biological responses to diphencyprone treatment
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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 hair follicles.

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 samples.

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 treatment.

3. Survivin-
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 of samples.

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 May;150(5):940-8. PMID: 15149507
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