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The Micro-Immunotherapy Medicine 2LARTH ® Reduces Inflammation and Symptoms of Rheumatoid Arthritis In Vivo

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Background: Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, which can cause cartilage and bone damages as well as pain and disability. In order to prevent disease progression, reduce pain, and major symptoms of RA, one good strategy consists in targeting proinflammatory cytokines that have the key role in the vicious circle of synovial inflammation and pain. The micro-immunotherapy medicine (MIM) 2LARTH® targets cytokines involved in inflammation. Aim: The aim of the study is to evaluate the effect of the MIM compared to vehicle in an in vivo model of RA, induced in mice after immunization with articular bovine type II collagen. Methods: Vehicle and MIM were dissolved in pure water (1 capsule in 100 ml) and 100 µl was given by gavage daily for 14 days. To evaluate the severity of arthritis, wrist and ankle thickness was determined, paw edema was measured, and a clinical score from 0 to 4 was established. Furthermore, histological analysis was performed. To evaluate systemic inflammation, circulating levels of IL-1β and TNF-α were measured by ELISA. Results: Ankle thickness was found to be significantly reduced in MIM-treated mice compared to vehicle-treated mice (P < 0.05) and compared to untreated me (P < 0.05) and compared to untreated me (P < 0.05) and compared to untreated me (β and TNF-α were measured by ELISA. P < 0.05) and compared to untreated me (. Conclusion: The results indicate that the tested medicine reduces inflammation, histological, and clinical signs of RA in a CIA model.
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Research Article
The Micro-Immunotherapy Medicine 2LARTH® Reduces
Inflammation and Symptoms of Rheumatoid Arthritis
In Vivo
Ilaria Floris ,1 Víctor García-González,2 Belen Palomares,3,4,5 Kurt Appel ,6
and Beatrice Lejeune 7
1Preclinical & Clinical Development and Regulatory Affairs, Labo’Life France, 1 Rue François Bruneau, 44000 Nantes, France
2Innohealth Group, Madrid, Spain
3Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain
4Departamento de Biología Celular, Fisiología e Inmunología, Universidad de Córdoba, Córdoba, Spain
5Hospital Universitario Reina Sofía, Córdoba, Spain
6VivaCell Biotechnology GmbH, Denzlingen 79211, Germany
7Labo’Life Belgium, Parc Scientifique Crealys, Rue Camille Hubert 11, 5032 Gembloux, Belgium
Correspondence should be addressed to Ilaria Floris; ilaria.
Received 23 May 2019; Accepted 29 October 2019; Published 23 January 2020
Academic Editor: Tim Jansen
Copyright © 2020 Ilaria Floris et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background. Rheumatoid arthritis (RA) is a chronic inammatory joint disease, which can cause cartilage and bone damages as well
as pain and disability. In order to prevent disease progression, reduce pain, and major symptoms of RA, one good strategy consists
in targeting proinammatory cytokines that have the key role in the vicious circle of synovial inammation and pain. e micro-
immunotherapy medicine (MIM) 2LARTH® targets cytokines involved in inammation. Aim. e aim of the study is to evaluate the
eect of the MIM compared to vehicle in an in vivo model of RA, induced in mice aer immunization with articular bovine type II
collagen. Methods. Vehicle and MIM were dissolved in pure water (1 capsule in 100 ml) and 100 µl was given by gavage daily for 14
days. To evaluate the severity of arthritis, wrist and ankle thickness was determined, paw edema was measured, and a clinical score
from 0 to 4 was established. Furthermore, histological analysis was performed. To evaluate systemic inammation, circulating levels
of IL-1β and TNF-α were measured by ELISA. Results. Ankle thickness was found to be signicantly reduced in MIM-treated mice
compared to vehicle-treated mice (
 < 0.05
) and compared to untreated me (
 < 0.01
). Paw edema was reduced, as well as clinical
score attributed to MIM-treated mice in comparison with vehicle-treated mice and untreated CIA mice (
 < 0.01
). In line with
these results, histological analysis conrmed that MIM reduced inammation and joint destruction in comparison to controls. No
signicant changes were found in plasmatic IL-1β levels between CIA and controls, while the levels of TNF-α signicantly increased
in the CIA group, and were lowered in MIM-treated mice (
 < 0.05
vs. vehicle and vs. CIA). Conclusion. e results indicate that
the tested medicine reduces inammation, histological, and clinical signs of RA in a CIA model.
1. Introduction
Rheumatoid arthritis (RA) is an immune-mediated, systemic
inammatory disease that aects mainly the synovial joints, char-
acterized by intraarticular inammation, synovial hyperplasia,
and progressive degradation of cartilage and bone. RA is one of
the most prevalent chronic inammatory diseases; its prevalence
is around 1% of the population, and the disease is more frequent
(~2 : 1) and more severe in women than in men [1].
Genomic studies have identied more than a hundred loci
associated with RA risk, mostly implicated in immune
mechanisms and chronic inammatory diseases. In particular,
the human leukocyte antigen (HLA) system is strongly linked
to susceptibility of RA [2]. Furthermore, some HLA polymor-
phisms are associated to a more aggressive form of RA and
higher mortality [3].
Joint inammation in RA is at the apex of clinical events:
pain, swelling, and stiness, development of adhesions, ero-
sion of joint surfaces, bone resorption, loss of function, and
joint deformation [4].
e major therapeutic goal in RA consists in reversing the
chronic inammation. ere have been clear advances in the
International Journal of Rheumatology
Volume 2020, Article ID 1594573, 9 pages
International Journal of Rheumatology2
pharmacological management of RA over the past decade,
nevertheless many patients still do not tolerate or do not
respond well to the available therapies. e current approach
consists of tight control of disease activity through dis-
ease-modifying anti-rheumatic drug (DMARD). is therapy
targets the inammation and is aimed at blocking disease
progression and joint damage. Both the American College of
Rheumatology and the European League against Rheumatism
recommend that DMARDs treatment should start as soon as
a diagnosis has been made [5]. However, it is not easy because
no diagnostic criteria exist for RA. Traditional nonsteroidal
anti-inammatory drugs are also widely used to reduce pain
and stiness, yet those drugs do not interfere with joint dam-
age and, as glucocorticoids, oer rapid symptomatic relief, but
are associated with serious long-term side eects [6, 7].
Despite the improvements made in the past two decades
in management of RA symptoms, many issues remain to be
addressed. Mainly, we cannot predict optimal responses nei-
ther toxic, nor side eects for a given treatment.
Micro-immunotherapy (MI) is a therapeutic approach
which can be used alone or in association with other therapies
to oer clinical benets without side eects. MI uses molecules
at low doses (LD) and ultralow doses (ULD) impregnated on
lactose-saccharose pillules for oromucosal administration, to
target the immune system and regulate immune responses in
diseases. e active substances used in MI medicines (MIM)
are cytokines, hormones, growth factors, neuropeptides,
nucleic acids, and specic nucleic acids (SNAs®). SNAs® consist
of single strands of DNA molecules, ranging from 16 to 34
bases, designed to target specically 1 or more genes (European
Patent: EP0670164B1).
2LARTH® is a MIM notied as a homeopathic medicine
under notication number 1507CH36 F1 by the Federal
Agency for Medicines and Health Products in Belgium. It has
been developed to counteract the over-expression of interleu-
kin (IL)-1β and tumor necrosis factor (TNF)-α, once the
inammatory cascade is activated and the chronic inamma-
tory status is established. Furthermore, the tested MIM aims
at targeting HLA system and IL-2. e anti-inammatory
eect of the medicine has been demonstrated in vitro on
human enriched monocytes exposed to lipopolysaccharide [8].
To evaluate its ecacy in vivo, authors planned a research
project in collagen-induced arthritis (CIA). CIA is induced in
mice with articular bovine type II collagen (CII) and the
resulting disease is essentially mediated by an autoimmune
response. CIA is commonly used to study in vivo the patho-
logical mechanisms of RA and to evaluate new antiarthritic
drugs as it shares clinical, histological, and immunological
features of human RA [9, 10].
e sublingual administration is the mode of administra-
tion of MIM, including for the tested MIM. Because rodents
have their buccal mucosa keratinized, pillules were dissolved
in pure water, then administered orally by gavage. e oral
route performed by gavage is convenient because it allows the
use of lower doses, it minimizes side eects, and may provide
enhanced ecacy [11]. Indeed, the gastrointestinal tract con-
tains innate immune system cells (macrophages, monocytes,
neutrophils, and dendritic cells), and cells of the adaptive
immune system (T and B lymphocytes and their secreted
mediators, cytokines, and chemokines). Organized lymphoid
structures are present in the gut: small lymphoid nodules in
the upper small intestine, and large organized aggregates of
lymphoid tissue (Peyer’s patches) in the ileum [12]. Other
studies demonstrated that orally administered peptides may
be detected by immune cells present in the intestinal lumen,
and induce a specic immune response [13, 14].
e aim of the study is to evaluate in vivo the ecacy of
MIM in treating clinical signs and chronic inammation of
arthritis, in a well-known animal model of RA.
2. Materials and Methods
2.1. Materials. e active substances present in the tested
MIM composition are: IL-1β, TNF-α, IL-2, SNAs® targeting
genomic regions, and transcripts of HLA class I, class II, and
human IL-2. Each active substance is singularly prepared
to obtain specic LD or ULD, following a “Serial Kinetic
Process” (SKP), consisting of a 1/100 dilution process followed
by vertical shaking. SKP is reproduced a dened number of
times; number that is reported in composition as number of
Centesimal Hahnemannian dilutions (CH).
e composition of the tested medicine is as follows: IL-1β
at 10 CH, or at 17 CH, TNF-α at 10 CH, or at 17 CH, IL-2 at
10 CH, or at 12 CH, SNA-HLA-I at 10 CH, or at 16 CH, SNA-
HLA-II at 10 CH, or at 16 CH, and SNA-ARTH at 10 CH, or
at 16 CH. e excipient consists of lactose-sucrose pillules,
also called globules (about 380 mg per capsule), which were
impregnated with ethanolic preparation of active substances,
at LD or ULD.
e tested MI medicine is a sequential drug and the com-
position of each capsule is specic. Indeed, capsules in blister
are numbered from 1 to 10 and the intake should respect the
sequential order.
e vehicle capsules used as controls contain lactose-sac-
charose pillules impregnated with the ethanolic preparation
lacking active substances.
2.2. Animal Care and CIA Induction. DBA/1 mice (male 8
weeks-old) were purchased from Janvier Labs (France). All
animals were kept on a 12 h light-dark cycle, at a controlled
temperature (23 ± 1°C) and 40–50% relative humidity with free
access to standard food and water. Experimental procedure
was performed under the guidelines for care and use of
animals specied by the Animal Research Ethic Committee
of Córdoba University (Spain).
CIA was induced in 32 mice, while 6 mice were let as a
control group. Briey, this is the protocol used for mice
(i) Bovine collagen nasal septum type II (CII) (C7806,
Sigma-Aldrich) was dissolved at 2 mg/ml in 10 mM
acetic acid by stirring overnight at 4°C. CII was
kept cold while being dissolved, to prevent CII
(ii) Heat-killed Mycobacterium tuberculosis (H37RA)
(231141, BD) was combined with incomplete
Freund’s adjuvant (F5506, Sigma-Aldrich) at a
4 mg/ml nal concentration.
3International Journal of Rheumatology
(iii) CII was emulsied in an equal volume (1 : 1)
of CFA just prior to immunization using a
high-speed homogenizer (Ultraturrax T8, IKA
Labortechnik). Collagen was kept cold throughout
the emulsication.
During the phase of mice immunization, animals were awake
and physically restrained. DBA/1 mice (8 weeks-old) were
immunized with 100 µl of CII/CFA emulsion by an intrader-
mal injection at the base of the tail. At day 0 mice received the
rst immunization, then at day 21 the animals were reimmu-
nized with a booster injection of the same composition and
volume and at the same site (Figure 1). Among the 32 mice,
only 15 showed clear evidences of disease induction before
starting the treatments. Hence, mice with CIA were redistrib-
uted in three groups: CIA (
), CIA + vehicle (
), and
2.3. Treatment. Starting from day 30 until day 44, animals were
treated with vehicle (CIA + vehicle) or MIM (CIA + MIM), as
represented in Figure 1. Both types of granules were dissolved
in pure water (the content of 1 capsule in 100 ml) and 100 µl
was given daily for 14 days by gavage starting from day 30. e
daily dose of sugar corresponds to 0.380 mg.
e order of administration respected the MIM sequen-
tiality and the order from 0 to 10 indicated on the blister.
2.4. Clinical Evaluation of Arthritis Signs. To evaluate wrist
and ankle inammation, thickness (mm) was measured every
3-4 days using a Vernier caliper.
Every 3-4 days, paw edema was also measured using a ple
thysmometer (LE7500, Panlab). Basically, the water displace-
ment produced by the immersion of the animal paw in the
measuring tube is reected into the second tube, inducing a
change in the conductance between the two electrodes which
generates an output signal indicating the volume displacement
e scoring system for subjective evaluation of arthritis
severity used the following scale: 0 = normal; 1 = detectable
swelling in one joint or toe; 2 = swelling in two types of toes
or joints but not entire paw inamed; 3 = entire paw inamed
and swollen; 4 = severe swelling in the entire paw or
2.5. Histological Analysis. At 44 days, mice were sacriced and
the knee and paws tissues were extracted and prepared for
histological analysis. Histopathology studies were performed
in 3 representative animals of each group.
e joints (knee and paws) were xed in 4% paraformal-
dehyde for 2 days and then decalcied in 7% nitric acid for 5
days. Aer decalcication, samples were processed for light
microscopy; these were dehydrated in alcohol and embedded
in paran. Sagittal serial sections were cut (5 µm) and stained
with hematoxylin-eosin (H&E) and Safranin-O.
2.5.1. H&E Staining. To determine whether the treatment was
eective, the joints were examined for the presence or absence of
synovitis, pannus formation, cartilage loss, bone erosions, and
disruption of joint architecture. e histologic slides stained
with H&E were scored from 0 to 4 according to the following
gradation of arthritis: 0 = normal ankle joint; 1 = normal
synovium with occasional mononuclear cells; 2 = denite
arthritis, a few layers of at to rounded synovial lining cells
and scattered mononuclear cells; 3 = clear hyperplasia of the
synovium with three or more layers of loosely arranged lining
cells and dense inltration with mononuclear cells; 4 = severe
synovitis with pannus and erosions of articular cartilage and
subchondral bone. Images were taken using a Leica DM2000
microscope and Leica MC190 camera.
2.5.2. Safranin-O Staining. Sagittal sections were deparanized
in xylol, hydrated with decrescent concentrations of ethanol
(100%, 96%, and 80%), and stained sequentially with Weigerts
iron hematoxylin, 0.002% fast green, 1% acetic acid, and 1%
Schema of the project
First collagen injection
DAY 21
Second collagen injection
DAY 14
Collagen-induced arthritis
(CIA) model
Start the examination of:
Wrist and ankle thickness
Paw inammation
Clinical score
(every 3-4 days)
DAY 30 DAY 44
Oral daily treatment (gavage) with:
→ MIM (N = 5)
Blood collection
Sacrice and
samples collection for histology
→ Vehicle (N = 5)
F 1:Experimental schema of the in vivo stu dy.
International Journal of Rheumatology4
Standards, control, and samples are pipetted into the wells and
the immobilized antibody binds specic proteins such as TNF-
α, or IL-1β. Aer washing away any unbound substances, an
enzyme-linked polyclonal antibody specic for mouse protein
of interest is added to the wells. Following washing to remove
any unbound antibody-enzyme reagent, a substrate solution
is added to the wells. e enzyme reaction yields a blue prod-
uct that turns yellow when the stop solution is added. e
intensity of the color measured is proportional to the amount
of protein bound in the initial step. e sample values are then
read o the standard curve.
2.7. Statistical Analysis. Data are expressed as mean ± SEM. To
test whether the mean values were signicantly dierent between
groups, we used one-way ANOVA test followed by Tukey’s post
hoc test. To analyze dierences between two groups, unpaired
T test was used. e
value ≤0.05 was considered statistically
Safranin-O, washed in running tap water, dehydrated by
crescent concentrations of ethanol (80%, 96%, and 100%),
incubated in xylol, and nally mounted with Eukitt mounting
medium. Images were taken using a Leica DM2000 microscope
and Leica MC190 camera. e cartilage area was determined
in pixel using image J soware.
2.6. Enzyme-Linked Immunosorbent Assay. Blood was
collected at the end of treatment and plasma was obtained by
centrifuging the samples in heparinized tubes (BD Vacutainer
Heparin tubes) at 2000 × g for 20 min.
e expression of circulating IL-1β and TNF-α was meas-
ured by quantitative sandwich enzyme immunoassay tech-
nique according to the instructions of the manufacturers
(R&D systems). Briey, the technique employs the monoclonal
antibody specic for mouse protein of interest (TNF-α, or
IL-1β). Antibody has been precoated onto a microplate.
14 22 27 34 38 44
14 22 27 34 38 44
Ankle thickness (mm)
F 2:Variations in wrist and ankle thickness. (a) Wrist and (b) ankle thickness (mm) were measured every 3-4 days using a caliper. e
black arrow indicates the reimmunization day, while the yellow arrow the beginning of the treatment with vehicle or MIM in their respective
 < 0.05
vs. vehicle-treated group; $
 < 0.01
vs. untreated CIA group; #
 < 0.05
vs. control group.
Control CIA
14 22 27 34 38 44
Increase of hind paw volume (ml)
14 22 27 34 38 44
∗∗ ∗∗
F 3:Variations in paw edema and clinical score. (a) Paw edema was measured as an increase in volume (ml), every 3-4 days using a
plethysmometer. e black arrow indicates the reimmunization day, while the yellow arrow the beginning of the treatments.
 ≤ 0.05
vehicle-treated group;
 < 0.05
vs. control group. (b) Clinical score was attributed every 3-4 days for subjective evaluation of arthritis severity.
e black arrow indicates the reimmunization day, while the orange arrow the beginning of the treatments.
 < 0.01
vs. vehicle-treated
group; §
 < 0.01
vs. untreated CIA group; #
 < 0.05
vs. control group.
5International Journal of Rheumatology
arthritis. Nevertheless, aer 11–14 days of treatment, the paw
edema was lower in mice treated with tested MIM than vehicle
 < 0.05
), or untreated CIA mice (
 < 0.05
) (Figure3(a)).
e clinical score attributed at the end of treatment to
active pillules-treated mice is lower than score of vehicle-
treated mice (
 < 0.01
) or untreated CIA mice (
 < 0.01
swelling was observed only in some toes or joints, but not the
entire paw was inamed. (Figure 3(b)). MIM-treated mice
showed improvement of arthritis signs throughout the study,
signicant at the end of treatment (day 41 and 44).
3.4. Histological Analysis of the Hind Paws. e H&E staining of
normal mice knee joints showed no inammatory symptoms.
On the other hand, the knee joint histology of CIA and
CIA + vehicle groups presented pathological features of arthritis
such as inltration of lymphocytes, synovial hyperplasia, pannus,
and articular cartilage loss with connective tissue replacement.
In CIA + MIM mice, inammation and joint destruction are
lower in comparison to arthritic mice. In fact, the synovial
membrane in the joints was almost like normal synovium,
with few signs of synovial hyperplasia or other characteristics
of inammation. According to the score mentioned in materials
and methods section for quantication of arthritis in the H&E
samples, MIM-treated mice had a lower score than mice treated
with vehicle (
 < 0.05
), and lower than untreated CIA mice
 < 0.05
) (Figure 4(a), Figure S1 in Supplementary Materials).
signicant. Statistical analysis was performed using GraphPad
Prism version 8 (GraphPad, San Diego, CA, USA).
3. Results
3.1. Body Weight Measurement. All mice with collagen-
induced arthritis, CIA, CIA + vehicle, and CIA + MIM, showed
slight reduction in body weight compared to control mice. No
signicant changes have been found between mice which were
treated with vehicle and MIM (data not shown).
3.2. Wrist and Ankle ickness Determination. e results
of the wrist thickness measurement did not show signicant
variations between the dierent experimental groups
during the treatment (Figure 2(a)). However, mice groups
that developed CIA showed an increase in ankle thickness
starting from day 31 (
 < 0.05
vs control). Aer 11–14 days
of treatment, the ankle thickness was found to be signicantly
reduced in MIM-treated mice in comparison with vehicle
-treated mice (
 < 0.05
) and in comparison with CIA
untreated mice (
 < 0.01
) (Figure 2(b)).
3.3. Evaluation of Hind Limbs Edema and Severity Score. CIA
and CIA + vehicle groups had a progressive increase in
inammation in the hind paw aer the booster injection
administered at day 21, in accordance with the induction of
200 µm
Histological score
Cartilage area in pixel (1
× 105)
F 4:Histological examination of joints. (a) Histological score was determined based on arthritis severity observed in the sections
of knee joints stained with H&E. (b) Quantication of cartilage area in pixel and representative histologic sections of knee joints of three
animals per group stained with Safranin-O. e stained cartilage appeared red in color. Representative histologic sections for CIA + vehicle
and CIA + MIM are shown next to each graph. More representative histologic sections are shown in Supplementary Materials, see Figures S1
and S2. C = cartilage layer; JS = joint space; black circles indicate inammation and synovial hyperplasia (pannus formation). Bars in graphs
represent mean ± SEM of 3 mice per group.
 < 0.05
 < 0.001
International Journal of Rheumatology6
levels between untreated CIA and controls (Figure 5(a)). In
contrast, plasma levels of TNF-α increased signicantly in
the CIA group (
 = 0.01
, vs. control), but were signicantly
reduced in the mice treated with tested MIM,
 < 0.05
vs. CIA
groups (Figure 5(b)).
4. Discussion
CIA is one of the most useful models to study chronic inam-
mation occurring during RA, indeed autoreactivity of T and
B cells, joint inammation, cartilage, and bone damage, are
Safranin-O staining results showed an evident reduction of
cartilage in the knee joint of untreated CIA mice (
 = 0.01
and vehicle treated mice (
 = 0.01
), while the aspect of cartilage
in mice treated with MIM is quite similar to the control group
(Figure 4(b), Figure S2 in Supplementary Materials). e sub-
sequent quantication shows that the mice which received MIM
present clearly reduced morphological alterations characteristic
of arthritis, corroborating the previous H&E results.
3.5. Determination of Proinflammatory Cytokines Levels in
Plasma. No signicant changes were found in IL-1β plasma
IL-1β (pg/ml)
TNF-α (pg/ml)
F 5:Inammatory cytokines levels in plasma analyzed by ELISA. e optical density of each sample was measured at 450 nm, using the
microplate reader TECAN. (a) IL-1-β levels (pg/ml), and (b) TNF-α levels (pg/ml) are represented in graphs,
 < 0.05
(a) (b)
F 6:Mode of action of tested MIM. (a) Oral administered MIM can interact with cells of the innate and adaptive immune system
present in the gut, reducing systemic inammation and in turn, local inammation of the synovial compartment. (b) RA is characterized by
the presence of cytokines in the synovial compartment, broblasts assume an aggressive inammatory phenotype, and chondrocytes enhance
their catabolism promoting articular destruction. e tested MIM aimed at reducing levels of proinammatory cytokines involved in pain
and clinical signs of RA.
7International Journal of Rheumatology
characteristics are the joint swelling and the leucocytes inl-
tration, which both reect synovial membrane inammation
consequent to immune activation.
Histological analysis conrmed the presence of inamma-
tion, immune inltration, and joint destruction in CIA mice,
while the aspect of joints in CIA + MIM group was almost like
the one observed in control mice, with only few signs of syn-
ovial hyperplasia and inammation. (Figure 4(a), Figure S1).
e joint cartilage destruction detected in Safranin-O staining
sections of CIA and CIA + vehicle groups, was not present in
MIM-treated mice, in which the aspect of cartilage was more
similar to those of the control group (Figure 4(b), Figure S2).
To check the impact on systemic levels of targeted
cytokines, plasmatic levels of IL-1β. and TNF-α were analyzed
at day 44, before the animal sacrice. IL-1β levels were not
found to be increased in CIA mice compared to control mice,
and no dierence were found between treated and CIA group,
as reported in another animal [25] and human studies [26]
(Figure 5(a)), while plasmatic levels of TNF-α increased sig-
nicantly in CIA group compared to control group, and were
found to be lowered in MIM-treated mice in comparison to
the rest of the mice with CIA (
 < 0.05
vs. CIA and vs vehicle
groups) (Figure 5(b)).
A crosstalk between the mucosal surfaces (buccal, intes-
tinal, nasal, etc.) and the systemic immune system exists [27,
28] and oral immunotherapy, including MI, use this concept
to target immune system imbalances.
e intestinal Peyer’s patches are connected with lymphoid
tissues to coordinate immune responses to pathogens in the gut,
as well as to maintain food tolerance, bacterial, and virus
defenses. Immune cells can be found inside the matrix of Peyer’s
patches, including T and B cells, macrophages, dendritic cells,
and specialized phagocytic cells known as M cells [12]. e
hypothesized mode of action of the tested MIM can be explained
with the involvement of those immune cells and structures pres-
ent in the gut, which are able to communicate with the systemic
immune system and in turn, with the cells present in the inam-
matory milieu of the synovial compartment (Figure 6).
At the molecular level, the mode of action of ULD and
MIM, might be explained with the implication of the hormesis
[8]. e concept of hormesis, which was introduced for the
rst time 130 years ago by Schulz, is now becoming prominent
in toxicology, pharmacology, and in many biomedical areas.
Indeed, hormetic dose responses are widely present in micro-
bial, plant, animal models, and humans [29].
5. Conclusion
e results obtained in this study showed that the oral admin-
istration of MIM eectively reduces the clinical score and the
degree of edema, and inammation caused by collagen-in-
duced arthritis in DBA/1 mice. Furthermore, the treatment
attenuates the CIA pathological features since it was able to
reduce the inltration of inammatory cells, synovium hyper-
plasia, and cartilage loss in comparison to the vehicle treat-
ment and untreated CIA groups. In addition, plasmatic levels
of the proinammatory cytokine TNF-α were decreased by
the tested MIM.
similar to the human disease. In CIA model, TNF-α, IL-1β,
and IL-6 play a key role in the early phase of the disease and
continue to be important during the transition to the chronic
phase [15]. TNF-α, IL-1β, and IL-6 are also osteolytic cytokines
that are involved in the destruction of bone and joints in RA
as implicated in promoting the expression of receptor activator
of nuclear factor kappa-B ligand or RANKL in synovial bro-
blasts, in the dierentiation of osteoclasts and also in the
induction of matrix metalloproteinase production [16, 17].
e gene inactivation of those three cytokines can par-
tially/totally protect against RA [18–20]. IL-2 is also implicated
in the early onset of RA [21]. e transition from acute to
chronic inammation is decisive in the shi of the immune
system responses from defensive to tissue-damaging responses.
Cytokines can directly and indirectly generate pain. e
direct eects on nociceptive neurons have been explained by:
(i) the presence of nociceptive sensory neurons which express
receptors for cytokines; (ii) in vitro studies on primary sensory
neurons demonstrating that cytokines can change the excita-
bility of neurons, modify ion currents, and regulate molecules
involved in nociception; (iii) the injection of cytokines like
TNF-α or IL-1β into normal tissue produces pain and enhances
the responsiveness of nociceptive sensory bers [22].
Hence, cytokines contribute to pain indirectly through the
generation of inammation and the production of prostaglan
dins and other regulators associated with pain. Targeting those
cytokines can reduce inammation-associated pain [22–24].
In particular, the neutralization of TNF-α rapidly reduced pain
and inammatory hyperalgesia in the absence of any other
antinociceptive drugs. e eect of TNF-α on nerve bers can
be more easily reversed than the eect of other pain-related
cytokines; indeed, each cytokine has a specic potential to
induce chronic pain states [22].
e tested MIM aimed at inhibiting the mentioned
cytokines involved in RA during early and chronic phases.
MIM consists of lactose-saccharose pillules impregnated with
LD or ULD of 3 human recombinant proinammatory
cytokines (IL-1β, TNF-α, and IL-2), LD or ULD of SNA® tar-
geting genomic regions and transcripts of molecules belonging
to HLA class I and class II, LD or ULD of SNA® targeting
human IL-2.
In Vitro studies demonstrated the specic anti-inamma-
tory eect of tested MIM on human primary enriched mono-
cytes, activated with lipopolysaccharide: MIM treated cells
secreted lower levels of IL-1β, TNF-α, and IL-6 than vehicle
and untreated cells [8].
e in vivo study described here demonstrates the ecacy
of the tested MIM to treat CIA, starting from the early phase
of the disease. e mice were daily treated with vehicle or
MIM, following the sequential administration indicated on
the blister. CIA + MIM mice were less impacted by ankle
inammation (Figure 2(b)) and paw edema (Figure 3(a)) com-
pared to CIA + vehicle and untreated mice. In line with those
results, clinical score attributed to MIM treated mice was
found to be lower compared to vehicle control mice, only some
toes or joints were inamed, but not the entire paw as for CIA
and CIA + vehicle groups (Figure 3(b)).
e most signicant characteristic of RA is the chronic
and intensive inammation that is out of control. Other
International Journal of Rheumatology8
Víctor García-González and Ilaria Floris analyzed the data;
Ilaria Floris wrote the paper; Beatrice Lejeune revised the
e authors thank Mrs. Anne Naedts for having prepared
and provided the vehicle pillules, and Mr. Cedric Wolf for
having provided and sent the medicine to Innohealth S.L.
Parque Cientíco de Madrid. e research study was funded
by Labo’Life France.
Supplementary Materials
Figure S1. Representative histologic sections of knee joints
of three animals per group stained with H&E from normal
(1a–3c), CIA (4a–6c), vehicle (7a–9c) and MIM-treated mice
(10a–12c) are shown. e magnication of the images in the
rst row was taken at 4x, whereas in the second and third
row were taken at 10x. Black circles indicate inammation
and synovial hyperplasia. Green circles show articular car-
tilage loss. T=Tibia; F=Femur; C=Cartilage layer; JS=Joint
space; E=Epiphyseal growth plate. Figure S2. Representative
histologic sections of knee joints of three animals per group
stained with Safranin-O from normal (1–3), CIA (4–6),
placebo-treated (7–9) and 2LARTH-treated mice (10–12)
are shown. e magnication of the images was taken at 4x
and 10x. e areas with cartilage appear red colour stained.
(Supplementary Materials)
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In conclusion, we demonstrated that the tested medicine
reduces the signs of arthritis and can be used in the pharma-
cological management of RA. Due to the chronic nature of
RA, the pain and the disabilities related with the disease and,
considering the side eects associated with the actual clinical
treatments of RA, the tested MIM is a promising therapy, com-
patible with other therapeutic agents, and safe because no
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medicine, as well as clinical studies to validate the ecacy
appreciated in CIA mice model also in human.
RA: Rheumatoid arthritis
HLA: Human leukocyte antigen
DMARD: Disease-modifying anti-rheumatic drug
MI: Micro-immunotherapy
LD: Low doses
ULD: Ultra-low doses
MIM: Micro-immunotherapy medicine
SNA®: Specic nucleic acids
IL: Interleukin
TNF-α: Tumor necrosis factor-α
CIA: Collagen-induced arthritis
CII: Type II collagen
SKP: Serial kinetic process
CH: Centesimal Hahnemannian dilutions
CFA: Complete Freund’s adjuvant
H&E: Hematoxylin and eosin
ELISA: Enzyme linked immunosorbent assay.
Data Availability
e raw datasets analyzed during the current study are avail-
able from the corresponding author on reasonable request.
Conflicts of Interest
Ilaria Floris works for Labo’Life France, the company service
provider of Labo’Life, specialized in preclinical, clinical, and
regulatory aairs. Beatrice Lejeune is employed by Labo’Life
Belgium, the pharmaceutical company that commercializes
the micro-immunotherapy medicines tested in the study. is
professional relationship does not imply any misconduct on
the part of both authors. Víctor García-González works for
Innohealth Group, a biotechnological company (Scientic
Park of Madrid, Spain). Kurt Appel works for VivaCell
Biotechnology GmbH, a contract research organization (CRO)
specialized in preclinical research. Belen Palomares works for
IMIBIC and Córdoba University.
Authors’ Contributions
Ilaria Floris, Belen Palomares, and Kurt Appel conceived
and designed the experiments; Víctor García-González and
Belen Palomares performed the experiments; Kurt Appel,
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... These assumptions are supported by the fact that IL-1β (27 CH) and TNF-α (27 CH) both exhibit anti-inflammatory effects, as each could reduce the secretion of IL-1β and TNF-α in a model of LPS-stimulated human primary monocytes and the monocyte-like THP-1 cell line [81]. Moreover, a MI formulation that includes ULD of IL-1β and TNF-α as a part of its active ingredients also displayed anti-inflammatory properties in a model of LPS-stimulated human primary monocytes and in vivo, in a model of rheumatoid arthritis [82,83]. ...
... Overall, the strong involvement of IL-1β in allergic diseases ( Figure 3) means that treatments aimed at down-regulating this cytokine and its signaling pathways are attractive therapeutic options during acute and chronic manifestations to reduce the symptoms of IgEmediated inflammation. Furthermore, it is also worth mentioning that, in addition to the context of an allergy, MI has previously shown IL-1β-related anti-inflammatory potential and the capability to down-regulate IL1-β expression when using this same cytokine in ULD [81][82][83]. ...
... treatments aimed at down-regulating this cytokine and its signaling pathways are attractive therapeutic options during acute and chronic manifestations to reduce the symptoms of IgE-mediated inflammation. Furthermore, it is also worth mentioning that, in addition to the context of an allergy, MI has previously shown IL-1β-related antiinflammatory potential and the capability to down-regulate IL1-β expression when using this same cytokine in ULD [81,82,83]. Nevertheless, numerous other ILs and immune factors also play a role in the complex etiopathogenesis of the atopic march, and a multi-target therapeutic strategy that has several regulatory effects on those factors, such as that employed in MI, could be of great interest and beneficial for patients. ...
Full-text available
Allergic diseases consist of improper inflammatory reactions to antigens and are currently an important healthcare concern, especially considering their increasing worldwide development in recent decades. The “atopic march” defines the paradigm of allergic diseases occurring in chronological order and displaying specific spatial manifestations, as they usually start as atopic dermatitis (AD) and food allergies during infancy and progressively evolve into allergic asthma (AA) and allergic rhinitis (AR) or rhino-conjunctivitis in childhood. Many immune cell subtypes and inflammatory factors are involved in these hypersensitivity reactions. In particular, the T helpers 2 (Th2) subset, through its cytokine signatures made of interleukins (ILs), such as IL-4, IL-5, IL-10, and IL-13, as well as mast cells and their related histamine pathways, contribute greatly to the perpetuation and evolution of the atopic march. By providing low doses (LD) and ultra-low doses (ULD) of ILs and immune factors to the body, micro-immunotherapy (MI) constitutes an interesting therapeutic strategy for the management of the atopic march and its symptoms. One of the aims of this review is to shed light on the current concept of the atopic march and the underlying immune reactions occurring during the IgE-mediated responses. Moreover, the different classes of traditional and innovative treatments employed in allergic diseases will also be discussed, with a special emphasis on the potential benefits of the MI medicine 2LALERG® formulation in this context.
... 3 This route of administration will, in turn, elicit systemic and local effects, as we could observe in previous published preclinical in vivo studies. [4][5][6] Through their exclusive preparation, MIM aim at gently modulating the expression and the related signaling pathways of cytokines and immune factors by providing to the organism these molecules at low doses (LD) and ultra-low doses (ULD) (Figure 1). These LD/ULD are expressed in Hahnemannian dilution (CH) or in Korsakovian dilutions (K). ...
... Moreover, other in vitro and in vivo preclinical data have demonstrated the anti-inflammatory properties of a MI formulation employing ULD of TNF-α and IL-1β in association with other immune regulators, 4,10,11 providing encouraging results about the potential of some MIM in slowing down the pro-inflammatory responses in the context of chronic inflammation. Based on these previous results, the first aim of this study was to assess the effects of these two ULD-based u-MIMs in another specific inflammatory context, an intestinal inflammation model (Figure 2A, red box). ...
... Previous publications described how these vehicle controls are produced, in order to provide a suitable control for preclinical research. 4,6,7,9,10 Regarding the in vitro experiments performed in the current study, u-MIM, c-MIM or the Veh. pillules were freshly diluted in 100 mL of culture medium to reach the final sucrose-lactose concentration of 11 mM. ...
Full-text available
Introduction: Chronic inflammation is a pernicious underlying status, well-known for its contribution to the progressive development of various diseases. In this regard, Micro-immunotherapy (MI) might be a promising therapeutic strategy. MI employs low doses (LD) and ultra-low doses (ULD) of immune regulators in their formulations. In particular, as both IL-1β and TNF-α are often used at ULD in MI medicines (MIM), a special emphasis has been made on formulations that include these factors in their compositions. Methods: Several in vitro models have been employed in order to assess the effects of two unitary MIM consisting of ULD of IL-1β and TNF-α (u-MIM-1 and u-MIM-2, respectively), and four complex MIM (c-MIM-1, -2, -3 and -4) characterized by the presence of ULD of IL-1β and TNF-α amongst other factors. Thus, we first investigated the anti-inflammatory effects of u-MIM-1 and u-MIM-2 in a model of inflamed colon carcinoma cells. In addition, the anti-inflammatory potential of c-MIM-1, -2, -3 and -4, was assessed in in vitro models of intestinal and neuronal inflammation. Results: The results revealed that u-MIM-1 and u-MIM-2 both induced a slight decrease in the levels of IL-1β and TNF-α transcripts. Regarding the c-MIMs' effects, c-MIM-1 displayed the capability to restore the altered transepithelial electrical resistance in inflamed-HCoEpiC cells. Moreover, c-MIM-1 also slightly increased the expression of the junction-related protein claudin-1, both at the mRNA and protein levels. In addition, our in vitro investigations on c-MIM-2 and c-MIM-3 revealed their immune-modulatory effects in LPS-inflamed human monocytes, macrophages, and granulocytes, on the secretion of cytokines such as TNF-α, PGE2, and IL-6. Finally, c-MIM-4 restored the cell viability of LPS/IFN-γ-inflamed rat cortical neurons, while reducing the secretion of TNF-α in rat glial cells. Discussion: Our results shed the light on the potential role of these MIM formulations in managing several chronic inflammation-related conditions.
... A specific MI formulation, hereafter referred to as MIM-seq, is the investigational product of this preclinical study. As featured in all of the MI medicines [25,27,28], MIMseq is a sequential medicine. It thus consists of a sequence of five different capsules containing pillules (MIM-1, MIM-2, MIM-3, MIM-4, and MIM-5), each one containing a unique active substances combination. ...
... Moreover, from a mechanistic standpoint, MIM-seq also employs mitogenic factors, such as epidermal growth factor (EGF), transforming growth factor-β (TGF-β) and fibroblast growth factor (FGF) 2 at 27 CH (ULD). Previous preclinical evidence has highlighted the inhibitory effects induced by ULD-based MI medicines [20,[25][26][27]30]. Accordingly, it is possible that the active substances present in MIM-seq at the ULD of 6 CH and beyond, may reduce the expression of those mitogenic factors, which are crucial cancer-related signaling pathways, rightly overexpressed in several cancers, including CRC [50]. ...
... The Veh. pillules used in the study as controls are manufactured by Labo'Life Belgium. Previous publications describe how those vehicle controls are produced, in order to provide a suitable control for preclinical research (for both in vitro and in vivo evaluations) [20,[26][27][28][29]. For all the in vitro experiments, MIM-3, MIM-4, or the Veh. ...
Full-text available
In this study, the immunomodulatory effects of a sequential micro-immunotherapy medicine, referred as MIM-seq, were appraised in human primary M1 and M2 macrophages, in which the secretion of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, IL-12, IL-23, and tumor necrosis factor (TNF)-alpha, was inhibited. In addition, the potential anti-proliferative effects of MIM-seq on tumor cells was assessed in three models of colorectal cancer (CRC): an in vitro two-dimensions (2D) model of HCT-116 cells, an in vitro tri-dimensional (3D) model of spheroids, and an in vivo model of subcutaneous xenografted mice. In these models, MIM-seq displayed anti-proliferative effects when compared with the vehicle. In vivo, the tumor growth was slightly reduced in MIM-seq-treated animals. Moreover, MIM-seq could slightly reduce the growth of our spheroid models, especially under serum-deprivation. When MIM-seq was combined with two well-known anti-cancerogenic agents, either resveratrol or etoposide, MIM-seq could even further reduce the spheroid’s volume, pointing up the need to further assess whether MIM-seq could be beneficial for CRC patients as an adjuvant therapy. Altogether, these data suggest that MIM-seq could have anti-tumor properties against CRC and an immunomodulatory effect towards the mediators of inflammation, whose systemic dysregulation is considered to be a poor prognosis for patients.
... Selective cyclooxygenase-2 antagonists; nonsteroidal anti-inflammatory drugs; and anti-rheumatic drugs such as methotrexate, sulfasalazine, and auranofin have been adopted at the early onset of rheumatoid arthritis to arrest disease progression [9][10][11]. Meanwhile, TNF-α antagonists and IL-1 and IL-6 receptor antagonists can also prevent novel anti-rheumatic rheumatoid arthritis [12][13][14][15][16][17]. Proteoglycan-4 (PRG4) is a mucous glycoprotein secreted from synoviocytes and is known to exhibit strong anti-inflammatory activity in binding with TLR2 and TLR4 in activated cells isolated from patients with rheumatoid arthritis and osteoarthritis [18]. ...
... Based on the in vitro results, more diverse inflammatory cytokines such as TNF-α, IL-6, and IL-1β were considered in an in vivo model. Among the various cytokines expressed in the synovial membrane, TNF-α and IL-1β, which are produced by macrophages and are known as upper cytokines that induce lower inflammatory mediators such as IL-6, various chemokines, and proteases amplify inflammation and cause damage to joints by promoting the proliferation of synovial cells [12][13][14][15][16][17]. TNF-α and IL-1β cytokines can be observed at high concentrations in the synovial fluid of rheumatoid arthritis patients and promote the production and secretion of metalloproteinase, prostaglandin, and NO in various cells while inhibiting the production of matrix components, thereby reducing inflammation and damage in joints [7][8][9]. ...
... In addition to stimulating the secretion of inflammatory mediators from synovial cells, it triggers systemic effects such as fever, muscle wasting, and loss of appetite. The expression levels of IL-6, TNF-α, and IL-1β among inflammatory cytokines induced by LPS were significantly decreased with the administration of the sample [10][11][12][13]. Among the study samples, it was found that the CTE/SKE 2:1 ratio displayed the best anti-rheumatoid arthritis effects both in vitro and in vivo. ...
Full-text available
Cudrania tricuspidata extracts (CTE) and Stewartia koreana extracts (SKE) are viable drugs for managing inflammation. We investigated the nitric oxide levels of CTE and a mixture of CTE/SKE (CTE mix) against lipopolysaccharide-induced RAW264.7 cells. In addition, we administered the CTE and CTE mix to mice with collagen-induced arthritis to confirm an anti-inflammatory effect against rheumatoid arthritis. We analyzed arthritis symptoms by oral administration of CTE mix using a CIA-induced animal model and analyzed the inhibitory activity of NO production with in vitro experiments. Both the CTE and CTE mix decreased nitric oxide levels, and a 2:1 ratio of CTE mix was most effective in vivo among the varying ratios of CTE mix tested. The spleen size increased by about 2.1 times, and the lymph node size decreased by about 2.5 times relative compared to the vehicle group. In blood biochemical analyses, tumor necrosis factor–α levels decreased by about three times, interleukin-1β and interleukin-6 levels were reduced by about eight times and three times, and PRG4 expression levels were increased by about 2.5 times relative to the vehicle group. We suggest that the CTE mix was superior to CTE alone and has potential as an anti-inflammatory treatment for patients with rheumatoid arthritis.
... Micro-immunotherapy (MI) medicines are homeopathic medicinal products that include signaling molecules, such as cytokines, in their formulations [20][21][22]. All MI medicines have their composition expressed as centesimal Hahnemannian dilution (CH). ...
... That process, also called SKP, has been repeated until reaching the desired centesimal dilution used to impregnate the pillules (4 CH, in the current study). Labo'Life employs the SKP process to manufacture MI medicines, both unitary and complex medicines, as previously described [21][22][23]68]. ...
Full-text available
As a cytokine, gamma-interferon (IFN-γ) is considered a key player in the fine-tuned orchestration of immune responses. The extreme cellular sensitivity to cytokines is attested by the fact that very few of these bioactive molecules per cell are enough to trigger cellular functions. These findings can, at least partially, explain how/why homeopathically-prepared cytokines, and especially micro-immunotherapy (MI) medicines, are able to drive cellular responses. We focused our fundamental research on a unitary MI preparation of IFN-γ, specifically employed at 4 CH, manufactured and impregnated onto sucrose-lactose pillules as all other MI medicines. We assessed the IFN-γ concentration in the medium after dilution of the IFN-γ (4 CH)-bearing pillules and we evaluated in vitro drug responses in a wide range of immune cells, and in endothelial cells. Our results showed that IFN-γ (4 CH) stimulated the proliferation, the activation and the phagocytic capabilities of primary immune cells, as well as modulated their cytokine-secretion and immunity-related markers’ expression in a trend that is quite comparable with the well-recognized biological effects induced by IFN-γ. Altogether, these data provide novel and additional evidences on MI medicines, and specifically when active substances are prepared at 4 CH, thus suggesting the need for more investigations.
... In this context, micro-immunotherapy (MI), which is a therapeutic approach aiming at gently modulating the body's homeostasis by maintaining and/or restoring immune system functions [11][12][13][14], could be a great asset in the management of respiratory infectious diseases. All the MI medicines' formulations combine different types of active ingredients that can be classified according to their roles and characteristics as follows: (1) chemical mediators directly involved in immune responses, such as cytokines; (2) other signaling molecules playing important roles in cell-cell communication under physiological and pathological conditions, such as growth factors, hormones and neuropeptides; and (3) nucleic acids, either under their original state (total DNA and RNA, extracted from Pinus halepensis and Foeniculum vulgare, respectively) or under the form of specific nucleic acids (SNA) [11][12][13][14][15][16]. ...
... In this context, micro-immunotherapy (MI), which is a therapeutic approach aiming at gently modulating the body's homeostasis by maintaining and/or restoring immune system functions [11][12][13][14], could be a great asset in the management of respiratory infectious diseases. All the MI medicines' formulations combine different types of active ingredients that can be classified according to their roles and characteristics as follows: (1) chemical mediators directly involved in immune responses, such as cytokines; (2) other signaling molecules playing important roles in cell-cell communication under physiological and pathological conditions, such as growth factors, hormones and neuropeptides; and (3) nucleic acids, either under their original state (total DNA and RNA, extracted from Pinus halepensis and Foeniculum vulgare, respectively) or under the form of specific nucleic acids (SNA) [11][12][13][14][15][16]. The SNA therapeutic innovation developed by Labo'Life and protected by the European Patent EP0670164B1 consists of single-stranded DNA molecules (16-44 bases) specifically designed to target one or more gene and/or transcript sequences, according to paired-base complementarity. ...
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This study aimed at evaluating the effects of the micro-immunotherapy medicine (MIM) 2LEID, both in vitro and in vivo, on several components of the innate and adaptive immune system. MIM increased the phagocytic activity of macrophages, and it augmented the expression of the activation markers CD69 and HLA-DR in NK cells and monocytes/macrophages, respectively. The effect of MIM was evaluated in a model of respiratory infection induced by influenza A virus administration to immunocompetent mice in which it was able to improve neutrophil recruitment within the lungs (p = 0.1051) and slightly increased the circulating levels of IgM (p = 0.1655). Furthermore, MIM stimulated the proliferation of CD3-primed T lymphocytes and decreased the secretion of the immunosuppressive cytokine IL-10 in CD14+-derived macrophages. Human umbilical vein endothelial cells were finally used to explore the effect of MIM on endothelial cells, in which it slightly increased the expression of immune-related markers such as HLA-I, CD137L, GITRL, PD-L1 and ICAM-1. In conclusion, the present study suggests that MIM might be a promising nonspecific (without antigen specificity) immunostimulant drug in preventing and early treating respiratory infections, but not only exclusively, as it would gently support several facets of the immune system and host defenses.
... Eight of the remaining 36 studies were excluded, 10-17 leaving 17 publications related to GUNA products, 18-34 eight to LABO'LIFE products, [35][36][37][38][39][40][41][42] two to Boiron products 43,44 and one to a product manufactured by Laboratorios Medicor 45 as the principal basis for this scoping review. ...
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Background Signaling molecules such as cytokines and interleukins are key mediators for the immune response in responding to internal or external stimuli. Homeopathically prepared signaling molecules have been used therapeutically for about five decades. However, these types of products are not available in many countries and their usage by homoeopaths is also infrequent. The aim of this scoping review is to map the available pre-clinical and clinical data related to the therapeutic use of homeopathically prepared signaling molecules. Methods We conducted a scoping review of clinical and pre-clinical studies of therapeutically used signaling molecules that have been prepared in accordance with an officially recognized homeopathic pharmacopoeia. Articles in peer-reviewed journals reporting original clinical or pre-clinical research of homeopathically prepared signaling molecules such as interleukins, cytokines, antibodies, growth factors, neuropeptides and hormones, were eligible. Non-English language papers were excluded, unless we were able to obtain an English translation. An appraisal of eligible studies took place by rating the direction of the outcomes on a five-point scale. The quality of the papers was not systematically assessed. Results Twenty-eight eligible papers, reporting findings for four different manufacturers' products, were identified and reviewed. Seventeen papers reported pre-clinical studies, and 11 reported clinical studies (six experimental, five observational). A wide range of signaling molecules, as well as normal T-cell expressed specific nucleic acids, were used. A majority of the products (21 of 28) contained two or more signaling molecules. The most common clinical indications were psoriasis, vitiligo, rheumatoid arthritis, respiratory allergies, polycystic ovary syndrome, and herpes. The direction of the outcomes was positive in 26 papers and unclear in two papers. Conclusion This scoping review found that there is a body of evidence on the use of homeopathically prepared signaling molecules. From a homeopathy perspective, these substances appear to have therapeutic potential. Further steps to explore this potential are warranted.
... Moreover, the secreted level of TNF-α was also reduced at concentrations ranging from 2.25 to 22 mM in the same conditions, as well as the IL-6 secretion, at 11 and 22 mM. Furthermore, the effects of the treatment have been evaluated in vivo, in a CIA mice model, in which a daily treatment respecting the sequential order of 2LARTH ® was administered by oral gavage, starting 30 days after the first immunization [95]. Results showed its efficacy in reducing the clinical score, the degree of edema and the inflammation in treated animals, compared with the control ones. ...
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Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are two cytokines involved in the perpetuation of the chronic inflammation state characterizing rheumatoid arthritis (RA). Significant advances in the treatment of this pathology have been made over the past ten years, partially through the development of anti-TNF and anti-IL-1 therapies. However, major side effects still persist and new alternative therapies should be considered. The formulation of the micro-immunotherapy medicine (MIM) 2LARTH® uses ultra-low doses (ULD) of TNF-α, IL-1β, and IL-2, in association with other immune factors, to gently restore the body’s homeostasis. The first part of this review aims at delineating the pivotal roles played by IL-1β and TNF-α in RA physiopathology, leading to the development of anti-TNF and anti-IL-1 therapeutic agents. In a second part, an emphasis will be made on explaining the rationale of using multiple therapeutic targets, including both IL-1β and TNF-α in 2LARTH® medicine. Particular attention will be paid to the ULD of those two main pro-inflammatory factors in order to counteract their overexpression through the lens of their molecular implication in RA pathogenesis.
... [9][10][11] Micro-immunotherapy (MI), is a therapeutic approach that uses active substances such as cytokines, hormones, growth factors, neuropeptides, nucleic acids, and specific nucleic acids (SNAs) at LD and ultra-low doses (ULD) to target the immune system and regulate immune responses in diseases, thereby reducing side effects and safety concerns. [17][18][19][20][21] This therapy is administered sublingually in the oral cavity, which could be interesting as adjunctive treatment in the maintenance phase of supportive periodontal therapy or as preventive treatment 22,23 , due to the proximity to gingival tissues, allowing to act both locally and systemically. ...
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Background: We aimed to evaluate the effect of low doses (LD) bone morphogenetic protein-2 (BMP2) and BMP4 micro-immunotherapy (MI) in two in vitro models of periodontal wound healing/regeneration. Methods: We first evaluated the effect of LD of BMP2 and BMP4 MI on a 2D cell culture using human gingival fibroblasts (hGF) under inflammatory conditions induced by IL1β. Biocompatibility, inflammatory response (Prostaglandin E2 (PGE2) release), collagen deposition and release of extracellular matrix (ECM) organization-related enzymes (matrix metalloproteinase-1 (MMP1) and metalloproteinase inhibitor 1 (TIMP1)) were evaluated after short (3 days) and long-term (24 days) treatment with BMP2 or BMP4 MI. Then, given the results obtained in the 2D cell culture, LD BMP4 MI treatment was evaluated in a 3D cell culture model of human tissue equivalent of gingiva (GTE) under the same inflammatory stimulus, evaluating the biocompatibility, inflammatory response and effect on MMP1 and TIMP1 release. Results: LD BMP4 was able to decrease the release of the inflammatory mediator PGE2 and completely re-establish the impaired collagen metabolism induced by IL1β treatment. In the 3D model, LD BMP4 treatment improved tissue viability compared to the vehicle, with similar levels to 3D tissues without inflammation. No significant effects were observed on PGE2 levels nor MMP1/TIMP1 ratio after LD BMP4 treatment, although a tendency to decrease PGE2 levels was observed after 3 days. Conclusions: LD BMP4 MI treatment shows anti-inflammatory and regenerative properties on human gingival fibroblasts, and improved viability of 3D gingiva under inflammatory conditions. LD BMP4 MI treatment could be used on primary prevention or maintenance care of periodontitis. This article is protected by copyright. All rights reserved.
The organ systems are specialized tissues that perform discrete functions. They are vital and thus have interdependencies. For instance, the heart and renal system both need and influence each other. When deficiency requirements of health and damaging factors lead to a weakened state, then the beginning of disease commences. This is at first hypofunction, then disordered circulation and communication in that tissue or organ. This further leads to inflammation, immune involvement, and eventually, fibrosis and drastic decline of function. Therapy involves addressing deficient requirements of health. More specific therapy involves supporting biochemical function, hormetic stimulation in the low-dose zone, and whole person therapy. These all help to increase adaptive responses of the body. Alternatively and often in combination, there are ways to dampen maladaptive responses and even to introduce some measure of homeostasis by external means. This is why standard medical therapy can often be successful, as it can externally create a balance or at least dampen maladaptive responses. But this may not lead to healing if the body is severely depleted and dysregulated.KeywordsDysfunctionNaturopathic treatmentDiagnosisWhole person approaches
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Background: Tumor necrosis factor-α (TNF-α) and IL-1β are 2 pro-inflammatory cytokines known to be involved in rheumatic diseases. The therapeutic strategy used in micro-immuno- therapy (MI) to reduce chronic inflammation and attenuate pain consists in mainly targeting these 2 cytokines. 2LARTH® is a sublingually administered medicine consisting of lactose- saccharose globules impregnated with ethanolic preparations of immune mediators and nucleic acids at ultra-low doses. Purpose: The aim of the study is to explore the effect of the MI medicine on TNF-α and IL-1β secretion in human primary enriched monocytes exposed to lipopolysaccharide (LPS). Materials and methods: Placebo and active globules were diluted in culture medium to test 5 lactose-saccharose globules concentrations (from 1.75 to 22 mM). Freshly isolated enriched monocytes from 6 healthy donors were treated with or without LPS (10 ng/mL), LPS+ placebo, or LPS+ 2LARTH® for 24 hours. IL-1β, TNF-α, and IL-6 release were evaluated by ELISA. Results: The medicine has significantly decreased the level of IL-1β secretion compared with placebo at these concentrations: 22 mM (P<0.0001), 11 mM (P=0.0086), 5.5 mM (P= 0.0254), and compared with untreated LPS control at these concentrations: 22 mM, 11 mM (P=0.0008), and 5.5 mM (P=0.002). The effect of active globules on the reduction of TNF-α release is sig- nificant compared with placebo at these concentrations: 22 mM (P=0.0018), 11 mM (P=0.0005), 5.5 mM (P=0.0136), and compared with untreated LPS control at these concentrations: 22 mM (P=0.0021), 11 mM (P=0.0017), 5.5 mM (P=0.0052) and 2.25 mM (P=0.0196). Besides, IL-6 secretion decreased compared with placebo at 22 mM (P=0.0177) and 11 mM (P=0.0031). Conclusion: The results indicate that the tested product exerts significant anti-inflammatory effects on human LPS-stimulated monocytes. Keywords: ultra-low doses, hormesis, chronic inflammation, rheumatic diseases
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This paper tells the story of how hormesis became recognized as a fundamental concept in biology, affecting toxicology, microbiology, medicine, public health, agriculture, and all areas related to enhancing biological performance. This paper assesses how hormesis enhances resilience to normal aging and protects against a broad spectrum of neurodegenerative, cardiovascular, and other diseases, as well as trauma and other threats to health and well-being. This paper also explains the application of hormesis to several neurodegenerative diseases such as Parkinson’s and Huntington’s disease, macrophage polarization and its systematic adaptive protections, and the role of hormesis in enhancing stem cell functioning and medical applications.
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Inflammatory bowel diseases (IBD) are associated with an altered systemic immune response leading to inflammation-mediated damage to the gut and other organs. Oral immune therapy is a method of systemic immune modulation via alteration of the gut immune system. It uses the inherit ability of the innate system of the gut to redirect the systemic innate and adaptive immune responses. Oral immune therapy is an attractive clinical approach to treat autoimmune and inflammatory disorders. It can induce immune modulation without immune suppression, has minimal toxicity and is easily administered. Targeting the systemic immune system via the gut immune system can serve as an attractive novel therapeutic method for IBD. This review summarizes the current data and discusses several examples of oral immune therapeutic methods for using the gut immune system to generate signals to reset systemic immunity as a treatment for IBD.
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Background: The activity of one of the major catechins in Green Tea, the polyphenol (-)-epigallocatechin-3-gallate (EGCG), has been shown to have a variety of health benefits. Recent studies suggest that EGCG can modulate both the innate and adaptive arms of the immune system. The goal of the current studies was to examine the immunomodulatory effects and mechanisms of action of EGCG on experimental arthritis in mice. Methods: EGCG (10 mg/kg) was administered by oral gavage after CIA induction, while control mice were administered phosphate buffered saline (PBS). Disease mechanisms were studied in both groups of mice. Phenotypes were examined using repeated measure analysis of variance (ANOVA) and data from in vitro and ex vivo experiments were analyzed for significance using the Mann-Whitney U test. Results: EGCG treatment ameliorated clinical symptoms and reduced histological scores in arthritic mice. Serum type-II collagen-specific immunoglobulin (Ig) IgG2a antibodies were significantly lower in EGCG-fed mice compared to PBS-treated mice. EGCG significantly suppressed T cell proliferation and relative frequencies of CD4 T cells, CD8 T cells and B cell subsets including marginal zone B cells, T1 and T2 transitional B cells, while increasing the frequency of CD4(+) Foxp3(+) regulatory T cells (Tregs) and indoleamine-2,3-dioxygenase (IDO) expression by CD11b(+) dendritic cells (DC). Splenic CD11b(+) DC from EGCG fed mice induced an increased frequency of Tregs via an IDO-dependent mechanism in in vitro cultures. Importantly, joint homogenates from EGCG-fed mice exhibited significantly increased levels of Nuclear Factor, Erythroid 2-Like 2 (Nrf-2) and Heme oxygenase-1 (HO-1) compared with PBS-fed mice. Conclusions: This is the first report of upregulation of the Nrf-2 antioxidant pathway in EGCG-mediated immunoregulation. EGCG ameliorated experimental arthritis in mice by eliciting IDO-producing DCs, increasing frequencies of T regs and inducing the activation of the Nrf-2 antioxidant pathway. It remains to be established whether EGCG is useful for the prevention and treatment of rheumatoid arthritis and other inflammatory disorders.
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Advances have been made in identifying genetic susceptibility loci for autoimmune diseases, but evidence is needed regarding their association with prognosis and treatment response. To assess whether specific HLA-DRB1 haplotypes associated with rheumatoid arthritis (RA) susceptibility are also associated with radiological severity, mortality, and response to tumor necrosis factor (TNF) inhibitor drugs. The Norfolk Arthritis Register (NOAR; 1691 patients and 2811 radiographs; recruitment: 1989-2008; 2008 as final follow-up) was used as a discovery cohort and the Early Rheumatoid Arthritis Study (421 patients and 3758 radiographs; recruitment: 1986-1999; 2005 as final follow-up) as an independent replication cohort for studies of radiographic outcome. Mortality studies were performed in the NOAR cohort (2432 patients; recruitment: 1990-2007; 2011 as final follow-up) and studies of treatment response in the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate cohort (1846 patients enrolled at initiation of TNF inhibitor; recruitment: 2006-2010; 2011 as final follow-up). Longitudinal statistical modeling was performed to integrate multiple radiograph records per patient over time. All patients were from the United Kingdom and had self-reported white ancestry. Sixteen HLA-DRB1 haplotypes defined by amino acids at positions 11, 71, and 74. Radiological outcome using the Larsen score (range: 0 [none] to 200 [severe joint damage]) and erosions of the hands and feet on radiographs, all-cause mortality, and treatment response measured by change in Disease Activity Score based on 28 joint counts and European League Against Rheumatism (EULAR) response. Patients with RA and valine at position 11 of HLA-DRB1 had the strongest association with radiological damage (OR, 1.75 [95% CI, 1.51-2.05], P = 4.6E-13). By year 5, the percentages of patients with erosions of the hands and feet were 48% of noncarriers (150/314) of valine at position 11, 61% of heterozygote carriers (130/213), and 74% of homozygote carriers (43/58). Valine at position 11 also was associated with higher all-cause mortality in patients with inflammatory polyarthritis (hazard ratio, 1.16 [95% CI, 1.03-1.31], P = .01) (noncarriers: 319 deaths in 1398 patients over 17 196 person-years, mortality rate of 1.9% per year; carriers: 324 deaths in 1116 patients in 13 208 person-years, mortality rate of 2.5% per year) and with better EULAR response to TNF inhibitor therapy (OR, 1.14 [95% CI, 1.01-1.30], P = .04) (noncarriers: 78% [439/561 patients] with moderate or good EULAR response; heterozygote carriers: 81% [698/866]; and homozygote carriers: 86% [277/322]). The risk hierarchy defined by HLA-DRB1 haplotypes was correlated between disease susceptibility, severity, and mortality, but inversely correlated with TNF inhibitor treatment response. Among patients with RA, the HLA-DRB1 locus, which is associated with disease susceptibility, was also associated with radiological severity, mortality, and treatment response. Replication of these findings in other cohorts is needed as a next step in evaluating the role of HLA-DRB1 haplotype analysis for management of RA.
Rheumatoid Arthritis (RA) is an autoimmune systemic disorder of unknown etiology and is characterized by chronic inflammation and synovial infiltration of immune cells. RA is associated with decreased life expectancy and quality of life. The research on RA is greatly simplified by animal models that help us to investigate the complex system involving inflammation, immunological tolerance and autoimmunity. The animal models of RA with a proven track record of predictability for efficacy in humans include: collagen type II induced arthritis in rats as well as mice, adjuvant induced arthritis in rats and antigen induced arthritis in several species. The development of novel treatments for RA requires the interplay between clinical observations and studies in animal models. However, each model features a different mechanism driving the disease expression; the benefits of each should be evaluated carefully in making the appropriate choice for the scientific problem to be investigated. In this review article, we focus on animal models of arthritis induced in various species along with the genetic models. The review also discussed the similarity and dissimilarities with respect to human RA.
Objectives: To determine possible differences in serious adverse effects among the 10 currently approved biological and targeted synthetic DMARDs (b/ts-DMARDs) for RA. Methods: Systematic review in bibliographic databases, trial registries and websites of regulatory agencies identified randomized trials of approved b/ts-DMARDs for RA. Network meta-analyses using mixed-effects Poisson regression models were conducted to calculate rate ratios for serious adverse events (SAEs) and deaths between each of the 10 drugs and control (i.e. no b/ts-DMARD treatment), based on subjects experiencing an event in relation to person-years. Confidence in the estimates was assessed by applying the Grading of Recommendations Assessment, Development and Evaluation approach (GRADE). Results: A total of 117 trials (47 615 patients) were included. SAEs were more common with certolizumab compared with abatacept (rate ratio = 1.58, 95% CI: 1.18, 2.14), adalimumab (1.36, 95% CI: 1.02, 1.81), etanercept (1.60, 95% CI: 1.18, 2.17), golimumab (1.45, 95% CI: 1.00, 2.08), rituximab (1.63, 95% CI: 1.16, 2.30), tofacitinib (1.44, 95% CI: 1.03, 2.02) and control (1.45, 95% CI: 1.13, 1.87); and tocilizumab compared with abatacept (1.30, 95% CI: 1.03, 1.65), etanercept (1.31, 95% CI: 1.04, 1.67) and rituximab (1.34, 95% CI: 1.01, 1.78). No other comparisons were statistically significant. Accounting for study duration confirmed our findings for up to 6 months' treatment but not for longer-term treatment (6-24 months). No differences in mortality between b/ts-DMARDs and control were found. Based on the GRADE approach, confidence in the estimates was low due to lack of head-to-head comparison trials and imprecision in indirect estimates. Conclusion: Despite low confidence in the estimates, our analysis found potential differences in rates of SAEs. Our data suggest caution should be taken when deciding among available drugs. Systematic review registration number: PROSPERO CRD42014014842.
Objective: To develop a new evidence-based, pharmacologic treatment guideline for rheumatoid arthritis (RA). Methods: We conducted systematic reviews to synthesize the evidence for the benefits and harms of various treatment options. We used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology to rate the quality of evidence. We employed a group consensus process to grade the strength of recommendations (either strong or conditional). A strong recommendation indicates that clinicians are certain that the benefits of an intervention far outweigh the harms (or vice versa). A conditional recommendation denotes uncertainty over the balance of benefits and harms and/or more significant variability in patient values and preferences. Results: The guideline covers the use of traditional disease-modifying antirheumatic drugs (DMARDs), biologic agents, tofacitinib, and glucocorticoids in early (<6 months) and established (≥6 months) RA. In addition, it provides recommendations on using a treat-to-target approach, tapering and discontinuing medications, and the use of biologic agents and DMARDs in patients with hepatitis, congestive heart failure, malignancy, and serious infections. The guideline addresses the use of vaccines in patients starting/receiving DMARDs or biologic agents, screening for tuberculosis in patients starting/receiving biologic agents or tofacitinib, and laboratory monitoring for traditional DMARDs. The guideline includes 74 recommendations: 23% are strong and 77% are conditional. Conclusion: This RA guideline should serve as a tool for clinicians and patients (our two target audiences) for pharmacologic treatment decisions in commonly encountered clinical situations. These recommendations are not prescriptive, and the treatment decisions should be made by physicians and patients through a shared decision-making process taking into account patients' values, preferences, and comorbidities. These recommendations should not be used to limit or deny access to therapies.
Collagen-induced arthritis (CIA) is a common autoimmune animal model used to study rheumatoid arthritis (RA). The development of CIA involves infiltration of macrophages and neutrophils into the joint, as well as T and B cell responses to type II collagen. In murine CIA, genetically susceptible mice (DBA/1J) are immunized with a type II bovine collagen emulsion in complete Freund's adjuvant (CFA), and receive a boost of type II bovine collagen in incomplete Freund's adjuvant (IFA) 21 days after the first injection. These mice typically develop disease 26 to 35 days after the initial injection. C57BL/6J mice are resistant to arthritis induced by type II bovine collagen, but can develop arthritis when immunized with type II chicken collagen in CFA, and receive a boost of type II chicken collagen in IFA 21 days after the first injection. The concentration of heat-killed Mycobacterium tuberculosis H37RA (MT) in CFA also differs for each strain. DBA/1J mice develop arthritis with 1 mg/ml MT, while C57BL/6J mice require and 3-4 mg/ml MT in order to develop arthritis. CIA develops slowly in C57BL/6J mice and cases of arthritis are mild when compared to DBA/1J mice. This protocol describes immunization of DBA/1J mice with type II bovine collagen and the immunization of C57BL/6J mice with type II chicken collagen.