Biomedical subjects

P Guldberg

Publications and source records attributed to P Guldberg.

At least 19 recordsLinked to original sources

PubMed2005

G(2) chromosomal radiosensitivity in Danish survivors of childhood and adolescent cancer and their offspring.

In order to investigate the relationship between chromosomal radiosensitivity and early-onset cancer, the G(2) chromosomal radiosensitivity assay was undertaken on a group of 23 Danish survivors of childhood and adolescent cancer, a control group comprising their partners and a group of 38 of their offspring. In addition, the previously reported in-house control group from Westlakes Research Institute (WRI) was extended to 27 individuals. When using the 90th percentile cutoff for the WRI control group, the proportion of individuals with elevated radiosensitivity was 11, 35, 52 and 53% for the WRI control, partner control, cancer survivor and the offspring groups, respectively, with significant differences between the WRI control group and the cancer survivor group (P=0.002) and the offspring group (P<0.001). However, while the comparisons with the WRI control group support an association of chromosomal radiosensitivity with cancer predisposition, when the partner control group was used to define the radiosensitivity cutoff point, no significant differences in radiosensitivity profiles were found between the partner control group and either the cancer survivor group or the offspring group. The failure to distinguish between the G(2) aberration profiles of the apparently normal group of partners and the cancer survivor group suggests that any association with cancer should be viewed with caution, but also raises questions as to the suitability of the partners of cancer survivors to act as an appropriate control group. Heritability of the radiosensitive phenotype was examined by segregation analysis of the Danish families and suggested that 67.3% of the phenotypic variance of G(2) chromosomal radiosensitivity is attributable to a putative major gene locus with dominant effect.

Adolescent↗

PubMed2004

Loss of heterozygosity at 9q33 and hypermethylation of the DBCCR1 gene in oral squamous cell carcinoma.

The DBCCR1 gene at chromosome 9q33 has been identified as a candidate tumour suppressor, which is frequently targeted by promoter hypermethylation in bladder cancer. Here, we studied the possible involvement of DBCCR1 in the development of oral squamous cell carcinoma. DNA from 34 tumours was examined for loss of heterozygosity (LOH) at three markers surrounding DBCCR1 and for hypermethylation of the DBCCR1 promoter, using methylation-specific PCR and methylation-specific melting-curve analysis. LOH was found in 10 of 31 cases (32%), and DBCCR1 hypermethylation was present in 15 of 34 cases (44%). Hypermethylation of DBCCR1 was also present in three of seven epithelial tissues adjacent to the tumours, including two hyperplastic and one histologically normal epithelia. Furthermore, of four oral leukoplakias with dysplasia, one showed LOH at 9q33 and two showed DBCCR1 hypermethylation. These data suggest that LOH at 9q33 and hypermethylation of the DBCCR1 promoter are frequent and possibly early events in oral malignant development.

Adult↗

PubMed2004

Screening for dysplasia and TP53 mutations in closed rectal stumps of patients with ulcerative colitis or Crohn disease.

BACKGROUND: Patients who undergo colectomy due to intractable chronic inflammatory bowel disease (IBD) may keep a closed rectal stump for several years, which may be at increased risk of malignant transformation owing to residual inflammatory activity. We examined a hospital series of patients with ulcerative colitis or Crohn colitis to describe the clinical, endoscopical and histological features of the closed rectal stump and to screen for dysplasia and mutations in the TP53 tumour suppressor gene. METHODS: During rigid proctoscopy, rectal mucosal biopsy specimens and rectal lavage fluid were collected from 42 patients. Biopsy specimens were examined histologically, and genomic DNA extracted from frozen biopsies and lavage fluid was analysed for mutations in TP53 exons 4-9. RESULTS: The median disease duration was 8.5 years (range 1.3-34 years). No endoscopic or histological signs of dysplasia or carcinoma were seen and no mutations in the TP53 gene were detected in any biopsy or lavage fluid specimens. Histological moderate to severe mucosal inflammation was present in 78% (33/42) of the patients, however, and rectal stump involution was noted in 43% (18/42). CONCLUSION: No signs of malignancy or premalignant degeneration were detected in this prospective series of IBD patients with a closed rectal stump. Although this is reassuring for patients, the presence of moderate to severe inflammation in the majority of rectal stumps indicates a role for adjuvant molecular markers to improve colorectal cancer surveillance on this subgroup of IBD patients.

Adult↗

PubMed2001

Methylation-dependent silencing of the reduced folate carrier gene in inherently methotrexate-resistant human breast cancer cells.

The molecular basis of methotrexate resistance was studied in human MDA-MB-231 breast cancer cells, which are inherently defective in methotrexate uptake and lack expression of the reduced folate carrier (RFC). Transfection of MDA-MB-231 cells with RFC cDNA restored methotrexate uptake and increased methotrexate sensitivity by approximately 50-fold. A CpG island in the promoter region of RFC was found to be methylated in MDA-MB-231 cells, but was unmethylated in RFC expressing, methotrexate-sensitive MCF-7 breast cancer cells. Chromatin immunoprecipitation with antibodies against acetylated histones H3 and H4 showed that the RFC promoter was enriched for acetylated histones on expressed, unmethylated alleles only. Treatment of MDA-MB-231 cells with 5-aza-2'-deoxycytidine restored RFC expression but also led to increased methotrexate efflux and did not reverse methotrexate resistance. This suggests that 5-aza-2'-deoxycytidine up-regulates both methotrexate uptake and some methotrexate-resistance mechanism(s). Reverse transcription-polymerase chain reaction analysis showed increased expression levels of several ATP-dependent efflux pumps in response to 5-aza-2'-deoxycytidine treatment, including P-glycoprotein and members of the multidrug resistance-associated protein family. Up-regulation of P-glycoprotein in response to 5-aza-2'-deoxycytidine was associated with demethylation of a CpG island in the MDR1 promoter, whereas the mechanism(s) for 5-aza-2'-deoxycytidine-induced up-regulation of multidrug resistance-associated proteins is probably indirect. Dipyridamole inhibited methotrexate efflux and reversed methotrexate resistance in 5-aza-2'-deoxycytidine-treated MDA-MB-231 cells.

Acetylation↗

PubMed2001

Characterization of Sp1, AP-1, CBF and KRC binding sites and minisatellite DNA as functional elements of the metastasis-associated mts1/S100A4 gene intronic enhancer.

The mts1/S100A4 gene encodes a small acidic calcium-binding protein that is expressed in a cell-specific manner in development, tumorigenesis and certain tissues of adult mice. A composite enhancer that is active in murine mammary adenocarcinoma cells was previously identified in the first intron of the mts1/S100A4 gene. Here we present a detailed analysis of the structure and function of this enhancer in the Mts1/S100A4-expressing CSML100 and non-expressing CSML0 mouse adenocarcinoma cell lines. In CSML100 cells the enhancer activity is composed of at least six cis-elements interacting with Sp1 and AP-1 family members and CBF/AML/PEBP2 and KRC transcription factors. In addition, a minisatellite-like DNA sequence significantly contributes to the enhancer activity via interaction with abundant proteins, which likely have been described previously under the name minisatellite-binding proteins. Extensive mutational analysis of the mts1/S100A4 enhancer revealed a cooperative function of KRC and the factors binding minisatellite DNA. This is the first example of an enhancer where two nuclear factors earlier implicated in different recombination processes cooperate to activate transcription. In Mts1/S100A4-negative CSML0 cells the strength of the enhancer was 7- to 12.5-fold lower compared to that in CSML100 cells, when referred to the activities of three viral promoters. In CSML0 cells the enhancer could be activated by exogenous AP-1 and CBF transcription factors.

Allosteric Site↗

PubMed2001

Missense mutations in the N-terminal domain of human phenylalanine hydroxylase interfere with binding of regulatory phenylalanine.

Hyperphenylalaninemia due to a deficiency of phenylalanine hydroxylase (PAH) is an autosomal recessive disorder caused by >400 mutations in the PAH gene. Recent work has suggested that the majority of PAH missense mutations impair enzyme activity by causing increased protein instability and aggregation. In this study, we describe an alternative mechanism by which some PAH mutations may render PAH defective. Database searches were used to identify regions in the N-terminal domain of PAH with homology to the regulatory domain of prephenate dehydratase (PDH), the rate-limiting enzyme in the bacterial phenylalanine biosynthesis pathway. Naturally occurring N-terminal PAH mutations are distributed in a nonrandom pattern and cluster within residues 46-48 (GAL) and 65-69 (IESRP), two motifs highly conserved in PDH. To examine whether N-terminal PAH mutations affect the ability of PAH to bind phenylalanine at the regulatory domain, wild-type and five mutant (G46S, A47V, T63P/H64N, I65T, and R68S) forms of the N-terminal domain (residues 2-120) of human PAH were expressed as fusion proteins in Escherichia coli. Binding studies showed that the wild-type form of this domain specifically binds phenylalanine, whereas all mutations abolished or significantly reduced this phenylalanine-binding capacity. Our data suggest that impairment of phenylalanine-mediated activation of PAH may be an important disease-causing mechanism of some N-terminal PAH mutations, which may explain some well-documented genotype-phenotype discrepancies in PAH deficiency.

Amino Acid Sequence↗

PubMed2001

In situ cytokine therapy: redistribution of clonally expanded T cells.

Immunity to tumors relies on recirculating antigen-specific T cells. Whilst induction of antigen-specific T cells by immunotherapy has been convincingly proven, direct evidence for recirculation of such cells is still lacking. Here, employing a recently established in situ immunotherapy model for murine melanoma we directly demonstrate the redistribution of clonally expanded T cells. In this model IL-2 is targeted to the tumor microenvironment by means of specific antibody-IL-2 fusion proteins resulting in the expansion of T cells. The therapeutic effect of the fusion protein is not restricted to tumors expressing the targeted antigen, but extends to antigen negative variants of the tumor if present in the same animal. Analysis of the T cell infiltrate by quantitative reverse transcription-PCR revealed the presence of highly expressed TCR BV regions in both tumor variants. TCR clonotype mapping revealed that the high expressions of these regions were caused by clonal expansions and, notably, that these specific clonotypic TCR transcripts were identical in both tumors. Thus, T cell clones activated locally by targeted IL-2 therapy recirculate and mediate eradication of distant tumor sites not subjected to in situ cytokine therapy.

Amino Acid Sequence↗

PubMed2001

In vitro expression of 34 naturally occurring mutant variants of phenylalanine hydroxylase: correlation with metabolic phenotypes and susceptibility toward protein aggregation.

Phenylalanine hydroxylase (PAH) is a homotetrameric enzyme that catalyzes the conversion of phenylalanine to tyrosine, the rate-limiting step of phenylalanine disposal in humans. Primary dysfunction of PAH caused by mutations in the PAH gene results in hyperphenylalaninemia, which may impair cognitive development unless corrected by dietary restriction of phenylalanine. The mechanism(s) by which PAH missense mutations cause enzyme impairment has been studied in detail only in a small number of cases, but existing evidence points to a major role of enhanced proteolytic degradation due to aberrant folding of mutant polypeptides. We have used two heterologous in vitro expression systems (a mammalian cell-free transcription-translation system and the pET system of Escherichia coli) to examine 34 mutations that have been associated with PAH deficiency in the Danish population. These mutations represent a broad range of amino acid substitutions, functional enzyme domains, and metabolic phenotypes. In both systems, residual in vitro activities correlated broadly with metabolic phenotypes, however, with significant discrepancies. Analysis of E. coli extracts by nondenaturing polyacrylamide gel electrophoresis and storage experiments showed that (i) in general, mutations in the N-terminal regulatory domain are associated with relatively stable proteins compared to most mutations in the central catalytic domain, and (ii) for mutations in the catalytic domain, high levels of protein aggregation do not always correspond with a severe phenotype. Our data support and extend previous evidence that PAH mutations exert their pathogenic effects by several distinct mechanisms that may operate individually or in concert.

Animals↗

PubMed2001

A phenylalanine hydroxylase amino acid polymorphism with implications for molecular diagnostics.

Mutations in the gene encoding phenylalanine hydroxylase (PAH, EC 1.14.16.1) are associated with various degrees of hyperphenylalaninemia, including classical phenylketonuria (PKU). We examined the PAH gene in a Brazilian PKU family of African origin and identified three missense variants, R252W (c.754C --> T), K274E (c.820A --> G), and I318T (c.953T --> C), the two latter of which were transmitted in cis. Expression analyses in two different in vitro systems showed that I318T is associated with profoundly decreased enzyme activity, whereas the enzyme activity of K274E is indistinguishable from that of the wild-type protein. Detailed kinetic analyses of PAH expressed in E. coli showed that the K274E mutant protein has kinetic properties similar to that of the wild-type protein. Population studies have suggested that the K274E variant occurs on approximately 4% of African-American PAH alleles, whereas the neonatal screening incidence of PKU among African Americans is only 1:100,000. This is to our knowledge the first demonstration of a PAH missense variant with no apparent association to PAH deficiency. Awareness of this common variant may be helpful to laboratories that perform molecular diagnosis of PAH deficiency in populations of African origin.

Alleles↗

PubMed2001

Mutation analysis of phenylketonuria in Yamagata prefecture, Japan.

BACKGROUND: We have screened 309,914 newborns in Yamagata prefecture, Japan, since 1977 and have detected four patients with phenylketonuria (PKU). We analyzed the phenylalanine hydroxylase (PAH) gene of the four patients to study the genetic background in this area and the genotype-phenotype relationship in these patients. METHODS: Mutations of the PAH gene were screened by denaturing gradient gel electrophoresis analysis and the sequences were determined. RESULTS: Three cases were compound heterozygotes of six different mutations of the PAH gene and the remaining case was a homozygote. Of the six detected mutations, K115fs is novel, whereas the others have been previously detected among Chinese and/or Japanese patients. CONCLUSIONS: The incidence and genetic basis in Yamagata prefecture was similar to that of other parts of Japan. Analysis of the genotype is useful to understand the clinical variation in some families.

Adolescent↗

PubMed2001

Normal clinical outcome in untreated subjects with mild hyperphenylalaninemia.

ABSTRACT There is international consensus that patients with phenylalanine (Phe) levels <360 microM on a free diet do not need Phe-lowering dietary treatment whereas patients with levels >600 microM do. Clinical outcome of patients showing Phe levels between 360 and 600 microM in serum on a free nutrition has so far only been assessed in a small number of cases. Therefore, different recommendations exist for patients with mild hyperphenylalaninemia. We investigated in a nationwide study 31 adolescent and adult patients who persistently displayed serum Phe levels between 360 and 600 microM on a normal nutrition with a corresponding genotype. Because of limited accuracy of measurements, Phe levels should be looked on as an approximation, but not as an absolute limit in every instance. In addition to serum Phe levels, the assessment program consisted of comprehensive psychological testing, magnetic resonance imaging of the head, (1)H magnetic resonance spectroscopy, and genotyping. We found a normal intellectual (intelligence quotient, 103 +/- 15; range, 79-138) and educational (school performance and job career) outcome in these subjects as compared with healthy control subjects (intelligence quotient, 104 +/- 11; range, 80-135). Magnetic resonance imaging revealed no changes of cerebral white matter in any patient, and (1)H magnetic resonance spectroscopy revealed brain Phe levels below the limit of detection (<200 microM). In the absence of any demonstrable effect, dietary treatment is unlikely to be of value in patients with mild hyperphenylalaninemia and serum Phe levels <600 microM on a free nutrition, and should no longer be recommended. Because of a possible late-onset phenylketonuria, Phe levels of untreated patients should be monitored carefully at least during the first year of life. Nevertheless, problems of maternal phenylketonuria should still be taken into account.

Adolescent↗

PubMed2001

Congenital heart disease in maternal phenylketonuria: report from the Maternal PKU Collaborative Study.

The frequency and types of congenital heart disease in offspring from pregnancies in women with hyperphenylalaninemia were examined in the international prospective Maternal Phenylketonuria Collaborative Study. Relationships of congenital heart disease in offspring to the basal blood phenylalanine level in the mother, metabolic control through diet during pregnancy, and phenylalanine hydroxylase mutations in mother and offspring were determined. The 416 offspring from 412 maternal phenylketonuria pregnancies that produced live births and 100 offspring from the 99 control pregnancies were included in this examination. Thirty-four of the 235 offspring (14%; 95% CI, 10.2 to 19.6%) from pregnancies in phenylketonuric women with a basal phenylalanine level > or = 900 microM (15 mg/dL) [normal blood phenylalanine < 120 microM (2 mg/dL)] and not in metabolic control [phenylalanine level < or = 600 microM (10 mg/dL)] by the eighth gestational week had congenital heart disease compared with one control offspring (1%) with congenital heart disease. One offspring among the 50 (2%) from mothers with non-phenylketonuria mild hyperphenylalaninemia also had congenital heart disease. Coarctation of the aorta and hypoplastic left heart syndrome were overrepresented compared with expected percentages among those with congenital heart disease in the general population. A basal maternal phenylalanine level > 1800 microM (30 mg/dL) significantly increased the risk for bearing a child with congenital heart disease (p = 0.003). Phenylalanine hydroxylase mutations in the mothers and offspring did not have an independent relationship to congenital heart disease but were related through the basal maternal phenylalanine levels. The data in this study indicate that a basal maternal phenylalanine level of 900 microM may be a threshold for congenital heart disease, that women with the most severe degree of phenylketonuria are at highest risk for bearing such a child, and that prevention of the congenital heart disease requires initiation of the low phenylalanine diet before conception or early in pregnancy with metabolic control no later than the eighth gestational week.

Female↗

PubMed2001

In-tube DNA methylation profiling by fluorescence melting curve analysis.

BACKGROUND: Most PCR assays for detection of 5-methylcytosine in genomic DNA entail a two-step procedure, comprising initial PCR amplification and subsequent product analysis in separate operations that usually require manual transfer. These methods generally provide information about methylation of only a few CpG dinucleotides within the target sequence. METHODS: An in-tube methylation assay is described that integrates amplification of bisulfite-treated DNA and melting analysis by using a thermal cycler coupled to a fluorometer (LightCycler). DNA melting curves were acquired by measuring the fluorescence of a double-stranded DNA-binding dye (SYBR Green I) during a linear temperature transition. RESULTS: Analysis of a region comprising 11 CpG sites at the SNRPN promoter CpG island showed that the melting temperature (T(m)) differed by approximately 3 degrees C between unmethylated and fully methylated alleles. This assay could easily distinguish patients with Prader-Willi syndrome or Angelman syndrome from individuals without these conditions. Melting curve analysis also allowed resolution of methylation "mosaicism" at the p15(Ink4b) promoter in bone marrow samples from patients with acute myeloid leukemia (AML). AML samples representing pools of heterogeneously methylated p15(Ink4b) alleles showed broadened melting peaks with overall T(m)s between those of the unmethylated and fully methylated alleles. CONCLUSIONS: Integration of PCR and fluorescence melting analysis may be useful for simple and cost-effective detection of aberrant methylation patterns.

Acute Disease↗

PubMed2000

Aberrant p27Kip1 promoter methylation in malignant melanoma.

p27Kip1 is a regulator of the mammalian cell cycle and a putative tumor suppressor. Distinct altered patterns of p27Kip1 protein expression are found in a variety of human carcinomas, and p27Kip1 expression levels usually correlate directly with disease-free survival. The mechanism(s) by which p27Kip1 expression is reduced or lost during tumorigenesis remains unclear. Specific alterations of the p27Kip1 gene, including mutations and homozygous deletions, are exceedingly rare in human cancers. We have used methylation-specific PCR and bisulfite genomic sequencing to examine the methylation status of p27Kip1 in 61 primary and metastatic tumors and 35 cell lines from patients with malignant melanoma. Dense methylation of a CpG island in the promoter region of p27Kip1 was detected in four of 45 metastatic tumors (9%) and three of the cell lines (9%), including two cell lines established from two different metastases from the same patient. Examination of a naturally occurring, allele-specific sequence variant demonstrated that p27Kip1 methylation is associated with transcriptional silencing in situ. Cell lines with p27Kip1 methylation showed retention of the wild-type allele and detectable p27Kip1 protein whose abundance was reduced compared with normal melanocytes. Collectively, our data suggest that DNA methylation may be a cause of monoallelic p27Kip1 silencing in malignant melanoma, which would support a role for p27Kip1 haploinsufficiency in human cancer.

Amino Acid Substitution↗

PubMed2000

Cell cycle arrest by the PTEN tumor suppressor is target cell specific and may require protein phosphatase activity.

PTEN, a tumor suppressor commonly targeted in human cancer, possesses phosphatase activities toward both protein and lipid substrates. While PTEN suppresses gliomas through cell cycle inhibition which requires its lipid phosphatase activity, PTEN's effects on other tumor types and the role of its protein phosphatase activity are controversial or unknown. Here we show that exogenous wild-type PTEN arrests some, but not all human breast cancer cell lines in G1, in a manner independent of endogenous PTEN. Unexpectedly, the G129E mutant of PTEN selectively deficient in the lipid phosphatase activity still blocked the cell cycle of MCF-7 cells, while the G129R and H123Y mutants lacking both phosphatase activities were ineffective. These results suggest that PTEN's protein phosphatase activity likely contributes to its tumor suppressor function in subsets of tumors and that elucidation of downstream targets which dictate cellular responses to PTEN may have important implications for future cancer treatment strategies.

Blotting, Western↗

PubMed2000

Tumor infiltrating lymphocytes in melanoma comprise high numbers of T-cell clonotypes that are lost during in vitro culture.

Melanoma is generally accepted as being an antigenic tumor capable of eliciting T-cell responses that, however, in most cases are inadequate to control tumor growth. Tumor-infiltrating lymphocytes (TIL) in melanoma lesions comprise clonotypic T cells, indicating the in situ recognition of melanoma-associated peptide epitopes. Cultured TIL have been studied in order to unveil characteristics of TIL and the interactions of TIL and melanoma cells. Whether in vitro cultured TIL mirrors the in situ situation has, however, been questioned. In the present study we have taken advantage of T-cell receptor clonotype mapping methodology to conduct a full and detailed analysis of the T-cell clonotypes in melanoma lesions and in corresponding lines of TIL established in vitro. All melanoma lesions and the corresponding TIL cultures comprised high numbers of T-cell clonotypes, typically in the range of 40 to more than 60. The subsequent comparison of T-cell clonotypes present in the original lesions and in the corresponding T-cell lines established in vitro demonstrated that a very limited number of the T-cell clonotypes established in vitro are identical to the T-cell clonotypes expanded in situ. These results demonstrate that in situ T-cell clonotypes in melanoma are not readily expanded in vitro and that the majority of T-cell clonotypes present in cultured TIL are not present in situ.

Cell Count↗

PubMed2000

Mutation analysis anticipates dietary requirements in phenylketonuria.

UNLABELLED: Phenylalanine hydroxylase (PAH) deficiency is inherited as an autosomal recessive trait and the associated hyperphenylalaninaemia phenotype is highly variable, primarily due to a great allelic heterogeneity at the PAH locus (approximately 400 disease-associated mutations are known). The arbitrary classification of PAH deficiency on the basis of clinical parameters has been complicated by the lack of international guidelines, leading to a wide confusion in both methodology and terminology. Recently, significant improvements in methods for detection of mutations have paved the way for an alternative system for classification of PAH deficiency, which is based solely on PAH genotypes. This paper gives a summary of the recent progress made in establishing a direct correlation between individual PAH mutations and biochemical and metabolic phenotypes, including the use of "functionally hemizygous" patients to classify both common and rare mutant alleles, and a simple and general model to predict the combined phenotypic effect of two mutant PAH alleles. CONCLUSION: Genotype-based prediction of the biochemical phenotype is now feasible in the majority of newborns with hyperphenylalaninemia, which may be useful for refining diagnosis and anticipating dietary requirements. A recent observation suggests that the genotype also determines cognitive development if dietary therapy is discontinued at 6 years of age.

Adolescent↗

PubMed2000

Comparative delineation of T cell clonotypes in coexisting syngeneic B16 melanoma.

B16 is a murine melanoma of C57B1/6 origin, which rapidly develops as a tumor when inoculated into syngeneic immunocompetent hosts. Nevertheless, B16 tumors are considered to be immunogenic since tumor regression can be induced by means of immunotherapeutic intervention. Furthermore, B16 melanoma cells express several melanoma-associated antigens that may serve as targets for autologous T cells. To study the in vivo T cell response against B16, with particular emphasis on diversity and systemic involvement, we examined the spectra of T cell clonotypes in coexisting B16 melanoma lesions in C57B1/6 mice. Three tumors from each animal (n = 8) were examined for the presence of clonotypic T cells using the highly sensitive T cell receptor (TCR) clonotype mapping technology. Systematic analysis of the TCRB variable regions 1-16 revealed from 19 to more than 30 clonotypic TCR transcripts in each tumor. To study intra- and inter-individual variations in the T cell response further, more than 600 clono-typic TCR transcripts were compared for sequence identity. Overall, approximately 2% of the T cell clonotypes were detected in more than one tumor from the same animal. Furthermore, none of the detected clonotypes was present in more than one animal, arguing against recurrent or "public" T cell responses against B16 melanoma. Our data strongly suggest that anti-melanoma T cell responses in this murine model encompass mainly localized T cells, and that systemic involvement is limited.

Animals↗