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a Study from Miami Children Hospital demonstrating action of Homeopathic Formulas (Phase 6 and Flu Terminator) at the cellular level (increase of Cytokines)

a Study from Miami Children Hospital demonstrating action of Homeopathic Formulas (Phase 6 and Flu Terminator) at the cellular level (increase of Cytokines)

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  • 1. THE JOURNAL OF ALTERNATIVE AND COMPLEMENTARY MEDICINEVolume 13, Number 4, 2007, pp. 403–407© Mary Ann Liebert, Inc.DOI: 10.1089/acm.2007.6292 ORIGINAL PAPERS Investigation of Cytokine Expression in Human Leukocyte Cultures with Two Immune-Modulatory Homeopathic Preparations CHEPPAIL RAMACHANDRAN, Ph.D., M.B.A.,1,2 P.K. RAVEENDRAN NAIR, Ph.D.1, RICHARD T. CLÈMENT, M.D.,3 and STEVEN J. MELNICK, Ph.D., M.D.2 ABSTRACT Background: The efficacy of homeopathic medicines for maintaining human health and treating disease hasbeen extensively examined in clinical trials. However, there is a paucity of preclinical evaluations of the ef-fects of homeopathic medicinal preparations on cellular signaling pathways relevant to the applications of thesepreparations. Materials and methods: In this study, the immune-modulatory effects of Phase 6 (for the stimulation of thenonspecific defense system) and Flu Terminator® (for influenza and viral diseases) (Be Well HomeopathicsInc. Miami, FL), two homeopathic preparations developed for the purpose, were evaluated in normal humanleukocyte cultures in vitro. Results: Both Phase 6 and Flu Terminator stimulated the production of pro- and anti-inflammatory cytokinesby human leukocytes, although higher doses often produced a weaker response than lower doses. The carriersolvent (20% ethanol) failed to elicit any cytokine synthesis. Conclusions: The results of the in vitro studies suggested that ultralow concentrations of ingredients in Phase6 and Flu Terminator were capable of eliciting a human immune response. INTRODUCTION detection (i.e., clinical trials, observational studies, statisti- cal evaluations, computerized storage programs, and instru- mental or laboratory testing). Some innovative models haveH omeopathy stands out as an experimental discipline, as is evidenced by the vast amount of homeopathic datacollected over more than 2 centuries. However, homeopathy been used to contribute to the debate about the efficacy of homeopathic medications, including demonstration of theremains controversial from the perspective of Western med- efficacy of ultrahigh dilutions of IgE on basophil degranu-icine because of the apparent inadequacy or inconsistency of lation, inhibition of basophil degranulation by ultrahigh di-data to support use of the ultralow concentrations in which lutions of histamine, and other models that incorporate thehomeopathic medications are given. Consequently, the pre- measurement of cell viability and cytokine induction, whichvailing attitude does not consider the small amount of ingre- may be diagnostic laboratory techniques for determining thedients used in homeopathy as being capable of inducing sig- immunologic potency of various homeopathic preparationsnificant changes in classic pharmacologic models.1 and may help clarify the clinical benefit of therapies with Homeopathic research has evolved over the past 20 years these substances.2–5with the advent of comprehensive biomedical research For the past 10 years, homeopathic research has venturedmethodologies that make subtle effects more amenable to into the evaluation of combination formulas used to treat a 1ResearchInstitute, Miami Children’s Hospital, Miami FL. 2Department of Pathology, Miami Children’s Hospital, Miami, FL. 3Be Well Homeopathics Inc., Miami, FL. 403
  • 2. 404 RAMACHANDRAN ET AL.variety of clinical conditions.6–9 In many respects, this is a 7considerable challenge, given the controversies surround- 6 p<0.01ing research on single-component homeopathic prepara- 5 TNF-a (pg/mL)tions. However, by focusing on well-established endpoints 4and using conventional research methodologies, it is possi-ble to elicite meaningful data from well-designed preclini- 3cal studies. The field of allergy and immunology has been 2viewed as a bridge into homeopathy since both disciplines 1share the concept that modulations in biologic systems aresensitive to very low concentrations of natural or endoge- 0 0 2 5 10 20 50 100 200nous substances.1 Therefore, appropriate models for evalu- Phase 6 (µL)ating multicomponent homeopathic preparations and deter-mining their effects on immune function or mediators of FIG. 1. TNF-␣ induction in human leukocytes by Phase 6 (for-immunity are essential. Traumeel® S (Heel GmbH, Baden- mulated and manufactured by Be Well Homeopathics Inc., Miami,Baden, Germany), an extensively evaluated commercial FL).homeopathic preparation with a combination of ingredients,has been shown to modulate the secretion of interleukin(IL)-1␤, tumor necrosis factor (TNF)-␣ and IL-8 in immunecells.10 Thus, modulation of mediators of immunity may MATERIALS AND METHODSrepresent an opportunity to assess appropriate homeopathicmixtures using in vitro models. In the present study we at- Preparations and reagentstempted to further validate this hypothesis by exploring the Phase 6 and Flu Terminator are homeopathic preparationseffects of 2 multicomponent homeopathic formulas, Flu that were obtained from the manufacturer. Roswell ParkTerminator® and Phase 6, on a panel of pro- and anti-in- Memorial Institute (RPMI) cell culture medium, fetal bovineflammatory cytokines in human leukocyte cultures. serum (FBS), phosphate-buffered saline (PBS), and anti- biotics were purchased from Invitrogen, Inc., Corporation, TABLE 1. CONSTITUENTS OF FLU TERMINATOR AND PHASE 6a Carlsbad, CA. Enzyme-linked immunosorbent assayFlu Terminator® (ELISA) reagent kits for human-specific cytokines (IL-1, Gelsemium 3ϫ IL-4, IL-10, and interferon [IFN]-␥ and TNF-␣) were pur- Zincum gluconium 3ϫ chased from BD Biosciences, San Diego, CA. The con- Zincum metallicum 3ϫ stituent ingredients of Flu Terminator and Phase 6 are listed Aconitum napellus 4ϫ Eupatorium perfoliatum 4ϫ in Table 1. Sulphur 4ϫ Phosphorus 5ϫ Asclepias vincetoxicum 6ϫ Isolation of human leukocytes and incubation with Influenzinum 12ϫ Phase 6 and Flu Terminator Anas barbariae hepati and cordis extractum 200KbPhase 6 Leukocytes were isolated from human blood samples col- Taraxacum officinalis 1ϫc lected from healthy volunteers using Histopaque 1066 Galium aparine 1ϫ 3ϫ (Sigma Chemical Co., St. Louis, MO) gradient centrifuga- Thuja occidentalis 3ϫ tion.9 Cells were washed with PBS and 106 cells/mL were Aloe vera 3ϫ, 6ϫ, 12ϫ, 30ϫ incubated for 24 hours with varying doses of Phase 6 or Flu Phytolacca decandra 3ϫ, 11ϫ, 19ϫ, 27ϫ, Euphorbium officinarum 6ϫ Terminator (0–200 ␮L/mL) in RPMI medium supplemented Zincum metallicum 6ϫ with 10% FBS and antibiotics at 37°C in a 5% humidified Arsenicum album 6ϫ, 14ϫ, 22ϫ, 30ϫ CO2 incubator. Human leukocytes treated with 0–200 ␮L Viscum album 8ϫ, 16ϫ, 24ϫ of 20% ethanol (the carrier) served as controls in order to Conium maculatum 30ϫ determine the carrier effects of Phase 6 and Flu Terminator. Histaminum hydrochloricum 200ϫ Formicum acidum 200ϫ Analysis of cytokine synthesis Note: Plant-based substances are indicated with italics. aBoth products are formulated and manufactured by Be Well After 24 hours of drug incubation, cell-culture mediumHomeopathics Inc. Miami, FL. was removed by centrifugation at 400 g for 10 min. Cy- bK, Korsakovian dilution, which uses the same container for each tokines (IL-1, IL-4, IL-10, IFN-␥, and TNF-␣) synthesizedserial dilution. The potency numbers that follow the homeopathicremedies are indicative of the number of dilutions and successions and released into the medium by leukocytes were estimatedfrom the original mother tincture. by ELISA, using reagent kits from BD Biosciences. Briefly, cDilution of common dandelion. 100 ␮L of medium was used for a multiwell ELISA proto-
  • 3. CYTOKINE CHANGES CAUSED BY HOMEOPATHIC MEDICINES 405col according to the manufacturer’s recommendations, and 35the synthesis of cytokines (in concentrations of pg/mL) by 30 p<0.01the leukocytes was quantified.11 The experiment was re- 25 IL-10 (pg/mL)peated thrice for estimation of standard deviation values, and 20the estimates of cytokine concentrations were analyzed sta- 15tistically through analysis of variance (ANOVA). 10 5 RESULTS AND DISCUSSION 0 0 10 20 50 100 200 Flu Terminator ® (µL) Phase 6 induced the production of TNF-␣ (Fig. 1) andIFN-␥ (Fig. 2) at doses of Flu Terminator and Phase 6 rang- FIG. 3. IL-10 induction in human leukocytes by Flu Termina-ing from 10 ␮L to 100 ␮L, with a decrease in production tor® (formulated and manufactured by Be Well Homeopathics Inc.,at 200 ␮L (p Ͻ 0.01). However IL-1, IL-4, and IL-10 were Miami, FL).not induced by Phase 6. The cells treated with increasingquantities of 20% ethanol failed to show any cytokine pro- starting material to high dilutions with concentrations be-duction, indicating that the effect of the two homeopathic yond (smaller than) Avogadro’s number (6.23 ϫ 1023),preparations was not due to the solvent carrier. which corresponds roughly to a 24ϫ dilution. Both prepa- Flu Terminator induced production of IL-10 and TNF-␣ rations are recommended for general immune defense and(Figs. 3 and 4), apparently in a dose-dependent manner (p Ͻ for the treatment of viral illnesses, respectively. They were0.01). However, Flu Terminator failed to induce synthesis therefore appropriate preparations for evaluating their ef-of IL-4 and IFN-␥. The cells treated with increasing quan- fects on mediators of immunity, which in our study con-tities of 20% ethanol failed to show any cytokine produc- sisted of pro- and anti-inflammatory cytokines.tion, indicating that the effect was not due to the solvent car- The results obtained in our study were reproducible andrier. statistically significant. While it is not possible to draw a Although a growing body of clinical research supports specific conclusion other than to note the presence of a bi-the existence of biologic effects of homeopathic treatments, ological effect, several points are noteworthy. In the case ofthe principles governing these interventions remain elusive Phase 6, two important proinflammatory cytokines associ-despite abundant theories. The principles of homeopathy ated with the T-helper 1 (Th1) pathway showed a “U”-will not achieve mainstream medical acceptance without shaped response pattern, and not in the dose–response man-homeopathic preparations being subjected to stringent evi- ner typical for a pharmaceutical agent. None of the cytokinesdence-based research using conventional methods. How- associated with the Th2 pathway that were studied were el-ever, it is equally essential to recognize that bias by some evated. This is an important observation, suggesting that thescientists who dismiss outright the effects of homeopathic effects of the two homeopathic preparations that we studiedpreparations as incredible or owing to placebo because the are not random. Rather they seem to reflect a selective ef-dilutions are so great is not an evidence-based approach to fect on specific biologic pathways in the activation of proin-their efficacy. flammatory cytokines. Both Phase 6 and Flu Terminator are mixtures of highly In the case Flu Terminator, the concentrations of TNF-␣,diluted extracts and minerals that range from 1/10 of the a proinflammatory cytokine, and IL-10, an anti-inflamma- tory cytokine, were both elevated again, with a “U”-shaped response pattern. While this may appear contradictory, it is 6 important to be aware that the Th1 and Th2 pathways of im- mune response act in a complementary manner in response 5 p<0.01 to various infections, thus providing a mechanism for im-INF-g (pg/mL) 4 mune modulation. The data in our study in no way address 3 the timing of these responses, but do suggest that Flu Ter- minator may play a role in activation and modulation of the 2 immune response associated with infection. 1 In either case, skepticism associated with the possibility 0 that ultralow concentrations of homeopathic medications 0 2 5 10 20 50 100 200 can modulate the expression of these cytokines, irrespective Phase 6 (µL) of the theoretical mechanism by which they do this, mustFIG. 2. INF-␥ induction in human leukocytes by Phase 6 (for- be reconsidered. Examination of principles of immunologymulated and manufactured by Be Well Homeopathics Inc., Miami, may provide some support for these observations. StudiesFL). of the effects of ultralow concentrations of various sub-
  • 4. 406 RAMACHANDRAN ET AL. 250 then will more reliable models for evaluating effects of homeopathic dilutions be established and expand our knowl- 200 edge of the physical and biological basis of these interac- p<0.01TNF-a (pg/mL) tions. 150 100 REFERENCES 50 1. Bellavite P, Conforti A, Piasere V, Ortolani R. Immunology 0 and homeopathy: 1. Historical background. eCAM 2005;2: 0 10 20 50 100 200 441–452. Flu Terminator ® (µL) 2. Brizzi M, Lazzarato L, Nani D, et al. Biostatistical insight into the As2O3 high dilutions effects on the rate and variability ofFIG. 4. TNF-␣ induction in Human Leukocytes by Flu Termi-nator® (formulated and manufactured by Be Well Homeopathics wheat seedling growth. MACROBUTTON HtmlResAnchorInc., Miami, FL). Forsch Komplementarmed Klass Naturheilkd. 2005;12: 277–283. 3. Elsasser-Beile U, Voss M, Schuhle R, Wetterauer U.stances have clearly demonstrated reproducible biologic ef- Biological. Effects of natural and recombinant mistletoe lectin and an aqueous mistletoe extract on human monocytes andfects in vitro and in vivo.2–10,12,13 The concentrations of cy- lymphocytes in vitro. J Clin Lab Anal 2000;14:255–259.tokines required to induce effects in vivo have been found 4. Bellavite P, Conforti A, Pontarollo F, Ortolani R. Immunol-to be in the femtomolar range (10Ϫ15 M), clearly within the ogy and homeopathy: 2. Cells of the immune system and in-range of homeopathic preparations.14–16 Such concentra- flammation. Evidence-Based Complement Alternat Med 2006;tions are at the lower limits of detection by standard labo- 3:13–24.ratory assays, and whether these preparations can demon- 5. Belon P, Cumps J, Ennis M, et al. Histamine dilutions modu-strate biologic effects unequivocally without inferring a late basophil activation. Inflamm Res 2004;53:181–188.concentration (theoretical determination) is therefore un- 6. Wiesenauer M, Gaus W, Bohnacker U, Haussler S. Efficiencyclear. However, there are examples of concentrations of cy- of homeopathic preparation combinations in sinusitis: Resultstokines that demonstrate biologic effects clearly, which have of a randomized double blind study with general practitioners.been indirectly determined. These include the inhibition of Arzneimittelforschung 1989:39:620–625. 7. Maiwald VL, Weinfurtner T, Mau J, Connert WD. Therapy ofbasophil deregulation by ultralow concentrations of hista- common cold with a homeopathic combination preparation inmine, with confirmation of receptor specificity, and other comparison with acetylsalicylic acid: A controlled, random-effects on immune function in vitro of other bioactive com- ized double-blind study. Arzneimittelforschung 1988;38:pounds at ultralow concentrations.4,5 It is also noteworthy 578–582.that antigens that have immunostimulatory effects may elicit 8. Jacobs J, Guthrie BL, Montes GA, et al. Homeopathic com-these effects at femtomolar or even subfemtomolar concen- bination remedy in the treatment of acute childhood diarrheatrations.17–19 Clearly, at least in the case of immune func- in borders. J Altern Complement Med 2006;12:723–732.tion, it does appear that cells in vitro normally respond to 9. Samsonavae EI, Smirnova TN, Poliakov VE. Effective com-signals associated with ultralow concentrations of bioactive plex homeopathic outpatient treatment of a child with allergicmolecules, thus lending some support to the principles of rhinitis and neutropenia. Vestn Otorinolaringol 2006;2:57–59.homeopathy. 10. Porozov S, Cahalon L, Weiser M, et al. Inhibition of IL-1␤ and TNF-␣ secretion from resting and activated human lym- phocytes by the homeopathic medication Traumeel® S. Dev Immunol 2004;11:143–149. CONCLUSIONS 11. Nair PK, Rodriguez S, Ramachandran R, et al. Immune stim- ulating properties of a novel polysaccharide from the medici- The impact on cytokine expression of Phase 6 and Flu nal plant Tinospora cordifolia. Int ImmunopharmacolTerminator in our study lends additional support to the grow- 2004;4:1645– body of evidence that dilutions of extracts and minerals 12. Bellavite P, Ortolani R, Conforti A. Immunology and home-do affect biologic systems. Furthermore, such studies also opathy: 3. Experimental studies on animal models. Evidence- Based Complement Alternat Med 2006;3:171– the possibility that homeopathic agents, as in the 13. Walchli C, Baumgartner S, Bastide M. Effects of low dosescase of pharmaceuticals, may affect specific molecular tar- and high homeopathic potencies in normal and cancerous hu-gets, thus defining potential medical applications for these man lymphocytes: An in vitro study. J Altern Complementagents. The controversy over the efficacy of low concen- Med 2006;12:421–427.trations of homeopathic agents will, however, probably re- 14. Collins DP, Luebering BJ, Shaut DM. T-lymphocyte func-main until more adequate techniques become available to tionality assessed by analysis of cytokine receptor expression,assess molecular interactions at such concentrations; only intracellular cytokine expression, and femtomolar detection of
  • 5. CYTOKINE CHANGES CAUSED BY HOMEOPATHIC MEDICINES 407 cytokine secretion by quantitative flow cytometry. Cytometry 19. Lichtenberger III FJ, Montague CR, Hunter M, et al. NAC 1998;33:249–255. and DTT promote TGF-␤ 1 monomer formation: Demon-15. Bank U, Reinhold D, Kunz D, et al. Effects of interleukin-6 stration of competitive binding. Journal of Inflammation (IL-6) and transforming growth factor-␤ (TGF-␤) on neu- 2006;3:7–15. trophil elastase release. Inflammation 1995;19:83–99.16. Curfs JHAJ, Meis JFGM, Hoogkamp-Korstanje JAA. A Address reprint requests to: primer on cytokines: Sources, receptors, effects and inducers. Cheppail Ramachandran, Ph.D., M.B.A. Clin Microbiol Rev 1997;10:742–780. Miami Children’s Hospital17. Klinman NR. The secondary immune response to a haptogen 3100 SW 62nd Avenue in vitro. J Exp Med 1971;133:963–973. Miami, FL 3315518. Dejda A, Sokolowska P. Nowak JZ. Neuroprotective potential of three neuropeptides PACAP, VIP and PHI. Pharmacol Rep 2005;57:307–320. E-mail: