Biological responses to diphencyprone treatment
Diphencyprone is a potent contact allergen which is used
as an immunotherapeutic in treating Alopecia areata. This study
is focused on the effectiveness of this treatment and how the
hair follicles behave to the immunotherapy. The effects of Diphencyprone
on the expression patterns of VEGF, factor (F) VIII, surviving,
p16, CD4, CD8, CLA and CCL27 were studied pre and post treatment
from the biopsy samples of the skin of alopecic patients.
VEGF, or the vascular endothelial growth factor, plays an important role in
maintaining the integrity around the hair follicle. The vasculature or formation
of new blood vessels and their permeability are taken care of by this factor.
Histological studies have proved that in alopecia areata, the vasculature and
the associated elements are progressively disintegrated and hence study of VEGF
may be important in formulating new drugs.
Alopecic patients show progressive patterns of cell death that
lead to hair loss. Survivin and p16 are cell death inhibitors
and their levels in the cells increase with cell stimulation from
some drugs and treatments.
Cutaneous lymphocyte-associated antigen (CLA) and CCL27, a chemokine,
are responsible for maintaining the movement of lymphocytes into
the skin. Lymphocyte activation and cytokine release is affected
by Diphencyprone, the reason which these two parameters were included
in the study.
CD4+ and CD8+ are receptors carried on the surface of certain
T lymphocyte cells, B cells and macrophages. CD4 cells are T-cells
with CD4 receptors. They normally recognize antigens on the surface
of virus infected cells and secrete lymphokines that stimulate
B cells and killer T cells. CD8 cells are T–cells with CD8
receptors that recognize antigens on the surface of virus infected
cells and bind to the infected cells to kill them. The direct
association of these receptors to T-cells makes them a major focus
in alopecia areata as the condition involves increased T-lymphocyte
activity in the peripheral blood cells and in the skin around
The study involved 14 patients [5 men and 9 women] of whom 12
had patchy alopecia areata, 1 had reticular alopecia areata or
50-90% hair loss and 1 with alopecia totalis. The patients were
sensitized to Diphencyprone in acetone 2 weeks prior to actual
start of the treatment which lasted 3 months. According to the
patient’s reactivity to the allergen, they were treated
weekly once with Diphencyprone in such a way that the allergic
reaction could be maintained for 48 hrs after treatment. 5-6 mm
diameter pieces of skin were removed by punch biopsy using lidocaine
as the local anesthesia. Skin samples from 5 controls were used
for comparison. They were frozen in an embedding compound, cooled
in liquid nitrogen.
The immunohistochemical studies involved-
1. sectioning of the specimens on a cryotome
2. fixing in acetone for 10 minutes
3. incubating the sections at 4 deg centigrade overnight in different
monoclonal antibodies such as anti-VEGF 165, anti-FVIII, antisurvivin,
anti-p16, anti-CLA, anti-CD4 and anti-CD8.
4. Using the streptavidin-biotin peroxidase technique and incubating
with 3,3’-diaminobenzidine, all the sections were immunostained.
5. Counter staining was carried out using hematoxylin and the
specimens mounted on slides using paramount.
6. 3-amino-9-ethyl-carbazole was used as a substrate to evaluate
the expressions of CD4 and CD8. Colon tumor sections that had
already reacted with primary antibodies were used as positive
controls and non immune sera for negative controls.
7. All samples were evaluated using a light microscope.
8. The positive epithelial cells in all antibodies were counted
in different fields under the microscope while the immunological
expression was determined by the intensity of the staining patterns
and categorized as negative, no staining, faint, moderate, good
and strong staining.
9. The results were analyzed for statistical significance.
The study revealed the following:
1. VEGF –
In normal individuals, the staining pattern was heavy in the outer
sheath region of the hair follicle and the percentage of positive
cells was high. The staining of basal and suprabasal interfollicular
keratinocytes was good.
In alopecic individuals, the staining of the outer sheath was
poor and the percentage of positive staining cells low.
After treatment with Diphencyprone, the outer sheath showed good
staining and there was significant increase in the percentage
of positive stained cells, almost similar to the levels in normal
2. Factor VIII –
Vascular density of the skin was determined by the number of FVIII
and capillary vessels found in the samples. In comparison with
a normal individual the alopecic patients had highly downsized
vasculature which was restored to a great extent after Diphencyprone
The normal skin cells showed no immunostaining for surviving in
the cytoplasm or interfollicular keratinocytes of the healthy
hair follicle cells and its expression in endothelial cells
was minimal. In the group of 14 patients, 10 showed immunopositivity
in the outer sheath cells while in 4 others, the results were
distributed. Only 9 patients had some expression of survivin
in the interfollicular keratinocytes. After treatment, there
was a marked increase in survivin expression in the capillary
endothelial cells. There was no difference in the expression
of survivin on the follicular keratinocytes in the three categories
4. p16 –
While very little expression was seen in the cells lining the
hair follicle in normal individuals, alopecic patients showed
p16 in the cells of the outer root sheath and its levels increased
after treatment. However, the intensity of labeling decreased
in the interfollicular keratinocytes.
5. CD4 –
CD4 was present in the follicular epithelium both before and after
treatment while a high level was seen in the perifollicular
area as well. The levels of CD4 cells were high in affected
individuals and post treatment levels showed a marked decrease.
6. CD8 –
In alopecia areata, the number of CD8 cells was low (though still
higher than in non-alopecia areata affected skin) and were localized
in the inner and outer root sheath and the perifollicular dermis.
The numbers increased after treatment and the cells were found
predominantly in the outer sheath and the perifollicular dermis.
7. Cutaneous lymphocyte-associated antigen –
In normal individuals, the CLA positive lymphocytes were very
few around the hair follicles. The majority of the alopecic
patients were CLA positive and had 30% or more of lymphocyte
infiltration. Post treatment showed a further increase in CLA.
8. CCL27 –
Cytoplasm of the outer root sheath and the basal layer keratinocytes
in the interfollicular epidermis showed signs of CCL27 immunoreactivity.
In the patients 6 had mild positivity in the keratinocytes and
the rest were positive in the outer root sheath but the positivity
was very low in comparison with the normal scalp skin sections.
Only half the number of patients was seen to have immunoreactivity
in the basal interfollicular keratinocytes. Post treatment studies
showed a significant increase in the labeling intensity and
there was an increase in the expression distribution as well.
What does this study indicate?
It is a known fact that the hair follicles undergo a cyclic
growth pattern with anagen, catagen and telogen phases. For normal
cycles to occur, it is necessary that the follicles are supplied
with a good source of blood. Decrease in VEGF is a bad sign mainly
because it’s presence in normal levels is a stimulating
one for the formation of new blood vessels in the scalp area.
Factors such as Transforming growth factor-alpha and beta, tumor
necrosis factor alpha, Interleukin (IL)– 1 beta, IL-6, and
platelet derived growth factor-beta influence the expression of
VEGF. Since the treatment with Diphencyprone helped increase the
level of VEGF in alopecic patients, it can be inferred that a
pathway in regulating hair growth is resuscitated similar to the
one seen in normal individuals.
Similarly, an increase in FVIII after treatment indirectly indicated
that survivin protects the viability of the endothelial cells
while the angiogenesis process is underway, but Diphencyprone
had no direct role on the expression of survivin. The role of
p16 however still needs clarity and hence nothing much can be
inferred except consider a further study to identify the mechanism
with which Diphencyprone increases p16 in outer root sheath.
The CD4/CD8 ratio in alopecia areata was 4 before treatment
and after treatment it reduced to below 1 which indicates that
Diphencyprone does affect T-cell activation. CCL27 has a role
to play in cutaneous inflammation but how it helps in leukocyte
trafficking needs to be studied.
The way Diphencyprone reacted in different groups of individuals
gives us a general view of how the allergen can be considered
as a therapeutic in this highly complex disease and which keys
areas of alopecia areata need further study.
Biological responses to diphencyprone treatment references
- Simonetti O, Lucarini G, Bernardini ML, Simoncini C, Biagini
G, Offidani A. Expression of vascular
endothelial growth factor, apoptosis inhibitors (survivin and
p16) and CCL27 in alopecia areata before and after diphencyprone
treatment: an immunohistochemical study. Br J Dermatol. 2004