Ultra low doses

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147918

ULTRA LOW DOSES

au

2n

Ultra Low Doses Edited by

C. Doutremepuich Université Bordeaux France

Taylor & Francis London - Washington, DC 1991

UK

Taylor & Francis Ltd., 4 John St. London WCIN 2ET

USA

Taylor & Francis Inc., 1900 Frost Road, Suite 101, Bristol, PA 19007

Copyright © Taylor & Francis Ltd., 1991

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, electrostatic, magnetic tape, mechanical,

photocopying, recording or otherwise, without the prior permission

: \DERBIL PaERvELIRBRtARY ICAL CENT E SHVILLE, TENNESSE 37232

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Available on request

Library of Congress Cataloging-in-Publication Data

Available on request

Printed in Great Britain by Burgess Science Press, Basingstoke on paper which has a specified pH value on final paper manufacture of not less than 7.5 and is therefore ‘acid free’.

Contents

Preface

I EXPERIMENTAL PHARMACOLOGY First experimental arguments in favor of the effect of very weak doses of copper on digestive motricity in mice and rabbits Santini, R., Tessier,M., Belon, P., Pacheco, H.

The effects of some regulatory peptides in femtomolar concentrations on the contraction of lymphatic vessels (LVs) Ashmarin,I.P., Levekova, L.Ts., Sanzhieva.

and

lower

11

Modulation of experimental rat liver carcinogenesis by ultra low doses of the carcinogens De Gerlache, J., Lans, M.

17

Overcoming tumor cell drug resistance by low doses of recombinant tumor necrosis factor and drug Bonavida, B., Safrit, J., Tsuchitani, T., Zighelboim, J.

Rs

Effect of acetylsalicylic acid at ultra low dose on the interaction platelets/ vessel wall Lalanne, M.C., De Seze, O., Doutremepuich, C.

45

II BIOPHYSICS A quantum chemical study of some model anti-inflammatory compounds: the preferred conformations and their electrostatic similarities Bhattacharjee, Apurka, K.

55

Influence of several physical factors on the activity of ultra low doses Cazin,J.C., Cazin, M., Chaoui,A., Belon,P.

69

vi

Contents

III BIOCHEMISTRY — TOXICOLOGY Uranyl nitrate induced corpuscular indication of renel dysfunctioning Gojer, M., Sawant, V.

derangement

in rat,

an

early

Degranulation of mesenteric mast cells as ‘spot test’ in toxicology Rathinam, K., Mohanan, P.V., Lizzy Michael

81

89

IV CELL BIOLOGY Simultaneous measurement of oxidative metabolism and adhesion of human neutrophils and evaluation of multiple doses of agonists and inhibitors Bellavite, P., Chirumbolo,S., Signorini, A., Bianchi,I., Dri,P.

93

Non-stimulatory concentrations of concanavalin A can modulate subsequent stimulation by the same mitogen Eskinazi, D.P., Molinaro, G.A.

119

Biological activity of ultra low doses: I/ Effect of ultra low doses of histamine on human basophil degranulation triggered by D. pteronyssinus extract Sainte-Laudy, J., Sambucy, J.L., Belon,P.

127

Biological activity of ultra doses: II/ Effect of ultra low doses of histamine on human basophil degranulation triggered by anti-IgE Sainte-Laudy, J., Belon,P.

139

V CLINICAL PHARMACOLOGY A study of the effectiveness of ultra low doses of copper in the treatment of hemodialysis-related muscle cramps Hariveau, E., Nolen, P., Holtzscherer, A. 145 Action of aspirin after ingestion at ultra low doses in healthy volunteers Lalanne, M.C., De Seze, O., Le Roy,D.,Doutremepuich, C., Boiron, J.

151

Index

15

Preface

Research on Ultra Low Doses (ULD) is a multidisciplinary concept, situated at the crossroads of medical, human, biological and physical sciences. Determination of the mechanism of action is at present being widely developed, with numerous physico-chemical findings. Many questions about ULD efficacy now have a proven answer. However, other questions have arisen for which there are not enough experimental findings to provide definite conclusions. It is therefore of interest to convene an assembly of all people working in this field, to compare their results and conclusions. Fifteen countries were represented in Bordeaux in September 1990, for the first ‘International Congress on Ultra Low Doses’. The proceedings of these communications are summarized in this issue. The scientific committee was professors Aran (INSERM, France), Bonavida (USA), Dufourcq (CNRS, Roberfroid (Belgium) and Turner (GB).

France),

The editors wish to thank the authors for their contribution. They are also indebted to M. Claire Lalanne, for supporting the copy preparation. They hope that these proceedings may serve as a source of material for all investigators, researchers and students, who will be attracted by this highly interesting area. The kind cooperation and help from the publisher, Taylor & Francis, is hereby acknowledged. Professor C. Doutremepuich

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NON-STIMULATORY CONCENTRATIONS OF CONCANAVALIN A CAN MODULATE SUBSEQUENT STIMULATION BY THE SAME MITOGEN

D.P.

Eskinazi,

*Columbia,

MD,

D.D.S., *Loma

Ph.D*

Linda

and

A.

University,

Molinaro7,M.D. Loma

Linda,

CA

We evaluated whether concentrations of biologically-active substances too low to induce a direct effect can modulate a response induced by a higher concentration of the same substance. We took as experimental model the concanavalin A (Con A)dependent mitogenesis of mononuclear’ cells as assessed by 3H-thymidine incorporation. In seven consecutive

experiments,

we

observed

treatment with non-mitogenic doses of Con the proliferation induced by suboptimal concentrations of Con A.

INTRODUCTION Recent

that _ pre-

A modulated stimulatory

work has confirmed that various biologically-active factors can be effective at very low concentrations. Thus, some lymphokines can exert an effect at concentrations as low as 10-15 M (Klein, 1990) and endogenous opioids can enhance cellular cytotoxicity at similar concentrations (Sibinga and Goldstein, 1988). Further, it has been shown that two or more factors such as lymphokines can act in synergy (Durum and Oppenheim, 1989). In this context, factor concentrations too low to produce a direct effect could still modulate the response to stimulatory concentrations of synergistic substances. The present studies were designed to extend this concept to self-modulation and evaluate whether a substance too low to promote direct effects could still modulate effects induced by higher concentrations of the same substance. 119

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MATERIALS

AND

HSKinNaZe

eb al.

METHODS

Isolation of Peripheral Mononuclear Cells Ten to 100 ml of freshly drawn blood was diluted 1:2 in RPMI 1640. Two volumes of diluted blood were layered onto one volume of Ficoll-Paque (Pharmaceutical LKB, Uppsala, Sweden). After centrifugation at 400 x g at room temperature for 30 minutes, the cells at the plasma/Ficoll interface were collected, diluted in 10 volumes of RPMI 1640, and centrifuged for 200 x g at room temperature for 10 minutes. The pelleted cells were washed two more times in a similar fashion, and finally resuspended in RPMI 1640 supplemented with heat inactivated fetal

bovine serum (10%), streptomycin (50 wg/ml).

penicillin

(50

U/ml)

and

Mononuclear

Cell Stimulation Assays 1 x 105 cells in 180 wl of medium were pipetted into wells of a microtiter culture plate containing 20 wl of appropriate modulatory concentrations of Con A and cultured at 37°C for one-hour in a 5% CO2 humidified chamber. Then 20 wl of either of four stimulatory concentrations of Con A was added to triplicate wells to provide final concentrations of 3.0 to 0.1 wg/ml and the plates were reincubated. After three’ "days; 1 °pyCi of 3H-Thy tin 20 plo of REMI 1640 was added to each well. After incubation at 37°C for four hours, the cells were harvested on glass strips and the 3HR-Thy incorporation was counted ina beta scintillation radioactivity counter.

Statistical Analysis Data were subjected

(ANOVA).

to

the

analysis

of

variance

RESULTS

The effectiveness of preincubations with dilutions of Con A to modulate the response of peripheral blood mononuclear cells to subsequent stimulatroy doses of Caon A was evaluated. In each experiment four stimulatory doses of Con A were used ; prior to being incubated with each of the stimulatory doses, cells were preincubated either with medium not containing Con A or containing Con A concentrations ranging from

1 x

10-3

to

3 x 10-7

wg/ml.

In experiments

using doses of Con A inducing maximal stimulation of the lymphocytes, we found no modulatory effect of the preincubations. In seven successive experiments using one or more doses of Con A inducing submaximal proliferation, we found that preincubation with dilutions of Con A resulted, within eah experiment,

Coneanavalin

A modulatton

HDI

in a statistically significant modulation of the proliferation, with p values ranging between 0.05 and 0.0005 depending on the experiment (Fig.1). To test for a reproducible operator-dependent artifact,

in one of these experiments, the blood sample was divided in two equal aliquots that were handled independently by two technicians. Thus in this experiment, cell isolation, stimulatory Con A dilutions, modulatory dilutions, and experimental set up were prepared in parallel but independently. In the microtest plates handled by either technician, two of the stimulatory doses of Con A resulted in submaximal

level of dissimilar versus

stimulation.

For

each

of

unmodulated stimulation in the microtest plates

those

doses,

the

was somewhat handled by one

the other technician ; the level of modulation observed was concomitantly different. For the second stimulatory dose of Con A that induced submaximal stimulation, the level of proliferation was quite similar in the microtest plates handled by either technician ; the level of modulation was concomitantly very similar (Fig.2). To test for a reproducible design-dependent artifact, an experiment was conducted with alternating control (Gises,. preincubation not containing modulatory doses of Con A) and experimental (i.e., preincubation containing modulatory doses of Con A) triplicates. Proliferation in control wells was observed to be significantly lower than in the experimental ones (p;

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