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Biomedical subjects

M G Tilanus

Publications and source records attributed to M G Tilanus.

At least 19 recordsLinked to original sources
PubMed2007

Role of human leucocyte antigen DQ in the presentation of T cell epitopes in the major cow's milk allergen alphas1-casein.

BACKGROUND: Little is known about the association between human leucocyte antigen (HLA) and cow's milk allergy (CMA). The aim of the present study was to determine the HLA restriction of T cell clones (TCCs) specific to alphas1-casein, the most abundant milk protein, and to study possible HLA class II allele associations with CMA. METHODS: alphas1-Casein-specific TCCs were derived from 6 children with CMA, 9 atopic children without CMA and 5 non-atopic children. T cell epitope specificity was defined by stimulation with overlapping peptides, spanning the alphas1-casein molecule. HLA restriction was determined in proliferation assays using antibodies blocking either HLA-DP, HLA-DQ or HLA-DR. HLA genotyping was performed in 32 subjects with CMA, 23 atopic and 22 non-atopic individuals. RESULTS: Ten TCCs were restricted to HLA-DQ, 6 TCCs to HLA-DR and 4 TCCs to HLA-DP. The sequence in alphas1-casein that was most immunogenic to T cells from children with CMA contained T cell epitopes restricted to DQB1*0201, DPB1*0401 and DRB1*1501. The DQB1*0501 allele frequency was lower in children with CMA than in non-atopic children, but this difference could not be confirmed in an additional group of subjects with and without CMA. CONCLUSIONS: HLA-DQ plays a substantial role in the presentation of T cell epitopes in alphas1-casein. However, HLA class II allele frequencies do not show major differences between cow's milk allergic, atopic and non-atopic subjects. T cell epitopes in the most immunogenic region are presented by various abundantly present HLA genotypes. Therefore, this sequence may be a suitable target for peptide immunotherapy.

Adolescent↗
PubMed2004

A multicenter international evaluation of single-tube amplification protocols for sequencing-based typing of HLA-DRB1 and HLA-DRB3,4,5.

We have described previously a novel single-tube polymerase chain reaction (PCR) amplification (STAmp) protocol for the efficient sequencing-based typing (SBT) of human leukocyte antigen (HLA)-DRB1. The PCR amplification mix includes primers to each of seven allele group-sequence motifs. We have applied this principle to the simultaneous SBT of HLA-DRB3, -DRB4, and -DRB5 using locus specific primers. We report here a multicenter international evaluation of the STAmp protocols performed as a component of the 13th International Histocompatibility Workshop. Identical amplification primer mixes, sequencing primers, and DNA were sent to participating laboratories. The primer mixes contained the amplification primers and the PCR buffer. Each laboratory was requested to amplify the DNA with the primer mixes and perform SBT on the resulting PCR protocols, using their own protocols, and return the typing results for analysis. The reported results indicated that the expected sequence could be obtained with a variety of PCR amplification and sequencing platforms and protocols. There were difficulties but these seemed unrelated to STAmp reagents and suggest that optimal SBT results can be obtained if bi-directional sequencing is performed and software is used for sequence verification and editing. This indicates that SBT by STAmp can be applied in many laboratories for high-throughput HLA-DRB1 and HLA-DRB3,4,5 SBT.

Alleles↗
PubMed2003

HLA-DPB1*0401 is associated with dominant protection against type 1 diabetes in the general Saudi population and in subjects with a high-risk DR/DQ haplotype.

The human leukocyte antigen (HLA) class II DQB1*0201/0202-DRB1*04 genotype has been identified as predisposing to type 1 diabetes [insulin-dependent diabetes mellitus (IDDM)] in the Saudi Arabian population (P = 0.0002; odds ratio = 0.67; 95% confidence interval = 0.009-0.381). In this study, we searched for a factor at the DPB1 locus by analysing DPB1 polymorphism using sequence-based typing in 86 Saudi IDDM patients and control subjects, all carrying the HLA-DRB1*04/DQB1*02 haplotype or the known susceptibility allele DQB1*0201/0202. Significant protection was conferred by DPB1*0401, which was observed in 17 of 50 control subjects (55%) and 2 of 36 IDDM patients (5%) with the DQB1*0201/0202 allele (P = 0.0012; odds ratio = 8.75; confidence interval = 1.72-59.70). Our data showing a high frequency of the DPB1*0401 allele even in the presence of the predisposing DQB1*02 allele in healthy subjects may indicate a protective effect of this combination of HLA alleles against type 1 diabetes. This finding supports the hypothesis that protective HLA class II genes can override the risk conferred by HLA-DQ susceptibility alleles. Further studies using larger cohorts of control subjects and patients should be undertaken to confirm this observation.

Diabetes Mellitus, Type 1↗
PubMed2001

No association between IL-10 promoter gene polymorphism and heart failure or rejection following cardiac transplantation.

Expression of interleukin (IL)-10 influences the frequency of rejection events after organ transplantation. Therefore, 70 heart transplant patients were genotyped for three single nucleotide polymorphisms and a microsatellite polymorphism in the promotor region of the IL-10 gene. The promoter region was amplified by polymerase chain reaction and genotyped by a colorometric oligo ligation assay and gene scan analysis, respectively. Patient groups consisted of patients suffering from dilated cardiomyopathy or ischaemic heart disease. Cardiac donors served as control group. No correlation was found between genotypes and heart failure or rejection after heart transplantation. This may indicate that in heart transplantation, the total balance of cytokine production is more important for post-transplant rejection activities than the levels of IL-10 as such.

Gene Expression↗
PubMed2001

Polymorphism of human HLA-DRB1 antigens generated by genetic exchange between DR2 (DRB1*15011) and DR6 (DRB1*1405) alleles: a novel DRB1 allele (DRB1*1437) identified in a Paiwan tribe member of Taiwan.

We report herein the identification of a new DRB1 allele using sequence-based typing (SBT). This novel allele, HLA-DRB1*1437, was found in an aboriginal individual from the Paiwan tribe in the southern part of Taiwan. This individual was typed by SBT method as having an HLA genotype of HLA-A*02011/0203, HLA-B*15011/3901, HLA-DRB1*11011/1437, HLA-DRB3*0202/0202, and HLA-DPB1*0501/1301. This new allele differs from DRB1*1309 in the 5'-end nucleotide sequence of polymorphic exon 2 at codon 16 (CAT-->CAA; H16Q), codon 37 (AAC-->TTC; R37F), codon 47 (TTC-->TAC; F47Y), and codon 58 (GCC-->GCT; both specify alanine). By sequence comparison, it was found that this new allele has a 5'-end sequence (from amino acid residues 7 to 66) identical to that found in the DRB1*1405 allele and a 3'-end sequence (from amino acid residues 58 to 94) identical to that found in the DRB1*15011 allele. Both DRB1*1405 and DRB1*15011 alleles have been identified among the Paiwan members (Note).

Alleles↗
PubMed2001

Identification of human ccn2 (connective tissue growth factor) promoter polymorphisms.

BACKGROUND: Connective tissue growth factor (CCN2; CTGF) is a newly identified growth factor, which is involved in the regulation of wound repair and fibrosis. Because there is variation among individuals with respect to tissue response to injury, genetic factors might be involved in the final outcome of tissue repair or scarring. For example, polymorphisms in the promoter region of genes, such as those encoding transforming growth factor beta1 (TGF-beta1), interleukin 10 (IL-10), and tumour necrosis factor alpha (TNF-alpha), influence transcriptional responses and are thought to contribute to the dysregulation of these genes in pathological conditions. AIM: To investigate whether the promoter region of the ccn2 (ctgf) gene contains polymorphic sequences that might account for differential expression. MATERIALS/METHODS: Seventy seven human DNA samples were sequenced-45 were from healthy controls and 32 were from patients with ischaemic heart disease (IHD)-using M13 tailed sequence specific ccn2 (ctgf) primers for amplification of a 600 bp fragment upstream of the transcription start site. Amplicons were bidirectionally sequenced with a dye primer M13 forward and reverse sequencing kit. RESULTS: A C to G substitution was identified at position -132 in one of the patients with IHD. Moreover, in five of the 32 patients with IHD and in six of the 45 healthy controls, a G to C polymorphism was found at position -447. These substitutions at -132 and -447 are thought to lie within predicted binding domains for the transcription factors Pbx-1 and MZF1, respectively. In addition, insertions at position -43 (G), -47 (C), -71 (G) and a C to T substitution at position -198 were found in all DNA samples compared with the published ccn2 (ctgf) promoter sequence. These corrections do not involve sequences predicted to function as transcription factor binding sites. CONCLUSION: Sequence analysis of the ccn2 (ctgf) promoter of 77 human DNA samples has revealed corrections and polymorphic sites. The latter lie within putative regulatory elements.

Alleles↗
PubMed2001

Sensitive detection of p53 mutation: analysis by direct sequencing and multisequence analysis.

In a wide variety of tumors, p53 mutations may have prognostic and diagnostic value. However, mutational screening methods often are restricted to the core domain and, therefore, do not detect all mutations. We improved existing sequencing-based mutation analysis methods consisting of direct sequencing of all exons of the p53 gene and RNA. Multisequence analysis software was developed and applied to increase the sensitivity of mutation identification. Multisequence analysis compares a large number of sequences and identifies profiles with additional small peaks, potentially indicating a mutation. Concordance between blood and tumor sequences indicates polymorphism, whereas discordance indicates a mutation. We could detect mutations with a limit of approximately 5% to 10% mutated DNA. In our ongoing studies, we applied sequencing-based mutation analysis for more than 50 patients with head and neck squamous cell carcinoma and identified mutations in more than 95% of all tumors. We encountered some differences between the previously published reference p53 sequence and all our sequences and identified polymorphism in six regions.

Base Sequence↗
PubMed2000

The origins of multiple squamous cell carcinomas in the aerodigestive tract.

BACKGROUND: Chemoprevention and cessation of smoking and alcohol may prevent development of multiple tumors (MTs) in the aerodigestive tract if new MTs arise independently, but they are of no benefit if MTs are due to migration of an already transformed clone of tumor cells. This issue was addressed in this study by investigation of the clonality among MTs. METHODS: Mutation analysis of the entire coding region of p53 and loss of heterozygosity (LOH) pattern analysis of microsatellite markers on chromosome arms 3p, 9p, and 17p are promising for the investigation of clonality. In the first part of this study, the authors established the variability and stability of these clonal markers by comparing primary head and neck squamous cell carcinomas (HNSCCs) with their metastases. In the second part of this study, the authors evaluated nine patients with multiple HNSCCs using these markers. In the final part, the authors illustrate the use of these clonal markers in 11 patients for whom there was diagnostic uncertainty as to whether their second squamous cell carcinoma was either a new primary tumor, a metastasis, or a recurrence. RESULTS: Both p53 gene mutations and LOH patterns were stable during tumor progression. Furthermore, the variability of p53 gene mutations was high. More than 90% of the tumors contained a p53 mutation. A particular mutation never occurred more than twice in a total of 69 primary HNSCCs. Five of 69 cases presented a common mutation. In contrast, LOH patterns showed less variability; they were identical in 5 of 16 cases. The metachronous HNSCCs from nine patients all showed different p53 mutations, and in the three cases that were subjected to LOH analysis different patterns were observed. All 11 patients for whom there was diagnostic uncertainty about the origin of their second squamous cell carcinoma could be categorized as having multiple primary tumors, disseminated disease, or recurrent disease. CONCLUSIONS: Metachronous HNSCCs at different locations are not clonally related and thus have not developed from the migration of tumor cells.

Carcinoma, Squamous Cell↗
PubMed2000

IgE production after antigen-specific and cognate activation of HLA-DPw4-restricted T-cell clones, by 78% of randomly selected B-cell donors.

The frequency of expression of the MHC class II antigen, HLA-DPw4, in the caucasoid population is approximately 78%, and is unmatched by phenotypic frequencies of other HLA class II molecules. Here we describe three human Der-P1-specific T-cell clones (TCC), restricted by the HLA-DPw4-variant HLA-DPB1*0401, of which two TCC also responded to antigen, presented on HLA-DPB1*0402. Thus, randomly selected caucasoid donors present a 78% chance for a correct match with these HLA-DPw4-restricted TCC. This allows comparative in vitro antigen presentation studies with various antigen presenting cells (APC) from different (healthy or diseased) donors without the variable influence of responding T cells. It was subsequently demonstrated that the TCC can be used to study antigen-induced IgE production in randomly selected primary B cells. Cognate HLA-DPw4-restricted antigen presentation caused enhanced immunoglobulin production of IgE, IgG1, IgA and IgM, of which only IgE induction was reversed by addition of anti-IL-4 antibodies.

Antigen-Presenting Cells↗
PubMed2000

A novel HLA-A24 (A*2420) allele identified in the Atayal tribe of Taiwan.

The Taiwan indigenous population groups are classified into different tribes according their linguistic classification and cultural anthropology. One of the tribes, the Atayal, showed a high frequency of A24 alleles by SSOP analysis. High-resolution sequencing based typing identified a A*2402 variant "A*2420" which was found in 6 unrelated individuals. High-resolution typing is required to identify HLA polymorphism in the Taiwanese minority groups.

Alleles↗
PubMed2000

DPB1*8501, a novel DPB1 variant in the US Black population.

We describe a new DPB1 allele, DPB1*8501, which was identified by sequencing-based typing (SBT) in the UCLA exchange. DPB1*8501 is similar to DPB1*2701 with a difference at position 272, (G to A). This difference leads to an amino-acid change of codon 91 from arginine (CGC) to histidine (CAC). Until now this position has been considered conserved. This substitution is located at the 3' site of exon 2, and may interfere with typing strategies using primers or probes located in this region.

Alleles↗
PubMed2000

Two new HLA DPB1 alleles identified by sequence-based typing: DPB1*8201 and DPB1*8301.

Two new HLA DPB1 alleles were identified by sequence-based typing and are reported. Both alleles differ from DPB1*0402 by a single nucleotide: DPB1*8201 has a difference at position 359 (codon 91) leading to an amino acid change from arg to his, making this position a new polymorphic site; DPB1*8301 has a difference at position 280 (codon 65) changing the amino acid from ile to phe.

Alleles↗
PubMed2000

Monitoring of residual disease and guided donor leucocyte infusion after allogeneic bone marrow transplantation by chimaerism analysis with short tandem repeats.

In this study, we analysed the chimaeric status of peripheral blood leucocytes (PBLs) in recipients of allogeneic bone marrow transplantation (BMT) with the use of short tandem repeat (STR) microsatellite markers for monitoring the efficacy of BMT and donor leucocyte infusions (DLIs). A set of four STR markers was used with a highly discrimative capacity between individuals. STRs were detected by polymerase chain reaction (PCR) and were analysed by gene scanning (STR-GS). Between June 1990 and December 1998, 52 patients treated with BMT for chronic myeloid leukaemia (CML) were analysed. Seventeen patients relapsed after BMT and two patients never achieved remission after BMT. Fourteen of the 17 patients achieved a complete donor chimaerism after BMT, as detected by the presence of only donor STR-GS fragments, and in three cases a weak recipient STR-GS signal remained persistently detectable after BMT. A reappearance or increase of recipient STR-GS signals was indicative of relapse, which was mostly detected by STR-GS several months before relapse was diagnosed clinically. Nineteen patients were treated with DLI for reappearance of CML after BMT which resulted in complete remission in 17 patients, concordant with the disappearance of recipient STR-GS signals. More importantly, DLI treatment could be guided based upon the STR-GS data, which prevented unnecessary extra DLI courses that could cause toxicity. This study indicates that STR-GS is an effective and reliable method for monitoring BMT recipients.

Adult↗
PubMed2000

Human oesophageal adenocarcinoma cell lines JROECL 47 and JROECL 50 are admixtures of the human colon carcinoma cell line HCT 116.

In two recently described human oesophageal adenocarcinoma cell lines JROECL 47 and JROECL 50, derived from one tumour, we detected identical E-cadherin and beta-catenin gene mutations as in colon carcinoma cell line HCT 116. We demonstrate by HLA-typing, mutation analysis and microsatellite analysis that cell lines JROECL 47 and JROECL 50 are admixtures of the human colon adenocarcinoma cell line HCT 116.

Adenocarcinoma↗
PubMed2000

Bioinformatics: analysis of HLA sequence data.

High resolution HLA typing is a requirement for the selection of matched donors in hematopoietic cell transplantation. The high resolution typing method that provides the most accurate and complete identification of HLA genotypes is sequencing-based typing (SBT). For each sample being tested, SBT defines the exact nucleotide sequence of the coding regions of both alleles at a given HLA locus. Identification of the underlying genotype of the sample can then be made by computerized sequence comparison with all possible HLA allele combinations at that locus. The use of SBT to identify the complete nucleotide sequence of a given HLA gene also enables the direct detection of previously undefined alleles. Since different HLA alleles may differ by a single nucleotide, the accurate assignment of an HLA genotype by SBT is absolutely dependent on the correct identification of the nucleotide at each position for a given sample. However, automated sequence analysis of heterozygous samples may result in the ambiguous assignment of nucleotides at a given position. In addition, ambiguous assignments may result from the sequencing of two different samples that express different HLA alleles but whose sequence profiles appear exactly the same. Both of these ambiguous situations can be resolved by the application of the multi-sequence analysis (MSA) method described here.

Alleles↗
PubMed1999

p53 tumor suppressor gene as a clonal marker in head and neck squamous cell carcinoma: p53 mutations in primary tumor and matched lymph node metastases.

In order to define the diagnostic value of p53 tumor suppressor gene as a clonal marker in head and neck squamous cell carcinoma (HNSCC), we investigated p53 mutations in primary tumors (PT) and matched lymph node metastases (LNM); the underlying question being whether differentiation between metastatic disease of a known PT or (a metastasis of) a synchronous or metachronous second tumor is possible by means of p53 sequencing-based mutation analysis. In 15 PT, the p53 status was analyzed, following RNA isolation, cDNA synthesis and polymerase chain reaction amplification, by direct sequencing full-length mRNA. Mutations thus found were confirmed by DNA sequencing analysis of the corresponding exon in the PT. When RNA isolation was defective, DNA sequencing analysis of exons 1 through 11 was performed. In the matched LNM, DNA analysis of the corresponding exon was performed to prove the presence of the same p53 mutation. In the event of small clones not detectable by direct sequencing, an oligo ligation assay was developed to detect a specific mutation. The presence of germline mutations was excluded by DNA sequencing analysis of the corresponding exon of peripheral blood leucocytes. In 14 PT (94%), a mutation was identified. In one PT, no p53 mutation could be identified either after full-length mRNA sequencing or after sequencing exons 1 through 11. In all cases of PT and matched LNM, the mutations proved to be identical. We conclude that p53 mutations develop in carcinogenesis before metastases occur and are maintained during metastasis. Consequently, p53 may serve as a clonal marker not susceptible to change during tumor metastasis. This merits further exploration of the application of p53 mutation analysis in differentiating between metastatic disease from a known PT versus a metastasis of another second PT.

Adult↗
PubMed1999

Characterization of a novel HLA-A2 variant, A*02012, in the Southern Thai-Muslim population.

Southern Thai Muslims (STM)--from Nakon Si Thammarat, whose ancestors come mainly from Malaysia--constitute more than half of all Thai Muslims which, in total, represent approximately 10% of the country's population. The most common A2 subtypes in STM were A*0203 (n=15), A*0201 (n=8) and A*0207 (n=7). In this study, samples with unexpected amplification patterns were sequenced. Three individuals were indicative of a novel A2 allele, now known as A*02012.

Base Sequence↗