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Homeopathy and experimental infections: In vivo and in vitro experiments with bacteria, fungi and protozoan

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Abstract

It is common sense in the homeopathic community that treating patients with diverse infections generates satisfactory clinical responses by enhancing the immune response. But experimental laboratory studies show exactly what happens in each case and how the immune response dynamically adjusts to the conditions of the infection in question. In this article, we aim to describe a brief review of the latest studies conducted by our group on animal and cellular models on immune cell activity against different infectious agents following treatment with specific homeopathic preparations. The studies dealt with infections induced by bacteria, fungi and protozoa.

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... For these reasons, newer less aggressive treatment options are sought, mainly for the production of organic milk (Busanello et al., 2017). Among these options, homeopathy has gained ground in conventional and organic systems because homeopathic products used as preventives do not require disposal of milk (Bonamin, 2019). Studies report the use of homeopathic products for the treatment of mastitis (Doehring and Sundrum, 2016;Mathie and Clausen, 2015;Werner and Sundrum, 2006); nevertheless, it remains unknown scientifically as to whether these products effectively prevent control mastitis. ...
... According to the literature, homeopathy encourages healing mechanisms through immune stimulation to fight viruses, bacteria, fungi, tumors, and other diseases, allowing the restoration of balance in the animal's system and encouraging organic responses that can lead to a reduction of stress (Da Costa Filho et al. 2014), as well as stimulating the proliferation of leukocytes in healthy dogs that consumed a homeopathic product daily (Marchiori et al., 2019). Homeopathic medicines can decrease microbial infection by strengthening immunity (Bonamin, 2019). We believe that the lower contamination by microorganisms of the mammary gland (lower TBC) reduced the inflammatory response, explaining the lower counts of total leukocytes, lymphocytes, and neutrophils in the blood of cows that consumed the homeopathic. ...
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The aim of this study was to determine whether the addition of a homeopathic product to dairy cow feed would improve health and production efficiency, as well as composition and quality of milk. The product tested here was produced to stimulate immunity and indirectly to prevent the colonization of the mammary gland by bacteria, thereby decreasing somatic cell counts and bacteria in cow’s milk. Fifty dairy cows were used, divided into two groups: Control (n = 25) and Treated (n = 25). For 90 consecutive days, 50 grams of homeopathic product (treated group) was added to the concentrate; 50 g of the product vehicle (limestone) was added to the concentrate of the control cows. We performed composition and quality analysis on the milk (days 1, 15, 30, 45, 60, 75, and 90) as well as blood collection to carry out hematological and metabolism analyses (days 1, 30, 60, and 90). We found that fat content in the milk cows consuming the homeopathic agent was greater than that of the control group. Because of this increased fat content, there was a tendency towards higher total solids content in treated cows. A treatment effect was found in terms of total bacterial count (TBC); that is, there were lower TBCs in milk of treated animals than in control animals. There was a trend of a treatment effect versus day for somatic cell count (SCC); that is, there were lower SCC in animals that consumed the homeopathic on days 15 and 30 of the experiment. There was no difference between groups in terms of milk production; however, there was a negative correlation between milk production and TBC or SCC in the milk of cows that consumed homeopathic product. Animals in the treated group had lower total leukocyte and lymphocyte counts than did the control group, as well as a tendency toward lower neutrophil counts in these animals. Several bacteria were isolated from the cows' milk during the experimental period, with no treatment effect. In particular, we isolated Corynebacterium spp., Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus hyicus in greater numbers of cows compared to other etiologic agents. Based on these results, we conclude that consumption of the homeopathic product by dairy cows had positive effects on milk quality.
... These findings suggest that the use of homeopathic has the potential to decrease microbial infection by strengthening immunity. According to Bonamin [29], animals with strengthened immune systems can more effectively rid themselves of pathogens; homeopathy is not aimed at microbicide; rather, it aims to enhance immune stimulation in the animal. ...
... Our findings reaffirm those described by Halberstein [18], who found that homeopathics can be used as exclusive or complementary therapies, in addition to substantial prophylactic potential against diseases. The association of homeopathics with allopathics may generate greater potency, because one acts on the immune system and the other acts on pathogens [29]. Homeopathic agents are also important for reducing bacterial resistance to antibiotics, curative possibilities [37], as well as serving as alternatives for antimicrobial effects. ...
Article
Escherichia coli is a bacterium normally found in the gastrointestinal tract of domestic animals that can usually control the infection. Nevertheless, some factors (high exposure, stress conditions, animal category, among others) can favor the exacerbation of E. coli infection and cause of disease. Because it is a zoonotic bacterium, it is important to control the infection, avoiding contamination of home interiors in the case of pets. There are various forms of treatment for E. coli; nevertheless, there are few options for prevention. In the present study, we evaluated homeopathy. Thus, the objective of this study was to determine whether administration of a prophylactic homeopathic in water would minimize the negative effects of E. coli infection, as well as reducing bacterial counts in the feces of a experimental model. Forty mice were divided into four experimental groups (n = 10/group). Groups NC (negative control) and PC (positive control) were not treated; In group T1, the animals received 0.002 mL/day/animal of the homeopathic in water, and animals in group T2 0.004 mL/day/animal. The experiment lasted 54 days, and on the 31st day, mice of T1, T2 and PC groups were infected orally a 0.2 mL inoculum of 1.5 × 10⁸ CFU of E. coli. Euthanasia and sample collection were performed on the 40th and 54th days of the experiment (n = 5/group/time point). Blood, liver, spleen, intestine, and feces samples were collected from the final portion of the intestine. There was no significant difference in animal weight between groups at the end of the experiment. Neutrophil count was lower in PC group animals on day 40, while on day 54, the counts were lower in T2 and PC. Lymphocyte counts were lower only in the PC group than in the NC group on day 54. Globulins were lower in the NC and PC groups than in T1 and T2 on day 40, remaining lower the PC group and higher in T1 on day 54; levels of immunoglobulin IgG and IgM were higher in groups T1 and T2, which differed from PC and NC. TNF-α levels were higher in the T1 and T2 groups at 40 and 54 days. INF-γ levels were higher in T1, T2, and PC compared to NC on day 40, remaining higher than NC in groups T1 and T2 on day 54. Total bacterial count, total coliforms and E. coli counts were lower in group T1 and higher in NC and PC on days 40 and 54, when they were lower for T1 and T2. Histologically, no lesions were observed in extra-intestinal tissues; however the height of intestinal crypts in the PC group was smaller than the others on day 40. On day 54, villi and crypts of all infected groups were larger in T1 and T2 than in NC; sizes in the PC group were higher than those of all other groups. These data suggest that the homeopathic agent in the drinking water improved health of the mice.
... Pre-clinical studies involving homeopathic medicines have gained increasing attention from researchers 16 : there are studies with animal models for diseases, [17][18][19] as well as in-vitro research, that test the effects of homeopathic medicines directly on infectious agents or on cell cultures that exhibit reduced growth. 11,[20][21][22] Special attention is required when analyzing pre-clinical studies with homeopathic medicines, especially regarding the interpretation of the effect of the vehicles used in their manipulation. Bonamin 23 suggests preparing the three last potencies only with sterile water. ...
Article
Background Myiasis by Cochliomyia hominivorax (Diptera: Calliphoridae) is a serious problem in animal health in tropical and sub-tropical regions. Ointment-type preparations are a good option of formulation in cases of myiasis in farm and pet animals. Sulphur and Pyrogenium have already shown in-vitro efficacy on C. hominivorax. This article describes an in-vitro experiment to test the inhibition of development from exposing larvae of C. hominivorax to two homeopathic ointments (prepared individually with Sulphur or Pyrogenium). Methods The homeopathic ointments were produced by mixing sterile lanolin, tocopherol and homeopathic medicine on a hydroalcoholic basis according to the Brazilian Homeopathic Pharmacopoeia. Larvae were obtained from naturally occurring myiases in sheep (wild larvae) or from a laboratory colony. The test consisted of exposing a group of 10 third-stage C. hominivorax wild larvae in contact with Sulphur or Pyrogenium ointment, or a group of 15 laboratory-propagated larvae in contact with the alcoholic vehicle of the ointment or homeopathic medicines prepared in sterile water (Sulphur or Pyrogenium), and observing the effect on the development, longevity and fertility of the blow-fly specimens. Results The C. hominivorax larval inhibition rate was 90.0% for the Sulphur ointment group and was 86.0% for the Pyrogenium ointment group. The non-alcoholic vehicle and the alcoholic vehicle inhibited the development of 24.0% and 22.08% of the larvae respectively. Sulphur prepared in sterile water inhibited the development of 74.67% and Pyrogenium in sterile water inhibited 73.33% of larvae. Specimens that survived contact with homeopathic ointments had their longevity decreased and did not reproduce. Conclusion Ointments of Sulphur or Pyrogenium were able to inhibit the development of C. hominivorax larvae. The ointment vehicle was harmless.
... Lots of experiments are needed to find out the most suitable preparations. There is plenty of work left [21,22]! ...
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IN VITRO EFFECTS OF HOMEOPATHIC DRUGS Christer Sundqvist Petrafoundation, Helsinki, Finland https://petrafoundation.com/en/blog/scientific-evidence-for-homeopathy-part-4 Published: 05.30.2020 The well-known and respected Finnish homeopath Jouni Jämsä (http://www.jounijamsa.fi/ ) continues his homeopathy research with us. In the first part, we talked to him about homeopathy in general and diluted homeopathic preparations in particular [1]. In the next section we investigated how plants respond to homeopathic preparations [2]. In the third part, we went through studies where homeopathy has been used for cancer patients [3]. In this fourth section, we investigate the effect of homeopathic preparations at the cellular level. Highly diluted homeopathic preparations are showing measurable effects in cell cultures [4]. Several studies have found that homeopathic agents give rise to apoptosis (programmed cell death). This phenomenon is a normal part of individual tissue development and regeneration. It is controlled by genes that regulate cell division [5, 6]. Apoptosis plays an important role in fetal development, regulation of the immune response, elimination of infected and transformed cells, and regulation of tissue size. Excessive apoptosis can lead to developmental disorders and degenerative diseases, while its absence can lead to autoimmune diseases, long-term viral infections and cancer. Utilization of drug apoptosis is already routine in the treatment of cancer. Drugs for the treatment of degenerative diseases that prevent apoptosis are likely to emerge in the market in the near future [7]. Apoptosis is something you want to achieve when treating cancer. Because cancer is a disease that affects many people and is very serious, researchers have been interested in different types of treatment options. The effect of homeopathy in cancer can also be studied with cell models. The potent homeopathic drug Lycopodium clavatum (5C and 15C) has anti-cancer activity on HeLa cells in vitro. This has been studied in the Laboratory of Cell Genetics and Molecular Biology at the University of Kalyan, India. The purpose of this study was to evaluate whether homeopathic highly diluted and potent preparations of Lycopodium Clavatum (LC-5C and LC-15C) have anti-cancer effects on HeLa cells. Cells were exposed to either LC-5C (diluted below Avogadro's number) or diluted above Avogadro's number (LC-15C). The results revealed that administration of Lycopodium had little or no toxic effects on the cells in the bloodstream, but caused marked apoptosis in cancer cells (HeLa), which appeared in the form of initial degradation of DNA. Highly diluted, dynamic homeopathic drugs, both below and above the Avogadro's number, caused cell death in cancer cells, suggesting that these drugs could potentially be used as supportive cancer care [8]. Frenkel's team at the University of Texas (M.D. Anderson Cancer Center, Texas, USA) performed cell experiments with highly diluted Indian homeopathic preparations (Carcinos, Phytolacca, Conium and Thuja). The preparations were tested with different breast cancer cell lines. The preparations were toxic to the cells, leading to cell cycle breakdown and cell death. Thus, these natural, diluted preparations had biological effects that should be further investigated [9]. The same was also supported by Psorinum 6 ×, which triggers apoptosis signals in human lung cancer cells. Studies of the effects of homeopathic Psorinum 6x on cell survival were initially performed in several cancer cell lines, including A549 (lung cancer cell line), HepG2 (liver cancer cell line) and MCF-7 (breast cancer cell line). The experiment investigated the therapeutic effects on cell cycle breakdown, cell death, reactive oxygen radical formation (ROS), and changes in mitochondrial membrane potential (MMP) using flow cytometry and fluorescence microscopy. It was found that treatment of cancer cell lines with Psorinum resulted in increased anti-cancer effects in A549 cells (lung cancer cell line) to a greater extent than in others. Psorinum prevented cell division after 24 hours of treatment and retained the cells in the G1 phase. It also caused e.g. ROS formation, MMP depolarization, morphological changes and DNA damage. The researchers concluded that Psorinum 6 × triggered apoptosis in A549 cells (lung cancer cells) via signaling proteins [10]. Condurango 6C and 30C also trigger apoptosis in lung cancer cells. The more diluted preparation was more effective [11]. The effects of homeopathy on programmed cell death have been extensively studied. Further support for this effect on apoptosis, which is advantageous in the treatment of cancer, has been obtained in e.g. Shagun Arora laboratory at Jaypee University of India. It was found that undiluted and diluted homeopathic preparations were toxic to cultured cancer cells. Homeopathic Sarsaparilla preparations were tested in isolated renal adenocarcinoma cell cultures, the Ruta graveolens preparation in cultured colon cancer cells and the Phytolacca decandra preparation in breast cancer cells. The results showed that all of the homeopathic preparations showed toxic effects in said cell cultures. The undiluted preparations had the best effect, but diluted preparations also served as a starting point of apoptosis. Homeopathic preparations have been shown to be anti-cancer drugs and further research can be encouraged [12]. The homeopathic preparation Calcarea carbonica caused apoptosis in cell cultures from cancerous mice. The study showed evidence of the so-called immunomodulatory mechanism of cell death [13]. In leukemia, homeopathic dilutions of Amanita phalloides have been successfully tested in cell cultures [14]. There is no placebo effect in cultured cancer cells, so here we have evidence that homeopathy is not just a placebo. Should homeopathic significance be increased in established medicine, more evidence from well-controlled and high-quality studies is needed [15].
Article
Motivated by conventional medicines' ineffectiveness and the appearance of bacterial resistance, homeopathic medicines are being increasingly demanded by the veterinary market. Dog feces contain high bacterial and pathogenic loads, which can cause diseases in animals and facilitate zoonotic transmission. Adequate animal immunity contributes to the elimination of potential pathogens. This work's objective was to evaluate the effects of using a homeopathic medication on blood cell counts, serum protein levels, and fecal bacterial counts in dogs. Two groups were used, each including five 19-month-old Beagle dogs. The homeopathic product Orgainfecto® was sprayed on the two daily meals in the treated group (T), at 0.5 mL/animal/day for 60 days; the group that did not receive the homeopathic was considered the control (C), who were fed only the vehicle used in the production of the homeopathic as a placebo. On days 1, 30, and 60, individual stool samples were collected for bacterial counts in the stool, blood samples for blood counts, and serum for biochemical tests. Total bacterial counts in feces were lower in group T on days 30 and 60 of the experiment, while total coliforms and E coli. were lower in this group on day 60. Total leukocyte counts were lower on day 60 in group T, unlike the neutrophil count, higher in group T on day 30, and eosinophils on day 60. However, the lymphocyte count was lower in group T on days 30 and 60. There was an increase in globulin and total protein in group T on day 60. We conclude that the tested homeopathic causes neutrophils and eosinophils' stimulation and increases globulins levels even with reduced lymphocytes. The daily consumption of homeopathic by dogs decreases the intestinal bacterial load, resulting in less environmental contamination and animal and human colibacillosis risks.
Article
Colibacillosis is a disease caused by Escherichia coli that manifests itself when there are homeostatic imbalances or in the context of increased exposure, in which case the organism displays opportunistic behavior. To control this problem in poultry, antibiotics are used in the feed, because E. coli is component of the intestinal microbiota of birds. However, because of the changing dietary habits of the human population that seeks out healthier foods without antimicrobial residues, there have been many studies of alternatives to replace conventional antimicrobials as performance enhancers. Thus, the objective of the present study was to determine whether daily consumption of a homeopathic product (immune stimulator) by broilers stimulates immune responses and thereby minimizes the negative effects of experimental E. coli infection. We used 320 1-day-old Cobb 500 chicks, distributed in two groups with eight repetitions each, and 20 birds per repetition: control (CG) and homeopathy (HG). HG birds consumed doses of 0.02 mL/bird/day (1–7 d) via water, 0.01 ml/bird (8–21 d), 0.02 ml/bird (22–28 d), 0.01 mL/bird (29–35 d), and 0.02 mL/bird (35–45 d), as recommended by the manufacturer. At day 22 of the birds' life, the two groups were divided into four subgroups, with four repetitions per subgroup. On day 22, birds in CG1 and HG1 groups were infected intraperitoneally with 0.5 mL of inoculum containing 1.0 × 10⁸ CFU of E. coli/mL. During the experimental period, data were collected for analysis of performance. On days 21 and 45 of age, we collected blood and feces. During the first 21 days of the experiment, we found that birds that consumed the immunostimulator had lower neutrophil counts and higher levels of globulins, however without significant difference between groups in terms of performance. Uninfected birds that consumed the homeopathic product in the water had less feed conversion (HG2) between days 1–35 and 1 to 45 compared to the other treatments. Mortality was higher in groups experimentally infected with E. coli (HG1 and CG1) from 22 to 35 days of life. There were greater numbers of lymphocytes in the HG2 group on day 45 than in CG1 and CG2; while numbers of neutrophils were lower at 42 days in birds of groups HG1 and HG2 than in CG1. Lower total bacterial counts, total coliforms and E. coli were observed in the feces of birds in the HG2 group compared to the other groups. Taken together, these findings suggest that inclusion of homeopathic product in the water of broilers had positive effects on the modulation of the immune response and on feed conversion in birds not challenged with E. coli. But the preventive protocol used in this study was not able to minimize the negative effects caused by the experimental E. coli intraperitoneal infection in broilers, featuring a substantial infectious challenge.
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In previous studies [1,2] we showed that treatment of mice with Antimonium crudum (Ant-c) 30cH was able to significantly reduce monocyte migration to the infection site after injection of Leishmania (L) amazonensis into the subcutaneous tissue, resulting in clinical improvement. Follow up was performed with an in vitro model, which showed that treatment of co-cultures of RAW 264.7 macrophages and parasites with Ant-c 30cH inhibited two parasite-induced CCL2 peaks 48 and 120 hours after infection together with early inhibition of lysosome activity. These findings explained the results previously obtained in vivo. In turn, treatment with Ant-c 200cH resulted in an early and transitory peak of cell spreading at 48 hours. The coherence between the in vivo and in vitro results indicates that this is a good model to study more thoroughly the mechanisms of action of homeopathic medicines, being the first step to establish correlations between the biological effects and the physical and chemical features of Ant-c 30cH and 200cH. In the present study, the same experimental model was replicated, through comparison of vehicle (30% cereal alcohol), Ant-c 200cH, Zincum metallicum (Zinc) 200cH and Arsenicum album (Ars) 200cH, to confirm the specificity of Ant-c effects. In addition, Ant-c 200cH was ultra-centrifuged, and only the superficial phase was applied to the culture medium. This procedure intended to separate the heavier particles from the lighter ones suspended in the homeopathic medicine. The physical-chemical profile of the medicines was assessed. Solid contaminants (microparticles) in the suspension were analyzed. Conductivity was assessed through measurement of the electron current induced by a micro-amperimeter (Ryodoraku®) connected to 2 clean electrodes immersed in the samples, prepared immediately before the analysis, diluted in pure water (MilliQ, Millipore®) and filtered in 22-µ filter (Millipore®). Pure water was used as control. The device was calibrated immediately before measurements. The microparticle profile was assessed with a scanning electronic microscope - SEM (JEOL JSM 6510®) coupled to an energy dispersive spectroscopy (EDS) system to identify the nature of the elements present in each particle. The size and the number of particles were analyzed from the images generated by electronic microscopy with an automatic image analysis system (Metamorph®). For this purpose, all materials used was cleansed through immersion in pure acetone and subjected to 30-minute sonication before insertion into the microscope to avoid secondary contamination. The samples of medicines were subjected to ultra-centrifugation (10000rpm for 60 minutes) to induce particle sedimentation in the bottom of microtubes. 10 microliters of each sample were collected from the bottom of tubes and placed on a copper stub and kept in a closed recipient until the material was fully dry. The samples were directly analyzed with the microscope. Metallization was not necessary, because the analyzed particles had metallic nature. The biological effects of Ant-c 200cH reproduced the previous ones: spreading and phagocytosis index were significantly higher in the co-cultures treated with Ant-c 200cH compared to vehicle and other, non-specific treatments (Ars 200cH and Zinc 200cH) (p=0.05). However, these results were not exhibited by centrifuged Ant-c 200cH. Analysis of the supernatant after 48-hour incubation revealed increase of the GM-CSF content only in cultures treated with Ant-c 200cH and centrifuged-Ant-c 200cH. No change was observed in the cytokine profile in the cultures treated with Ars 200cH or Zinc 200cH. Morphological analysis of Ant-c samples on SEM showed that the microparticles in Ant-c 30cH were smaller compared to Ant-c 6cH, most of them having half-moon shape. Curiously, agglomerates of particles were detected in Ant-c 200cH. Contaminant particles suspended in pure water contained Pb, Zn, Ca, Na, Au, Hg, Nb and Si, therefore, not related to any specific biological effect of Ant-c. P was identified only in Ant-c 30cH (6.51%) and Ant-c 200cH (13.56%). This wide-range profile of different microparticles did not change after centrifugation, which indicates that the weight of these particles is not conditioned by the nature of their component elements. Conductivity was lower in the vehicle (30% alcohol) compared to Ant-c 6, 30 and 200cH (p=0.0001); the conductivity of Ant-c 200cH was the highest (p=0.008). Also Ars 200cH exhibited higher conductivity (p=0.001) compared to the vehicle. Taken together, these data suggest that the biological effect of Ant-c 200cH on macrophage spreading and phagocytosis might be partially related to the size of the microparticles found in suspension. However, specific effects relative to cytokine production did not depend on microparticle size or content. The changes in conductivity changes exhibited correlation with presence of some elements, such as P, but not with any biological effect. To summarize, the results point to the relevance of eventual false-positive effects relative to phagocytosis in macrophages treated with homeopathic medicines in vitro, due to the interference of larger sized microparticles. They also points to the specificity of GM-CSF expression after 48-hours of co-culture exposure to Ant-c 200cH, centrifuged or not, which suggests it was independent from microparticle content and conductivity. The physical-chemical features of homeopathic medicines related to their specific biological effects are still unknown. Additional studies are needed in this regard.
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Previous studies have reported that two single nucleotide polymorphisms (SNPs) in the RANTES gene promoter region, -403G/A and -28C/G, are associated with a slower rate of decline in CD4+ T cell count. In addition, as a ligand of the major HIV coreceptor CCR5, it is known to block HIV-CCR5 interactions in the course of the HIV infection cycle. This study was carried out with the aim of determining the occurrence of single nucleotide polymorphisms (SNPs) -403G > A and -28C > G in the promoter region of RANTES, in a subset of the Kenyan population. Genomic DNA was extracted from peripheral blood monocular cells and used to amplify the RANTES gene region. Restriction fragment length polymorphism was used to determine the genotypes of the RANTES gene. Out of 100 HIV infected individuals, 19% had G1 genotypes (403G/G, 28C/G), 30% (403A/A, 28C/C), and 50% (403G/A, 28C/C), while in healthy blood donors 13% had G4 (403G/A, 28C/C) genotypes, 22% (403A/A, 28C/C), and 54% (403G/A, 28C/C). HIV negative blood donors (54%) had higher risk of alteration to risk of HIV transmission compared to those who were HIV infected (50%). However, the risk to transmission and distribution differences was not significant ( P=0.092 ). The study showed that RANTES polymorphisms -403 and -28 alleles do exist in the Kenyan population.
Article
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.
Article
Infection in humans with Leishmattia manifests into a spectrum of diseases. The manifestations of the disease depend on the resultant evasion of the parasite to immune responses namely macrophages, which is an exclusive host of leishmania. The B cells valiantly mount antibody responses, however to no avail as the Leishmattia parasites occupy the intracellular niches of the macrophages. Extensive studies have been documented on the role of cell-me- diated immunity (CMI) in protection and counter survival strategies of the parasites leading to down-regulation of CMI. The present review attempts to discuss the cytokines in progression or resolution of visceral form of leishmaniasis or kala-azar, predominantly affecting the Indian subcontinent. The components/cytokine(s) responsible for the regulation of the critical balance of Thl/Th2/Th9/Thl7/Treg cells has been discussed in the perspective. Therefore, any strategy involving the treatment of VL needs to consider the balance and regulation of CD4+ T cell function.
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Studies show that highly diluted medications demonstrate benefits in treating infections, constituting an alternative for their treatment. The present study evaluated the effects of Lycopodium clavatum, dynamization 13c, in Wistar rats infected with T. cruzi. In this study 42 male rats were intraperitoneally inoculated with T. cruzi - Y strain and allocated into groups: IC (infected control group) and Ly (treated with L. clavatum 13c). The cytokines dosage (IFN-γ, IL-12, IL-10, IL-4), quantification and morphometry of myenteric neurons were evaluated. The treatment with L. clavatum modifies the immune response, with increase of IFN-γ on day 10 a.i. and IL-12 on day 24 a.i., decrease of IL-10 concentration on day 10 a.i. and subsequent increase of this cytokine and IL-4 on day 24 a.i., affording a bigger number of myenteric neurons compared to IC group. Thus, L. clavatum 13c promoted on rats infected with T. cruzi a beneficial immunomodulatory action reducing the pathogenic progression of digestive Chagas disease.
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Leishmaniasis is a term referring to a range of clinical conditions caused by protozoan parasites of the genus Leishmania, Trypanosomatidae family, Kinetoplastida order that is transmitted by the bite of certain species of mosquitoes Phlebotominae subfamily. These parasites infect hosts wild and domestic mammals, considered as natural reservoirs and can also infect humans. Leishmania are obligate intramacrophage protozoa that have exclusively intracellular life style. This suggests that the amastigotes possess mechanisms to avoid killing by host cells. Cutaneous leishmaniasis, the most common form of the disease, causes ulcers on exposed parts of the body, leading to disfigurement, permanent scars, and stigma and in some cases disability. Many studies concluded that the cytokines profile and immune system of host have fundamental role in humans and animals natural self-healing. Conventional treatments are far from ideals and the search for new therapeutic alternatives is considered a strategic priority line of research by the World Health Organization. A promising approach in the field of basic research in homeopathy is the treatment of experimental infections with homeopathic drugs prepared from natural substances associations highly diluted, which comprise a combination of several different compounds considered as useful for a symptom or disease. Therefore, this study aimed to evaluate the effect of M1, a complex homeopathic product, in macrophage-Leishmania interaction in vitro and in vivo. It was used RAW cells lineage and BALB/c mice as a host for the promastigotes of L. amazonensis (WHOM/BR/75/Josefa). Several biochemical and morphological parameters were determined. Together, the harmonic results obtained in this study indicate that, in general, the highly diluted products trigger rapid and effective responses by living organisms, cells and mice, against Leishmania, by altering cytokines profile, by NO increasing (p < 0.05), by decreasing parasitic load (p < 0.001), and modifying classical maturation and biogenesis of parasitophorous vacuoles (p < 0.001). M1 complex decreased endocytic index (p < 0.001), and the % of infected macrophages (p < 0.05), preventing the development of lesions (p < 0.05) caused by L. amazonensis by increasing Th1 response (p < 0.05). Therefore the M1complex can be a good candidate for a complementary therapy to conventional treatments, since all the parameters observed in vitro and in vivo improved. It could be an interesting clinical tool in association to a classical anti-parasitic treatment, maybe resulting in better quality of life to the patients, with less toxicity.
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Recent evidence includes apoptosis as a defense against Trypanosoma cruzi infection, which promotes an immune response in the host induced by T cells, type 1, 2 and 17. Currently, there is no medicine completely preventing the progression of this disease. We investigated the immunological and apoptotic effects, morbidity and survival of mice infected with T. cruzi and treated with dynamized homeopathic compounds 13c: Kalium causticum (GCaus), Conium maculatum, (GCon), Lycopodium clavatum (GLy) and 7% alcohol solution (control, vehicle compounds, GCI). There was significant difference in the increase of apoptosis in the treated groups, compared with GCI, which might indicate action of the compounds in these cells. Infected animals treated with Lycopodium clavatum presented better performance compared with other groups. GLy showed a higher amount of hepatocytes and splenocytes undergoing apoptosis, higher number of apoptotic bodies in the liver, predominance of Th1 response, increased TNF-α and decreased IL-6, higher survival, lower morbidity, higher water consumption, body temperature, tendency to higher feed intake and weight gain compared with GCI. Conium maculatum had worse results with increased Th2 response with increased IL-4, worsening of the infection with early mortality of the animals. Together, these data suggest that highly diluted medicines modulate the immune response and apoptosis, affecting the morbidity of animals infected with a highly virulent strain of T. cruzi, being able to minimize the course of infection, providing more alternative approaches in the treatment of Chagas disease.
Article
The prevalence of Th1/Th2 response, spleen changes and megakaryocytes were investigated in BALB/c mice (n = 138) infected with Leishmania infantum, and treated with Leishmania infantum 30× (10⁻³⁰) biotherapy – BioLi30×. We performed controlled experiments using 8-to-12-week-old mice, infected with 5 × 10⁷L. infantum promastigotes, divided into eight groups: G1 (healthy), G2 (infected with L. infantum), G3 (BioLi30× pre-treated), G4 (BioLi30× pre/post-treated), G5 (BioLi30× post-treated), G6 (Water 30× post-treated), G7 (Antimonium crudum 30× post-treated) and G8 (Glucantime® post-treated). G3–G7 groups were orally treated with their respective drugs diluted in filtered water (1:10), and G8 received Glucantime® (0.6 mg/100 µl of PBS), intraperitoneally. Spleen fragments were submitted to double blind histopathological evaluation and the number of megakaryocytes was counted. Besides, animals’ serum was measured after 49 days of infection, and cytokines (IFN-γ, IL-4, IL-10, IL-12), as well as the Th1/Th2 correlation (IFN-γ/IL-4 and IFN-γ/IL-10), were analyzed. Spleen histological parameters were classified as: healthy appearance (G1); discreet (G3–G7), moderate (G2) and moderate to severe (G8) white pulp hyperplasia; proliferation of megakaryocytes (G2–G8), and intense disruption (G2–G8). All groups, except for G7, showed higher percentages of megakaryocytes per field ranging from 87% to 15%, when compared to healthy animals (G1). Th1 predominance in IFN-γ/IL-4 ratio (comparing to G2) was detected in G4, G5, G6 and G7. Finally, pre/post (BioLi30x) and post-treatment (Antimonium crudum 30x) presented reduction of megakaryocytes/spleen changes due to immunomodulation animal process, controlling the infection process, probably by the Th1 cytokine predominance.
Article
Objective: This is a random blinded placebo controlled murine experimental model to study the effects of Cantharis 6 CH, a homeopathic medicine, on E coli-induced cystitis. Methods: 24 adult susceptible female BALB/c mice were inoculated with E coli - UPEC O4:K-:H5 by a transurethral catheter. Cantharis 6cH or vehicle (placebo) was offered to mice by free access into the drinking water (1:100), during 24 h after infection. Spleen, bladder and kidneys were processed for quantitative histopathology after immunohistochemistry, using anti-CD3, CD79, MIF, NK and VEGF antibodies; the cytokines present in the bladder washing fluid were measured using a LUMINEX-Magpix KIT. Mann-Whitney and Fisher exact test were used as statistical analysis. Results: Cantharis 6 CH increased IL12p40, IFN-γ and decreased IL10 concentrations in the bladder fluid (p⩽0.05); in the bladder mucosa, it increased the ratio between B and T lymphocytes (31%) and between B lymphocytes and MIF+ macrophages (57%, p⩽0.05). In the pelvis, instead, it decreased the B/T cells ratio (41%, p⩽0.05) and increased the M1/M2 macrophage ratio (42%, p⩽0.05). No differences were seen in the kidney and spleen analysis. Conclusion: The inverted balance of inflammatory cells and cytokines in bladder and pelvis mucosa shows specific local immune modulation induced by Cantharis 6cH.
Article
Background: In previous results mice treated with high dilutions of antimony presented reduction of monocyte migration to the site of infection with increase in B lymphocytes population in the local lymph node. Aims: To know the mechanisms involved, a series of in vitro studies was done, using co-cultures of macrophages (RAW 264.7) and Leishmania (L.) amazonensis treated with different dilutions of antimony (Antimonium crudum or AC), in different times. Methodology: Spreading, phagocytosis, the oxidative activity of macrophages, the viability of free promastigotes and the cytokines/chemokines concentration in the supernatant were evaluated. The assays were performed in quadruplicate. Results: Cells treated with AC 30cH (10-58M) and AC 200cH (10-398M) presented a temporary reduction of the spreading after 02h of incubation, followed by increase after 48h, being the most significant increase observed after the AC 200cH treatment. However, the percentage of internalized parasites at 48, 96 and 120h of incubation was also higher in cells treated with AC 200cH. It is suggested that the AC 200cH improves the ability of phagocytes to internalize the parasites, but not to digest them. The cytokines-chemokines panel corroborated these results. Both dilutions potentiated the parasite-induced reduction of cytokines production, especially IL-6, IL 12 p40 and γ-IFN, after 48h of incubation. In addition, the production of MIP-1 beta (CCL4), a chemokine involved in chronic inflammation, was also reduced after 120h. A specific effect of AC 30cH was seen by the inhibition of two peaks of CCL2 (MCP-1) observed in infected macrophages, at 24 and 120h. Since this cytokine is an important chemokine for monocytes, it explains the results obtained formerly in vivo. The morphology of macrophages after acridine orange staining revealed that the treatment with AC 30cH reduced substantially the acid vacuoles in the cytoplasm, indicating a certain inability of these cells to digest the parasites. On the other hand, a large peak of VEGF-A, associated with increase of internalized parasites was observed after 120h of treatment with AC 200cH, which could be associated to the regulation of the chronic inflammation events by M1-M2 polarization. There was no statistical difference among groups regarding the production of TNF, NO and H2O2, showing that the drugs do not alter macrophage cytotoxic activity. A clear quantitative and qualitative variation of the modulatory effects of AC 30cH and 200cH was seen, in function of time. Conclusions: Both dilutions were able to potentiate the decrease of most of cytokines and chemokines induced by the parasite infection in vitro, which explains the clinical improvement seen previously in vivo, however, the mechanisms involved and the epidemiological significance of these findings are still under discussion.
Article
Aim: To evaluate the effects of Kalium causticum, Conium maculatum, and Lycopodium clavatum 13cH in mice infected by Trypanosoma cruzi. Materials and methods: In a blind, controlled, randomized study, 102 male Swiss mice, 8 weeks old, were inoculated with 1400 trypomastigotes of the Y strain of T. cruzi and distributed into the following groups: CI (treated with 7% hydroalcoholic solution), Ca (treated with Kalium causticum 13cH), Co (treated with Conium maculatum 13cH), and Ly (treated with Lycopodium clavatum 13cH). The treatments were performed 48 h before and 48, 96, and 144 h after infection. The medication was repertorized and prepared in 13cH, according to Brazilian Homeopathic Pharmacopoeia. The following parameters were evaluated: infectivity, prepatent period, parasitemia peak, total parasitemia, tissue tropism, inflammatory infiltrate, and survival. Statistical analysis was conduced considering 5% of significance. Results: The prepatent period was greater in the Ly group than in the CI group (p = 0.02). The number of trypomastigotes on the 8th day after infection was lower in the Ca group than in the CI group (p < 0.05). Total parasitemia was significantly lower in the Ca, Co, and Ly groups than in the CI group. On the 12th day after infection, the Ca, Co, and Ly groups had fewer nests and amastigotes/nest in the heart than the CI group (p < 0.05). Decreases in the number of nests and amastigotes in the intestine were observed in the Ly group compared with the CI group (p < 0.05). In the liver (day 12), Ly significantly prevented the formation of inflammatory foci compared with the other groups. In skeletal muscle, Co and Ly decreased the formation of inflammatory foci compared with CI (p < 0.05). Ly afforded greater animal survival compared with CI, Ca, and Co (p < 0.05). The animals in the Co group died prematurely compared with the CI group (p = 0.03). Conclusions: Ly with 13cH potency had significantly more benefits in the treatment of mice infected with T. cruzi, reducing the number of blood parasites, amastigote nests in tissue, and the number of amastigotes per nest and increasing animal survival.
Article
Background Leishmaniasis is a zoonotic disease caused by protozoan parasites of the mononuclear phagocytic system. The modulation activity of these cells can interfere in the host/parasite relationship and influences the prognosis. Methods We evaluated the effects of the homeopathic preparation Antimonium crudum 30cH on experimental infection induced by Leishmania (L.) amazonensis. Male Balb/c mice were inoculated with 2 × 106Leishmania (L.) amazonensis promastigotes into the footpad and, after 48 h (acute phase) or 60 days (chronic phase), cell population of lymphocytes and phagocytes present in the peritoneal washing fluid and spleen were analyzed by flow cytometry and histopathology, with histometry of the subcutaneous primary lesion, local lymph node and spleen. Immunohistochemistry was performed to quantify CD3 (T lymphocyte), CD45RA (B lymphocyte) and CD11b (phagocytes) positive cells. Results In treated mice, during the acute phase, there was significant increase of the macroscopic lesion, associated to inflammatory edema, as well increase in the number of free amastigotes and B lymphocytes inside the lesion. Increase of B lymphocytes (predominantly B-2 cells) was also seen in the local lymph node, spleen and peritoneum. In the chronic phase, the inflammatory process in the infection focus was reduced, with reduced phagocyte migration and peritoneal increase of B-1a cells (precursors of B-2 immunoglobulin producers cells) and T CD8+ cells. Conclusion The treatment of mice with Antimonium crudum 30cH induced a predominantly B cell pattern of immune response in Leishmania (L.) amazonensis experimental infection, alongside the increase of free amastigote forms number in the infection site. The clinical significance of this study is discussed, further studies are suggested.
Homeopathy and tropical diseases: exploring the effects of homeopathy on murine infection with Trypanosoma cruzi
  • F N Ferraz
  • L Ciupa
  • D L Aleixo
  • S M Araújo
Ferraz FN, Ciupa L, Aleixo DL, Araújo SM. Homeopathy and tropical diseases: exploring the effects of homeopathy on murine infection with Trypanosoma cruzi. In: Bonamin LV, Waisse S, editors. Transdisciplinarity and Translationality in High Dilution Research. Signals and Images GIRI Series. Cambridge Scholars Publishing; 2019193-210.
Farmacopéia Homeopática Brasileira, 3a
  • Anvisa Brasil
BRASIL, ANVISA. Farmacopéia Homeopática Brasileira, 3a. ed 2011, http://portal.anvisa.gov.br/documents/33832/259147/ 3a_edicao.pdf/cb9d5888-6b7c-447b-be3c-af51aaae7ea8.
Homeopathy and tropical diseases: exploring the effects of homeopathy on murine infection with Trypanosoma cruzi
  • Ferraz
Searching for the mechanisms involved in Antimonium crudum action on macrophage - Leishmania interaction in vitro. Abstract of conference presentation. Proceedings of the XXXI GIRI Meeting, 2017. Krakow, Poland
  • Bonamin