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

Abstract

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.

Full text

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.oris@labolife.com
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
Hindawi
International Journal of Rheumatology
Volume 2020, Article ID 1594573, 9 pages
https://doi.org/10.1155/2020/1594573

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
immunization:
(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
denaturation.
(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 (
=5
), CIA + vehicle (
=5
), and
CIA + MIM (
=5
).
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
measured.
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
ankylosed.
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
DAY 0
First collagen injection
(immunization)
DAY 21
Second collagen injection
(re-immunization)
DAY 14
Collagen-induced arthritis
(CIA) model
Start the examination of:
Weight
(i)
(ii)
(iii)
(iv)
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
𝑝
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.
1.0
1.5
2.0
2.5
3.0
3.5
14 22 27 34 38 44
Days
Wrist thickness (mm)
Control CIA
(a)
14 22 27 34 38 44
Days
VehicleMIM
#
$
$
1.5
2.0
2.5
3.0
3.5
4.0
∗∗
Ankle thickness (mm)
(b)
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
group.
∗
 < 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
Days
0.0
0.1
0.2
0.3
0.4
0.5
Increase of hind paw volume (ml)
#
∗∗
(a)
VehicleMIM
14 22 27 34 38 44
Days
#
0
1
2
3
4
Score
∗∗ ∗∗
(b)
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
vs.
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
MIM
JS
JS
C
C
C
C
200 µm
Vehicle
Control
CIA
Vehicle
MIM
0
1
2
3
4
Histological score
(a)
JS
JS
C
CC
C
MIM
Vehicle
Control
CIA
Vehicle
MIM
5
4
3
2
1
Cartilage area in pixel (1
× 105)
(b)
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
0
5
15
10
20
25
Co
ntrol
CIA
Vehicle
MI
M
IL-1β (pg/ml)
NS
(a)
0
10
20
30
40
CIA
Vehicle
Co
ntrol
MI
M
TNF-α (pg/ml)
(b)
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
paper.
Acknowledgments
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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Abbreviations
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
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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,

9International Journal of Rheumatology
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