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The effects of homoeopathic potencies of house-dust mite on the migration of house-dust-sensitive human leucocytes
Complementary Therapiesin Medicine (1996) 4, 169 171 © Pearson Professional Ltd 1996
RESEARCH
The effects of homoeopathic potencies of house-dust mite on the migration of house-dust-sensitive human leucocytes S.L.M. Gibson and R.G. Gibson
S U M M A R Y . Suspensions of leucocytes from house-dust-sensitive individuals were incubated with 25 different homoeopathic potencies of house-dust and assayed by means of leucocyte migration inhibition. It was found that the homoeopathic preparations produced significant enhancement or inhibition of leucocyte migration compared with the control preparation, and that not all the potencies tested were equally effective. This study corroborates previous reports of the activity of homoeopathic preparations in in vitro and in vivo systems.
INTRODUCTION
of less than 80% or greater than 120% are considered abnormal and indicative of a reaction of the cells to the antigen. 13,14 In the present study, doubling dilutions of antigen were replaced by various homoeopathic potencies of house dust, i.e. solutions prepared by serial centesimal dilution plus the addition of repeated mechanical shock at each dilution stage. Two questions were asked:
A current stumbling block to the acceptance of homoeopathy as a valid therapeutic system is the belief that infinitesimal potentized dilutions of remedies can have no measurable physiological effect. Despite research carried out on the effects of a number of homoeopathic preparations on a variety of biological systems1 12the controversy continues. One of the difficulties in testing homoeopathic potencies in vitro is the problem of selecting a suitable study model. Clinically, homoeopathic remedies are prescribed according to the clinical symptoms and signs produced by the substance in healthy human volunteers, and this is obviously impossible to determine for an isolated in vitro system. However, the field of allergy offers a promising model since, homoeopathically, allergies can be treated using the specific potentized allergen. For the present study, patients showing a positive skin-prick test to housedust mite and symptoms of house-dust allergy were selected, and the evaluating system used was the production by their leucocytes in the presence of the appropriate antigen, of leucocyte migration inhibition factor, (LIF), with consequent alteration in the migration pattern. In an orthodox setting if a mixed culture of leucocytes is incubated with the sensitizing antigen, migration inhibition factor is produced, with a subsequent inhibition of migration of the sensitized white cells. 13,14 In practice, either inhibition or stimulation of migration may be produced, and migration indices
1. In this system, do homoeopathic potencies produce a significantly altered migration pattern of the leucocytes? 2. Do all homoeopathic potencies of house dust produce a similar effect?
MATERIALS
AND METHODS
Twenty five different centesimal potencies of housedust in 20% ethyl alcohol were prepared and supplied by A. Nelson and Company, and labelled 1 25. A control solution, 20% ethyl alcohol only, was also supplied. The code of the potencies was kept by Nelson's Laboratories and was not available to the experimenters. The patients selected for the study were new referrals to the homoeopathic outpatient department at the Glasgow Homoeopathic Hospital. None had previously been treated with homoeopathy and all had positive reactions (weal equal to or greater than 10 mm in diameter) to the modified skin-prick test for house-dust allergy using house-dust mite and control solutions supplied by Bencard. The patients were 18 years old or over for ease of collecting blood (a 30-ml sample was required from each patient). Any patients currently on steroids or who had had large doses of
Sheila L.M. Gibson MD BSc,MFHom,ResearchPhysicianand Robin G. Gibson MB, FRCP,DCH, BDS,FFHom,Consultant
Physician,GlasgowHomoeopathicHospital, 1000Great Western Road, GlasgowG12 ONR, UK. 169
170
Complementary Therapies in Medicine
1614E 120 c
10I--
"6 6~:
4-
z
2-
HHIIHNIHIHIInH' I
5
I
I
I
10 15 Potency Number
20
I
I
25 control
Fig.
1 Number of abnormal tests obtained for each of the 25 homoeopathic potencies of house-dust, and for the controls
16-
g .Q .<
141210-
I--
"6
E= Z
The areas thus obtained were measured with a planimeter. The quadruplicate values for each sample were meaned and the migration index calculated by dividing each mean value by the meaned controls and converting to percentage. Migration indices were calculated for both the test and control samples. From these figures, the percentage inhibition or enhancement of migration was calculated. This method has been used in our laboratory since 1978 and has proved reliable. There was no valid reason for changing it. The results were analysed by the Z2 test. 15
864-
20
I
1'oc
1ooc
1o'ooc (1 M)
Potency of House Dust Fig. 2 Graph of the relative activities of the 25 homoeopathic potencies of house-dust.
steroids in the recent past, were rejected. Blood from 25 patients, 7 male and 18 female, age range 18-55, was tested. Migrational activity was assayed by the method of Cochran et al. 13J4 The leucocytes were separated from heparinized blood using Dextran 110, washed twice with phosphate buffered saline, pH 7.2-7.3, and suspended in minimal Eagle's medium containing 10% fetal calf serum and 1% antibiotic/antimycotic solution. Two hundred microlitre aliquots of this suspension were incubated at 37°C for one hour with 20-gl of each of the homoeopathic potencies and a number of control samples (on average 10-12, depending on the availability of the cell suspension). At the end of the incubation period, quadruplicate samples were taken from each 200 gl aliquot, using micropipettes which were then sealed at one end. The samples were centrifuged at 2000 r.p.m, for 5 min and the sections of the microcapillary tubes containing the resulting cell button were detached and mounted in leucocyte plates. These were then filled with the Eagle's medium/fetal calf serum/antibiotic - antimycotic culture medium, sealed with a cover-slip and left to migrate at 37°C overnight. The migration areas were drawn using a light microscope with a drawingarm attachment.
RESULTS Twenty five potencies studied on 25 patients gives a total of 625 tests of potencies, of which 244 (39%) were outwith the normal limits of 80-120% migration, 132 enhancements, and 112 inhibitions. Concurrently, there were 266 control samples of which 42 (15.8%) were outwith the normal limits. The test samples taken as a whole, therefore, produced effects outwith the normal limits 2.5 times oftener than the control samples. The Z2 test of Goodness of Fit demonstrates that the test samples were significantly different to the controls Z2= 15.6; P< 0.01). Figure 1 plots the number of abnormal tests for each of the 25 potencies, prior to breaking the code, and for the controls, calculated to the same scale. The number of abnormal results ranged from 4 14 out of a possible number of 25. The control samples also scored 4. Figure 1 demonstrates that not all the potencies were equally effective in producing either inhibition or enhancement of migration, but gives no indication as to which potencies were active and which were not. Further analysis by the Z2 test revealed that 18 of the 25 potencies produced effects significantly different from the controls, while 7 did not. Those producing a statistically significant result were potency numbers 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 16, 17, 19, 21, 22, 23,24. Potencies 4,13,14,15,18,20 and 25 did not significantly alter migrational activity. On breaking the code, it was found that the potencies displaying statistically significant activity were the 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 18, 27, 50, 200, 300, 400, 500 and 1 M centestimal dilutions. Those which did not show significant activity were the 14, 15, 20, 22, 25, 30 and 100 centesimal dilutions. Figure 2, produced after breaking the code, graphs the relative activities of the 25 potencies. The number of abnormal tests is shown on the y-axis and the potency number on the logarithmic x-axis. The eight most active potencies were found to be the 5, 6, 8, 11, 24, 200, 400 and 500 centesimal dilutions.
Homoeopathic potencies of House-dust mite and migration of House-dust-sensitive human leucocytes DISCUSSION
The results of this study demonstrate that homoeopathic potencies of house-dust have a demonstrable effect on the migration of leucocytes from housedust-sensitive individuals, and that not all potencies are equally active. Remedy effects have previously been demonstrated in plants by Pelikan and Unger, 1 and by Jones and Jenkins,2,3 in yeasts by Steffen4 and chlorella by Boiron and Marin. 8 The climbing activity of frogs was used as a model by Endler et al? and effects on rat brain by Boiron et al. 1° Isolated systems such as neutrophils,6 basophils 7 and cultured human fibroblasts H have demonstrated activity of homoeopathic remedies in vitro. While some workers have used low potencies, ~,4,9 11 with concentrations of the starting material below Avogadro's number (10-23), others have used both high and low potencies. 2,3,5-7 Although Chirumbolo et al 6 obtained better results with low potencies than with high potencies, Jones and Jenkins] ,3 Endler et al 5 and Davenas et al7 found activity with potencies beyond Avogadro's number. In the present study both the lowest and highest potencies tested (4C and 1M) produced significant effects on the migrational activity of the sensitized leucocytes and there were no obvious differences in the activities of potencies below and above Avogadro's number. It is apparent that not all potencies are equally active. This has been observed by previous workers and is a phenomenon well known to homoeopathic practitioners. As long ago as 1941, Heintz ~6 showed similar fluctuations in infra-red absorption spectra with a series of homoeopathic potencies. In this connection, it is of interest that HD 200, the standard potency used in the treatment of house-dust allergy in the Glasgow Homoeopathic Hospital, was one of the eight most active potencies, whereas HD 30, which in our hands is ineffective clinically, scored no better than placebo (Fig. 1). This study shows that homoeopathic potencies, even at very high dilutions (1M) do have significant in vitro effects when tested in an appropriate biological system. It also confirms that different potencies have different activities with some being apparently inactive. It is beyond the scope of this paper to discuss the various possible mechanisms underlying the activity of homoeopathic preparations. This work adds to the
171
literature which already exists demonstrating in vitro and in vivo effects of homoeopathic remedies and may encourage further exploration of this field.
ACKNOWLEDGEMENTS The authors wish to thank A. Nelson and Company for preparing the house-dust potencies and the control solution, and Dr Harper Gilmour of the Department of Community Medicine, University of Glasgow, for statistical advice.
REFERENCES
1. Pelikan W, Unger G. The activity of potentised substances. Br Hom J 1971; 60: 233-266. 2. Jones RL, Jenkins MD. Plant responses to homoeopathic remedies. Br Horn J 1981; 70:120 128. 3. Jones RL, Jenkins MD. Comparison of wheat and yeast as in vitro models for investigating homoeopathic remedies. B r H o m J 1 9 8 3 ; 7 2 : 1 4 3 147. 4. Steffen WA. Growth of yeast cultures as in vitro model for investigating homoeopathic medicines. Br Horn J 1985; 74:132 139. 5. Endler PC, Pongratz W, Kastberger G e t al. Climbing activity in frogs and the effect of highly diluted snccussed thyroxine. Br Horn J 1991; 80:194 200. 6. Chirumbolo S, Signorini A, Banchi I e t al. Effects of homoeopathic preparations of organic acids and minerals on the oxidative metabolism of human neutrophils. Br Horn J 1993; 82: 237-244. 7. Davenas E, Beauvais F, Amara J e t al. Human basophil degranualtion triggered by very dilute antiserum against IgE. Nature 1988; 333:816 818. 8. Boiron J, Marin M. Action d'une 15CH de sulfate de cuivre sur la culture de Chlorella vulgaris. Assisses Scientifiques Homoeopathiques 1970; 9:25 32. 9. Boiron J, Abecassis J, Belon R A pharmacological study of the retention and mobilisation of arsenic, as caused by Hahnemannian potencies of arsenicum album. Aspects Res Horn 1983; 1:19 26. 10. Boiron J, Abecassis J, Belon R The action of gelsemium sempervirens tincture upon the capture of neurotransmitters by synaptosomal preparations of various fractions of rat brain. Aspects Res Horn 1983; 1: 39-50. 11. Boiron J, Abecassis J, Belon P. The effects of mercurius corrosivus Hahnemannian potencies upon the multiplication of cultured fibroblasts poisoned with mercury chloride. Aspects Res Hom 1983; 1: 51-60. 12. Boiron J, Abecassis J, Belon R The effects of 7C Histaminum and 7C Apis mellifica Hahnemannian potencies upon basophil degranulation in allergic patients. Aspects Res Hom 1983; 1; 61-66. 13. Cochran AJ, Sprig WGS, Mackie RM et al. Post-operative depression of tumour-directed cell-mediated immunity in patients with malignant disease. BMJ 1972; iv: 67 70. 14. Cochran A J, Mackie RM, Ross CE et al. Studies of the immunology of melanoma patients. Pigment Cell 1976; 2: 182-190. 15. Siegel S. Non-parametric statistics for the behavioral sciences. New York: McGraw-Hill, 1956. 16. Heintz E. The physical effect of highly diluted, potentised substances. Die Naturwissenschaften 1941: 29: 713-725.
RESEARCH
The effects of homoeopathic potencies of house-dust mite on the migration of house-dust-sensitive human leucocytes S.L.M. Gibson and R.G. Gibson
S U M M A R Y . Suspensions of leucocytes from house-dust-sensitive individuals were incubated with 25 different homoeopathic potencies of house-dust and assayed by means of leucocyte migration inhibition. It was found that the homoeopathic preparations produced significant enhancement or inhibition of leucocyte migration compared with the control preparation, and that not all the potencies tested were equally effective. This study corroborates previous reports of the activity of homoeopathic preparations in in vitro and in vivo systems.
INTRODUCTION
of less than 80% or greater than 120% are considered abnormal and indicative of a reaction of the cells to the antigen. 13,14 In the present study, doubling dilutions of antigen were replaced by various homoeopathic potencies of house dust, i.e. solutions prepared by serial centesimal dilution plus the addition of repeated mechanical shock at each dilution stage. Two questions were asked:
A current stumbling block to the acceptance of homoeopathy as a valid therapeutic system is the belief that infinitesimal potentized dilutions of remedies can have no measurable physiological effect. Despite research carried out on the effects of a number of homoeopathic preparations on a variety of biological systems1 12the controversy continues. One of the difficulties in testing homoeopathic potencies in vitro is the problem of selecting a suitable study model. Clinically, homoeopathic remedies are prescribed according to the clinical symptoms and signs produced by the substance in healthy human volunteers, and this is obviously impossible to determine for an isolated in vitro system. However, the field of allergy offers a promising model since, homoeopathically, allergies can be treated using the specific potentized allergen. For the present study, patients showing a positive skin-prick test to housedust mite and symptoms of house-dust allergy were selected, and the evaluating system used was the production by their leucocytes in the presence of the appropriate antigen, of leucocyte migration inhibition factor, (LIF), with consequent alteration in the migration pattern. In an orthodox setting if a mixed culture of leucocytes is incubated with the sensitizing antigen, migration inhibition factor is produced, with a subsequent inhibition of migration of the sensitized white cells. 13,14 In practice, either inhibition or stimulation of migration may be produced, and migration indices
1. In this system, do homoeopathic potencies produce a significantly altered migration pattern of the leucocytes? 2. Do all homoeopathic potencies of house dust produce a similar effect?
MATERIALS
AND METHODS
Twenty five different centesimal potencies of housedust in 20% ethyl alcohol were prepared and supplied by A. Nelson and Company, and labelled 1 25. A control solution, 20% ethyl alcohol only, was also supplied. The code of the potencies was kept by Nelson's Laboratories and was not available to the experimenters. The patients selected for the study were new referrals to the homoeopathic outpatient department at the Glasgow Homoeopathic Hospital. None had previously been treated with homoeopathy and all had positive reactions (weal equal to or greater than 10 mm in diameter) to the modified skin-prick test for house-dust allergy using house-dust mite and control solutions supplied by Bencard. The patients were 18 years old or over for ease of collecting blood (a 30-ml sample was required from each patient). Any patients currently on steroids or who had had large doses of
Sheila L.M. Gibson MD BSc,MFHom,ResearchPhysicianand Robin G. Gibson MB, FRCP,DCH, BDS,FFHom,Consultant
Physician,GlasgowHomoeopathicHospital, 1000Great Western Road, GlasgowG12 ONR, UK. 169
170
Complementary Therapies in Medicine
1614E 120 c
10I--
"6 6~:
4-
z
2-
HHIIHNIHIHIInH' I
5
I
I
I
10 15 Potency Number
20
I
I
25 control
Fig.
1 Number of abnormal tests obtained for each of the 25 homoeopathic potencies of house-dust, and for the controls
16-
g .Q .<
141210-
I--
"6
E= Z
The areas thus obtained were measured with a planimeter. The quadruplicate values for each sample were meaned and the migration index calculated by dividing each mean value by the meaned controls and converting to percentage. Migration indices were calculated for both the test and control samples. From these figures, the percentage inhibition or enhancement of migration was calculated. This method has been used in our laboratory since 1978 and has proved reliable. There was no valid reason for changing it. The results were analysed by the Z2 test. 15
864-
20
I
1'oc
1ooc
1o'ooc (1 M)
Potency of House Dust Fig. 2 Graph of the relative activities of the 25 homoeopathic potencies of house-dust.
steroids in the recent past, were rejected. Blood from 25 patients, 7 male and 18 female, age range 18-55, was tested. Migrational activity was assayed by the method of Cochran et al. 13J4 The leucocytes were separated from heparinized blood using Dextran 110, washed twice with phosphate buffered saline, pH 7.2-7.3, and suspended in minimal Eagle's medium containing 10% fetal calf serum and 1% antibiotic/antimycotic solution. Two hundred microlitre aliquots of this suspension were incubated at 37°C for one hour with 20-gl of each of the homoeopathic potencies and a number of control samples (on average 10-12, depending on the availability of the cell suspension). At the end of the incubation period, quadruplicate samples were taken from each 200 gl aliquot, using micropipettes which were then sealed at one end. The samples were centrifuged at 2000 r.p.m, for 5 min and the sections of the microcapillary tubes containing the resulting cell button were detached and mounted in leucocyte plates. These were then filled with the Eagle's medium/fetal calf serum/antibiotic - antimycotic culture medium, sealed with a cover-slip and left to migrate at 37°C overnight. The migration areas were drawn using a light microscope with a drawingarm attachment.
RESULTS Twenty five potencies studied on 25 patients gives a total of 625 tests of potencies, of which 244 (39%) were outwith the normal limits of 80-120% migration, 132 enhancements, and 112 inhibitions. Concurrently, there were 266 control samples of which 42 (15.8%) were outwith the normal limits. The test samples taken as a whole, therefore, produced effects outwith the normal limits 2.5 times oftener than the control samples. The Z2 test of Goodness of Fit demonstrates that the test samples were significantly different to the controls Z2= 15.6; P< 0.01). Figure 1 plots the number of abnormal tests for each of the 25 potencies, prior to breaking the code, and for the controls, calculated to the same scale. The number of abnormal results ranged from 4 14 out of a possible number of 25. The control samples also scored 4. Figure 1 demonstrates that not all the potencies were equally effective in producing either inhibition or enhancement of migration, but gives no indication as to which potencies were active and which were not. Further analysis by the Z2 test revealed that 18 of the 25 potencies produced effects significantly different from the controls, while 7 did not. Those producing a statistically significant result were potency numbers 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 16, 17, 19, 21, 22, 23,24. Potencies 4,13,14,15,18,20 and 25 did not significantly alter migrational activity. On breaking the code, it was found that the potencies displaying statistically significant activity were the 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 18, 27, 50, 200, 300, 400, 500 and 1 M centestimal dilutions. Those which did not show significant activity were the 14, 15, 20, 22, 25, 30 and 100 centesimal dilutions. Figure 2, produced after breaking the code, graphs the relative activities of the 25 potencies. The number of abnormal tests is shown on the y-axis and the potency number on the logarithmic x-axis. The eight most active potencies were found to be the 5, 6, 8, 11, 24, 200, 400 and 500 centesimal dilutions.
Homoeopathic potencies of House-dust mite and migration of House-dust-sensitive human leucocytes DISCUSSION
The results of this study demonstrate that homoeopathic potencies of house-dust have a demonstrable effect on the migration of leucocytes from housedust-sensitive individuals, and that not all potencies are equally active. Remedy effects have previously been demonstrated in plants by Pelikan and Unger, 1 and by Jones and Jenkins,2,3 in yeasts by Steffen4 and chlorella by Boiron and Marin. 8 The climbing activity of frogs was used as a model by Endler et al? and effects on rat brain by Boiron et al. 1° Isolated systems such as neutrophils,6 basophils 7 and cultured human fibroblasts H have demonstrated activity of homoeopathic remedies in vitro. While some workers have used low potencies, ~,4,9 11 with concentrations of the starting material below Avogadro's number (10-23), others have used both high and low potencies. 2,3,5-7 Although Chirumbolo et al 6 obtained better results with low potencies than with high potencies, Jones and Jenkins] ,3 Endler et al 5 and Davenas et al7 found activity with potencies beyond Avogadro's number. In the present study both the lowest and highest potencies tested (4C and 1M) produced significant effects on the migrational activity of the sensitized leucocytes and there were no obvious differences in the activities of potencies below and above Avogadro's number. It is apparent that not all potencies are equally active. This has been observed by previous workers and is a phenomenon well known to homoeopathic practitioners. As long ago as 1941, Heintz ~6 showed similar fluctuations in infra-red absorption spectra with a series of homoeopathic potencies. In this connection, it is of interest that HD 200, the standard potency used in the treatment of house-dust allergy in the Glasgow Homoeopathic Hospital, was one of the eight most active potencies, whereas HD 30, which in our hands is ineffective clinically, scored no better than placebo (Fig. 1). This study shows that homoeopathic potencies, even at very high dilutions (1M) do have significant in vitro effects when tested in an appropriate biological system. It also confirms that different potencies have different activities with some being apparently inactive. It is beyond the scope of this paper to discuss the various possible mechanisms underlying the activity of homoeopathic preparations. This work adds to the
171
literature which already exists demonstrating in vitro and in vivo effects of homoeopathic remedies and may encourage further exploration of this field.
ACKNOWLEDGEMENTS The authors wish to thank A. Nelson and Company for preparing the house-dust potencies and the control solution, and Dr Harper Gilmour of the Department of Community Medicine, University of Glasgow, for statistical advice.
REFERENCES
1. Pelikan W, Unger G. The activity of potentised substances. Br Hom J 1971; 60: 233-266. 2. Jones RL, Jenkins MD. Plant responses to homoeopathic remedies. Br Horn J 1981; 70:120 128. 3. Jones RL, Jenkins MD. Comparison of wheat and yeast as in vitro models for investigating homoeopathic remedies. B r H o m J 1 9 8 3 ; 7 2 : 1 4 3 147. 4. Steffen WA. Growth of yeast cultures as in vitro model for investigating homoeopathic medicines. Br Horn J 1985; 74:132 139. 5. Endler PC, Pongratz W, Kastberger G e t al. Climbing activity in frogs and the effect of highly diluted snccussed thyroxine. Br Horn J 1991; 80:194 200. 6. Chirumbolo S, Signorini A, Banchi I e t al. Effects of homoeopathic preparations of organic acids and minerals on the oxidative metabolism of human neutrophils. Br Horn J 1993; 82: 237-244. 7. Davenas E, Beauvais F, Amara J e t al. Human basophil degranualtion triggered by very dilute antiserum against IgE. Nature 1988; 333:816 818. 8. Boiron J, Marin M. Action d'une 15CH de sulfate de cuivre sur la culture de Chlorella vulgaris. Assisses Scientifiques Homoeopathiques 1970; 9:25 32. 9. Boiron J, Abecassis J, Belon R A pharmacological study of the retention and mobilisation of arsenic, as caused by Hahnemannian potencies of arsenicum album. Aspects Res Horn 1983; 1:19 26. 10. Boiron J, Abecassis J, Belon R The action of gelsemium sempervirens tincture upon the capture of neurotransmitters by synaptosomal preparations of various fractions of rat brain. Aspects Res Horn 1983; 1: 39-50. 11. Boiron J, Abecassis J, Belon P. The effects of mercurius corrosivus Hahnemannian potencies upon the multiplication of cultured fibroblasts poisoned with mercury chloride. Aspects Res Hom 1983; 1: 51-60. 12. Boiron J, Abecassis J, Belon R The effects of 7C Histaminum and 7C Apis mellifica Hahnemannian potencies upon basophil degranulation in allergic patients. Aspects Res Hom 1983; 1; 61-66. 13. Cochran AJ, Sprig WGS, Mackie RM et al. Post-operative depression of tumour-directed cell-mediated immunity in patients with malignant disease. BMJ 1972; iv: 67 70. 14. Cochran A J, Mackie RM, Ross CE et al. Studies of the immunology of melanoma patients. Pigment Cell 1976; 2: 182-190. 15. Siegel S. Non-parametric statistics for the behavioral sciences. New York: McGraw-Hill, 1956. 16. Heintz E. The physical effect of highly diluted, potentised substances. Die Naturwissenschaften 1941: 29: 713-725.