Vitiligo: characterization of melanocytes in repigmented skin after punch grafting.

BACKGROUND: Punch grafting is a surgical technique mainly applied in therapy-resistant, stable and circumscribed vitiligo. OBJECTIVE: (i) To characterize in detail the features of the repigmented skin among punch grafts; and (ii) to correlate the ex vivo results with clinical data and punch grafting outcome. METHODS: We evaluated by immunohistochemistry and image analysis the expression of a panel of specific melanocyte markers including HMB45, MITF, c-kit, MART-1 and TRP1, the proliferation marker Ki67 and the cell-cell adhesion molecule E-cadherin in tissue samples collected from nine patients after punch grafting. RESULTS: Cells positive for MITF, c-kit, MART-1 and TRP1 were detected in the repigmented skin of all biopsies, whereas no reactivity was observed for HMB45. Melanocytes were identified along the entire length of the sections, and their mature state was assessed by the immuno-reactivity for the differentiation marker MART-1, the absence of cells positively stained for Ki67 and by the co-expression of c-kit and TRP1, a marker of a differentiated and pigmented state. Clinically, smaller punch grafts aimed at repigmenting lesional areas on the face gave the faster clinical results with no side-effects. Patients subjected to bigger punch grafts on the knee exhibited a longer repigmentation time and presented cobble stoning. CONCLUSION: Our results suggest that the repigmentation observed in the areas between the grafts is due to the activation of the melanocytes located in the donor sites. These cells start to horizontally migrate towards the lesional skin thanks to successively the enlargement of intercellular spaces in relation to a decrease of E-cadherin reactivity and the up-modulation of pro-melanogenic mediators. Production and transfer of melanin in the surrounding keratinocytes and their persistence were assessed by the reactivity for MITF, c-kit, MART-1 and TRP1 but not for the pre-melanosome marker (HMB45).

A scanning electron microscopy study of the invasion of leaflets of a bloat-safe and a bloat-causing legume by rumen microorganisms.

A newly developed technique using ruthenium red to detect foci of bacterial digestion in mounts of whole leaflets that had been incubated with rumen bacteria was used to compare the digestion of alfalfa, a bloat-causing legume, and sainfoin, a bloat-safe legume. When whole leaflets were suspended in an artificial rumen medium and inoculated with rumen bacteria, massive bacterial adhesion and proliferation were noted at the stomata of alfalfa leaflets after 6 h of incubation, whereas only a few isolated bacteria adhered near the stomata of sainfoin leaflets After 22 h of incubation, the epidermal layers of alfalfa leaflets had peeled away in many areas, revealing an extensive bacterial invasion of the underlying mesophyll tissue in which large bacterial microcolonies had formed in intercellular spaces, and in intracellular spaces in several areas where plant cell walls had broken down. After 22 h of incubation, the surface of sainfoin leaflets resembled that of alfalfa leaflets at 6 h, with bacterial microcolonies adhering to the area surrounding the stomata, but without sloughing of the epidermis. Uninoculated control leaflets of both species showed no surface alteration but part of their normal bacterial flora had proliferated to form microcolonies on the surface after 22 h incubation. Dry matter loss due to leaching or bacterial digestion when whole leaflets of legumes were suspended in an artificial rumen medium, alone or with rumen bacteria, was significantly higher in the bloat-causing group. Values of leaching and of bacterial digestion were positively correlated. We conclude that reported differences in plant anatomy, and in cell wall chemistry, produce distinct rates or organic nutrient release from legume leaflets, and that these same differences produce an equally distinct susceptibility of leaflets to bacterial invasion, plant cell rupture, and the consequent release of intracellular plant components. The rate of release of organic nutrients from legume leaflets may be important in the etiology of foamy pasture bloat. This technique of in vitro digestion of whole leaflets followed by ruthenium red staining shows some promise of providing a rapid and qualitative test to distinguish, within a species, cultivars that may differ in their bloat-related characteristics.

In vitro digestion of bloat-safe and bloat-causing legumes by rumen microorganisms: gas and foam production.

Leaves of three bloat-safe legumes -- birdsfoot trefoil (Lotus corniculatus L.), sainfoin (Onobrychis viciaefolia Scop.), and cicer milkvetch (Astralagus cicer L.) -- and of three bloat-causing legumes -- alfalfa (Medicago sativa L.), red clover (Trifolium pratense L.), and white clover (Trifolium repens L.) -- were incubated with strained rumen fluid or with mixed rumen fluid and solids. Gas released was measured during the early period (0 to 22 h) of this in vitro digestion. Gas volume was greater with a 1:1 (wt/vol) mixture of solid and fluid rumen contents than with rumen fluid alone. It was greater with whole and chewed leaves from the bloat-causing legumes than with whole leaves from the bloat-safe legumes. However, when leaves were homogenized, volumes of gas from bloat-causing and bloat-safe legumes were similar. More gas was released from homogenized leaves than from the same weight of whole leaves. The amount of foam produced on chewed herbage and homogenized leaves of bloat-causing legumes was greater than on those of bloat-safe legumes. These results are consistent with the rate of disintegration and digestion of legumes by rumen bacteria being an important determinant in pasture bloat. Measurement of gas produced early in in vitro digestion may provide a useful bioassay for evaluating the bloat-causing potential of legumes in breeding selections if variability of the method can be reduced.