Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2011, Article ID 212459, 10 pages
doi:10.1093/ecam/nep119
Original Article
Treatment with at Homeopathic Complex Medication Modulates
Mononuclear Bone Marrow Cell Differentiation
Beatriz Cesar,1 Ana Paula R. Abud,1 Carolina C. de Oliveira,1
Francolino Cardoso,2 Raffaello Popa Di Bernardi,1 Fernando S. F. Guimarães,1
Juarez Gabardo,1 and Dorly de Freitas Buchi1
1 Departamento
2 Instituto
de Biologia Celular, Setor de Ciências Biológicas, Universidade Federal do Paraná (UFPR), Curitiba, PR, Brazil
de Tecnologia do Paraná, Tecpar, Curitiba, Paraná, Brazil
Correspondence should be addressed to Dorly de Freitas Buchi, buchi@ufpr.br
Received 18 August 2008; Accepted 21 July 2009
Copyright © 2011 Beatriz Cesar et al. This 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.
A homeopathic complex medication (HCM), with immunomodulatory properties, is recommended for patients with depressed
immune systems. Previous studies demonstrated that the medication induces an increase in leukocyte number. The bone marrow
microenvironment is composed of growth factors, stromal cells, an extracellular matrix and progenitor cells that differentiate into
mature blood cells. Mice were our biological model used in this research. We now report in vivo immunophenotyping of total
bone marrow cells and ex vivo effects of the medication on mononuclear cell differentiation at different times. Cells were examined
by light microscopy and cytokine levels were measured in vitro. After in vivo treatment with HCM, a pool of cells from the new
marrow microenvironment was analyzed by flow cytometry to detect any trend in cell alteration. The results showed decreases,
mainly, in CD11b and TER-119 markers compared with controls. Mononuclear cells were used to analyze the effects of ex vivo
HCM treatment and the number of cells showing ring nuclei, niche cells and activated macrophages increased in culture, even in
the absence of macrophage colony-stimulating factor. Cytokines favoring stromal cell survival and differentiation in culture were
induced in vitro. Thus, we observe that HCM is immunomodulatory, either alone or in association with other products.
1. Introduction
Recently, great advances in conventional medicine have
included the discovery of powerful new drugs the requested
bibliographies such as 2-chloro-2-deoxyadenosine (CdA) for
use in patients with various types of cytopenia and opportunistic infections [1], and Decitabine, a cytosine analog
used at lower doses for the treatment of myelodysplastic
syndrome [2]. In addition, biomarker and drug studies have
aided in the development of personalized medicines [3].
Such new medications act to assist rapid patient recovery,
but with side effects, which reduce quality-of-life. In this
context, Conforti and collaborators in 2007 [4], comment
the action of conventional anti-inflammatory drugs and
compare with, homeopathic treatment who is used to
regulate the pathological excess of inflammation expressing
the natural healing dynamics and the efficacy of high dilution
of active natural substances. Previous studies demonstrated
that a homeopathic complex medication (HCM) activates
macrophages (MΦ) both in vivo and in vitro. It was observed
that the in vitro production of tumor necrosis factor-α
(TNF-α) by MΦ was significantly reduced when HCM
was administered [5]. NADPH oxidase activity increased
after HCM ingestion, as did that of inducible nitric oxide
synthetase (iNOS), resulting in production of reactive oxygen
species (ROS) and nitric oxide (NO), respectively [6].
HCM stimulated the endosomal/lysosomal system and the
phagocytic activity of MΦ interacting with Saccharomyces
cerevisiae and Trypanosoma cruzi epimastigotes [7]. The
modulatory effects of HCM were also observed both in vivo
and in vitro in experimental infections with Leishmania amazonensis and Paracoccidioides braziliensis; HCM controlled
infection progression and limited pathogen dissemination
[8, 9]. Moreover, HCM is neither toxic nor mutagenic [10].
Similarly, improvement in the immune response of mice
bearing Sarcoma-180 tumors was seen after HCM treatment.
In the cited study, a reduction in sarcoma size was shown
and lymphoid cells significantly infiltrated the tumors;
granulation tissue and fibrosis surrounded the sarcomas. All
animals of the treated group survived, and, in 30% of mice,
2
Evidence-Based Complementary and Alternative Medicine
complete tumor regression was observed. The total number
of leukocytes was increased by HCM treatment. Among
lymphocyte classes, T-CD4, B and natural killer (NK) cells
increased in number [11]. These results suggested that HCM
affected hematopoiesis either directly or indirectly. Bone
marrow cells were treated with HCM in vitro and examined
by light, scanning electron, and confocal microscopy. These
modalities, and also flow cytometry, indicated that cells of
the monocytic lineage (CD11b) and stromal cells (adherent
cells) were activated by HCM treatment, which also increased
cell clusters over adherent cells, suggesting that cell proliferation and differentiation were taking place [12, 13].
The microenvironment influences growth and differentiation of hematopoietic cells. Adherent cell layers elaborate
soluble factors and deposit extracellular matrix which, in
turn, influence hematopoietic proliferation and differentiation [14, 15]. The differentiation of monocytic cells
into monoblasts, promonocytes, and monocytes (Mo), is
stimulated by macrophage colony-stimulating factor (MCSF) [16]. This factor acts principally to stimulate the proliferation of progenitors committed to MΦ lineages [17–20].
Mo, tissue MΦ, dendritic cells (DCs), microglia and
osteoclasts all contribute to maintenance of tissue homeostasis and provide a first line of defense against invading
pathogens. These cell types are produced in bone marrow,
and undergo differentiation therein before being released to
peripheral blood. The recovery of myelopoiesis to normal
levels would contribute significantly to increased life-span
by preventing delayed severe side effects (e.g., secondary
infections) after chemotherapy. Provision to the periphery of
more Mo/MΦ, and normalization of bone marrow cellularity, might also permit more intensive patient treatment.
Thus, the aim of this study was to use in vitro, in vivo
and ex vivo techniques to examine whether HCM stimulates
a preferential response of the Mo/MΦ lineage in the place of
origin, the microenvironment surrounding progenitor cells.
2. Methods
2.1. Animals. Male Swiss mice of the Rockefeller lineage
(10–12 weeks of age) were kindly supplied by the Instituto
de Tecnologia do Paraná (TECPAR). Animals had free
access to food and water. All recommendations of the
National Law (No. 6.638; 5 November 1979) for scientific
animal management were observed and the Institutional
Animal Care Committee of the Universidade Federal do
Paraná approved all relevant practices. Experiments were
carried out at the Laboratory of Research in Neoplastic and
Inflammatory Cells, which has a management program for
animal residues.
2.2. Animal Treatment. The following groups were established:
Group 1: control group; mice did not receive any
treatment;
Group 2: mice treated with HCM
Group 3: mice treated with both HCM and M-CSF;
Group 4: positive control; mice treated with M-CSF.
Mice were subcutaneously treated at daily intervals for 7
days. Animals receiving HCM were given doses of 7 µL g−1
body weight per day. Mice of group 3 received 2.8 µL g−1
of HCM and 4.2 µL g−1 of M-CSF per day. These amounts
were calculated to reflect concentrations used in in vitro
experiments [9]. After 7 days of treatment animals were
euthanized by cervical dislocation and bone marrow cells
were obtained as described below. Cells were processed
following routine protocols. Experiments were performed at
least three times in quadruplicate, independently. A total of
100 animals were used.
2.3. Bone Marrow Preparation (In Vitro and In Vivo). After
cervical dislocation femurs were dissected and cleaned.
Epiphyses were removed and the marrow was flushed with
Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) and 1 µg mL−1
ciprofloxacin (all from Sigma Pharma, St. Louis, MO). Some
cells were used for immunophenotyping and the remaining
cells were purified on 1.077 Ficoll-Hypaque (FH) (Sigma).
This product is a solution of Ficoll and sodium diatrizoate
adjusted to a density of 1.077 g mL−1 . When blood is
overlaid and the solution is centrifuged, mononuclear cells
concentrate at the plasma-reagent interface [21]. For FH
purification, 10 mL of flushed bone marrow aspirate was
layered onto 3 mL of FH solution in a sterile 15 mL centrifuge
tube. The tube was capped and centrifuged in a tabletop
centrifuge at 1500 g for 40 min at room temperature. A
diffuse band of leukocytes (mononuclear cells) formed above
the erythrocytes and polymorphonuclear cells, which were
together in the pellet. This diffuse cell layer was aseptically
removed with a pipette and transferred to a sterile 15 mL
centrifuge tube. Cells were washed with phosphate buffered
saline (PBS) because Ficoll is toxic to cells. Mononuclear
cells were counted in a Neubauer chamber and suspended in
DMEM with 10% (v/v) FBS supplemented with 1 µg mL−1
ciprofloxacin and 4 mM l-glutamine (Sigma).
2.4. The HCM. HCM is a new immunomodulatory therapy
and formulation follows Hahnemann’s ancient homeopathic
techniques. HCM is an aqueous, colorless and odorless solution produced and sold in authorized drugstores in Brazil.
Mother tinctures are purchased from agencies authorized
by the Brazilian Health Ministry. These agencies assure the
quality (endotoxin-free) and physicochemical composition
of the product. Starting from the original mother tincture
(e.g., a plant ethanolic extract), several dynamizations
(succussion, or shaking, and dilution in distilled water) are
performed. Decimal dilutions (dH) are prepared. The final
commercial product is composed of 11 dH Aconitum napellus
(Ranunculaceae), 19 dH Thuya occidentalis (Cupressaceae),
18 dH Bryonia alba (Curcubitaceae), 19 dH Arsenicum album
(arsenic trioxide), 18 dH Lachesis muta (Viperidae), and
<1% (v/v) ethanol, in distilled water. We used the commercial product of Narciso da Lozzo Neto, batch number
CRF-PR 5604. A homeopathy principle is that substance
effects become stronger with dilution and dynamization.
Thus, dilution followed by succussion should increase drug
Evidence-Based Complementary and Alternative Medicine
potency. The medication was always vigorously succussed
immediately before use.
2.5. Preparation of L929-Conditioned Medium. M-CSF is
highly expressed by the L929 mouse cell line [22] and L929
cell-conditioned medium was used as a source of M-CSF for
cells cultured in plastic dishes in DMEM with 10% (v/v) FBS;
L-cell conditioned medium was added to 30% (v/v). As MCSF was not a mother tincture, a high dilution (as used for
HCM) was not employed, nor was succussion necessary.
To obtain L929 cell-conditioned medium, L929 cells were
seeded in culture bottles (150 cm2 ) to a density of 1 × 106
cells per bottle, and cultivated in DMEM, which is rich in
glucose, supplemented with 5% (v/v) FBS. The conditioned
medium was collected after 7 days, at which time the cells
were confluent. The medium was centrifuged to remove cells
and the supernatant was stored at −20◦ C until use. M-CSF
was stable under these conditions for more than 6 months.
Once thawed, aliquots were stored at 0◦ C to avoid the M-CSF
degradation that occurs during freeze-thaw cycles.
2.6. Liquid Culture (In Vitro). Cells were adjusted to a
concentration of 2.5 × 105 /mL, plated in 24-well culture
plates, and maintained at 37◦ C under a 95% air/5% CO2
atmosphere for 96 h. All experiments were performed at least
three times in quadruplicate; there were four animal groups.
Controls received no treatment, because our previous results
showed no statistical differences between a control group and
an ethanolic aqueous solution group. Cells of group 2 were
treated with 20% (v/v) HCM. The M-CSF groups received
30% (v/v) M-CSF medium and, in group 3, cells were treated
with both M-CSF medium and 20% (v/v) HCM. Groups
2 and 3 also received 1% (v/v) HCM daily, at precise 24 h
intervals.
2.7. Cytokine Quantification (In Vitro). After 96 h of culture adherent cells had increased in number. Supernatant
cytokine levels were measured using a mouse Th1/Th2
cytokine CBA kit (BD Pharmingen, USA), according to
the manufacturer’s instructions. The kit contains antibodies
against TNF-α, interferon-γ (IFN-γ), and interleukins 2,
4 and 5 (IL-2, IL-4, IL-5). Cytokine concentrations were
obtained by comparison of experimental data with standard
curves of the CBA program (Becton Dickinson). Fluorescence was measured using a FACSCalibur flow cytometer
(Becton Dickinson), equipped with an argon ion laser
(488 nm).
2.8. Immunophenotyping. This experiment was preliminary
in nature, and was performed to guide further work. After in
vivo treatment immunophenotyping was performed as soon
as cells were collected. Cells (106 ) were fixed with 1% (v/v)
paraformaldehyde, washed, and incubated with 0.5 µg mL−1
of the biotinylated antibodies listed below, in PBS for 40 min.
Cells were washed with PBS and incubated with 0.5 µg mL−1
phycoerythrin (PE)-labeled secondary antibody in PBS for
30 minutes [23]. Fluorescence was analyzed according to
3
Table 1
Antibody
CD11b (Mac-1)
Ly-6G
CD45R
CD11c
CD3
TER-119
Principal cell types marked
Monocytes/macrophages
Granulocytes
B-lymphocytes
Dendritic cells
T-lymphocytes
Erythrocytes
standard procedures using a FACSCalibur flow cytometer.
Data were analyzed by Cell Quest.
2.9. Surface Markers. All antibodies used were from a mouse
lineage panel, specific to bone marrow, and were purchased
from BD Pharmingen (see Table 1).
2.10. Ex Vivo Culture Conditions. Animals were treated as
described above and bone marrow cells were collected.
Mononuclear cells were purified, concentrated to 2.5 ×
105 cells/mL, plated in 24-well culture plates with glass
coverslips for adherent cell experiments, and maintained at
37◦ C under 95% air/5% CO2 for 24, 48, and 72 h. Groups 2
and 3 received 1% (w/v) HCM daily, precisely 24 h after the
preceding dose.
2.11. Morphological Assay. Cells (2.5 × 105 ) were plated into
culture plates with coverslips for morphological analysis [24]
and maintained as described above. After 24, 48 and 72 h,
cells were rinsed with PBS, fixed in Bouin, stained with
Giemsa, dehydrated and mounted with Entellan. Adherent
cells were observed by light microscopy using a Nikon Eclipse
E200. Structural characteristics of lymphocytes, resident
macrophages, activated macrophages, cell niches and cells
with ring-shaped nuclei, were sought. On each cover slip,
100 cells were examined. Ten cover slips for each treatment
and timepoint were prepared and analyzed. Mean data, in
percentages, were transformed as described below.
2.12. Statistical Analysis. In cytokines experiments, data
were analyzed with Cell Quest software, according to the
manufacturer’s instructions. ANOVA and the Tukey test
(significance: P < .05) were used for intergroup comparisons.
Percentage data, obtained
from light microscopy analysis,
√
were transformed to x + 0.5 values, to give normal distributions. Data were submitted to analysis of variance (ANOVA)
with a factorial diagram, randomly delineated, to determine
statistical significance. The Tukey test was performed when
an interaction effect was significant. The levels of significance
were taken to be ∗ P < .05 and ∗∗ P < .01.
3. Results
3.1. Cytokine Quantification (In Vitro). After 96 h of culture,
we evaluated the capacities of cells to produce TNF-α, IFN-γ,
IL-2, IL-4 and IL-5. TNF-α was significantly higher in groups
2 and 3 than in groups 1 and 4 (Figure 1).
4
Evidence-Based Complementary and Alternative Medicine
70
∗
Immunophenotyping in vivo total markers used
60
50
∗
(%)
Cytokines liberation in pg/mL
Cytokines quantification (in vivo)
200
180
160
140
120
100
80
60
40
20
0
40
30
20
10
0
Cd11b
Group 1
TNF-a
IFN-g
IL-5
Group 2
Group 3
Group 4
IL-4
IL-2
Figure 1: Cytokine quantification in vitro. Mice were euthanized
and mononuclear cells were obtained by Ficoll separation. Cells
were cultured and in vitro treated. After 96 h, supernatants were
collected and cytokine levels analyzed. These results are presented as
mean ± SEM. The significance of differences between mean values
was evaluated by two-way analysis of variance (ANOVA) followed
by Tukey test. ∗ P < .05 were considered statistically significant. Only
the TNF-α data are statistically significant at ∗ P < .05. Data are
representative of three independent experiments.
Group 1
Group 2
Cd11c
Cd3
Cd45r
Ly6G
Ter119
Group 3
Group 4
Figure 2: Immunophenotyping. An aliquot was used to analyze the
cells just after collecting. Mice were euthanized and bone marrow
cells were removed. Aliquots of freshly collected cells were analyzed
by flow cytometry. Group 1: control group; Group 2: mice treated
with HCM; Group 3: mice treated with HCM and MCSF; Group
4: mice treated with M-CSF. We show data on all markers used
in analysis. All bone marrow cells after in vivo treatment were
subjected to flow cytometry to detect any trend in cell alteration.
Decreases in CD11b and TER-119 markers in test groups, compared
to controls, may be noted.
3.3.1. Lymphocytes. Lymphocytes were characterized by their
classical morphology, showing rounded small nuclei with
condensed chromatin and a thin cytoplasm (Figures 3(a)
and 4(a)). After lymphocyte statistical analysis we showed
differences only when treatment and culture times were
separately compared. Lymphocyte numbers decreased over
48 h and the lower numbers were maintained to 72 h
(Figure 4). Group 3 tended to show fewer lymphocytes than
did other groups.
3.2. Immunophenotyping. After in vivo treatment, bone marrow cells were analyzed by flow cytometry. Cell populations
were similar to those seen by Civin and Loken in 1987
[25]. The mean percentage of each population is shown in
Figure 2. We observed that in vivo treatment with HCM
decreased the number of CD11b cells (in the Mo lineage).
Group 4 (M-CSF treatment) showed more CD11b cells than
did group 1 (control); this was expected, as M-CSF is a
growth factor specific for this lineage. When HCM was given
together with M-CSF (group 3), the data were similar to
those of group 2 treated only with HCM.
Dendritic cells (CD11c+ ) were reduced by HCM treatment compared to the control group (group 1). The other
groups (3 and 4) showed the same tendency. Granulocytes
(Ly6G+ ) and T-lymphocytes (CD3+ ) were apparently downregulated by HCM treatment, but the reduction was very
subtle. When HCM was administered together with M-CSF,
the reduction was not observed. B-lymphocytes (CD45R+ )
were diminished by HCM treatment in group 2, although
groups 3 and 4 also showed slight reductions. Erythrocytes
(TER-119+ ) were reduced only in the group treated with
HCM alone (group 2).
3.3.2. Resident MΦ. Resident MΦ were characterized by
classical fibroblast-like morphology with a central nucleus,
little cytoplasm with few extensions, resulting in an elongated (so not a spread) appearance. Such cells had small,
condensed and “kidney”-shaped nuclei (Figures 3(b) and
4(b)). As for lymphocytes, resident MΦ data analysis showed
differences only when treatment and culture times were
separately compared. We found that group 1 (control) had
more resident MΦ (mostly round, with fewer membrane
extensions); such cells were rarer in groups 2, 3 and
4. This corroborates previous work showing that HCM
activates MΦ, thus decreasing the number of resident MΦ.
Although growth factors were absent, we found that after
72 h fewer resident MΦ remained adherent to glass coverslips
(Figure 4).
3.3. Morphological Assay. Mononuclear cells obtained from
bone marrow of in vivo treated animals were observed
by light microscopy and characterized by cell morphology.
Each cell type was counted, and the data obtained were
analyzed statistically. Analysis was performed for each cell
type, treatment, and culture time. Adherent cells were
characterized as follows.
3.3.3. Activated MΦ. The morphological characteristics of
activated MΦ are increased membrane ruffling, increased
spreading and large and euchromatic nuclei (Figures 3(a),
3(b), and 4(c)). When we examined both treatment and
culture time, we observed that the numbers of activated MΦ
increased with time in all groups, but there was no significant
difference between treatment groups. The addition of HCM,
Evidence-Based Complementary and Alternative Medicine
5
LY
AM
AM
RM
(a)
(b)
MNC-LR
MNC-LR
PMNC-LR
(c)
(d)
NICHES
NICHES
(e)
(f)
Figure 3: Microphotography of mononuclear bone marrow cells. Animals were treated in vivo and bone marrow cells were collected.
Mononuclear cells were separated, concentrated to 2.5 × 105 cells/mL, plated on 24-well culture plates with glass coverslips as for the
adherent cell experiments, and maintained at 37◦ C under 95% air/5% CO2 for 24, 48 and 72 h. Cells were stained with Giemsa. Original
magnification: 40×. In (c) and (d), we show ring cells and in (e) and (f), we emphasize niches found in groups 2 and 3 after 48 h, respectively.
In (a) it is possible to compare lymphocytes (Ly) and activated macrophages (Am) in the positive control (group 4) and in (b) (group 1; the
negative control), resident macrophages (Rm) and active macrophages (Am) are seen (Bar = 10 Gm).
M-CSF, or both, to cultures, increased cell spreading and cell
density.
3.3.4. Cells with Ring-Shaped Nuclei. We found cells with
constricted ring-shaped nuclei and wide cytoplasmic centers.
We observed two such types of cells: polymorphonuclear-like
ring cells (PMN-LR) with lobular or constricted ring-shaped
nuclei and a cytoplasmic center larger than the width of
the ring (Figures 3(d) and 5(a)), and mononuclear-like ring
cells (MNC-LR) with ring-shaped nuclei of smooth contour
and cytoplasmic centers smaller than the width of the ring
(Figures 3(c), 3(d), and 5(b)). With this cell type, ANOVA
showed that all interactions were significant and the Tukey
test was performed. In groups 2 and 3 these cells appeared at
24 h and increased in number over the next 24 h. After 72 h
the numbers of such cells decreased in all groups these dates
are showed in Figure 5.
3.3.5. Cell Niches. Cell niches were associated with adherent
layers in small foci over stromal cells (Figures 3(e), 3(f), and
6(a)). After ANOVA analysis we found that all interactions
were significant and the Tukey test was performed to
6
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Lymphocytes (Ly), resident macrophages (RM) and active d macrophages (Am)
Am
Ly
Rm
100
(a)
90
(c)
Cellular percentual
80
(b)
70
60
50
40
30
20
10
0
G1-24 h G2-24 h G3-24 h G4-24 h
G1-48 h G2-48 h G3-48 h G4-48 h
G1-72 h G2-72 h G3-72 h G4-72 h
Figure 4: Lymphocytes (Ly), resident macrophages (Rm), and activated macrophages (Ac). Adherent cells were counted at three culture
times: 24, 48 and 72 h. The ex vivo treatments (G1-group1, G2-group2, G3-group 3 and G4-group 4) showed that lymphocyte and resident
macrophage numbers decreased with time of culture. However, activated macrophages had a tendency to increase. These results are presented
as mean ± SEM.
Cells with ring-shaped nuclei
∗∗
6
∗∗
PMN-LR
MNC-LR
5
4
(a)
2
(b)
∗∗
3
∗ ∗
∗
1
0
24 h
48 h
72 h
Cellular percentual
Cellular percentual
7
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Cellular niches
∗
∗
∗
∗
∗ ∗
24 h
Group 1
Group 2
Group 3
Group 4
Figure 5: Cells with ring-shaped nuclei (Ring-shaped nuclei cells
was removed). These adherent cells were counted. The ex vivo
treatment showed maintenance of this cell type upon culture and
the levels of such cells increased in 48 h of culture. The numbers
are perceptual means; ∗ P < .05 and ∗∗ P < .01. These results are
presented as mean ± SEM.
compare the means. Groups 2 and 3 showed a higher level
of cell niches at 24 h and 48 h, but this decreased with culture
time. These dates are showed in Figure 6.
For better representation of our results, we constructed a
schematic representation (Figure 7) to show the effect of the
medicament in all aspects analyzed.
4. Discussion
Hematopoietic cells proliferate in vivo and develop in
association with bone marrow stromal cells. Proliferation
(a)
48 h
72 h
Group 1
Group 3
Group 2
Group 4
Figure 6: Cellular niches. Cells in niches were counted among
adherent cells. We emphasize that this type of cell arrangement was
seen immediately upon plating after in vivo treatment. The niches
were maintained for 48 h, decreasing in number after 72 h. The
numbers are perceptual means; ∗ P < .05. These results are presented
as mean ± SEM.
and differentiation also occur in vitro, either in association
with stromal cells or in response to soluble growth factors.
Our in vitro studies have shown that HCM promotes growth
and differentiation of myelomonocytic cells in normal mice
[12, 13]. Stromal cells produce different types of growth
factors leading to the formation of microenvironments that
allow cell differentiation into specific lineages.
Homeopathic regulation can be obtained through the
similia principle in health body of what constitutes the
vital force and its possible dynamic alterations and could
Evidence-Based Complementary and Alternative Medicine
7
Bone marrow after HCM treatment
Differentiated
cell migration
to peripheral
blood
TNF-α
Cell niches and ringshaped nuclei cells
decreased after 72
hours
Cell niches (multiplication/
differentiation areas)
Stromal cells
CD45R+ cells
TER-119+ cells
Ring-shaped nuclei cells
(Progenitor cells)
CD3+ cells
CD11c+ cells
Ly6G+ cells
CD11b+ cells
Figure 7: After 96 h of in vitro treatment the TNF-α concentration has shown a significant increase. After in vivo treatment, it was decreased
the number of monocyte/macrophages cells (CD11b+), dendritic cells (CD11c+), granulocytes (Ly6G+), B lymphocytes (CD45R+), T
lymphocytes (CD3+) and specifically in group 2, erythrocytes (TER-119+). Cell niches and cells with ring-shaped nuclei were present in
the first 48 h, and after this diminished.
be translated in today’s terms as both homeodynamic and
communication disorders. Bellavite and collaborators [26]
mentioned that, when a factor of imbalance occurs a signal
is triggered and the system increases its activity producing a
greater amount of signal (e.g., IL-1, cytokines, interferons),
released from inflammatory exudate, thus bringing the
effector system (phagocytes or complement) back to its
normal homeodynamic, by eliminating a signal excess and
re-establishing condition (healing).
First, several proinflammatory cytokines and chemokines are simultaneously induced by viral and bacterial
products in infected or inflamed tissue, and cytokines
can co-operate even within a single cell type [27]. Subsequently, host-derived cytokines act synergistically to enhance
chemokine induction [28]. Alternatively, some inflammatory
chemokines may cooperate with constitutively produced
chemokines to influence the distribution of both immature
and mature hematopoietic cells, including cell release into
the circulation or homing to the bone marrow. Our results
showed an increase in TNF-α production when HCM
was added to cultures. TNF-α is an important cytokine
with hematopoietic-regulating activities, targeting specific
population(s) of bone marrow cells. TNF-α has pleiotropic
effects on hematopoiesis, depending on the target cells
involved, the developmental stage of target cells, or both [29].
Leukocyte production in the bone marrow is controlled
by inflammatory cytokines. Acting in concert, TNF-α and IL1 induce the loss of bone marrow lymphocytes by emigration while significantly expanding granulocyte production.
Zhang and collaborators, in 1995 [30], established that TNFα is a bifunctional regulator of hematopoiesis. A single dose
of the cytokine stimulated the growth of immature murine
myeloid progenitors, whereas daily injections induced a
slight decrease in such progenitors, accompanied by neutrophilia and lymphopenia. The effects of TNF-α on the
growth of hematopoietic stem and progenitor cells may
change, depending on the phase of the cell cycle. This
cytokine can suppress the growth of early hematopoietic progenitor cells in vivo and in vitro. We found elevated numbers
of cells with ring nuclei in groups 2 and 3, suggesting that
HCM stimulates the movement of granulocyte precursors, as
well as Mo, from bone marrow to the circulation.
Cells with ring nuclei were described as Mo/MΦ lineage
precursors by Biermann and colleagues in 1999 [31], not
only in rodents, but also in hamsters and humans. Patients
with mononucleosis and myeloproliferative disorders also
present with these cell types. Different types of cells with
ring nuclei were found within 24 h of HCM treatment,
including polymorphonuclear-like ring cells (PMN-LR) and
mononuclear-like ring cells (MNC-LR). The number of
8
cells with ring nuclei increased significantly after 48 h of
treatment, but diminished with time thereafter (Figure 5).
Similarly, it is important to note that immunophenotyping
showed that the specific granulocytic lineage marker (Ly6G)
increased in group 3 (Figure 2). Adhesion, during at least
one period of culture, is a feature differentiating Mo/MΦ
and PMN cells from other leukocytes. As ring nuclei cells
are of myeloid origin and are Mo/MΦ and PMN precursors,
we suggest that, after adhesion for a period between 24 and
48 h, differentiated PMN detach to the supernatant. The
MΦ precursors remain adherent, increasing the number of
activated MΦ in cultures treated for 72 h (Figure 4).
In ours morphological results, we observed cell clusters
over the adherent cells (Figure 6), suggesting sites of multiplication and differentiation called cell niches. These niches
are known to contain stem cells [32] and/or leukocytes committed with some cellular lineage [33, 34]. The adherent cell
clusters are dependent on direct cell-to-cell communication,
as well as cytokines and growth factors (colony forming)
[35]. In bone marrow erythropoiesis, erythroid cells are
organized in small anatomic units termed erythroblastic
islets, the islet cells differentiate into cells of the erythroid
series, and with MΦ, create these peculiar anatomic units.
Soni and co-workers, in 2007 [36], showed that the erythroblast macrophage protein (EMP) is responsible for
erythroblast linkage to MΦ on the erythroblastic islets. Each
islet is composed of one MΦ surrounded by erythroblasts
at different stages of maturation. Mature erythroblasts are
considered to move along the cytoplasmic extension of a
central MΦ toward the sinusoid [37].
Belon and collaborators, in 2007 [38], mentioned the
Arsenicum album administered in homeopathic doses
(Arsenicum album-30 and Arsenicum album-200) to
revert the alarmingly high incidence of elevated ANA
titers observed in random populations of high-risk arsenic
contaminated villages in West Bengal, India. Positive modulation was observed along with changes in certain relevant
hematological parameters, namely total count of red blood
cells and white blood cells, packed cell volume, hemoglobin
content, erythrocyte sedimentation rate and blood sugar
level, mostly within 2 months of drug administration. A
complex homeopathic medicine is produced from Aconitum
napellus, Thuya occidentalis, Bryonia alba, Lachesis muta
and Arsenicum album. It seems to enhance the individual’s
own immunity to trigger a particular immunologic response
against several pathological conditions [5–9, 11].
Even the Immunophenotyping results, were our preliminary experiment, it was demonstrated that HCM treatment
decreased expression of the TER-119 marker specific to the
erythrocytic lineage (Figure 2). There are two possible explanations for this reduction. Either the maturation process was
accelerated by HCM treatment (thus allowing cell migration
to the peripheral circulation) or immature cells adhered in
niches formed over stromal cells, thus evading detection
by flow cytometry. Chen and collaborators [39], genomic
approach (microarray analysis) found up-regulated genes are
the genes coding for heme hoxygenase HMOX-1 in responses
to feverfew extracts. HMOX-1 (heme oxygenase 1, also called
HO-1) not only is an essential enzyme in heme catabolism
Evidence-Based Complementary and Alternative Medicine
but also plays a critical role in cell migration. HMOX-1 can
mediate cell migration by regulating the expression of adhesion molecules. De Oliveira and co-workers [40] have shown
that HMOX-1 up-regulated in the microarray analysis.
Cells from the medullar microenvironment and MΦ
appear to establish close contact and to form a functional
relationship with erythrocytes. This is a key feature in
progenitor regulation and differentiation of all lineages,
including erythroid precursors [41]. Recent work showed
that after HCM treatment such cellular niches could be
seen in vitro cultures. Scanning electron microscopy demonstrated that MΦ maintained direct contact with the cells,
promoting cellular interaction with the extracellular matrix.
The contact regions were observed by transmission electron
microscopy as adhesion areas between cellular membranes,
and the space between any two cells in an adhesion area contained irregularly distributed extracellular particles, forming
a septate-like zone with electron-dense points, evidencing
the presence of molecules facilitating adhesion between
stromal cells [12, 13].
In 1996, Barbé and colleagues [42] showed that the
absence of resident MΦ resulted in immature erythrocyte
release to the bloodstream. It was also observed that MΦ
removal from bone marrow impeded erythroblast adhesion, corroborating the idea that MΦ are important in
erythroblast adhesion and differentiation. The identification
of niches in the present paper shows that HCM may
immunomodulate medullar erythropoiesis caused by MΦ
and stromal cells. The arrangement of cells in niches (islets)
over MΦ may be important for iron turnover, hemoglobin
synthesis, erythropoietin production, and phagocytosis of
expelled erythroblastic nuclei [33].
5. Conclusion
Our results show that in vitro, in vivo and ex vivo treatment
with HCM stimulated cytokines important in cell differentiation and survival of specific monocytic lineage cells and their
precursors. We hope these data will find clinical application
as a safe and feasible pharmacological treatment of isolated
mononuclear cells, to enhance therapeutic efficacy.
Funding
Coordenação de Aperfeiçoamento de Pessoal de Ensino
Superior-Capes, Secretaria do Estado da Ciência Tecnologia
e Ensino Superior-Seti e Instituto de Tecnologia do ParanáTecpar.
Acknowledgments
The authors thank TECPAR, SETI (PR) and CAPES for
financial support.
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