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Biomedical subjects

R Kaufmann

Publications and source records attributed to R Kaufmann.

At least 127 records · Page 7Linked to original sources
PubMed1998

Direct identification of major histocompatibility complex class I-bound tumor-associated peptide antigens of a renal carcinoma cell line by a novel mass spectrometric method.

Melanoma and renal cell carcinoma (RCC) are thought to be the most immunogenic human tumors. Presently a series of tumor-specific peptides of melanoma is being tested in clinical trials with different immunotherapy protocols. In contrast, only one decameric peptide (SPSSNRIRNT) derived from one (ORF2) of three possible open reading frames (ORFs) of a gene named RAGE (Renal tumor AntiGEn) was shown to be the target for tumor-specific CTLs on renal carcinoma cells. One reason for the lack of identification of tumor antigens on RCC compared with melanoma may be the difficulty in generating tumor-specific CTLs as screening instruments. Therefore, our approach was directly to isolate and identify peptides bound to HLA class I molecules of the HLA-A2 and -B8 homozygous RCC line A-498. High performance liquid chromatography-fractionated peptides eluted with acid from immunoaffinity-purified HLA class I-peptide complexes were sequenced and identified for the first time by the novel and highly sensitive mass spectrometric method matrix-assisted laser desorption ionization-post source decay (MALDI-PSD) from minute amounts of 100 fmol to 1.5 pmol of the fractionated peptide samples. Fourteen peptide sequences first deduced from interpretations of the mass spectra were also shown to fulfill other reliability criteria such as matching the mass spectra of the respective synthetic peptides. Some peptides were identified to be derived from genes preferentially activated in malignant tissues or resulted from a possibly mutated gene. The most promising candidate for a CTL epitope is a decameric peptide (PASKKTDPQK) derived from another possible ORF (ORF5) of the RAGE gene and probably presented in association with HLA-B8. This peptide was synthesized and used for the in vitro induction of CTLs that lysed the A-498 cells and another HLA-B8-positive RCC line significantly more strongly than either other RAGE-positive but HLA-B8-negative RCC lines or K562 cells. Sensitive sequencing by MALDI-PSD thus may provide a powerful method of identifying potentially tumor-specific and HLA-restricted antigens, even on native malignant cells and tissues.

Antigens, Neoplasm↗
PubMed1998

Primary staging and follow-up of high risk melanoma patients with whole-body 18F-fluorodeoxyglucose positron emission tomography: results of a prospective study of 100 patients.

BACKGROUND: Positron emission tomography (PET) has been retrospectively reported to be a sensitive method for detecting malignant melanoma metastases. METHODS: One hundred consecutive patients with high risk melanoma (tumor thickness > 1.5 mm) were prospectively evaluated (52 at primary diagnosis, comprising Group A, and 48 during follow-up, comprising Group B) by whole-body PET and conventional diagnostics (CD). RESULTS: In Group A, the sensitivity of PET was 100% and the specificity was 94%, whereas CD did not identify any of the 9 lymph node metastases and demonstrated a lower specificity (80%). In Group B, 121 lesions were detected, 111 by PET and 69 by conventional imaging. On the basis of patients, the sensitivity, specificity, and accuracy of PET were 100%, 95.5%, and 97.9%, respectively (91.8%, 94.4%, and 92.1%, respectively, on the basis of single metastases). Prospectively, CD did not identify all patients with progression (sensitivity, 84.6%) and detected significantly fewer metastases (sensitivity, 57.5%) with much lower specificity (68.2% on the basis of patients, 45% on the basis of single lesions); therefore, the accuracy of CD was 77.1% on the basis of patients and only 55.7% on the basis of single metastases. Results also depended on specific sites: while PET yielded a higher sensitivity in detecting cervical metastases (100% vs. 66.6%) and abdominal metastases (100% vs. 26.6%), computed tomography proved to be superior in detecting small lung metastases (87% vs. 69.6%). CONCLUSIONS: PET is a highly sensitive and specific technique for melanoma staging. With the exception of the brain, one single whole-body 18F-fluorodeoxyglucose-PET scan could replace the standard battery of imaging tests currently performed on high risk melanoma patients.

Adult↗
PubMed1998

[Laser applications in dermatology].

Over the past decades continuous wave laser systems have been used in dermatology for the photocoagulation and vaporization of vascular neoformations and malformations and to remove epithelial lesions. However, their mainly thermal effect can have negative impact on wound healing and scar formation due to thermal damage of adjacent tissue. More recent laser systems now permit more selective and more restrictive destruction of pathological tissue. They include pulsed and Q-switched lasers matched to the absorption characteristics of the target structures, which permit skin ablation and selective photothermolosysis of pigmented or vascular lesions. These technical innovations have expanded the therapeutic spectrum in dermatology and improved the quality of the results achieved. Nevertheless, these systems should be used only for carefully selected indications, in particular when corrective-aesthetic considerations are of importance. Furthermore, prior to treatment, it is mandatory for the dermatologist, to establish the proper diagnosis, especially in the case of pigmented lesions and skin changes of uncertain dignity.

Humans↗
PubMed1998

Functional thrombin receptor PAR1 in primary cultures of human glioblastoma cells.

In this study we investigated primary cultures obtained from two glioblastomas surgically removed from a 64-year-old man and a 50-year-old woman, respectively. The presence of the tethered ligand thrombin receptor PAR1 (protease-activated receptor 1) in these cells was demonstrated at the level of receptor binding by using immunofluorescence studies with the monoclonal anti-PAR1 antibody Mab 31-2. Stimulation of human glioblastoma cells both with alpha-thrombin and the thrombin receptor activating peptide TRAP-6 resulted in a series of [Ca+]i spikes as shown by confocal laser fluorescence microscopy with fluo-3 as calcium sensitive fluorescence indicator. This effect was completely blocked with the thrombin receptor antagonist peptide T1. Our results demonstrate functional thrombin receptors (PAR1) in primary cultures of human glioblastomas for the first time.

Brain Neoplasms↗
PubMed1998

Adenosine(5') oligophospho-(5') guanosines and guanosine(5') oligophospho-(5') guanosines in human platelets.

We isolated and identified nucleoside(5') oligophospho-(5') nucleosides containing adenosine and guanosine (ApnG; n = 3-6) as well as diguanosine polyphosphates (GpnG; n = 3-6) in human platelets. For identification, UV spectrometry, matrix-assisted laser desorption/ionization, postsource decay matrix-assisted laser desorption/ionization mass spectrometry, and enzymatic cleavage experiments were used. The adenosine(5') oligophospho-(5') guanosines act as vasoconstrictors and growth factors. The diguanosine polyphosphates are potent modulators of growth in vascular smooth muscle cells, but do not affect vascular tone.

Adenosine↗
PubMed1998

Derivation of insertin.

Insertin is an actin-binding protein that has been isolated from chicken gizzard smooth muscle that has been shown to be highly homologous to amino acids 962-1292 of tensin [Weigt et al., 1992]. Because of the high homology, we investigated the question whether the mRNAs of insertin and of tensin are derived from the same gene by alternative splicing, whether insertin and tensin are encoded by two different genes, or whether insertin is a proteolytic fragment of tensin. In a Northern blot analysis, mRNA from chicken gizzard was hybridized with oligonucleotides specific for tensin and for the insertin domain of tensin. The tensin-specific oligonucleotide hybridized only with the previously reported 8- and 10-kbp RNAs. However, the insertin domain-specific oligonucleotide hybridized with a 1.2 and a 1.6 kbp RNA in addition to the 8 and 10 kbp RNA. The 1.2- and 1.6-kbp RNA occurred in small amounts, as compared with the 8- and 10-kbp RNA. Southern blot analysis of DNA cleaved by the restriction endonucleases BamH1 and HindIII demonstrated that only one gene for the insertin and tensin exists. Insertin isolated from chicken gizzard smooth muscle was investigated by mass spectrometry. The N-termini of three isolated peptides were found to begin at adjacent amino acids and were likely to be formed from tensin by proteolysis. The results suggest that, for insertin, an mRNA exists that is derived from one gene common for insertin and tensin. However, the insertin-specific mRNA contributes relatively little to expression of insertin domains in cells. Insertin preparations from chicken gizzard contain mainly insertin domains formed from tensin by proteolysis.

Amino Acid Sequence↗
PubMed1998

Proteinase-activated receptor-2-mediated signaling and inhibition of DNA synthesis in human pancreatic cancer cells.

CONCLUSION: Proteinase-activated receptor-2 (PAR-2)-mediated effects contribute to the intracellular signaling network in pancreatic tumor cells. A role of PAR-2 as negative regulator in human pancreatic tumor growth might be implied. BACKGROUND: Using the human pancreatic tumor cell line MIA PaCa-2, we evaluated cellular effects of trypsin and the PAR-2-activating peptide SLIGRL on [Ca2+]i mobilization, Ins(1,4,5)P3 level, and protein kinase (PKC) activation. Furthermore, PAR-2 involvement in the regulation of cell proliferation has been estimated by measurement of [3H]thymidine incorporation in MIA PaCa-2 cells. RESULTS: Trypsin and the PAR-2 synthetic peptide agonist SLIGRL induced [Ca2+]i mobilization, transient increase in inositol (1,4,5) triphosphate level, and PKC translocation in MIA PaCa-2 cells. In addition, SLIGRL induced a decrease in DNA synthesis in MIA PaCa-2 cells.

Biological Transport↗
PubMed1998

[Coincidence of balloon cell melanoma with balloon cells in a dermal nevus].

We report a coincidence of balloon cell melanoma and balloon cell transformation in a dermal nevus of a 56 years old female patient. The foamy cells in both tumors with different dignity expressed S-100 and Vimentin. HMB45 was expressed in all balloon cells of the melanoma. Detection of HMB45 in some of the nevus-associated balloon cells was considered as an expression of cellular activation.

Antigens, Neoplasm↗
PubMed1998

Protein kinase C is involved in cholecystokinin octapeptide-induced proliferative action in rat glioma C6 cells.

Chotecystoknin octapeptide (CCK-8) has been shown to stimulate DNA synthesis in rat glioma C6 cells by activation of CCKB type receptors. However, the signalling pathways contributing to this proliferative action in C6 cells have not been investigated thus far. This study demonstrated that stimulation of rat glioma C6 cells with CCK-8S resulted in activation of protein kinase C isozymes betaI, betaII, gamma and zeta. The participation of protein kinase C in the CCK-8S-induced effect on C6 cell growth was demonstrated by measurement of [3H]thymidine incorporation and estimation of cell number. The data indicate that CCK-8S stimulates growth in rat glioma C6 cells by a protein kinase C-dependent mechanism.

Animals↗
PubMed1998

Nonspecific immunity in pregnancy: monocyte surface Fcgamma receptor expression and function.

The state of pregnancy is an immunological enigma during which the body must prevent rejection of the antigenically foreign fetus while at the same time maintain sufficient maternal host defense mechanisms to combat infection. Although most studies on the immunology of pregnancy focus on immune suppression, several studies have shown an increase in nonspecific host defense, which is postulated to be a compensatory mechanism for decreased specific immunity during pregnancy. Studies in this laboratory have shown that monocyte surface FcgammaRI (CD64) and FcgammaRII (CD32) expression progressively increase throughout pregnancy, while surface MHC class II expression remains unchanged. Functional studies revealed that the number of phagocytic monocytes which could be isolated from pregnant women was increased. These cells exhibited an increased capacity to ingest IgG-opsonized human erythrocytes. This study shows for the first time that monocyte surface FcgammaR expression and FcgammaR-mediated functions are increased during pregnancy. These results support the hypothesis that nonspecific immunity as represented by FcgammaR expression and function is increased during pregnancy.

Adult↗
PubMed1998

Investigation of PAR-1-type thrombin receptors in rat glioma C6 cells with a novel monoclonal anti-PAR-1 antibody (Mab COR7-6H9).

Rat glioma C6 cells have been demonstrated to be a suitable model in the investigation of PAR-1-type thrombin receptors in brain. However, anti-PAR-1 antibodies, which should be very helpful tools in studying PAR-1 in rat cells, have not been available up until now. Therefore, we prepared a monoclonal anti-thrombin receptor antibody (Mab COR7-6H9) directed against the peptide sequence GRAVYLNKSRFPPMPPPPFISEDASG in the N-terminus below the thrombin cleavage site of the rat PAR-1-type thrombin receptor. Using this antibody, we demonstrated the presence of PAR-1 binding sites on the plasma membrane of rat glioma C6 cells both with confocal laser fluorescence and with scanning electron microscopy. In addition, Mab COR7-6H9 was shown to block PAR-1-mediated transmembranal signaling as demonstrated by measurement of free intracellular calcium and cyclic AMP. This novel anti-PAR-1 antibody is therefore likely to be a very helpful tool in studying PAR-1-type thrombin receptors in rat brain.

Amino Acid Sequence↗
PubMed1998

Quantification of tyrosinase, TRP-1, and Trp-2 transcripts in human melanocytes by reverse transcriptase-competitive multiplex PCR--regulation by steroid hormones.

We have introduced a reverse transcriptase polymerase chain reaction based method to measure mRNA levels of the melanogenesis enzymes tyrosinase, tyrosinase-related-protein 1 (TRP-1), and tyrosinase-related-protein 2 (TRP-2). Expression was determined by reverse transcriptase-competitive multiplex polymerase chain reaction of (i) melanogenesis enzyme transcripts and the "housekeeping" gene glyceraldehyde-3-phosphate dehydrogenase, and (ii) two internal standards consisting of mutated melanogenesis enzyme cDNA and mutated gene glyceraldehyde-3-phosphate dehydrogenase cDNA. This was investigated on in vitro cultured melanocytes in the presence of three different steroids; one glucocorticoid (betamethasone-17-valerate) and two sex steroids (diethylstilbestrol and estradiol). All three steroids lead to an increase of about 1.5-2.5-fold of tyrosinase transcripts. The amount of TRP-1 transcripts was likewise enhanced, but only moderately (approximately 1.5-fold). In contrast, TRP-2 transcripts were reduced by approximately 40% in number after betamethasone-17-valerate treatment, whereas the two sex steroids, diethylstilbestrol and estradiol, caused an upregulation of about 20-fold of the initial TRP-2 transcript level. We therefore suggest that hyperpigmentation during pregnancy or under contraceptive treatment is mediated by a direct induction of melanogenesis via sex steroids.

Betamethasone↗
PubMed1998

Dermatomyositis presenting as plaque-like mucinosis.

We describe two women, aged 65 and 37 years, who presented with mucinous plaques as the initial skin signs of dermatomyositis. Other cutaneous manifestations were lacking, but typical myositis appeared later in the course of the disease.

Adult↗
PubMed1998

Hepatocyte growth factor/scatter factor (HGF/SF) induces vascular permeability factor (VPF/VEGF) expression by cultured keratinocytes.

Skin expression of the endothelial cell-specific vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) as an outstanding mediator of physiologic and pathologic angiogenesis has been previously demonstrated to be subject to regulation by distinct stimuli. We explored whether the multifunctional hepatocyte growth factor/scatter factor (HGF/SF) may mediate its angiogenic properties in part through paracrine induction of cutaneous VPF/VEGF synthesis. In these studies, we demonstrate that HGF/SF functions as a potent inducer of VPF/VEGF expression by human epidermal keratinocytes and by different epithelial-derived cells in vitro. VPF/VEGF mRNA and protein expression are regulated by HGF/SF in both a concentration- and a time-dependent fashion. Examination of mRNA half-lives does not reveal an increase in VPF/VEGF mRNA stability after HGF/SF stimulation. Thus, HGF/SF-induced VPF/VEGF mRNA expression appears to be largely dependent on enhanced gene transcription. In analyses of transiently transfected 5'-deletional reporter gene constructs, we identified a GC-rich VPF/VEGF promoter element that conveys transcriptional activation in response to HGF/SF. This sequence, located between nucleotides -88 and -70, is critical for both constitutive and HGF/SF-induced transcriptional activity. Together, our observations support a model in which HGF/SF mediates angiogenic properties in part through paracrine induction of VPF/VEGF synthesis by keratinocytes. In addition to cutaneous inflammation and wound healing, our findings have potential significance for vascular hyperpermeability and angiogenesis in tumor growth.

5' Untranslated Regions↗
PubMed1998

Grafting of in vitro cultured melanocytes onto laser-ablated lesions in vitiligo.

A variety of grafting procedures using autologous melanocytes have achieved promising results in the treatment of vitiligo. We here report on the preparation of an adequate graft recipient bed by pulsed Erbium-YAG laser skin ablation. In particular, for irregular lesions on delicate sites, which cannot be approached by utilization of suction blisters or dermabrasion, this technique may offer a distinct advantage.

Adult↗
PubMed1998

Characterization of functional thrombin receptors in human pancreatic tumor cells (MIA PACA-2).

In this article, the "tethered ligand" thrombin receptor was identified on human pancreatic tumor cells, MIA PaCa-2, using immunofluorescence studies with a monoclonal anti-thrombin receptor antibody. Pharmacological characterization, using 3H-labeled thrombin receptor activating peptide-6 (TRAP-6) as radioligand, demonstrated a single class of high-affinity binding sites (KD = 9.1+/-1.8 x 10(-7) M) and a binding capacity of 13.9+/-0.7 fmol/mg protein. These binding sites represent functional thrombin receptors, as shown by alpha-thrombin- and TRAP-6-induced mobilization of free intracellular calcium, protein kinase C translocation from cytosol to the cell membrane, and stimulation of DNA synthesis in MIA PaCa-2 cells. These results provide the first identification of tethered ligand thrombin receptor in human pancreatic cancer cells and suggest thrombin receptor involvement in mechanisms of human pancreatic tumor progression.

Calcium↗