Lesional activation of Tc 17 cells in Behçet disease and psoriasis supports HLA class I-mediated autoimmune responses.

BACKGROUND: Behçet disease (BD) presents with lymphocytic and neutrophilic vasculitis of unknown aetiology. HLA-B*51, the endoplasmic reticulum aminopeptidase 1 (ERAP1), and interleukin 23 receptor (IL23R)/IL12R are genetic risk factors. IL-23 regulates IL-17A, which controls the recruitment and activation of neutrophils. OBJECTIVES: To determine pathological changes in BD skin lesions related to the complex genetic predisposition. METHODS: We characterized the expression of IL-17A and IL-23A in various cell types by immunohistological double staining of sections from papulopustular skin lesions of acute attacks of BD and psoriasis vulgaris lesions, another HLA-class I-associated T-cell-mediated autoimmune disease in which excessive T-cell-derived IL-17A production promotes neutrophil activation. RESULTS: We found that in BD lesions, as in psoriasis, actively expanding CD8+ T cells were the predominant source of IL-17A. IL-17A+ CD8+ T (Tc 17) cells outnumbered infiltrating IL-17A+ CD4+ T cells. Unlike the epidermal localization of CD8+ T cells in psoriasis, Tc 17 cells in BD lesions mainly infiltrated the perivascular tissue and the blood vessel walls of dermis and subcutaneous tissue. They co-localised with a marked IL-23A expression by CD11c+ dendritic cells and CD68+ macrophages. IL-17A expression was associated with extensive recruitment of neutrophils around blood vessels that formed neutrophil extracellular traps (NETs). CONCLUSIONS: In BD, the genetic predisposition may mediate antigen-specific activation and differentiation of a Tc 17 response, possibly targeting endothelial (auto)antigens. Neutrophils recruited by IL-17A in this process may enhance tissue damage by extensive NET formation (NETosis). Thus, the IL-23/IL-17 axis presumably controls neutrophilic inflammation in BD vasculitis in the context of a predominant antigen-specific CD8+ T-cell response.

Detection of Merkel cell polyomavirus DNA in atypical fibroxanthoma in correlation to clinical features.

A clear etiopathogenetic concept for atypical fibroxanthoma (AFX) is not established yet. Nevertheless, AFX is known as a pleomorphic but indolent tumor primarily of the elderly and/or immunosuppressed patient occurring in severely sun- or radiation-damaged skin. These risk factors are almost identical to those of Merkel cell carcinoma (MCC), a highly malignant skin tumor being thought to be pathogenetically associated with the recently discovered Merkel cell polyomavirus (MCPyV). Because AFX and MCC share risk factors, the aim of this study was to evaluate presence of MCPyV DNA in 23 cases of AFX by PCR and direct DNA sequencing. Subsequently, we correlated clinical features with MCPyV DNA status in AFX. We detected MCPyV DNA in 4 of 23 AFX. All patients with MCPyV DNA-positive tumors were men. The mean age of patients with MCPyV DNA-positive AFX was 84.8 ± 8.7 years (vs. 75.2 ± 7.8 years of MCPyV DNA-negative AFX), the mean duration of tumor growth was 4.5 ± 2.3 months (vs. 5.1 ± 2.8 months) and the mean tumor diameter was 1.2 ± 0.3 cm (vs. 1.3 ± 0.7 cm). Ulceration was present in 75% of MCPyV DNA-positive tumors (vs. 65.2%). In conclusion, MCPyV DNA is present in 17% of AFX, in this cohort affecting predominantly male patients with higher age (>80 years). Clinical features seem to be independent of MCPyV DNA status. Although the role of MCPyV is unclear in this setting, it may act as a cofactor in the tumorigenesis of AFX in a subset of cases.

[Immunohistochemical and molecular-pathologic investigations in dermatohistology]. / Immunhistochemische und molekularpathologische Untersuchungen in der Dermatohistologie.

Despite sophisticated diagnostic algorithms, pure morphologic diagnosis has reached its limits in many areas of general and dermatologic pathology, especially in the wake of advances in basic sciences. Modern microscopic diagnosis, especially when evaluating lymphocytic and mesenchymal tumors, depends greatly on identifying the expression of surface markers (for example CD3 as T-cell surface receptor), signal proteins (cyclin D in cell cycle control) or structural proteins in tumor cells (actin in myogenous cells). Molecular biological methods include those techniques which make it possible to identify cellular and extracellular macro-molecules such as proteins and nucleic acids. At the protein level, the selective identification of proteins on sections via immunohistochemical methods is a widely used and essential component of modern pathologic-anatomic diagnosis.

[In situ PCR and PCR in situ hybridization of paraffin-embedded tissue. New diagnostic possibilities in pathology]. / In-situ-PCR und PCR-in-situ-Hybridisierung am Paraffingewebe. Neue diagnostische Möglichkeiten in der Pathologie.

In situ polymerase chain reaction (PCR) and PCR in situ hybridization are new diagnostic tools in dermatopathology. These techniques can combine the sensitivity of PCR with the advantage of in situ hybridization to localize specific cellular structures. With optical control of the PCR reaction product in these techniques, false-positive results due to contamination can be minimized. Several working protocols have been established which allow detection of DNA and RNA sequences in a rapid and reproducible manner. Two different methods have been established to detect the amplification product, the indirect PCR in situ hybridization (PCRisHyb), and the direct in situ PCR (isPCR). An easy and reproducible method for PCRisHyb and isPCR in formalin-fixed and paraffin-embedded tissue is described and the two techniques are compared. Using both isPCR and PCRisHyb, the amplification product can be visualized with an optimal morphological preservation of the tissue. Indirect PCRisHyb showed a slightly higher specificity whereas direct isPCR was the quicker, easier and less expensive method.

Automated high-resolution polymerase chain reaction fragment analysis: a method for detecting T-cell receptor gamma-chain gene rearrangements in lymphoproliferative diseases.

Southern blot analysis and the polymerase chain reaction (PCR) are powerful tools for detecting clonal antigen receptor gene rearrangements. However, a number of limitations restrict the predictive value of the results obtained by these techniques as they are commonly used. We describe a new method, automated high-resolution PCR fragment analysis, that can partially overcome many of the limitations of analyzing the T-cell receptor (TCR) gamma-chain gene. Analysis of TCR-gamma is performed using PCR with four sets of primers, previously described by others, specific for all variable (V) and joining (J) regions of the TCR gamma-chain gene. In addition, the four V region primers are 5' end-labeled with a fluorescent compound, 5-carboxyfluorescein. After amplification, the labeled PCR products are separated with an automated sequencing system, ABI 373 (Applied Biosystems, Weiterstadt, Germany). With the help of the Gene-Scan software ABI 672 (Applied Biosystems) and fluorescent-labeled DNA length markers, the exact size of each peak can be displayed and analyzed. The resolution of this method allows separation of PCR products differing in length by as little as 1 bp. Semiquantitative estimation of specific clones also can be performed. Infiltrate-specific gene rearrangement patterns can be identified and recognized in different tissue specimens at the time of diagnosis or in subsequent biopsy specimens. We conclude that automated high-resolution PCR fragment analysis allows more accurate and convenient analysis of the TCR gamma-chain gene.