Country differences and determinants of yield in programmatic migrant TB screening in four European countries.

INTRODUCTION The WHO End TB Strategy emphasises early diagnosis and screening of TB in high-risk groups, including migrants. We analysed TB yield data from four large migrant TB screening programmes to inform TB policy.METHODS We pooled routinely collected individual TB screening episode data from Italy, the Netherlands, Sweden and the United Kingdom under the European Union Commission E-DETECT.TB grant, described characteristics of the screened population, and analysed TB case yield.RESULTS We collected data on 2,302,260 screening episodes among 2,107,016 migrants, mostly young adults aged 18-44 years (77.8%) from Asia (78%) and Africa (18%). There were 1,658 TB cases detected through screening, with substantial yield variation (per 100,000): 201.1 for Sweden (95% confidence intervals CI 111.4-362.7), 68.9 (95% CI 65.4-72.7) for the United Kingdom, 83.2 (95% CI 73.3-94.4) for the Netherlands and 653.6 (95% CI 445.4-958.2) in Italy. Most TB cases were notified among migrants from Asia (n = 1,206, 75/100,000) or Africa (n = 370, 76.4/100,000), and among asylum seekers (n = 174, 131.5/100,000), migrants to the Netherlands (n = 101, 61.9/100,000) and settlement visa migrants to the United Kingdom (n = 590, 120.3/100,000).CONCLUSIONS We found considerable variations in yield across programmes, types of migrants and country of origin. These variations may be partly explained by differences in migration patterns and programmatic characteristics.

Assessment of in vivo mast cell reactivity in patients with systemic mastocytosis.

BACKGROUND: Patients with systemic mastocytosis (SM) have clinical signs of mast cell (MC) activation and increased levels of MC mediators. It is unclear whether the increased mediator levels are caused by increased numbers of tissue MCs, or whether these cells in affected individuals have a hyperactive phenotype. OBJECTIVE: To determine reactivity of the skin and the airways to directly acting mediators and indirectly acting mast cell secretagogues in subjects with SM. METHODS: Skin reactivity to morphine and histamine, and airway responsiveness to mannitol and methacholine, was assessed in 15 patients with SM, 11 patients with allergic asthma (A) and 13 healthy controls (HC). Serum tryptase and urinary metabolites of the MC mediators histamine and prostaglandin D2 were measured, as well as ex vivo basophil histamine release. RESULTS: Mast cell mediators in the blood and urine were significantly higher in patients with SM than in HC and A controls. Responsiveness to local activation of skin MCs (by morphine) and airway MCs (by mannitol) was similar in SM and HC groups. Likewise, end-organ responsiveness in the skin to histamine, and in the airways to methacholine, was similar in all three subject groups. There was no evidence of increased basophil reactivity in SM patients. CONCLUSIONS AND CLINICAL RELEVANCE: Mast cells in the skin and airways of subjects with SM do not exhibit hyper-reactivity towards the MC-activating stimuli morphine and mannitol, respectively. Therefore, the highly elevated baseline levels of MC mediators in SM are most likely due to increased MC numbers, rather than altered MC responsiveness. The underlying mechanisms could involve leakage of MC mediators, or dysfunctions in mediator synthesis, storage and release. One clinical implication of our study is that there is no contraindication to perform skin tests using morphine in subjects with mastocytosis.

A quantitative LC/MS method targeting urinary 1-methyl-4-imidazoleacetic acid for safety monitoring of the global histamine turnover in clinical studies.

Anaphylaxis is a potentially life-threatening condition triggered mainly by the release of inflammatory mediators, notably histamine. In pharmaceutical research, drug discovery, and clinical evaluation, it may be necessary to accurately assess the potential of a compound, event, or disorder to promote the release of histamine. In contrast to the measurement of plasma histamine, determination of the stable metabolite 1-methyl-4-imidazoleacetic acid (tele-MIAA) in urine provides a noninvasive and more reliable methodology to monitor histamine release. This study presents a repeatable high-performance liquid chromatography coupled to electrospray mass spectrometry (LC-ESI-MS) method where tele-MIAA is baseline separated from its structural isomer 1-methyl-5-imidazoleacetic acid (pi-MIAA) and an unknown in human urine. The ion-pairing chromatography method, in reversed-phase mode, based on 0.5 mM tridecafluoroheptanoic acid demonstrated high repeatability and was applied in a clinical development program that comprised a large number of clinical samples from different cohorts. The inter- and intra-run precision of the method for tele-MIAA were 8.4 and 4.3%, respectively, at the mean urinary concentration level, while method accuracy was between -16.2 and 8.0% across the linear concentration range of 22-1,111 ng mL(-1). Overall, method precision was greater than that reported in previously published methods and enabled the identification of gender differences that were independent of age or demography. The median concentration measured in female subjects was 3.0 µmol mmol(-1) of creatinine, and for male subjects, it was 2.1 µmol mmol(-1) of creatinine. The results demonstrate that the method provides unprecedented accuracy, precision, and practicality for the measurement of tele-MIAA in large clinical settings.

Quantitative real-time PCR analysis and microarray-based RNA expression of HER2 in relation to outcome.

BACKGROUND: Our aim was to use quantitative real-time PCR (Q-PCR) and RNA expression profiles (RNA-EPs) to investigate HER2 status in relation to outcome. PATIENTS AND METHODS: Cut-off levels for Q-PCR and RNA-EP were established in relation to immunohistochemistry (IHC) validated by FISH in a test set of frozen tissue samples from 40 primary breast cancers. The HER2 status was subsequently studied in another validation set of 306 tumors, where Q-PCR and RNA-EP results were compared with previously carried out IHC that we had validated by chromogenic in situ hybridization (CISH). RESULTS: Q-PCR and RNA-EP offered similar sensitivity (90% versus 77%), specificity (93% versus 95%), and negative (99% versus 98%) and positive (63% versus 61%) predictive values for HER2 determinations. Analyses of relapse-free survival (RFS) and overall survival on the basis of 5 and 10 years of follow-up indicated equivalent hazard ratios for all three techniques. In contrast to IHC/CISH, both Q-PCR and RNA-EP analyses of HER2 also gave statistically significant results regarding RFS and breast cancer-corrected survival after 10 years of follow-up. CONCLUSION: The use of RNA-EP and Q-PCR to analyze HER2 in frozen and formalin-fixed breast cancer samples may be an alternate approach to IHC in combination with FISH/CISH.

Pro-inflammatory mediator leukotriene D4 induces transcriptional activity of potentially oncogenic genes.

The inflammatory mediator LTD4 (leukotriene D4) is present at high levels in many inflammatory conditions, and areas of chronic inflammation have an increased risk for subsequent cancer development. We demonstrate here that following LTD4 stimulation, beta-catenin is translocated to the nucleus, triggering the transcriptional activity of the TCF (T-cell factor)/LEF (lymphoid enhancer factor) family of transcription factors. These events are dependent on phosphoinositide-3 kinase activation and glycogen synthase kinase inhibition. Our data suggest that, similar to Wnt signalling, LTD4 increases free beta-catenin and targets it to the nucleus.

Worse survival for TP53 (p53)-mutated breast cancer patients receiving adjuvant CMF.

BACKGROUND: TP53 has been described as a prognostic factor in many malignancies, including breast cancer. Whether it also might be a predictive factor with reference to chemo- and endocrine therapy is more controversial. PATIENTS AND METHODS: We investigated relapse-free (RFS), breast cancer-corrected (BCCS) and overall survival (OS) related to TP53 status in node-positive breast cancer patients that had received polychemotherapy [cyclophosphamide, methotrexate, 5-fluorouracil (CMF)] and/or endocrine therapy (tamoxifen). Sequence analyses of the whole TP53 coding region was performed in 376 patients operated on for primary breast cancer with axillary lymph node metastases between 1984 and 1989 (median follow-up time 84 months). RESULTS: TP53 mutations were found in 105 patients (28%). We found 90 (82%) of the 110 mutations in the more frequently analysed exons 5-8, while the other 20 (18%) were located in exons 3-4 and 9-10, respectively. Univariate analyses showed TP53 to be a significant prognostic factor with regard to RFS, BCCS and OS in patients who received adjuvant CMF. CONCLUSIONS: TP53 mutations might induce resistance to certain modalities of breast cancer therapy. Sequence-determined TP53 mutation was of negative prognostic value in the total patient population and in the CMF treated patients.

Leukotrienes induce cell-survival signaling in intestinal epithelial cells.

BACKGROUND & AIMS: Inflammatory bowel conditions, particularly ulcerative colitis, are associated with an increased incidence of neoplastic transformation. High levels of proinflammatory leukotrienes (LTs) and up-regulated expression of cyclooxygenase (COX)-2 are characteristic of inflammation. Moreover, COX-2 has been implicated in cell survival and early colon carcinogenesis. Other aspects of interest for intestinal cell viability are the levels of beta-catenin and the antiapoptotic protein Bcl-2. We investigated the possibility that LTs participate in the regulation of these survival factors. METHODS: We used the human intestinal epithelial cell line Int 407 and the rat intestinal epithelial cell line IEC-6. Immunoblotting was applied to ascertain protein expression and distribution, and enzyme immunoassay methodology was used to measure prostaglandin E(2) (PGE(2)) production. Apoptotic ability was assessed by trypan blue exclusion, Hoechst staining, DNA fragmentation, and a caspase-3 activity assay. RESULTS: LTD(4) and LTB(4), but not LTC(4), caused a time- and dose-dependent increase in expression and/or membrane accumulation of COX-2, beta-catenin, and Bcl-2, as well as PGE(2) production. Apoptosis assays showed that the effects of LTs on these transformation-associated proteins correlated well with the ability of these LTs to reduce programmed cell death. CONCLUSIONS: The results suggest that inflammatory conditions are associated with the expression and distribution of proteins that are characteristic of transformed cells; such conditions may involve a signaling mechanism comprising an altered rate of apoptosis.

Leukotriene D4-induced signalling events in human epithelial cells: G alpha i3 activation and translocation.

Our model of LTD4-induced signal transduction in epithelial cells is summarised in Figure 2. Extending what is already known about LTD4 signalling in epithelial cells, we identified the Gi3-protein as the crucial PTX sensitive G-protein and found that it is translocated to what might be a cytoskeletal fraction. This finding suggests a subtle response to LTD4, mediated via the bifurcation at the alpha/beta gamma junction. Although little is known about the role of epithelial cells in inflammation, it has been shown that such cells produce the potent chemoattractant LTB4 and the proinflammatory 5-HETE in response to intracellular accumulation of Ca2+ 24. The target protein(s) and the effect(s) of the translocation of the activated G alpha i3-proteins, as well as the possible role of the beta/gamma-subunits of Gi3, remain to be elucidated.

Leukotriene D4-induced activation and translocation of the G-protein alpha i3-subunit in human epithelial cells.

The present results show that stimulation of Intestine 407 epithelial cells with LTD4 (Leukotriene D4) triggers a rapid activation of the pertussis-toxin-sensitive Gi3-protein and a simultaneous translocation of its alpha-subunits to a crude cytoskeletal fraction. The activation of G alpha i3, which was measured as the GTP/ GDP exchange ratio, peaked about 15 s after the addition of LTD4. Western blot analyses of subcellular fractions showed that G alpha i3-subunits accumulated in the cytoskeletal fraction and decreased in the membrane fraction, and the decrease was most marked 15 s after the exposure to LTD4. None of the other pertussis-toxin-sensitive G-proteins (Gi3-Z and G alpha 0) were activated or translocated upon stimulation with LTD4. This agonist was also found to reduce the GTP/GDP exchange ratio of Gi-proteins without affecting the subcellular distribution of its alpha-subunits. These findings imply that the Gi3-protein is the pertussis-toxin-sensitive G-protein previously found to mediate several downstream LTD4-stimulated signalling events. Furthermore, the translocation of G alpha i3-subunits to the cytoskeleton and the simultaneous inhibition of G3-proteins indicate that the cytoskeleton might participate in the signalling process of human epithelial cells.