):  
Peipei Liu ◽  
Zhongzong Pan ◽  
Chunyin Gu ◽  
Xiaodan Cao ◽  
Xiaowu Liu ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Half Life ◽  
Critical Role ◽  
Allergic Diseases ◽  
High Binding ◽  
Improved Stability ◽  
Acid Isomerization ◽  
High Binding Affinity

The critical role of IgE in allergic diseases is well-documented and clinically proven. Omalizumab, a humanized anti-IgE antibody, was the first approved antibody for the treatment of allergic diseases. Nevertheless, omalizumab still has some limitations, such as product instability and dosage restriction in clinical application. In this study, we attempted to develop an omalizumab biobetter antibody with the potential to overcome its limitations. We removed two aspartic acid isomerization hotspots in CDRs of omalizumab to improve antibody candidate’s stability. Meanwhile, several murine amino acids in the framework region of omalizumab were replaced with human source to reduce the potential immunogenicity. Yeast display technology was then applied to screen antibody candidates with high binding affinity to IgE. Moreover, YTE mutation in Fc fragment was introduced into the candidates for extending their serum half-life. A lead candidate, AB1904Am15, was screened out, which showed desired biophysical properties and improved stability, high binding affinity and elevated potency in vitro, prolonged half-life in human FcRn transgenic mouse, and enhanced in vivo efficacy in cynomolgus monkey asthma model. Overall, our study developed a biobetter antibody of omalizumab, AB1904Am15, which has the potential to show improved clinical benefit in the treatment of allergic diseases.

scholarly journals Advanced oxidation protein products downregulate CYP1A2 and CYP3A4 expression and activity via the NF-κB-mediated signaling pathway in vitro and in vivo

2021 ◽  
Author(s):  
Tianrong Xun ◽  
Zhufen Lin ◽  
Xiaokang Wang ◽  
Xia Zhan ◽  
Haixing Feng ◽  
...  
Keyword(s):  
Liver Microsomes ◽  
Advanced Oxidation ◽  
Uremic Toxins ◽  
Uremic Toxin ◽  
Dependent Manner ◽  
Advanced Oxidation Protein Products ◽  
Protein Levels ◽  
Cyp3a4 Expression

AbstractUremic toxin accumulation is one possible reason for alterations in hepatic drug metabolism in patients with chronic kidney disease (CKD). However, the types of uremic toxins and underlying mechanisms are poorly understood. In this study, we report the role of advanced oxidation protein products (AOPPs), a modified protein uremic toxin, in the downregulation of cytochromes P450 1A2 (CYP1A2) and P450 3A4 (CYP3A4) expression levels and activities. We found that AOPP accumulation in plasma in a rat CKD model was associated with decreased protein levels of CYP1A2 and CYP3A4. CYP1A2 and CYP3A4 metabolites (acetaminophen and 6β-hydroxytestosterone, respectively,) in liver microsomes were also significantly decreased. In human hepatocytes, AOPPs significantly decreased CYP1A2 and CYP3A4 protein levels in a dose- and time-dependent manner and downregulated their activities; however, bovine serum albumin (BSA), a synthetic precursor of AOPPs, had no effect on these parameters. The effect of AOPPs was associated with upregulation of p-IKKα/β, p-IκBα, p-NF-κB, and inflammatory cytokines protein levels and increases in p-IKKα/β/IKKα, p-IκBα/IκBα, and p-NF-κB/NF-κB phosphorylation ratios. Further, NF-kB pathway inhibitors BAY-117082 and PDTC abolished the downregulatory effects of AOPPs. These findings suggest that AOPPs downregulate CYP1A2 and CYP3A4 expression and activities by increasing inflammatory cytokine production and stimulating NF-κB-mediated signaling. Protein uremic toxins, such as AOPPs, may modify the nonrenal clearance of drugs in patients with CKD by influencing metabolic enzymes.

scholarly journals RaptRanker: in silico RNA aptamer selection from HT-SELEX experiment based on local sequence and structure information

2020 ◽  
Vol 48 (14) ◽  
pp. e82-e82 ◽  
Author(s):  
Ryoga Ishida ◽  
Tatsuo Adachi ◽  
Aya Yokota ◽  
Hidehito Yoshihara ◽  
Kazuteru Aoki ◽  
...  
Keyword(s):  
Binding Affinity ◽  
In Silico ◽  
Rna Aptamer ◽  
Affinity Reagents ◽  
Structure Information ◽  
High Binding ◽  
Local Sequence ◽  
Exponential Enrichment ◽  
High Binding Affinity

Abstract Aptamers are short single-stranded RNA/DNA molecules that bind to specific target molecules. Aptamers with high binding-affinity and target specificity are identified using an in vitro procedure called high throughput systematic evolution of ligands by exponential enrichment (HT-SELEX). However, the development of aptamer affinity reagents takes a considerable amount of time and is costly because HT-SELEX produces a large dataset of candidate sequences, some of which have insufficient binding-affinity. Here, we present RNA aptamer Ranker (RaptRanker), a novel in silico method for identifying high binding-affinity aptamers from HT-SELEX data by scoring and ranking. RaptRanker analyzes HT-SELEX data by evaluating the nucleotide sequence and secondary structure simultaneously, and by ranking according to scores reflecting local structure and sequence frequencies. To evaluate the performance of RaptRanker, we performed two new HT-SELEX experiments, and evaluated binding affinities of a part of sequences that include aptamers with low binding-affinity. In both datasets, the performance of RaptRanker was superior to Frequency, Enrichment and MPBind. We also confirmed that the consideration of secondary structures is effective in HT-SELEX data analysis, and that RaptRanker successfully predicted the essential subsequence motifs in each identified sequence.

Coumarins and Quinolones as Effective Multiple Targeted Agents Versus Covid-19: An in Silico Study

2021 ◽  
Vol 17 ◽  
Author(s):  
Mojgan Nejabat ◽  
Razieh Ghodsi ◽  
Farzin Hadizadeh
Keyword(s):  
Binding Affinity ◽  
In Silico ◽  
Oral Absorption ◽  
Antiviral Agent ◽  
In Vivo Studies ◽  
Reference Drug ◽  
Viral Proteases ◽  
In Silico Study

Background: The Covid-19 virus emerged a few months ago in China and infections rapidly escalated into a pandemic. Objective: To date, there is no selective antiviral agent for the management of pathologies associated with covid-19 and the need for an effective agent against it is essential. Method: In this work two home-made databases from synthetic quinolines and coumarins were virtually docked against viral proteases (3CL and PL), human cell surface proteases (TMPRSS2 and furin) and spike proteins (S1 and S2). Chloroquine, a reference drug without a clear mechanism against coronavirus was also docked on mentioned targets and the binding affinities compared with title compounds. Result: The best compounds of synthetic coumarins and quinolines for each target were determined. All compounds against all targets showed binding affinity between -5.80 to -8.99 kcal/mol in comparison with the FDA-approved drug, Chloroquine, with binding affinity of -5.7 to -7.98 kcal/mol. Two compounds, quinoline-1 and coumarin-24, were found to be effective on three targets – S2, TMPRSS2 and furin – simultaneously, with good predicted affinity between -7.54 to -8.85 kcal/mol. In silico ADME studies also confirmed good oral absorption for them. Furthermore, PASS prediction was calculated and coumarin-24 had higher probable activity (Pa) than probable inactivity (Pi) with acceptable protease inhibitory as well as good antiviral activity against Hepatitis C virus (HCV), Human immunodeficiency virus (HIV) and influenza. Conclusion: Quinoline-1 and Coumarin-24 have the potential to be used against Covid-19. Hence these agents could be useful in combating covid-19 infection after further in vitro and in vivo studies.

The Efficient Engraftment of Human Hematopoiesis in the Balb/c Strain Is Mounted by Balb/c-Specific SIRPA Polymorphism That Enhances Binding Affinity to Human CD47,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4008-4008
Author(s):  
Chika Iwamoto ◽  
Katsuto Takenaka ◽  
Shingo Urata ◽  
Takahiro Shima ◽  
Katsuhiko Itoh ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Affinity ◽  
Hela Cells ◽  
Hematopoietic Cells ◽  
Human Cells ◽  
Murine Macrophages ◽  
High Binding ◽  
High Binding Affinity ◽  
Intermediate Effect

Abstract Abstract 4008 Various immunodeficient mouse lines for human hematopoietic assay have been developed. It is well recognized that in addition to disruption of the lymphoid system, the strain background such as NOD and Balb/c is critical to achieve efficient xenotransplantation. We have reported that in the NOD strain, NOD-specific polymorphism of SIRPA allows the mouse SIRPA to bind human CD47, preventing activation of host macrophages to engulf human hematopoietic cells and/or to cytokine production, and therefore can inhibit graft rejection of human cells (Takenaka et al., Nature Immunology, 2007). Here we tested whether the efficient xenotransplantation capability in the Balb/c strain is decided also by the SIRPA-CD47 self-recognition system. We have also reported that the efficiency of human to mouse xenotransplantation in hematopoiesis can be measured by maintenance of human hematopoiesis in vitro in chimeric long-term culture (LTC) assay, where human hematopoietic cells are cultured in the presence of murine macrophages. Different doses of macrophages were seeded on MS-5 stromal cells with lineage negative cord blood cells, and 4–5 weeks after the initiation of culture, the number of colony-forming cells (CFCs) was counted. In this system the addition of B6-derived murine macrophages severely suppressed human LTC-initiating cells (LTC-IC), whereas replacement of B6-derived macrophages with NOD-derived macrophages significantly enhanced the maintenance of human LTC-IC in vitro. Interestingly, Balb/c-derived macrophages had an intermediate effect between those of B6 and NOD-derived macrophages. These results are reasonably corresponding to the efficiency of human hematopoietic cell engraftment in immunodeficient mice of each background strain. To test if this effect is derived from binding of murine SIRPA and human CD47, we performed protein-binding assays of these molecules. B6 SIRPA never binds to human CD47. NOD SIRPA showed a very high binding affinity, while Balb/c SIRPA showed an intermediate affinity (Kd=2.501 for NOD, 3.076E+2 for Balb/c, and 4.974E+10 for B6). We then cloned the SIRPA cDNA from Balb/c and NOD strains, and enforced to express these molecules in HeLa cells by using lentivirus vectors. HeLa cells with enforced NOD and Balb/c SIRPA also showed the similar strong and intermediate affinity to human CD47, respectively, indicating that strain-specific SIRPA decides its binding capability with human CD47. We then enforced to express SIRPA derived from B6, NOD, or Balb/c strains into B6 macrophages, and tested their inhibitory effect on the human LTC-IC assay. Again, the addition of macrophages with enforced B6 SIRPA into cultures inhibited human LTC-IC, whereas those with enforced NOD SIRPA promoted maintenance of human LTC-IC. The addition of macrophages with Balb/c SIRPA again showed an intermediate effect. Because we have also reported the high binding affinity to human CD47 in the NOD strain is caused by alteration of amino-acid sequences of the IgV domain of SIRPA caused by the NOD-specific single nucleotide polymorphisms (SNPs), DNA sequencing of the SIRPA domain in each strain was performed. We found 2 independent Balb/c-specific SNPs. One of the SNPs causes the exchange of the 29th amino acid leucine with valine (L29V). We then transduced the SIRPA with L29V mutation into HeLa cells, performed the binding assay for human CD47, and found that cells expressing L29V SIRPA had the affinity equivalent to HeLa cells transduced with Balb/c SIRPA. Thus, the Balb/c-specific L29V polymorphism of SIRPA allows moderate binding to human CD47, and SIRPA signaling evoked by this binding maintains human LTC-IC activity presumably by inhibiting Balb/c macrophages to engulf cultured human cells. These data collectively suggest that the efficiency of mouse to human xenotransplantation is decided by “macrophage tolerance” based on the binding affinity of human CD47 to strain-specific murine SIRPA. Our findings should be useful to establish a novel immunodeficient strain with more efficient xenotransplantation capability for human cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Studies on Agranulocytosis. II. The Effect of Chlorpromazine upon the Influx of S35 L-Cystine and L-Methionine into Human Leukocytes

Blood ◽  
1960 ◽  
Vol 16 (4) ◽  
pp. 1456-1468 ◽  
Author(s):  
ANTHONY V. PISCIOTTA ◽  
SHIRLEY N. EBBE ◽  
MARY DALY ◽  
MONA RUWALDT ◽  
MILTON GLASER ◽  
...  
Keyword(s):  
Whole Blood ◽  
Blood Cells ◽  
The Other ◽  
In Vivo Studies ◽  
Human Leukocytes ◽  
In Vivo Administration

Abstract 1. When whole blood was incubated in vitro with S-35 L-cystine and L-methionine, the blood cells became radioactive. 2. Preincubation of whole blood from normals and from patients susceptible to agranulocytosis with chlorpromazine showed no effect upon uptake of S-35 L-cystine and L-methionine by leukocytes. 3. The in vivo administration of S-35 L-cystine was followed by the appearance of radioactive leukocytes. Peak radioactivity occurred in leukocytes in 5 to 12 days. 4. Pretreatment of test subjects with large doses of chlorpromazine did not block the uptake of S-35 L-cystine by leukocytes in vivo. Leukocytes of women showed an increase in the incorporation of S-35 L-cystine, in vivo. Studies performed in vivo on two persons during recovery from agranulocytosis showed enhanced uptake of L-cystine in one and a normal uptake in the other.

scholarly journals In Vitro and In Vivo Studies of the RNA Conformational Switch in Alfalfa Mosaic Virus

2009 ◽  
Vol 84 (3) ◽  
pp. 1423-1429 ◽  
Author(s):  
Shih-Cheng Chen ◽  
René C. L. Olsthoorn
Keyword(s):  
Mosaic Virus ◽  
Binding Affinity ◽  
Linear Array ◽  
Alfalfa Mosaic Virus ◽  
In Vivo Studies ◽  
Conformational Switch ◽  
Stem Loop ◽  
Viral Replicase

ABSTRACT The 3′ termini of Alfalfa mosaic virus (AMV) RNAs adopt two mutually exclusive conformations, a coat protein binding (CPB) and a tRNA-like (TL) conformer, which consist of a linear array of stem-loop structures and a pseudoknot structure, respectively. Previously, switching between CPB and TL conformers has been proposed as a mechanism to regulate the competing processes of translation and replication of the viral RNA (R. C. L. Olsthoorn et al., EMBO J. 18:4856-4864, 1999). In the present study, the switch between CPB and TL conformers was further investigated. First, we showed that recognition of the AMV 3′ untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding enzyme) in vitro is more efficient when the distribution is shifted toward the TL conformation. Second, the recognition of the 3′ UTR by the viral replicase was similarly dependent on the ratio of CBP and TL conformers. Furthermore, the addition of CP, which is expected to shift the distribution toward the CPB conformer, inhibited recognition by the CCA-adding enzyme and the replicase. Finally, we monitored how the binding affinity to CP is affected by this conformational switch in the yeast three-hybrid system. Here, disruption of the pseudoknot enhanced the binding affinity to CP by shifting the balance in favor of the CPB conformer, whereas stabilizing the pseudoknot did the reverse. Together, the in vitro and in vivo data clearly demonstrate the existence of the conformational switch in the 3′ UTR of AMV RNAs.

scholarly journals Vasculopathy in the setting of cardiorenal syndrome: roles of protein-bound uremic toxins

2017 ◽  
Vol 313 (1) ◽  
pp. H1-H13 ◽  
Author(s):  
Jingbin Guo ◽  
Lu Lu ◽  
Yue Hua ◽  
Kevin Huang ◽  
Ian Wang ◽  
...  
Keyword(s):  
Vascular System ◽  
Current Knowledge ◽  
Cardiorenal Syndrome ◽  
Left Ventricular ◽  
Uremic Toxins ◽  
Uremic Toxin ◽  
Contributing Factors ◽  
Clinical Observations

Chronic kidney disease (CKD) often leads to and accelerates the progression of cardiovascular disease (CVD), while CVD also causes kidney dysfunction. This bidirectional interaction leads to the development of a complex syndrome known as cardiorenal syndrome (CRS). CRS not only involves both the heart and the kidney but also the vascular system through a vast array of contributing factors. In addition to hemodynamic, neurohormonal, mechanical, and biochemical factors, nondialyzable protein-bound uremic toxins (PBUTs) are also key contributing factors that have been demonstrated through in vitro, in vivo, and clinical observations. PBUTs are ineffectively removed by hemodialysis because their complexes with albumins are larger than the pores of the dialysis membranes. PBUTs such as indoxyl sulfate and p-cresyl sulfate are key determinate and predictive factors for the progression of CVD in CKD patients. In CRS, both vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) exhibit significant dysfunction that is associated with the progression of CVD. PBUTs influence proliferation, calcification, senescence, migration, inflammation, and oxidative stress in VSMCs and ECs through various mechanisms. These pathological changes lead to arterial remodeling, stiffness, and atherosclerosis and thus reduce heart perfusion and impair left ventricular function, aggravating CRS. There is limited literature about the effect of PBUT on the vascular system and their contribution to CRS. This review summarizes current knowledge on how PBUTs influence vasculature, clarifies the relationship between uremic toxin-related vascular disease and CRS, and highlights the potential therapeutic strategies of uremic vasculopathy in the setting of CRS.

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