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HIGH  DILUTIONS OF ANTIGENS MODULATE
THE IMMUNE RESPONSE TO KLH

Modulation of the Specific Immune Response

 

 

Z. WEISMAN, M. OBERBAUM, R. TOPPER, N. HARPAZ,

Z. BENTWICH

R. Ben Ari Institute of Clinical Immunology, Kaplan Hospital, Rehovot; The Hebrew University Hadassah Medical School, Jerusalem, Israel

 

 
1 - Introduction
2 - Material and methods
3 - Results and Discussion
4 - References

 

1. Introduction

 

Homeopathy, despite being practiced by large number of people around the world (Einsenberg et al., 1993; Kewal, 1995; Fisher and Ward, 1994) is not acceptable by the established medical and scientific community  mainly because of the lack of acceptable scientific explanation, or theory, that would account for its claimed effects (Delbanco, 1994; Campion, 1993; Skrabanek, 1986). The possibility that extremely diluted solutions, that do not contain any trace of the solute, would still retain any biological effect of that solute is practically considered heretical, or at least unacceptable, according to the existing scientific paradigms. It is therefore of utmost importance to try and address this general issue by accepted conventional methods, and this has motivated the present study. In this study we have tested the possibility that highly diluted "homeopathic" antigen solutions retain the ability to generate, or influence, a specific immune response to such antigen in an animal model.

 

 

2. Material and Methods

 

2.1 MICE

 

C57Bl/6J, pathogen-free 6-8 wk old females, were obtained from The Jackson Laboratory (Bar Harbor, ME). For some experiments inbred mice were used (Ehud Hepher, Hulda, Israel). The animals were permitted access to food and water ad libitum, and 12 h light (0700-1900), 12 h dark / day .

 

2.2 ANTIGENS

 

Keyhole Limpet Hemocyanin (KLH) (Calbiochem Corporation, La Jolla, CA 92037, cat. no. 3748411) and Porcine Thyroglobulin (Tg) (Sigma, Israel, cat. no.T-1126) were dialyzed , and sterile stock solutions of 3mg/ml in saline were kept at 4°C (sterilized by  using 0.45  filters).   

 

2.2.1 Dilutions of the antigens

KLH, Tg, and saline (as a control solution) were diluted in steps of 1:100. For the first dilution, designated 1 C, 5 ml of the stock solution was added to 5 ml sterile water (pyrogen free) in conical 10ml plastic sterile tubes (Sterilin). The tubes were shaken vigorously for 3 min by a Dolisos dynamisator, followed for 1 min. by a Boiron dynamisator. This procedure was repeated until one dilution before the last. The last dilution was prepared similarly using sterile saline instead of water.

 

2.3 EXPERIMENTAL DESIGN

 

2.3.1 Preconditioning of the mice

The animals were  divided at random into groups of 8-10 animals per cage. The mice were injected intraperitoneally (i.p) with 0.3 ml of either antigen or saline, at different dilutions, 3 times per week during 8 weeks. In some experiments the animals were preconditioned orally by adding antigens or saline dilutions to the drinking water (1:100) and mixing vigorously for 2 min by dynamisator.

 

2.3.2 Immunization

At the end of the preconditioning period all the mice, including sex and age matched naive controls that were kept untreated during the preconditioning time, were immunized with 1 mg KLH per mouse in Complete or Incomplete Freund's adjuvant (CFA, ICFA) diluted  v/v with Phosphate Buffer Saline (PBS). Immunization with CFA was given in the tail base while ICFA was injected in the foot pads.

 

2.3.3 Bleedings

The mice were bled, by orbital sinus puncture, at intervals during the preconditioning period, and after immunization. Serum was separated and kept at -20 °C until assayed for antibodies titer.

 

2.4 IMMUNOLOGICAL ASSAYS

 

KLH IgM and IgG antibodies titers were assayed by ELISA in serum of individual mice. Flat bottom microtiter plates  (Nunc-Immuno Plate MaxiSorp) were coated overnight at 4 °C with 5 mg/100 ml/well of KLH, Tg or Rabbit Serum Albumin (RSA) (Sigma, Israel) dissolved in PBS. After blocking with 2.5% fetal calf serum in DMEM medium containing 0.02% Hepes and 0.05% sodium azide, serum dilutions (1:1000 or 1:10000 for IgM and IgG respectively) were added, and the plates were incubated for 2 h. The reaction was developed with alkaline phosphatase-conjugated goat anti mouse IgM or IgG ( m or g-chain specific respectively)(Sigma, Israel), followed by alkaline phosphatase substrate (Sigma 9389).  After 30 min incubation the optical density was recorded at 405 nm. The results are represented by the mean value of each experimental group, and are expressed in optical density units or as percentages of normal untreated mouse serum (NMS).

            Lymphocyte proliferation was determined by plating 5 x 105 spleen cells in 0.2ml RPMI medium enriched with 1% fresh NMS, 2 mM glutamine, 1% Non Essential Amino Acids, 1 mM Sodium Pyruvate, 5 x 10-5M 2-Mercaptoethanol, and antibiotics in flat bottom microtiter plates (Nunc). The cells were cultured for 5 days in medium alone or in medium containing one of the following: Concavalin A (con A) 0.8mg/ml, lipopolysacharide (LPS) 4 and 8 mg/ml, KLH/Tg 5, 25, and 50 mg/ml. The cultures were set in triplicates. 3H Thymidine (0.5 Ci/well) was added to the cultures for the last 18 h, the cells were harvested, and the radioactivity was counted by a b-counter (cells with con A and LPS were labeled and harvested at day 3 ). Proliferation was expressed as stimulation index (S.I). S.I = The maximum counts per minute (cpm) in the presence of antigen divided by the cpm in medium alone.

 

2.5 STATISTICAL ANALYSIS

 

The Anova, Multiple measurements analysis of variance (Anova) and the Student's t-test were used. P < 0.05 was considered as significant.

 

 

3.Results and discussion

 

3.1 THE CONCENTRATION OF ANTIGENS

 

The concentration of antigens and the total number of molecules injected to each mice during the 8 weeks of preconditioning with the various dilutions are summarized in table 1. As can be seen in the table, no antigen is present any more in the 15 C dilution which is beyond the Avogadro number.

 

 

 

TABLE 1: The number of molecules of antigen injected to each mice at different dilutions

 

            Dilution                       Molarity                      # of injections total # of molecules/mouse

            ____________________________________________________________

 

            4 C                  10-14                             24                    48,106

            6 C                  10-18                             24                    4800   

            7 C                  10-20                             24                    48                   

            15 C                10-36                             24                    0

            ____________________________________________________________        

 

 

 

3.2 THE EFFECTS OF PRECONDITIONING BY INJECTIONS

 

3.2.1 Antibody generation during preconditioning

Repeated i.p injections of high dilutions of KLH, Tg or saline induced generally a similar increase in serum IgM (Fig.1). The response was not antigen specific since similar titers were observed with assays for antibodies to KLH, RSA or Tg (Data not shown). There was no induction of IgG antibodies by the preconditioning treatment.

 

 3.2.2 Antibody response to immunization with KLH

            IgM response. In 2/6 experiments the IgM response to immunization with KLH was significantly different in mice preconditioned by injections of high KLH dilutions. In the first experiment (immunization with CFA), all the KLH pretreated mice  had showed significantly lower IgM titers compared with the control naive mice, 14 days post immunization (P<0.05 student 's t-test) (data not shown). These results could not be repeated in a following similar experiment in which all the mice, whether pretreated with KLH or not, responded similarly to immunization with KLH. Therefore, the immunization procedure was changed, and ICFA was used instead of CFA, hoping to slow down the immune response of the animals, so it will be able to distinguish between variations in the kinetics of the immune response. However, in only 1/4 such experiments did we find a significant difference in the kinetics of IgM response by KLH preconditioned mice (P<0.05 anova). As can be seen in Figure 2, mice that were pretreated  with the 7 C and 15 C KLH dilutions show an earlier and higher IgM anti KLH response compared with saline injected mice or naive mice.

 

 

Figure 1. Anti KLH IgM response. Mice were treated  by repeating i.p injections of saline, KLH and Tg. The results represent the mean antibody titers of each group.

           

 

 

            In all the other experiments the IgM response was similar in all the groups. Since repeated i.p injections of mice evoked mostly a polyclonal IgM response (see figure 1), a possible modulation of a specific anti KLH response by the treatment could be obscured.

            IgG response. In contrast to the IgM response, preconditioning with high diluted antigen solutions clearly affected the specific anti KLH secondary response of the animals in 4/6 experiments. A very dramatic effect was observed in the first experiment. As can be seen in Figure 3, at 14 days post immunization, most of the mice that were preconditioned by injections of high dilutions of KLH switched to IgG response, while the control naive mice still show low titers of IgG and high titers of IgM (data not shown).

            In 3/4 additional experiments, in which ICFA was used for immunization, a significant increase in IgG response was observed in the 15C KLH preconditioned group, and in two experiments also in the 7C KLH preconditioned group (Figures 4 and 5).

 

 

Figure 2. Kinetics of anti KLH IgM post immunization response. Mice were preconditioned for 2 mo. by i.p injections with 7 and 15 C KLH and saline dilutions. The treated and naive mice (control) were then  immunized in the foot pads. The results are expressed as percentage of IgM titer of untreated mice.

* significant different kinetics from control and saline groups (Anova).

 

 

 

 

         

 

Figure 3. Anti KLH  IgG response 14 days post immunization. Mice were  injected i.p  with several dilutions of KLH for 2 months. The mice were immunized with KLH/CFA in the tail. Each bar represent one animal.  

 

 

 

To check for the specificity of the effect of KLH, the antigen Tg was included in some experiments. Different groups of mice were treated with 7 C and 15 C dilutions of KLH and Tg, as well as with the 7C dilution of Saline (Figures 4 and 5).

 

 

 

Figure 4. Kinetics of anti KLH IgG response. Mice were preconditioned with repeated i.p injections of saline, KLH and Tg dilutions for 2 months and subsequently immunized with KLH/ICFA.

 * - significantly different from the control and saline preconditioned mice  (Anova).

**- Student t-test.

 

 

 

 

 

In Figure 4, it can be seen that the kinetics of the IgG response was significantly different in the 15 C KLH pretreated mice (P=0.055, anova multiple measurements analysis of variance,  and p<0.01 student's t-test at day 15). The effect of preconditioning by 7 C KLH dilution was evident only 30 days post immunization. Saline or Tg dilutions did not affect the subsequent immune response to KLH. The response was anti KLH specific since the ELISA assays on RSA or Tg coated plates were negative. However, as can be seen in Figure 5,  preconditioning with 15 C Tg also increased significantly the IgG response similar to that of the KLH preconditioned mice. The differences in the kinetics of the response of all the preconditioned groups, with the exception of 7 C Tg , compared to the control groups, were highly significant (P<0.02  Anova).

            Taken together, it can be summarized that preconditioning with high dilution of antigen accelerated significantly the secondary IgG response to the antigen. The central issue of the specificity of this response is not yet resolved. It is possible that KLH and Tg cross react with each other, both being very large molecules. Such experiments need to be repeated, using additional antigens both in the preconditioning treatment and in immunization.

 

 

Figure 5.  Kinetics of anti KLH IgG response. Mice were preconditioned with repeated i.p injections of saline, KLH and Tg dilutions for 2 mo.nths and subsequently immunized with KLH/ICFA. Group " KLH 7d " was orally preconditioned by adding 7 C KLH to the drinking water.

* - significantly different from the control and saline preconditioned mice (Anova).

 

 

 

3.3 THE EFFECT OF ORAL PRECONDITIONING

 

Oral antigen treatment was given through the drinking water (3 experiments).

 

3.3.1 IgM response

There was no IgM response in the animals during the pretreatment period. After immunization with KLH, there was no difference between the different groups, whether preconditioned or not.

 

3.3.2 IgG response

In 2/3 experiments the kinetics of the anti KLH IgG response was different in the preconditioned groups compared with the control naive mice or the saline pre-conditioned one. In Figure 5 it can be seen that mice receiving the 7 C KLH in the drinking water ("KLH 7d") showed a significantly faster increase in IgG titers compared with the control groups, similarly to the kinetics shown by the groups which were preconditioned by injections. In another experiment (Figure 6) oral treatment by 7 C KLH , as well as by 7 C Tg , increased  significantly the IgG response to immunization with KLH at day 11 post immunization (the increase in IgG in the 15 C KLH was not significant).

Taken together, these results highly suggest that preconditioning of mice by high dilution of antigens, delivered orally, can transfer signals to the immune system and modulate its response to subsequent immunization.

 

 

 

Figure 6. Kinetics of anti KLH IgG response. Mice were preconditioned orally with KLH and Tg dilutions in the drinking water for 2 months and subsequently immunized with KLH/ICFA.

* significantly different from the control naive and saline preconditioned mice (p<0.001, student's t- test).

 

 

3.4 THE EFFECT OF PRECONDITIONING ON THE PROLIFERATIVE RESPONSE

 

A different approach to study the effects of preconditioning by high dilutions of antigens is by testing the proliferative responses of lymphoid cells, in vitro, to the immunizing antigen. The results of two such experiments are described.

            Spleen cells, obtained from animals preconditioned with high dilutions of antigens and from naive animals, 10 days post immunization with KLH/ICFA were cultured in the presence of KLH and Tg for 5 days. (con A and LPS were added to the cultures as positive controls for testing the activity of the cells).The proliferative response was measured by the uptake of labeled thymidine, and was expressed as stimulation index; a stimulation index higher than 2 was regarded as a positive response. In Figure 7 it can be seen that spleen cells from naive or saline preconditioned mice (4 mice) did not respond to KLH or Tg added to the cultures. However, 5/6 KLH (7 C and 15 C) preconditioned mice responded to KLH and 1 responded also to Tg. It can be also seen that 1/3 mice preconditioned with Tg (7 C) responded to both KLH and Tg.

            In the second experiment the animals were preconditioned by oral treatment. The results are summarized in Figure 8. In this experiment, at 10 days post immunization with KLH, 15/16 animals responded already to KLH stimulation in vitro (S.I > 2). However, the highest proliferative responses to both antigens were observed in preconditioned mice.

 

 

Figure 7. Proliferative response of spleen cells to KLH and Tg. Mice were preconditioned for 2 mo by repeated i.p injections of high dilutions of KLH, Tg and saline, and then immunized with  KLH/ICFA. Ten  days post immunization the spleen cells were cultured in the presence of the antigens KLH and Tg. The results are expressed as stimulation index, each bar represent the response of one mouse.

 

 

 

 

 

Figure 8. Proliferative responses of spleen cells to KLH and Tg. Mice were oral preconditioned for 2 mo by adding high dilutions of KLH (7 and 15C), Tg (7C) and saline (7C) to the drinking  water, and then  immunized with  KLH/ICFA. Ten days post immunization the spleen cells were cultured in   the presence  of the antigens KLH and Tg. The results are expressed as stimulation index, each bar represent the response of one mouse.

 

           

            In both experiments, preconditioning of the mice with high dilution of antigen probably modulated the response of the mice to subsequent immunization. This was reflected by an increased  response of the spleen cells to the secondary challenge with the antigen in vitro. The significance of the response to Tg by spleen cells from KLH immunized mice is not clear. It may suggest cross reaction between the 2 antigens, but it can also be the effect of preconditioning with high dilution of the Tg. More experiments are needed to define the best conditions for this type of experiments, and the kinetics of the response.

            The cumulative results of the experiments described above strongly support the possibility that antigen administered in high diluted form, whether orally or  parenterally, does have a modulating and specific effect on the immune response to that antigen. This effect was observed for both humoral  (IgG) and cellular responses, and was observed in animals preconditioned even with antigen diluted beyond Avogadro's number. This in itself is of great importance in lending support to the basic concept , that highly diluted substances retain their biological activity even in this form. Similar results in other experimental systems have been published in the last decade (Bastide et al., 1985; Bastide et al., 1987; Daurat et al., 1988; Davenas et al., 1987; Endler et al., 1991; Fisher et al.,, 1987; Sukul et al., 1986). Furthermore, our studies show that immunological information can be transferred by very high diluted substances, and can have an imprint in the form of antigen memory. The issue of the specificity of this response, though highly suggestive, is not clearly resolved and requires further studies. Especially due to possible cross reactivity between antigens like thyroglobulin and KLH. It would be advantageous to use other, and possibly smaller, antigens to try and resolve this issue. Also, adoptive transfer experiments to show definitively the transfer of immunological memory, will be most critical for better definition of these effects.

            Another clearly bothersome aspect of these studies is the variation in the response and reproducibility of the observed effects. The reasons for these variations could probably be the results of a number of variables. Since the system developed by us is very likely to be extremely sensitive to small environmental and experimental changes, such results may not be that surprising (Doucet-Jaboeuf et al., 1984; Carrière et al., 1989). These type of experiments have to be repeated many times in order to get reliable statistical results.

 

 

4 References

 

Bastide, M., Daurat, V., Doucet-Jaboeuf, M., Pelegrin, A., Dorfman, P. (1987) Immunomodulator activity of very low doses of thymulin in mice, Int.J.Immunother. 3, 191-200.

Bastide, M., Doucet-Jaboeuf , M., Daurat, V. (1985) Activity and chronopharmacology of very low doses of  physiological  immune inducer, Immunol.Today 6, 234-235.

Campion, E.,W. (1993) Why unconventional medicine?, New England J. Med. 328, 282-283.

Carrière,V., Dorfman, P., and Bastide, M. (1989) Evaluation of various factors influencing the action of mouse ab interferon on the chemiluminescence of mouse peritoneal macrophages, Annual Rev. of Chronopharmacol . 5, 9-12.

Daurat, V., Dorfman, P., and Bastide, M. (1988) Immunomodulatory activity of low doses of ab interferon in mice, Biomed. & Pharmacother. 42, 197-206.

Davenas, E., Poitevin, B., Benveniste, J. (1987) Effect on mouse peritoneal macrophages of orally administered very high dilutions of silica, Europ. J.Pharmacol .135, 313-319.

Delbanco, T., L. (1994) Bitter Herbs: Main stream, Magic, and Menace, Ann. Intern. Med. 121, 803-804.

Doucet-Jaboeuf, M., Pelegrin, A., Cot, M.C., Guillemain, J., and Bastide, M. (1984) Seasonal variations in the humoral immune response in mice following administration of thymic hormones, Annual Rev. of Chronopharmacol .1, 231-234.

Eisenberg, D.M., Kessler, R.C., Foster, C., Norlock, F.E., Calkins, D.R., and Delbanco,T.L. (1993) Unconventional medicine in the United States. Prevalence, costs, and patterns of use, The New England J. of Medicine 328, 246-252.

Endler, P.C., Pongratz, W., Van Wijk, R., Kastberger, G., Haidvogl, M. (1991) Effects of highly diluted succussed thyroxine on metamorphosis of highland frogs, Berlin J.Res. Hom. 1,151-160.

Fisher, P., House, I., Belon, P., and Turner, B. (1987) The influence of the homoeopathic remedy Plumbum metallicum on the excretion kinetics of Lead in rats, Human Toxicology 6, 321-324.

Fisher, P.,and Ward, A. (1994) Complementary medicine in Europe, B.M.J. 309,107-111.

Kewal, J. (1995)  Swiss embrace complementary Medicine, Nature Medicine 1, 107.

Skrabanek, P. (1986) Demarcation of the absurd, The Lancet, April 26, 960-961.

Sukul, N.C., Bala, S.K., and Bhattacharyya, B. (1986)  Prolonged cataleptogenic effects of potentized homoeopathic drugs, Psychopharmacology 89, 338-339.

 

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