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  • 1.
    Fransén, Karin
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Klintenäs, Maria
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Österström, Anna
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Dimberg, Jan
    Department of Natural Science and Biomedicine, University College of Health Sciences, Jönköping, Sweden.
    Monstein, Hans-Jürg
    Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Söderkvist, Peter
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas2004In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 25, no 4, p. 527-533Article in journal (Refereed)
    Abstract [en]

    Colorectal cancer is a multi-step process characterized by a sequence of genetic alterations in cell growth regulatory genes, such as the adenomatous polyposis coli, KRAS, p53 and DCC genes. In the present study mutation analysis was performed with SSCA/direct sequencing of the hot-spot regions in exons 11 and 15 for the BRAF gene and exons 1–2 for the KRAS gene in 130 primary colorectal cancer tumors and correlated with clinico-pathological and mutational data. We also performed mutation analysis of the corresponding conserved regions in the ARAF and RAF-1 genes. Mutations in the BRAF and KRAS genes were found in 11.5 and 40% of the tumors, respectively. One germline exonic and nine germline intronic genetic variants were found in the ARAF and RAF-1 genes. All of the BRAF mutations were located in the kinase domain of the conserved region 3 in exon 15 of the BRAF gene. One novel somatic mutation was also identified in the BRAF gene. The majority of the BRAF mutations were found in colon compared with rectal tumors (P = 0.014). In agreement with others, a statistically significant correlation between BRAF mutations and microsatellite instability could be found. A negative correlation was also evident between mutations in the BRAF and KRAS genes, which supports earlier studies where somatic mutations in these genes are mutually exclusive. Collectively, our results provide support for the idea that activation of the MAP kinase pathway, especially via BRAF and KRAS mutations, is of critical importance for the development of colorectal cancer.

  • 2. Grahn, Niclas
    et al.
    Hmani-Aifa, Mounira
    Fransén, Karin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Söderkvist, Peter
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Monstein, Hans-Jurg
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Molecular identification of Helicobacter DNA present in human colorectal adenocarcinomas by 16S rDNA PCR amplification and pyrosequencing analysis2005In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 54, no 11, p. 1031-1035Article in journal (Refereed)
    Abstract [en]

    Seroepidemiological studies have indicated that Helicobacter pylori infection might be a possible risk factor for colorectal adenocarcinoma (CRC) development. However, limited information is available as to whether or not Helicobacter species are present in CRC tissues. In this study the presence of Helicobacter DNA in 77 CRC biopsies was investigated by means of a Helicobacter species-specific 16S rDNA PCR assay and real-time DNA pyrosequencing of the 16S rDNA variable V3 region. Pyrosequencing revealed the presence of Helicobacter DNA sequences in 21 of 77 biopsy specimens (27%). 16S rDNA sequences corresponding to H. pylori 26695 and H. pylori J99 were most commonly found. Intriguingly, one sequence belonged to Helicobacter mustelae, previously identified in ferrets. No significant correlations were found in the prevalence of Helicobacter DNA between colon and rectum tumour biopsies (P = 0.815), nor between Dukes' classes A/B and C/D (P = 0.262). 16S rDNA PCR amplification combined with pyrosequencing analysis of 16S rDNA variable V3 regions provides a powerful molecular tool to identify Helicobacter species in human biopsy specimens. © 2005 SGM.

  • 3.
    Jonasson, Jon
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Monstein, Hans-Jurg
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Commentary: Classification, identification and subtyping of bacteria based on pyrosequencing and signature matching of 16S rDNA fragments APMIS 110, 263-270: Commentary2007In: APMIS: Acta pathologica, microbiologica et immunologica Scandinavica. Supplementum, ISSN 0903-465X, E-ISSN 1600-5503, Vol. 115, no 5, p. 678-679p. 678-679Article in journal (Other academic)
    Abstract [en]

    [No abstract available]

  • 4.
    Jonsson, K.
    et al.
    Jönsson, K., Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Hospital, S-171 76 Stockholm, Sweden.
    Guo, B.P.
    Dept. of Microbiol./Molec. Genet., Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, United States.
    Monstein, Hans-Jurg
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology . Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Mekalanos, J.J.
    Dept. of Microbiol./Molec. Genet., Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, United States.
    Kronvall, G.
    Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Hospital, S-171 76 Stockholm, Sweden.
    Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins2004In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 101, no 7, p. 1852-1857Article in journal (Refereed)
    Abstract [en]

    Helicobacter pylori binds a number of host cell proteins, including the plasma protein plasminogen, which is the proenzyme of the serine protease plasmin. Two H. pylori plasminogen-binding proteins have been described, however, no genes were identified. Here we report the use of a phage display library to clone two genes from the H. pylori CCUG 17874 genome that mediate binding to plasminogen. DNA sequence analysis of one of these genes revealed 96.6% homology with H. pylori 26695 HP0508. A subsequent database search revealed that the amino acid sequence of a lysine-rich C-terminal segment of HP0508 is identical to the C terminus of HP0863. Recombinant proteins expressed from HP0508 and HP0863 bound plasminogen specifically and in a lysine-dependent manner. We designate these genes pgbA and pgbB, respectively. These proteins are expressed by a variety of H. pylori strains, have surface-exposed domains, and do not inhibit plasminogen activation. These results indicate that pgbA and pgbB may allow H. pylori to coat its exterior with plasminogen, which subsequently can be activated to plasmin. The surface acquisition of protease activity may enhance the virulence of H. pylori.

  • 5. Kolak, M.
    et al.
    Karpati, F.
    Stockholm Cystic Fibrosis Centre, Huddinge University Hospital, Stockholm, Sweden.
    Monstein, Hans-Jurg
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology . Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Jonasson, Jon
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Molecular and Immunological Pathology . Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Molecular typing of the bacterial flora in sputum of cystic fibrosis patients2003In: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 293, no 4, p. 309-317Article in journal (Refereed)
    Abstract [en]

    Despite recent advances in therapy, lower airway infections remain the major cause of morbidity and mortality in cystic fibrosis (CF) patients. Bacterial colonisation of the lower airways in CF is limited to a few bacterial species, commonly Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae. Burkholderia cepacia colonisation is much rarer, but it has been thought to be associated with more advanced lung disease and increased mortality. A rapid characterisation of the bacterial flora in sputum of CF patients is of great importance for proper treatment. The aim of this study was to establish bacterial profiles and to identify pathogenic bacteria in respiratory specimens by means of molecular methods including temporal temperature gradient gel electrophoresis (TTGE) and DNA sequencing of PCR amplicons derived from 16S rDNA variable V3 and V6 regions. Sputa of 13 CF patients (7 males/6 females, age 19-59 years) collected at the Stockholm CF centre were analysed. TTGE revealed the presence of complex bacterial profiles in all samples. The V3 and V6 PCR amplicons were cloned and sequenced by real-time DNA PyrosequencingTM. DNA from Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa, respectively, was identified together with sequences from normal oral cavity flora. The results were in reasonable agreement with those obtained by conventional bacterial culture, considering that only known CF pathogens are included in routine reports. However, the methodology seems too elaborate to be introduced into daily routine.

  • 6.
    Monstein, Hans-Jurg
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Balkhed Östholm, Åse
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Infectious Diseases in Östergötland.
    Nilsson, MV
    Nilsson, M
    Dornbusch, Kathrine
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology.
    Nilsson, Lennart
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology.
    Multiplex PCR amplification assay for the detection of blaSHV, blaTEM and blaCTX-M genes in Enterobacteriaceae2007In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 115, no 12, p. 1400-1408Article in journal (Refereed)
    Abstract [en]

    Extended-spectrum β-lactamases (ESBLs) are often mediated by bla-SHV, blaTEM and blaCTX-M genes in Enterobacteriaceae and other Gram-negative bacteria. Numerous molecular typing methods, including PCR-based assays, have been developed for their identification. To reduce the number of PCR amplifications needed we have developed a multiplex PCR assay which detects and discriminates between bla-SHV, blaTEM and blaCTX-M PCR amplicons of 747, 445 and 593 bp, respectively. This multiplex PCR assay allowed the identification of bla-SHV, blaTEM and blaCTX-M genes in a series of clinical isolates of Enterobacteriaceae with previously characterised ESBL phenotype. The presence of blaSHV, blaTEM and blaCTX-M genes was confirmed by partial DNA sequence analysis. Apparently, the universal well-established CTX-M primer pair used here to reveal plasmid-encoded blaCTX-M genes would also amplify the chromosomally located K-1 enzyme gene in all Klebsiella oxytoca strains included in the study.

  • 7.
    Monstein, Hans-Jurg
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology . Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Ellnebo-Svedlund, Katarina
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery.
    Molecular typing of Helicobacter pylori by virulence-gene based multiplex PCR and RT-PCR analysis2002In: Helicobacter, ISSN 1083-4389, E-ISSN 1523-5378, Vol. 7, no 5, p. 287-296Article in journal (Refereed)
    Abstract [en]

    Background. In recent years, numerous PCR amplification typing methods have been developed for the identification of H. pylori specific virulence genes. To reduce the number of PCR amplifications needed we have previously developed virulence gene based multiplex PCR and RT-PCR assays. Aim. The aim of the present study was to characterise Helicobacter pylori strains by means of virulence-gene based multiplex PCR and reverse-transcription PCR. Subjects. Helicobacter pylori clinical reference strains HP-HJM 1-25 originally obtained from routine cultures of clinical gastric biopsy specimens from dyspeptic patients were used in the present study. Methods. Helicobacter pylori multiplex PCR and RT-PCR assays were carried out using primer pairs targeting the cagA, vacA, ureA, ureI, hspA (hsp60), and sodB genes and their cDNA. Results. Unexpected multiplex PCR and RT-PCR patterns were observed by ethidium bromide staining of agarose gels and Southern blot hybridisation analysis. DNA sequence analysis revealed a complex pattern of point mutations, deletions and insertions in the sodB, cagA and vacA genes, respectively. Mutations occurring in the PCR and hybridisation primer binding sites might explain some of the discrepancies previously observed in the expression of these genes. Furthermore, the present data show that coexpression of cagA and vacA transcripts of different sizes may take place in the same strain. Conclusions. The multiplex PCR and RT-PCR assay described allows rapid characterisation of H. pylori virulence genes at the DNA and RNA (cDNA) levels. However, extensive DNA sequence analysis seems necessary if one wants to reveal details of mutations occurring in the cagA and vacA genes.

  • 8.
    Monstein, Hans-Jurg
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Grahn, Niclas
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Ohlsson, Bodil
    Proenkephalin-A mRNA is widely expressed in tissues of the human gastrointestinal tract2006In: European Surgical Research, ISSN 0014-312X, E-ISSN 1421-9921, Vol. 38, no 5, p. 464-468Article in journal (Refereed)
    Abstract [en]

    Background: It has been shown that the non-opioid effects of Met-enkephalin, which is derived from proenkephalin-A, are mediated through a specific opioid growth factor (OGF) receptor which is assumed to be involved in the control of cell growth. The expression and tissue location of proenkephalin-A mRNA in the gastrointestinal tract remains largely unknown. Methods: In this study we have analyzed the expression of proenkephalin-A mRNA in the human esophagus, gastrointestinal tract and surrounding organs by means of reverse-transcriptase PCR (RT-PCR). Results: The present study demonstrates proenkephalin-A mRNA expression in the human esophagus, gastrointestinal tract, pancreas, and gallbladder. Conclusion: The present study demonstrates proenkephalin-A mRNA expression in regions of the human esophagus, gastrointestinal tract, pancreas, and gallbladder tissues which provides information for the future mapping of proenkephalin-A mRNA and protein expression/co-expression at the cellular level. Copyright © 2006 S. Karger AG.

  • 9.
    Monstein, Hans-Jurg
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology . Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Grahn, Niclas
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Truedsson, M.
    Department of Internal Medicine, University Hospital Malmö, S-205 02 Malmö, Sweden.
    Ohlsson, B.
    Department of Internal Medicine, University Hospital Malmö, S-205 02 Malmö, Sweden.
    Oxytocin and oxytocin-receptor mRNA expression in the human gastrointestinal tract: A polymerase chain reaction study2004In: Regulatory Peptides, ISSN 0167-0115, E-ISSN 1873-1686, Vol. 119, no 1-2, p. 39-44Article in journal (Refereed)
    Abstract [en]

    Background/aim: Oxytocin (OT) has a wide range of effects throughout the body. However, the role of OT on the gastrointestinal (GI) tract has to be settled. So far, the few studies performed reveal no conclusive results. The aim of this study was to examine the expression of OT and OT-receptor mRNA in the human GI tract. Material and methods: Full-thickness biopsies from all segments of the GI tract and the gallbladder were collected during operations at the Department of Surgery, Malmö University Hospital. Biopsies were taken and put immediately into fluid nitrogen and stored at -70°C until total RNA was extracted after mechanical tissue homogenization. Subsequently, poly A + mRNA was isolated from the total RNA extract using an automated nucleic acid extractor and converted into single-stranded cDNA. PCR amplifications were carried out using gene-specific OT and OT-receptor primers. The specificity of the PCR amplicons was further confirmed by Southern blot analyses using gene specific OT and OT-receptor hybridization probes. Results: Expression of OT and OT-receptor mRNA was detected in nearly all segments of the GI tract analyzed. In most of the biopsy specimens analyzed, co-expression of both OT and OT-receptor mRNA appeared to take place. Conclusion: The present study demonstrates that OT and OT-receptor mRNAs are expressed throughout the GI tract. A possible physiological and/or pathophysiological role of OT and OT-receptor expression in the human GI tract and the cellular location of its expression remain to be shown. © 2004 Elsevier B.V. All rights reserved.

  • 10.
    Monstein, Hans-Jurg
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Grahn, Niclas
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Truedsson, Mikael
    Ohlsson, Bodil
    Progastrin-releasing peptide and gastrin-releasing peptide receptor mRNA expression in non-tumor tissues of the human gastrointestinal tract2006In: World Journal of Gastroenterology, ISSN 1007-9327, E-ISSN 2219-2840, Vol. 12, no 16, p. 2574-2578Article in journal (Refereed)
    Abstract [en]

    Aim: To investigate the expression of gastrin-releasing peptide (GRP) and GRP-receptor mRNA in non-tumor tissues of the human esophagus, gastrointestinal tract, pancreas and gallbladder using molecular biology techniques. Methods: Poly A+ mRNA was isolated from total RNA extracts using an automated nucleic acid extractor and, subsequently, converted into single-stranded cDNA (ss-cDNA). PCR amplifications were carried out using gene-specific GRP and GRP-receptor primers. The specificity of the PCR amplicons was further confirmed by Southern blot analyses using gene-specific GRP and GRP-receptor hybridization probes. Results: Expre ssion of GRP and GRP-receptor mRNA was detected at various levels in nearly all segments of the non-tumor specimens analysed, except the gallbladder. In most of the biopsy specimens, co-expression of both GRP and GRP-receptor mRNA appeared to take place. However, expression of GRP mRNA was more prominent than was GRP-receptor mRNA. Conclusion: GRP and GRP-receptor mRNAs are expressed throughout the gastrointestinal tract and provides information for the future mapping and determination of its physiological importance in normal and tumor cells. © 2006 The WJG Press. All rights reserved.

  • 11.
    Monstein, Hans-Jurg
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology . Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Ohlsson, B
    Axelson, J
    Malmo Univ Hosp, Dept Surg, MAS, SE-20502 Malmo, Sweden Linkoping Univ Hosp, LMO, Mol Biol Lab, S-58185 Linkoping, Sweden.
    Differential expression of gastrin, cholecystokinin-A and cholecystokinin-B receptor mRNA in human pancreatic cancer cell lines2001In: Scandinavian Journal of Gastroenterology, ISSN 0036-5521, E-ISSN 1502-7708, Vol. 36, no 7, p. 738-743Article in journal (Refereed)
    Abstract [en]

    Background: It has been assumed that gastrin stimulates the growth of pancreatic cancer in an autocrine way through co-expression of gastrin and the cholecystokinin-B receptor (CCK-BR). However, pancreatic cancer cell lines established directly from patients have revealed a great heterogeneity in cell proliferation when exposed to CCK, gastrin and their receptor antagonists. The aim of this study was therefore to examine co-expression of CCK-A and CCK-B receptor (CCK-AR and CCK-BR), and gastrin mRNA as well as the secretion of CCK and gastrin peptides in these cell lines. Methods: Fourteen cell lines were established from primary pancreatic cancers or their metastases. Total RNA was isolated from the cell lines and reverse-transcribed into single-stranded cDNA. A PCR technique based on Tag polymerase-antibody interaction and CCK-AR, CCK-BR and gastrin-specific primers, followed by Southern blot analysis, were the methods used. The incubation mediums were analysed for the presence of secreted CCK/proCCK and gastrin/progastrin peptides by specific radioimmunoassays (RIA). Results: By means of nested Reverse-Transcribed Polymerase Chain Reaction (nested RT-PCR), combined with Southern blot analysis of the PCR amplified products, CCK-AR and gastrin mRNA co-expression was detected in cell Lines LPC-6p and LPC-10m, whereas CCK-BR and gastrin mRNA could be detected in cell lines LPC-8p and LPC-12m. A low level of secreted CCK peptides was detected in cell line LPC-6p. which also expressed CCK-AR mRNA. In no other cases were CCK or gastrin peptides detected in the cell culture mediums. Conclusion: The lack of CCK-BR and gastrin mRNA co-expression, and not detectable levels of secreted CCK and gastrin in culture media, does not lend support to the hypothesis that concomitant gene-expression of CCK receptors and gastrin or CCK are essential to maintaining pancreatic cancer cell proliferation.

  • 12.
    Monstein, Hans-Jurg
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Olsson, Crister
    Nilsson, Isabelle
    Linköping University, Department of Medicine and Care. Linköping University, Faculty of Health Sciences.
    Grahn, Niclas
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Benoni, Cecilia
    Ahrné, Siv
    Multiple displacement amplification of DNA from human colon and rectum biopsies: Bacterial profiling and identification of Helicobacter pylori-DNA by means of 16S rDNA-based TTGE and pyrosequencing analysis2005In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 63, no 3, p. 239-247Article in journal (Refereed)
    Abstract [en]

    Amplifying bacterial DNA by PCR from human biopsy specimens has sometimes proved to be difficult, mainly due to the low amount of bacterial DNA present. Therefore, nested or semi-nested 16S rDNA PCR amplification has been the method of choice. In this study, we evaluate the potential use of whole genome amplification of total DNA isolated from human colon and rectum biopsy specimens, followed by 16S rDNA PCR amplification of multiple displacement amplified (MDA)-DNA. Subsequently, a H. pylori-specific 16S rDNA variable V3 region PCR assay was applied directly on MDA-DNA and, combined with pyrosequencing analysis, the presence of H. pylori in some biopsies from colon in patients with microscopic colitis was confirmed. Furthermore, temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA amplicons using primers flanking variable regions V3, V4, and V9, was used to establish bacterial profiles from individual biopsies. A variation of the bacterial profiles in the colonic mucosa in microscopic colitis and in normal rectal mucosa was observed. In conclusion we find the MDA technique to be a useful method to overcome the problem of insufficient bacterial DNA in human biopsy specimens. © 2005 Elsevier B.V. All rights reserved.

  • 13.
    Monstein, Hans-Jurg
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology . Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Truedsson, M.
    Department of Clinical Sciences Malmö University.
    Ryberg, Anna
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine.
    Ohlsson, M.
    Department of Clinical Sciences Malmö University.
    Vasopressin receptor mRNA expression in the human gastrointestinal tract2008In: European Surgical Research, ISSN 0014-312X, E-ISSN 1421-9921, Vol. 40, no 1, p. 34-40Article in journal (Refereed)
    Abstract [en]

    Background/Aim: Vasopressin and oxytocin are closely related peptides, and both exert effects on the gastrointestinal function. In the present study, we wanted to map the expression of vasopressin receptor mRNAs (V1a, V1b/V3, and V2) in nontumorous tissue biopsy specimens of human gastrointestinal tract and surrounding tissues. Methods: Total and polyA+ RNAs were isolated from human tissue biopsy specimens using an automated nucleic acid extractor and, subsequently, converted into single-stranded cDNA. Seminested PCR amplifications were carried out, using gene-specific V1a, V1b/V3, and V2 receptor primers. The PCR amplicons were partially sequenced to confirm their identity. Results: The present study demonstrated the expression of vasopressin receptor mRNAs in human gastrointestinal tract, pancreas, kidney, lung, brain, and ovary. The expression pattern varied between different parts of the gastrointestinal tract. In the colon ascendens, V1a receptor mRNA expression could not be detected in 3 out of 4 analyzed tissue biopsy specimens. On the other hand, all the vasopressin receptor mRNAs were expressed in all colon transversum biopsy samples. Conclusions: V1a, V1b/V3, and V2 receptor mRNAs are widely expressed throughout human gastrointestinal tract and surrounding tissues. The data obtained provide information for further mapping and determination of the physiological role of the vasopressin receptor mRNA expression in normal and tumorous tissues.

  • 14.
    Nilsson, Isabelle
    et al.
    Linköping University, Department of Medicine and Care. Linköping University, Faculty of Health Sciences.
    Shabo, Ivan
    Linköping University, Department of Biomedicine and Surgery. Linköping University, Faculty of Health Sciences.
    Svanvik, Joar
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of surgery. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Surgery in Östergötland.
    Monstein, Hans-Jurg
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Multiple displacement amplification of isolated DNA from human gallstones: Molecular identification of Helicobacter DNA by means of 16S rDNA-based pyrosequencing analysis2005In: Helicobacter, ISSN 1083-4389, E-ISSN 1523-5378, Vol. 10, no 6, p. 592-600Article in journal (Refereed)
    Abstract [en]

    Background. Molecular typing of Helicobacter spp. in clinical biopsy specimens has become increasingly important. By means of nested polymerase chain reaction (PCR) amplification and Southern blot analysis of the PCR amplicons, we have shown that Helicobacter spp. DNA is present in human gallstones. In this study we have investigated the possibility of using multiple displacement amplification (MDA) of isolated gallstone DNA and pyrosequencing analysis for the molecular identification of Helicobacter spp. Materials and Methods. DNA isolated from the nucleus of 33 human gallstones and one control strain were used in a MDA assay. Subsequently, pyrosequencing analysis was performed either directly on MDA-DNA using primers flanking the Helicobacter spp. 16S rDNA variable V3 region or on PCR amplicons derived from broad-range primers flanking the 16S rDNA variable V3, V4, and V9 regions. Results. Pyrosequencing analysis of 16S rDNA derived from MDA-DNA revealed that Helicobacter spp.-like DNA was present in 25 of 33 (approximately 76%) gallstones. Using an H. pylori-specific Southern blot analysis, Helicobacter spp.-like DNA was present in 20 of 33 [approximately 61%] of the gallstones. Using MDA-DNA directly in pyrosequencing analysis, Helicobacter spp.-like DNA was present in 13 of 33 [approximately 39%] gallstones. Conclusions. We conclude that multiple displacement amplification combined with pyrosequencing enables a rapid and accurate molecular typing of Helicobacter spp. from small and precious biopsy specimens. © 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd.

  • 15.
    Nowrouzian, F.L.
    et al.
    Department of Clinical Bacteriology, Göteborg University, Göteborg, Sweden, Department of Clinical Bacteriology, Göteborg University, Guldhedsgatan 10, S-413 46 Göteborg, Sweden.
    Monstein, Hans-Jurg
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology . Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Wold, A.E.
    Department of Clinical Bacteriology, Göteborg University, Göteborg, Sweden.
    Adlerberth, I.
    Department of Clinical Bacteriology, Göteborg University, Göteborg, Sweden.
    Effect of human milk on type 1 and P-fimbrial mRNA expression in intestinal Escherichia coli strains2005In: Letters in Applied Microbiology, ISSN 0266-8254, E-ISSN 1472-765X, Vol. 40, no 1, p. 74-80Article in journal (Refereed)
    Abstract [en]

    Aims: Escherichia coli from breastfed infants express more type 1 fimbriae and less P fimbriae than E. coli from bottle-fed infants. In this study we investigated the effect of human milk on production of mRNA for fimA (type 1 fimbriae) and papC (P fimbriae) in E. coli. Methods and Results: Production of adhesin gene mRNA was estimated using a reverse transcriptase polymerase chain reaction in E. coli strains under different culture conditions. More type 1 fimbrial mRNA was produced after culture in human milk (P = 0.001) or Luria broth (P = 0.014) than after culture on agar, whereas P-fimbrial mRNA production was similar under all tested growth conditions. When cultured on agar, E. coli strains carrying both the fim and pap operons produced less type 1 and P-fimbrial mRNA than strains that had only the fim or pap operons, respectively (P = 0.03 and 0.056). Significance and Impact of the Study: Environmental regulation of adhesin expression may be influenced by cross-talk between fimbrial operons.

  • 16.
    Sun, Yi-Qian
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of surgery.
    Monstein, Hans-Jurg
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Ryberg, Anna
    Borch, Kurt
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of surgery. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Surgery in Östergötland.
    Multiple strand displacement amplification of DNA isolated from human archival plasma/serum: Identification of cytokine polymorphism by pyrosequencing analysis2007In: Clinica Chimica Acta, ISSN 0009-8981, E-ISSN 1873-3492, Vol. 377, no 1-2, p. 108-113Article in journal (Refereed)
    Abstract [en]

    Background: DNA isolation from formalin-fixed paraffin-embedded tissue appears to be problematic due to degradation caused by fixative. Our aim was to investigate if the isolated genomic DNA from archival plasma/serum, combined with multiple strand displacement amplification (MDA) can be used for genotyping. Methods: Nine archival plasma/serum samples and freshly frozen gastric biopsies from the same nine H. pylori-infected subjects were used for DNA isolation. Subsequently, MDA-DNA derived from the plasma/serum samples and DNA isolated from the antrum biopsies were analyzed by PCR amplification and pyrosequencing for the presence of interleukin-1beta gene (IL-1B) single nucleotide polymorphism (SNP). In addition, Southern blot and pyrosequencing analysis of H. pylori-specific PCR amplicons were performed. Results: IL-1B SNP profiles obtained from the plasma/serum MDA-DNA and antrum biopsy DNA were identical. A C/C genotype was observed in 7 of 9 samples, and 2 of 9 revealed a C/T genotype for IL-1B - 511. Similarly, 7 of 9 had a T/T, and 2 of 9 had a C/T genotype for IL-1B - 31, 4 of 9 had a C/C, 4 of 9 had a C/T, and 1 of 9 had a T/T genotype, respectively, for IL-1B + 3954. Moreover, pyrosequencing analysis revealed the presence of H. pylori 26695 and J99-like 16S rDNA variable V3 region sequence motifs in the antrum biopsies but not in the plasma/serum samples. Conclusions: We conclude that MDA combined with pyrosequencing enables a rapid and accurate molecular typing of cytokine single nucleotide polymorphisms from archival plasma/serum samples. © 2006 Elsevier B.V. All rights reserved.

  • 17.
    Östholm Balkhed, Åse
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Infectious Diseases in Östergötland.
    Tärnberg, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
    Nilsson, Maud
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
    Johansson, Anita
    Östergötlands Läns Landsting, Centre for Diagnostics, Department of Clinical Microbiology.
    Hanberger, Håkan
    Linköping University, Department of Clinical and Experimental Medicine, Infectious Diseases. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Medicine, Department of Infectious Diseases in Östergötland.
    Monstein, Hans-Jurg
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Nilsson, Lennart E
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
    Prevalence of extended-spectrum beta-lactamase-producing Enterobacteriaceae and trends in antibiotic consumption in a county of Sweden2010In: Scandinavian Journal of Infectious Diseases, ISSN 0036-5548, E-ISSN 1651-1980, Vol. 42, no 11-12, p. 831-838Article in journal (Refereed)
    Abstract [en]

    In the last decade extended-spectrum beta-lactamase (ESBL)-producing bacteria have become an increasing problem. Our aims were to investigate the prevalence of ESBL-producing Enterobacteriaceae and trends in antibiotic use in the county of Ostergotland, Sweden. From 2002 through 2007 there were 224 ESBL-producing Escherichia coli and 23 Klebsiella pneumoniae isolates with an ESBL-phenotype identified among all Enterobacteriaceae isolated at the clinical laboratory. Trends in antibiotic consumption expressed as defined daily doses (DDD) per 1000 inhabitants and day (DID) were studied. The prevalence of ESBL-producing isolates among Enterobacteriaceae in our region is still low (andlt; 1%). Patients with ESBL-producing E. coli increased significantly (p andlt; 0.001) from 5 in y 2002 to 47 in y 2007. CTX-M group 1 was the dominant enzyme group in both E. coli and K. pneumoniae. Antibiotic susceptibility testing of ciprofloxacin, gentamicin and trimethoprim-sulfamethoxazole revealed that 58% of E. coli and 50% of K. pneumoniae isolates were multi-resistant. Antibiotic use remained unchanged from 2001 through 2009, but there was a trend towards increased use of drugs with low ESBL selection potential, which was probably due to the increased prevalence of ESBL producers.

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