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G Burg

Publications and source records attributed to G Burg.

At least 163 records · Page 9Linked to original sources
PubMed1998

Interleukin-15 is an autocrine/paracrine viability factor for cutaneous T-cell lymphoma cells.

In this study we investigated the role of interleukin-15 (IL-15) in the immunobiology of cutaneous T-cell lymphoma (CTCL) cells. Using cell culture techniques, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunhistochemistry we found that IL-15, like IL-7, is a growth factor for the Sézary cell line SeAx and that both cytokines prolonged the survival of malignant T cells directly isolated from Sézary syndrome (SS) patients. Both IL-15 and IL-7 were more potent than IL-2. IL-4 and IL-9, whose receptors share the same gamma chain with the receptors of IL-2, IL-7, and IL-15, did not sustain the growth of CTCL cells, indicating that signaling through the common gamma chain (gammac) is not sufficient for continuous growth. IL-13 and tumor necrosis factor-alpha (TNF-alpha) had no effect. IL-7 and IL-15 also supported the growth of SeAx cells in the presence of the apoptosis inducing agents dexamethasone and retinoic acid. The analysis of patient Sézary cells and three CTCL cell lines by RT-PCR showed that all these cells expressed IL-15 mRNA, but only a few (25%) produced IL-7 mRNA. Immunohistological analyses of skin biopsy samples of SS and Mycosis fungoides patients showed immunoreactivity for IL-15 in basal cell layer keratinocytes and in the infiltrating lymphocytes. We conclude that IL-15 is a growth or viability factor for CTCL-derived cell lines or shortly cultivated Sézary cells. The findings that IL-15 mRNA can be detected in Sézary syndrome peripheral blood mononuclear cells and that the IL-15 protein is detected in skin sections from CTCL patients suggest that IL-15 plays an important role in the biology of CTCL.

Autocrine Communication↗
PubMed1998

Ultraviolet-induced acute histological changes in irradiated nevi are not associated with allelic loss.

BACKGROUND: Transformed melanocytes in atypical nevi, which are thought to be precursors of melanoma, are frequently deleted on chromosomes 1p, 9q, and 9p21 (p16 locus). Single UV irradiation induces histological changes that are similar to those of atypical nevi and, in part, of melanoma in situ. OBJECTIVE: To determine the effects of UV irradiation on benign melanocytic nevi in vivo. DESIGN: We investigated one half of a symmetric nevus 1 week after a single UV exposure with 4 times the patient's minimal erythema dose and compared it with the nonirradiated, shielded half of the same nevus. Two to 3 areas containing 5 to 30 melanocytes in 7 nevi were microdissected (a total of 18 areas in each nonirradiated and irradiated part), followed by a single-step DNA extraction. Extracted genomic DNA was amplified using a polymerase chain reaction with polymorphic markers D1S450 (1p), D9S12 (9q), IFNA, and D9S171 (9p21) and subjected to autoradiography. OBSERVATIONS: Two, 3, 2, and 2 of 18 areas were homozygous for D1S450, D9S12, IFNA, and D9S171, respectively. No allelic loss could be demonstrated in either nonirradiated or irradiated nevi. CONCLUSIONS: Acute histological changes demonstrated in melanocytic nevi after UV irradiation are not followed by allelic loss on identical chromosomal areas found in dysplastic melanocytes of atypical nevi. This finding supports the hypothesis that initial nonspecific genetic events may occur after UV irradiation, followed by an increase in various repair mechanisms potentially leading to specific genetic damage and loss of heterozygosity; however, loss of heterozygosity is not detectable at an early stage.

Cell Nucleus↗
PubMed1998

Immune stimulatory potential of B7.1 and B7.2 retrovirally transduced melanoma cells: suppression by interleukin 10.

The immunostimulatory capacities of B7.1-and B7.2- expressing melanoma cells were investigated. A365, 960306 and 950504 melanomas, established from nodular melanoma lesions, were retrovirally transduced. Irradiated B7-, B7.1+ and B7.2+ melanoma cells were co-cultured with autologous or allogeneic peripheral blood mononuclear cells (PBMCs). Proliferation was assessed by [3H]thymidine uptake. mRNA encoding for interleukin 2 (IL-2), IL-4, IL-10 and interferon gamma (IFN-gamma) was determined. IFN-gamma, IL-2, IL-4 and IL-10 secretion were quantitated by ELISA. B7.1+ and B7.2+ melanomas induced proliferation of PBMCs and mRNA for IL-2 and IFN-gamma. After co-incubation of transduced melanoma cells with PBMCs, high levels of IL-10 were detectable in the supernatant. The presence of neutralizing anti-IL-10 antibodies resulted in enhanced proliferation and IL-2 and IFN-gamma secretion. Our data indicate that B7.1- and B7.2-transduced melanoma cells trigger lymphocytic proliferation with transcription of IL-10, IL-2 and IFN-gamma. Blocking of IL-10 augments these effects. Gene therapy protocols using tumour cells as a vaccine have to consider the adverse effects of IL-10.

Adult↗
PubMed1998

Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells.

Melanoma is the main cause of death in patients with skin cancer. Cytotoxic T lymphocytes (CTLs) attack melanoma cells in an HLA-restricted and tumor antigen-specific manner. Several melanoma-associated tumor antigens have been identified. These antigens are suitable candidates for a vaccination therapy of melanoma. Dendritic cells (DCs) are antigen-presenting cells (APCs) specialized for the induction of a primary T-cell response. Mouse studies have demonstrated the potent capacity of DCs to induce antitumor immunity. In the present clinical pilot study, DCs were generated in the presence of granulocyte/macrophage-colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) and were pulsed with tumor lysate or a cocktail of peptides known to be recognized by CTLs, depending on the patient's HLA haplotype. Keyhole limpet hemocyanin (KLH) was added as a CD4 helper antigen and immunological tracer molecule. Sixteen patients with advanced melanoma were immunized on an outpatient basis. Vaccination was well tolerated. No physical sign of autoimmunity was detected in any of the patients. DC vaccination induced delayed-type hypersensitivity (DTH) reactivity toward KLH in all patients, as well as a positive DTH reaction to peptide-pulsed DCs in 11 patients. Recruitment of peptide-specific CTLs to the DTH challenge site was also demonstrated. Therefore, antigen-specific immunity was induced during DC vaccination. Objective responses were evident in 5 out of 16 evaluated patients (two complete responses, three partial responses) with regression of metastases in various organs (skin, soft tissue, lung, pancreas) and one additional minor response. These data indicate that vaccination with autologous DCs generated from peripheral blood is a safe and promising approach in the treatment of metastatic melanoma. Further studies are necessary to demonstrate clinical effectiveness and impact on the survival of melanoma patients.

Adult↗
PubMed1998

A novel culturing and grafting system for the treatment of leg ulcers.

The purpose of this study was to develop and test an efficient culturing and grafting system for the treatment of leg ulcers. The culturing system consisted of a Petriperm culture vessel (20 cm2) aseptically placed in a larger standard Petri dish (60 cm2). Skin cultures were established and cultivated in the Petriperm dish. The cells grew on the bottom of the Petriperm dish, which was made of a gas-permeable 25-micron thick transparent Teflon film. Grafts were produced simply by cutting the film from the bottom of the Petriperm dish with a scalpel. The system was used to produce subconfluent epidermal autografts which were used to heal a 32 cm2 chronic rheumatoid arthritis leg ulcer. The cultured autografts were transferred cell side down on to the cleaned wound bed without an enzymatic digestion. The grafts consisted of autologous keratinocytes, melanocytes and fibroblasts. Caution was taken not to disturb the wound bed for 7-9 days at which time the Teflon film was removed. The wound closed 2 weeks after the last grafting and has remained closed for more than a year post-treatment. The culturing and grafting system presented here will make it possible to develop cellular-based therapies that were previously not possible.

Arthritis, Rheumatoid↗
PubMed1998

Chromosomal allele loss in primary cutaneous melanoma is heterogeneous and correlates with proliferation.

Loss of heterozygosity at specific loci in neoplastic cells suggests the presence of a tumor suppressor gene within the deleted region. Using the microdissection technique, loss of heterozygosity has been identified in paraffin-embedded primary melanomas on chromosomes 1p and 9q. Our purpose was to determine loss of heterozygosity in primary cutaneous melanomas and to relate chromosomal alterations with cell morphology and proliferation of the tumor. Two to seven morphologic different areas in 12 primary cutaneous high risk melanomas (Breslow > 1.5 mm), as well as adjacent normal tissue, were microdissected and subjected to single step DNA extraction. Extracted genomic DNA was amplified by polymerase chain reaction using two polymorphic markers on chromosomes 1p (D1S450, between D1S548 and D1S228) and 9q (D9S12). Proliferation was evaluated by MIB-1 (Ki67) immunoreactivity and cell morphology, pigmentation and inflammation surrounding microdissected areas were investigated by microscopic inspection of hematoxylin and eosin stained sections. Twelve of 34 different areas (35%) showed loss of heterozygosity with at least one marker. Two of 32 areas showed loss of heterozygosity with D1S450 (two areas noninformative), seven of 30 with D9S12 (four areas noninformative), and three areas showed loss of heterozygosity at both loci. In three of these cases, analysis of different tumor foci revealed areas with/without loss of heterozygosity. In these cases, the percentage of MIB-I-positive cells was at least four times higher in areas with loss of heterozygosity compared with areas without loss of heterozygosity. Most areas with loss of heterozygosity consisted of small cuboidal to epitheloid cells. Spindle shaped and large anaplastic cells showed loss of heterozygosity less frequently. Neither melanization of tumor cells nor the presence of inflammation had an influence on the frequency of loss of heterozygosity. Primary cutaneous melanomas show intratumoral morphologic and chromosomal heterogeneity. Loss of heterozygosity on chromosomes 1p and 9q correlated with cell proliferation, suggesting that selected cell clones are responsible for tumor progression.

Alleles↗
PubMed1998

Human dermal dendritic cells process and present soluble protein antigens.

Recently, a novel type of dendritic antigen-presenting cell has been identified in the dermis of normal human and mouse skin. These dermal dendritic cells (DDC) occur in higher numbers than epidermal Langerhans cells, represent a distinct differentiation pathway of dendritic cells, and are as potent as Langerhans cells in the activation of superantigen specific T cells. As yet, nothing is known about their capacity to take up, process, and present soluble protein antigens. We used the model of tetanus toxoid (TT) driven T cell proliferation to address these questions. To test for active internalization of TT protein, gold labeled TT was incubated with Langerhans cells and DDC and could be traced to multivesicular endo-lysosomal compartments. DDC internalize TT through a receptor-mediated, clathrin-independent pathway, whereas Langerhans cells predominantly use macropinocytosis. To verify that DDC process TT by the exogenous pathway of antigen presentation, we pulsed DDC with TT protein or TT peptide after preincubation with chloroquine. Preincubation with chloroquine diminished the capacity of DDC to induce TT protein specific T cell proliferation (70-80%), but was not effective to suppress TT peptide induced T cell responses. DDC were as potent as Langerhans cells and 5-10 x more potent than plastic adherent monocytes in the presentation of TT to autologous resting T cells. Furthermore, as few as 50 DDC (stimulator:responder ratio of 1:1000) were able to induce a significant TT specific T cell proliferation. Because a subpopulation of DDC expresses low levels of CD1a, a phenotypic marker of Langerhans cells, sorting of CD1a positive and negative DDC was performed. On a per cell basis, CD1a positive and negative DDC were equally potent at mediating TT specific T cell proliferation. Thus, DDC are able to internalize, process, and present soluble protein antigens such as TT and may therefore play an important role in the regulation of skin immune responses.

Antigen-Presenting Cells↗
PubMed1998

Higher frequency of selective losses of HLA-A and -B allospecificities in metastasis than in primary melanoma lesions.

Expression of HLA class I molecules is essential for the recognition of tumor cells by CD8+ T cells. In this study, 48 bioptic samples of 42 patients in all stages of melanoma were investigated after short-time cultivation of tumor cells. To confirm melanocytic origin of cultured cells, samples were screened for mRNA expression of melanoma markers gp100, tyrosinase, MAGE-3, MelanA, and MUC18 by reverse transcriptase-polymerase chain reaction. Surface expression of specific HLA-A and -B allospecificities on melanoma cells were analyzed with a standard microcytotoxicity assay after stimulation with interferon (IFN)-alpha and compared with the background found in peripheral blood mononuclear cells from the corresponding patients. Immunohistochemistry and flow cytometry confirmed specific losses in cases where the appropriate monoclonal antibodies were available. The level of expression of HLA-I, HLA-II, and intercellular adhesion molecule 1 antigens on melanoma cells cultured in the presence or absence of IFN-alpha and IFN-gamma was determined cytofluorometrically. All cell cultures tested were found to be positive for one or more melanocytic markers by reverse transcriptase-polymerase chain reaction. The specific HLA-I alleles on the cultured cells were detectable in 45 of 48 samples. In 11 cases a specific loss of one HLA-I allele was observed (2 x A2, B7, B8, B18, 4XB44, B47, B49). Ten of these samples were derived from locoregional lymphnode metastases or from distant metastatic tumors. Only one sample from a primary melanoma showed a specific loss of HLA-I (B47). IFN-alpha upregulated expression of HLA-I up to 4-fold. IFN-gamma enhanced the appearance of HLA-II up to 35-fold and the expression of intercellular adhesion molecule 1 up to 40-fold. Selective loss of HLA-I allospecificities might be a major step in tumor progression.

Antigen-Antibody Reactions↗
PubMed1998

Experts and gold standards in dermatopathology: qualitative and quantitative analysis of the self-assessment slide seminar at the 17th colloquium of the International Society of Dermatopathology.

The diagnosis of lymphoproliferative and melanocytic skin lesions is one of the most vexing problems in dermatopathology, a problem that is compounded by the far-reaching therapeutic and psychosocial consequences of the diagnosis for both patient and physician. On the occasion of a self-assessment slide seminar held during a dermatopathology meeting, 30 unusual lymphoproliferative and melanocytic lesions, each provided with four differential diagnoses, were evaluated by "expert pathologists" and other participants ("nonexperts") of the slide seminar. The final diagnosis was pinpointed by the majority of the experts in 16 of 30 cases (56%). The group of experts returned an unanimous decision on the diagnosis in only 2 of the 30 cases (7%). In contrast to the expert group, the preferred diagnoses given by the nonexperts showed a wider range. In 20 of 30 cases (66%), the final diagnosis could only be established after consideration of clinical, histologic, immunophenotypic, and molecular features. Our findings agree with the results of recent studies indicating quite a high degree of discordance among expert pathologists. The discordance between experts and, to a higher extent, nonexperts may have some crucial consequences for dermatopathology. Full agreement on diagnosis, particularly in unusual skin lesions, cannot be achieved only by an accumulation of expertises. Instead of relying on one single finding or diagnostic procedure ("gold standard") as the main criterion upon which to base a diagnosis, the diagnoses become more reliable if based on the integration of several factors including an evaluation of clinical and histomorphologic features and immunophenotypic and molecular findings ("diagnostic elements"), particularly in the field of lymphoproliferative and melanocytic lesions. In addition, a continuous retrospective work-up of difficult or unusual cases is recommended to ensure a long-term improvement in diagnostic reliability.

Biomarkers↗
PubMed1998

The PTEN tumour suppressor gene and malignant melanoma.

A candidate tumour suppressor gene, PTEN, has recently been identified within chromosome 10q23, the locus of the Cowden syndrome/Lhermitte Duclos disease susceptibility gene. Cowden disease is an autosomal dominant cancer predisposition syndrome associated with tumours of the breast, thyroid and, less frequently, malignant melanoma. Based on the identification of mutations in sporadic breast, brain and prostate tumours, we decided to examine the potential role of PTEN in sporadic malignant melanoma. Frozen tissue from primary cutaneous melanomas (n = 23) and metastases (n = 17) were microdissected, and microsatellite markers D10S541 and D10S547, flanking the gene on both sides, were used to search for loss of heterozygosity (LOH) in the PTEN gene locus. To identify mutations within the putative tumour suppressor gene, we performed single strand conformation polymorphism (SSCP) analysis using intronic primers to amplify exons 5, 6, 7 and 8 of the PTEN gene. No LOH was detected using the polymorphic markers D10S541 and D10S547. SSCP analysis revealed no aberrant bands in the tumour specimen. Our results suggest that the PTEN gene does not play a major role in the initiation and progression of melanoma.

Chromosomes, Human, Pair 10↗
PubMed1998

Histopathologic and ultrastructural study of lupus-like skin lesions in a patient with Bloom syndrome.

The histopathology of the lupus-like skin lesions associated with Bloom syndrome has been sporadically described. Skin biopsies from a 2-year-old boy with the classical features of Bloom syndrome, including lupus-like skin lesions, demonstrated marked interface changes with basal liquefaction degeneration, a moderate superficial mononuclear infiltrate, pigmentary incontinence, and capillary dilation in the papillary dermis. Immunophenotyping of the dermal infiltrate revealed predominance of T-cells. Basement membrane thickening on periodic acid-Schiff examination was not seen. Direct immunofluorescence failed to demonstrate deposits of immunoglobulin other than nonspecific IgM deposition along the basement membrane zone of lesional skin. Ultrastructurally, the most striking findings were disintegration of basal cell cytoplasm and tubuloreticular inclusions in vascular endothelia. Taken together, the histologic and ultrastructural features of lupus-like lesions associated with Bloom syndrome mimic those of cutaneous lupus erythematosus, with the exception of paucity of immune deposits at the dermoepidermal junction.

Basement Membrane↗