Apmis

MARQUARD, LENA; PETERSEN, KAMILLE DUMONG; PERSSON, MORTEN; HOFF, KIRSTEN DAMGAARD; JENSEN, PETER BUHL; SEHESTED, MAXWELL

doi: 10.1111/j.1600-0463.2008.00957.xpmid: 18452428

Histone deacetylase (HDAC) inhibition is a novel entity in medical oncology, and several HDAC inhibitors are in clinical trials. One of them is the hydroxamic acid belinostat (PXD101) that has demonstrated therapeutic efficacy for several clinical indications. Acetylation of histones is a key event after treatment with HDAC inhibitors, and could thus be used as a marker for monitoring cellular response to HDAC inhibitor treatment. Here we describe the utility of a newly described monoclonal antibody against acetylated H4 for immunohistochemistry on paraffin‐embedded fine needle biopsies from nude mice carrying A2780 human ovarian cancer xenografts. Acetylated H4 was monitored in vivo by immunohistochemistry during treatment with belinostat, and compared with pharmacokinetics in plasma and tumor tissue. We found an increased level of acetylated H4 15 min after a single treatment (200 mg/kg i.v.) with maximum level reached after 1 h. H4 acetylation intensity reflected the belinostat concentration in plasma and tumor tissue. The threshold level for belinostat activity, indicated by acetylated H4, correlated with belinostat plasma concentrations above 1,000 ng/ml. In conclusion, examination of H4 acetylation in fine needle biopsies using the T25 antibody may prove useful in monitoring HDAC inhibitor efficacy in clinical trials involving humans with solid tumors.

ALLUNANS, JURIS; KRISTIANSEN, KNUT IVAN; ASSALKHOU, REZA; BJØRÅS, MAGNAR

doi: 10.1111/j.1600-0463.2008.00850.xpmid: 18452423

In the Neisseria meningitidis strain MC58 (serogroup B; ET‐5 complex) genome three putative islands of horizontally transferred DNA (IHTs) have been identified. IHT‐A2 codes for eight hypothetical proteins and two disrupted open reading frames with similarity to a secretion protein (NMB0097) and an ABC transporter (NMB0098). The strains MC58 and 44/76 (shown here) are meningocin resistant/weakly sensitive. None of these strains are meningocin producers. However, NMB0097 and NMB0098 homologues with open reading frames are found in meningocin producers (N. meningitidis P241 (serogroup A; systemic isolate) and BT878 (serogroup B; carrier isolate), and also in strain FAM18 (serogroup C; ET‐37 complex). Knocking out either of the two genes in the strain BT878 yielded mutants that did not secrete meningocin. A similarly disrupted tolC mutant in strain BT878 still released meningocin. Among systemic meningococcal isolates prior to and at the onset (mid‐1973 to the end of 1974) of the epidemic peaking in 1975 in North Norway, 12 of 30 (40%) isolates of serogroup A were meningocin producers. However, the rate for serogroup B was 1 of 45 (2.2%). Serogroup B meningocin‐resistant/weakly sensitive non‐producers dominated in the region from mid‐1975 and spread to the rest of the country from then on. No producers were found in selected pharyngeal isolates from healthy carriers collected in Svalbard in the early spring of 1975. Our results suggest that meningocinogeny has played a part in the change from serogroup A to serogroup B among isolates in North Norway during the first half of 1975.

EDVINSSON, BENJAMIN; LUNDQUIST, JESSICA; LJUNGMAN, PER; RINGDÉN, OLLE; EVENGÅRD, BIRGITTA

doi: 10.1111/j.1600-0463.2008.00871.xpmid: 18452424

Active infection with Toxoplasma gondii in immunocompromised transplant recipients can lead to toxoplasmosis, which may have a rapid disease course and in some cases be fatal. It is of paramount importance to diagnose toxoplasmosis at an early stage, and to initiate specific treatment to improve the outcome. Polymerase chain reaction (PCR) is today the primary diagnostic tool to diagnose toxoplasmosis in immunocompromised patients. Timely diagnosis may, however, be difficult if toxoplasmosis is at first asymptomatic. To investigate the magnitude of toxoplasmosis after bone marrow transplantation (BMT), we conducted a screening study by PCR where 21 autologous and 12 allogeneic BMT recipients were included. Peripheral blood samples were taken one week prior to BMT; thereafter, blood samples were drawn weekly for the first 6 months, and monthly up to one year after BMT. The samples were analyzed by conventional PCR and real‐time PCR. T. gondii DNA was detected in peripheral blood from one patient 5 days post allogeneic BMT. There were no clinical signs of toxoplasmosis. Medical records were reviewed and showed a previously undiagnosed eye infection in another allogeneic BMT recipient. These two patients were seropositive for T. gondii. We concluded that monitoring for T. gondii DNA in peripheral blood samples using PCR might be a valuable method for identifying toxoplasma‐seropositive stem cell transplant recipients.

EBDRUP, LOTTE; KROG, JAN; GRANFELDT, ASGER; LARSEN, PERNILLE Ø.; VESTERGAARD, CHRISTIAN; HOKLAND, MARIANNE; TØNNESEN, ELSE

doi: 10.1111/j.1600-0463.2008.00982.xpmid: 18452425

Systemic administered lipopolysaccharide (LPS) induces a cytokine response in peripheral blood without correlations with cytokine content at the organ level. We hypothesised (1) that cytokine mRNA expression in peripheral blood mononuclear cells (PBMCs) preceded the plasma cytokine increase during endotoxaemia and (2) that statins as anti‐inflammatory agents modified the LPS‐induced cytokine responses. 30 pigs were randomised into 3 groups: placebo (I) or atorvastatin 80 mg (II) for 21 days, followed by LPS‐infusion on day 22, or controls (III). LPS was infused at increasing concentrations (2.5 to 15 μg/kg/h) for 30 min, followed by sustained infusion (2.5 μg/kg/h) for 330 min. We measured plasma IL‐6, IL‐10, and TNF‐α, and their mRNA expression in PBMCs during the LPS‐infusion, and the cytokine content in kidney and heart biopsies at 360 min. LPS reduced TNF‐α mRNA in PBMCs at 60 min, whereas IL‐6 mRNA increased at 240 min. There were no correlations with plasma cytokines, which peaked at 60 min (IL‐10 and TNF‐α) and 240 min (IL‐6). Cytokine content did not increase in organs, and no effects of statins could be demonstrated. In conclusion, LPS‐infusion reduced leukocyte TNF‐α mRNA and increased IL‐6 mRNA, whereas plasma TNF‐α, IL‐6, and IL‐10 increased markedly.

KRISTIANSEN, SØREN; BJARNSHOLT, THOMAS; ADELTOFT, DINAH; IFVERSEN, PETER; GIVSKOV, MICHAEL

doi: 10.1111/j.1600-0463.2008.00966.xpmid: 18452426

Pseudomonas aeruginosa uses acyl‐homoserine lactones to coordinate gene transcription in a process called quorum sensing (QS). The QS molecules C4‐HSL and C12‐oxo‐HSL are synthesized from the universal precursor S‐adenosyl methionine, which is also a precursor of polyamines in human cells. Polyamines are required for mitotic cell division and peak during this phase. The polyamine putrescine is synthesized by ornithine decarboxylase (ODC) as a rate‐limiting step. The ODC enzyme concentration also peaks during the mitotic phase. This peak is mediated by translation of ODC mRNA by the ITAF45 protein, which translocates from the nuclear compartment to the cytoplasm in a phosphorylation‐dependent manner. We observed that C12‐HSL‐treated human epidermal cells had a higher cytoplasm‐to‐nuclear ITAF45 protein concentration and this translocation was dependent on the dephosphorylation of ITAF45. Finally, C12‐HSL‐treated cells also had a time‐course‐dependent higher concentration of ODC mRNA. Based on these mitotic markers, more human cells were apparently trapped in the mitotic phase when treated with C12‐HSL. This should normally imply higher levels of putrescine. However, C12‐HSL‐treated human cells had a significantly lower concentration of putrescine and displayed a lower cell proliferation rate. In conclusion, the P. aeruginosa autoinducer C12‐oxo‐HSL apparently arrests human cells in the mitotic phase by lowering the concentration of putrescine.

JIANG, HONG; GAO, XUE; LI, YUAN; XU, ZHI‐KAI; WANG, LI‐MEI; BAI, XUE‐FAN; XUE, YING

doi: 10.1111/j.1600-0463.2008.00975.xpmid: 18452427

Ferric uptake regulator B (FurB) of Mycobacterium tuberculosis, which belongs to the Fur superfamily, is principally responsible for maintaining iron and zinc homeostasis in prokaryotes. This common feature of FurB and the role of FurB in iron and zinc metabolism contribute to research on the pathogenesis of mycobacteria. In this study, three novel mouse monoclonal antibodies were generated using the prokaryotically expressed FurB protein as immunogen. The FurB gene of M. tuberculosis H37Rv was inserted into a bacterial expression vector of pQE‐80L and was effectively expressed in Escherichia coli DH5α. The expressed fusion protein existed as the insoluble form (inclusion bodies) in cell lysate and was purified on an Ni‐NTA column. Using the fusion protein to immunize BALB/c mice, three monoclonal antibodies (DD12, BH1, and DH8) were produced. As shown by Western blot analysis and indirect immunofluorescence assay, the three respective antibodies could recognize the FurB protein. These results suggest that the antibodies against FurB may provide a powerful tool for investigating the function of FurB in the pathogenesis of tuberculosis.

TJERNBERG, IVAR; SCHÖN, THOMAS; ERNERUDH, JAN; WISTEDT, ANNIKA CARLSSON; FORSBERG, PIA; ELIASSON, INGVAR

doi: 10.1111/j.1600-0463.2008.00842.xpmid: 18452429

The aim of this study was to evaluate the usefulness of borrelia serology (Quick ELISA C6 Borrelia assay kit) as a diagnostic tool in cases of suspected neuroborreliosis. A retrospective patient material consisting of 124 paired serum and cerebrospinal fluid samples with a positive anti‐borrelia antibody index (AI) using the IDEIA Lyme Neuroborreliosis test was compared with 124 AI‐negative matched control subjects. The patients were divided into four groups based on presence of pleocytosis and age above or below 12 years. The presence of positive C6 serology in AI‐positive patients with pleocytosis was 89% (83/93), significantly different (p<0.01) from in patients without pleocytosis (58%, 18/31). In AI‐positive patients aged ≥12 years with pleocytosis, 94% (51/54) had a positive C6 serology. Of AI‐positive patients with a symptom duration of more than 30 days, 93% (27/29) were positive by the C6 test. We conclude that the C6 serum test, together with clinical evaluation, is a powerful diagnostic tool in adult (≥12 years) European patients with suspected neuroborreliosis with a symptom duration of more than 30 days. Patients with suspected neuroborreliosis and positive C6 results should be further investigated by lumbar puncture for definite diagnosis.

HØGDALL, ESTRID V. S.; CHRISTENSEN, LISE; HØGDALL, CLAUS K.; FREDERIKSEN, KIRSTEN; GAYTHER, SIMON; BLAAKAER, JAN; JACOBS, IAN J.; Kjaer, Susanne K.

doi: 10.1111/j.1600-0463.2008.00917.xpmid: 18452430

The clinical roles played by normal and altered p53 in cancer are under intensive investigation, but larger studies describing the pattern as well as the prognostic value are still needed. The aim of this study was, using tissue array (TA), to examine the overexpression of p53 protein in 774 epithelial ovarian tumour tissues from Danish women and to evaluate whether p53 tissue expression levels correlate with clinicopathological parameters and prognosis. The distribution of p53 expression levels at different stages of disease, in different histological subtypes, and the prognostic value of p53 tissue expression were examined. Overall, p53 was expressed in 24/189 (13%) low malignant potential ovarian tumours (LMP) and in 278/585 (48%) ovarian cancers (OC). No significant difference in frequency of p53 tissue expression in LMP tissue was noted with increasing tumour stage (p=0.98). By contrast, there was a significant increase in the frequency of p53 tissue expression in OC with increasing FIGO stage (p<0.00001). Multivariate Cox regression analysis found that less than 20% tissue expression of p53 was associated with longer OC disease‐specific survival. Tissue p53 expression may be of prognostic value in women with OC.

doi: 10.1111/j.1600-0463.2008.001165.xpmid: N/A