Research

 

The impact of ultra-high dilutions of antigen on Dendritic Cell activation and Immune response initiation

Research and words by Dimitris Zienkiewicz

Introduction

Our lab focuses mainly on dendritic cells (DC) and their role in initiating, maintaining and regulating the immune system. Dendritic cells reside at sites where pathogen entry is most prevalent such as the skin, lungs, gut and liver. They recognise invading pathogens through a wide range of surface molecules. These bind to evolutionary conserved structures shared between different categories of pathogens such as viruses, bacteria, helminths, protozoa as well as allergens and “self”. Once DCs have encountered and internalised an invading organism they present parts of it on their surface. During this process they migrate from the periphery to the secondary lympoid organs, where they present those parts (antigen) of the pathogen they encountered in the periphery to effector cells such as T cells. The unique ability of dendritic cells to recognise and present novel antigen to effector cells has established them as the professional antigen presenting cell of the immune system (APC).

To date, the scientific community has chacterised at least 5 distinct types of immune responses, however, numerous variations of the immunological profile in each category exist, depending on the individual pathogen. The type and subtype of the immune reaction mounted against an invading organism is predominantly decided during the initial encounter of the DC with the pathogen. Since the dendritic cell is such an excellent cell type in recognising antigen, as well as the most crucial cell type in deciding the direction of the immune response, we hypothesised that possible biological effects conferred by ultra-high dilutions of antigen could occur via DCs.

Questions

  • During the last three years we asked, amongst others, two main questions:
  • Can ultra-high dilutions of antigen alter the DC pheontype?
  • Can ultra-high dilutions of antigen alter the ability of DC to initiate immune responses?

DC activation

In order to address the first question we incubated DC with a 30CH dilution of heat-killed Propionibacterium acnes or 30CH water dilution for 24h, in vitro. A stimulatory dose of the same antigen was then added for a further 18 hours. Several markers of stimulation were assessed to compare DC that had been treated with a 30CH water dilution to those treated with a 30CH P. acnes dilution. The same markers were used to compare DC that had been pre-treated with a 30CH dilution of water and then were incubated with the stimulatory dose of P. acnes, to DC that had been pre-treated in 30CH P. acnes dilution and then given a stimulatory dose of the same antigen. These two methods were used to address whether DC can be affected by ultra-high dilutions of antigen alone, or whether ultra-high dilutions of antigen conditioned DC to respond differently to exposure to the antigen in question.

We found that ultra-high dilutions of antigen did not activate DC in any way. Similarly, ultra high dilutions of antigen did not condition DC to respond differently to stimulatory doses of antigen in vitro.

Immune responses

Next, we wanted to address whether DC that had been pre-treated in ultra-high dilutions of antigen prior to being given a stimulatory dose of the same antigen could have a measurable biological effect in vivo. DC were pre-treated in either 30CH water or 30CH P. acnes before being incubated in a stimulatory dose of P. acnes (exactly in the same way as for our earlier experiments described above). The dendritic cells were then transferred into wild type mice. The mice were left for a week to develop a primary immune reaction against the bacterium. At the 7th day after the transfer the ability of animals to respond to P. acnes was measured with a well known and widely used within the scientific community splenocyte restimulation assay.

Preliminary results had shown that the immune responses against P. acnes in animals that had received DC pre-treated in 30CH P. acnes dilutions and then stimulated with a stimulatory dose of P. acnes were characterised by a release of a larger amount of inflammatory molecules than the response in animals that had received DC that had been pre-treated with 30CH water before being incubated in a stimulatory dose of P. acnes. Since this was a very surprising result we decided to repeat the experiment several times. There was, however, some variation in the observed effect throughout experimental repeats. We were unable to find the reason for this variation despite the fact that we addressed several factors that could be contributing to this effect, such as water contamination, method of dynamisation and the source of water. We performed meta analysis on 10 experimental repeats that were performed in exact conditions, and found that overall ultra-high dilutions of P. acnes conditioned DC to induce stronger immune responses in mice than those induced by DC pre-treated in 30CH dilutions of water. The size of the effect was nevertheless small, which makes it hard to ascribe a biological role for such ultra-high dilutions.

Conclusion

The results so far suggest that there is a biological role for ultra-high dilutions of antigen in the initiation of immune responses. The size of the effect is, however, too small to convincingly ascribe biological significance to this observation. Furthermore, we hoped that by addressing factors that could possibly be contributing to the variability we could learn something more about the nature of such extreme dilutions. Unfortunately, we did not find any correlation between the variables that we examined and the ability of DC to initiate immune responses. In conclusion, although it is hard to ascribe a role for ulra-high dilutions of water due to the lack of a mechanism of action at the biological level as well as the physical level, our results are promising for further investigation into these two matters.

If you would like any further information please contact The Trust directly.

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The use of homeopathic products in childhood: data generated over 8.5 years from the Avon Longitudinal Study of Parents and Children (ALSPAC).

Research and words by Jackie Bishop
Research by Kate Northstone, Elizabeth Thompson, Professor Jean Golding.

Introduction
The Blackie Foundation Trust funded the University of Bristol to research homeopathic use by children participating in the Avon Longitudinal Study of Parents and Children (ALSPAC), otherwise known as ‘the Children of the 90s’. The population-based cohort study is investigating the development and health of children during childhood and into adulthood. The study began in 1991 by inviting pregnant women within the former county of Avon in the south-west of England to enroll. A total of 14541 pregnant women in that area signed up to take part.

The study has gathered a wealth of different information regarding many aspects of the families’ health, well being, social, demographic, environmental and genetic features. One of the main information sources has been self-completion questionnaires sent to the parents to fill in either about themselves or their study child. These questionnaires, completed since pregnancy and throughout the life of the child, have provided a valuable source of homeopathic medicine use including use during pregnancy and over time by the children (now aged 16-17 years) and their parents.

This study concentrated on the use of homeopathic products at various time points throughout childhood, starting with use at 18 months to use at 8.5 years of age. The research focused on how often children used homeopathic products, what childhood conditions were treated, who had prescribed those products and how the products were confused with other forms of complementary medicines.

Results
The research team found that 11.8% of the children had used a homeopathic product at least once up to 8.5 years of age. The most commonly used products throughout that time were Chamomilla (a remedy traditionally used for teething), Arnica (in either tablet or cream form traditionally used for soft tissue bruising), Belladonna (most commonly used for high fevers), Calendula (usually administered as a cream for healing cuts and scrapes), and Pulsatilla (most commonly used for ear complaints, coughs and colds).

The number of children using homeopathic products varied across time with the greatest use at 18 months of age (n=891; 8%) and the lowest use at 78 months of age (n=444; 5%). The greatest use at 18 months reflected the fact that children were given homeopathic products to treat teething complaints. At all later time points children were most likely to be treated for injuries and first aid type conditions.

When the child was 81 months of age the mothers were asked who had prescribed the homeopathic product to the child. The results show that the parents were most likely to select a homeopathic product for their child themselves (46.3% of all users). A qualified homeopath prescribed for 14.8% of children, a specialist homeopathic doctor for 12% of children, a GP for 10.1%, a family, friend or neighbour for 7.3% and a chemist for 6.3%. Mothers also took advice about homeopathic products from a Health Food Store, a Dermatologist, a homeopathic seminar/talk, a newspaper/magazine article and a school nurse.

The parents were most likely to treat their child’s first aid type, self-limiting complaints themselves such as injuries, bruising, coughs and colds, whereas they were more likely to consult a qualified homeopath or specialist homeopathic doctor for their child’s on-going, chronic complaints of eczema or asthma. The range of conditions treated were mainly of a physical nature, however some parents/carers used homeopathy for their child’s psychological problems such as insomnia, nightmares, stress, anxiety, fear and shock.

Homeopathic products can sometimes be mistaken for other products, and the team found that as many as 7% of the descriptions given as homeopathic were in fact herbal products. The herbal products most likely to be confused as homeopathic ones were Matricaria (or German Chamomile, most popularly used as an ingredient in herbal teething products) and Echinacea (or Purple Coneflower, most popularly used as a preventative treatment to ward off infections). Quite a high number also confused Aromatherapy oils as being homeopathic.

Conclusion
It is hoped that the knowledge and understanding of the extent of homeopathic product use in childhood in a general population, why it is used, what it is used for and the influences around the choices of homeopathic products will give valuable insight into the planning, direction and focus of future homeopathic research projects.

If you would like any further information please contact The Trust directly.

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Homeopathic Treatment for Malaria: Results from a Pilot study sponsored by the Blackie Foundation Trust*

Research and words by Dr. Jacob Minah and Professor Florence Margai

The efficacy of homeopathy in reducing malaria burden in poor communities was the subject of a recent pilot study that was sponsored by the Blackie Foundation Trust. Malaria is a prevalent parasitic disease that kills nearly 1 million people around the world each year, with 90% of those deaths occurring in Sub-Saharan Africa. The changing global climate, armed regional conflicts, the HIV/AIDs pandemic, and rising levels of global poverty have all contributed to the persistence of this disease in tropical regions. These conditions, along with increasing globalization and international migration, have heightened concerns about the potential emergence or re-emergence of the disease in temperate regions such as Europe and North America.

Efforts to eradicate malaria have been centered largely on conventional drugs some of which are known for harmful side effects and parasitic resistance particularly for plasmodium falciparum, the fatally transmitted form of malaria. The drugs are also relatively expensive for residents in poor, endemic regions with some treatments costing up to 50 Euros per malaria episode.

In searching for safe, affordable and effective treatment alternatives, the pilot study led by Dr. Jacob Minah and Professor Florence Margai, was undertaken in the Kroo Bay Community in Freetown, Sierra Leone. Following ethical approval of the research protocol, the project was implemented in four phases, at four month intervals, beginning in June 2006. Eligible participants included those who were 18 years or older with no major health problems. Of the 731 participants who registered for the project, the average age was 38.6 years, and 73% were females. The participants were first required to complete a questionnaire, detailing their personal case histories. This was followed by a comprehensive physical exam including the collection of blood samples to test for malaria. After analyzing the blood samples, 534 subjects with negative or very low parasitic burden were formally enrolled in the trial. These subjects were randomized into two groups, with the first group of 289 subjects receiving five pellets of Tropica Malaria Nosode, a homeopathic prophylaxis developed exclusively for P. falciparum. The second group of 245 subjects received five placebo pellets that were similar in form to the homeopathic therapy. All drugs were administered on site and patients were advised to report any potential health problems, specifically malaria symptoms, to the nursing staff at the health center. Over the course of the next four months, community health workers visited the neighborhoods regularly to check on the health status of the registrants.

The procedures established in Phase I were repeated four months later in Phase II, and thereafter in Phase III, and Phase IV. In each phase, patients completed a questionnaire, a health exam, and submitted a blood sample. Patients with negative test results were re-enrolled in phases, II (404 persons), III (347 persons) and IV (297 persons). Participants who tested positive with clinical signs of high temperatures and high levels of parasitemia were treated using Quinine and Paracetamol, the state recommended therapy. Overall, the distribution of patients was fairly consistent across the phases with 55% of the participants receiving homeopathic prophylaxis and 45% receiving inactive pellets.

The pilot study ended in June 2007, following which all of the data were compiled and analyzed. The first set of analyses involved a phase-by-phase evaluation of the data comparing the risks of malaria diagnosis among the homeopathic and placebo groups. The results from Phase I confirmed that malaria was prevalent in the community. Self reports from patients suggested an average of 3.24 episodes per year. Evidence from the blood test results showed that 29% tested positive for malaria with a mean parasitic density of 401/ul. However, only eight clinical cases were identified based on the diagnostic criteria established in the study. In analyzing the data in subsequent phases, the most significant effect of the homeopathic therapy was observable in the second phase when the risk of getting malaria was 3.6 times higher among those in the placebo group than those in the homeopathic group. Thereafter, the results in Phases III and IV were marginal and no statistically significant differences were observed between the two groups.

The second set of analyses was completed at the end of the pilot project during which a data subset was compiled, comprising of only the participants for whom we had complete information across all four phases. The analytical findings were more hopeful, suggesting potential benefits of the homeopathic Tropica nosodes. Over a one year period, the mean density of malaria parasites decreased from 76.35/ul to 7.74/ul among all patients. A test of the differences observed between the pre- and post-intervention phase confirmed that the observed reduction in malaria parasitic density. Further, a comparison of the mean parasitic density between the two treatment groups suggested that the parasitic burden was statistically lower in the homeopathic group (38.1/ul) than those in the placebo group (24.7/ul) during the one year period.

Conclusions
Overall, the results of this pilot study are promising and provide some indication of the likely benefits of homeopathic prophylaxis in reducing the burden of malaria in endemic communities. However, several challenges were encountered during the implementation of the project that would warrant a cautionary interpretation of the results. In addition to the homeopathic prophylaxis, the observed reduction in malaria burden could also have been due to the educational campaigns and outreach in the community, greater public awareness of the dangers and symptoms of malaria, more testing, diagnosis and treatment for malaria, and greater access and utilization of the health care system, all of which were critical components of the malaria project. Clearly, further research is needed to corroborate the findings and confirm the full potential of the homeopathic therapy in reducing the burden of malaria. The research protocol is now being revised in preparation for the replication of the study over a more extended time period.

For more information, contact Dr Jacob Minah, Pediatrician and Homeopathic Specialist in Steinheim, Germany: drminah@t-online.de or Professor Florence Margai, Binghamton University, New York: margai@binghamton.edu.

*Report compiled by Professor Florence Margai
If you would like any further information please contact The Trust directly.

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Improving homeopathy by scientific research

Text and Research by Lex Rutten MD, Committee for Methods and Validation of the VHAN (Dutch homeopathic doctors association)

Many people think that medical scientific research is nothing else than Randomised Controlled Trial (RCT), where patients receive either a medicine that is active or one that is not active (placebo) and nor the patient nor the doctor knows who gets what. But there are many other methods of research and some of them are better suited to investigate daily clinical practice. Such a method is Bayesian research, which is frequently applied to assess diagnostic instruments in conventional medicine, but seldom to enhance the effectiveness of prescribing medicines. This method suits homeopathy perfectly because it can handle the fact that a homeopathic medicine is not prescribed on indication alone.
Thomas Bayes (1702-1761) was a clergyman from Tunbridge Wells. His posthumous publication in 1763 still has an increasing influence on all kinds of decisions made by humans and computers. The strength of his statistical method is that we can combine new information with what we already know. In card playing you know the cards in your hand and the cards on the table. With this knowledge you can make a better guess what your opponents will do than without this knowledge.

A sad example of the difference between ‘classical’ statistics and Bayesian statistics is the wrongful imprisonment of Sally Clark, who lost two babies shortly after birth. Classical statisticians calculated that two deaths by natural causes in a row was very unlikely, so the mother must have killed both babies. With Bayesian statistics it was possible to account for other possible causes like genetic defects. After more than three years in prison Sally was released because of these new calculations, but she died in 2007, aged 42.

Back to our card-players, it would help very much if you knew the cards in the hands of your opponents. The research we performed revealed just that, but not as a game. We collected all experience of ten homeopathic doctors during three and a half years. These doctors recorded the presence of six symptoms in each new patient and the result after each prescribed medicine. Instead of the cards we wanted to know which symptoms were most frequently present in patients who responded well to various medicines.

We evaluated more than 4,000 homeopathic prescriptions and the presence of the six symptoms in a similar number of patients. This evaluation learned e.g. that the symptom ‘diarrhoea from anticipation’- like going to a theatre or the dentist – was present in many patients that respond well to the homeopathic medicine Argentum nitricum, eleven times more than in the remainder of all patients. With this knowledge we can predict that the next patient before you with the symptom ‘diarrhoea from anticipation’ will have a fair chance to benefit from Argentum nitricum. There is even a formula connected to Bayes’ theory, which tells us what that symptom implies. Suppose that the doctor already considered Argentum nitricum and gave it a chance of, say, 10% to work. With Bayes’ formula the doctor can calculate that the chance that Argentum nitricum works rises up to 55% if the patient has diarrhoea from anticipation.

Who would have thought that homeopathy could be explained mathematically? At least part of it, it is of course vital that we ask the right questions and understand what is wrong with the patient. But homeopathy will gain much credit if we can show that we handle our experience in a scientific way and that the method is perfectly reproducible.

A group of ten experienced Dutch homeopathic doctors systematically recorded information about all new patients between June 2004 and December 2007. They recorded the presence of six homeopathic symptoms: ‘Diarrhoea from anticipation’, ‘Fear of death’, ‘Recurrent herpes lips’, ‘Grinding teeth during sleep’, ‘Sensitivity to injustice’ and ‘Loquacity’. Furthermore they recorded every prescribed homeopathic medicine and the results from each medicine.

In the end more than 4,000 patients entered the study and a similar number of prescriptions was evaluated. Half of the prescriptions gave the desired result, which means that not only the complaint is ameliorated but there should also be an improvement of well-being. We counted 26 patients with good results on Argentum nitricum and 12 of these patients had ‘diarrhoea from anticipation’. This was eleven times as frequently as in all other patients. The statistical expression for this is that the Likelihood Ratio (LR) is eleven. The same way we calculated 50 (statistically significant) likelihood ratios for other homeopathic medicines and for other symptoms.

The results were partly reassuring, but partly also disquieting. The facts that all homeopathic doctors agree on, e.g. that ‘diarrhoea from anticipation’ is an indication for Argentum nitricum, is indeed confirmed by our research. But about half of our other results does not correspond with the existing information in the homeopathic repertory. In fact homeopathic doctors already knew that much of the information in the homeopathic repertory is not reliable, but with Bayes’ theory and this kind of research we can distinguish which information is wrong and why.

For the future we should make a research program based Bayes’ and other statistical techniques. The enormous amount of symptoms in homeopathy shouldn’t discourage us because we use a limited amount of symptoms on a daily basis. There are about six hundred symptoms that characterise the most frequently used homeopathic medicines. If we could compose thirty research groups spread over the world we can improve the homeopathic method considerably in a period of ten years. The costs will be much less than the development of just one conventional medicine.

More information about this research can be found on the website: http://www.dokterrutten.nl/Inc.html

If you would like any further information please contact The Trust directly.

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Data collected from a 12 month study of the Homeopathic treatment of women attending a menopausal service in Sheffield. This work was funded by a grant from The Blackie Foundation Trust.

Researcher Dr E P Strong MB BS MFHom.

Introduction and background
A homeopathic service was available at the Menopause Clinic in Sheffield for over eight years. This service ran alongside conventional medical treatment, usually Hormone Replacement Therapy (HRT), for those women who attended for treatment of severe menopausal symptoms. Homeopathy was offered to those women who were unable to use HRT or to those who found it difficult, sometimes impossible, to withdraw from HRT treatment. A clinical audit was undertaken, in which women completed the Measure Yourself Medical Outcome Profile (MYMOP): they reported significant benefit from the service.

The Sheffield PCT had funded the homeopathic service, but in September 2006 this was discontinued due to financial constraints placed upon the PCT.

In January 2007 an application was made to The Blackie Foundation Trust for funds to enable the service to be continued, albeit at a reduced level. This was to enable the service provision to continue during the proposed amalgamation of Sheffield’s four PCTs. When this had come about, an application was made for this valuable service to be reinstated.

The Trust granted sufficient money for one year enabling two new patients to be seen a month. Each patient would also receive five follow-up appointments.

The study and results
There was no difficulty recruiting patients for this study. The Clinical team at the Sheffield Menopause Clinic was co-operative and helpful.
The need for alternatives to HRT in treating women with severe menopausal symptoms is becoming widely acknowledged, and it is hoped that this study will add to previous work that reported positive findings – a randomised controlled trial in Germany and a clinical outcomes study in France.
The results from this study show that women suffering in the menopausal period have a variety of symptoms, the commonest being hot flushes.
Patients who did well on the scores appeared to be in better general health and were less likely to be on other medication. Those patients who had been suffering with symptoms since coming off HRT also did well on their scores.
The patients who were most difficult to treat were on chemotherapy or anti-oestrogen therapy.

Conclusion
Generally there was an acceptance of the therapy on offer and an acknowledgement that homeopathy was helpful.
More research in this field is indicated.

If you would like any further information please contact The Trust directly.