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Biomedical subjects

Herbert Auer

Publications and source records attributed to Herbert Auer.

At least 19 recordsLinked to original sources

Accurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform.

DNA methylation is the best-studied epigenetic modification and describes the conversion of cytosine to 5-methylcytosine. The importance of this phenomenon is that aberrant promoter hypermethylation is a common occurrence in cancer and is frequently associated with gene silencing. Various techniques are currently available for the analysis of DNA methylation. However, accurate and reproducible quantification of DNA methylation remains challenging. In this report, we describe Bio-COBRA (combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform), as a novel approach to quantitative DNA methylation analysis. The combination of a well-established method, COBRA, which interrogates DNA methylation via the restriction enzyme analysis of PCR-amplified bisulfite treated DNAs, with the Bioanalyzer platform allows for the rapid and quantitative assessment of DNA methylation patterns in large sample sets. The sensitivity and reproducibility of Bio-COBRA make it a valuable tool for the analysis of DNA methylation in clinical samples, which could aid in the development of diagnostic and prognostic parameters with respect to disease detection and management.

Azacitidine↗

20q11.1 amplification in giant-cell tumor of bone: Array CGH, FISH, and association with outcome.

The goal of this study was to identify recurrent regions of genomic gain or loss in giant-cell tumor of bone (GCTb). Array comparative genomic hybridization (aCGH) was performed for 20 frozen tumor samples of GCTb. A separate subset of 59 GCTb with outcome data was used for validation. The most frequent region of change identified by aCGH was gain of a 1-Mbp region at 20q11.1. In the validation arm of 59 cases the minimal common region of copy number gain at 20q11.1, seen in 54% of the samples, was BAC clone RP11-4O9, which contained the genes TPX2 and BCL2L1. For most cases, amplification was restricted to the mononuclear component and was not present in the multinucleated giant cells. Southern blot for TPX2 and BCL2L1 identified the former as the gene with the highest level of amplification for these two proposed candidate genes of importance. Immunohistochemistry for TPX2 expression correlated with amplification, while BCL2L1 expression was not identified. Kaplan-Meier curves for progression-free survival showed a statistically significant difference for cases with 20q11.1 amplification (P = 0.0001). Univariate analysis involving Cox proportional hazards models did not show a significant difference for initial treatment type (curettage versus resection) (P = 0.575), age (</=50 vs. >50) (P = 0.543), or sex (P = 0.268), but did correlate with 20q11.1 amplification (P = 0.001). By multivariate analysis, it was found that 20q11.1 amplification (P = 0.001) was the only factor to reach statistical significance. 20q11.1 amplification can be used as a marker of prognostic importance in GCTb. We propose TPX2 as a candidate oncogene in the core-amplified region at 20q11.1.

Bone Neoplasms↗

[Relevance of parasitological examinations for the clinical course, epidemiology and prevention of alveolar echinococcosis - experiences of more than two decades in Austria].

Alveolar echinococcosis has been known in Austria since 1854. At the beginning of the 20th century the Austrian researcher on echinococcosis, Adolf Posselt, has realized already the enormous medical relevance of serological tests for the diagnosis of alveolar echinococcosis as well as for preventive purposes. Since the beginning of the 1980ies a long-term study about the epidemiology and serodiagnosis of alveolar echinococcosis has started in the Department of Medical Parasitology of the Clinical Institute of Hygiene and Medical Microbiology of the University of Vienna. Today a highly sensitive enzyme-linked immunosorbent assay as well as a highly specific westernblot system are available for the serological diagnosis of human alveolar echinococcosis cases, for the serological monitoring of the disease as well as for seroepidemiological and preventive medical studies. In addition, molecular biological tools for the detection of E. multilocularis (as well as E. granulosus strain) specific DNA in surgical resected and bioptic materials complete our diagnostic spectrum of methods.

Animals↗

Cercarial dermatitis in Austria: questionnaires as useful tools to estimate risk factors?

Cercarial dermatitis is a worldwide occurring skin disease characterized by itching and skin papulation. It is caused by cercariae of the fluke family Schistosomatidae. In the tropics and subtropics species of the genus Schistosoma can cause severe diseases of man. However, several genera (e.g. Trichobilharzia, Bilharziella) of medical significance are prevalent in Europe as well; they are also known as "bird schistosomes", because waterfowl is the final host. Pulmonate snails act as intermediate hosts. Humans are accidental hosts; they get infested by penetration of the cercariae into the skin of swimmers/bathers in ponds and lakes. They can not mature in humans, but die shortly after penetration. Cercarial dermatitis is known in Austria since 1969, with regularly occurrences nearly every summer. In early 2003 we created a homepage to provide information about the causative agents for the public/patients, to document the occurrences and to get data about the distribution of this parasitic disease. We therefore created a questionnaire and asked people for the following parameters: personal data, information about the waters, activity in the water and details about the dermatitis itself. A total of 34 questionnaires were returned. The results will be discussed according to their relevance as risk factors. The way how people interact with the water seems to be essential, but not demographic features. In addition, this approach revealed a new segment of the public that is at risk - owners/users of swimming ponds.

Adolescent↗

Paradigm Burgenland: risk of Borrelia burgdorferi sensu lato infection indicated by variable seroprevalence rates in hunters.

BACKGROUND: This study concerns the prevalence of antibodies against B. burgdorferi sensu lato as an indicator of previous borrelial infection among hunters, a group of occupationally exposed persons. In order to define associated risk factors and preparing data for future comparisons, a study was performed in the eight districts of Burgenland, the most eastern state of Austria. METHODS: Blood samples of 1214 men (median age 51 years, range 18 to 89 years) and 39 women (median age 44 years, range 21 to 69 years) were collected during autumn 2002 and winter 2003. Demographic data regarding age, sex, profession, residence, duration of employment (hunters), hunting-ground, animals in the environment, and history of tick bite were obtained by a questionnaire. A two-step testing strategy was used in which sera were screened for antibodies against B. burgdorferi sensu lato by a commercially available recombinant enzyme-linked immunosorbent assay (Biotest Anti-Borrelia IgG ELISA; Biotest AG, Dreieich, Germany). Reactive sera were then subjected to immuno blot testing (recomBlot Borrelia; Mikrogen, Munich, Germany) for confirmation of specificity. RESULTS: A total of 673 (54%) sera tested positive for IgG antibodies to Borrelia burgdorferi sensu lato: 663 (55%) men and 10 (26%) women. Seropositivity was clearly related to age and duration of hunting activity; it was 33% among persons younger than 29 years and 83% in those older than 70 years. Further, there was also a difference in the distribution of seroprevalence within the districts; the highest was found in hunters from the most southern district of Burgenland, Jennersdorf, (69%) and the lowest was noticed in the most northern district, Neusiedl (39%). CONCLUSIONS: We found an overall seroprevalence of 54% in asymptomatic hunters of Burgenland. Infectious risk exists in the entire state but the prevalence rate differs in the various districts indicating a variable risk which peaks in the south. The nearly linear increase of seroprevalence with age and duration of hunting activity reflects repeated tick exposure.

Adult↗

ADOA3R as a therapeutic target in experimental colitis: proof by validated high-density oligonucleotide microarray analysis.

Adenosine A3 receptors (ADOA3Rs) are emerging as novel purinergic targets for treatment of inflammatory diseases. Our goal was to assess the protective effect of the ADOA3R agonist N(6)-(3-iodobenzyl)-adenosine-5-N-methyluronamide (IB-MECA) on gene dysregulation and injury in a rat chronic model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)--induced colitis. It was necessary to develop and validate a microarray technique for testing the protective effects of purine-based drugs in experimental inflammatory bowel disease. High-density oligonucleotide microarray analysis of gene dysregulation was assessed in colons from normal, TNBS-treated (7 days), and oral IB-MECA-treated rats (1.5 mg/kg b.i.d.) using a rat RNU34 neural GeneChip of 724 genes and SYBR green polymerase chain reaction. Analysis included clinical evaluation, weight loss assessment, and electron paramagnetic resonance imaging/spin-trap monitoring of free radicals. Remarkable colitis-induced gene dysregulation occurs in the most exceptional cluster of 5.4% of the gene pool, revealing 2 modes of colitis-related dysregulation. Downregulation occurs in membrane transporter, mitogen-activated protein (MAP) kinase, and channel genes. Upregulation occurs in chemokine, cytokine/inflammatory, stress, growth factor, intracellular signaling, receptor, heat shock protein, retinoid metabolism, neural, remodeling, and redox-sensitive genes. Oral IB-MECA prevented dysregulation in 92% of these genes, histopathology, gut injury, and weight loss. IB-MECA or adenosine suppressed elevated free radicals in ex vivo inflamed gut. Oral IB-MECA blocked the colitis-induced upregulation ( 90% of genes tested (33 of 37 genes). We conclude that our validated high-density oligonucleotide microarray analysis is a powerful technique for molecular gene dysregulation studies to assess the beneficial effects of purine-based or other drugs in experimental colitis. ADOA3R is new potential therapeutic target for inflammatory bowel disease.

Adenosine↗

Chronic long-standing headache due to neurocysticercosis.

A 35-year-old Chinese woman presented with a 26-year history of persistent headache, relieved only by diuretics. Characteristic CT findings, peripheral eosinophilia, lymphocytic CSF pleocytosis, elevated CSF IgG, positive oligoclonal bands, antibody-positive ELISA, and Western blot results with Taenia solium antigen, and a favorable response to albendazole led to the diagnosis of neurocysticercosis.

Adult↗

The ABRF MARG microarray survey 2005: taking the pulse of the microarray field.

Over the past several years, microarray technology has evolved into a critical component of any discovery-based program. Since 1999, the Association of Biomolecular Resource Facilities (ABRF) Microarray Research Group (MARG) has conducted biennial surveys designed to generate a profile of microarray service laboratories and, more importantly, an overview of technology development and implementation. Survey questions addressed instrumentation, protocols, staffing, funding, and work flow in a microarray facility. Presented herein are the results of the MARG 2005 survey; where possible, trends in the field are discussed and compared to data collected from previous surveys.

Animals↗

MYC amplification and polysomy 8 in chondrosarcoma: array comparative genomic hybridization, fluorescent in situ hybridization, and association with outcome.

PURPOSE: To identify recurrent regions of genomic gain or loss in chondrosarcoma in a clinically relevant and statistically valid fashion. MATERIALS AND METHODS: Array comparative genomic hybridization (CGH) results of 15 frozen tumor samples of high-grade chondrosarcoma for chromosome 8 are presented. A separate subset of 116 cartilaginous tumors with outcome data was used for validation. RESULTS: Array CGH identified gain at 8q24.12-q24.13, the region of the MYC (c-Myc) oncogene, as a frequent change in high-grade chondrosarcoma. In the validation arm of 116 cartilaginous tumors, MYC was frequently amplified in G2 (15%), G3 (20%), and dedifferentiated (21%) chondrosarcomas. No amplification was identified in samples of enchondroma and grade 1 chondrosarcoma. In samples without MYC amplification, polysomy 8 was a frequent finding in grade 1 (18%), grade 2 (31%), grade 3 (80%), and dedifferentiated (29%) chondrosarcomas, but was not found in any samples of enchondroma. MYC protein expression was identified in all samples with amplification, but was also frequent in the remaining samples without amplification or polysomy 8. Kaplan-Meier survival curves for overall survival showed a statistically significant difference for patients with MYC amplification or polysomy 8 (P = .034). Univariate analysis involving Cox proportional hazards models showed that grade (P = .003), polysomy 8 (P = .045), and MYC amplification (P = .053) correlated with shorter overall survival. By multivariate analysis, grade of chondrosarcoma (P = .026) was the only factor to reach statistical significance. CONCLUSION: MYC amplification and polysomy 8 can be used as markers of prognostic importance in chondrosarcoma. Molecular targeting of MYC expression may have therapeutic potential in the future for subsets of chondrosarcoma.

Aneuploidy↗

Reduced stress tolerance of glutamine-deprived human monocytic cells is associated with selective down-regulation of Hsp70 by decreased mRNA stability.

In critically ill patients, clinicians observe a reverse correlation of survival and a decreased plasma concentration of the most abundant free amino acid, glutamine (Gln). However, in this context, the role of Gln remains largely elusive. Gln is used as an energy substrate by monocytes. Gln deprivation of these cells results in an increased susceptibility to cell stress and apoptosis, as well as in a reduced responsiveness to pro-inflammatory stimuli. We performed a systematic study to elucidate the molecular mechanism by which Gln depletion affects the heat stress response of the monocytic cell line U937. Proteomic analysis revealed that Gln depletion was associated with specific changes in the protein expression pattern. However, the overall level of tRNA-bound Gln remained unaffected. The stress protein heat shock protein (Hsp) 70 showed the highest reduction in protein synthesis. This was due to enhanced mRNA decay during Gln starvation while the transcriptional and the translational control of Hsp70 expression remained unchanged. A physiological Gln concentration and above was found to be necessary for maximum Hsp70 accumulation upon heat shock. Thus, the study shows a specific link between Gln metabolism and the regulation of heat shock proteins.

Cells, Cultured↗

Toxocara-infestations in Austria: a study on the risk of infection of farmers, slaughterhouse staff, hunters and veterinarians.

A total of 585 persons from several occupational groups (farmers, slaughterhouse staff, hunters, veterinarians) exposed to Toxocara infestations and 50 persons of a control group were tested for the presence of specific antibodies to the Toxocara canis antigen using an enzyme-linked immunosorbent assay and a western blot. Farmers showed the highest seroprevalence (44%), followed by veterinarians (27%), slaughterhouse staff (25%) and hunters (17%), whereas only 2% of the individuals of the control group were seropositive. Thus, the risk to Toxocara infestation is 39, 18, 16 and 9 times higher for farmers, veterinarians, slaughterhouse staff (some workers were part-time farmers) and for hunters, respectively, when compared to the control group. The main source of infection in rural areas seems to be (roaming) farm cats and dogs that have not been dewormed. The results are discussed with a view to potential risk factors and preventive measures, in terms of veterinary and human medicine.

Abattoirs↗

[Toxoplasmosis round-robin test in Austria: results and limits of the toxoplasmosis screening].

In 1983 the Osterreichische Gesellschaft für Qualitätssicherung und Standardisierung medizinisch-diagnostischer Untersuchungen (OQUASTA) has invited the Department of Medical Parasitology of the Clinical Institute of Hygiene and Medical Microbiology, University of Vienna (today: Medical University of Vienna), to establish an external quality assessment service on the detection of specific antibodies against Toxoplasma gondii, one of the most prevalent protozoic parasites in the world. The objective of this project was the support of Austrian laboratories in standardising their test methods for the detection of specific antibodies against T. gondii. Between 1983 and 2004, 45 collaborating studies were carried out. During this period, the number of participating laboratories has increased from 10 in 1983 to about 50 in recent years. In total, the test results produced by the laboratories matched with the nominal values in more than 90%. On the examples of three human cases we demonstrate that externally validated serological methods alone, despite their great benefit, are not enough for a sufficient serodiagnosis of toxoplasmosis; unusually complicated serological situations do arise and can only be met by the knowledge about special tests, the application of an appropriate examination strategy and -- last but not least -- by many years of experience.

Austria↗

Role of cancer-associated stromal fibroblasts in metastatic colon cancer to the liver and their expression profiles.

The cancer microenvironment and interaction between cancer and stromal cells play critical roles in tumor development and progression. The molecular features of cancer stroma are less well understood than those of cancer cells. Cancer-associated stromal fibroblasts are the predominant component of stroma associated with colon cancer and its functions remain unclear. Fibroblast cell cultures were established from metastatic colon cancer in liver, liver away from the metastatic lesions, and skin from three patients with metastatic colorectal cancer. We generated expression profiles of cancer-associated fibroblasts using oligochip arrays and compared them to those of uninvolved fibroblasts. The conditioned media from the cancer-associated fibroblast cultures enhanced proliferation of colon cancer cell line HCT116 to a greater extent than cultures from uninvolved fibroblasts. In microarray expression analysis, cancer-associated fibroblasts clustered tightly into one group and skin fibroblasts into another. Approximately 170 of 22,000 genes were up-regulated in cancer-associated fibroblasts (fold change > 2, P < 0.05) as compared to skin fibroblasts, including many genes encoding cell adhesion molecules, growth factors, and COX2. By immunohistochemistry in-vivo, we confirmed COX2 and TGFB2 expression in cancer-associated fibroblasts in metastatic colon cancer. The distinct molecular expression profiles of cancer-associated fibroblasts in colon cancer metastasis support the notion that these fibroblasts form a favorable microenvironment for cancer cells.

Cell Line, Tumor↗

Bone morphogenetic protein 3B silencing in non-small-cell lung cancer.

Bone morphogenetic protein 3B (BMP3B) is a member of the TGF-beta superfamily. The BMP3B promoter sequence was previously identified as a target for aberrant DNA methylation in non-small-cell lung cancer (NSCLC). Aberrant DNA hypermethylation in the BMP3B promoter is associated with downregulation of BMP3B transcription in both primary human lung cancers as well as lung cancer cell lines. In order to understand the mechanisms of BMP3B silencing in lung cancer, a sample set of 91 primary NSCLCs was used to detect aberrant BMP3B promoter methylation, mutations in the coding sequence of BMP3B, and loss of heterozygosity (LOH). Our results showed that 45 of 91 (or 49.5%) tested primary NSCLCs exhibited increased promoter methylation, and 40% demonstrated LOH in at least one of the flanking microsatellite markers sJRH and D10S196 (63 kb upstream or 3.338 Mbp downstream of BMP3B). The lung cancer cell line A549, a type II alveolar epithelial human lung cancer cell line, is characterized by aberrant DNA promoter methylation. We used retroviral vector constructs containing the BMP3B cDNA to re-express the gene in A549 cells and to investigate the effects on cell growth. No change in the cell growth rate was observed after BMP3B re-expression, as compared to the vector controls. Although the number of colonies formed in anchorage-dependent assays was only slightly decreased, the colony-forming ability of A549 cells after BMP3B expression in anchorage-independent assays in soft agar was significantly reduced to 10% (P<0.005, t-test). Moreover, the in vivo tumorigenicity assay in nude mice indicated that cells re-expressing BMP3B grew significantly slower than cells not expressing BMP3B (P<0.05, t-test). In conclusion, this study provides evidence that BMP3B expression is repressed by different mechanisms in lung cancer, and that the silencing of BMP3B promotes lung tumor development.

Bone Morphogenetic Protein 3↗

Acute myeloid leukemia with complex karyotypes and abnormal chromosome 21: Amplification discloses overexpression of APP, ETS2, and ERG genes.

Molecular mechanisms of leukemogenesis have been successfully unraveled by studying genes involved in simple rearrangements including balanced translocations and inversions. In contrast, little is known about genes altered in complex karyotypic abnormalities. We studied acute myeloid leukemia (AML) patients with complex karyotypes and abnormal chromosome 21. High-resolution bacterial artificial chromosome (BAC) array-based comparative genomic hybridization disclosed amplification predominantly in the 25- to 30-megabase (MB) region that harbors the APP gene (26.3 MB) and at position 38.7-39.1 MB that harbors the transcription factors ERG and ETS2. Using oligonucleotide arrays, APP was by far the most overexpressed gene (mean fold change 19.74, P = 0.0003) compared to a control group of AML with normal cytogenetics; ERG and ETS2 also ranked among the most highly expressed chromosome 21 genes. Overexpression of APP and ETS2 correlated with genomic amplification, but high APP expression occurred even in a subset of AML patients with normal cytogenetics (10 of 64, 16%). APP encodes a glycoprotein of unknown function previously implicated in Alzheimer's disease, but not in AML. We hypothesize that APP and the transcription factors ERG and ETS2 are altered by yet unknown molecular mechanisms involved in leukemogenesis. Our results highlight the value of molecularly dissecting leukemic cells with complex karyotypes.

Acute Disease↗

Tissue-wide expression profiling using cDNA subtraction and microarrays to identify tumor-specific genes.

With the objective of discovering novel putative intervention sites for anticancer therapy, we compared transcriptional profiles of breast cancer, lung squamous cell cancer (LSCC), lung adenocarcinoma (LAC), and renal cell cancer (RCC). Each of these tumor types still needs improvement in medical treatment. Our intention was to search for genes not only highly expressed in the majority of patient samples but which also exhibit very low or even absence of expression in a comprehensive panel of 16 critical (vital) normal tissues. To achieve this goal, we combined two powerful technologies, PCR-based cDNA subtraction and cDNA microarrays. Seven subtractive libraries consisting of approximately 9250 clones were established and enriched for tumor-specific transcripts. These clones, together with approximately 1750 additional tumor-relevant genes, were used for cDNA microarray preparation. Hybridizations were performed using a pool of 16 critical normal tissues as a reference in all experiments. In total, we analyzed 20 samples of breast cancer, 11 of LSCC, 11 of LAC, and 8 of RCC. To select for genes with low or even no expression in normal tissues, expression profiles of 22 different normal tissues were additionally analyzed. Importantly, this tissue-wide expression profiling allowed us to eliminate genes, which exhibit also high expression in normal tissues. Similarly, expression signatures of genes, which are derived from infiltrating cells of the immune system, were eliminated as well. Cluster analysis resulted in the identification of 527 expressed sequence tags specifically up-regulated in these tumors. Gene-wise hierarchical clustering of these clones clearly separated the different tumor types with RCC exhibiting the most homogeneous and LAC the most diverse expression profile. In addition to already known tumor-associated genes, the majority of identified genes have not yet been brought into context with tumorigenesis such as genes involved in bone matrix mineralization (OSN, OPN, and OSF-2) in lung, breast, and kidney cancer or genes controlling Ca(2+) homeostasis (RCN1,CALCA, S100 protein family). EGLN3, which recently has been shown to be involved in regulation of hypoxia-inducible factor, was found to be highly up-regulated in all RCCs and in half of the LSCCs analyzed. Furthermore, 42 genes, the expression level of which correlated with the overall survival of breast cancer patients, were identified. The gene dendogram clearly separates two groups of genes, those up-regulated such as cyclin B1, TGF-beta 3, B-Myb, Erg2, VCAM-1, and CD44 and those down-regulated such as MIG-6, Esp15, and CAK in patients with short survival time.

Adenocarcinoma↗

[Nosology and epidemiology of human toxocarosis--the recent situation in Austria].

Toxocara canis and T. cati are not only ubiquitously distributed parasites of dogs, foxes and cats, but may also infest humans, causing a great variety of symptoms and sometimes also severe diseases: the visceral larva migrans syndrome, the ocular larva migrans syndrome, covert toxocarosis, common toxocarosis, neurological toxocarosis, and some other clinical pictures (asthma bronchiale, epilepsy, rheumatoid arthritis) are considered to be induced by Toxocara species. Both Toxocara species are also widely distributed in Austrian dog, fox and cat populations; seroepidemiological studies carried out in Austria revealed seroprevalence rates of 3.7% among the normal human population and up to 44% among persons particularly exposed to those parasites (i.e. veterinarians, farmers). Although many Toxocara infestations do not cause severe clinical manifestations, a few dozens of toxocarosis patients have been registered every year during the last years; in reality, however, we have to assume that several hundreds of patients suffer from toxocarosis. This paper tries to give a synoptic overview of the nosology of this (still) largely almost unknown helminthozoonosis, moreover it summarizes the most important epidemiologic parameters, and presents the diagnostic and therapeutic possibilities available today.

Adolescent↗

Definition of TCR epitopes for CTL-mediated attack of cutaneous T cell lymphoma.

Therapeutic vaccination against cutaneous T cell lymphoma (CTCL) requires the characterization of cancer cell-specific CTL epitopes. Despite reported evidence for tumor-reactive cytotoxicity in CTCL patients, the nature of the recognized determinants remains elusive. The clonotypic TCR of CTCL cells is a promising candidate tumor-specific Ag. In this study, we report that the clonotypic and framework regions of the TCRs expressed in the malignant T cell clones of six CTCL patients contain multiple peptides with anchor residues fitting the patients' MHC class I molecules. We demonstrate that TCR peptide-specific T cells from the blood of healthy donors and patients can be induced to become cytotoxic effectors after repeated stimulation with 6 of 11 selected peptides with experimentally proven affinity for HLA-A*0201. Importantly, 4 of these 6 CTL lines reproducibly recognize and lyse autologous primary CTCL cells in MHC class I/CD8-dependent fashion. These tumoricidal CTL lines are directed against epitopes from V, hypervariable, and C regions of TCRalpha. We therefore conclude that recombined as well as V framework regions of the tumor cell TCRs contain predictable epitopes for CTL-mediated attack of CTCL cells. Our data further suggest that such peptides represent valuable tools for future anti-CTCL vaccination approaches.

Amino Acid Sequence↗