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hemokines are important in the development and progression of tumors. We investigated the expression of CXCL14 and CXCL16 in colon cancer. Expression of mRNA was assessed in primary tumors and lymph nodes and CXCL16 mRNA levels were correlated to patient's survival. Protein expression was investigated by two-color immunofluorescence and immunomorphometry. CXCL14 and CXCL16 mRNA levels and protein expression were significantly higher in colon cancer primary tumors compared to apparently normal colon tissue. Positive cells were tumor cells, as revealed by anti-CEA and anti-EpCAM staining. CXCL16, but not CXCL14, mRNA levels were significantly higher in hematoxylin and eosin positive (H&E(+)) compared to H&E(−) colon cancer lymph nodes or control nodes (P < 0.0001). CXCL16 mRNA was expressed in 5/5 colon cancer cell lines while CXCL14 was expressed significantly in only one. Kaplan-Meier analysis revealed that colon cancer patients with lymph nodes expressing high or very high levels (7.2 and 11.4 copies/18S rRNA unit, respectively) of CXCL16 mRNA had a decreased mean survival time of 30 and 46 months at the 12-year follow-up (P = 0.04, P = 0.005, respectively). In conclusion, high expression of CXCL16 mRNA in regional lymph nodes of colon cancer patients is a sign of a poor prognosis.
BACKGROUND AND PURPOSE CYP1B1 and CYP1A1 are important extra-hepatic cytochromes, expressed in the colon and involved in the metabolism of dietary constituents and exogenous compounds. CYP1B1 expression is increased by pro-inflammatory cytokines, and it has been recently implicated in regulation of blood brain barrier function. We investigated its involvement in the increased permeability of the intestinal epithelial barrier observed in inflammatory conditions. EXPERIMENTAL APPROACH Epithelial monolayers formed by human T84 colon carcinoma cells cultured on transwells, were disrupted by incubation with IFN gamma (10 ng.mL(-1)). Monolayer integrity was measured using transepithelial electrical resistance. CYP1A1 and CYP1B1 inhibitors or inducers were applied apically. Potential mechanisms of action were investigated using RT-qPCR. KEY RESULTS IFN gamma disrupts the barrier integrity of the T84 monolayers and increases CYP1B1 and HIF1 alpha mRNA expression. CYP1B1 induction is inhibited by the NF-kappa B inhibitor ammonium pyrrolidinedithiocarbamate (100 mu M) but not by the HIF1 alpha inhibitor 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (50 mu M). Inhibition of CYP1B1 with the selective inhibitor 2,4,3,5-tetramethoxystilbene (100 nM) partly reverses the effects of IFN gamma on epithelial permeability. CONCLUSIONS AND IMPLICATIONS These data suggest that increased expression of CYP1B1 is involved in the effects of IFN gamma on epithelial permeability. Inhibition of CYP1B1 counteracts the alterations of epithelial barrier integrity induced by IFN gamma and could thus have a therapeutic potential in disorders of intestinal permeability associated with inflammation.
The ontogeny of the immune system and the effect thereon by type of infant feeding is incompletely understood. We analyzed frequencies and composition of immune cells in blood of breastfed (BF) and formula-fed (FF) infants at 1.5, 4, and 6 mo of age. Three formulas with the same protein concentration but with varying levels of alpha-lactalbumin and caseinoglycomacropeptide were compared. Twenty-nine exclusively BF infants served as reference, and 17 infants in each formula group completed the study. Whole blood and PBMCs were analyzed by flow cytometry and immunoflow cytometry, respectively. Leukocyte count of BF infants increased with time due to increased frequency of neutrophils. Lymphocyte count was high at 1.5 mo and was unchanged over time, as were the relative proportions of CD4+ alphabetaT cells, CD8+ alphabetaT cells, B cells, NK cells, and gammadeltaT cells. Most CD45R0+CD3+ cells were HLA-DR- and hence memory cells. Compared with breastfeeding, formula feeding resulted in a significant decrease in proportion of NK cells, but a significant increase in naive CD4+ alphabetaT cells and an elevated CD4-to-CD8 ratio, that is, 3.3 in the combined FF groups compared with 2.6 in the BF group. No significant differences were found between the three groups of FF infants. In conclusion, blood cells of lymphoid lineage did not change significantly in frequencies or composition from 1.5 to 6 mo of age in BF infants. In contrast, FF infants displayed an ongoing maturation of adaptive immunity cells and a delayed recruitment of innate immunity cells as compared with BF infants.
BACKGROUND: Coeliac disease is a small intestine enteropathy caused by permanent intolerance to wheat gluten. Gluten intake by patients with coeliac disease provokes a strong reaction by intestinal intraepithelial lymphocytes (IELs), which normalises on a gluten-free diet. AIM: To investigate whether impaired extrathymic T cell maturation and/or secondary T cell receptor (TCR) gene recombination in IELs are features of coeliac disease which could contribute to the failure of establishing tolerance to gluten.
METHODS: Expression levels of the four splice-forms of recombination activating gene-1 (RAG1) mRNA and preT alpha-chain (preTalpha) mRNA were determined in IEL-subsets of children with coeliac disease and controls. Frequencies of RAG1 expressing IELs were determined by immunomorphometry.
RESULTS: In controls, the RAG1-1A/2 splice-form selectively expressed outside the thymus, was dominant and expressed in both mature (TCR(+)) and immature (CD2(+)CD7(+)TCR(-)) IELs ( approximately 8 mRNA copies/18S rRNA U). PreTalpha was expressed almost exclusively in CD2(+)CD7(+)TCR(-) IELs ( approximately 40 mRNA copies/18S rRNA U). By contrast, RAG1 and preTalpha mRNA levels were low in patients with coeliac disease compared to controls, both with active disease and with inactive, symptom-free disease on a gluten-free diet (p values <0.01 for mature and <0.05 for immature IELs). Similarly, the frequencies of RAG1+ IELs were significantly lower in patients with coeliac disease compared to controls (p<0.001).
CONCLUSIONS: Patients with coeliac disease appear to have an impaired capacity for extrathymic TCR gene rearrangement. This is an inherent feature, which probably plays a pivotal role in the failure to efficiently downregulate the T cell response to gluten.
The accuracy of 18S rRNA, β-actin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as indicators of cell number when used for normalization in gene expression analysis of T lymphocytes at different activation stages was investigated. Quantitative real-time reverse transcriptase-polymerase chain reaction was used to determine the expression level of 18S rRNA, β-actin mRNA, GAPDH mRNA and mRNA for six cytokines in carefully counted samples of resting human peripheral blood mononuclear cells (PBMCs), intestinal lymphocytes and PBMCs subjected to polyclonal T-cell activation. The 18S rRNA level in activated and resting PBMCs and intestinal lymphocytes was essentially the same, while the levels of β-actin and GAPDH mRNAs fluctuated markedly upon activation. When isolated γδTCR+, CD4+ and CD8+ subpopulations were studied, 18S rRNA levels remained unchanged after 21 h of activation but increased slightly after 96 h. In contrast, there was a 30–70-fold increase of GAPDH mRNA/cell in these cell populations upon activation. Cytokine analysis revealed that only normalization to 18S rRNA gave a result that satisfactorily reflected their mRNA expression levels per cell. In conclusion, 18S rRNA was the most stable housekeeping gene and hence superior for normalization in comparative analyses of mRNA expression levels in human T lymphocytes.
Two new 5'-untranslated region (5'UTR) exons were identified in the human gene for the lymphocyte-specific endonuclease recombination activating gene-1 (RAG1) required for the somatic recombination yielding functional Ag receptors. These 5'UTR exons were used in three different splice forms by jejunal lymphocytes of the T cell lineage. RAG1 mRNA containing the previously described 5'UTR exon was not expressed in these cells. Conversely, one of the new 5'UTR exons was not expressed in thymus. The new RAG1 mRNA splice forms were all expressed in immature T cells (CD2(+)CD7(+)CD3(-)). This cell population also expressed high levels of mRNA for the pre-T alpha-chain. In situ hybridization demonstrated jejunal cells expressing the new splice forms of RAG1 mRNA, both intraepithelially and in lamina propria. Pre-T alpha-chain mRNA-expressing cells were detected at the same sites. These results strongly suggest ongoing TCR gene rearrangement in human small intestinal mucosa, yielding T cells specially adapted for this environment. This seems to be achieved by two parallel processes, extrathymic T cell development and peripheral Ag-driven TCR editing.
The small intestinal epithelium of Vibrio cholerae infected patients expresses the immunomodulatory microRNAs miR-146a and miR-155 at acute stage of disease. V. cholerae release outer membrane vesicles (OMVs) that serve as vehicles for translocation of virulence factors including V. cholerae cytolysin (VCC). The aim was to investigate whether OMVs, with and/or without VCC-cargo could be responsible for induction of microRNAs in intestinal epithelial cells and thereby contribute to immunomodulation. Polarized tight monolayers of T84 cells were challenged with OMVs of wildtype and a VCC deletion mutant of the non-O1/non-O139 (NOVC) V. cholerae strain V:5/04 and with soluble VCC. OMVs, with and without VCC-cargo, caused significantly increased levels of miR-146a. Increase was seen already after 2 hours challenge with OMVs and persisted after 12 hours. Challenge with soluble VCC caused significant increases in interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α), CCL20, IL-1β, and IRAK2 mRNA levels while challenge with OMVs did not cause increases in expression levels of any of these mRNAs. These results suggest that V. cholerae bacteria release OMVs that induce miR-146a in order to pave the way for colonization by reducing the strength of an epithelial innate immune defence reaction and also preventing inflammation in the mucosa that factors like VCC can evoke.
The potential immunomodulatory role of microRNAs in small intestine of patients with acute watery diarrhea caused by Vibrio cholerae O1 or enterotoxigenic Escherichia coli (ETEC) infection was investigated. Duodenal biopsies were obtained from study-participants at the acute (day 2) and convalescent (day 21) stages of disease, and from healthy individuals. Levels of miR-146a, miR-155 and miR-375 and target gene (IRAK1, TRAF6, CARD10) and 11 cytokine mRNAs were determined by qRT-PCR. The cellular source of microRNAs in biopsies was analyzed by in situ hybridization. The ability of V. cholerae bacteria and their secreted products to cause changes in microRNA- and mRNA levels in polarized tight monolayers of intestinal epithelial cells was investigated. miR-146a and miR-155 were expressed at significantly elevated levels at acute stage of V. cholerae infection and declined to normal at convalescent stage (P<0.009 versus controls; P = 0.03 versus convalescent stage, pairwise). Both microRNAs were mainly expressed in the epithelium. Only marginal down-regulation of target genes IRAK1 and CARD10 was seen and a weak cytokine-profile was identified in the acute infected mucosa. No elevation of microRNA levels was seen in ETEC infection. Challenge of tight monolayers with the wild type V. cholerae O1 strain C6706 and clinical isolates from two study-participants, caused significant increase in miR-155 and miR-146a by the strain C6706 (P<0.01). One clinical isolate caused reduction in IRAK1 levels (P<0.05) and none of the strains induced inflammatory cytokines. In contrast, secreted factors from these strains caused markedly increased levels of IL-8, IL-1β, and CARD10 (P<0.001), without inducing microRNA expression. Thus, miR-146a and miR-155 are expressed in the duodenal epithelium at the acute stage of cholera. The inducer is probably the V. cholerae bacterium. By inducing microRNAs the bacterium can limit the innate immune response of the host, including inflammation evoked by its own secreted factors, thereby decreasing the risk of being eliminated.
The small intestinal epithelium of Vibrio cholerae infected patients expresses the immunomodulatory microRNAs miR-146a and miR-155 at acute stage of disease. V. cholerae release outer membrane vesicles (OMVs) that serve as vehicles for translocation of virulencefactors including V. cholerae cytolysin (VCC). The aim was to investigate whether OMVs, with and/or without VCC-cargo could be responsible for induction of microRNAs in intestinal epithelial cells and thereby contribute to immunomodulation. Polarized tight monolayers of T84 cells were challenged with OMVs of wild-type and a VCC deletion mutant of the non-O1/non-O139 (NOVC) V. cholerae strain V:5/04 and with soluble VCC. OMVs, with and without VCC-cargo, caused significantly increased levels of miR-146a. Challenge with soluble VCC caused significant increases in interleukin-8 (IL-8), tumour necrosis factor-α(TNF-α), CCL20, IL-1β, and IRAK2 mRNA levels while challenge with OMVs did not causeany changes. Notably, OMVs from the VCC deficient mutant caused significant decreases in CCL20 and IL-18 mRNA levels. These results suggest that V. cholerae bacteria release OMVs that induce miR-146a in order to pave the way for colonization by reducing thestrength of an epithelial innate immune defence reaction and also preventing inflammation inthe mucosa that factors like VCC can evoke.
Four carcinoembryonic antigen-related cell adhesion molecule (CEACAM)s, i.e. CEA, CEACAM1, CEACAM6 and CEACAM7, are localized to the apical glycocalyx of normal colonic epithelium and have been suggested to play a role in innate immunity. The expression of these molecules in colon carcinoma cells was studied at the mRNA and protein levels after treatment with interferon-gamma (IFN-gamma), interleukin-1beta, live bacteria or lipopolysaccharide. The colon carcinoma cell lines LS174T and HT-29 were studied in detail using real-time quantitative reverse transcriptase-polymerase chain reaction, immunoflow cytometry and immunoelectron microscopy. IFN-gamma, but not the other agents, modified expression of CEA, CEACAM1 and CEACAM6. None of the agents upregulated CEACAM7 expression. Two expression patterns were seen. HT-29 cells, which initially showed low quantities of mRNAs and proteins, displayed marked upregulation of both mRNAs and proteins. LS174T cells transcribed stable high levels of mRNA before and after treatment. Additionally, IFN-gamma induced increased cell surface expression of CEA, CEACAM1 and CECAM6. IFN-gamma has two important effects on the expression levels of the CEA family molecules in colon epithelial cells: direct upregulation of CEACAM1 and promotion of cell differentiation resulting in increased expression of CEA and CEACAM6 and decreased expression of CEACAM7.
mRNA expression of two recently described human beta-defensins (hBD-3 and hBD-4) in epithelial cells of normal small and large intestine and the impact of chronic intestinal inflammation on their expression levels was investigated. Intestinal specimens from patients with ulcerative colitis (UC), Crohn's disease (CD) and controls with no history of inflammatory bowel disease were studied. hBD-3 and hBD-4 mRNAs were determined in freshly isolated epithelial cells by real-time quantitative reverse transcription-polymerase chain reaction (QRT-PCR) and by in situ hybridization. The effect of proinflammatory cytokines on hBD-3 and hBD-4 mRNA expression in colon carcinoma cells was also investigated. Purified epithelial cells of normal small and large intestine expressed both hBD-3 and hBD-4 mRNA, with higher expression levels of hBD-3 mRNA. In situ hybridization revealed higher levels of mRNA expression in the crypt- compared to the villus/luminal-compartment. Interferon (IFN)-gamma, but not tumour necrosis factor (TNF)-alpha or IL-1beta, augmented hBD-3 mRNA expression. None of these agents stimulated hBD-4 expression. Colonic epithelial cells from patients with UC displayed a significant increase in hBD-3 and hBD-4 mRNA compared to epithelial cells of controls. In contrast, small intestinal epithelial cells from CD patients did not show increased expression levels compared to the corresponding control cells. Moreover, Crohn's colitis did not show increased expression of hBD-4 mRNA, while the data are inconclusive for hBD-3 mRNA. We conclude that the chronic inflammatory reaction induced in the colon of UC patients enhances hBD-3 and hBD-4 mRNA expression in the epithelium, whereas in CD this is less evident.
OBJECTIVES: Exposure to gliadin and related prolamins and appropriate HLA-DQ haplotype are necessary but not sufficient for contracting celiac disease (CD). Aberrant innate immune reactions could be contributing risk factors. Therefore, jejunal biopsies were screened for bacteria and the innate immune status of the epithelium investigated.
METHODS: Children with untreated, treated, challenged CD, and controls were analyzed. Bacteria were identified by scanning electron microscopy. Glycocalyx composition and mucin and antimicrobial peptide production were studied by quantitative RT-PCR, antibody and lectin immunohistochemistry.
RESULTS: Rod-shaped bacteria were frequently associated with the mucosa of CD patients, with both active and inactive disease, but not with controls. The lectin Ulex europaeus agglutinin I (UEAI) stained goblet cells in the mucosa of all CD patients but not of controls. The lectin peanut agglutinin (PNA) stained glycocalyx of controls but not of CD patients. mRNA levels of mucin-2 (MUC2), alpha-defensins HD-5 and HD-6, and lysozyme were significantly increased in active CD and returned to normal in treated CD. Their expression levels correlated to the interferon-gamma mRNA levels in intraepithelial lymphocytes. MUC2, HD-5, and lysozyme proteins were seen in absorptive epithelial cells. beta-defensins hBD-1 and hBD-2, carcinoembryonic antigen (CEA), CEA cell adhesion molecule-1a (CEACAM1a), and MUC3 were not affected.
CONCLUSIONS: Unique carbohydrate structures of the glycocalyx/mucous layer are likely discriminating features of CD patients. These glycosylation differences could facilitate bacterial adhesion. Ectopic production of MUC2, HD-5, and lysozyme in active CD is compatible with goblet and Paneth cell metaplasia induced by high interferon-gamma production by intraepithelial lymphocytes.
Celiac disease (CD) is a small intestinal enteropathy caused by permanent intolerance to wheat gluten. Active disease is characterized by a prominent cytokine response of intraepithelial lymphocytes (IELs) to gluten-containing diet with concomitant increase in expression of pro-inflammatory IFN-gamma and down-regulatory IL-10 without increase in tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1). The aim was to understand the local immune reaction by determining which intraepithelial T cell subsets produce the different cytokines. The three major IEL-subsets gammadeltaIELs, CD4(+)alphabetaIELs and CD8(+)alphabetaIELs, as well as CD94(+)CD8(+)alphabetaIELs, selectively expanded in active CD, were retrieved from small intestinal biopsies of children with active CD and controls and analyzed quantitatively for cytokine mRNA expression. In active CD, CD8(+)alphabetaIELs showed a significant increase in expression levels of both IFN-gamma and IL-10. CD8(+)alphabetaIELs were also the IEL subset with highest expression level per cell of both cytokines and constituted the cellular source for almost all IFN-gamma and most IL-10. Expression levels of both cytokines were higher in CD94(-)CD8(+)alphabetaIELs than CD94(+)CD8(+)alphabetaIELs. TNF-alpha levels were only increased in CD4(+)alphabetaIELs, which also showed the highest expression level per cell and constituted the major source of this cytokine. Interestingly, IL-10 was increased also in CD4(+)alphabetaIELs. Cytokine levels were low in gammadeltaIELs. 'Classical' CD94(-)CD8(+)alphabeta T cells within the epithelium are responsible for the excessive production of IFN-gamma, believed to drive the formation of intestinal lesions in active CD. Production of IL-10 may be a common feature of IELs producing pro-inflammatory cytokines, thereby attempting to limit inflammation in an autocrine fashion.
V gamma 9+ T cells from malaria non-exposed donors make proliferative responses to Plasmodium falciparum on in vitro stimulation. V gamma 9+ cells are strongly activated by components of the schizont stage of the parasite and by antigens released into the culture upon schizogony, while CD4+V gamma 9- cells are stimulated by the earlier stages of the parasite. Using reverse transcriptase-polymerase chain reaction (RT-PCR) we determined mRNA expression for 14 cytokines in highly purified V gamma 9+ cells enriched by positive selection after in vitro stimulation with P. falciparum schizont antigens. Interferon-gamma (IFN-gamma) and Tumor Necrosis Factor-alpha (TNF-alpha) were detected in all samples tested. The majority of samples also expressed TNF-beta, transforming growth factor-beta (TGF-beta) and Interleukin-8 (IL-8). Only occasional samples expressed IL-2, IL-5 and IL-10. Using the ELISPOT assay we found that a large fraction of the reactive V gamma 9+ cells produced IFN-gamma and that gamma delta T cells are the major producers of IFN-gamma in cultures stimulated with schizont antigens. The majority of V gamma 9+ cells in these cultures also express the membrane-bound form of TNF-alpha. Expression of these cytokines speaks for a cytolytic and/or inflammatory role of gamma delta cells in the response to malaria in non-exposed individuals.
Five obligately anaerobic, Gram-stain-negative, saccharolytic and proteolytic, non-spore-forming bacilli (strains CD3 :27, CD3 :28(T), CD3 :33, CD3 :32 and CD3 :34) are described. All five strains were isolated from the small intestine of a female child with coeliac disease. Cells of the five strains were short rods or coccoid cells with longer filamentous forms seen sporadically. The organisms produced acetic acid and succinic acid as major metabolic end products. Phylogenetic analysis based on comparative 16S rRNA gene sequence analysis revealed close relationships between CD3 : 27, CD3 :28(T) and CD3 :33, between CD3 :32 and Prevotella histicola CCUG 55407(T), and between CD3 :34 and Prevotella melaninogenica CCUG 4944B(T). Strains CD3 : 27, CD3 :28(T) and CD3 :33 were clearly different from all recognized species within the genus Prevotella and related most closely to but distinct from P. melaninogenica. Based on 16S rRNA, RNA polymerase) beta-subunit (rpoB) and 60 kDa chaperonin protein subunit (cpn60) gene sequencing, and phenotypic, chemical and biochemical properties, strains CD3 :27, CD3 :28(T) and CD3 :33 are considered to represent a novel species within the genus Prevotella, for which the name Prevotella jejuni sp. nov. is proposed. Strain CD3 : 28(T) (=CCUG 60371(T)=DSM 26989(T)) is the type strain of the proposed novel species. All five strains were able to form homologous aggregates, in which tube-like structures were connecting individual bacteria cells. The five strains were able to bind to human intestinal carcinoma cell lines at 37 degrees C.
Two new obligately anaerobic Gram-positive, saccharolytic and non-proteolytic spore-forming bacilli (strain CD3:22 and N1) are described. Strain CD3:22 was isolated from a biopsy of the small intestine of a child with celiac disease and strain N1 from the saliva of a healthy young man. The cells of both strains were observed to be filamentous with lengths of approximately 5 to >20 µm, some of them curving and with swellings. The novel organisms produced H2S, NH3, butyric acid and acetic acid as major metabolic end products. Phylogenetic analyses, based on comparative 16S rRNA gene sequencing, revealed close relationships (98 % sequence similarity) between the two isolates, as well as the type strain of Eubacterium saburreum CCUG 28089T and four other Lachnospiraceae bacterium/E. saburreum-like organisms. This group of bacteria were clearly different from any of the 19 known genera in the family Lachnospiraceae. While Eubacterium spp. are reported to be non-spore-forming, reanalysis of E. saburreum CCUG 28089T confirmed that the bacterium, indeed, is able to form spores. Based on 16S rRNA gene sequencing, phenotypic and biochemical properties, CD3:22 (CCUG 58757T) and N1 (CCUG 60305T) represent new species of a new and distinct genus, named Lachnoanaerobaculum, in the family Lachnospiraceae [within the order Clostridiales, class Clostridia, phylum Firmicutes]. Strain CD3:22 is the type strain of the type species, Lachnoanaerobaculum umeaense gen. nov., sp. nov., of the proposed new genus. Strain N1 is the type strain of the species, Lachnoanaerobaculum orale gen. nov., sp. nov. Moreover, E. saburreum CCUG 28089T is reclassified as Lachnoanaerobaculum saburreum comb. nov.
OBJECTIVE: To determine whether use of steroid hormone contraceptives modifies immune defense parameters of the vaginal epithelium in humans. METHODS: Vaginal biopsies were collected during the follicular and luteal phases in regularly menstruating women (controls) and in women using combined oral contraceptives, depot-medroxyprogesterone acetate injections, or levonorgestrel implants. Fifteen healthy women (aged 20-34 years) were enrolled in each group. Biopsies were analyzed in a blinded manner. Epithelial thickness was estimated by morphometry. Immune cells were analyzed by immunomorphometry with cell-type-specific monoclonal antibodies. RESULTS: The epithelium of controls harbored 241+/-35 leukocytes (CD45+ cells) per mm2 (mean+/-1 standard error of the mean), and the thickness was 261+/-16 microm. T lymphocytes (CD3+) dominated, and cytotoxic or suppressor T cells (CD8+) were more frequent than T helper cells (CD4:CD8 ratio: 0.7+/- 0.1). Macrophages (CD68+) constituted the second-largest population, followed by Langerhans cells (CD1a+). B cells, natural killer cells, monocytes, and granulocytes were generally absent. There were no significant differences between the follicular and luteal phases. The epithelium was significantly thicker in all three groups that used hormonal contraceptive (333+/-9 microm) compared with controls, and it exhibited superficial hyperplasia. The frequency of intraepithelial leukocytes (CD45+) was increased in depot-medroxyprogesterone acetate (P<.001) and levonorgestrel implant users (P<.04). In depot-medroxyprogesterone acetate users, this was explained by an increased frequency of the CD8+ T lymphocyte subset. CONCLUSION: Hormonal contraceptives induce hyperplasia of the vaginal epithelium. The significant changes in the intraepithelial leukocyte population in depot-medroxyprogesterone acetate and levonorgestrel implant users most probably reflect altered local immune capacity.
The functional properties of intraepithelial lymphocytes (IEL) in normal human jejunum, ileum, and colon were investigated. Cytokine mRNA expression in IEL and enterocytes was determined by reverse transcriptase-PCR and IFN-gamma+ IEL by immunohistochemistry. Polyclonal activators were used to study proliferation and IFN-gamma secretion of IEL, and an anti-CD3-mediated redirected cytotoxicity assay was used to determine the lytic potential of IEL. Freshly isolated IEL at all three gut levels expressed mRNA for IL-1 beta, IL-2, IL-8, IFN-gamma, and TNF-alpha. Approximately 10% of IEL produced IFN-gamma, suggesting that IEL are immunologically active in vivo, performing similar functions along the intestine. IEL could be stimulated further in vitro to express IL-10, TNF-beta, and TGF-beta 1, while no Th2-type cytokines were induced, suggesting suppressive and cytolytic functions for IEL. All three jejunal IEL subpopulations (CD4-CD8-TCR-gamma delta+, CD4+TCR-alpha beta+, CD8+TCR-alpha beta+) expressed the same four cytokines, IL-2, IL-8, IFN-gamma, and TNF-alpha, indicating that CD4+TCR-alpha beta+ IEL are Th1 cells and that TCR-gamma delta+ IEL and CD8+TCR-alpha beta+ IEL include cytotoxic effector cells. Indeed, freshly isolated jejunal IEL displayed cytolytic activity. IEL were induced to proliferation by anti-CD3/TCR complex mAbs and leukoagglutinin, but not by Con A. There was no correlation between the magnitude of the proliferative response and the amounts of secreted IFN-gamma. Enterocytes expressed IL-1 beta and IL-8, and sometimes TNF-alpha. Although jejunal enterocytes express HLA-DR and hsp60, Ag presentation by these cells may induce anergy since their cytokine profile is different from that of classical APCs.
The human gut epithelium is a unique immunological compartment, containing substantial amounts of intra-epithelial lymphocytes (IEL) with unknown functions. In this study we show that distinct and unusual subpopulations of IEL are present at different levels of human intestine. IEL phenotypes in normal jejunum, ileum and colon were compared using immunoflow cytometry and immunohistochemistry. The expression of mRNA for recombination-activating gene-1 (RAG-1) in IEL from all three levels was compared using reverse-transcription polymerase chain reaction, and the morphology of IEL in situ was determined using immunoelectron microscopy. Surface marker profiles of isolated intestinal epithelial cells at all three levels were also investigated. On average the proportion of TCR gamma delta IEL was comparable in jejunum than ileum and colon and varied in phenotype with gut level. CD4-CD8-TCR alpha beta IEL dominated in colon but were absent in jejunum. CD8+ TCR alpha beta IEL were present at all levels but only in jejunum did they constitute the majority of all IEL. CD4+ TCR alpha beta IEL were present in similar frequencies at all levels of the gut. In general, the majority of IEL had an activated phenotype (CD45RO+, alpha E beta 7+). Furthermore, IEL exhibited phenotypes which are rare in peripheral blood. The thymocyte markers CD1a and CD1c as well as the NK cell marker CD56 were expressed on a fraction of TCR alpha beta and TCR gamma delta IEL. A small population of 'null' cells (CD45+ TCR/CD#-CD20-CD14-CD15- cells) was also present at equal proportions along the gut. Jejunal but not colonic IEL expressed RAG-1 mRNA suggesting that extrathymic T cell maturation occurs in the epithelium of small intestine. RAG-1 was expressed in CD2+TCR/CD3- and CD3+/TCR-IEL. Ultrastructurally, IEL often formed small clusters and intimate contacts with epithelial cells, suggesting cell cooperation within the epithelium. Some IEL had pseudopodium-like extensions penetrating the epithelial basement membrane suggesting transmigration. Epithelial cells in small intestine but not colon expressed heat shock protein 60 and HLA-DR. CD1a, CD1b and CD1c were not expressed on intestinal epithelial cells at any level. The distinct surface marker profiles of IEL and epithelial cells along small and large intestine suggest functional regional specialization and are compatible with the hypothesis that TCR alpha beta IEL participate in immune reactions to lumenal antigens while TCR gamma delta IEL perform surveillance of the epithelium.
We have shown that gamma delta T cells in human gingiva have an intraepithelial location and, that in the chronic inflammatory disease periodontitis, the expression of CD45RO and CD8 or CD4 is induced on gamma delta T cells. To study the role of gamma delta T cells in local antibacterial responses, we determined the cytokine profiles of isolated human gingival cells. Different T cell subpopulations, isolated by positive selection with mAb-coated magnetic beads and macrophages, as well as epithelial cells, were analyzed for expression of mRNA for 15 cytokines by reverse transcriptase-PCR. The ultrastructure of gingival gamma delta T cells was also studied. The gamma delta T cells expressed mRNA for IFN-gamma, TNF-alpha, TGF-beta 1, and IL-6. Expression of IFN-gamma was a consequence of inflammation. CD4+ gamma delta T cells expressed IFN-gamma only, whereas CD8+ gamma delta T cells expressed all four cytokines. CD8+ cells expressing IFN-gamma, TNF-alpha, and IL-6 in combination suggest a cytotoxic effector function. Gingival gamma delta T cells contained cytoplasmic electron-dense membrane-bound granules and multivesicular bodies that are ultrastructural characteristics of cytotoxic cells. Epithelial cells from inflamed gingiva expressed HLA-DR, CD1a, CD1c, and heat shock protein 60 on the cell surface. They also expressed mRNA for IL-1 beta, IL-6, IL-8, TNF-alpha, and TGF-beta 1. Thus, epithelial cells may function as accessory cells in immune activation and, at the same time, be target cells for CD8+ gamma delta T cells reactive with CD1 Ag or heat shock protein. These results suggest that gamma delta T cells constitute a first line of defense in gingiva, preventing entrance of pathogens by cytotoxicity against infected and stressed epithelial cells, and by control of epithelial cell growth through secretion of regulatory cytokines.
The phenotypic profile of leucocytes in diseased and normal gingival tissue was studied in situ and in isolated gingival mononuclear cell (GMC) preparations. T-cell receptor (TcR)gamma delta + cells showed preferential localization to epithelium, both in normal and inflamed gingiva, and were present in crevicular as well as oral epithelium. In normal gingiva > or = 30% of the isolated leucocytes expressed TcR gamma delta, of which the majority were CD4- CD8-, and expressed CD45RA. The proportion of TcR gamma delta + cells in GMC from periodontitis tissue varied between 2 and 32%. In contrast to normal gingiva the majority of TcR gamma delta + cells in diseased tissue were CD8+ and expressed CD45RO. Thus expression of the CD8 antigen on gingival TcR gamma delta + cells is probably a consequence of immune activation. Numerous Langerhans' cells and keratinocytes expressing the major histocompatibility complex (MHC) class I-like antigen, CD1, were present within normal and inflamed gingival epithelium in close proximity to the TcR gamma delta + cells. Most CD1a+ cells were scattered within oral epithelium. CD1c+ cells were localized close to the basal layer of crevicular epithelium. No CD1b+ cells were found. TcR alpha beta + cells, CD4+ and B cells were restricted to lamina propria of periodontitis lesions. The presence of intraepithelial TcR gamma delta + cells in normal gingiva suggests that they constitute the 'first line of defence' against the potentially harmful microflora in the oral cavity. Induction of CD8 and CD45RO antigens on TcR gamma delta + cells in periodontitis tissue indicate that they play a significant role in the disease. CD1 molecules on Langerhans' cells and keratinocytes may be the restriction elements for the CD8+ TcR gamma delta + cells.
A mild purification method has been developed for the isolation of human intraepithelial lymphocytes (IEL) and enterocytes from the same individual. The isolation procedure includes mechanical disruption of the mucosal layer, treatment with reducing agent and sedimentation followed by Percoll gradient centrifugation. Finally, epithelial cells are removed from the IEL fraction using magnetic beads coated with the anti-epithelial antigen monoclonal antibody (mAb) BerEP4. Leucocytes are removed from the enterocyte fraction using magnetic beads coated with mAbs directed against common leucocyte antigen (CD45). Using this procedure IEL and enterocytes have been isolated from apparently normal jejunal, ileal and colonic tissue specimens. Recoveries of IEL were 7 x 10(5), 4 x 10(5) and 1 x 10(5)/cm2 mucosa from jejunum, ileum and colon respectively. 1-2 x 10(6) enterocytes/cm2 mucosa were recovered from small intestine while the corresponding value for colonic biopsies was approximately 2 x 10(5) enterocytes/cm2. The IEL fraction was pure as judged by the low percentages of B cells, macrophages and BerEP4 positive cells (less than 4%) present in the purified fraction. The enterocyte fraction contained less than 2% CD45+ cells. The two cell fractions were viable and expanded in vitro. Enterocytes expanded spontaneously while IEL required initial stimulation with mitogens. The isolation procedure described here will make it possible to study the function of human IEL, interactions between IEL and enterocytes and the role of both cell types in local immunity.
Cell-mediated lymphocyte cytotoxicity in ileum and colon of patients with ulcerative colitis (UC), Crohn's disease (CD) and controls was investigated. Frequencies of cells expressing perforin and Fas-ligand (FasL) were determined by immunomorphometry. mRNA expression of perforin, granzyme B and FasL in T cells and subsets was assayed by reverse transcriptase-polymerase chain reaction. Cytotoxicity of intraepithelial and lamina propria lymphocytes was analysed without ex vivo activation in three functional assays: (1) anti-CD3-dependent T-cell receptor (TCR)-/CD3-mediated redirected cytotoxicity, (2) Fas-/FasL-mediated TCR-/CD3-independent cytotoxicity and (3) natural killer (NK) cell cytotoxicity. Inflammation in ileum of CD patients caused increased frequency of perforin-expressing cells and enhanced perforin-dependent TCR-/CD3-mediated cytotoxicity. In contrast, lymphocytes in the inflamed colon of UC or Crohn's colitis patients did not display this cytotoxicity nor did lymphocytes of normal colon. Normal colon lymphocytes showed spontaneous Fas-/FasL-mediated cytotoxicity. This activity was retained but not enhanced in inflamed UC colon. In contrast, a significant increase of FasL-expressing cells was seen in situ. Inflammation did not induce NK cell activity in colonic lymphocytes. Intestinal lymphocytes comprise effectors active in two different cytolytic processes. 'Classical' cytotoxic T lymphocytes in small intestine and lymphocytes executing TCR-/CD3-independent FasL-/Fas-mediated killing of unknown biological role present throughout the intestinal mucosa. Ongoing normal cytolytic processes seem to be enhanced by chronic inflammation.
Ulcerative colitis (UC), a chronic inflammatory bowel disease, exhibits pronounced increase of T lymphocytes in the inflamed mucosa. To understand the role of intestinal T lymphocytes in the pathogenesis of UC their cytokine production in the mucosa was analysed. Intestinal T lymphocytes of UC, Crohn's disease and control patients were analysed for cytokine mRNA levels by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) directly after isolation without in vitro stimulation. Frequencies of cytokine positive cells were determined in UC and control colon by immunomorphometry. T lymphocytes in normal colon expressed interleukin (IL)-2, interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1, but not IL-4, IL-5 or IL-10. In UC, a highly significant increase in IL-10 mRNA levels in T lymphocytes and an increased frequency of IL-10 positive cells was seen in colon. IL-10 mRNA levels were also elevated in T lymphocytes of the non-inflamed ileum and correlated with disease activity at both locations. CD4+ T lymphocytes were the major source of IL-10 mRNA. IL-2, IFN-gamma and TNF-alpha mRNA levels were decreased in colonic T lymphocytes, and virtually no IL-2, IFN-gamma, TNF-alpha or TGF-beta positive cells were detected in basal lymphoid aggregates. However, scattered IL-10 positive cells were found here. Lamina propria outside the aggregates contained IL-10-, IFN-gamma, TNF-alpha and TGF-beta but not IL-2 positive cells. T cells of UC patients did not express IL-4 or IL-5. Taken, together the data suggest a generalized activation of IL-10 producing CD4+ T cells along the intestine of UC patients. The local environment seems to determine the biological consequences of elevated IL-10.
Human decidual lymphocytes from early, normal pregnancy were characterized in situ with respect to ultrastructure and distribution of subsets. The ultrastructure of isolated decidual gamma delta T cells was also studied. CD45+ cells comprised 11 +/- 2% of all decidual cells. The majority were localized in large lymphoid cell clusters (LCC), near endometrial glands, or as intraepithelial lymphocytes (IEL) in glandular epithelium. The major cell populations in LCC were CD56+TCR-gamma delta+ cells, CD56+ cells, TCR-alpha beta+CD4+ cells, and TCR-alpha beta+CD8+ cells. All expressed activation markers (CD45RO, Kp43, and/or HML-1) and MHC class II Ag (HLA-DR, HLA-DP, and/or HLA-DQ). No B cells were found. Almost all IEL were activated TCR-gamma delta+ cells (CD56+ and CD56-). The glandular epithelial cells expressed heat shock protein 60 at the basolateral side facing the TCR-gamma delta+ IEL. Decidual lymphocytes displayed cytoplasmic processes, microvilli, characteristic cytoplasmic granules, and had intimate contact with neighboring cells. Lymphocytes in the outer rim of LCC and the stroma showed signs of cellular movement. Two main morphotypes of gamma delta T cells could be distinguished. One had single microvilli, membrane-bound granules, and nuclear inclusions. The other had many microvilli, nonmembrane-bound granules and cytoplasmic multivesicular bodies. Our data suggest that LCC are centers of immune reactivity where T and NK cells become activated. The activated cells may guard against infections and undue trophoblast invasion and/or be involved in modulating the local maternal immune system toward unresponsiveness against the semiallogeneic fetus.
The modulatory effect of human milk proteins from colostrum and late milk on the proliferative response of human T lymphocytes activated by mitogens (OKT3 and leucoagglutinin from Phaseolus vulgaris) and alloantigens was studied. High concentrations (10-100 micrograms/ml) of crude colostral milk proteins had an inhibitory effect on T cell growth while low concentrations (0.1-1 microgram/ml) enhanced T cells growth. In contrast, proteins from late milk did not inhibit T lymphocyte proliferation while the enhancing effect was retained. Colostrum was fractionated by ammonium sulphate precipitation and gel filtration on sepharose 6B. The inhibitory activity was recovered in a protein fraction containing lactoferrin as its major component. Lactoferrin was, however, not responsible for the observed inhibition. On the contrary, lactoferrin in most cases augmented the proliferative response induced by polyclonal activators. The inhibitory activity was found to bind concanavalin A-sepharose suggesting an association with glycoprotein. Inhibitory fractions contained glycoproteins of the following molecular sizes 26, 74/76 (doublet), 84, 145 and 160 kD under reducing conditions. The inhibitory effect appeared to be lymphocyte specific since the active fraction did not inhibit the growth of tissue culture cells (HeLa cells and human fibroblasts) or bacteria. Furthermore, the fraction was not toxic for lymphocytes. The inhibitory colostrum factor may prevent the newborn from overreacting immunologically against the environmental antigens encountered at birth.
Lactoferrin (Lf), an iron-binding protein in milk, mucosal secretions and neutrophil granules has bactericidal properties and is a source of iron for breast-fed infants. In this paper the authors show that most in vivo activated lymphocytes, i.e. freshly isolated lymphocytes from first trimester human decidua, and most in vitro activated human blood lymphocytes, express lactoferrin receptors (Lf-R), while unstimulated blood lymphocytes do not. All major lymphocyte subsets, i.e. alpha beta T cells, gamma delta T cells, CD8+ T cells, CD4+ T cells, B cells and NK cells, express Lf-R after activation. The proportion of Lf-R expressing activated gamma delta T cells is significantly larger than that of activated alpha beta T cells. Lf-R and transferrin receptors (Tr-R/CD71) show the same kinetics of appearance on activated blood lymphocytes and are, to a large extent, expressed on the same cells. However, 35% of decidual lymphocytes and 15% of activated blood lymphocytes express Lf-R only. Addition of Lf to cultures containing an optimal concentration of Tr augments the proliferative response to polyclonal T cell activators and alloantigens, suggesting that presently used standard culture conditions for in vitro activation are suboptimal in particular for gamma delta T cells. Lf-R on decidual lymphocytes contain bound Lf, which probably is produced locally. The results suggest that Lf is a growth-supporting factor, especially important in local immune responses in the mucosa.
The mononuclear lymphoid cell population in human pregnant uterus mucosa, decidua, from early normal pregnancies was studied phenotypically and functionally. The phenotype was determined in situ by immunohistochemistry, and in isolated decidual mononuclear cell preparations by immunofluorescence and flow cytometry. A mild isolation procedure of gentle mechanical disruption followed by density gradient centrifugation was used. Leukocytes comprised a large part of the decidual tissue. They were present in aggregates mainly situated adjacent to the glandular epithelium. In addition, individual leukocytes were present intraepithelially, as well as scattered between the stromal cells and around vessels and lacunes. Four lymphocyte populations of approximately the same size were identified: TCR gamma delta+/CD56+ cells, TCR gamma delta+/CD56- cells, TCR gamma delta-/CD56+ cells, and TCR alpha beta+/CD8+ cells. TCR gamma delta- expressing cells comprised about 60% of the T cells. They were CD4-/CD8-, and about half of the TCR gamma delta+ cells expressed the memory/activation marker CD45RO. The Kp 43 Ag, earlier described on activated CD56+ and TCR gamma delta+ cells in peripheral blood, was essentially only expressed on the TCR gamma delta-/CD56+ cell population in decidua. At least 50% of the TCR alpha beta+ cells were CD8+. The function(s) of either one of these populations might be to prevent immunologic reactions against the fetus, to protect the uterus from unwanted extensive invasion of trophoblasts, or to protect the uteroplacental unit from infection. Decidual T cells did not respond to stimulation by alloantigens or mitogenic anti-CD3 mAb but responded to the same extent as PBMC to mitogenic lectins. The surface density of the TCR/CD3 complex was low on freshly isolated decidual lymphocytes, but could be up-regulated upon stimulation with PMA/Ionomycin. Local selective down-regulation of surface expression of the TCR/CD3 complex and of activation involving this complex might be one of the mechanisms by which a maternal immunologic reaction against the semiallogeneic fetus is prevented.
The uterine mucosa in pregnancy, the decidua, allows placenta formation and survival of the fetus despite the fact that it is semiallogeneic. Decidua contains large numbers of lymphocytes, of which CD56+ cells dominate, followed by T cells expressing either alpha beta or gamma delta TCR. We have investigated the developmental relationship between the CD56- and TCR gamma delta-expressing cells in early pregnancy decidua using dual labeling immunoelectron microscopy, immunoflow cytometry, and cell fractionation. Lymphocyte subpopulations were, in addition, analyzed for expression of the cytokine receptor for IL-7 and c-kit and for mRNA expression of recombinase-activating genes 1 and 2. Four different cell populations could be distinguished: CD56+bright, CD56+dim/TCR gamma delta+low, CD56+dim/TCR gamma delta+high, and TCR gamma delta+low. Recombinase-activating genes 1 and 2 were expressed in the CD56+bright cells and to a limited degree in CD56+dim/TCR gamma delta+low cells. c-kit was preferentially expressed on the CD56+bright cells, while IL-7R was preferentially expressed on CD56+dim/TCR gamma delta+low and CD56+dim/TCR gamma delta+high cells. The CD56+dim TCR gamma delta+low and CD56+dim/TCR gamma delta+high cells displayed the characteristic morphology of large granular lymphocytes, while single positive TCR gamma delta+low cells were usually smaller and did not contain cytoplasmic granules. The gamma delta 1 gene segment was almost exclusively used in the TCR. Gamma delta T cells in mitosis were seen. We suggest that human early pregnancy decidua is a transient site for extrathymic maturation and that the progenitors of TCR gamma delta+ cells are bone marrow-derived immature cells expressing the CD56 (neural cell adhesion molecule) homing receptor.
The immune compromise in decidua allows a semiallogeneic fetus to survive without impairing the ability of the maternal immune system to fight infections. Cytotoxic mechanisms are likely to be important in this compromise. Using RT-PCR, immunoflow cytometry and immunoelectron microscopy, the cytotoxic potential of isolated human decidual gammadelta T cells was studied. mRNA for perforin (Pf), granzymes A and B, granulysin and Fas ligand (FasL) was simultaneously expressed in decidual gammadelta T cells. Pf and FasL were not expressed on the cell surface. However, the cells constitutively synthesized Pf and stored it in cytolytic granules. Within the granules Pf mainly resided in the granule core formed by Pf-containing microvesicles. Ultrastructurally, three groups of Pf-containing granules were distinguished. They probably represent different stages of granule maturation in a process where Pf-containing microvesicles first attach to the core cortex and then are translocated across the cortex into the core. Presynthesized FasL was also stored in the core and microvesicles of the cytolytic granules. Upon degranulation by ionomycin/Ca(2+) treatment, FasL was rapidly translocated to the cell surface, demonstrating that its surface expression was not controlled by de novo biosynthesis. Thus decidual gammadelta T cells appear to perform Pf- and FasL-mediated cytotoxicity utilizing a common secretory mechanism based on cytolytic granule exocytosis. The first cytochemical visualization of lipids in the cytolytic granules is provided. These intragranular lipids probably wrap up the core and participate in packaging of the cytotoxic proteins as well as in the killing process. An ultrastructural model of a cytolytic granule is presented.
OBJECTIVES: To investigate if changes in the national Swedish vaccination program coincided with changes in the celiac disease (CD) incidence rate in infants (ie, the Swedish CD Epidemic), and to assess the potential association between these vaccinations and CD risk.
METHODS: All studies were based on the National Swedish Childhood Celiac Disease Register. Using an ecological approach, we plotted changes over time in the national vaccination program in the graph displaying CD incidence rate. A population-based incident case-referent study of invited infants was performed. Exposure information was received through a questionnaire and child health clinic records. Vaccines explored were diphtheria/tetanus, pertussis (acellular), polio (inactivated), Haemophilus influenzae type b (conjugated), measles/mumps/rubella, and live attenuated bacillus Calmette-Guerin (BCG) in children with increased tuberculosis risk. Findings were subjected to a birth cohort analysis.
RESULTS: Introduction of pertussis vaccine coincided in time with decreasing CD incidence rates. In the infant case-referent study, however, neither vaccination against pertussis (odds ratio 0.91; 95% confidence interval 0.60-1.4), nor against Haemophilus influenzae type b or measles/mumps/rubella was associated with CD. Coverage for the diphtheria/tetanus and polio vaccines was 99%. BCG was associated with reduced risk for CD (adjusted odds ratio 0.54; 95% confidence interval 0.31-0.94). Discontinuation of general BCG vaccination did not affect the cumulative incidence of CD at age 15 years.
CONCLUSIONS: Early vaccinations within the national Swedish program were not associated with CD risk, nor could changes in the program explain the Swedish epidemic. A protective effect by BCG was suggested, which could be subject to further studies. Pediatrics 2012;130:e63-e70
BACKGROUND: Celiac disease is defined as a 'chronic small intestinal immune-mediated enteropathy precipitated by exposure to dietary gluten in genetically predisposed individuals'. Sweden has experienced an "epidemic" of celiac disease in children below two years of age. Celiac disease etiology is considered multifactorial; however, little is known regarding potential risk- or protecting factors. We present data on the possible association between early infectious episodes and celiac disease, including their possible contribution to the Swedish celiac disease epidemic.
METHODS: A population-based incident case-referent study (475 cases, 950 referents) with exposure information obtained via a questionnaire (including family characteristics, infant feeding, and the child's general health) was performed. Celiac disease cases were diagnosed before two years of age, fulfilling the diagnostic criteria of the European Society for Pediatric Gastroenterology, Hepatology and Nutrition. Referents were randomly selected from the national population register after fulfilling matching criteria. The final analyses included 954 children, 373 (79%) cases and 581 (61%) referents, with complete information on main variables of interest in a matched set of one case with one or two referents.
RESULTS: Having three or more parental-reported infectious episodes, regardless of type of infection, during the first six months of life was associated with a significantly increased risk for later celiac disease, and this remained after adjusting for infant feeding and socioeconomic status (odds ratio [OR] 1.5; 95% confidence interval [CI], 1.1-2.0; P=0.014). The celiac disease risk increased synergistically if, in addition to having several infectious episodes, infants were introduced to dietary gluten in large amounts, compared to small or medium amounts, after breastfeeding was discontinued (OR 5.6; 95% CI, 3.1-10; P<0.001).
CONCLUSION: This study suggests that having repeated infectious episodes early in life increases the risk for later celiac disease. In addition, we found a synergistic effect between early infections and daily amount of gluten intake, more pronounced among infants for whom breastfeeding had been discontinued prior to gluten introduction. Regarding contribution to the Swedish celiac disease epidemic, which partly was attributed to concurrent changes in infant feeding, early infections probably made a minor contribution via the synergistic effect with gluten amount.
The binding specificities of 52 well-characterized monoclonal antibodies (Mabs) against carcinoembryonic antigen (CEA) from 12 different research groups were studied by immunohistochemistry and immuno flow cytometry. In addition, the binding constant for the interaction between Mab and CEA was determined by a solution-phase assay. Cryostat sections of colon carcinoma and normal colon, stomach, liver, pancreas, and spleen were studied by immunohistochemistry. Peripheral blood granulocytes, monocytes, and lymphocytes were assayed by immuno flow cytometry. The Mabs used here have previously been classified into five essentially nonoverlapping epitope groups (GOLD 1-5) (Cancer Res., 49: 4852-4858, 1989). Most Mabs cross-reacted with different normal tissues, ranging from highly cross-reactive Mabs (positive reaction with 8 of 9 discriminating tissues) to relatively specific Mabs (positive reaction with 1 of 9 discriminating tissues). Five Mabs (10%) were specific, reacting only with colon carcinoma, normal colon mucosa, and normal gastric foveola. There was a correlation between epitope group and binding specificity. Mabs with a high degree of CEA specificity almost exclusively belonged to epitope groups 1, 2, and 3, while highly cross-reactive Mabs belonged to epitope groups 4 and 5. There was no correlation between antibody specificity and affinity for CEA. Specific Mabs with high as well as low affinity were found.
Accurate identification of lymph node involvement is critical for successful treatment of patients with colorectal carcinoma (CRC). Real-time quantitative RT–PCR with a specific probe and RNA copy standard for biomarker mRNA has proven very powerful for detection of disseminated tumour cells. Which properties of biomarker mRNAs are important for identification of disseminated CRC cells? Seven biomarker candidates, CEA, CEACAM1-S/L, CEACAM6, CEACAM7-1/2, MUC2, MMP7 and CK20, were compared in a test-set of lymph nodes from 51 CRC patients (Dukes' A–D) and 10 controls. Normal colon epithelial cells, primary tumours, and different immune cells were also analysed. The biomarkers were ranked according to: (1) detection of haematoxylin/eosin positive nodes, (2) detection of Dukes' A and B patients, who developed metastases during a 54 months follow-up period and (3) identification of patients with Dukes' C and D tumours using the highest value of control nodes as cutoff. The following properties appear to be of importance; (a) no expression in immune cells, (b) relatively high and constant expression in tumour tissue irrespective of Dukes' stage and (c) no or weak downregulation in tumours compared to normal tissue. CEA fulfilled these criteria best, followed by CK20 and MUC2.
Background: The novel chemokine CXCL17 acts as chemoattractant for monocytes, macrophages and dendritic cells. CXCL17 also has a role in angiogenesis of importance for tumour development. Methods: Expression of CXCL17, CXCL10, CXCL9 and CCL2 was assessed in primary colon cancer tumours, colon carcinoma cell lines and normal colon tissue at mRNA and protein levels by real-time qRT-PCR, immunohistochemistry, two-colour immunofluorescence and immunomorphometry. Results: CXCL17 mRNA was expressed at 8000 times higher levels in primary tumours than in normal colon (P<0.0001). CXCL17 protein was seen in 17.2% of cells in tumours as compared with 0.07% in normal colon (P = 0.0002). CXCL10, CXCL9 and CCL2 mRNAs were elevated in tumours but did not reach the levels of CXCL17. CXCL17 and CCL2 mRNA levels were significantly correlated in tumours. Concordant with the mRNA results, CXCL10-and CXCL9-positive cells were detected in tumour tissue, but at significantly lower numbers than CXCL17. Two-colour immunofluorescence and single-colour staining of consecutive sections for CXCL17 and the epithelial cell markers carcinoembryonic antigen and BerEP4 demonstrated that colon carcinoma tumour cells indeed expressed CXCL17. Conclusions: CXCL17 is ectopically expressed in primary colon cancer tumours. As CXCL17 enhances angiogenesis and attracts immune cells, its expression could be informative for prognosis in colon cancer patients.
Colorectal cancer is ranked third in worldwide incidence for women and fourth for men representing ͌ 9% of the world cancer or approximately 1 million new cases for 2002 (Parkin et al., 2005). Two thirds of colorectal cancers are located in the colon and one third in the rectum. At diagnosis approximately one third of all patients with colorectal cancer has lymph node positive disease, one third has lymph node-negative disease, and one third has distant metas-tases (Benson et al., 2004). The principal curative treatment for colorectal cancer is surgery. Adjuvant chemotherapy given to lymph node positive colon cancer patients has been shown to increase the survival rate (Haydon, 2003). In rectal cancer patients preoperative irradiation therapy is given to reduce local recurrences and has also been shown to improve survival (Folkesson et al., 2005). Still with these improved treatment modalities only approximately half the number of patients will survive for 5 years. For example, Swedish results for the time period 1995–1999 show a 5-years relative survival of ≈ 57% for both genders (Birgisson et al., 2005).
Tumor stage, based on histopathologi-cal examination of the resected specimen, and perioperative findings predict survival. Relative 5-year survival in Dukes' A (T1-2N0M0, Stage I) is 90–95%, Dukes' B (T3-4N0M0, Stage II) 60–80%, Dukes' C (anyTN1-2M0, Stage III) 40–60% and Dukes' D (anyTN0-2M1, Stage IV) < 5% (Staib et al., 2002). Besides distant metas-tases the most important prognostic indicator is the status of the regional lymphatic field showing presence or absence of tumor cells in regional lymph nodes. Given the importance of correctly identifying Dukes' C patients, i.e., patients with lymph node involvement who are eligible for chemotherapy, we have focused on improving the methods for detecting disseminated tumor cells in regional lymph nodes.
The aim was to explore the utility for staging and prognostic impact of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20), guanylyl cyclase C (GCC), CUB domain protein 1 (CDCP1) and mucin 2 (MUC2) mRNA levels in mesenteric lymph nodes of colorectal cancer (CRC) patients. Lymph nodes were collected at surgery and bisected; one half was subjected to biomarker mRNA analysis using real-time quantitative RT-PCR and the other half to routine histopathology. Lymph nodes from 174 CRC patients and 24 controls were analyzed. The median follow-up time was 59 (range 17-131) months. Cut-off levels were defined by analyzing quintiles by Cox regression model. CEA mRNA showed the best discriminating power between patients with recurrence in CRC after surgery and patients who were apparently disease-free (P=0.015). The risk of recurrence for the CEA(+) patients was 4.6 times greater than for the CEA(-) patients (P<0.0001). The other biomarkers gave lower hazard ratios. Cumulative survival analysis demonstrated that the average survival time was 99 months for CEA(-) patients compared to 39 months for CEA(+) patients, a difference of 60 months (P<0.0001). Six to nine percent of the stage I and II patients [H&E(-)] had CEA(+), CK20(+), GCC(+) and/or MUC2(+) lymph nodes. Two of these patients died from recurrent CRC. Low lymph node MUC2/CEA mRNA ratio identified patients with high risk for recurrence (P=0.011). Thus, qRT-PCR of CEA mRNA is a sensitive method to identify tumor cells in lymph nodes of CRC patients and, in combination with MUC2 mRNA, allows improved prediction of clinical outcome. © 2011 Wiley-Liss, Inc.
Background:A most important characteristic feature for poor prognosis in colorectal cancer (CRC) is the presence of lymph node metastasis. Determination of carcinoembryonic antigen (CEA) mRNA levels in lymph nodes has proven powerful for quantification of disseminated tumour cells. Here, we investigate the utility of human tissue kallikrein-related peptidase 6 (KLK6) mRNA as a progression biomarker to complement CEA mRNA, for improved selection of patients in need of adjuvant therapy and intensified follow-up after surgery.Methods:Lymph nodes of pTNM stage I-IV CRC- (166 patients/503 lymph nodes) and control (23/108) patients were collected at surgery and analysed by quantitative RT-PCR.Results:Lymph node KLK6 positivity was an indicator of poor outcome (hazard ratio 3.7). Risk of recurrence and cancer death increased with KLK6 lymph node levels. Patients with KLK6 lymph node levels above the 90th percentile had a hazard ratio of 6.5 and 76 months shorter average survival time compared to patients with KLK6 negative nodes. The KLK6 positivity in lymph nodes with few tumour cells, that is, low CEA mRNA levels, also indicated poor prognosis (hazard ratio 2.8).Conclusion:In CRC patients, lymph node KLK6 positivity indicated presence of aggressive tumour cells associated with poor prognosis and high risk of tumour recurrence.
Using quantitative morphometric analysis of immunohistochemically stained tissue sections we compared hypertrophic obstructive adenoids (HOA, n = 10) from children without middle ear disease with chronically infected adenoids (CIA, n = 10) from children with middle ear disease. gamma delta T cell receptor (TCR)+ cells constituted the dominating T cell population in the surface epithelium of HOA, while alpha beta TCR+ cells were the dominating intraepithelial T cell population in CIA. Intraepithelially CD8+ cells dominated over CD4+ cells in both diseases. Intraepithelially B cells were not detected. The cellular composition of follicles, with B cells dominating followed by activated CD4+ alpha beta TCR+ cells, was the same in both groups. However, the number of follicles in CIA was twice as many as in HOA. In the deeper interfollicular areas granulocytes were more abundant in CIA than in HOA. The latter two findings suggest a more pronounced inflammatory response in the adenoids of patients with middle ear disease. There was no significant difference with regard to pathogenic bacterial strains colonizing the adenoid surface when comparing the two patient groups. These results suggest that in patients with HOA gamma delta TCR+ T cells help to maintain the integrity of the surface epithelium, thereby preserving its protective function. On the basis of our results we speculate that CIA have a malfunctioning defence, thereby facilitating long-standing infections deep in the adenoid. This may be the main reason for development of middle ear disease and an indication for adenoidectomy in patients with CIA.


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The UK's airspace needs to be better organised by the government in order to increase the country's capacity.
Speaking at a reception at the Houses of Parliament organised by trade body Airlines UK, Airport Operators Association chairman Ed Anderson said better management of the airspace would help the country.
He added: "Our airspace needs to be modernised to enable the full use of all the technological advantages that these (modern) aircraft offer and that requires government leadership."
Anderson also used the speech to again demand the end of Air Passenger Duty which he believes is hampering the UK's route development.
He added: "Many more destinations could be unlocked if we weren't charging more than double the amount of tax than some of our neighbouring countries."
Anderson said increased connectivity was vital for the country, pointing out that for every 10% increase in aviation connectivity, the UK's GDP grows by 0.5%.
He added the British public's appetite for flying was also growing with more than half the population having taken a flight in 2017, a 10% increase since 2011.
His comments were supported by Airlines UK chairman Jane Middleton who also welcomed the recent decision by the government to build a third runway at Heathrow.
However, she warned against the expansion being used as an excuse to raise the cost of flying in and out of the west London airport.
She also urged the industry to continue focusing on remaining sustainable buy keeping noise and emissions to a minimum.
The government's aviation minister Baroness Sugg agreed the vote to expand the airport had come at the right time as Britain prepares for Brexit.
She added: "Heathrow expansion will finally deliver much capacity at a time when air links are more needed than ever."
While she agreed that the growth was a positive thing, she added the caveat that it must always be within the limits of strict environmental standards.

subset: commoncrawl


text: I've been waking at 3,4am in pain and unable to get back to sleep. I took meds at 5am after flip flopping for a couple of hours which did help but the day is wasted and I now I feel just bleak. Tried to refill Plaqyinyl but pharmacist said the dr wants me in for a visit first. An expensive visit which I truly don't need at the moment. Last time I was there I complained about ever increasing joint pain in my hands and he gave me a handout for a special can opener and commented on my extreme prednisone sensitivity. I can find nifty gadgets on my own for less than $350 an hour. He didn't even see the incidental finding on my mri report. He's an okay guy really but I wish I had a dr I felt better about. I'm nervous about the thyroid biopsy Tuesday--if anyone has had I would appreciate knowing how it was. I don't know if this app was updated recently but I had to register like new a couple of days ago. I don't know why I feel so bleak, maybe it's holidays and preparing to be alone which isn't that horrible it's just being reminded of it every second. I'm going to try and walk around the block. Maybe I'll chase my tail.


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You don't need a reason to be romantic. Being romantic is surprising your beloved with a wonderful evening when he or she least expects it. Don't wait for birthdays and anniversaries to do something special.
While movie and dinner is the popular agenda for a date, try to be creative in coming up with ideas for a romantic evening. It doesn't have to be an expensive affair. The simple things in life are often those which give the greatest joy.
The moon and the stars
Nature has blessed us with many beautiful marvels to behold. Take advantage of a splendid full moon night by planning a romantic evening of gazing at the sky.
You can use a garden or the terrace of your home. Carry along some wine and food and enjoy the company of your loved one under the brightly lit skies.
Dinner for two
The dreamy quality of a candlelight dinner has never failed to charm couples in love. Add a unique touch to this tradition by having the dinner at the poolside or beach. Even the terrace of your own home can be transformed into a magical place with flowers, candles and soft music.
Dream destination
Take a break from your weekend tasks and go on a mini-vacation with your beloved. Select a place that you two have visited before and relive old memories.
Plan out fun activities that both of you will enjoy. Buy gifts for each other that you can treasure for a long time in memory of the time you spent together.
Dinner at home
Prepare your dinner together and have fun trying to follow a new recipe. Even if your efforts don't pay off, you will have had a good time in each other's company, laughing and talking.
When you truly feel the need to do something special for your sweetheart, you can think of many ideas for a romantic evening. Do your best to create a memorable evening for both of you.


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In Code Name Bella Dawn, we deal with money and in particular gold being used as a weapon of war. After all, South Africa has 74% of the world's gold reserves. It is a much more interesting topic than first realised. Like many others, I was concerned about "perpetual small wars" that benefit only the Merchants of Death and cause tremendous problems for the future generations. We love history in GMJ Books, we always go back in time to see what we can learn, and I made a few interesting discoveries. Did you know, for instance, that when Adolf Hitler seized Czechoslovakia he also got hold of their gold reserves and not only their tanks (excellent for the time - they formed a significant part of General Heinz Guderian's victorious panzer divisions in 1940) and formidable weapons industry? Yet, the gold bullion so stolen was in London, not in Czechoslovakia. The gold was kept in London to safeguard it against abuse. In later years, West Germany did the same, leaving their bullion outside Germany for safekeeping. After the 1940 defeats, all of the UK's and the British Empire's wealth including gold reserves went to Canada. The world's biggest gold producer, the Union of South Africa, had to give all her reserves to the US, a US Navy cruiser fetched the bullion in Cape Town, to help pay for the war. So did India, once the jewel in the British crown, she was abused to pay 1 million pounds a day in war loans to London during World War Two (1940 money, much more today, billions). That almost broke India financially besides sending hundreds of thousands of her well-trained soldiers to die and you must wonder for what? Sufficient to say that the Burma Theatre and to a lesser extent the North African and Italian Theatres would not have succeeded as they did without the tough lads of the Indian Army standing fast. They paid a terrible price for this loyalty. In Bengal, a State in India, starvation was taking place but still that arch liar, Winston Churchill to you, refused to stop food exports from Bengal. He showed his true racist colours by asking "if the famine was so horrible, why had Gandhi not yet died of starvation?" Leo Amery, the Indian Secretary of State bitterly replied he regarded that comment as "Hitler-like." A great insult but then the man was probably drunk, he often was during Cabinet meetings. What the Indians or Mr Gandhi said I don't know but we took note of it. It reminded us of the concentration camps on a much larger scale and showed what the Pommies will do to survive. They are terrible people to go to war with since they never fight alone and expect their friends to do the dying for them (yes, see the World War Two fatality statistics discussed in Code Name Caribbean, Angelique). A large portion of France's gold bullion was already kept on a small island in the West Indies (some say St Martinique) out of Hitler's reach and kept hostage there by the watching Royal Navy. Thus, you may say that the politicians of the day well knew the value of gold (or money) as a weapon of war or they would not have taken the above measures. Yet, Sir Montagu Norman, the Governor of the Bank of England, released all the Czechoslovakian gold to Nazi Germany on request. Winston S Churchill spoke out, bitterly but alone. In today's money (2016, when this book was published first), that gold was worth just over 100-million-pound Sterling. You may be sure that many British and Commonwealth soldiers died because of this. Code Name Bella Dawn is full of such historical examples to enjoy. Our conclusion is most definitive that money can be used as a weapon of war. We see it today with "blood diamonds" being used to fuel wars – something we deal with in a later book, Code Name Dawn. Currently, the fact that no Western country, not the USA, not the UK, not one, has the gold reserves to back their currencies and are totally overextended with loans to China, should worry you. This will be abused in any future war as it was abused before in history. What is also fascinating is that there are only two countries buying gold physically and at enormous quantities, China and Russia. We also know that since the 1990s China has been buying silver mines in Africa, a place where they are rapidly replacing America and often warned about in GMJ Books.
On a personal note, Code Name Bella Dawn was sabotaged as far as I am concerned by Amazon's Kindle Scout Program – let me explain. The author is invited to hand over a book that was never published before, the first chapter, about ten pages, are then placed on their website, your mates and whoever else votes on it (thus you do the advertising at your cost). If you can generate enough interest in your book, they give you a dreadfully bad contract, but money is money and even an author must eat at times. Fair enough, that is the system we are subjected to but you will note at Chapter 1, that I cited the following: –
"We have contained them," Barack Obama to ABC News in an interview broadcast to the US citizens and the world, talking about ISIS. "I don't think they're gaining strength."
The very next day, November 13, 2015, ISIS hit Paris with simultaneous bombing and shooting attacks, 130 civilians died as did the 7 terrorists. France responded decisively, counter attacking in no uncertain terms, there were no lines drawn in the sand. They are not interested in "containing" ISIS but terminating them. And so is Russia, a country much more often attacked by Muslim radicals than the USA or UK combined.
The above was surprisingly and outright unlawfully removed from the published manuscript. Note, you are never allowed to change a manuscript without the author's permission – you are breaking international copyright laws, end of legal arguments, that is the law. Nothing that you read in a book is not there for a reason. Let me explain as follows. When Hemingway wrote "The Old Man and the Sea" he rewrote the first sentence more than sixty times according to some reports. Those reading the short extract of this book would never have seen the above quote because it was dishonestly and illegally removed. Yet those that would have appreciated the quote, as I did, would be seen as GMJ readers (good conservative folk, all of them) and the ones most likely voting for the book. I worry about the USA, my late wife's people. Do you know that the crime of lying to the FBI, called at times in my books "The Clinton Whitewash Bureau" for obvious reasons, does not exist in any other country in the world? Not even the Apartheid Police, KGB or Nazi Gestapo had such powers to be abused. It is not a normal state of affairs, and it is abused by Deep State against whoever they dislike for personal / political reasons. When freedom of speech is interfered with, you are on the road to doom, I cannot really express to you how worrisome this is. I asked Kindle Scout in writing why. Why was that specific quote removed? I am still waiting for an adequate answer and ran into red tape designed to shrug the answer off. Mysteriously, my other Kindle Scout book, Code Name Casselberry, GMJ 18, was not subjected to the same censorship but also did not have a quote like that on the first page – the quote there was published. There is no way that I will believe that it was by chance. No way, but you make up your own mind. I assure you that since then I have never supported Kindle Scout again and advised every other conservative author to stay away. It is stupid, you may suppress the quotes but that alone does not change history one tiny bit. The quoted words were spoken by Obama, the terrorist attacks did take place the very next day. The man looked like an utter fool and so did America because he spoke for his country, end of story. He was in my opinion the worst disaster for the War on Terror ever, the only question remaining is, why did he act the way he did? That I cannot answer yet.
Description: Spymaster extraordinaire, Angelique Dawson and her team uncovered a plot involving an unholy alliance between white supremacists and Muslim radicals. Oddly, the Muslim radicals are protected by counter-espionage intelligence officials inside South Africa. It is clear that the white supremacists somehow found the lost Transvaal Republic's Kruger millions. They are using the gold to pay for weapons and training. She tracks them down to an isolated game ranch right on the edge of the Zimbabwean border with South Africa, a place that is being used as an entrance point by the radicals, coming and going unofficially. Against her future husband and longtime bodyguard, former Police Special Forces Company Commander, Geoffrey Foxtrot's, advice, Angelique infiltrates the place and is captured with Foxtrot. The white supremacists threaten to hand her over to the Zimbabweans, a sure and slow death by torture awaits them if they cannot escape. Angelique makes one of the best speeches of her life regarding right-wing terrorism in South Africa... If you wish to read about Covert and Special Forces Operations in sub-Saharan Africa, the GMJ Books are the place to start. You will learn about Special Forces techniques and military history not known outside the select few. Code Name Bella Dawn is the nineteenth book of the popular GMJ Series.
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"Matière première" desktop accessories
Chesnut tree
3 logs of 26 x 4 cm(diam.)
This desk set referring to stacks of forestry in the Pyrenees is composed of three logs: a card holder (2 slots), a pencil box, a magnet log for paper clips and other metal elements.
Series of pull toys representing 4 of the emblematic figures of the Pyrenean fauna bears, marmots, wolves and chamois.
Born from the collaboration between Richard Fournier, a carpenter and Emilie Cazin, designer, The "Pyrene" collection is made in Midi-Pyrénées (South of France) and aims to promote the wood industry of the Pyrenean mountains.
It represents few iconic images of this relief as bears, marmots, markings of hiking trails or stacking of tree trunks logging … to give a second life to loss pieces from the workshop. Types of wood used evolves depending of what is done in the workshop at that time. An other way to highlight the uniqueness of the pieces.


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|Paul Carrack
|Paul has had a brilliant career and it was nice to talk with the guy who created some
of my favourite hits including HOW LONG by his band ACE 28th May 2008
|Career Overview The 1970s: Warm Dust to Ace to Roxy Music Carrack's
recording career began with the progressive rock band Warm Dust, who released
3 albums of original material between 1970 and 1972. Carrack was the keyboard
player for Warm Dust, and occasionally played other instruments as well, but he
was not the band's lead vocalist. The lead vocalist was Les Walker. After Warm
Dust broke up, Carrack and Warm Dust bassist Tex Comer helped to found the
pub rock band Ace. Carrack wrote and sang "How Long", Ace's debut single and
a hit on both sides of the Atlantic in 1975. However, no further Ace recordings
caught on. In 1977 Ace disbanded and Carrack began working as a backing
musician for Frankie Miller. This was followed by a turn as a member of Roxy
Music, playing keyboards on their 1979 reunion album Manifesto. Carrack would
also play on the next two Roxy Music albums, but as a session musician, not a
band member. The 1980s: Squeezing a solo career around other band
commitments The band Squeeze during its East Side Story period. L-R: John
Bentley, Glenn Tilbrook, Paul Carrack, Gilson Lavis, Chris DiffordShortly after
leaving Roxy Music full time, Carrack issued his first solo album, 1980s Nightbird.
It was not a commercial success. 1n 1981, Glenn Tilbrook recruited Carrack to
join Squeeze as a replacement for long-time keyboardist Jools Holland. This new
Squeeze line-up achieved international success with the album East Side Story, on
which Carrack sang their biggest US hit to that time, "Tempted." But by 1982,
Carrack had left the band, and had been replaced in Squeeze by keyboardist Don
Snow. Beginning in late 1981, Carrack had joined up with Nick Lowe in a band
that featured Lowe, Carrack, Martin Belmont on guitar, James Eller on bass and
Bobby Irwin on drums. This band, referred to as Noise To Go, existed to back
both Carrack on his solo recordings, and Lowe on his. (This was similar to the
arrangement Lowe had with Dave Edmunds and Rockpile in the late 1970s.)
"Noise To Go" also backed Lowe's wife Carlene Carter on her 1981 album Blue
Nun. After Carrack's 1982 solo album Suburban Voodoo, Eller left Noise To Go,
and Nick Lowe took over bass duties within the group. The group was
rechristened Nick Lowe and His Cowboy Outfit, and recorded two albums from
1983 to 1985, with Lowe handling all the lead vocals. They also played behind
John Hiatt on side two of Hiatt's 1983 album Riding With The King. During this
era, Carrack also kept himself busy with session work for The Smiths and The
Pretenders. Nick Lowe and His Cowboy Outfit dissolved in 1985, but around the
same time, Carrack was contacted by Mike Rutherford (of Genesis) and asked to
join Rutherford's new side project, Mike + The Mechanics. Initially, Carrack was
one of two alternating lead vocalists for the group. He sang lead on their 1985 hit
"Silent Running (On Dangerous Ground)". In 1986, Carrack was a member of
Roger Waters' newly-formed backing band, "The Bleeding Hearts Band",
recording tracks for the soundtrack of the film When the Wind Blows. The
following year, Waters (still accompanied by Carrack and the Bleeding Hearts)
released a solo album, Radio KAOS, and toured the US and Europe. Carrack
often filled in as an unofficial support act on the tour, usually playing "Tempted" to
warm up the crowd. Leveraging the success of Mike + The Mechanics, Carrack
was able to reestablish his solo career in 1987 with the hit album One Good
Reason, and the accompanying hit single "Don't Shed A Tear", which reached No.
9 on the Billboard Hot 100. From this point forward, Carrack usually managed to
maintain both a solo career and a career in Mike + The Mechanics, a band which
recorded and toured irregularly due to Rutherford's commitments to Genesis. In
1989, Mike + The Mechanics had a UK No. 2 and US No. 1 hit with the "The
Living Years", on which Carrack again sang lead. Also in 1989, Carrack recorded
a duet with Terri Nunn from the Los Angeles-based synth-pop band Berlin entitled
"Romance". The song appeared on the soundtrack to the movie Sing, starring Peter
Dobson and Lorraine Bracco. The 1990s: Sessions, solo work, Squeeze (again)
and Spin 1ne 2wo In the 1990s, Carrack's solo career went into abeyance for a
few years, although he maintained a career as a session musician for hire. He also
continued working with Mike + The Mechanics, who continued to record through
the 1990s. During this era, Carrack assumed keyboard duties within the band, and
also began to co-compose some of the band's songs with Rutherford. In 1990,
Carrack was recruited to perform in the groundbreaking live stage show of The
Wall in Berlin by Roger Waters. He performed "Hey You" to the crowd of
250,000. In 1993, Carrack joined with bassist Tony Levin, drummer Steve
Ferrone, guitarist Phil Palmer and keyboardist/producer Rupert Hine to form a
band known as Spin 1ne 2wo. They released one album, a self-titled project,
made up of classic rock covers including songs by Jimi Hendrix, The Who, Led
Zeppelin, Blind Faith, Steely Dan and Bob Dylan. Also in 1993, Squeeze had
Carrack return for their album Some Fantastic Place and a subsequent tour. With
Carrack on vocals, the band re-recorded their hit "Tempted" for the soundtrack to
the movie "Reality Bites" (1994). However, Carrack once again left Squeeze after
about a year with the band. Following his second stint with Squeeze, Carrack
joined forces with Timothy B. Schmit and Don Felder of The Eagles for an
ambitious but ultimately unrealized recording project. Schmit and Felder soon
reunited with the rest of the Eagles and their Hell Freezes Over album, bringing
with them one of the songs Carrack co-wrote, "Love Will Keep Us Alive." It was
recorded by The Eagles, and won an award as being the most-played single in
America in 1995. Finally in 1996, Carrack resumed his solo career with the album
Blue Views. [edit] Into the 21st century Carrack maintains his active solo career,
as well as his career as a session musician and songwriter.Paul toured solo in 2002,
mostly opening for bands such as Supertramp. He also became Mike + The
Mechanics' only lead vocalist after the death of Paul Young in 2000. In 2004, the
band was officially re-named Mike + The Mechanics & Paul Carrack. After a
period of comparative quiet in Carrack's career, in 2007 The Eagles covered
Carrack's "I Don't Want To Hear Anymore" on their long-awaited reunion album
Long Road Out Of Eden. The album went to #1 in the US, UK, Australia, New
Zealand and several other countries around the world. [edit] Session Work
Carrack's distinctive voice and keyboard skills have kept him in demand as a
session musician on many projects. Some of his credits include: Keyboards on The
Smiths' eponymous debut album, The Smiths (1984) Piano on The Pretenders
album Learning To Crawl (1983) Keys and vocals on Roger Waters' "Radio
K.A.O.S." (1987) and subsequent tour Organ on Elton John's Made in England
(1996) and The Big Picture (1997) Organ and vocals on B.B. King's Deuces Wild
(1997) Touring with Ringo Starr's All-Starr Band (2003) In 1997, Carrack played
organ for Elton John on the single "Something About The Way You Look Tonight".
Coupled with "Candle In The Wind '97", this became the best selling single of all
time. [edit] Discography


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DPMP leads AOD treatment review
A review of Alcohol and Other Drug (AOD) treatment services being conducted by the Drug Policy Modelling Program at NDARC has interviewed around 300 sector representatives in eight states and territories since it began in July 2013. Eight consultation papers have been published to date and are available for comment on the DPMP website. The review is being carried out on behalf of the federal Department of Health and is due to report later this year.
Significant findings to date include:
- There are 1.6 million AOD treatment contacts, episodes of care or encounters accessed by around 200,000 individuals. Working Paper Number 8.
- Australia spends around $1.1 billion a year on alcohol and other drug (AOD) treatment. Working Paper Number 7.
- Pay for performance schemes (P4P) which have been widely introduced in the UK and US should be regarded as experimental. The authors warn that P4P is not a quick fix for healthcare or alcohol and other drug treatment systems and if it is introduced in Australia it should be seen as an experiment rather than an adoption of a proven method. For more information and to comment see Working Paper Number 5.
- A true estimate of the extent of current provision of AOD treatment in Australia is challenging because of the existence of unrecorded or hard to count treatment episodes which are not recorded in current administrative data sets. The authors identify eleven hard to count categories including telephone and internet/computer based interventions; self‐help; other AOD medical treatment; services provided in education settings; workplace treatment interventions; services provided as part of research trials; and complementary/alternative treatments. Working Paper Number 6.
- Overcoming barriers to the utilisation of AOD services is crucial in achieving equitable outcomes for Australians living in rural and remote areas. While there is not a 'one size fits all' service model a systematic approach is needed to address reform of the AOD service system in rural and remote Australia. Working Paper Number 2.
For full details of the project, the six working papers and responses published to date, please visit the project website.


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A Virginia Tech offer wasn't in the cards for Brock Hoffman a couple of years ago. Hoffman turned heads at a Hokies camp during his high school recruitment, but as an early enrollee the timing for a scholarship wasn't quite right. However, Vance Vice never lost track of him, and in the end he will strap it up for Virginia Tech. Hoffman announced his transfer from Coastal Carolina to Virginia Tech on Friday.
"Once summer comes, [Vice] wants me to hit the ground running," said Hoffman.
A trio of reasons factored into the move: CC head coach Joe Moglia stepped down, his desire to compete against NFL talent, and primarily to move closer to and care for his mom. The latter point is the thesis for the waiver to gain immediate eligibility. Blacksburg is about 2 hours closer to the Statesville, NC native's home than Conway, SC.
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Farbridge (Visit website)
It's your day, your choice and Farbridge is exclusively your venue. Set in rural landscape in the Sussex Downs, you create your perfect occasion to suit your style in beautifully restored barns designed specifically for wedding receptions and ceremonies for 60-150 guests. With modern facilities and 10 contemporary bedrooms, Farbridge is a flexible venue, ideal for weddings all year round.
Special Offer
2014 Special Offer
£1,000 discount off venue hire in 2014
Special Offer: Farbridge Mar 2 2010 12:36PM
To check available dates, please call us on 01243 776776
* Offer not available to previously confirmed bookings or transfers of date.
Wedding Services:
Business Name: Farbridge
Business Address: Farbridge, Preston Farm, West Dean, Chichester, PO18 0JT
View Location Map
Website: http://www.farbridge.org.uk
Telephone: 01243776776
Send an email to Farbridge


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Claiming Tax Rebate in the UK
As the economic crisis deepens and uncertainty grows, sorting out taxes becomes even more important. Identifying any overpaid taxes may save you hundreds of pounds and may soften the blow of unemployment. You may be due a tax rebate if:
- You have worked on the wrong tax code
- You have not worked the full tax year (worked only casually as a student or been made redundant)
- You have other income taxed through your tax code
- Your tax-related circumstances have changed
If you paid tax through PAYE and are no longer employed, it is likely that you could reclaim some of your income tax. If you are a self-employed worker and your earnings have lowered, you can apply for lower payment than usual. Getting professional help can maximize your tax savings and ensure that your taxes are filed in compliance with all current regulations.


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Widespread flooding in the region forces hundreds of families from their homes just days after historic blackout.
Three thousand people in Uruguay and 250 people in Entre Ríos were forced from their homes on Monday amid intense rainfall and flooding. Twelve national highways in Uruguay were also cut off.
Uruguay and Argentina on Sunday suffered a historic blackout which left most of both countries without electricity for most of the day.
In Rosario del Tala in Entre Ríos province, dozens of families were evacuated on Monday and Tuesday with the assistance of provincial authorities, the Army and volunteer fire fighters.
A weather alert was in place in Buenos Aires City, including warnings of hail.
Entre Ríos Governor Gustavo Bordet told Diario Uno newspaper that evacuees were receiving assistance from the provincial government.
In Uruguay, authorities were focusing on "mitigating" the impact of "leaving one's home and seeing their belongings damaged", Presidential Secretary Juan Andrés Roballo told El País de Montevideo.
Buenos Aires City officials warned residents on Monday to avoid walking in flooded streets and leaving home amid strong winds, as well as leaving objects on balconies and terraces.
Residents are also advised against "touching cables or electricity supplies in the street", the government urged.
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Top 3 beach holiday destinations in PNG
When you think of PNG is it culture, thick jungles and trekking adventures that spring to mind? PNG is actually an island destination with over 600 islands of largely untouched beauty.BY KARRYON: With some of the
When you think of PNG is it culture, thick jungles and trekking adventures that spring to mind? PNG is actually an island destination with over 600 islands of largely untouched beauty.
BY KARRYON: With some of the most spectacular diving spots, lively marine life, impressive fishing and superb surfing, PNG is one of the world's most undiscovered coastal holiday destinations.
So where to go and stay and what to do when you get there? We've listed three of the best coastal holiday locations in PNG:
1. Milne Bay Province – For History Buffs
Located at the bottom of the main island of PNG to the east, is Milne Bay Province, home to seven different groups of islands with a fascinating war history, (the Battle of Milne Bay was the first land defeat suffered by the Japanese in World War II) and unbeatable diving of un-disturbed reefs and sunken ships – Milne Bay is one of the country's best spots for coastal adventures.
Snorkel with manta-rays, go dolphin and dugong spotting or take in the annual canoe festival in November each year.
Stay at Driftwood Resort or for a more authentic and remote stay, try Nuli Sapi on the island of Logeia.
2. Rabaul/Kokopo – For Scuba Divers + Snorkellers
Famous for the volcanic eruption in 1994 which covered the town of Rabaul in ash and forced many to move to the neighbouring town of Kokopo, the area offers turquoise waters lapping at white sandy beaches of tiny islands; practically guaranteed dolphin spotting – by the dozen; wreck diving and plenty of spots for a snorkel or swim.
Stay at Rapopo Plantation Resort or Kokopo Beach Bungalows.
3. Madang – For Surfers
Keen surfer? Grab your board and head north on the main island of PNG, to the area known as Madang. Set on a peninsula, with a backdrop of steep mountains and lush tropical vegetation, the town of Madang looks across a scattering of perfect islands, dotted through a tropical deep water harbour.
Not far north of the town of Madang is Ulingan Bay, one of the most consistent swell magnets in PNG, with waves straight out the front, great culture and awesome food. For divers, the deep harbour is home to over 30 sunken war ships just waiting to be discovered. Stay Tupira Surf Club for a great surf trip or Madang Resort, for a relaxing break.
This article was brought to you by Papua New Guinea Tourism