Homeopathy 2019; 108(03): 188-200
DOI: 10.1055/s-0039-1678700
Original Research Article
The Faculty of Homeopathy

Effects of Homeopathic Phosphorus on Encephalitozoon cuniculi-Infected Macrophages In-Vitro

Mirian Yaeko Nagai
1   Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
,
Luciane Costa Dalboni
1   Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
,
Thayná Neves Cardoso
1   Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
,
Michelle Sanchez Correia
1   Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
,
Sandra Augusta G. Pinto
1   Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
,
Andreia A. G. Pinto
1   Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
,
Cideli de Paula Coelho
2   Universidade de Santo Amaro – UNISA, São Paulo, São Paulo, Brazil
3   HD Science, São Caetano do Sul, São Paulo, Brazil
,
Anuska Alvarez-Saraiva
1   Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
,
Giovani B. Peres
1   Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
,
Maria Anete Lallo
1   Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
,
1   Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
› Institutsangaben
Weitere Informationen

Publikationsverlauf

07. November 2018

07. Januar 2019

Publikationsdatum:
18. April 2019 (online)

Abstract

IntroductionEncephalitozoon cuniculi (E. cuniculi), a fungus that acts as an intracellular pathogen, causes a marked neurological syndrome in many host species and is a zoonotic concern. Although no well-established treatment for this syndrome is known, previous successful clinical experience using homeopathic phosphorus has been described in which symptom remission with no mortality occurred in 40/42 animals by means of unknown immunological mechanisms. The latter observation was the main motivation for this study.

Objective To verify, in an in-vitro model, if macrophages infected with E. cuniculi can change in function after treatment with different potencies of phosphorus.

Materials and Methods RAW 264.7 macrophages were infected with E. cuniculi in-vitro and treated with various homeopathic potencies of phosphorus. The vehicle was used as a control solution (0.06% succussed ethanol). After 1 and 24 hours, the following parameters were analyzed: parasite internalization (by the Calcofluor staining method), lysosome activity (by the acridine orange method), cytokine/chemokine production (by the MAGPIX system), and cell ultrastructure. Automatic image analysis was used when applicable, and the experiments were performed in triplicate.

Results Treatment with vehicle alone increased interleukin (IL)-6, tumor necrosis factor alpha and monocyte chemotactic protein -1 production (p ≤ 0.05) and reduced the number of internalized parasites (p ≤ 0.001). A progressive and time-dependent increase in RANTES (regulated on activation, normal T-cell expressed and secreted) and lysosome activity (p ≤ 0.002) was observed only after treatment with the highest potency of phosphorus (Phos 200cH), together with decreased apoptosis rate, intense parasite digestion, and the presence of non-internalized spores.

ConclusionsPhos 200 cH has a modulatory action on the activity of infected macrophages, especially a specific increase in RANTES, a key element in the prognosis of E. cuniculi-infected and of immunosuppressed patients bearing infections.

Highlights

• Successful previous clinical experience in rabbits diagnosed with encephalitozoonosis and homeopathically treated with high dilutions of phosphorus was the primary motivation for this study.


• An in-vitro model using infected macrophages was used in an attempt to identify the possible mechanisms involved.


• The results suggest non-specific, vehicle-related inflammatory action and that specific modulatory effects occurred at the highest potency of phosphorus (Phos 200 cH).


• The specific modulatory effects were related to an increase in RANTES production, decrease in apoptosis rate and an alteration in the balance between the phagocytic and endocytic pathways of parasite internalization by macrophages.


 
  • References

  • 1 Rodríguez-Tovar LE, Villarreal-Marroquín A, Nevárez-Garza AM. , et al. Histochemical study of Encephalitozoon cuniculi spores in the kidneys of naturally infected New Zealand rabbits. J Vet Diagn Invest 2017; 29: 269-277
  • 2 Neumayerová H, Juránková J, Jeklová E. , et al. Seroprevalence of Toxoplasma gondii and Encephalitozoon cuniculi in rabbits from different farming systems. Vet Parasitol 2014; 204: 184-190
  • 3 Francisco Neto A, Dell'Armelina Rocha PR, Perez EC. , et al. Diabetes mellitus increases the susceptibility to encephalitozoonosis in mice. PLoS One 2017; 12: e0186954
  • 4 Nell B, Csokai J, Fuchs-Baumgartinger A, Maaß G. Encephalitozoon cuniculi causes focal anterior cataract and uveitis in dogs. Tierarztl Prax Ausg K Klientiere Heimtiere 2015; 43: 337-344
  • 5 Desoubeaux G, Peschke R, Le-Bert C. , et al. Seroprevalence survey for microsporidia in common bottlenose dolphin (Tursiops truncatus): example of a quantitative approach based on immunoblotting. J Wildl Dis 2018; 54: 870-873
  • 6 Wasson K, Peper RL. Mammalian microsporidiosis. Vet Pathol 2000; 37: 113-128
  • 7 Abu-Akkada SS, Oda SS. Prevention and treatment of Encephalitozoon cuniculi infection in immunosuppressed rabbits with fenbendazole. Iran J Vet Res 2016; 17: 98-105
  • 8 Bonamin LV, Endler PC. Animal models for studying homeopathy and high dilutions: conceptual critical review. Homeopathy 2010; 99: 37-50
  • 9 Bonamin LV, Cardoso TN, de Carvalho AC, Amaral JG. The use of animal models in homeopathic research--a review of 2010-2014 PubMed indexed papers. Homeopathy 2015; 104: 283-291
  • 10 Bonamin LV. The soundness of homeopathic fundamental research. Revista de Homeopatia 2017; 80: 82-89
  • 11 Jordan CN, Zajac AM, Lindsay DS. Encephalitozoon cuniculi infection in rabbits. Compend Contin Educ Vet 2006; 28: 108-116
  • 12 Künzel F, Gruber A, Tichy A. , et al. Clinical symptoms and diagnosis of encephalitozoonosis in pet rabbits. Vet Parasitol 2008; 151: 115-124
  • 13 de Santana FR, Dalboni LC, Nascimento KF. , et al. High dilutions of antimony modulate cytokines production and macrophage – Leishmania (L.) amazonensis interaction in vitro. Cytokine 2017; 92: 33-47
  • 14 Nascimento KF, de Santana FR, da Costa CRV. , et al. M1 homeopathic complex trigger effective responses against Leishmania (L.) amazonensis in vivo and in vitro. Cytokine 2017; 99: 80-90
  • 15 Rodrigues de Santana F, de Paula Coelho C, Cardoso TN. , et al. Modulation of inflammation response to murine cutaneous Leishmaniasis by homeopathic medicines: Antimonium crudum 30cH. Homeopathy 2014; 103: 264-274
  • 16 Cajueiro APB, Goma EP, Dos Santos HAM. , et al. Homeopathic medicines cause Th1 predominance and induce spleen and megakaryocytes changes in BALB/c mice infected with Leishmania infantum . Cytokine 2017; 95: 97-101
  • 17 Olioso D, Marzotto M, Bonafini C, Brizzi M, Bellavite P. Arnica montana effects on gene expression in a human macrophage cell line. Evaluation by quantitative Real-Time PCR. Homeopathy 2016; 105: 131-147
  • 18 Marzotto M, Bonafini C, Olioso D. , et al. Arnica montana stimulates extracellular matrix gene expression in a macrophage cell line differentiated to wound-healing phenotype. PLoS One 2016; 11: e0166340
  • 19 de Paula Coelho C, Motta PD, Petrillo M. , et al. Homeopathic medicine Cantharis modulates uropathogenic E. coli (UPEC)-induced cystitis in susceptible mice. Cytokine 2017; 92: 103-109
  • 20 Bonamin LV, Bellavite P. Immunological models in high dilution research following M Bastide. Homeopathy 2015; 104: 263-268
  • 21 Rodrigues de Santana F, Coelho CdeP, Cardoso TN, Laurenti MD, Perez Hurtado EC, Bonamin LV. Modulation of inflammation response to murine cutaneous Leishmaniosis by homeopathic medicines: thymulin 5cH. Homeopathy 2014; 103: 275-284
  • 22 Bonamin LV, Sato C, Zalla Neto R. , et al. Immunomodulation of homeopathic thymulin 5CH in a BCG-induced granuloma model. Evid Based Complement Alternat Med 2013; 2013: 686018
  • 23 Sato C, Listar VG, Bonamin LV. Development of broiler chickens after treatment with thymulin 5cH: a zoo technical approach. Homeopathy 2012; 101: 68-73
  • 24 ANVISA. Farmacopeia Homeopática Brasileira 3ª edição 2011. Available at: http://portal.anvisa.gov.br/documents/33832/259147/3a_edicao.pdf/cb9d5888-6b7c-447b-be3c-af51aaae7ea8
  • 25 Bell IR, Muralidharan S, Schwartz GE. Nanoparticle characterization of traditional homeopathically-manufactured Gelsemium sempervirens medicines and placebo controls. J Nanomedine Biotherapeutic Discov 2015; 5: 136 . doi:10.4172/2155-983X.1000136
  • 26 Chikramane PS, Suresh AK, Bellare JR, Kane SG. Extreme homeopathic dilutions retain starting materials: a nanoparticulate perspective. Homeopathy 2010; 99: 231-242
  • 27 Chikramane PS, Suresh AK, Kane SG, Bellare JR. Metal nanoparticle induced hormetic activation: a novel mechanism of homeopathic medicines. Homeopathy 2017; 106: 135-144
  • 28 Temgire MK, Suresh AK, Kane SG, Bellare JR. Establishing the interfacial nano-structure and elemental composition of homeopathic medicines based on inorganic salts: a scientific approach. Homeopathy 2016; 105: 160-172
  • 29 Saha SK, Roy S, Khuda-Bukhsh AR. Ultra-highly diluted plant extracts of Hydrastis canadensis and Marsdenia condurango induce epigenetic modifications and alter gene expression profiles in HeLa cells in vitro. J Integr Med 2015; 13: 400-411
  • 30 Desoubeaux G, Franck-Martel C, Caille A. , et al. Use of Calcofluor-blue brightener for the diagnosis of Pneumocystis jirovecii pneumonia in bronchial-alveolar lavage fluids: a single-center prospective study. Med Mycol 2017; 55: 295-301
  • 31 John DE, Nwachuku N, Pepper IL, Gerba CP. Development and optimization of a quantitative cell culture infectivity assay for the microsporidium Encephalitozoon intestinalis and application to ultraviolet light inactivation. J Microbiol Methods 2003; 52: 183-196
  • 32 Marzotto M, Olioso D, Brizzi M, Tononi P, Cristofoletti M, Bellavite P. Extreme sensitivity of gene expression in human SH-SY5Y neurocytes to ultra-low doses of Gelsemium sempervirens . BMC Complement Altern Med 2014; 14: 104
  • 33 Olioso D, Marzotto M, Moratti E, Brizzi M, Bellavite P. Effects of Gelsemium sempervirens L. on pathway-focused gene expression profiling in neuronal cells. J Ethnopharmacol 2014; 153: 535-539
  • 34 Khuda-Bukhsh AR, Sikdar S. Condurango 30C induces epigenetic modification of lung cancer-specific tumour suppressor genes via demethylation. Forsch Komplement Med 2015; 22: 172-179
  • 35 Mondal J, Das J, Shah R, Khuda-Bukhsh AR. A homeopathic nosode, Hepatitis C 30 demonstrates anticancer effect against liver cancer cells in vitro by modulating telomerase and topoisomerase II activities as also by promoting apoptosis via intrinsic mitochondrial pathway. J Integr Med 2016; 14: 209-218
  • 36 Ghosh K, Weiss LM. T cell response and persistence of the microsporidia. FEMS Microbiol Rev 2012; 36: 748-760
  • 37 Scanlon M, Leitch GJ, Shaw AP, Moura H, Visvesvara GS. Susceptibility to apoptosis is reduced in the microsporidia-infected host cell. J Eukaryot Microbiol 1999; 46: 34-35
  • 38 Han B, Polonais V, Sugi T. , et al. The role of microsporidian polar tube protein 4 (PTP4) in host cell infection. PLoS Pathog 2017; 13: e1006341
  • 39 Desoubeaux G, Piqueras MDC, Pantin A. , et al. Application of mass spectrometry to elucidate the pathophysiology of Encephalitozoon cuniculi infection in rabbits. PLoS One 2017; 12: e0177961
  • 40 Fischer J, West J, Agochukwu N, Suire C, Hale-Donze H. Induction of host chemotactic response by Encephalitozoon spp. Infect Immun 2007; 75: 1619-1625
  • 41 Crawford A, Angelosanto JM, Nadwodny KL, Blackburn SD, Wherry EJ. A role for the chemokine RANTES in regulating CD8 T cell responses during chronic viral infection. PLoS Pathog 2011; 7: e1002098
  • 42 Kawai T, Seki M, Hiromatsu K. , et al. Selective diapedesis of Th1 cells induced by endothelial cell RANTES. J Immunol 1999; 163: 3269-3278
  • 43 Mutuiri SP, Kutima HL, Mwapagha LM. , et al. RANTES gene polymorphisms associated with HIV-1 infections in Kenyan population. Dis Markers 2016; 2016: 4703854
  • 44 Witt CM, Bluth M, Albrecht H, Weisshuhn TE, Baumgartner S, Willich SN. The in vitro evidence for an effect of high homeopathic potencies--a systematic review of the literature. Complement Ther Med 2007; 15: 128-138
  • 45 Demangeat JL. Gas nanobubbles and aqueous nanostructures: the crucial role of dynamization. Homeopathy 2015; 104: 101-115
  • 46 Madl P, Del Giudice E, Voeikov VL. , et al. Evidence of coherent dynamics in water droplets of waterfalls. Water 2013; 5: 57-68
  • 47 Voeikov VL. Fundamental role of water in bioenergetics. In: Belousov VL, Voeikov VK, Martynyuk VS. , eds. Dordrecht: Springer; 2007: 89-104
  • 48 de Oliveira CC, Abud AP, de Oliveira SM. , et al. Developments on drug discovery and on new therapeutics: highly diluted tinctures act as biological response modifiers. BMC Complement Altern Med 2011; 11: 101
  • 49 Gonçalves JP, Dos Santos MLF, Rossi GR, Costa Gagosian VS, de Oliveira CC. Differential effects of Zincum metallicum on cell models. Homeopathy 2017; 106: 171-180
  • 50 Bonafini C, Marzotto M, Bellavite P. In vitro effects of zinc in soluble and homeopathic formulations on macrophages and astrocytes. Homeopathy 2017; 106: 103-113
  • 51 Barreto CS, Homsani F, Holandino C, Silva NCB. Plant tissue culture and ultra high diluted studies: suggesting a novel model using in vitro techniques. In: Proceedings of the XXX GIRI meeting; Sep 9–11, 2016; Amsterdam, The Netherlands. Int J High Dilution Res 2016;15:45–49
  • 52 Otsuka I. Nanoscale cluster structure of O2 gas-filled nanobubbles is O2 gas-supersaturated alkaline solution. In: Proceedings of the XXIX GIRI meeting; June 3–5, 2015; Verona, Italy. Int J High Dilution Res 2015;14:40