# Citrus/Cydonia Comp. Can Restore the Immunological Balance in Seasonal Allergic Rhinitis-Related Immunological Parameters In Vitro

Authors: E. W. Baars; H. F. J. Savelkoul
Journal: Mediators of Inflammation
 (2008)

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## Abstract

In two in vitro studies, we examined the immunological (pathways of the) effects of Citrus/Cydonia comp. from, respectively, a healthy and an allergic donor; peripheral blood mononuclear cells (PBMCs) were isolated out of peripheral blood and analyzed in vitro after polyclonal stimulation of T-cells. The differentiation capacity and the influence with regard to Th1 (IFN-γ) and Th2 (IL-5) cells were examined. Citrus/Cydonia comp. has a selective effect on the differentiation of T-cells by producing relatively more IL-10 than IL-12. By that, it also seems to have an effect on the induction of regulatory (IL-10 producing) T-cell subsets. It is in vitro capable of neutralizing (to some extent) the changes, characteristic to allergic rhinitis, with regard to the maturation, differentiation, and activity of the immune system. Thus, Citrus/Cydonia comp. can potentially restore the disturbed immune state of rhinitis patients, which essentially could be sufficient to make allergic symptoms disappear permanently.


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## Body

## 1. Introduction

Allergic  rhinitis is  a condition characterized by sneezing, watery nasal
discharge, and nasal obstruction and itching. It is an increasingly prevalent
condition, particularly in the Western world where it affects around 20% of the
adult population. Allergic rhinitis is divided into seasonal allergic rhinitis
(hay fever) which is triggered by pollens and moulds, and perennial allergic
rhinitis in which house dust mites and pet dander are the predominant triggers.
The spectrum of severity is wide and includes a significant number of sufferers
with severe symptoms that are resistant to treatment with usual pharmacotherapy
(antihistamines and topical nasal corticosteroids) [1]. The mean prevalence of
allergic rhinitis in several Western countries is 12% [2].Seasonal allergic
rhinitis or hay fever is a type I immediate hypersensitivity reaction
mediated by specific IgE antibody to a seasonal allergen, leading to
mucosal inflammation characterized by sneezing, itching, rhinorrhoea,
and nasal blockage. Pollens (6–40μm in
diameter) from wind-pollinated grasses, trees, weeds, and spores
from fungi are the most common aeroallergens. Grass pollen is the
most common cause of seasonal allergic rhinitis. The highest levels of pollen
in the atmosphereare found in May to July and pollen concentrations
of 50 grains/mm3 are associated with symptoms in all
susceptible people. The treatment of choice of seasonal allergic rhinitis is
the symptomatic treatment with local or oral antihistamines and/or local corticosteroids.
Immunotherapy, including sublingual immunomodulation therapy, is indicated in a
limited subpopulation of patients [3].Citrus/Cydonia
comp. is an anthroposophic medicine, which contains extracts of lemon (Citrus
lemon) and quince (Cydonia oblongata) [4]. For over eighty years now, the
medicine “Citrus/Cydonia comp.” is being prescribed as a subcutaneous injection
or as a nasal spray for patients who suffer from seasonal allergic rhinitis. A
survey on clinical experiences, carried out among a group of 39 active Dutch
general practitioners [4], indicates that the subcutaneous treatment with
Citrus/Cydonia comp. ampoules is profoundly effective. Firstly, a permanent
effect from the treatment with Citrus/Cydonia comp. tends to be experienced,
which indicates that the patients in question are claiming to lastingly suffer
less from hay fever or even that they are free from complaints. Secondly, the
effect is occurring within a period of two weeks, up to three months, after the
actual treatment. Thirdly, the effect is optimal after a treatment of several
years. Moreover, the survey pointed out that preventive administration before
the start of the pollen season with Citrus/Cydonia comp. may be even more
efficacious to the patients in question. Recently, positive effects by Citrus/Cydonia comp. were obtained among a
group of 13 patients with the following characteristics: (a) allergic to grass
pollen, (b) suffering from hay fever, on average, for nine years, and (c) the
necessity for the use of antihistamines with regard to the nature of the
complaints [5]. In addition, a
prospective, observational study on the effect of Citrus/Cydonia comp. nasal
spray on hay fever symptoms reported positive results without side effects in
140 patients [6]. We now performed in vitro studies to study the possible immunological (pathways
of the) effects of Citrus/Cydonia comp.


## 2. Materials and Methods

### 2.1. Blood Donors

From a healthy and an allergic
individual, 8 mL of blood was collected in sodium heparinate-coated vacutainers
(BD Biosciences, San Diego, Calif, USA). The allergic individual was sensitized for birch pollen (RAST 6+) and grass pollen (RAST 4+), and he was also food allergic with a positive skin prick test on apple and cherry.
The blood was subsequently diluted 1:1 with IMDM containing GlutaMAX (IMDM;
Gibco-BRL, Paisley, Scotland)
before the density gradient centrifugation on Ficoll-Paque PLUS (Amersham
Biosciences, Uppsala, Sweden). The PBMC layer was washed
twice with IMDM and the cell viability and cell concentration were determined by
Trypan blue exclusion. An informed consent was obtained before the sample
collection and the performed experiments were approved by the local ethical
committee.


### 2.2. Culture Conditions

PBMCs were cultured in Yssel’s
medium at37C° in a humidified atmosphere with 5%CO2 at a density
of 1⋅106 viable cells/mL.  Yssel’s medium consisted of
IMDM supplemented with 1% Penicillin-Streptomycin (Gibco BRL), extra additions according to Yssel et al. [1984], and 1% human AB serum
(Gibco BRL). Cells
were plated out in 48 well plates at a concentration of 1⋅106cells/mL and cultured at 37C°. After five hours of adaptation to the culture conditions, the various
stimuli or a matching volume of medium were added. Cultures were
stimulated with 150 ng/mL anti-CD3 plus 100 ng/mL anti-CD28monoclonal
antibodies (BD Pharmingen, San Diego, Calif, USA)
or cultured in medium only [7].


### 2.3. Citrus/Cydonia Comp. Stimulation

Both conditions (negative and
positive controls) took place in the presence of Citrus/Cydonia comp. 100μL/1 mL culture in two dilutions
(undiluted and 1:3 dilution in culture medium). The extract (Gencydo) was
obtained from (Weleda, Zoetermeer, The Netherlands).


### 2.4. Cell Viability

Half a million PBMCs were
washed and subsequently incubated with 2μL Annexin V-APC (BD Biosciences) in 200 μL
Annexin V buffer according to the manufacturer’s protocol. After an incubation
period of 15 minutes on ice, the cells were spun down (400 g for 10 minutes) and resuspended in 200 μL Annexin V buffer and 2 μL PI (1 mg/mL;
Sigma, St. Louis, Mo, USA). The cells
were then analyzed on a flow cytometer (FACS array, BD Biosciences).


### 2.5. Immunological Phenotype

The
immunological phenotype of PBMC subsets was determined by staining the surface
antigens with the following two monoclonal antibody (α) mixtures: (1) α-hCD3 (PE-Cy7), α-hCD4 (PE), α-hCD8 (APC), and α-hCD25 (APC-Cy7); (2) α-hCD3 (PE-Cy7), α-hCD14 (APC), α-hCD16 (PE), α-hCD19 (APC-Cy7), and α-hCD56 (PE). All antibodies were purchased at BD Biosciences.Per well,5⋅105cells were spun down in a 96 wells U-bottom plate. The cells were
incubated with staining buffer (1% FCS and 0.1 M NaN3 in PBS)
containing the surface markers or the matching isotype controls for 30 minutes
on ice in the dark. The cells were washed once with PBS and resuspended in PBS
for flow cytometry. The four-color flow cytometric acquisition was performed on
an FACS array, using the BD FACS-array software. An electronic gate was set to
exclude debris and at least 10 000 events/samples were acquired. The percentages
of positive cells were corrected for the isotype control.


### 2.6. Proliferation Capacity

The proliferation capacity of
the PBMC was studied by intracellular expression of the nuclear Ki-67 antigen
(Ki-67; BD Pharmingen). The Ki-67 antigen is absent in the nuclei of resting cells, but
present in all other phases of the cell division cycle as well as in the
mitosis phase [8]. In each well, 5⋅105PBMCs were
incubated with 100 μL Cytofix/Cytoperm (BD Pharmingen) for 15–20 minutes on ice
to fix and permeabilize the cells. Cells were washed twice with perm/wash
buffer (BD Pharmingen) and incubated with anti-Ki-67 PE
antibody, or the matched isotype control, diluted in perm/wash buffer for 30
minutes on ice in the dark. Hereafter, the cells were washed with perm/wash
buffer, resuspended in PBS, and measured on the flow cytometer. Values are
expressed as cells positive for the Ki-67 mAb corrected for the isotype
control.


### 2.7. Cytokines

PBMC culture supernatants were
analyzed for their IL-1β, IL-12, IFN-γ, TNF-α, IL-4, IL-5, IL-10, and IL-13 contents. The cytokine production was measured with
Cytometric Bead Assay Flex Sets (BD Pharmingen). All buffers used in this protocol were obtained
from the BD CBA Soluble Protein Master Buffer Kit (BD Pharmingen). Supernatants were collected, stored at −20C°, and tested
within 2 weeks. The procedure was performed according to the manufacturer’s
protocol. The samples were measured on the FACS array, using the FCAP software.
The sensitivity limits for quantitative determinations, according to the
manufacturer, were 1.1 pg/mL for IL-1β, 0.3 pg/mL for IL-4 and IFN-γ, 0.5 pg/mL for IL-5, 2.3 pg/mL for IL-10, 2.2 pg/mL for IL-12, 0.6 pg/mL for IL-13, and
0.7 pg/mL for TNF-α.


## 2.1. Blood Donors

From a healthy and an allergic
individual, 8 mL of blood was collected in sodium heparinate-coated vacutainers
(BD Biosciences, San Diego, Calif, USA). The allergic individual was sensitized for birch pollen (RAST 6+) and grass pollen (RAST 4+), and he was also food allergic with a positive skin prick test on apple and cherry.
The blood was subsequently diluted 1:1 with IMDM containing GlutaMAX (IMDM;
Gibco-BRL, Paisley, Scotland)
before the density gradient centrifugation on Ficoll-Paque PLUS (Amersham
Biosciences, Uppsala, Sweden). The PBMC layer was washed
twice with IMDM and the cell viability and cell concentration were determined by
Trypan blue exclusion. An informed consent was obtained before the sample
collection and the performed experiments were approved by the local ethical
committee.


## 2.2. Culture Conditions

PBMCs were cultured in Yssel’s
medium at37C° in a humidified atmosphere with 5%CO2 at a density
of 1⋅106 viable cells/mL.  Yssel’s medium consisted of
IMDM supplemented with 1% Penicillin-Streptomycin (Gibco BRL), extra additions according to Yssel et al. [1984], and 1% human AB serum
(Gibco BRL). Cells
were plated out in 48 well plates at a concentration of 1⋅106cells/mL and cultured at 37C°. After five hours of adaptation to the culture conditions, the various
stimuli or a matching volume of medium were added. Cultures were
stimulated with 150 ng/mL anti-CD3 plus 100 ng/mL anti-CD28monoclonal
antibodies (BD Pharmingen, San Diego, Calif, USA)
or cultured in medium only [7].


## 2.3. Citrus/Cydonia Comp. Stimulation

Both conditions (negative and
positive controls) took place in the presence of Citrus/Cydonia comp. 100μL/1 mL culture in two dilutions
(undiluted and 1:3 dilution in culture medium). The extract (Gencydo) was
obtained from (Weleda, Zoetermeer, The Netherlands).


## 2.4. Cell Viability

Half a million PBMCs were
washed and subsequently incubated with 2μL Annexin V-APC (BD Biosciences) in 200 μL
Annexin V buffer according to the manufacturer’s protocol. After an incubation
period of 15 minutes on ice, the cells were spun down (400 g for 10 minutes) and resuspended in 200 μL Annexin V buffer and 2 μL PI (1 mg/mL;
Sigma, St. Louis, Mo, USA). The cells
were then analyzed on a flow cytometer (FACS array, BD Biosciences).


## 2.5. Immunological Phenotype

The
immunological phenotype of PBMC subsets was determined by staining the surface
antigens with the following two monoclonal antibody (α) mixtures: (1) α-hCD3 (PE-Cy7), α-hCD4 (PE), α-hCD8 (APC), and α-hCD25 (APC-Cy7); (2) α-hCD3 (PE-Cy7), α-hCD14 (APC), α-hCD16 (PE), α-hCD19 (APC-Cy7), and α-hCD56 (PE). All antibodies were purchased at BD Biosciences.Per well,5⋅105cells were spun down in a 96 wells U-bottom plate. The cells were
incubated with staining buffer (1% FCS and 0.1 M NaN3 in PBS)
containing the surface markers or the matching isotype controls for 30 minutes
on ice in the dark. The cells were washed once with PBS and resuspended in PBS
for flow cytometry. The four-color flow cytometric acquisition was performed on
an FACS array, using the BD FACS-array software. An electronic gate was set to
exclude debris and at least 10 000 events/samples were acquired. The percentages
of positive cells were corrected for the isotype control.


## 2.6. Proliferation Capacity

The proliferation capacity of
the PBMC was studied by intracellular expression of the nuclear Ki-67 antigen
(Ki-67; BD Pharmingen). The Ki-67 antigen is absent in the nuclei of resting cells, but
present in all other phases of the cell division cycle as well as in the
mitosis phase [8]. In each well, 5⋅105PBMCs were
incubated with 100 μL Cytofix/Cytoperm (BD Pharmingen) for 15–20 minutes on ice
to fix and permeabilize the cells. Cells were washed twice with perm/wash
buffer (BD Pharmingen) and incubated with anti-Ki-67 PE
antibody, or the matched isotype control, diluted in perm/wash buffer for 30
minutes on ice in the dark. Hereafter, the cells were washed with perm/wash
buffer, resuspended in PBS, and measured on the flow cytometer. Values are
expressed as cells positive for the Ki-67 mAb corrected for the isotype
control.


## 2.7. Cytokines

PBMC culture supernatants were
analyzed for their IL-1β, IL-12, IFN-γ, TNF-α, IL-4, IL-5, IL-10, and IL-13 contents. The cytokine production was measured with
Cytometric Bead Assay Flex Sets (BD Pharmingen). All buffers used in this protocol were obtained
from the BD CBA Soluble Protein Master Buffer Kit (BD Pharmingen). Supernatants were collected, stored at −20C°, and tested
within 2 weeks. The procedure was performed according to the manufacturer’s
protocol. The samples were measured on the FACS array, using the FCAP software.
The sensitivity limits for quantitative determinations, according to the
manufacturer, were 1.1 pg/mL for IL-1β, 0.3 pg/mL for IL-4 and IFN-γ, 0.5 pg/mL for IL-5, 2.3 pg/mL for IL-10, 2.2 pg/mL for IL-12, 0.6 pg/mL for IL-13, and
0.7 pg/mL for TNF-α.


## 3. Results

### 3.1. Effect of Citrus/Cydonia Comp. on Healthy Donor PBMC

PBMCS were isolated and analyzed
for their subset composition. The percentages of the subsets PBMC were 63% CD3+
T-cells (with 50.4% CD4+ Th-cells and 12.6% CD8+ Tc-cells), 8% CD19+ B cells,
5% CD14+ monocytes, and 12% CD16/CD56+ NK cells. After one day (Table1) and
four days (Table 2) of culture, the results showed that Citrus/Cydonia comp. not only induced T-cell
proliferation directly, but also activated monocytes resulting in a selective
cytokine production (TNF-α, IL-1β, IL-10, and IL-12). However, Citrus/Cydonia
comp. induced more IL-10 than IL-12 production most likely derived from
monocytes, thereby stimulating the outgrowth of immunoregulatory (IL-10)
monocytes more than the immunoreactive (IL-12) subsets of monocytes. These monocyte-related
effects of Citrus/Cydonia comp. were detectable within one day and were found
in cultures with a normal therapeutic dose. Subsequently, T-cell activation and
proliferation were suprastimulated by Citrus/Cydonia comp. over the polyclonal
stimulation alone. Citrus/Cydonia comp. had no effect on cell survival and did
appear to be toxic for PBMC cell
subpopulations.Table 1
Mean scores after one
day (healthy donor).
MediumMedium + Citrus/Cydonia
comp. 1Medium + Citrus/Cydonia
comp. 1:3Proliferation (%)3(1)*13
(3)1
(1)Cell death (%)88
(12)82
(11)87
(5)Cytokines (pg/mL)TNF-α13
(4)3315
(129)13
(1)IL-1β25
(6)3535
(147)15
(2)IL-1022
(9)918
(52)12
(1)IL-1212
(3)46
(12)12
(2)IFN-γ15
(2)55
(8)25
(3)IL-410
(2)12
(1)10
(1)IL-512
(2)28
(3)10
(1)Percentages of proliferating
cells and cells in apoptosis of PBMC cultures of a healthy donor stimulated
for one day with medium or Citrus/Cydonia undiluted and 1:3 diluted. In the
supernatants of these cultures, cytokines were measured by flow cytometric
analysis in the Bead Assay Flex Sets system. Cytokine levels in pg/mL.*All
results are described with standard deviations (SDs).Table 2
Mean scores after four
days (healthy donor).
MediumAnti-CD3/28Medium + Citrus/Cydonia comp. 1Medium + Citrus/Cydonia comp. 1:3Stimulation + Citrus/Cydonia comp. 1Stimulation + Citrus/Cydonia comp. 1:3Proliferation (%)3(1)*42
(8)3
(1)1
(1)48
(11)40
(2)Cell death (%)88
(12)61
(11)87
(8)92
(11)65
(12)60
(6)Cytokines (pg/mL)TNF-α13
(2)8483
(987)15
(2)13
(2)9647
(733)8117
(566)IL-1β15
(3)413435
(11)15
(1)4858
(247)4037
(138)IL-1012
(2)955318
(8)12
(2)12276
(566)5662
(931)IL-1212
(1)8426
(9)12
(2)238
(87)185
(77)IFN-γ15
(2)3435515
(1)15
(2)48800
(12778)37750
(12501)IL-412
(2)13412
(1)12
(2)55
(12)106
(28)IL-512
(2)37512
(2)12
(1)118
(45)266
(56)Percentages of proliferating
cells and cells in apoptosis of PBMC cultures of a healthy donor stimulated
for four days with medium alone, polyclonal stimulation with anti-CD3 plus
anti-CD28 antibodies, or polyclonal
stimulation in the presence of Citrus/Cydonia preparation undiluted and 1:3
diluted. In the supernatants of these cultures, cytokines were measured by
flow cytometric analysis in the Bead Assay Flex Sets system. Cytokine levels
in pg/mL.*All results are described
with standard deviations (SDs).


### 3.2. Effect of Citrus/Cydonia Comp. on an Allergic Donor PBMC

After four days (Table3), the
results demonstrated that Citrus/Cydonia comp. was able to restore the reduced
IL-10 production in PBMC cultures of the allergic individuals. The stronger
immunoregulatory balance (IL-10) and a curbed augmented Th2 response (a
decrease of IL-4 and IL-5 production) was accompanied by an increased
production of IL-12 and a reduced production
of IFN-γ. Even after 4 days, monocyte
stimulation by Citrus/Cydonia comp. was detected by the production of TNF-α and IL-10.Table 3
Mean scores after four
days (allergic donor).
MediumStimulation with anti-CD3/28Stimulation + Citrus/Cydonia comp.Proliferation (%)1(1)*48
(12)57
(12)Cell death (%)78
(8)59
(16)55
(8)CytokinesTNF-α10
(1)5782
(154)6893
(738)IL-1β10
(1)2275
(339)3772
(665)IL-1010
(1)2331
(452)7634
(1299)IL-1210
(1)134
(26)335
(89)IFN-γ10 (1)9778
(452)11668
(1638)IL-410
(1)456
(87)138
(18)IL-510
(1)667
(154)227
(85)Percentages of proliferating
cells and cells in apoptosis of PBMC cultures of an allergic donor stimulated
for four days with medium alone, polyclonal stimulation with anti-CD3 plus
anti-CD28 antibodies, or in the
presence of Citrus/Cydonia undiluted preparation. In the supernatants of
these cultures, cytokines were measured by flow cytometric analysis in the Bead
Assay Flex Sets system. Cytokine levels in pg/mL.*All results are described with standard
deviations (SDs).


## 3.1. Effect of Citrus/Cydonia Comp. on Healthy Donor PBMC

PBMCS were isolated and analyzed
for their subset composition. The percentages of the subsets PBMC were 63% CD3+
T-cells (with 50.4% CD4+ Th-cells and 12.6% CD8+ Tc-cells), 8% CD19+ B cells,
5% CD14+ monocytes, and 12% CD16/CD56+ NK cells. After one day (Table1) and
four days (Table 2) of culture, the results showed that Citrus/Cydonia comp. not only induced T-cell
proliferation directly, but also activated monocytes resulting in a selective
cytokine production (TNF-α, IL-1β, IL-10, and IL-12). However, Citrus/Cydonia
comp. induced more IL-10 than IL-12 production most likely derived from
monocytes, thereby stimulating the outgrowth of immunoregulatory (IL-10)
monocytes more than the immunoreactive (IL-12) subsets of monocytes. These monocyte-related
effects of Citrus/Cydonia comp. were detectable within one day and were found
in cultures with a normal therapeutic dose. Subsequently, T-cell activation and
proliferation were suprastimulated by Citrus/Cydonia comp. over the polyclonal
stimulation alone. Citrus/Cydonia comp. had no effect on cell survival and did
appear to be toxic for PBMC cell
subpopulations.Table 1
Mean scores after one
day (healthy donor).
MediumMedium + Citrus/Cydonia
comp. 1Medium + Citrus/Cydonia
comp. 1:3Proliferation (%)3(1)*13
(3)1
(1)Cell death (%)88
(12)82
(11)87
(5)Cytokines (pg/mL)TNF-α13
(4)3315
(129)13
(1)IL-1β25
(6)3535
(147)15
(2)IL-1022
(9)918
(52)12
(1)IL-1212
(3)46
(12)12
(2)IFN-γ15
(2)55
(8)25
(3)IL-410
(2)12
(1)10
(1)IL-512
(2)28
(3)10
(1)Percentages of proliferating
cells and cells in apoptosis of PBMC cultures of a healthy donor stimulated
for one day with medium or Citrus/Cydonia undiluted and 1:3 diluted. In the
supernatants of these cultures, cytokines were measured by flow cytometric
analysis in the Bead Assay Flex Sets system. Cytokine levels in pg/mL.*All
results are described with standard deviations (SDs).Table 2
Mean scores after four
days (healthy donor).
MediumAnti-CD3/28Medium + Citrus/Cydonia comp. 1Medium + Citrus/Cydonia comp. 1:3Stimulation + Citrus/Cydonia comp. 1Stimulation + Citrus/Cydonia comp. 1:3Proliferation (%)3(1)*42
(8)3
(1)1
(1)48
(11)40
(2)Cell death (%)88
(12)61
(11)87
(8)92
(11)65
(12)60
(6)Cytokines (pg/mL)TNF-α13
(2)8483
(987)15
(2)13
(2)9647
(733)8117
(566)IL-1β15
(3)413435
(11)15
(1)4858
(247)4037
(138)IL-1012
(2)955318
(8)12
(2)12276
(566)5662
(931)IL-1212
(1)8426
(9)12
(2)238
(87)185
(77)IFN-γ15
(2)3435515
(1)15
(2)48800
(12778)37750
(12501)IL-412
(2)13412
(1)12
(2)55
(12)106
(28)IL-512
(2)37512
(2)12
(1)118
(45)266
(56)Percentages of proliferating
cells and cells in apoptosis of PBMC cultures of a healthy donor stimulated
for four days with medium alone, polyclonal stimulation with anti-CD3 plus
anti-CD28 antibodies, or polyclonal
stimulation in the presence of Citrus/Cydonia preparation undiluted and 1:3
diluted. In the supernatants of these cultures, cytokines were measured by
flow cytometric analysis in the Bead Assay Flex Sets system. Cytokine levels
in pg/mL.*All results are described
with standard deviations (SDs).


## 3.2. Effect of Citrus/Cydonia Comp. on an Allergic Donor PBMC

After four days (Table3), the
results demonstrated that Citrus/Cydonia comp. was able to restore the reduced
IL-10 production in PBMC cultures of the allergic individuals. The stronger
immunoregulatory balance (IL-10) and a curbed augmented Th2 response (a
decrease of IL-4 and IL-5 production) was accompanied by an increased
production of IL-12 and a reduced production
of IFN-γ. Even after 4 days, monocyte
stimulation by Citrus/Cydonia comp. was detected by the production of TNF-α and IL-10.Table 3
Mean scores after four
days (allergic donor).
MediumStimulation with anti-CD3/28Stimulation + Citrus/Cydonia comp.Proliferation (%)1(1)*48
(12)57
(12)Cell death (%)78
(8)59
(16)55
(8)CytokinesTNF-α10
(1)5782
(154)6893
(738)IL-1β10
(1)2275
(339)3772
(665)IL-1010
(1)2331
(452)7634
(1299)IL-1210
(1)134
(26)335
(89)IFN-γ10 (1)9778
(452)11668
(1638)IL-410
(1)456
(87)138
(18)IL-510
(1)667
(154)227
(85)Percentages of proliferating
cells and cells in apoptosis of PBMC cultures of an allergic donor stimulated
for four days with medium alone, polyclonal stimulation with anti-CD3 plus
anti-CD28 antibodies, or in the
presence of Citrus/Cydonia undiluted preparation. In the supernatants of
these cultures, cytokines were measured by flow cytometric analysis in the Bead
Assay Flex Sets system. Cytokine levels in pg/mL.*All results are described with standard
deviations (SDs).


## 4. Discussion

Here, we show that
Citrus/Cydonia comp. has a selective effect on the differentiation of T-cells
with regard to the production of cytokines; the production of IL-10 is
relatively larger than that of IL-12. By that, Citrus/Cydonia comp. also seems
to have an effect on the induction of regulatory (IL-10 producing) T-cell
subsets. Hence, as a consequence, Citrus/Cydonia comp. might be producing an
allergy reducing effect, at which it does not concern Th1 induction and the
reduction of the allergen-specific Th2 response, bearing the risk of induction
of a chronic inflammation and very likely even an increased risk for
autoimmunity.Recent
developments mainly concern the field of allergen-specific immunotherapeutic
protocols. This immunotherapy is widely believed to occur through restoration
of the disturbed Th1-Th2 balance [9–12], either
linked to the induction of allergen-specific (blocking) IgG4 antibodies, or to the
induction of regulatory T-cell subsets. The exact role which the regulatory T-cell
subsets play with regard to these mechanisms is not yet indistinct. With regard
to the development of these immunotherapeutic protocols, special allergen
preparations, obtained from purified natural or recombinant produced allergens,
are necessary and
need to be developed.Citrus/Cydonia
comp. is likely to induce more regulatory T-cells, whether CD4+CD25+Fosp3+
natural or antigen-induced IL-10 and/or TGF-b producing Tr-cells, that are,
therefore, very immunosuppressive, and which are capable of reducing allergen specifically
activated Th2 cells. Our results imply that Citrus/Cydonia comp. does not induce a complete state of
immunosuppression, resulting in a diminished resistance against infections and
a reduced protection against tumors. This is consistent with the long-term
clinical experiences and the results of the empirical studies on the use of
Citrus/Cydonia comp.Based on these in vitro investigations,
we hypothesized that Citrus/Cydonia
comp. is capable of neutralizing (to some extent) the changes, characteristic
to allergic rhinitis, with regard to the construction, the maturation, the
differentiation, and the activity of the immune system. By that, it is possible
to explain the therapeutic positive effects on allergic rhinitis patients
treated with Citrus/Cydonia comp. found in previous studies and clinical
practice.The conclusions
based on this study are of great importance, since the standard treatment of
allergic rhinitis is based on the long-term use of antihistamines, potentially
in combination with a local application of corticosteroids, in case of
persisting and/or serious symptoms. Those treatments tend to reduce the symptoms,
but they do not possess any immunotherapeutic potency themselves. This implies
that it is compulsory for individual patients to keep on using such medicines
for many years. Based on our pilot data, indicating that in vitro Citrus/Cydonia comp. is capable of modulating the Th1-Th2 balance,
we actually expect Citrus/Cydonia comp. to have an immunotherapeutic potency.
This adds to the clinical therapeutic effect from Citrus/Cydonia comp., both as
an injection and as a topical application. This implies that a long-term treatment
with Citrus/Cydonia comp. injections during several years, before the start of
the pollen season, can potentially restore the disturbed immune state of
rhinitis patients,  which essentially could be sufficient to make the allergic
complaints disappear.



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*Source: 496467-2008-11-30.xml*