Expanded murine regulatory T cells: analysis of phenotype and function in contact hypersensitivity reactions.

Regulatory T cells (Treg) exert suppressive functions in the periphery of the body for the maintenance of tolerance. The functional analysis of Treg is hampered by the fact that only small numbers (5%-10% among the CD4(+) T cells) of Treg exist in peripheral blood and tedious isolation methods further reduce the yield of high-purity Treg. We therefore set out to expand isolated murine Treg in ex vivo cultures with help of anti-CD3/anti-CD28 antibodies in the presence of IL-2.

The oncogenic B-raf V599E mutation occurs more frequently in melanomas at sun-protected body sites.

Downstream of Ras, the serine/threonine kinase, B-raf, has recently been reported to be mutated, among other carcinomas, in a substantial subset of primary melanomas with a preponderance of the oncogenic V599E transition. As the risk of melanoma is enhanced by intermittent ultraviolet (UV) exposure but is less with chronic UV exposure, we here studied B-raf kinase domain (exon 15) mutations in primary cutaneous melanomas with respect to anatomical locations reflecting chronic versus intermittent UV exposure. Investigating a representative number of 101 primary melanoma resection specimens for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain by polymerase chain reaction and single-strand conformation polymorphism gel electrophoresis, followed by DNA cloning and sequencing, we found 32 cases (32%) which harbour somatic B-raf exon 15 mutations.

Preponderance of the oncogenic V599E and V599K mutations in the B-raf kinase domain is enhanced in melanoma lymph node metastases.

Downstream of Ras, the serine/threonine kinase B-raf has been reported to be mutated, amongst other carcinomas, in a substantial subset of primary melanomas, with a preponderance of mutations within the kinase domain, including the activating V599E and V599K transitions. We investigated a representative series of 54 resection specimens of melanoma lymph node metastases for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain by polymerase chain reaction (PCR) and single-strand conformational polymorphism (SSCP) gel electrophoresis. Sequencing of cloned PCR-SSCP amplicons resulted in 24 (44%) samples harbouring somatic mutations, which is not significantly different from the mutation frequency found in recently investigated primary cutaneous melanomas (Deichmann M, Thome M, Benner A, Näher H.

The chemoresistance gene ABCG2 (MXR/BCRP1/ABCP1) is not expressed in melanomas but in single neuroendocrine carcinomas of the skin.

Background: As the molecular mechanisms in melanoma chemoresistance remain unknown to date, we hypothesized these tumors to express the ATP-binding cassette (ABC) transporter G2 (ABCG2/MXR/BCRP1/ABCP1), a recently detected membrane transporter and putative stem-cell marker. Besides melanoma, we addressed the neuroendocrine carcinoma of the skin (Merkel cell carcinoma), another cutaneous cancer supposed to originate from neuroectoderm and usually chemoresistant.

Methods And Results: Upon semiquantitative reverse transcription polymerase chain reaction, ABCG2 mRNA expression was not upregulated in 18 melanoma resection specimens when compared with 19 acquired melanocytic nevi from which melanomas are known to often arise (Mantel-Haenszel test, p=0.

Preponderance of the oncogenic V599E and V599K mutations in B-raf kinase domain is enhanced in melanoma cutaneous/subcutaneous metastases.

Background: Downstream of Ras, the serine/threonine kinase B-raf has been reported to be mutated, among other carcinomas, in a substantial subset of primary melanomas with a preponderance of mutations within the kinase domain including the activating V599E and V599K transitions.

Methods: We here investigated a representative series of 60 resection specimens of cutaneous and subcutaneous melanoma metastases for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) gel electrophoresis.

Results: Sequencing of cloned PCR-SSCP amplicons resulted in 24 (40%) samples harbouring somatic mutations which is not exceeding the mutation frequency in recently investigated primary melanomas.

BRAF mutations distinguish anorectal from cutaneous melanoma at the molecular level.

Background & Aims: Anorectal melanoma (AM) is a rare but highly malignant tumor, displaying histologic and immunohistochemical features very similar to cutaneous melanoma (CM). Because BRAF mutations were recently identified in the majority of CM and nevi, we investigated AM for BRAF mutations and mutations of NRAS , an additional component of the MAPK-signalling pathway.

Methods: DNA from formalin-fixed and paraffin-embedded AM was PCR amplified and sequenced.

B-raf exon 15 mutations are common in primary melanoma resection specimens but not associated with clinical outcome.

Downstream of Ras, the serine/threonine kinase B-raf has recently been reported to be mutated, among other carcinomas, in a majority of melanoma cell lines with a preponderance of mutations within the kinase domain including the activating V599E transition. We therefore investigated a representative number of 50 primary melanoma resection specimens for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain. Applying polymerase chain reaction and single-strand conformation polymorphism gel electrophoresis, followed by DNA cloning and sequencing, we found 19 cases (38%) to harbor somatic B-raf exon 15 mutations.

Expression of the heat shock cognate protein HSP73 correlates with tumour thickness of primary melanomas and is enhanced in melanoma metastases.

Seeking to identify melanoma-associated genes by comparing gene expression in uncultured primary melanoma specimens with those in nevi, from which melanomas often are known to arise, we applied subtractive suppression hybridization. Generating a subtracted library of candidate genes up-regulated in primary melanomas, this library contained cDNA fragments of the genes encoding heat shock cognate protein (HSP73) and major histocompatibility complex (HLA-DR) which were overexpressed in further 19 independent melanoma resection specimens on cDNA Southern blots when compared to 19 acquired melanocytic nevi. Upon immunohistochemistry, HSP73 protein expression was detected in the cytoplasm of melanoma cells in primary tumours and metastases.

Melanoma metastasis is associated with enhanced expression of the syntenin gene.

Primary cutaneous melanomas and melanoma metastases were examined for differential gene expression using subtractive suppression hybridization in a search for any genes associated with metastasis. Generating a subtracted library of candidate genes up-regulated in melanoma metastases, this library contained 8 different cDNAs, among them a cDNA fragment of the syntenin gene which was overexpressed in further independent melanoma resection specimens on cDNA Southern blots when compared to acquired melanocytic nevi from which melanomas are known to arise. Upon immunohistochemistry, the syntenin protein expression was detected in the cytoplasm of primary cutaneous melanomas and melanoma metastases.

Somatic mitochondrial mutations in melanoma resection specimens.

As epidemiological data suggest ultraviolet (UV) radiation to be the major environmental factor for the development of melanoma, we screened this malignancy for UV-specific C right curved arrow T and CC right curved arrow TT DNA mutations as described in squamous and basal cell carcinomas of the skin. Mitochondrial (mt) DNA was addressed which is known to be highly more susceptible for mutations than nuclear DNA. In the mt genome we chose part of the non-coding displacement-loop (D-loop) containing two hypermutable (C)n tracts especially prone for C right curved arrow T and CC right curved arrow TT changes.