A randomized pilot study to compare hair follicle cell suspensions prepared using trypsin alone versus trypsin in combination with collagenase type I for transplantation in vitiligo.

BACKGROUND: Noncultured extracted hair follicle outer root sheath cell suspension (NC-EHF-ORS-CS) is an upcoming surgical technique to treat stable vitiligo. Conventionally it employs trypsin to tap the hair follicle (HF) reservoir for autologous melanocytes and their precursors for transplantation. However, a perifollicular dermal sheath composed of type 1 collagen encases the target 'bulge' region of the HF. Adding collagenase type 1 would digest the ORS, facilitating better release of cells. AIM: To compare the repigmentation achieved using trypsin and a combination of collagenase plus trypsin, respectively, with dermabrasion alone, and to compare cell counts, viability and composition of both suspensions. METHODS: This was a randomized, double-blind, comparative, therapeutic trial, conducted as a pilot study on 22 patients with stable vitiligo. Three similar patches were randomized into three parallel treatment arms [(A) trypsin plus collagenase, (B) trypsin alone and (C) dermabrasion with vehicle alone]. Each patient's HF sample was divided and digested by the two methods, and transplanted as suspensions onto dermabraded patches, while a third dermabraded patch received the vehicle only. Suspensions were sent for laboratory analysis. Repigmentation was assessed over a follow-up of 6 months. RESULTS: There was a significant increase in cell yield and comparable viability when collagenase was added. Immunohistochemical and flow cytometry studies showed a nonsignificant increase in HMB45+ melanocytes and their precursor stem cells in group A. This trend was reflected clinically in the extent of repigmentation [group A (33.22%) > B (24.31%) > C (16.59%); P = 0.13]. Adding collagenase induced significantly higher repigmentation than dermabrasion alone (P < 0.05). CONCLUSIONS: Incorporating collagenase type I into the conventional NC-EHF-ORS-CS technique resulted in enhanced retrieval of pigment-forming cells and subsequently improved repigmentation in vitiligo.

Alteration in regulatory T cells and programmed cell death 1-expressing regulatory T cells in active generalized vitiligo and their clinical correlation.

BACKGROUND: Vitiligo is an autoimmune depigmentation disease, and defects in regulatory T cells (Tregs) have been proposed in the pathogenesis of generalized vitiligo (GV). However, the role of programmed cell death (PD)1(+) Tregs has not been studied. OBJECTIVES: To investigate the status of Tregs, PD1(+) Tregs and associated parameters in active GV (aGV) during the first episode of disease attack and to establish the clinical correlation. METHODS: The percentages of circulating Tregs, PD1(+) Tregs and CD3(+) CD4(+) PD1(+) T cells were evaluated in 50 patients with aGV and 51 controls. Expression levels of FOXP3, TGFB1, CTLA4 and genes for chemokine receptors (CCR4, CCR7) and their ligands (CCL21, CCL22) were quantified in peripheral blood and in lesional, perilesional, nonlesional and normal skin sections. The corresponding proteins were immunolocalized in tissue of aGV. RESULTS: The percentage of Tregs was decreased (P = 0·001) and that of PD1(+) Tregs increased (P = 0·001) in peripheral blood of patients with aGV compared with controls. The abundance of TGFB1 and CCL21 mRNA was significantly decreased in the peripheral blood of patients with aGV. Significant differences in forkhead box P3, transforming growth factor-ß and CCL21 protein expression were found in skin sections. CONCLUSIONS: Deficiency in Treg frequency and decreased expression of Treg-associated parameters (TGFB and CCL21) suggested a possible defect in Tregs that may alter their suppression function and skin homing in aGV. The increased PD1(+) Tregs suggests that the PD1/PD ligand pathway may be involved in aGV and may have a role in Treg exhaustion. Further study is required to delineate the effect of PD1 in regulating Treg function in aGV.

T-helper and regulatory T-cell cytokines in the peripheral blood of patients with active alopecia areata.

BACKGROUND: Alteration in T-lymphocyte function and cytokines secreted by T-cell subsets has been proposed in the immunopathogenesis of alopecia areata (AA). The role of T-helper and regulatory T-cell cytokines in the pathogenesis of active AA has not been established. OBJECTIVES: To assess the role of hallmark cytokines of T-helper cells (Th1, Th2 and Th17) and regulatory T cells (Tregs) in the pathogenesis of AA, and its clinical correlation. METHODS: Fifty-one patients with AA and 45 age- and sex-matched healthy control subjects were included in the study. Serum interleukin (IL)-2, interferon (IFN)-γ, IL-10, IL-13, IL-17A and transforming growth factor (TGF)-ß1 were measured by enzyme-linked immunosorbent assay in both groups. Correlation of serum cytokine levels with age, sex, disease subtype and duration, number of patches on the scalp, associated autoimmune disorders and atopy was studied. RESULTS: The serum cytokine levels of IL-2, IFN-γ, IL-13 and IL-17A were significantly increased, and serum TGF-ß1 levels were significantly decreased (P < 0·05) in patients with AA compared with controls. Serum IL-2 levels were significantly different among AA subgroups (P < 0·05). IL-2 levels were positively correlated with the total disease duration and the number of patches on the scalp. CONCLUSIONS: The increased levels of serum IL-2, IFN-γ, IL-13 and IL-17A suggested altered T-helper cell function, and reduced serum TGF-ß1 levels suggested a defect in Treg function. Therefore, enhanced T-cell-mediated immunity and breakdown of immune tolerance due to deficiency in Tregs may facilitate the occurrence of AA.