Efeito da pigmentação extrínseca nas cerâmicas à base de dissilicato de lítio submetidas aos processos de desgaste e biodegradação nas propriedades mecânicas e aderência bacteriana / Effect of extrinsic pigmentation on lithium disilicate-based ceramics subjected to wear and biodegradation processes on mechanical properties and bacterial adherence

O objetivo deste estudo foi avaliar se há alteração no comportamento mecânico e na aderência microbiológica da cerâmica à base de dissilicato de lítio com a técnica de pigmentação extrínseca aplicada, após ser submetida a diferentes condições experimentais. Foram confeccionadas 160 amostras, divididas em grupos com e sem pigmentação (n=80). Destes, cada grupo foi subdividido em Controle, Desgate, Biodegradação e Desgaste com Biodegradação (n=20).15 amostras de cada subgrupo foram submetidas ao teste de resistência à flexão e 5 para o teste de aderência microbiológica. As amostras passaram anteriormente por testes complementares para caracterização da superfície (rugosidade, perfilometria volumétrica e microscopia eletrônica de varredura (MEV)). Os resultados foram submetidos a análise estatística descritiva (média e desvio padrão) e inferencial, mediante o teste paramétrico de análise de variância (ANOVA) dois fatores e teste de Tukey ( = 0,05). O fator pigmentação extrínseca influenciou negativamente no comportamento mecânico da cerâmica, apresentando significância estatística (p = 0,000), assim como a interação entre o tipo de condição experimental e a pigmentação (p = 0,020). Entretanto, na aderência microbiológica, foi a condição experimental que influenciou negativamente no comportamento microbiológico (p = 0,000), assim como a interação entre a condição experimental e a pigmentação (p = 0,000). Nas análises complementares, observou-se que a interação entre os fatores aumentou a rugosidade superfial (p = 0,000) e aumentou o volume perdido pelo desgatse (p = 0,040). As microscopias da superficie mostram as características de cada grupo, mostrando as diferenças entre as condições experimentais com e sem pigmentação extrínseca. E as microscopias da aderência microbiológica ilustram e confirmam os resultados obtidos no teste estatístico. Concluiu-se que a pigmentação extrínseca altera as propriedades mecânicas da cerâmica de dissilicato de lítio, reduzindo a resistência à flexão e aumentando a rugosidade superficial e o desgaste. Porém, a aderência microbiológica foi aumentada pela condição experimental. Entretanto, a interação entre os fatores contribuiu para esse aumento e para agravar a alteração nas propriedades mecânicas(AU)

The objective of this study was to evaluate the mechanical and microbiological behavior of the ceramics based on lithium disilicate with extrinsic characterization. For this, 160 discs were made, divided into two large groups, with extrinsec characterization and without, after which each was divided into four groups (n = 20): Control, Wear, Biodegradation and Biodegradation with Wear. Fifteen samples from each group were submitted to the flexural strength test and 5 submitted to the microbiological adherence test. Prior to the destructive test of flexural strength, the representative samples of each group underwent complementary tests for surface characterization. The results were submitted to descriptive statistical analysis (mean and standard deviation) and inferential, using the parametric analysis of variance (ANOVA) two way and Tukey test ( = 0,05). The extrinsec characterization factor influenced the mechanical behavior of the ceramic, presenting statistical significance (p = 0.000), as well as the interaction between the type of experimental condition and the extrinsec characterization (p = 0.020). However, in the microbiological adherence, it was the experimental condition that influenced the microbiological behavior (p = 0.000), as well as the interaction between the experimental condition and the extrinsec characterization (p = 0.000). It was concluded that the makeup influenced the mechanical behavior of the ceramic, and the experimental condition influenced the microbiological adherence. The interaction between the factors influenced both the mechanical behavior and the microbiological adherence(AU)

Dermal mesenchymal stem cells (DMSCs) inhibit skin-homing CD8+ T cell activity, a determining factor of vitiligo patients' autologous melanocytes transplantation efficiency.

We here investigated the efficiency of autologous melanocyte transplantation of 23 vitiligo patients by focusing on perilesional skin homing CD8+ T lymphocytes, and studied the potential effect of dermal mesenchymal stem cells (DMSCs) on CD8+ T cell activities in vitro. Out of 23 patients with the autologous melanocyte transplantation, 12 patients (52.17%) had an excellent re-pigmentation, 6 patients (26.09%) had a good re-pigmentation, 5 patients (21.74%) had a fair or poor re-pigmentation. CD8+ T cells infiltrating was observed in the perilesional vitiligo area of all patients. Importantly, the efficiency of the transplantation was closely associated with skin-homing CD8+ T cell activities. The patients with high number of perilesional CD8+ T cells or high level of cytokines/chemokines were associated with poor re-pigmentation efficiency. For in-vitro experiments, we successfully isolated and characterized human DMSCs and skin-homing CD8+ T cells. We established DMSCs and CD8+ T cell co-culture system, where DMSCs possessed significant inhibitory effects against skin homing CD8+ T lymphocytes. DMSCs inhibited CD8+ T cells proliferation, induced them apoptosis and regulated their cytokines/chemokines production. Our results suggest that vitiligo patients' autologous melanocytes transplantation efficiency might be predicted by perilesional skin-homing CD8+ T cell activities, and DMSCs might be used as auxiliary agent to improve transplantation efficacy.

Biolistic DNA vaccination against melanoma.

We describe here the use of particle-mediated gene transfer for the induction of immune responses against melanoma antigens in murine tumor models using the melanocyte differentiation antigen tyrosinase-related protein 2 (TRP2) as an antigen in a murine B16 melanoma model. We have utilized marker genes such as ß-galactosidase (ßgal) and EGFP, which can be readily detected, as control antigens to establish the gene delivery and to detect antigen-specific humoral and cellular immune responses. After biolistic DNA vaccination with plasmids encoding the TRP2 gene we observed the induction of TRP2-specific T-cells and antibodies associated with vitiligo-like fur depigmentation and tumor immunity against B16 melanoma cells. Here we describe the preparation of cartridges with DNA-coated gold beads and the in vivo gene transfer into skin using the Helios Gene Gun system. We also describe protocols for the measurement of humoral and cellular immune responses against the melanocyte differentiation antigen TRP2. These protocols can subsequently be adapted to other antigens.

First histopathological and immunophenotypic analysis of early dynamic events in a patient with segmental vitiligo associated with halo nevi.

Segmental vitiligo is often ascribed to the neurogenic theory of melanocyte destruction, although data about the initial etiopathological events are scarce. Clinical, histopathological and T-cell phenotypic analyses were performed during the early onset of a segmental vitiligo lesion in a patient with associated halo nevi. Histopathological analysis revealed a lymphocytic infiltrate, mainly composed of CD8+ T-cells and some CD4(+) T-cells around the dermo-epidermal junction. Flow cytometry analysis of resident T-cells revealed a clear enrichment of pro-inflammatory IFN-gamma producing CD8+ T-cells in lesional skin compared to the non-lesional skin. Using human leukocyte antigen-peptide tetramers (MART-1, tyrosinase, gp100), increased numbers of T cells, recognizing melanocyte antigens were found in segmental vitiligo lesional skin, as compared with the non-lesional skin and the blood. Our findings indicate that a CD8+ melanocyte specific T cell-mediated immune response, as observed in generalized vitiligo, also plays a role in segmental vitiligo with associated halo nevi.

Selection for cuticular melanism reveals immune function and life-history trade-offs in Spodoptera littoralis.

Several insect species show an increase in cuticular melanism in response to high densities. In some species, there is evidence that this melanism is correlated with an up-regulation of certain immune system components, particularly phenoloxidase (PO) activity, and with the down-regulation of lysozyme activity, suggesting a trade-off between the two traits. As melanism has a genetic component, we selected both melanic and nonmelanic lines of the phase-polyphenic lepidopteran, Spodoptera littoralis, in order to test for a causative genetic link between melanism, PO activity and lysozyme activity, and to establish if there are any life-history costs associated with the melanic response. We found that, in fact, melanic lines had lower PO activity and higher lysozyme activity than nonmelanic lines, confirming a genetic trade-off between the two immune responses, but also indicating a genetic trade-off between melanism and PO activity. In addition, we found that lines with high PO activity had slower development rates suggesting that investment in PO, rather than in melanism, is costly.

Autoimmune etiology of generalized vitiligo.

Vitiligo is characterized by progressive skin depigmentation resulting from an autoimmune response targeting epidermal melanocytes. Melanocytes are particularly immunogenic by virtue of the contents of their melanosomes, generating the complex radical scavenging molecule melanin in a process that involves melanogenic enzymes and structural components, including tyrosinase, MART-1, gp100, TRP-2 and TRP-1. These molecules are also prime targets of the immune response in both vitiligo and melanoma. The immunogenicity of melanosomal proteins can partly be explained by the dual role of melanosomes, involved both in melanin synthesis and processing of exogenous antigens. Melanocytes are capable of presenting antigens in the context of MHC class II, providing HLA-DR+ melanocytes in perilesional vitiligo skin the option of presenting melanosomal antigens in response to trauma and local inflammation. Type I cytokine-mediated immunity to melanocytes in vitiligo involves T cells reactive with melanosomal antigens, similar to T cells observed in melanoma. In vitiligo, however, T cell tuning allows T cells with higher affinity for melanocyte differentiation antigens to enter the circulation after escaping clonal deletion in primary lymphoid organs. The resulting efficacious and progressive autoimmune response to melanocytes provides a roadmap for melanoma therapy.

Coexistence and relationship of antikeratinocyte and antimelanocyte antibodies in patients with non-segmental-type vitiligo.

To test for autoantibodies in patients with vitiligo, skin biopsies from 16 patients with active vitiligo and 12 patients with stable vitiligo were examined by direct immunofluorescence. In periodate-lysine-paraformaldehyde-fixed biopsy specimens, the presence of IgG deposits in keratinocytes and the number of keratinocytes with focal IgG in active vitiligo were significantly greater than in stable vitiligo. To test whether the antibodies to normal human keratinocytes or melanocytes are present in vitiligo, we used an indirect immunofluorescent test and enzyme-linked immunosorbent assay to test the serum of 43 patients. With unfixed viable melanocytes, we found a granular pattern of IgG staining on the plasma membrane of melanocytes incubated with patients' sera but not in cells incubated with the control sera. With methanol-fixed melanocytes, however, we found a homogeneous pattern of IgG staining in the cytoplasm of melanocytes. With unfixed viable keratinocytes as targets, there was no deposit of IgG on the cells. A homogeneous pattern of IgG binding in the cytoplasm of methanol-fixed keratinocytes suggested the presence of antikeratinocyte autoantibodies to cytoplasmic keratinocyte components. The fluorescence staining for IgG binding was more prominent in active or extensive vitiligo. Vitiligo sera were cytotoxic for melanocytes but not for keratinocytes in vitro. Antimelanocytic antibodies may play a role in melanocytotoxicity, whereas antikeratinocyte antibodies may occur secondary to cellular damage.