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

Mirian Yaeko Nagai; Luciane Costa Dalboni; Thayná Neves Cardoso; Michelle Sanchez Correia; Sandra Augusta G. Pinto; Andreia A. G. Pinto; Cideli de Paula Coelho; Anuska Alvarez-Saraiva; Giovani B. Peres; Maria Anete Lallo; Leoni Villano Bonamin

doi:10.1055/s-0039-1678700

Homeopathy, Inhaltsverzeichnis

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

Authors

Mirian Yaeko Nagai

¹Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
Luciane Costa Dalboni

¹Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
Thayná Neves Cardoso

¹Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
Michelle Sanchez Correia

¹Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
Sandra Augusta G. Pinto

¹Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
Andreia A. G. Pinto

¹Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
Cideli de Paula Coelho

²Universidade de Santo Amaro – UNISA, São Paulo, São Paulo, Brazil

³HD Science, São Caetano do Sul, São Paulo, Brazil
Anuska Alvarez-Saraiva

¹Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
Giovani B. Peres

¹Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
Maria Anete Lallo

¹Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil
Leoni Villano Bonamin

¹Research Center, Graduating Program of Environmental and Experimental Pathology, Universidade Paulista – UNIP, São Paulo, São Paulo, Brazil

Abstract

Introduction Encephalitozoon 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.

Conclusions Phos 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.

Keywords

homeopathy - fungus - microsporidiosis - cytokines - chemokines

Volltext

Referenzen

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