The Experts below are selected from a list of 246 Experts worldwide ranked by ideXlab platform

Steven W. Graves - One of the best experts on this subject based on the ideXlab platform.

  • Digoxin Antibody fragment antigen binding fab treatment of preeclampsia in women with endogenous digitalis like factor a secondary analysis of the deep trial
    American Journal of Obstetrics and Gynecology, 2013
    Co-Authors: Moana Hopoatesitake, Donna Johnson, Christopher Robinson, Daryl Adair, Vardaman M. Buckalew, George R Saade, David F. Lewis, Steven W. Graves
    Abstract:

    Objective Endogenous digitalis-like factors (EDLFs) are elevated in women with preeclampsia, and the use of an anti-Digoxin Antibody Fab (DIF) in women with preeclampsia who were remote from term reduced maternal blood pressure and preserved renal function. The objective was to determine whether DIF treatment in women with severe preeclampsia in association with positive EDLFs in maternal serum improves maternal-perinatal outcomes. Study Design This was a planned secondary analysis from a randomized, placebo-controlled, double-blind study of DIF in women with severe preeclampsia with positive EDLF status that was managed expectantly between 23 weeks 5 days and 34 weeks' gestation (19 women received placebo, and 17 women received DIF). Primary outcome variables were a change in creatinine clearance and the use of antihypertensives. Secondary outcomes were maternal and perinatal complications. Results Women with positive EDLFs who received DIF had an attenuated decline in creatinine clearance from baseline compared with placebo (–4.5 ± 12.9 vs –53.2 ± 12.6 mL/min; P  = .005). In this same group, the use of antihypertensives (the other primary outcome) was lower but not significantly so (41% vs 63%; P  = .12). However, women who were treated with DIF had a lower rate of pulmonary edema (1/17 vs 6/19 women; P  = .035) and lower rates of neonatal intraventricular hemorrhage (DIF: 0/17 women vs placebo: 5/19 women; P  = .015). Conclusion In women with severe preeclampsia who were remote from term who were EDLF positive, the use of DIF was associated with improved maternal and neonatal outcome. These findings suggest the need for a large multicenter trial that would evaluate the benefits of DIF in the treatment of women with severe preeclampsia who are remote from term and with positive EDLF status.

  • Digoxin Antibody fragment, antigen binding (Fab), treatment of preeclampsia in women with endogenous digitalis-like factor: a secondary analysis of the DEEP Trial.
    American journal of obstetrics and gynecology, 2013
    Co-Authors: Moana Hopoate-sitake, Donna D Johnson, Vardaman M. Buckalew, C. David Adair, George R Saade, David F. Lewis, Christopher J. Robinson, Steven W. Graves
    Abstract:

    Endogenous digitalis-like factors (EDLFs) are elevated in women with preeclampsia, and the use of an anti-Digoxin Antibody Fab (DIF) in women with preeclampsia who were remote from term reduced maternal blood pressure and preserved renal function. The objective was to determine whether DIF treatment in women with severe preeclampsia in association with positive EDLFs in maternal serum improves maternal-perinatal outcomes. This was a planned secondary analysis from a randomized, placebo-controlled, double-blind study of DIF in women with severe preeclampsia with positive EDLF status that was managed expectantly between 23 weeks 5 days and 34 weeks' gestation (19 women received placebo, and 17 women received DIF). Primary outcome variables were a change in creatinine clearance and the use of antihypertensives. Secondary outcomes were maternal and perinatal complications. Women with positive EDLFs who received DIF had an attenuated decline in creatinine clearance from baseline compared with placebo (-4.5 ± 12.9 vs -53.2 ± 12.6 mL/min; P = .005). In this same group, the use of antihypertensives (the other primary outcome) was lower but not significantly so (41% vs 63%; P = .12). However, women who were treated with DIF had a lower rate of pulmonary edema (1/17 vs 6/19 women; P = .035) and lower rates of neonatal intraventricular hemorrhage (DIF: 0/17 women vs placebo: 5/19 women; P = .015). In women with severe preeclampsia who were remote from term who were EDLF positive, the use of DIF was associated with improved maternal and neonatal outcome. These findings suggest the need for a large multicenter trial that would evaluate the benefits of DIF in the treatment of women with severe preeclampsia who are remote from term and with positive EDLF status. Copyright © 2013 Mosby, Inc. All rights reserved.

  • Increasing Evidence for and Regulation of a Human Placental Endogenous Digitalis-Like Factor
    Reproductive Sciences, 2012
    Co-Authors: Jie Ma, M. Sean Esplin, Lorrie A. Mason, C. David Adair, Steven W. Graves
    Abstract:

    Endogenous digitalis-like factors (EDLFs) appear to be hypertensiogenic and increased in the serum and placenta of women with preeclampsia (PE), a complication of pregnancy. Digibind, an anti-Digoxin Antibody Fab fragment, reverses in vitro effects of EDLF and in vivo features of PE. We used Digibind in a radioimmunoassay to measure EDLF and compared this to a bio-functional assay of EDLF with good agreement. These methods confirmed that human placenta was a source of EDLF, synthesizing and releasing EDLF into the media of cultured human placental tissue. Ketoconazole, a steroid synthesis inhibitor, and 17-OH progesterone, a possible substrate of steroid synthesis, were shown to inhibit or increase EDLF release respectively, suggesting overlap of synthetic pathways. Abnormalities of PE such as placental hypoxia, increased reactive oxygen species and increased proinflammatory cytokines were demonstrated to increase placental EDLF release. These findings strongly support placental production of EDLF with increased release due to features of PE.

  • Digibind Reverses Inhibition of Cellular Rb^+ Uptake Caused by Endogenous Sodium Pump Inhibitors Present in Serum and Placenta of Women with Preeclampsia
    Reproductive Sciences, 2011
    Co-Authors: Moana Hopoate-sitake, Lorrie A. Mason, C. David Adair, Carlos Torres, Joseph Kipikasa, Steven W. Graves
    Abstract:

    Introduction Mechanisms mediating preeclampsia (PE) are unclear. Endogenous digitalis-like factors (EDLFs) are sodium pump (SP) inhibitors implicated in essential hypertension, but not fully explored in PE. This study asks whether EDLFs are present and increased in PE and considers their source. Methods EDLF in sera and placentas from third trimester women with uncomplicated pregnancies or PE was assessed by a Rb^+ uptake assay. A Digoxin Antibody Fab fragment (Digibind) known to inactivate EDLFs was also used to assess EDLFs. Results PE serum caused significantly more SP inhibition than serum from uncomplicated pregnancies. This inhibition was concentration-dependent and reversed by Digibind. Serum from uncomplicated pregnancies showed no concentration-dependence or reversal with Digibind. Placental homogenates from control women showed little SP inhibition, but homogenates from PE women showed marked SP inhibition reversed by Digibind. Conclusion These studies evidence EDLF in PE serum. Additionally, PE placentas have high EDLF and may represent a source.

Alexei Y. Bagrov - One of the best experts on this subject based on the ideXlab platform.

  • Interaction of Digibind with endogenous cardiotonic steroids from preeclamptic placentae
    2015
    Co-Authors: Olga V. Fedorova, Anton Bzhelyansky, Elena V. Frolova, Natalia I. Tapilskaya, R. Nikitina, Vitaly A. Reznik, Vladimir A. Kashkin, Alexei Y. Bagrov
    Abstract:

    Background—Preeclampsia (PE) is a major cause of maternal and fetal mortality, and its pathogenesis is not fully understood. Endogenous digitalis-like cardiotonic steroids (CTS) have been implicated in the pathophysiology of PE; this is illustrated by clinical observations that Digibind, a therapeutic Digoxin Antibody fragment which binds CTS, lowers blood pressure and reverses Na/K-ATPase inhibition in patients with PE. Recently we reported that plasma levels of marinobufagenin (MBG), a bufadienolide vasoconstrictor CTS, are increased four-fold in patients with severe PE. Methods—In the present study, we compared levels of MBG in normal and PE placentae, as well as the interactions of Digibind and antibodies against MBG and ouabain with material purified from PE placentae using high-performance liquid chromatography (HPLC). Results—Levels of endogenous MBG, but not that of endogenous ouabain, exhibited a four-fold elevation in PE placentae vs. normal placentae (13.6±2.5 and 48.6±7.0 nmoles/g tissue; P<0.01). The elution time of endogenous placental MBG-like immunoreactive material from reverse-phase HPLC column was identical to that of authentic MBG. A competitive immunoassay based on Digibind exhibited reactivity to HPLC fractions having retention times similar to that seen with MBG and other bufadienolides, but no to ouabain-like immunoreactive material. Conclusions—Our results suggest that elevated levels of endogenous bufadienolide CTS represent a potential target for immunoneutralization in patients with PE

  • Magnesium Sulfate Potentiates Effect of Digifab on Marinobufagenin-Induced Na/K-ATPase Inhibition
    American journal of hypertension, 2013
    Co-Authors: Irina E. Zazerskaya, C. David Adair, Olga V. Fedorova, Valentina V. Ishkaraeva, Elena V. Frolova, Nelly G. Solodovnikova, Yulia N. Grigorova, Alexei Y. Bagrov
    Abstract:

    BACKGROUND Immunoneutralization of elevated circulating levels of endogenous digitalis-like Na/K-ATPase inhibitors (i.e. cardiotonic steroids (CTS)) represents a novel approach in the treatment of preeclampsia (PE). Recently we demonstrated that DigiFab (Fab fragments of affinity-purified ovine Digoxin Antibody) restores PE-induced inhibition of Na/K-ATPase in erythrocytes ex vivo. Previously magnesium ions were shown to antagonize digitalis-induced toxicity, which is mediated by Na/K-ATPase inhibition. We hypothesized that magnesium sulfate would potentiate the effect of DigiFab in the reversal of CTS-induced Na/K-ATPase inhibition.

  • Antibody to marinobufagenin lowers blood pressure in pregnant rats on a high NaCl intake.
    Journal of hypertension, 2005
    Co-Authors: Olga V. Fedorova, Nikolai I. Kolodkin, Natalia I. Agalakova, Alexandra R Namikas, Anton Bzhelyansky, Jean St-louis, Edward G. Lakatta, Alexei Y. Bagrov
    Abstract:

    ObjectiveThe pathogenesis of pre-eclampsia (PE), a major cause of maternal and fetal mortality, is not fully understood. Digitalis-like sodium pump ligands (SPLs) are believed to be implicated in PE, as illustrated by clinical observations that DIGIBIND, a Digoxin Antibody that binds SPLs, lowers bl

  • Effect of endogenous Digoxin-like factor and Digoxin Antibody on myocardial Na+, K+-pump activity and ventricular arrhythmias in acute myocardial ischaemia in rats
    Cardiovascular research, 1993
    Co-Authors: Alexei Y. Bagrov, Olga V. Fedorova, Natalia I. Roukoyatkina, Eugenii P Zhabko
    Abstract:

    Objective: The aim was to study whether a circulating sodium pump inhibitor (endogenous Digoxin like factor) contributes to the genesis of early ventricular arrhythmias in acute myocardial ischaemia in rats. Methods: Effects of Digoxin Antibody (260 μg·kg−1) on the incidence of ventricular arrhythmias, plasma Digoxin like immunoreactivity (DELFIA immunoassay), Na+, K+, and Mg2+ ions, and activity of the ouabain sensitive Na+, K+-pump in different regions of myocardium have been studied in propranolol naive and propranolol pretreated rats exposed to acute coronary artery ligation. Adult male Wistar rats were divided into six experimental groups: (1) saline pretreated controls; (2) saline pretreated coronary artery ligated rats; (3) coronary artery ligated rats pretreated with 260 μg·kg−1 Digoxin Antibody; (4) propranolol pretreated controls; (5) propranolol pretreated rats with acute myocardial ischaemia; (6) rats with acute myocardial ischaemia pretreated with both propranolol and Digoxin Antibody. Results: Acute myocardial ischaemia in saline pretreated rats was associated with a twofold increase of plasma Digoxin like immunoreactivity and ventricular arrhythmias, but did not lead to changes in myocardial Na+, K+-pump activity. Pretreatment of coronary artery ligated rats with Digoxin Antibody reduced the total duration of ventricular tachycardia and ventricular fibrillation during a 15 minute postligation period from 201(SEM 34) to 46(18) seconds (p

  • effect of endogenous Digoxin like factor and Digoxin Antibody on myocardial na k pump activity and ventricular arrhythmias in acute myocardial ischaemia in rats
    Cardiovascular Research, 1993
    Co-Authors: Alexei Y. Bagrov, Olga V. Fedorova, Natalia I. Roukoyatkina, Eugenii P Zhabko
    Abstract:

    Objective: The aim was to study whether a circulating sodium pump inhibitor (endogenous Digoxin like factor) contributes to the genesis of early ventricular arrhythmias in acute myocardial ischaemia in rats. Methods: Effects of Digoxin Antibody (260 μg·kg−1) on the incidence of ventricular arrhythmias, plasma Digoxin like immunoreactivity (DELFIA immunoassay), Na+, K+, and Mg2+ ions, and activity of the ouabain sensitive Na+, K+-pump in different regions of myocardium have been studied in propranolol naive and propranolol pretreated rats exposed to acute coronary artery ligation. Adult male Wistar rats were divided into six experimental groups: (1) saline pretreated controls; (2) saline pretreated coronary artery ligated rats; (3) coronary artery ligated rats pretreated with 260 μg·kg−1 Digoxin Antibody; (4) propranolol pretreated controls; (5) propranolol pretreated rats with acute myocardial ischaemia; (6) rats with acute myocardial ischaemia pretreated with both propranolol and Digoxin Antibody. Results: Acute myocardial ischaemia in saline pretreated rats was associated with a twofold increase of plasma Digoxin like immunoreactivity and ventricular arrhythmias, but did not lead to changes in myocardial Na+, K+-pump activity. Pretreatment of coronary artery ligated rats with Digoxin Antibody reduced the total duration of ventricular tachycardia and ventricular fibrillation during a 15 minute postligation period from 201(SEM 34) to 46(18) seconds (p<0.002) but did not alter activity of the myocardial Na+, K+-pump. In rats pretreated with propranolol, acute myocardial ischaemia was associated with a twofold inhibition of the Na+, K+-pump in left atrial and left ventricular myocardium, and with a 69% increase in plasma K+ concentration. Administration of Digoxin Antibody to propranolol pretreated coronary artery ligated rats in parallel with the antiarrhythmic effect prevented the increase in plasma K+ concentration and inhibition of Na+, K+-pump in the left atrial, but not the left ventricular myocardium. Conclusions: A circulating Digoxin like factor contributes to the pathogenesis of myocardial ischaemia induced ventricular arrhythmias. As propranolol pretreatment of coronary artery ligated rats inhibited the Na+, K+-pump in myocardium, the inhibitory effect of endogenous Digoxin like factor on Na+, K+-ATPase was probably masked in propranolol naive animals by the stimulatory action of catecholamines on Na+, K+-ATPase described previously. Cardiovascular Research 1993; 27 :1045-1050

C. David Adair - One of the best experts on this subject based on the ideXlab platform.

  • Magnesium Sulfate Potentiates Effect of Digifab on Marinobufagenin-Induced Na/K-ATPase Inhibition
    American journal of hypertension, 2013
    Co-Authors: Irina E. Zazerskaya, C. David Adair, Olga V. Fedorova, Valentina V. Ishkaraeva, Elena V. Frolova, Nelly G. Solodovnikova, Yulia N. Grigorova, Alexei Y. Bagrov
    Abstract:

    BACKGROUND Immunoneutralization of elevated circulating levels of endogenous digitalis-like Na/K-ATPase inhibitors (i.e. cardiotonic steroids (CTS)) represents a novel approach in the treatment of preeclampsia (PE). Recently we demonstrated that DigiFab (Fab fragments of affinity-purified ovine Digoxin Antibody) restores PE-induced inhibition of Na/K-ATPase in erythrocytes ex vivo. Previously magnesium ions were shown to antagonize digitalis-induced toxicity, which is mediated by Na/K-ATPase inhibition. We hypothesized that magnesium sulfate would potentiate the effect of DigiFab in the reversal of CTS-induced Na/K-ATPase inhibition.

  • Digoxin Antibody fragment, antigen binding (Fab), treatment of preeclampsia in women with endogenous digitalis-like factor: a secondary analysis of the DEEP Trial.
    American journal of obstetrics and gynecology, 2013
    Co-Authors: Moana Hopoate-sitake, Donna D Johnson, Vardaman M. Buckalew, C. David Adair, George R Saade, David F. Lewis, Christopher J. Robinson, Steven W. Graves
    Abstract:

    Endogenous digitalis-like factors (EDLFs) are elevated in women with preeclampsia, and the use of an anti-Digoxin Antibody Fab (DIF) in women with preeclampsia who were remote from term reduced maternal blood pressure and preserved renal function. The objective was to determine whether DIF treatment in women with severe preeclampsia in association with positive EDLFs in maternal serum improves maternal-perinatal outcomes. This was a planned secondary analysis from a randomized, placebo-controlled, double-blind study of DIF in women with severe preeclampsia with positive EDLF status that was managed expectantly between 23 weeks 5 days and 34 weeks' gestation (19 women received placebo, and 17 women received DIF). Primary outcome variables were a change in creatinine clearance and the use of antihypertensives. Secondary outcomes were maternal and perinatal complications. Women with positive EDLFs who received DIF had an attenuated decline in creatinine clearance from baseline compared with placebo (-4.5 ± 12.9 vs -53.2 ± 12.6 mL/min; P = .005). In this same group, the use of antihypertensives (the other primary outcome) was lower but not significantly so (41% vs 63%; P = .12). However, women who were treated with DIF had a lower rate of pulmonary edema (1/17 vs 6/19 women; P = .035) and lower rates of neonatal intraventricular hemorrhage (DIF: 0/17 women vs placebo: 5/19 women; P = .015). In women with severe preeclampsia who were remote from term who were EDLF positive, the use of DIF was associated with improved maternal and neonatal outcome. These findings suggest the need for a large multicenter trial that would evaluate the benefits of DIF in the treatment of women with severe preeclampsia who are remote from term and with positive EDLF status. Copyright © 2013 Mosby, Inc. All rights reserved.

  • Increasing Evidence for and Regulation of a Human Placental Endogenous Digitalis-Like Factor
    Reproductive Sciences, 2012
    Co-Authors: Jie Ma, M. Sean Esplin, Lorrie A. Mason, C. David Adair, Steven W. Graves
    Abstract:

    Endogenous digitalis-like factors (EDLFs) appear to be hypertensiogenic and increased in the serum and placenta of women with preeclampsia (PE), a complication of pregnancy. Digibind, an anti-Digoxin Antibody Fab fragment, reverses in vitro effects of EDLF and in vivo features of PE. We used Digibind in a radioimmunoassay to measure EDLF and compared this to a bio-functional assay of EDLF with good agreement. These methods confirmed that human placenta was a source of EDLF, synthesizing and releasing EDLF into the media of cultured human placental tissue. Ketoconazole, a steroid synthesis inhibitor, and 17-OH progesterone, a possible substrate of steroid synthesis, were shown to inhibit or increase EDLF release respectively, suggesting overlap of synthetic pathways. Abnormalities of PE such as placental hypoxia, increased reactive oxygen species and increased proinflammatory cytokines were demonstrated to increase placental EDLF release. These findings strongly support placental production of EDLF with increased release due to features of PE.

  • Digibind Reverses Inhibition of Cellular Rb^+ Uptake Caused by Endogenous Sodium Pump Inhibitors Present in Serum and Placenta of Women with Preeclampsia
    Reproductive Sciences, 2011
    Co-Authors: Moana Hopoate-sitake, Lorrie A. Mason, C. David Adair, Carlos Torres, Joseph Kipikasa, Steven W. Graves
    Abstract:

    Introduction Mechanisms mediating preeclampsia (PE) are unclear. Endogenous digitalis-like factors (EDLFs) are sodium pump (SP) inhibitors implicated in essential hypertension, but not fully explored in PE. This study asks whether EDLFs are present and increased in PE and considers their source. Methods EDLF in sera and placentas from third trimester women with uncomplicated pregnancies or PE was assessed by a Rb^+ uptake assay. A Digoxin Antibody Fab fragment (Digibind) known to inactivate EDLFs was also used to assess EDLFs. Results PE serum caused significantly more SP inhibition than serum from uncomplicated pregnancies. This inhibition was concentration-dependent and reversed by Digibind. Serum from uncomplicated pregnancies showed no concentration-dependence or reversal with Digibind. Placental homogenates from control women showed little SP inhibition, but homogenates from PE women showed marked SP inhibition reversed by Digibind. Conclusion These studies evidence EDLF in PE serum. Additionally, PE placentas have high EDLF and may represent a source.

Michael N. Margolies - One of the best experts on this subject based on the ideXlab platform.

  • A single H:CDR3 residue in the anti-Digoxin Antibody 26-10 modulates specificity for C16-substituted Digoxin analogs.
    Protein engineering, 2001
    Co-Authors: Mary K. Short, Philip D. Jeffrey, Aram N. Demirjian, Michael N. Margolies
    Abstract:

    We constructed Fab libraries of bacteriophage-displayed H:CDR3 mutants in the high-affinity anti-Digoxin Antibody 26-10 to determine structural constraints on affinity and specificity for Digoxin. Libraries of mutant Fabs randomized at five or 10 contiguous positions were panned against Digoxin and three C16-substituted analogs, gitoxin (16-OH), 16-formylgitoxin and 16-acetylgitoxin. The sequence data from 83 different mutant Fabs showed highly restricted consensus patterns at positions H:100, 100a and 100b for binding to Digoxin; these residues contact Digoxin in the 26-10:Digoxin co-crystal structure. Several mutant Fabs obtained following panning on Digoxin-BSA showed increased affinity for Digoxin compared with 26-10 and retained the wild-type (wt) Trp at position 100. Those Fabs selected following panning on C16-substituted analogs showed enhanced binding to the analogs. Replacement of H:Trp100 by Arg resulted in mutants that bound better to the analogs than to Digoxin. This specificity change was unexpected, as C16 lies on the opposite side of Digoxin from H:CDR3. Substitution of wt Trp by Arg appears to alter specificity by allowing the hapten to shift toward H:CDR3, thereby providing room for C16 substituents in the region of H:CDR1.

  • an approach for preventing recombination deletion of the 40 50 anti Digoxin Antibody vh gene from the phage display vector pcomb3
    Gene, 2000
    Co-Authors: Seho Kim, Christian C Titlow, Michael N. Margolies
    Abstract:

    Abstract Phage display has been used extensively in Antibody (Ab) engineering. Sometimes, however, phage display vectors exhibit deletion of immunoglobulin (Ig) genes. As an approach to circumvent the recombination-deletion of the murine anti-Digoxin Fab 40–50 cloned into the pComb3 vector, the vector was modified with short synthetic oligonucleotides by replacing a pelB leader sequence with a gene 3 (g3) leader sequence and by using a single lacZ promoter sequence. By this means, the N-terminal amino acids of the L chain and Fd remained unchanged, and a random HCDR3 library built on this newly designed vector did not exhibit the recombination-deletion.

  • contribution of Antibody heavy chain cdr1 to Digoxin binding analyzed by random mutagenesis of phage displayed fab 26 10
    Journal of Biological Chemistry, 1995
    Co-Authors: Mary K. Short, Philip D. Jeffrey, Roufun Kwong, Michael N. Margolies
    Abstract:

    Abstract We constructed a bacteriophage-displayed library containing randomized mutations at H chain residues 30-35 of the anti-Digoxin Antibody 26-10 Fab to investigate sequence constraints necessary for high affinity binding in an Antibody of known crystal structure. Phage were selected by panning against Digoxin and three C-16-substituted analogues. All antigen-positive mutants selected using other analogues also bound Digoxin. Among 73 antigen-positive clones, 26 different nucleotide sequences were found. The majority of Fabs had high affinity for Digoxin (Ka ≥ 3.4 × 109M−1) despite wide sequence diversity. Two mutants displayed affinities 2- and 4-fold higher than the parental Antibody. Analysis of the statistical distribution of sequences showed that highest affinity binding occurred with a restricted set of amino acid substitutions at positions H33-35. All clones save two retained the parental Asn-H35, which contacts hapten and hydrogen bonds to other binding site residues in the parental structure. Positions H30-32 display remarkable diversity, with 10-14 different substitutions for each residue, consistent with high affinity binding. Thus complementarity can be retained and even improved despite diversity in the conformation of the N-terminal portion of the H-CDR1 loop.

  • Structure and specificity of the anti-Digoxin Antibody 40-50.
    Journal of molecular biology, 1995
    Co-Authors: Philip D. Jeffrey, Michael N. Margolies, Joel F. Schildbach, Chiehying Y. Chang, P. H. Kussie, Steven Sheriff
    Abstract:

    We determined the sequence, specificity for structurally related cardenolides, and three-dimensional structure of the anti-Digoxin Antibody 40-50 Fab in complex with ouabain. The 40-50 Antibody does not share close sequence homology with other high-affinity anti-Digoxin antibodies. Measurement of the binding constants of structurally distinct Digoxin analogs indicated a well-defined specificity pattern also distinct from other anti-Digoxin antibodies. The 40-50-ouabain Fab complex crystallizes in space group C2 with cell dimensions of a = 93.7 A, b = 84.8 A, c = 70.1 A, beta = 128.0 degrees. The structure of the complex was determined by X-ray crystallography and refined at a resolution of 2.7 A. The hapten is bound in a pocket extending as a groove from the center of the combining site across the light chain variable domain, with five of the six complementarity-determining regions involved in interactions with the hapten. Approximately three-quarters of the hapten surface area is buried in the complex; two hydrogen bonds are formed between the Antibody and hapten. The surface area of the Antibody combining site buried by ouabain is contributed equally by the light and heavy chain variable domains. Over half of the surface area buried on the Fab consists of the aromatic side-chains. The surface complementarity between hapten and Antibody is sufficient to make the complex specific for only one lactone ring conformation in the hapten. The crystal structure of the 40-50-ouabain complex allows qualitative explanation of the observed fine specificities of 40-50, including that for the binding of haptens substituted at the 16 and 12 positions. Comparison of the crystal structures of 40-50 complexed with ouabain and the previously determined 26-10 anti-Digoxin Fab complexed with Digoxin, demonstrates that the antibodies bind these structurally related haptens in different orientations, consistent with their different fine specificities. These results demonstrate that the immune system can generate antibodies that provide diverse structural solutions to the binding of even small molecules.

  • Effect of heavy chain signal peptide mutations and NH2-terminal chain length on binding of anti-Digoxin antibodies.
    The Journal of biological chemistry, 1993
    Co-Authors: J. Ping, Jirí Novotný, J. F. Schildbach, Shyh Yu Shaw, Thomas Quertermous, Robert E. Bruccoleri, Michael N. Margolies
    Abstract:

    In certain instances, Antibody variable region mutations outside of the antigen-combining site influence antigen binding. We reported previously that a heavy chain mutation (Ser-94-->Arg) decreased binding of the anti-Digoxin Antibody 40-150, whereas an additional signal peptide mutation at the -2 position (Gln-->Pro) causing NH2-terminal 2-residue truncation partially restored binding. To assess the combined effects on binding of two seemingly distant mutations, we constructed signal peptide mutations and NH2-terminal deletions in the presence of Ser-94 and Arg-94. Deletions of one to three amino acids had little effect on binding for Ser-94 mutants, whereas 2-residue truncations produced directly or by signal peptide mutation increased affinity approximately 40-fold for Arg-94 mutants. These observations are consistent with the reported computer-generated model of Antibody 40-150. Introduction of Pro at the signal peptide -3 position in 40-150 resulted in cleavage at alternative sites, with varying effects on affinity. Introduction of Pro at -2 into the anti-Digoxin Antibody 26-10 resulted, unexpectedly, in expression of heavy chains with 3 extra NH2-terminal residues, causing an approximately 100-fold reduction in affinity. Thus, both extensions and deletions of the heavy chain amino terminus can enhance or reduce antigen binding, depending on the structural context of specific antigen combining sites.

Olga V. Fedorova - One of the best experts on this subject based on the ideXlab platform.

  • Interaction of Digibind with endogenous cardiotonic steroids from preeclamptic placentae
    2015
    Co-Authors: Olga V. Fedorova, Anton Bzhelyansky, Elena V. Frolova, Natalia I. Tapilskaya, R. Nikitina, Vitaly A. Reznik, Vladimir A. Kashkin, Alexei Y. Bagrov
    Abstract:

    Background—Preeclampsia (PE) is a major cause of maternal and fetal mortality, and its pathogenesis is not fully understood. Endogenous digitalis-like cardiotonic steroids (CTS) have been implicated in the pathophysiology of PE; this is illustrated by clinical observations that Digibind, a therapeutic Digoxin Antibody fragment which binds CTS, lowers blood pressure and reverses Na/K-ATPase inhibition in patients with PE. Recently we reported that plasma levels of marinobufagenin (MBG), a bufadienolide vasoconstrictor CTS, are increased four-fold in patients with severe PE. Methods—In the present study, we compared levels of MBG in normal and PE placentae, as well as the interactions of Digibind and antibodies against MBG and ouabain with material purified from PE placentae using high-performance liquid chromatography (HPLC). Results—Levels of endogenous MBG, but not that of endogenous ouabain, exhibited a four-fold elevation in PE placentae vs. normal placentae (13.6±2.5 and 48.6±7.0 nmoles/g tissue; P<0.01). The elution time of endogenous placental MBG-like immunoreactive material from reverse-phase HPLC column was identical to that of authentic MBG. A competitive immunoassay based on Digibind exhibited reactivity to HPLC fractions having retention times similar to that seen with MBG and other bufadienolides, but no to ouabain-like immunoreactive material. Conclusions—Our results suggest that elevated levels of endogenous bufadienolide CTS represent a potential target for immunoneutralization in patients with PE

  • Magnesium Sulfate Potentiates Effect of Digifab on Marinobufagenin-Induced Na/K-ATPase Inhibition
    American journal of hypertension, 2013
    Co-Authors: Irina E. Zazerskaya, C. David Adair, Olga V. Fedorova, Valentina V. Ishkaraeva, Elena V. Frolova, Nelly G. Solodovnikova, Yulia N. Grigorova, Alexei Y. Bagrov
    Abstract:

    BACKGROUND Immunoneutralization of elevated circulating levels of endogenous digitalis-like Na/K-ATPase inhibitors (i.e. cardiotonic steroids (CTS)) represents a novel approach in the treatment of preeclampsia (PE). Recently we demonstrated that DigiFab (Fab fragments of affinity-purified ovine Digoxin Antibody) restores PE-induced inhibition of Na/K-ATPase in erythrocytes ex vivo. Previously magnesium ions were shown to antagonize digitalis-induced toxicity, which is mediated by Na/K-ATPase inhibition. We hypothesized that magnesium sulfate would potentiate the effect of DigiFab in the reversal of CTS-induced Na/K-ATPase inhibition.

  • Antibody to marinobufagenin lowers blood pressure in pregnant rats on a high NaCl intake.
    Journal of hypertension, 2005
    Co-Authors: Olga V. Fedorova, Nikolai I. Kolodkin, Natalia I. Agalakova, Alexandra R Namikas, Anton Bzhelyansky, Jean St-louis, Edward G. Lakatta, Alexei Y. Bagrov
    Abstract:

    ObjectiveThe pathogenesis of pre-eclampsia (PE), a major cause of maternal and fetal mortality, is not fully understood. Digitalis-like sodium pump ligands (SPLs) are believed to be implicated in PE, as illustrated by clinical observations that DIGIBIND, a Digoxin Antibody that binds SPLs, lowers bl

  • Effect of endogenous Digoxin-like factor and Digoxin Antibody on myocardial Na+, K+-pump activity and ventricular arrhythmias in acute myocardial ischaemia in rats
    Cardiovascular research, 1993
    Co-Authors: Alexei Y. Bagrov, Olga V. Fedorova, Natalia I. Roukoyatkina, Eugenii P Zhabko
    Abstract:

    Objective: The aim was to study whether a circulating sodium pump inhibitor (endogenous Digoxin like factor) contributes to the genesis of early ventricular arrhythmias in acute myocardial ischaemia in rats. Methods: Effects of Digoxin Antibody (260 μg·kg−1) on the incidence of ventricular arrhythmias, plasma Digoxin like immunoreactivity (DELFIA immunoassay), Na+, K+, and Mg2+ ions, and activity of the ouabain sensitive Na+, K+-pump in different regions of myocardium have been studied in propranolol naive and propranolol pretreated rats exposed to acute coronary artery ligation. Adult male Wistar rats were divided into six experimental groups: (1) saline pretreated controls; (2) saline pretreated coronary artery ligated rats; (3) coronary artery ligated rats pretreated with 260 μg·kg−1 Digoxin Antibody; (4) propranolol pretreated controls; (5) propranolol pretreated rats with acute myocardial ischaemia; (6) rats with acute myocardial ischaemia pretreated with both propranolol and Digoxin Antibody. Results: Acute myocardial ischaemia in saline pretreated rats was associated with a twofold increase of plasma Digoxin like immunoreactivity and ventricular arrhythmias, but did not lead to changes in myocardial Na+, K+-pump activity. Pretreatment of coronary artery ligated rats with Digoxin Antibody reduced the total duration of ventricular tachycardia and ventricular fibrillation during a 15 minute postligation period from 201(SEM 34) to 46(18) seconds (p

  • effect of endogenous Digoxin like factor and Digoxin Antibody on myocardial na k pump activity and ventricular arrhythmias in acute myocardial ischaemia in rats
    Cardiovascular Research, 1993
    Co-Authors: Alexei Y. Bagrov, Olga V. Fedorova, Natalia I. Roukoyatkina, Eugenii P Zhabko
    Abstract:

    Objective: The aim was to study whether a circulating sodium pump inhibitor (endogenous Digoxin like factor) contributes to the genesis of early ventricular arrhythmias in acute myocardial ischaemia in rats. Methods: Effects of Digoxin Antibody (260 μg·kg−1) on the incidence of ventricular arrhythmias, plasma Digoxin like immunoreactivity (DELFIA immunoassay), Na+, K+, and Mg2+ ions, and activity of the ouabain sensitive Na+, K+-pump in different regions of myocardium have been studied in propranolol naive and propranolol pretreated rats exposed to acute coronary artery ligation. Adult male Wistar rats were divided into six experimental groups: (1) saline pretreated controls; (2) saline pretreated coronary artery ligated rats; (3) coronary artery ligated rats pretreated with 260 μg·kg−1 Digoxin Antibody; (4) propranolol pretreated controls; (5) propranolol pretreated rats with acute myocardial ischaemia; (6) rats with acute myocardial ischaemia pretreated with both propranolol and Digoxin Antibody. Results: Acute myocardial ischaemia in saline pretreated rats was associated with a twofold increase of plasma Digoxin like immunoreactivity and ventricular arrhythmias, but did not lead to changes in myocardial Na+, K+-pump activity. Pretreatment of coronary artery ligated rats with Digoxin Antibody reduced the total duration of ventricular tachycardia and ventricular fibrillation during a 15 minute postligation period from 201(SEM 34) to 46(18) seconds (p<0.002) but did not alter activity of the myocardial Na+, K+-pump. In rats pretreated with propranolol, acute myocardial ischaemia was associated with a twofold inhibition of the Na+, K+-pump in left atrial and left ventricular myocardium, and with a 69% increase in plasma K+ concentration. Administration of Digoxin Antibody to propranolol pretreated coronary artery ligated rats in parallel with the antiarrhythmic effect prevented the increase in plasma K+ concentration and inhibition of Na+, K+-pump in the left atrial, but not the left ventricular myocardium. Conclusions: A circulating Digoxin like factor contributes to the pathogenesis of myocardial ischaemia induced ventricular arrhythmias. As propranolol pretreatment of coronary artery ligated rats inhibited the Na+, K+-pump in myocardium, the inhibitory effect of endogenous Digoxin like factor on Na+, K+-ATPase was probably masked in propranolol naive animals by the stimulatory action of catecholamines on Na+, K+-ATPase described previously. Cardiovascular Research 1993; 27 :1045-1050