PubMed Health⌕ Search

Biomedical subjects

Olfert Landt

Publications and source records attributed to Olfert Landt.

At least 19 recordsLinked to original sources
PubMed2006

H5N1 Oseltamivir-resistance detection by real-time PCR using two high sensitivity labeled TaqMan probes.

A single amino acid substitution, from histidine to tyrosine at position 274 of the neuraminidase gene has converted Oseltamivir sensitive H5N1 influenza A virus into a resistant strain. Currently, Oseltamivir is being stockpiled in many countries potentially affected by the influenza A virus subtype H5N1 epidemic. To identify this change in Oseltamivir-treated patients, a method based on real-time PCR using two labeled TaqMan probes was developed for its rapid detection. In order to validate the method, Oseltamivir specimen from treated (Oseltamivir-resistant strain from a Vietnamese patient, two Oseltamivir-treated tigers) and untreated subjects have been used for this study. The results thus obtained as well as those derived from clone selection and sequencing showed that TaqMan probes could clearly discriminate wild type H274 from the mutant 274Y variant. The sensitivity of this assay was as low as 10 copies/microl and allowed the detection of the mutation in a mixture of wild type and mutant. Overall, the assay based on real-time PCR with two labeled TaqMan probes described here should be useful for detecting Oseltamivir-resistant H274Y H5N1 influenza A virus in many species and various sources of specimens with high sensitivity and specificity. Such studies can address potential differences in the diagnostic outcomes between patients who develop detectable Oseltamivir resistance and those who retain only the wild type strain of H5N1.

Antiviral Agents↗
PubMed2006

Keratin 8 sequence variants in patients with pancreatitis and pancreatic cancer.

Keratin 8 (KRT8) is one of the major intermediate filament proteins expressed in single-layered epithelia of the gastrointestinal tract. Transgenic mice over-expressing human KRT8 display pancreatic mononuclear infiltration, interstitial fibrosis and dysplasia of acinar cells resulting in exocrine pancreatic insufficiency. These experimental data are in accordance with a recent report describing an association between KRT8 variations and chronic pancreatitis. This prompted us to investigate KRT8 polymorphisms in patients with pancreatic disorders. The KRT8 Y54H and G62C polymorphisms were assessed in a cohort of patients with acute and chronic pancreatitis of various aetiologies or pancreatic cancer originating from Austria (n=16), the Czech Republic (n=90), Germany (n=1698), Great Britain (n=36), India (n=60), Italy (n=143), the Netherlands (n=128), Romania (n=3), Spain (n=133), and Switzerland (n=129). We also studied 4,234 control subjects from these countries and 1,492 control subjects originating from Benin, Cameroon, Ethiopia, Ecuador, and Turkey. Polymorphisms were analysed by melting curve analysis with fluorescence resonance energy transfer probes. The frequency of G62C did not differ between patients with acute or chronic pancreatitis, pancreatic adenocarcinoma and control individuals. The frequency of G62C varied in European populations from 0.4 to 3.8%, showing a northwest to southeast decline. The Y54H alteration was not detected in any of the 2,436 patients. Only 3/4,580 (0.07%) European, Turkish and Indian control subjects were heterozygous for Y54H in contrast to 34/951 (3.6%) control subjects of African descent. Our data suggest that the KRT8 alterations, Y54H and G62C, do not predispose patients to the development of pancreatitis or pancreatic cancer.

Acute Disease↗
PubMed2006

Real-time PCR using hybridization probes for the rapid and specific identification of Francisella tularensis subspecies tularensis.

Tularemia is a plague-like infection caused by Francisella (F.) tularensis classified as a biological warfare agent. F. tularensis subsp. tularensis is the most virulent subspecies demanding rapid diagnosis. Typing systems for this fastidious bacterium to the subspecies level are laborious and time consuming. Therefore, the aim of this study was to develop a real-time PCR for the rapid and specific identification of F. tularensis subsp. tularensis. The specificity of the assay was determined using a comprehensive panel of Francisella strains, clinically relevant bacteria, and DNA preparations of potential hosts. F. tularensis subsp. tularensis was specifically detected but no other organisms. The range of linearity was determined to be 100 fg to 10 ng, the lower limit of detection was 25 fg of DNA (13 genome equivalents). An internal amplification control PCR system targeting lambda phage DNA was included. Neither the internal amplification control nor host DNA influenced the cycle threshold values obtained for F. tularensis subsp. tularensis. In conclusion, we have developed a highly sensitive and specific assay that can be integrated into real-time PCR-based identification procedures for biological agents. This is a major diagnostic improvement, as all other methods for the specific identification of F. tularensis subsp. tularensis are more time consuming.

Animals↗
PubMed2006

A degradation-sensitive anionic trypsinogen (PRSS2) variant protects against chronic pancreatitis.

Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 x 10(-8)). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.

Base Sequence↗
PubMed2006

DLG5 variants in inflammatory bowel disease.

OBJECTIVES: Genetic variants within DLG5 were recently reported to be associated with inflammatory bowel disease (IBD). The aim of our study was to test for allelic and haplotype associations of six DLG5 variants in 668 IBD patients from two European populations. Furthermore, we evaluated whether DLG5 variants alter gastrointestinal permeability in Crohn's disease (CD). METHODS: Six DLG5 variants (p.R30Q, p.P1371Q, p.G1066G, rs2289308, DLG_e26, p.D1507D) were genotyped in two study populations: (1) German IBD patients (CD n = 250; ulcerative colitis (UC) n = 150) and German healthy controls (n = 422); (2) Hungarian IBD patients (CD n = 144; UC n = 124) and Hungarian healthy controls (n = 205). Subtyping analysis was performed in respect of CARD15 mutations and clinical characteristics. We also tested for differences within DLG5 genotypes in German CD patients with respect to gastroduodenal and intestinal permeability measured by triple-sugar-test. RESULTS: Allele as well as genotype frequencies of DLG5 variants did not differ between IBD patients and controls in either study population. Indeed, the p.R30Q polymorphism was found more frequently in controls than in patients. The distribution of DLG5 genotypes in German and Hungarian CD patients with CARD15 mutations was not different from patients without mutated CARD15. We did also not observe any association between DLG5 variants and clinical parameters. Importantly, DLG5 variants were not associated with gastroduodenal or intestinal permeability. CONCLUSIONS: We could not replicate that DLG5 is a relevant disease susceptibility gene for IBD in German or Hungarian subjects. In addition, we have no evidence that DLG5 variants are involved in altered gastrointestinal permeability in CD.

Adult↗
PubMed2006

Fifteen polymorphisms in endothelin-1, endothelin-2 and endothelin-receptor-A genotyped by four duplex assays and seven simple assays on a LightCycler using hybridization probes.

Endothelins (EDNs) are peptides, produced by various tissues, with potent vasoactive and mitogenic properties. Endothelin actions are mediated via specific G protein-coupled receptors of two subtypes, endothelin-receptor-A (EDNRA) and endothelin-receptor-B (EDNRB). Some polymorphisms of the EDN1, EDN2 and EDNRA genes may influence susceptibility to vascular diseases. Thus, genotyping for polymorphisms of these genes may represent a tool for predicting individual susceptibility to vascular diseases. Here, we report 11 fluorescence resonance energy transfer (FRET) assays for the detection of 15 polymorphisms, because the assays used in previous studies (allele-specific PCR and restriction fragment-length polymorphism assays) for some of these polymorphisms are laborious and time-consuming. The newly developed assays are fast and work without expensive ready-to-use mixtures.

DNA Probes↗
PubMed2005

Distribution and effects of polymorphic RANTES gene alleles in HIV/HCV coinfection -- a prospective cross-sectional study.

AIM: Chemokines and their receptors are crucial for immune responses in HCV and HIV infection. RANTES gene polymorphisms lead to altered gene expression and influence the natural course of HIV infection. Therefore, these mutations may also affect the course of HIV/HCV coinfection. METHODS: We determined allele frequencies of RANTES-403 (G --> A), RANTES-28 (C --> G) and RANTES-IN1.1 (T --> C) polymorphisms using real-time PCR and hybridization probes in patients with HIV (n = 85), HCV (n = 112), HIV/HCV coinfection (n = 121), and 109 healthy controls. Furthermore, HIV and HCV loads as well as CD4(+) and CD8(+) cell counts were compared between different RANTES genotypes. RESULTS: Frequencies of RANTES-403 A, RANTES-28 G and RANTES-IN1.1 C alleles were higher in HIV infected patients than in healthy controls (-403: 28.2% vs 15.1%, P = 0.002; -28: 5.4% vs 2.8%, not significant; IN1.1: 19.0% vs 11.0%, P = 0.038). In HIV/HCV coinfected patients, these RANTES alleles were less frequent than in patients with HIV infection alone (15.4% P = 0.002; 1.7%; P = 0.048; 12.0%; not significant). Frequencies of these alleles were not significantly different between HIV/HCV positive patients, HCV positive patients and healthy controls. CONCLUSION: All three RANTES polymorphisms showed increased frequencies of the variant allele exclusively in patients with HIV monoinfection. The finding that the frequencies of these alleles remained unaltered in HIV/HCV coinfected patients suggests that HCV coinfection interferes with selection processes associated with these alleles in HIV infection.

Adolescent↗
PubMed2005

Rapid detection of the SPINK5 polymorphism Glu420Lys by real-time PCR technology.

BACKGROUND: The SPINK5 gene, encoding the serine protease inhibitor LEKTI maps to chromosome 5q32, and has been suggested to be a locus predisposing to atopic diseases in general. The Glu420Lys variant showed significant association with atopy, asthma and atopic dermatitis in recent studies. AIMS OF THE STUDY: Development of a high throughput assay to analyse the polymorphism G1258A (Glu420Lys) in exon 14 of the SPINK5 gene followed by the validation using samples of 235 latex-allergic health care workers (HCWs) with (N=63) and without asthma (N=172), and 80 non-atopic controls. METHODS: Twenty DNA samples were first analysed by a polymerase chain restriction fragment analysis (RFLP) using Hph I to generate defined control DNAs which were used for the development of the assay suitable for the detection of the Glu420Lys variant by LightCycler technology. RESULTS AND CONCLUSIONS: 315 samples were successfully screened with this new assay. The temperatures in the melting analysis of the SPINK5 exon 14 PCR product were characteristic to the probes hybridised to the mutant (AA) at 51.5 degrees C and to the wild-type (GG) at 59.5 degrees C. The fast and reliable mutation detection in the tested samples makes this high-speed method suitable for larger epidemiological studies.

Adult↗
PubMed2005

Rapid presumptive identification of Burkholderia pseudomallei with real-time PCR assays using fluorescent hybridization probes.

Burkholderia pseudomallei (the etiologic agent of melioidosis) can cause pyogenic or granulomatous lesions in almost any organ. Septicemia has a case fatality rate of >40%. Early diagnosis and appropriate antibiotic therapy are crucial for survival, but cultivation, biochemical identification, and conventional PCR of B. pseudomallei are time consuming. We established real-time PCR assays using fluorescent hybridization probes targeting the 16S rDNA, the flagellin C (fliC) and the ribosomal protein subunit S21 (rpsU) genes. The test sensitivity and specificity were assessed with a representative panel of 39 B. pseudomallei, 9 B. mallei, 126 other Burkholderia strains of 29 species, and 45 clinically relevant non-Burkholderia organisms. The detection limit for the 16S rDNA, fliC, and rpsU assay was 40, 40, and 400 genome equivalents per reaction, however, in spiked blood samples it was 300, 300, and 3000, respectively. Specificity, positive and negative predictive value of the assays was 100%. In conclusion, we recommend the use of the 16S rDNA and/or fliC real-time PCR assays for the rapid identification of B. mallei and B. pseudomallei in positive blood cultures or from suspicious bacterial colonies.

Burkholderia pseudomallei↗
PubMed2005

Evaluation of the -26G>A CC16 polymorphism in acute respiratory distress syndrome.

OBJECTIVE: Different risk factors are presumably involved in the pathogenesis of acute respiratory distress syndrome (ARDS) including genetic factors. Clara cell protein 16 (CC16) is a potential candidate gene for ARDS susceptibility because reduced levels of the anti-inflammatory CC16 have been observed in bronchoalveolar lavage fluids or serum of patients with different inflammatory lung diseases. Furthermore, CC16 potently inhibits phospholipase A2, which plays a major role in ARDS pathophysiology. A functional polymorphism (-26G>A) was previously identified and related to decreased CC16 levels, asthma, and asthma severity. DESIGN: Observational study. SETTINGS: Adults with ARDS were recruited from intensive care units in two university medical centers. SUBJECTS: We evaluated the role of this genetic variant in 117 German patients with ARDS and 373 German controls. MEASUREMENTS: The CC16 -26G>A polymorphism was analyzed by melting-curve analysis using a pair of fluorescence resonance energy transfer probes. MAIN RESULTS: CC16 genotype frequencies in ARDS patients did not differ from those seen in controls. Also, the allele frequencies were identical in patients compared with controls (0.66 and 0.34). Moreover, only one of the patients who died (n = 27) was homozygous for the -26A allele. CONCLUSIONS: The CC16 -26G>A polymorphism does not affect the susceptibility to and the outcome of ARDS.

Adolescent↗
PubMed2005

Use of a locked-nucleic-acid oligomer in the clamped-probe assay for detection of a minority Pfcrt K76T mutant population of Plasmodium falciparum.

Given the emergence of drug resistance and the high rate of polyclonal microorganism infections, the availability of a fast and sensitive test to detect minority mutant populations would be an improvement in the diagnosis of infectious diseases. A clamped-probe real-time PCR assay to diagnose the Plasmodium falciparum K76T mutation in clone populations was developed, using a wild-type-specific locked-nucleic-acid-containing oligomer to suppress wild-type PCR amplification and to enhance melting analysis with a mutation-specific detection probe.

Animals↗
PubMed2004

Polymorphic metabolic susceptibility genes and longevity: a study in octogonarians.

In order to investigate possible associations of genetic variants in genes of xenobiotic metabolism with longevity, we compared allele frequencies and genotype distributions of polymorphic genes between 205 octogenarians and a non-cancer reference group of 294 persons aged less than 80 years. We analyzed common sequence variations in the cytochrome P-450 genes CYP1A1 T461N, 3801 T > C and CYP1B1 V432L, and in the glutathione S-transferase genes GSTM1 (deletion), GSTT1 (deletion), and GSTP1 (I105V). In octogenarians, the CYP1B1 432L allele was less prevalent than in the reference group (allele frequency 0.49 versus 0.60; odds ratio, OR, 0.63, 95% confidence limits (CI) 0.40-1.00). Octogenarians turned out to have marginally significant more GSTM1 negatives (frequency 0.56 versus 0.48; OR 1.41, 95% CI 0.97-2.05), but less GSTT1 deficient genotypes (frequency 0.14 versus 0.21; OR 0.64; 95% CI 0.38-1.06). In octogenarians without cancer, GSTT1 negative carriers were less prevalent than in the aged with cancer (frequency 0.12 versus 0.27; OR 2.81; 95% CI 1.00-7.38). Polymorphic metabolic susceptibility genes could become relevant for processes of aging when toxic defense mechanisms decline.

Adolescent↗
PubMed2004

Guideline to reference gene selection for quantitative real-time PCR.

Today, quantitative real-time PCR is the method of choice for rapid and reliable quantification of mRNA transcription. However, for an exact comparison of mRNA transcription in different samples or tissues it is crucial to choose the appropriate reference gene. Recently glyceraldehyde 3-phosphate dehydrogenase and beta-actin have been used for that purpose. However, it has been reported that these genes as well as alternatives, like rRNA genes, are unsuitable references, because their transcription is significantly regulated in various experimental settings and variable in different tissues. Therefore, quantitative real-time PCR was used to determine the mRNA transcription profiles of 13 putative reference genes, comparing their transcription in 16 different tissues and in CCRF-HSB-2 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate and ionomycin. Our results show that "Classical" reference genes are indeed unsuitable, whereas the RNA polymerase II gene was the gene with the most constant expression in different tissues and following stimulation in CCRF-HSB-2 cells.

Base Sequence↗
PubMed2004

Transrenal DNA as a diagnostic tool: important technical notes.

A small portion of DNA from apoptotic cells escapes complete degradation, appears in blood as oligonucleosomal-size fragments, is excreted in the urine, and can be used for diagnostic purposes. More detailed study revealed that transrenal DNA (Tr-DNA) belongs to a relatively low molecular-weight (150-250 bp) fraction, thereby requiring more careful attention to methods employed for purification and analysis. For example, here it is demonstrated that the QIAamp blood kit purifies primarily high molecular-weight DNA from serum, whereas the Guanidine/Promega Wizard Resin (GITC/WR) method purifies primarily low molecular-weight DNA. As a result, sensitivity in detection of K-RAS mutations in serum of patients with colorectal tumors is significantly higher with DNA isolated with the GITC/WR method than with the QIAamp kit. Amplicon size is also extremely important in analysis of Tr-DNA, because the shorter the amplicon, the higher is the sensitivity of biomarker detection in Tr-DNA. One hundred fifty-seven and 87 bp amplicons were employed for detection of mutant K-RAS in DNA isolated from 0.1 mL of urine obtained from 15 patients with pancreatic cancer. Mutant K-RAS was found in Tr-DNA of 3 and 10 patients with the long and short amplicons, respectively. The sensitivity and specificity of detection of mutant sequences are reduced in the presence of high excess of a respective wild-type allele, but they can be significantly increased through application of enriched polymerase chain reaction (PCR), peptide nucleic acid (PNA) clamped PCR, and/or stencil-aided mutation analysis (SAMA), based on selective pre-PCR elimination of wild-type sequences.

Apoptosis↗