Amarnath Sen
40 Jadunath Sarbovouma Lane, Kolkata 700035, India
E-mail: amarns2@yahoo.co.in
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Abstract
The absence of any drug in the ultra-diluted homeopathic medicines coupled with unfavourable
clinical trial results has painted homeopathic remedies as placebos. Different mechanisms have
been forwarded to explain the anomalies but with little success. Here it is proposed that
homeopathy is a form of protein-based immunotherapy and the immunogenic proteins exist in
the microbial lysates, which are present in the homeopathic medicines. The microbial lysates
are formed in the homeopathic medicines during their preparation, when microbes from the
surrounding environment are unwittingly incorporated into the homeopathic medicines and the
microbial cell lysis is induced by alcohol, a component of the drug vehicle (water-alcohol
mixture), and augmented by powerful shaking. The drugs in the homeopathic medicines
modulate the conformations and, in essence, the immunogenicity of the proteins present in the
medicines. The modulated proteins act as immunostimulants and help in boosting and tuning
both innate and adaptive immunity. In addition, bystander T cell activation and trained
immunity are expected to play important roles in the therapeutic and prophylactic actions of
the homeopathic medicines. The importance of dilution in homeopathy vis-à-vis the ‘law of
infinitesimals’ can be appreciated by considering the effect of dilution on protein folding and
the immunogenicity of proteins. In the case of ultra-diluted homeopathic medicines devoid of
any drug molecule, it has been suggested that in the absence of drug-protein interaction,
protein-protein interaction leads to the conformational modulation of protein molecules, where
allosteric communication and synchronization of vibrating of the protein molecules play key
roles. The dictum ‘like cures like’ can be understood by considering the mimicry between the
antigens present on the invading pathogen and the antigens present on the proteins in the
selected homeopathic medicine. The discrepancies in the clinical trial results of homeopathic
medicines arising from the heterogeneities inherent in immunotherapy as well as from a strong
placebo response in the clinical trials in some diseases may partly be mitigated by conducting
modified clinical trials.
Keywords: Homeopathy, Dilution, Ultra-diluted, Immunotherapy, Microbial lysates, Bacterial
lysates, Proteins, Immunostimulants, Immunomodulants.
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1. Introduction
Two major issues plague homeopathy and portray it as a fringe science. First, as dictated by
the Avogadro’s number (6.023 x 1023), above a particular dilution (12C or 24X, equivalent
dilution 1 in 1024, C and X are the centesimal and decimal scales of dilution, respectively), the
probability of getting a drug molecule in the diluted medicines goes down dramatically and at
dilutions of 30C (1 in 1060) and above, the probability turns out to be vanishingly small and,
for all practical purposes, equals zero [1-3]. Accordingly, homeopathic medicines diluted
beyond the so-called ‘Avogadro’s limit’ (designated as ultra-diluted) should be devoid of any
drug molecule and, hence, unlikely to cure any disease. In order to solve the so-called ‘mystery’
of the ultra-diluted homeopathic medicines, different mechanisms [1-3] like persistent memory
of water, quantum entanglement, quantum coherence domains, water-ethanol cluster, glass
derived silica nanoparticles and nano bubbles have been proposed, which, so far, failed to get
any favourable response from the mainstream scientific community. Incidentally, among the
different models, ‘water memory’ has been in the limelight for more than three decades after
Benveniste’s team [4] claimed that basophils can show immune response in ultra-diluted
antibody solutions and proposed a kind of memory of the antibodies imprinted in water to
explain the unusual observation. However, a later study [5] failed to replicate the results.
Nevertheless, it is believed by many that during the preparation of homeopathic medicines by
‘attenuation’, the homeopathic pharmaceutical vehicle (also called drug vehicle or simply,
vehicle) containing a mixture of water and alcohol, can retain the memory of the drug
molecules. ‘Attenuation’ [6] in homeopathic parlance is a process of serial dilution and
potentization (also known as dynamization) of the drug in the water-alcohol vehicle (insoluble
substances are initially diluted and potentized in lactose powder by trituration). The
potentization is done in each step of dilution by vigorously shaking the water-alcohol mixture
containing the drug. Given the fact that the hydrogen bonds in water break and reform very
fast (lifetime of the order of 1 picosecond [7]), it is an open question how water can retain any
form of memory for a reasonable period of time. Also, it remains unexplained how
homeopathic medicines dispensed as a few drops of liquid on lactose/sucrose globules or
powder retain their medicinal properties, when the globules/powder become dry and bereft of
water or alcohol molecules, which are supposed to store the memory of the drug.
Second, the unfavourable clinical trial results of homeopathic medicines have further
trivialized homeopathy. Although some well-controlled studies [8-12] found weak therapeutic
effects of homeopathic medicines, a meta-analysis [13] and two other larger studies by the
Australian National Health and Medical Research Council [14] and European Academies’
Science Advisory Council [15] failed to get any effect of homeopathic medicines beyond
placebo.
Against such a backdrop, it is proposed that homeopathy is a form of protein-based oral
immunotherapy, where the immunogenic proteins are obtained from the microbial lysates
formed in the homeopathic medicines during their preparation. The proposition accounts for
the origin of the microbial lysates in the homeopathic medicines, the mechanism of action of
homeopathic medicines based on immunotherapy, the ‘law of infinitesimals’ in the context of
dilution, the so-called ‘mystery’ of the ultra-diluted homeopathic medicines and the ‘law of
similars’ (‘like cures like’). In addition, the proposition explains why the clinical trials
apparently fail to show any effect of homeopathic medicines beyond placebo.
2. What are microbial lysates and how are they related to the mucosal immune system?
Microbial lysates or extracts (mostly polyvalent containing extracts of multiple species) are the
products of bacterial and fungal cell lysis (cell disruption). Bacterial lysates in particular have
been studied over several decades and are being used as potent immunostimulators/
immunomodulators for enhancing both innate and adaptive immunity [16-22]. Bacterial lysates
are generally prepared by chemical (mainly alkaline cell lysis) or mechanical (lysis by high
pressure shear or in bead mills) methods and they demonstrate therapeutic and prophylactic
effects in the treatment of upper and lower respiratory tract infections (RTI) in both adults and
paediatric patients [17-20]. In addition to their predominant use in the treatment of respiratory
tract disorders like asthma, COPD and rhinitis, bacterial lysates have shown encouraging
results in the treatment of various diseases like diverticulitis, rheumatoid arthritis, type 1
diabetes, inflammatory bowel disease, urinary tract infections, periodontitis, relapse of
tuberculosis and food allergy [16-22]. Bacterial lysates are also effective against fungal and
viral infections [23-26]. Interestingly, fungal lysates like bacterial lysates contain many
immunogenic proteins, though a few studies on their role as immunostimulants have been
reported so far [23, 24, 27].
Bacterial lysates contain bacterial components like proteins, lipopolysaccharides,
peptidoglycans, ribosomes and nucleic acids [17-20], where proteins are the major component
[28]. Given the size and structural complexity, proteins are very potent [29] antigens and
immunogens too (immunogens are antigens, which elicit immune response after binding with
the receptors). Bacterial lysates are normally administered through oral, sublingual or
intranasal routes. When antigens come in contact with the epithelial surfaces and mucosa,
mucosal immune system [30-32] enters the picture and it practically works independently of
the systemic immune system, though they bear many similarities. The inductive sites, where
the immune response is initiated, are the organized aggregates of the lymphoid tissue (mucosaassociated lymphoid tissue, MALT) like tonsils, Peyer’s patches in the small intestine
and appendix.
Interestingly, the immune response activated by bacterial lysates is almost similar to the natural
immune response provoked by pathogens [19, 20]. When pathogens come in contact with the
mucosa, as the first manifestation of mucosal innate immunity, pattern recognition receptors
(PRRs) such as toll like receptors (TLRs) expressed on the host dendritic cells (DCs),
macrophages, monocytes, and mast cells recognize the pathogen assisted molecular patterns
(PAMPs), which are conserved molecular structures of the pathogens. PAMPs can be surface
proteins, polysaccharides, peptidoglycans and nucleic acids [33], and they differ a little from
one pathogen to another but are absent in the host. The PRR-PAMP interaction activates the
phagocytic macrophages, DCs, polymorphonuclear leukocytes (neutrophils, eosinophils,
basophils, and mast cells) and natural killer (NK) cells and promotes mitogen-activated protein
kinase (MAPK) / nuclear factor kappa-B (NF-kβ) signalling pathway leading to the production
of pro-inflammatory chemokines, cytokines and interferons, which are essential for microbial
killing. The antigen presenting cells (APCs) comprising macrophages and DCs present the
fragments of the foreign antigens (specifically, epitopes or antigenic determinants, which are
small components of the antigens that bind to proper receptors) along with major
histocompatibility complex II (MHC II) molecules on their surface to the T cells followed by
their activation. The cell adhesion molecules like integrins and co-stimulatory surface
molecules like CD 28 (Cluster of Differentiation 28), CD 80 and CD 86 (present on both the
APCs and T cells) also play important roles in the activation of T cells. Interestingly, the
lymphocytes activated by an antigen at a mucosal site migrate to other mucosa to protect all
the mucosal tissues against the same antigen generating the concept of common mucosal
immune system (CMIS) [32]. There are also a group of immune cells called innate lymphoid
cells (ILCs) including NK cells, which are a kind of innate counterpart of the adaptive T
lymphocytes but lacking antigen receptors and hence, devoid of specificity [34]. The ILCs are
present abundantly at the mucosal sites and they respond quickly by sensing any change in
cytokine microenvironment during pathogen attack and produce cytokines to clear the
pathogens and repair the tissue damage. In addition, host defence peptides (antimicrobial
peptides) [35] like defensins and cathelicidins are found in the mucus secretions from the
epithelial cells and granulocytes, which show antimicrobial and immunomodulatory effects.
3. How do microbial lysates work in preventing and alleviating different diseases?
Microbial lysates being a mixture of bacterial and fungal lysates are expected to show
immunostimulating/immunomodulating actions similar to those of bacterial lysates [16-22, 25,
26] augmented by a contribution from the fungal lysates, though, as stated earlier, a little is
known about the immunomodulating characteristics of fungal lysates [23, 24]. Most of the
studies on bacterial lysates are limited to their prophylactic use [16-22, 25, 26], though they
are quite effective in the management of acute episodes too [36, 37]. Bacterial lysates
strengthen both innate and adaptive immunity [16-22]. They promote broad-spectrum nonspecific innate immune response by activating the macrophages and DCs. They enhance the
formation of adhesion molecules, antimicrobial peptides and recruit neutrophils in sufficient
numbers. The lysates augment the expression of MHC II and co-stimulatory molecules like
CD40 and CD86. In addition, by activating the DCs and macrophages, bacterial lysates trigger
an increased production of pro-inflammatory cytokines like IL-1 (interleukin 1), IL-2, IL-6,
IL-8, IL-12, TNF-α (tumour necrosis factor α), IFN-γ (interferon γ), IFN-β and IFN-α, whereas
the levels of anti-inflammatory cytokines like IL-4 and IL-13 are reduced [16-22]. Also,
depending on the cytokine microenvironment, if the inflammatory tissue damage becomes
inordinate, bacterial lysates promote the synthesis of anti-inflammatory cytokines like IL-6 (IL-
6 exhibits both anti-inflammatory and pro-inflammatory activities depending on its amount and
the signalling pathway [38]), IL-1 (a cytokine of IL-6 family and can exhibit both anti- and
pro-inflammatory behaviour), IL-10 and transforming growth factor beta (TGF-β).
Bacterial lysates regulate the adaptive immunity (antigen-specific response) by boosting the
activation of the T and B cells. The CD8+ T cells after getting the signals from the antigen and
co-stimulatory molecules are activated to become cytotoxic T lymphocytes (CTLs), which kill
the infected cells and the CD4+ T cells, depending on the cytokine environment, become
different helper T (Th) cells and regulatory T (Treg) cells. Among the different types of Th
cells, Th1 and Th17 cells primarily secrete pro-inflammatory cytokines and Th2 cells secrete
anti-inflammatory cytokines [39]. It has been reported that bacterial lysates specifically
strengthen the Th1 immune response and help in maintaining the Th1/Th2 balance. The lysates
also recruit Tregs [19, 20], which secrete anti-inflammatory cytokines and are responsible for
homeostasis as well as immune suppression and tolerance.
Concurrently, the Th2 cells stimulate the naïve B cells and the latter, after maturing into
antibody-secreting plasma cells, secrete antigen-specific antibodies (immunoglobin, Ig).
Antibodies are present in the blood, mucus secretions, and breast milk and they bind pathogens
and kill them mainly by opsonization (enhanced phagocytosis by marking antigens with
opsonins, specialized biomolecules, which are mostly extracellular proteins). Antibody class
switching (switching the production of one type of immunoglobin to another) in mature B cells
depends on the nature of antigen stimulation, co-stimulatory signals and cytokine
microenvironment [40]. Bacterial lysates have an important role in immunoglobulin synthesis,
and class switching of IgM to IgA, IgG, and IgE [19, 20]. The secretory immunoglobin A
(SIgA) is a dimeric/polymeric form of IgA, which is found in large amounts in tear, saliva, and
in the mucus secretions in the respiratory epithelium and GI tract. The main function of SIgA
is to defend the host against pathogens by blocking the adhesion of the pathogens to the
mucosal surfaces [41]. Bacterial lysates increase both IgM (mainly responsible for
opsonization of antigens) and SIgA and lower IgE (associated with allergic and autoimmune
diseases) and eosinophil counts [19, 20].
In addition, it has been proposed that bystander T cells play an important role in bacteriallysate-based immunotherapy [42]. In contrast to conventional T-cell-receptor-dependent
(TCR-dependent), antigen-specific activation of T cells, bystander activation is a non-specific,
TCR-independent activation of T cells (including neighbouring heterologous T cells). The
bystander activation is triggered by inflammatory cytokines during the activation of antigenspecific T cells [43, 44]. Even in the absence of any antigenic stimulation, the bystanderactivated memory T cells respond swiftly to microbial attacks by secreting pro-inflammatory
cytokines so that the spread of the pathogens is restrained. However, unregulated activation of
bystander T cells can lead to inflammatory diseases in hosts, where Tregs are expected to
contain it [43, 44]. Indeed, in the case of allergen-specific immunotherapy, it has been
suggested that Treg-induced bystander suppression [43] in the presence of inhibitory cytokines
like TGF-β and IL-10 helps in curbing exaggerated inflammation and tissue damage in asthma.
It is also believed that bacterial lysates via trained immunity provide a broad-spectrum crossprotection against various pathogens (heterologous protection) [45, 46]. Trained immunity can
be understood as a non-specific memory (duration of several weeks to months) of the innate
immune system, where a stimulus from various agents [45-47] like pathogens, live vaccines,
microbial extracts, β-glucan and lipopolysaccharide modifies the immune system in such a way
that a robust immune response is obtained towards future challenges. Interestingly, trained
immunity provides protection against not only the initial mediator, but also other unrelated
agents/pathogens including bacteria, fungi, viruses and parasites (heterologous immunity).
Such trained immunity is achieved by epigenetic and metabolic modifications
(reprogramming) of the bone marrow myeloid cells, innate immune cells like monocytes,
macrophages, ILCs and even non-immune cells like stem cells and microglial cells.
In the present context, it may be mentioned that lesser-known autologous bacterial vaccines
bear some resemblance to bacterial lysates. Autologous/autogenous vaccines (also called
autovaccines) are personalized vaccines and are prepared from the cultures of pathogens
collected from individual patients followed by inactivation of the pathogens and subsequent
administration (by oral or subcutaneous route) to the respective patients so that each patient
gets the vaccine prepared from the pathogen which has inflicted disease on that patient [48,
49]. It has been observed that autovaccines are useful in both preventing and curing some
specific diseases, though a little is known about their mechanism of action. So far, it has been
found from limited studies that autovaccines downregulate Th1 lymphocytes and enhance the
production of IgG and IgM [48, 49].
4. What is the origin of microbial lysates in homeopathic medicines?
Bioaerosols are ubiquitous in the atmosphere and in normal conditions, the bacterial and fungal
counts reach 104 and 103 cells m-3, respectively, and there are seasonal variations too [50, 51].
Generally, homeopathic medicines are prepared in clean rooms (mostly, low-end clean rooms
like class 100, equivalent ISO 5) [52]. In the clean rooms, though the microbial population is
lower than that of the surrounding environment, bacteria, fungi and their spores are found in
the air, in the water (even in distilled water [53, 54]) and on the room/equipment surfaces. On
top of everything, human bodies are a perennial source of microbial contamination [53]. During
the preparation of homeopathic medicines, the microbes from the aforesaid sources in the clean
rooms are unknowingly included in the drug vehicle in each step of dilution and potentization.
Additionally, a lot of air bubbles are entrapped during vigorous shaking at each step of dilution
leading to an enhanced capturing of the microbes by the drug vehicle from the entrapped air.
The alcohol in the drug vehicle then lyses the microbial cells by fluidizing their membranes
[55] and vigorous shaking augments the disruption of the microbial cells (mechanical lysis
[17]). Hence, besides drug, the homeopathic drug vehicle contains microbial lysates
comprising proteins, protein fragments, nucleic acids, polysaccharides, peptidoglycans etc.
(Sec.2), and as mentioned earlier, the major constituent of the lysates is proteins [28].
Alcohol (ethanol), a component of the drug vehicle, can denature proteins (partially or
completely, depending on the concentration of alcohol and the characteristics of the proteins)
by disrupting their tertiary structure [56]. Even vigorous shaking denatures proteins [57].
Hence, the proteins in the homeopathic medicines should be present in the denatured and/or
partially denatured form(s). Normally, proteins remain in a stable folded three-dimensional
state (native form) and proper folding is necessary for their functionality [28, 29]. Denatured
proteins are unfolded proteins devoid of three-dimensional (3D) structure and functionality.
Proteins can be denatured by heat, radiation, sonication, acids, alkalis, oxidizing/reducing
agents etc. [58]. However, proteins are immunogenic in both native and denatured forms [29,
59] and the immunogenicity of proteins is determined by their conformations (shapes) as some
epitopes may be masked and some exposed when the conformations change [60]. The process
of denaturation/partial denaturation changes the conformation [58] and hence, the
immunogenicity of the proteins.
5. How do homeopathic medicines work and what is the specific role of the drugs?
Homeopathic medicines containing microbial lysates should act as potent
immunostimulants/immunomodulants like bacterial lysates [16-22, 25, 26] and fungal lysates
[23, 24]. Accordingly, homeopathic medicines are supposed to stimulate the innate and
adaptive immunity and the mechanism of action of homeopathic medicines should resemble
that of bacterial lysates (mechanism of action of fungal lysates is almost unexplored). Also, in
a similar way to bacterial lysates, bystander activation and trained immunity are likely to play
important roles in the prophylactic and therapeutic actions of homeopathic medicines. Hence,
irrespective of the presence of any drug in the medicine, microbial lysates present in the
homeopathic medicines should act as oral immunostimulant. Indeed, potentized ethyl alcohol
in the absence of any drug has been found to enhance the immune response in murine infection
with Trypanosoma cruzi [61].
In order to understand the specific role of drugs (different inorganic and organic substances) in
homeopathic medicines, the energy landscape of proteins should be taken into consideration.
The free energy landscape of proteins is in the form of a funnel [62, 63], where the X-axis is
the configurational entropy and the Y-axis is the free energy of the conformational states of the
protein. Such energy landscape funnel is used to describe the folding behaviour of proteins and
hence, it is also called ‘folding funnel’. In the energy landscape, the denatured states with high
entropy are located at the top of the funnel, native folded states with low entropy are at the
narrow end of the bottom and partially denatured states lie around the middle of the funnel [62-
64]. The energy landscapes are not smooth and contain many substates and, accordingly, the
thermal energy kBT (kB is the Boltzmann constant and T is the ambient temperature) drives the
protein molecules to shuffle the energy substates, provided the energy barriers do not exceed
kBT. As the different substates have different configurational entropy, the conformations of
protein molecules also fluctuate and, consequently, proteins vibrate (breathing mode
vibration). Hence, protein molecules need to be understood as dynamic ensembles of
conformational states having many conformational isomers, where the dominant or ensembleaveraged conformations correspond to the conformations at the local minima on the protein
energy landscape. As denatured proteins with high entropy lie at the top of the funnel, where
the width (conformational entropy) is maximum, they have a diverse set of conformational
isomers with varying shapes [64] and they are more susceptible to perturbations in comparison
to folded native proteins [65]. Hence, denatured (and partially denatured proteins) can assume
many conformations and therefore, can show a varied immunogenicity. The drugs in the
homeopathic medicines take advantage of the conformational flexibility of the
denatured/partially denatured proteins and modulate their conformations in different ways,
given the fact that proteins, because of their large, complex and multi-reactive structures, can
easily interact with a variety of substances [66-69]. As the immunogenicity of proteins is
sensitive to subtle changes in their conformation [60], effectively, a diverse set of homeopathic
medicines with a wide variation in their immunogenicity is obtained by tuning the modulation
of the proteins using different drugs.
6. Why is the ‘law of infinitesimals’ so important in homeopathy?
In homeopathy, the ‘law of infinitesimals’ states that the more is the dilution of the drug, the
more is the potency of the medicine. In order to understand the importance of dilution in
homeopathic medicines, it is necessary to know how the immunogenicity of proteins changes
with unfolding [29, 70, 71]. In the folded state, proteins have conformational B-cell epitopes,
which are recognized by the B cells and antibodies. However, in comparison to the B-cell
epitopes, the T-cell epitopes in the folded state are less abundant and, as a result, B-cell
activation is dampened because of weak support from the T cells [70]. On the other hand,
unfolded proteins (denatured proteins) have linear epitopes recognized by the T cells but the
conformational B-cell epitopes are nearly absent in the denatured proteins, which compromises
the antibody-mediated immune response [29, 70]. Hence, it is apparent that both native and
denatured proteins may not be very immunogenic. On the other hand, partially denatured
proteins can show very strong immunogenicity because of their heightened T-cell and B-cell
activities [70].
It is known that some chemicals, even in low concentrations (order of 1 ppm), can denature
proteins leading to loss of their immunogenicity [72]. In fact, different substances are used as
drugs in homeopathic medicines and, as discussed earlier (Sec.5), they are expected to
modulate the conformations of the protein molecules and, in effect, their immunogenicity.
However, as proteins are very sensitive to many chemicals [66-69, 72], the concentration of
the drugs in the medicines during the initial stages of preparation of homeopathic medicines
can be high enough to modulate the protein molecules inordinately resulting in the degradation
or loss of their immunogenicity. After adequate dilution in subsequent stages, when the
concentration of the drugs in the medicines goes below certain levels, the unfolded proteins
(denatured proteins) begin to refold and gain immunogenicity, provided, the denaturation is
not irreversible. It is now evident that i) without sufficient dilution of the drugs, the proteins
lose their immunogenicity leading to the degradation of the therapeutic potential of the
homeopathic medicines and ii) with increasing dilution, as the protein folding increases, the
immunogenicity of the partially denatured proteins and, correspondingly, the potency of the
homeopathic medicines increases, which substantiates the ‘law of infinitesimals’. However,
such increase in the immunogenicity of the proteins with increasing dilution should be valid
until the dilutions exceed certain levels, when the refolded states of the proteins approach their
native folded states having immunogenicity less than that of the partially folded (partially
denatured) proteins. The desired level of unfolding (partial denaturation) to get the optimum
immunogenicity should be protein-specific [70] as well as drug-specific and, above all, the
optimum immunogenicity should depend on the concentration of the drugs and, therefore, the
level of dilution of the drugs in the homeopathic medicines.
7. What happens in ultra-diluted homeopathic medicines having no drug molecule?
Ultra-diluted homeopathic medicines devoid of any drug molecule still remain an enigma. In
order to delve into it, a homeopathic medicine of dilution near the ‘Avogadro’s limit’ may be
chosen (the chosen medicine is designated as ‘chosen dilution’) for subsequent discussion. It
is assumed that the drug molecules present in the ‘chosen dilution’ is insufficient to modulate
the conformations of all the protein molecules present in the drug vehicle. Such situation should
arise at some stage of dilution during the preparation of homeopathic medicines because with
dilution, though the number of both protein and drug molecules goes on decreasing, the deficit
of the former is replenished by the protein molecules obtained from the freshly formed
microbial lysates in each step of dilution and potentization. Hence, given the paucity of the
drug molecules, the ‘chosen dilution’ should contain a mixture of drug-modulated and
unmodulated protein molecules. In the present context, it would be of interest to know how the
drug-modulated and unmodulated protein molecules interact between themselves in the drug
vehicle (water-alcohol mixture). Protein-protein interaction normally takes place in aqueous
media, when the protein molecules are in contact with each other, at least, through their
solvation shells [73, 74]. As during the preparation of homeopathic medicines, the inclusion of
microbes in the homeopathic drug vehicle from the ambient environment is accidental and not
by design, the amount of the microbial lysates formed and, therefore, the number of protein
molecules present in the drug vehicle may not be large. Hence, all the protein molecules in the
homeopathic drug vehicle may not be in contact with each other, which should restrict their
interactions. Intriguingly, a study on biopolymers revealed [75] that solvent-mediated
interactions among the biopolymer molecules are possible even when they are not in direct
contact with each other. Hence, solvent-mediated protein-protein interaction is conceivable and
can be understood by invoking allostery as described below.
Allostery [76-78] is a phenomenon where perturbations originated from an event like binding
of ligands, change in temperature, absorption of light, change in pH, mutation, interactions
with proteins and reaction with small molecules at specific sites of protein molecules (also,
DNA and RNA molecules), excluding fibrous proteins, regulate the functional states at the
distant sites. The perturbations are transmitted to the distant sites through allosteric networks
(generally, networks of connected residues or clusters of residues, where a residue is an amino
acid unit in a chain of amino acids in the protein molecules) as propagating
structural/conformational fluctuations [76-78]. Allosteric signals are not restricted within a
single protein molecule and can extend over a long distance across multiple protein-protein
linkages. Indeed, allosteric signals are ubiquitous and are associated with cell signalling,
enzyme catalysis, cooperativity etc. In the present context, it is interesting to note that thermalfluctuation-induced conformational fluctuations can give rise to a long-range allosteric
signalling in proteins [79]. As discussed earlier (Sec.5), the conformational fluctuations in
protein molecules generate low-frequency breathing modes of the order of 1MHz (may be
contrasted with the frequency of side-chain fluctuations in protein molecules ~ 1 GHz) [80].
When the protein or the hydrated protein molecules are in contact with each other, the
perturbations (vibrations of the protein molecules) are transmitted through the allosteric
networks, given the fact that allosteric signals can be transmitted through the networks
containing water molecules [74]. In case the protein molecules are insufficient to make direct
contacts with each other, it is argued that the perturbations can still be transmitted through the
medium as the attenuation(loss) coefficient of acoustic wave of 1MHz frequency in water is
extremely low (0.0022 dB/cm, in contrast to 12.0 dB/cm for air [81]). Hence, in the ‘chosen
dilution’, the perturbations (vibrations) generated from each protein molecule are allosterically
transmitted to other protein molecules present in the medium (water-alcohol mixture). As a
result, each vibrating protein molecule receives weak periodic forces from the transmitted
vibrations originated from other protein molecules. Such interactions among the vibrating
protein molecules lead to vibration synchronization and the latter should be facile for oscillators
having natural frequencies of vibration close to each other [82, 83]. In the case of ‘chosen
dilution’, the unmodulated and drug-modulated protein molecules of the same family (of
related conformation or shape) satisfy the aforesaid conditions leading to synchronization of
their vibrations. It is known that the synchronization frequency of two coupled oscillators lies
between their natural frequencies and in an ensemble of coupled oscillators, the
synchronization frequency depends on the distribution of the natural frequencies of the
oscillators, coupling strength etc. [82]. Therefore, the synchronization frequency of the
unmodulated and drug-modulated protein molecules in the ‘chosen dilution’ should be
intermediate between the natural frequencies of the unmodulated and drug-modulated protein
molecules. As the vibration frequency, which is the frequency of conformational fluctuation,
and the conformation of the protein molecules are interrelated, protein molecules of
conformation intermediate between the conformations of the unmodulated and drug-modulated
protein molecules should be realized after synchronization. Interestingly, the intermediate
conformation acquired by the unmodulated protein molecules suggests that they are indeed
modulated through protein-protein interaction, i.e., through the interaction between the
unmodulated protein and the drug-modulated protein molecules, followed by vibration
synchronization. The proteins modulated through protein-protein interaction may be called
protein-modulated proteins and it is worthy of note that the acquired modulation of the proteinmodulated protein molecules is partial in comparison to the modulation of the drug-modulated
protein molecules as the protein-modulated protein molecules get an intermediate
conformation through protein-protein interaction. Eventually, the ‘chosen dilution’ is expected
to contain both drug-modulated and protein-modulated protein molecules but no unmodulated
protein molecule.
From the above discussion it is now evident that even in the absence of any drug in the
homeopathic medicines, the unmodulated proteins can be modulated through protein-protein
interaction, followed by vibration synchronization leading to the generation of proteinmodulated proteins. It may be noted that the protein-modulated proteins can be proteins
modulated by the ‘drug’-modulated proteins (as happened in the case of ‘chosen dilution’ as
described above) and in the later stages of dilution, they can be proteins modulated by the
‘drug-modulated-protein’-modulated proteins and so on down the line as the dilution increases
and goes beyond the ‘Avogadro’s limit’. In reality, the protein-modulated proteins impart
medicinal properties to the ultra-diluted homeopathic medicines having no drug molecule,
given the fact that in each step of serial dilution and potentization during the preparation of
homeopathic medicines, the unmodulated protein molecules from the freshly formed microbial
lysates interact with and get modulated by the protein-modulated protein molecules available
from a portion of microbial lysates being transferred from the preceding dilution as per the
procedure of serial dilution. However, it should be noted that the rate of modulation of protein
molecules through protein-protein interaction decreases with increasing dilution steps, as the
modulation of protein molecules through protein-protein interaction in each step of dilution is
partial as discussed above. In essence, even in the absence of any drug molecule in the ultradiluted homeopathic medicines, the information of drug-induced conformational modulation
of protein molecules can partly be communicated from the modulated protein molecules to the
unmodulated protein molecules through their interaction (protein-protein interaction in the
drug vehicle) followed by vibration synchronization of the protein molecules. In the process,
the unmodulated protein molecules retain the information by getting themselves modulated and
during subsequent stages of dilution, they communicate the information in the same manner to
other unmodulated protein molecules.
8. How can the dictum ‘like cures like’ be valid in homeopathy?
The ‘law of similars’, also called ‘similia principle’ in homeopathy, states that ‘like cures like’,
and to appreciate the implication of the dictum, the method of ‘drug proving’ in homeopathy
should be understood first. In homeopathic ‘drug proving’ or pathogenetic trials [84],
homeopathic medicines (generally, in 12C potency) are administered repeatedly on healthy
volunteers as per a specified regimen [84], followed by observations and recording of the
symptoms experienced by the volunteers. The ‘drug-proving’ steps may be repeated after a
wash-out period of a few days, if the ‘drug picture’ (symptoms experienced by the volunteers
during ‘drug proving’) is not prominent. According to the dictum ‘like cures like’, a patient
can be cured by a specific homeopathic medicine if the ‘drug proving’ symptoms of the specific
medicine match the symptoms of the patient.
The manifestation of symptoms in the healthy volunteers during drug proving can be
understood as the immune response to repeated challenge by the immunogenic proteins present
in the homeopathic medicines. The importance of ‘similia principle’ lies in selecting specific
homeopathic medicines for boosting the adaptive immune response of the patients. In order to
generate a robust adaptive immune response from a homeopathic medicine, the specific
medicine should contain antigens from the pathogen, which has inflicted disease on the patient.
This is apparent from the studies on bacterial-lysate-based immunotherapies, where the lysates
for the treatment of respiratory tract infections and the urinary tract infections are made from
different sets of bacteria based on the bacteriological profiles of the diseases [18, 85], which is
reminiscent of autologous vaccines [Sec.3, refs 48, 49]. In the case of homeopathic medicines,
it is not known whether the antigens or the extracts of a specific pathogen exist in any
homeopathic medicine among a large number of medicines available for the treatment of the
patients. Here ‘similia principle’ is exploited to select the right remedy given the fact that if a
person suffers from a disease whose symptoms are similar to the ‘drug proving’ symptoms of
a particular homeopathic medicine, a mimicry between the antigens (precisely, the epitopes)
present on the disease producing pathogen and the antigens (epitopes) present on the proteins
in that homeopathic medicine is anticipated. Hence, if by matching symptoms homeopathic
medicines are prescribed, the selected homeopathic medicine is expected to contain epitopes
which are functionally equivalent to the epitopes of the invading pathogen. Consequently, an
enhanced adaptive immune response from the selected homeopathic medicine is obtained
though, in reality, the selected homeopathic medicine may not contain any antigen directly
from the invading pathogen. It may be noted that ‘mimicry’ in the present context is different
from the well-known ‘molecular mimicry’ or ‘antigenic mimicry’, which is the mimicry
between the foreign antigens and self-antigens (host antigens) leading to autoimmune diseases
under certain circumstances [86].
9. Why are the clinical trial results of homeopathic medicines controversial?
Homeopathy, as per the present proposition, is a form of immunotherapy and the clinical trial
results in immunotherapy in general are fraught with heterogeneities in data and replication
issues [87]. Such issues arise from different factors like variation in the immune response of
the patients as per their immune status, temporal variation in the immune status of the patients,
size and frequency of the dose, nature of the immunogens, method of preparation of the
immunogens, administration routes of the immunogens as well as age, sex, obesity, race and
gut microbiome of the patients [17-21, 87-89]. Also, proper selection of the patients (or subset
of patients) is important for the clinical trials in immunotherapy [90]. Hence, inconsistent
results in the clinical trials are not uncommon in immunotherapies and such inconsistencies
have been observed in allergen-specific immunotherapies for asthma and rhinitis [91] and
bacterial-lysate-based immunotherapies for respiratory tract disorders [17]. Homeopathy,
being a form of immunotherapy and related to bacterial-lysate-based immunotherapy, is
expected to encounter similar issues. The problems are further worsened in homeopathy
because of the fluctuations in the quality of homeopathic medicines arising from the variations
in the constituents and amounts of the microbial lysates present in the homeopathic medicines.
Such variations can arise from the spatial and temporal variations in the microbiome of the
rooms where the homeopathic medicines are prepared, given the fact that the microbes from
the ambient environment are unknowingly incorporated into the homeopathic medicines during
their preparation and, hence the nature and amount of the microbial contents in the homeopathic
medicines remain unregulated.
Though randomised controlled trials (RCTs) are considered the ‘gold standard’ of preventive,
diagnostic, and therapeutic interventions, the assumption of additivity [92, 93], where the drug
response is the sum of the pharmacological effect of the drug and placebo response, is being
debated in recent times. The placebo response in the clinical trials in some diseases [94] like
pain, depression, irritable bowel syndrome (IBS), osteoarthritis, ulcerative colitis, fatigue
(cancer related), cough, asthma and Parkinson’s disease can be substantially high and therefore,
can corrupt the clinical trial results, where the additivity rule of RCT breaks down leading to a
wrong evaluation of the efficacy of the drugs. Incidentally, a sizable fraction of patients visits
homeopathic clinics for the treatment of the aforementioned diseases, where clinical trials may
downplay the true efficacy of the homeopathic medicines because of high placebo response.
Hence, different strategies [95, 96] like enrichment and adaptive designs should be employed
in the clinical trials of homeopathic medicines.
10. Conclusion
Homeopathy can be understood as protein-based oral immunotherapy and the immunogenic
proteins are found in the homeopathic medicines as a component of microbial lysates, which
are present in the homeopathic medicines. The microbial lysates are formed during the
preparation of homeopathic medicines, where the microbes from the ambient environment are
unwittingly included in the homeopathic drug vehicle and, subsequently, lysed by alcohol
present in the drug vehicle under vigorous shaking. The drugs by modulating the conformations
of the protein molecules in various ways provide a wide range of homeopathic medicines with
varied immunogenicity. The significance of dilution and the associated ‘Law of infinitesimals’
can be appreciated by noting the fact that with increasing dilution, the immunogenicity of the
proteins can increase. The so-called ‘mystery’ of the ultra-diluted homeopathic medicines
devoid of any drug molecule may be solved by considering the fact that drug-protein interaction
is not essential for the modulation of protein molecules. The protein-protein interaction under
certain circumstances can partly play the role of drug-protein interaction in modulating the
protein molecules, where allosteric communication and synchronization of vibrations of
protein molecules are important actors. The similia principle (‘like cures like’) used for
selecting the proper medicine based on the similarities between the ‘drug proving’ symptoms
of the medicine and the patient’s symptoms can be accounted for by considering the mimicry
between the epitopes present on the invading pathogen and the epitopes present on the protein
molecules in the selected medicine. The inconsistencies in the clinical trial results of
homeopathic medicines arising from different sources like heterogeneities inherent in
immunotherapies, high placebo response in the clinical trials in some diseases and the
fluctuation in the characteristics of homeopathic medicines may partly be fixed by modifying
the methodology of the clinical trials. Finally, the proposed hypothesis should be put to the test
through rigorous experiments and the possible roles of other bacterial immunogens like
ribosomes, capsular polysaccharides, lipopolysaccharides and nucleic acids (in denatured
forms) as well as the role of fungal immunogens present in the homeopathic medicines should
be probed.
Declaration of Competing Interest
The author declares that he has no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Source of funding
Nil
References
1. Cukaci C, Freissmuth M, Mann C, Marti J, Sperl V. Against all odds – the persistent
popularity of homeopathy. Wien Klin Wochenschr. 2020; 132:232–242
2. Walach H, Jonas W B, Ives J, Van Wijk R, Weingartner O. Research on homeopathy:
State of the art. J Alternative Complementary Medicine. 2005; 11(5): 813–829
3. Bell I R, Koithan M. A model for homeopathic remedy effects: Low dose nanoparticles,
allostatic cross-adaptation, and time-dependent sensitization in a complex adaptive
system. BMC Complementary and Alternative Medicine. 2012; 12:191
4. Davenas E, Beauvais F, Amara J, Oberbaum M, Robinzon B, Miadonnai A, Tedeschi A,
Pomeranz B, Fortner P, Belon P, Sainte-Laudy J, Poitevin B, Benveniste J. Human basophil
degranulation triggered by very dilute antiserum against IgE. Nature. 1988; 333(6176):
816-8.
5. Hirst S J, Hayes N A, Burridge J, Pearce F L, Foreman J C. Human basophil degranulation
is not triggered by very dilute antiserum against human IgE. Nature. 1993; 366: 525–527
6. Homeopathic Pharmacopoeia Convention of the United States (HPCUS). Guidelines for
manufacturing homeopathic medicines. 2013, p 4
http://www.hpus.com/Draft-Guidelines-for-Manufacturing-Homeopathic-Medicines.pdf
7. Luzar A, Chandler D. Hydrogen-bond kinetics in liquid water. Nature.1996;
379(6560): 55-57
8. Linde K, Clausius N, Ramirez G, Melchart D, Eitel F, Hedges L V, Jonas W B. Are the
clinical effects of homoeopathy placebo effects? A meta-analysis of placebo-controlled
trials. Lancet. 1997; 350: 834–843
9. Kleijnen J, Knipschild P, Riet G. Clinical trials of homoeopathy. BMJ 1991; 302(6772):
316-23
10. Cucherat M, Haugh M C, Gooch M, Boissel J P. Evidence of clinical efficacy of
homeopathy. A meta-analysis of clinical trials. HMRAG Homeopathic Medicines
Research Advisory Group. Eur J Clin Pharmacol. 2000; 56(1): 27-33
11. Reilly D, Taylor M A, Beattie N G, Campbell J H, McSharry C, Aitchison T C, Carter
R, Stevenson R D. Is evidence for homoeopathy reproducible? Lancet. 1994; 344(8937):
1601-6
12. Mathie R T, Ramparsad N, Legg L A, Clausen J, Moss S, Davidson J R T, Messow C-M,
McConnachie A. Randomised, double-blind, placebo-controlled trials of nonindividualised homeopathic treatment: Systematic review and meta-analysis. Systematic
Reviews. 2017; 6: 63
13. Ernst E. A systematic review of systematic reviews of homeopathy. Br J Clin Pharmacol,
2002; 54(6): 577-82.
14. National Health and Medical Research Council. NHMRC information paper: Evidence
on the effectiveness of homeopathy for treating health conditions. Canberra, 2015
https://www.nhmrc.gov.au/about-us/resources/homeopathy
15. European Academies’ Science Advisory Council. Homeopathic products and practices:
assessing the evidence and ensuring consistency in regulating medical claims in the EU.
2017.
https://easac.eu/publications/details/homeopathic-products-and-practices/
16. Villa E, Garelli V, Braido F, Melioli G, Canonica G W. May we strengthen the human
natural defenses with bacterial lysates? WAO Journal, 2010; S17-S23
17. Suárez N, Ferrara F, Rial A, Dee V, Chabalgoity J A, Bacterial lysates as
immunotherapies for respiratory infections: Methods of preparation. Front in Bioeng and
Biotech, 2020; 8 :545
18. Rossi G A, Esposito S, Feleszko W, Melioli G, Olivieri D, Piacentini G, Scaglione F,
Vercelli D. Immunomodulation therapy – Clinical relevance of bacterial lysates OM-85.
European Respiratory & Pulmonary Diseases. 2019; 5(1): 17–23
19. Kaczynska A, Klosinska M, Janeczek K, Zarobkiewicz M, Emeryk A. Promising
immunomodulatory effects of bacterial lysates in allergic diseases. Frontiers in
Immunology, 2022; 13: 907149
20. Janeczek K, Kaczyńska A, Emeryk A, Cingi C. Perspectives for the use of bacterial
lysates for the treatment of allergic rhinitis: A systematic review. J Asthma Allergy 2022;
15: 839–850
21. Ahrens B, Quarcoo D, Buhner S, Matricardi P M, Hamelmann E. Oral administration of
bacterial lysates attenuates experimental food allergy. Int Arch Allergy Immunol. 2011;
156(2): 196-204.
22. Alyanakian M-A,Grela F, Aumeunier A, Chiavaroli C, Gouarin C, Bardel E, Normier G,
Chatenoud L, Thieblemont N, Bach J-F. Transforming growth factor and natural killer Tcells are involved in the protective effect of a bacterial extract on type 1 diabetes.
Diabetes, 2006; 55: 179-185
23. Rigg D, Miller M M, Metzger W J. Recurrent allergic vulvovaginitis: Treatment
with Candida albicans allergen immunotherapy. Am J Obstetrics Gynecology. 1990;
162(2): 332-336
24. Vrzal V, Bittner L, Nepereny J. Use of yeast lysate in women with recurrent
vulvovaginal candidiasis. Procedia in Vaccinology. 2015; 9: 35-37
25. Rossi G A, Pohunek P, Feleszko W, Ballarini S, Colin A A. Viral infections and
wheezing–asthma inception in childhood: Is there a role for immunomodulation by oral
bacterial lysates? Clin Transl Allergy 2020; 10: 17.
26. Roth M, Khameneh H J, Fang L, Tamm M, Rossi G A. Distinct antiviral properties of
two different bacterial lysates. Canadian Respiratory Journal. 2021; 2021: 8826645
27. Liu Y, Bastiaan-Net S, Wichers H J. Current understanding of the structure and
function of fungal immunomodulatory proteins. Frontiers in Nutrition, 2020; 7: 132
28. Milo R, Philips R. Cell biology by the numbers. 2015; Garland Science, p 106
29. Dennehy R, McClean S. Immunoproteomics: The key to discovery of new vaccine
antigens against bacterial respiratory infections. Current Protein and Peptide Science, 2012;
13: 807-815
30. McGhee J R, Fujihashi K. Inside the Mucosal Immune System. PLoS Biol 2012; 10:
e1001397
31. Jones R M, Neish A S. Recognition of bacterial pathogens and mucosal immunity
Cellular Microbiology. 201; 13(5): 670–676
32. Date Y, Ebisawa M, Fukuda S, Shima H, Obata Y, Takahashi D, Kato T, Hanazato
M, Nakato G, Williams I R, Hase K, Ohno H. NALT M cells are important for immune
induction for the common mucosal immune system. International Immunology, 2017;
29(10): 471-478
33. Li D, Wu M. Pattern recognition receptors in health and diseases. Signal Transduction
Targeted Therapy. 2021; 6: 291
34. Seo G-Y, Giles D A, Kronenberg M. The role of innate lymphoid cells in response to
microbes at mucosal surfaces. Mucosal Immunol, 2020;13(3): 399-412.
35. Tjabringa G S, Vos J B, Olthuis D, Ninaber D K, Rabe K F, Schalkwijk J, Hiemstra P S,
Zeeuwen P L J M. Host defense effector molecules in mucosal secretions. FEMS
Immunology and Medical Microbiology. 2005; 45: 151–158
36. Lanzilli G, Falchetti R, Tricarico M, Ungheri D, Fuggetta M P. In vitro effects of an
immunostimulating bacterial lysate on human lymphocyte function. International J
Immunopathology Pharmacology. 2005; 18 (2): 245-254
37. Tang Y, Zhao D, Sun W, Yang C. Clinical observation of bacterial lysates capsules in
the treatment of acute attack of asthma in children. China Pharmacy. 2017; 12: 4537- 4540
38. Borsini A, Di Benedetto M G, Giacobbe J, Pariante C M. Pro- and anti-Inflammatory
properties of interleukin (IL)6 in vitro: Relevance for major depression and human
hippocampal neurogenesis. Int J Neuropsychopharmacology. 2020; 23(11): 738–750
39. Su C, Yang T, Wu Z, Zhong J, Huang Y, Huang T, Zheng E. Differentiation of T-helper
cells in distinct phases of atopic dermatitis involves Th1/Th2 and Th17/Treg. Europ J
Inflammation. 2017; 15(1): 46–52
40. Stavnezer J. Immunoglobulin class switching. Curr Opin Immunol, 1996; 8(2): 199-205.
41. Corthésy B. Multi-faceted functions of secretory IgA at mucosal surfaces. Frontiers in
Immunology. 2013; 4:185
42. Bartkowiak‑Emeryk M, Emeryk A, Roliński J, Wawryk‑Gawda E, Markut‑Miotła E.
Impact of polyvalent mechanical bacterial lysate on lymphocyte number and activity in
asthmatic children: A randomized controlled trial. Allergy Asthma Clin Immunol 2021;
17: 10
43. Richardson N, Wraith D C. Advancement of antigen-specific immunotherapy:
knowledge transfer between allergy and autoimmunity. Immunotherapy Advances, 2021;
1(1): 1–16
44. Boyman O. Bystander activation of CD4+ T cells. Eur J Immunol. 2010; 40(4): 936-
939.
45. Brandi P, Conejero L, Cueto F J, Martı´nez-Cano S, Dunphy G, Go´mez M J, Relano C,
Saz-Leal P, Enamorado M, Quintas A, Dopazo A, Amores-Iniesta J, Fresno C, Nistal-Villa´ E,
Ardavı´n C, Nieto A, Casanovas M, Subiza J, Sancho D. Trained immunity induction by the
inactivated mucosal vaccine MV130 protects against experimental viral respiratory
infections. Cell Reports. 2022; 38: 110184
46. Salzmann M, Haider P, Kaun C, Brekalo M, Hartmann B, Lengheime Tr, Pichler R, Filip
T, Derdak S, Podesser B, Hengstenberg C, Speidl W S, Wojta J, Plasenzotti J R, Hohensinner
P J. Innate immune training with bacterial extracts enhances lung macrophage
recruitment to protect from betacoronavirus infection. J Innate Immun, 2022;14(4): 293-
305
47. Mulder W J M, Ochando J, Joosten L A B, Fayad Z A, Netea M G. Therapeutic targeting
of trained immunity. Nat Rev Drug Discov, 2019;18(7): 553-566.
48. Holtfreter S, Jursa-Kulesza J, Masiuk H, Verkaik N J, de Vogel C, Kolata J, Kolata M, Steil
L, van Wamel W, van Belkum A, Völker U, Giedrys-Kalemba S, Bröker B M. Antibody
responses in furunculosis patients vaccinated with autologous formalin-killed
Staphylococcus aureus. Eur J Clin Microbiol Infect Dis. 2011 (30): 707–717
49. Nakatsuji T, Gallo R L, Shafiq F, Tong Y, Chun K, Butcher A M, Cheng J Y, Hata T R.
Use of autologous bacteriotherapy to treat Staphylococcus aureus in patients with atopic
dermatitis: A randomized double-blind clinical trial. JAMA Dermatol. 2021;157(8): 978-
982.
50. Polymenakou P N. Atmosphere: A source of pathogenic or beneficial microbes?
Atmosphere. 2012; 3: 87-102.
51. Bowers R M, Clements N, Emerson J B, Wiedinmyer C, Hannigan M P, Fierer N. Seasonal
variability in bacterial and fungal diversity of the near-surface atmosphere. Environ. Sci.
Technol. 2013; 47 (21): 12097–12106
52. Government of india, Ministry of Ayush (Drug Control Cell). Guidelines for inspection
of gmp compliance by homoeopathic drug industry. March, 2015
http://www.e-aushadhi.gov.in/ayush/download/external/Notification/8536171420-
Guidelines%20For%20Inspection%20Of%20GMP%20Compliance%20By%20Homoeopathi
c%20Drug%20Industry%20%20%2012201902121950.pdf
53. Mora M, Mahnert A, Koskinen K, Pausan M R, Oberauner-Wappis L, Krause R, Perras A
Gorkiewic K G, Berg G, Moissl-Eichinger C. Microorganisms in confined habitats:
Microbial monitoring and control of intensive care units, operating rooms, cleanrooms
and the international space station. Frontiers in Microbiology. 2016; 7:15
54. Hirsch P. Microbial life at extremely low nutrient levels Adv Space Res. 1986; 6(12):
287-98.
55. Ingram L O. Mechanism of lysis of Escherichia coli by ethanol and other chaotropic
agents. J Bacteriol. 1981; 146(1): 331–336.
56. Charoensuk D, Brannan R G, Chaiyasit W, Chanasattru W. Physico-chemical and gel
properties of heat-induced pasteurized liquid egg white gel: Effect of alkyl chain length
of alcohol. Int J Food Properties. 2021; 24(1):1229-1243
57. Ohnishi T, Asakura T. Denaturation of oxyhemoglobulin S by mechanical shaking.
Biochim Biophys Acta, 1976; 453(1): 93-100.
58. Acharya V V, Chaudhuri (Chattopadhyay) P. Modalities of protein denaturation and
nature of denaturants. Int. J. Pharm. Sci. Rev. Res. 2021; 69(2): 19-24
59. Greenfield E W, DeCaprio J, Brahmandam J. Making weak antigens strong: Modifying
protein antigens by denaturation. Cold Spring Harb Protoc, 2018; 2018(5)
60. Arnon R. Conformational antigenic determinants in proteins. In: Celada F, Schumaker
V N, Sercarz E E. (eds) Protein conformation as an immunological signal. 1983; Springer,
Boston, MA., pp 157–164
61. Sandria P F, Portocarrerob A R, Ciupaa L, Ferrazb F N, Falkowski-Temporinib G J,
Rodriguesc W N S, Ferreirad E C, Aleixoe D L, de Araújo S M. Dynamized ethyl alcohol
improves immune response and behavior in murine infection with Trypanosoma cruzi.
Cytokine 2017; 99: 240–248
62. Wolynes P G. Evolution, energy landscapes and the paradoxes of protein folding.
Biochimie 2015; 119: 218-230
63. Liu S-Q, Ji X-L, Tao Y, Tan D-Y, Zhang K-Q, Fu Y-X. Protein Folding, Binding and
Energy Landscape: A Synthesis. In: Kaumaya P. (ed) Protein Engineering. InTech, 2012.
http://www.intechopen.com/books/protein-engineering/protein-foldingbinding-and-energy-landscape-a-synthesis
64. Chang J-Y. Conformational isomers of denatured and unfolded proteins: Methods of
production and applications. Protein J, 2009; 28(1): 44-56.
65. Uversky V N. Intrinsically disordered proteins and their “mysterious” (meta) physics.
Frontiers in Physics. 2019; 7:10
66. Zhang J. Protein-Protein Interactions in Salt Solutions. In: Cai W. (eds) Protein-Protein
interactions – Computational and experimental tools. 2012; 359-376. InTech
http://www.intechopen.com/books/protein-protein-interactions-computational-andexperimentaltools/protein-protein-interactions-in-salt-solutions
67. Gillard, R.D., Laurie, S.H. Metal-Protein interactions. In: Hudson, B.J.F. (eds)
Biochemistry of Food Proteins. 1992; 155–196. Springer, Boston, MA
68. Pérez-Fuentes L, Drummond C, Faraudo J, Bastos-González D. Interaction of organic
ions with proteins. Soft Matter. 2017; 13(6): 1120-1131
69. Feeney R E, Whitaker J R, Wong W S D, Osuga D T, Gershwin M E. Chemical reactions
of proteins. In: Richardson, T., Finley, J.W. (eds) Chemical changes in food during
processing.1985; 255–287. Basic Symposium Series. Springer, Boston, MA
70. Scheiblhofer S, Laimer J, Machado Y, Weiss R, Thalhamer J. Influence of protein fold
stability on immunogenicity and its implications for vaccine design. Expert Review
Vaccines. 2017; 16(5): 479-489
71. Ratanji K D, Derrick J P, Dearman R J, Kimber I. Immunogenicity of therapeutic
proteins: Influence of aggregation. J Immunotoxicology. 2014; 11(2): 99-109
72. Holley C K, Dobrovolskaia M A. Innate immunity modulating impurities and the
immunotoxicity of nanobiotechnology-based drug products. Molecules 2021; 26: 7308
73. Bellissent-Funel M-C, Hassanali A, Havenith M, Henchman R, Pohl P, Sterpone F, van
der Spoel D, Xu Y, Garcia A E. Water determines the structure and dynamics of
proteins. Chem Rev. 2016; 116(13): 7673–97
74. Leitner D M, Hyeon C, Reid K M. Water-mediated biomolecular dynamics and
allostery. J. Chem. Phys. 2020; 152: 240901
75. Völker J, Klump H H, Breslauer K J. Communication between noncontacting
macromolecules. PNAS. 2001; 98(1):7694–7699
76. Bu Z, Callaway D J E. Proteins move! Protein dynamics and long-range allostery in
cell signaling. Advances in protein chemistry and structural biology. 2011; 83:163-221
77. Liu J, Nussinov R. Allostery: An overview of its history, concepts, methods, and
applications. PLoS Comput Biol 2016; 12(6): e1004966.
78. Berlow R B, Jane Dyson H, Wright P E. Expanding the paradigm: Intrinsically
disordered proteins and allosteric regulation. J Mol Biol. 2018; 430(16): 2309–2320
79. Toncrova H, McLeish T C B. Substrate-modulated thermal fluctuations affect longrange allosteric signaling in protein homodimers: Exemplified in CAP. Biophysical
Journal. 2010; 98: 2317–2326
80. Xu Y, Havenith M. Perspective: Watching low-frequency vibrations of water in
biomolecular recognition by THz spectroscopy. J. Chem. Phys. 2015; 143:170901
81. Lacefield J C. Physics of ultrasound in diagnostic radiology physics: A handbook for
teachers and students. In: Christofides S, Dance D R, Maidment A D A, McLean I D, Ng K
H. (eds) Diagnostic radiology physics – A handbook for teachers and students. 2014; p 296.
International atomic energy agency, Vienna.
82. Pikovsky A, Rosenblum M. Synchronization. Scholarpedia. 2007; 2(12):1459
doi:10.4249/scholarpedia.1459
83. Majewski T. Vibratory forces and synchronization in physical systems. Ingeniería
Mecánica Tecnología Y Desarrollo. 2013; 4(4): 119-128
84. Teut M, Hirschberg U, Luedtke R, Schnegg C, Dahler J, Albrecht H, Witt C M.
Protocol for a phase 1 homeopathic drug proving trial. Trials 2010; 11: 80
85. Ahumada-Cota R E, Hernandez-Chiñas U, Milián-Suazo F, Chávez-Berrocal M E,
Navarro-Ocaña A, Martínez-Gómez D, Patiño-López G, Salazar-Jiménez E P, Eslava C A.
Effect and analysis of bacterial lysates for the treatment of recurrent urinary tract
infections in adults. Pathogens 2020; 9:102
86. Rojas M, Restrepo-Jiménez P, Monsalve D M, Pacheco Y, Acosta-Ampudia Y,
Ramírez-Santana C, Leung P S C, Ansari A A, Gershwin M E, Anaya J-M. Molecular
mimicry and autoimmunity. Journal of Autoimmunity. 2018; 95:100-123
87. Yee C S K, Rachid R. The heterogeneity of oral immunotherapy clinical trials:
Implications and future directions. Current Allergy and Asthma Reports. 2016; 16: 25
88. Baiden-Amissah R E M, Tuyaerts S. Contribution of aging, obesity, and microbiota on
tumor immunotherapy efficacy and toxicity. Int. J. Mol. Sci. 2019; 20: 3586
89. Klein S L, Morgan R. The impact of sex and gender on immunotherapy outcomes.
BMC Open Access 2020; 11: 24
90. Casale T B, Canonica G W, Bousquet J, Cox L, Lockey R, Nelson H S, Passalacqua G.
Recommendations for appropriate sublingual immunotherapy clinical trials. J Allergy
Clin Immunol. 2009; 124: 665-70.
91. Pfaar O, Alvaro M, Cardona V, Hamelmann E, Mosges R, Kleine-Tebbe J. Clinical trials
in allergen immunotherapy: Current concepts and future needs. Allergy. 2018; 73(9):
1775-1783
92. Lund K, Vase L, Petersen G L, Jensen T S, Finnerup N B. Randomised controlled trials
may underestimate drug effects: Balanced placebo trial design. PLOS ONE. 2014; 9 (1):
e84104
93. Boehm K, Berger B, Weger U, Heusser P. Does the model of additive effect in placebo
research still hold true? A narrative review. JRSM Open, 2017; 8(3): 2054270416681434
94. Khan A, Bhat A. Is the problem of a high placebo response unique to antidepressant
trials? J Clin Psychiatry. 2008; 69: 1979-1980
95. Park Y, Liu S. A randomized group sequential enrichment design for immunotherapy
and targeted therapy. Contemporary Clinical Trials. 2022; 116: 106742
96. Bhatt D L, Mehta C. Adaptive designs for clinical trials. N Engl J Med 2016; 375: 65-74.

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