325,266 research outputs found
Differential effects of low molecular weight inhibitors on the conformation and the immunologic function of the adhesion receptor LFA-1
No full textPrevious studies of our group 1,2 and by others 3 on the isolated ligand binding
domain of LFA-1 (αL I domain) have suggested that some LFA-1 inhibitors act
allosterically while other inhibitors were proposed to competitively block the LFA-
1/ligand interaction 4. We postulated that LMW LFA-1 inhibitors allosterically alter the
LFA-1 receptor conformation, resulting in shielding or neo-expression of epitopes
recognized by monoclonal antibodies (mAbs) mapping to regulatory domains of the
aL or b2 chains. Our data revealed that LFA-1 inhibitors can be differentiated according to their mode
of action on the receptor level.
The first group of lovastatin-derived LFA-1 inhibitors strongly induced conformational
changes within the aL I domain. This was detected by the potent inhibition of the
binding of the mAb R7.1 (anti CD11a, aL I domain specific) to either purified LFA-1 or
LFA-1 expressed on Jurkat T-cells. The degree of epitope reduction by the LFA-1
antagonists tested, correlated well with the potency in inhibition of the LFA-1/ICAM-1
interaction. These LFA-1 inhibitors had no effect on the binding of mAbs directed to
other domains within LFA-1.
In contrast, one lovastatin-derived inhibitor (LFA703) induced epitope changes in the
aL I domain and also in the b2 I-like domain, a regulatory domain located on the b2
chain of LFA-1. This effect became evident by the reduced binding of mAb IB4 (anti
CD18; b2 I-like domain specific) to cation-activated LFA-1 in the presence of
LFA703. These results demonstrated that amongst lovastatin-derived inhibitors
subclasses exit, which exert differential effects on the LFA-1 receptor conformation.
Moreover, the antibody binding patterns observed on native LFA-1 receptors in the
presence of various inhibitors demonstrated that upon receptor activation a
conformational interaction between the aL I domain and the b2 I-like domain is formed. These findings have meanwhile been confirmed by others in more
comprehensive biochemical studies 5.
For the first time our results provided strong evidence that the b2 I-like domain
embodies a target for allosteric LFA-1 inhibition similar to the well established
regulatory L-site in the aL I domain. XVA143, a suggested ICAM-1 mimetic, which
was proposed by the inventors to be a competitive aL I domain inhibitor 4, blocked
the binding of the β2 I-like domain specific mAb IB4 with nM potency. XVA143 had no
effect on the binding of mAb R7.1 or other anti CD11a mAbs under all experimental
conditions, and did not bind to the αL L-site as determined by NMR studies.
Furthermore, we showed that the target of XVA143 is most probably located on the
β2 chain, as the compound also blocked the binding of mAb IB4 to purified Mac-1
(αMβ2) and inhibited the interaction of purified Mac-1 with ICAM-1. The compound
typifies therefore a novel class of LFA-1 inhibitors with a distinct, probably allosteric
mode of action. These findings provided evidence that the β2 I-like domain could
represent a new target for potent inhibition of adhesion receptors of the β2 integrin
subgroup. Potent LMW inhibitors like XVA143 may open new opportunities for
specific intervention with the function of β2 integrins. These inhibitors could be
therapeutically useful in transplantation, autoimmune diseases and inflammatory
disorders.
Compellingly, the combined use of various LFA-1 inhibitors and selected monitoring
mAbs contributed to the understanding of the mode of action of LFA-1 inhibitors and
the function of β2 integrins on a molecular level. In addition, our findings show that
currently available LFA-1 inhibitors can be differentiated into two major groups
according to their mode of action on the receptor level: the αL L-site inhibitors and the
putative β2 I-like domain inhibitors. LMW LFA-1 inhibitors may soon enter clinical trials. For their pharmacological and
safety evaluation in clinical studies, it will be mandatory to provide, in addition to
pharmacokinetic (PK) measurements, insights in the pharmacodynamic (PD)
properties of these potentially immunosuppressive and anti-inflammatory
compounds. The aim of the studies described here was to develop the methodology
for studying the effect of LFA-1 inhibitors on receptor occupancy, receptor expression
and T-cell function in whole blood. These studies are intended as a basis for the
pharmacodynamic characterization of LFA-1 inhibitors in clinical trials. Furthermore,
the effect of LFA-1 inhibitors on T-cell function was compared to the
immunosuppressants cyclosporine A and everolimus. LFA-1 inhibitors of different chemical classes were tested in novel whole blood
receptor epitope monitoring assays (REMAs). We designate here REMAs as
cytometric methods which use target-specific mAbs to detect receptor occupancy by
LMW compounds in whole blood. The lovastatin-derived LFA-1 inhibitor LFA878 and
the experimental COMPOUND X, a non lovastatin-derived LFA-1 inhibitor, blocked
the binding of mAb R7.1 to leukocytes in undiluted blood with nM potencies. As
expected, the putative β2 I-like domain inhibitor XVA143 was unable to alter the
binding of mAb R7.1 to leukocytes in whole blood. In contrast, we found that LFA-1
receptor occupancy by XVA143 led to a significantly increased binding of the β2
chain, stalk region specific mAb MEM48 to whole blood leukocytes. These results
demonstrated for the first time that LFA-1 inhibitors with different modes of action can
interact with LFA-1 in undiluted human blood and that target occupancy can be
monitored by selected mAbs.
The REMA principle was validated ex vivo by measuring LFA-1 receptor occupancy
in blood of rabbits after i.v. administration of LFA878. LFA878 blocked the binding of
the mAb R7.1 with transient duration of action. Dependent on the dose administered the pharmacodynamic half-life was 0.6 h (11.5 mg/kg i.v.) or 3.3 h (50mg/kg i.v.).
These data showed for the first time that the REMA can be applied to study
pharmacodynamic effects of αL L-site inhibitors in rabbits ex vivo. Our results
furthermore suggested that the αL L-site and the mAb R7.1 epitope are conserved
between man and rabbit. The pharmacodynamic effects of XVA143 could not be
investigated because the mAb MEM48 did not cross-react with LFA-1 of other
species.
To allow the assessment of the effect of LFA-1 inhibitors on several T-cell
parameters, we developed an anti CD3 (OKT3) mAb stimulated T-cell activation
assay (CD69 readout) and combined it with the REMAs described above. The socalled
EA-REMAs allowed us to quantify simultaneously receptor occupancy by LFA-
1 inhibitors (REMA), the cell surface LFA-1 expression (E) and the upregulation of
the activation marker CD69 (A) on individual T-lymphocytes after in vitro stimulation
of 1:1 diluted blood with immobilized mAb OKT3.
LFA878, COMPOUND X and XVA143 completely blocked mAb OKT3 stimulated
CD69 upregulation with IC50s of 2 μM, 1 μM and 0.05 μM respectively, while
pravastatin, a statin that does not bind to LFA-1, was completely inactive at 50 μM.
The LFA-1 inhibitors tested were completely inactive in blood cultures stimulated with
a combination of mAbs OKT3 and anti CD28, demonstrating the specific inhibition of
LFA-1 dependent T-cell responses by the compounds tested.
An additional pharmacodynamic property of XVA143 was revealed by the EA-REMA.
22 h incubation of whole blood with XVA143 led to a partial (35-55%) downregulation
of LFA-1 cell surface receptors on T-cells, a phenomenon not observed for the αL Lsite
inhibitors tested. The compounds were then assessed on their effect on mAb OKT3 stimulated T-cell
proliferation in 1:10 diluted blood. All LFA-1 inhibitors blocked mAb OKT3 stimulated T-lymphocyte proliferation with nearly equal potencies than observed in the mAb
OKT3 stimulated T-cell activation assay.
Applying these protocols, experimental evidence was obtained for the first time that
LFA-1 receptor occupancy by LFA-1 inhibitors can translate into efficient blockade of
in vitro stimulated T-cell activation and proliferation in whole blood. The correlation
between receptor occupancy and blockade of T-cell activation and proliferation
(response) revealed that a >85% receptor occupancy in whole blood is required by
the αL L-site inhibitors tested for the suppression of T-cell responses in whole blood
cultures by 50%. In contrast, an almost 1:1 correlation between receptor occupancy
and the resulting suppression of T-cell responses was observed for the β2 I-like
domain inhibitor XVA143. A comparison of LFA-1 inhibitors with cyclosporin A (CsA) and everolimus in the
whole blood assays suggested that the structurally different LFA-1 inhibitors could be
useful as immunosuppressants. XVA143 blocked T-cell activation (0.05 μM) and
proliferation (0.02 μM) with higher potency than CsA (0.8 μM; 0.15 μM respectively)
and was nearly equipotent to everolimus (0.01 μM) in the whole blood proliferation
assay. In contrast, αL L-site inhibitors were nearly as potent as CsA in the CD69 Tcell
activation assay, but significantly less active in whole blood proliferation assays
(1-2 μM). As expected, CsA or everolimus did not interfere with LFA-1 expression or
the binding of the monitoring mAbs R7.1 or MEM48.
During the development of the EA-REMA we found that supplemental MgCl2 strongly
synergized with anti CD3 triggered T-cell activation in whole blood. This finding may
suggest a new role for magnesium cations in the regulation of integrin dependent Tcell
responses in vivo. We hypothesize that locally elevated (mM) concentrations of
Mg2+ may regulate integrin adhesiveness and thereby strengthen cell to cell contacts
leading to enhanced integrin dependent T-lymphocyte responses. Further investigations are ongoing, to elucidate the effect of magnesium on the activation and
function of immune cells.
In conclusion, we demonstrated that various LFA-1 inhibitors could occupy their
target on leukocytes in whole blood and that LFA-1 occupancy by these inhibitors
translated into potent suppression of in vitro stimulated blood T-lymphocytes. Our
data are strongly suggesting that LFA-1 inhibitors, in particular inhibitors with the
potency of XVA143, could be applicable as therapeutic immunosuppressants. In
addition, our array of novel methods allowed us to generate an “in vitro
pharmacodynamic “ profile of LMW LFA-1 inhibitors with different modes of action in
whole blood. These protocols may be applicable as pharmacodynamic assays for
LFA-1 inhibitors in clinical studies and may assist therapeutic dose finding
The Soviet Union and the Creation of the International Human Rights System
No full textThe following comments draw from the authors Eric D. Weitz´s upcoming book : A World Divided: The Global Struggle for Human Rights in the Age of the Nation-State (Princeton: Princeton University Press, forthcoming 2019)
Estimating the strength, speed, and final size of disease outbreaks: application to 2019-nCoV - presentation
No full textEstimating the strength, speed, and final size of disease outbreaks: application to 2019-nCoVA Presentation at the Center for Microbial Dynamics and Infection 'Coronavirus Forum', February 10, 2020, Georgia Institute of Technology.Presenter: Joshua S. Weitz, [email protected], http://ecotheory.biology.gatech.edu</div
THE EFFECT OF ORGANIC COSOLVENTS ON THE ENANTIOSELECTIVITY IN THE BOVINE SERUM-ALBUMIN CATALYZED WEITZ-SCHEFFER CONDENSATION
No full textWater miscible or immiscible organic cosolvents can strongly influence the enantioselectivity of the Weitz-Scheffer condensation catalyzed by bovine serum albumin, the enantiomeric excess (e.e.) being in the range 0-90%
Ist "Kollektiverziehung" heute möglich? Erfahrungen aus dem Bielefelder Oberstufen-Kolleg
No full textEmmerich O, Koch-Priewe B. Ist "Kollektiverziehung" heute möglich? Erfahrungen aus dem Bielefelder Oberstufen-Kolleg. In: Hillig G, Weitz S, eds. Stand und Perspektiven der Makarenko-Forschung. Marburger Beiträge zur Vergleichenden Erziehungswissenschaft und Bildungsforschung. Vol 27. München: Minerva-Publ.; 1994: 402
Supplemental Material - <b>Credit Claiming by Labeling</b>
No full textSupplemental Material for Credit Claiming by Labeling by Virginia Oliveros, Rebecca Weitz-Shapiro, Matthew S. Winters in Comparative Political Studies</p
Diffusive author(s), cohesive author: Analysis of S/N (1994)
No full textThis study indicates the ways in which various aspects of the author(s) are brought forth in Dumb type’s performance art, the S/N production. Previous research has suggested a non-hierarchical organization of Dumb type and the absence of a “privileged author” in Dumb type’s collaborative work, S/N. However, the results that I have investigated from member’s interviews on the creative process of S/N along with my analysis of the recorded images of S/N, indicate a different aspect of the author(s). First, S/N was created through, so to speak, the collective ideas of the members of Dumb type. Further, S/N has at least nine quotations from previous performances, installations, and printed writings, besides the work-in-progress technique. Explicating one of the “author functions” as given by Michel Foucault, each text has plural subjects of the author. However, it has been revealed from members’ interviews that Teiji Furuhashi had a decision-making role in selecting the members’ ideas within the performance. Since then, S/N has had plural subjects of creation; however, Furuhashi is one of the subjects of creation along with the “privileged author.” S/N has plural authors (diffusive authors) yet at the same time, it has a “privileged author,” Teiji Furuhashi (cohesive author)
Going Beyond Counting First Authors in Author Co-citation Analysis
No full textThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
The Brazilian experience shows that voters are more forgiving of incompetence than they are of corruption
No full textCorruption in the UK is perceived as being on the rise, with recent research showing that British citizens are increasingly concerned about the relationship between private financial interests and politicians. But are voters willing to forgive corrupt politicians if they are competent? New research which looks at the Brazilian case by Rebecca Weitz-Shapiro and Matthew S. Winters shows that voters are merciless when it comes to forgiving corrupt officials and politicians, and that their electoral success is usually a result of lower voter information
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