Proton pump inhibitor use is associated with an increased risk for microscopic colitis: a case-control study.

BACKGROUND: Microscopic colitis causes chronic watery diarrhoea. Recent studies have suggested an aetiological role for various medications, including proton pump inhibitors, in the pathogenesis of microscopic colitis. AIM: To determine whether an association exists between microscopic colitis and proton pump inhibitor use in patients with documented microscopic colitis vs. age- and gender-matched controls. METHODS: In this retrospective case-control study, cases of microscopic colitis from a secondary and tertiary referral medical centre diagnosed in the last 5 years were reviewed. Demographic characteristics, clinical, histological and endoscopic records, as well as exposure to PPIs and NSAIDs were assessed. Controls from the population were matched to cases by gender and by age. RESULTS: During the investigated period, 136 cases were identified in both hospitals. Of these, 95 cases of microscopic colitis were retrieved for detailed analysis. Exposure to proton pump inhibitors at the time of the histological diagnosis was significantly higher in patients with collagenous colitis than in controls [38% vs. 13%, P < 0.001; adjusted OR of 4.5 (95% CI 2.0-9.5)]. CONCLUSIONS: This observation confirms the presumed association between microscopic colitis and PPI use, and it supports the possible aetiological role of PPI exposure in the development of microscopic colitis.

Intervention with educational outreach at large scale to reduce antibiotics for respiratory tract infections: a controlled before and after study.

BACKGROUND: A multiple intervention targeted to reduce antibiotic prescribing with an educational outreach programme had proven to be effective in a randomized controlled trial in 12 peer review groups, demonstrating 12% less prescriptions for respiratory tract infections. OBJECTIVE: To assess the effectiveness of a multiple intervention in primary care at a large scale. METHODS: A controlled before and after study in 2006 and 2007 was designed. Participants were from general practices within a geographically defined area in the middle region of The Netherlands. Participants were GPs in 141 practices in 25 peer review groups. A control group of GP practices from the same region, matched for type of practice and mean volume of antibiotic prescribing. The multiple intervention consisted of the following elements: (i) group education meeting and communication training; (ii) monitoring and feedback on prescribing behaviour; (iii) group education for GPs and pharmacists assistants and (iv) patient education material. The main outcome measures are as follows: (i) number of antibiotic prescriptions per 1000 patients per GP and (ii) number of second-choice antibiotics, obtained from claims data from the regional health insurance company. The associations between predictors and outcome measurements were assessed by means of a multiple regression analyses. RESULTS: At baseline, the number of antibiotic prescriptions per 1000 patients was slightly higher in the intervention group than in the control group (184 versus 176). In 2007, the number of prescriptions had increased to 232 and 227, respectively, and not differed between intervention and control group. CONCLUSIONS: The implementation of an already proven effective multiple intervention strategy at a larger scale showed no reduction of antibiotic prescription rates. The failure might be attributed to a less tight monitoring of intervention and audit. Inserting practical tools in the intervention might be more successful and should be studied.

Serum-free transient protein production system based on adenoviral vector and PER.C6 technology: high yield and preserved bioactivity.

Stable E1 transformed cells, like PER.C6, are able to grow at scale and to high cell densities. E1-deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an DeltaE1/DeltaE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed DeltaE1/DeltaE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS-CoV), a gene coding for the SARS-CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS-CoV (SARS-AB). The DeltaE1/DeltaE2A-vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/cell) of DeltaE1/DeltaE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that DeltaE1/DeltaE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein.

The human cell line PER.C6 provides a new manufacturing system for the production of influenza vaccines.

Influenza viruses for vaccine production are currently grown on embryonated eggs. This manufacturing system conveys many major drawbacks such as inflexibility, cumbersome down stream processing, inability of some strains to replicate on eggs to high enough yields, and selection of receptor-binding variants with reduced antigenicity. These limitations emphasize the need for a cell line-based production system that could replace eggs in the production of influenza virus vaccines in a pandemic proof fashion. Here we present the efficient propagation of influenza A and B viruses on the fully characterized and standardized human cell line PER.C6.

The effect of promoter strength in adenoviral vectors in hyperplastic synovium.

OBJECTIVES: To compare the activity of the CytoMegaloVirus promoter (CMV) and the Major Late promoter (MLP) in synoviocytes in vitro and in vivo. To determine the phenotype of infected cells and the induction of inflammation. To investigate the effects of the cytomegalovirus (CMV) or major late (MLP) promoter on adenovirus-mediated reporter gene transduction of synoviocytes in vitro and in vivo. METHODS: After infection with adenoviral vectors harboring CMV- and MLP-driven luciferase and lacZ genes, gene expression was examined in cultured synoviocytes and in the synovium of rhesus monkeys with collagen-induced arthritis. Immunohistochemical staining for the macrophage-marker CD68 and lacZ expression was performed. Inflammation was scored in the synovial membrane of injected and non-injected joints. RESULTS: CMV-driven reporter gene expression was found to be 6 to 10 times higher than MLP-driven gene expression in both cultured synoviocytes and monkey synovium. Both CD68 positive and CD68 negative cells were lacZ positive. Inflammation in joints injected with CMV-driven adenoviral vectors was not higher than that in MLP-driven adenoviral vectors- or non-injected joints. CONCLUSION: These experiments show that the CMV promoter induces higher gene expression in synoviocytes than the MLP promoter. Both fibroblast-like and macrophage-like synoviocytes can be infected with adenoviral vectors. No deleterious effects of the CMV-promoter driven adenoviral vectors were observed.

Yoghurt enriched with Lactobacillus acidophilus does not lower blood lipids in healthy men and women with normal to borderline high serum cholesterol levels.

OBJECTIVE: To investigate whether intake of Lactobacillus acidophilus strain L-1 lowers serum cholesterol in healthy men and women. DESIGN: Randomised, placebo-controlled parallel trial. SETTING: Subjects were free-living. Blood sampling and distribution of yoghurts were administered at a local hospital. SUBJECTS: Seventy-eight adult men and women with cholesterol levels of 3.9-7.8 mmol/L (mean +/- s.d., 5.4+/-0.7). INTERVENTIONS: Subjects consumed 500 mL of control yoghurt daily for two weeks. They were then randomly allocated to receive 500 mL per day of control yoghurt or of yoghurt enriched with Lactobacillus acidophilus L-1 for another six weeks. The yoghurts were spiked with a trace of lithium; compliance as assessed by plasma lithium was excellent. RESULTS: Energy and nutrient intake was constant, and identical for the two groups. Mean body weight was stable. Baseline blood lipid concentrations in the control and treatment groups were highly similar. The effect of consumption of Lactobacillus acidophilus L-1 vs. control on total cholesterol was -0.02 mmol/L (95% CI, -0.18-0.15) after three weeks and 0.04 mmol/L (95% CI, -0.12-0.20) after six weeks. Serum LDL and HDL-cholesterol and triacylglycerol levels were also unaffected. CONCLUSIONS: Yoghurt enriched with Lactobacillus acidophilus L-1 does not lower serum cholesterol in men and women with normal to borderline high cholesterol levels.

Feasibility of adenovirus-mediated nonsurgical synovectomy in collagen-induced arthritis-affected rhesus monkeys.

Gene transfer to synovial tissue by adenoviral vectors (Ad) was studied in vitro in cultured human synoviocytes and in vivo in seven primates with arthritis. Hyperplastic synovium was efficiently transduced with Ad.lacZ in vitro and in vivo in rhesus monkeys with collagen-induced arthritis, whereas chondrocytes were not transduced. Intraarticular injection of recombinant Ad harboring the luciferase gene showed the presence of reporter gene products only in Ad-injected joints. In addition, the feasibility of synovectomy by Ad harboring the herpes simplex virus thymidine kinase gene (tk) was studied. In vitro infection of synovium from rheumatoid arthritis patients with Ad.TK, followed by administration of ganciclovir, resulted in death of >90% of the synoviocytes. By mixing Ad.TK-infected with noninfected cells, it appeared that the presence of 10% infected synoviocytes resulted in the killing of more than 85% of the synoviocytes, demonstrating a substantial bystander effect. Intraarticular injection of Ad.TK in the knees of rhesus monkeys with arthritis, followed by treatment with ganciclovir for 14 days, resulted in increased apoptotic cell death in the synovium of Ad.TK-injected as compared with noninjected joints and ablation of the synovial lining layer. The procedure revealed no toxic side effects. These data suggest that nonsurgical synovectomy by tK gene therapy is feasible.

Transposon Tc1 of the nematode Caenorhabditis elegans jumps in human cells.

The transposon Tc1 of the nematode Caenorhabditis elegans is a member of the widespread family of Tc1/mariner transposons. The distribution pattern of virtually identical transposons among insect species that diverged 200 million years ago suggested horizontal transfer of the elements between species. Thishypothesis gained experimental support when it was shown that Tc1 and later also mariner transposons could be made to jump in vitro , with their transposase as the only protein required. Later it was shown that mariner transposons from one fruit fly species can jump in other fruit fly species and in a protozoan and, recently, that a Tc1-like transposon from the nematode jumps in fish cells and that a fish Tc1-like transposon jumps in human cells. Here we show that the Tc1 element from the nematode jumps in human cells. This provides further support for the horizontal spread hypothesis. Furthermore, it suggests that Tc1 can be used as vehicle for DNA integration in human gene therapy.

IkappaB alpha is a target for the mitogen-activated 90 kDa ribosomal S6 kinase.

The activity of transcription factor NFkappaB is regulated by its subcellular localization. In most cell types, NFkappaB is sequestered in the cytoplasm due to binding of the inhibitory protein IkappaB alpha. Stimulation of cells with a wide variety of agents results in degradation of IkappaB alpha which allows translocation of NFkappaB to the nucleus. Degradation of IkappaB alpha is triggered by phosphorylation of two serine residues, i.e. Ser32 and Ser36, by as yet unknown kinases. Here we report that the mitogen-activated 90 kDa ribosomal S6 kinase (p90rsk1) is an IkappaB alpha kinase. p90rsk1 phosphorylates IkappaB alpha at Ser32 and it physically associates with IkappaB alpha in vivo. Moreover, when the function of p90rsk1 is impaired by expression of a dominant-negative mutant, IkappaB alpha degradation in response to mitogenic stimuli, e.g. 12-O-tetradecanoylphorbol 13-acetate (TPA), is inhibited. Finally, NFkappaB cannot be activated by TPA in cell lines that have low levels of p90rsk1. We conclude that p90rsk1 is an essential kinase required for phosphorylation and subsequent degradation of IkappaB alpha in response to mitogens.

Brightness constancy in a Ganzfeld environment.

In this paper, experiments are described in which brightness constancy was studied in a Ganzfeld environment. Luminance variation by means of neutral density filters was applied to stimuli consisting of a Ganzfeld with superimposed disks. To this end, a special-purpose apparatus was constructed. Sequential dichoptical brightness matches with a reference stimulus were carried out for the disks as well as the homogeneous surround. The results of these measurements indicate that (1) besides a clear tendency toward brightness constancy, small but systematic effects of the average luminance level are present and (2) the brightness of the Ganzfeld is hardly affected by the presence of the disks. Finally, it is shown that the experimental results can be modeled adequately in terms of a concept that involves an accumulation of contrast information.

Brightness indention: a novel compression mechanism in the luminance-brightness mapping.

In a previous study, Schouten and Blommaert (1995) explicitly showed that brightness constancy implies a substantial compression in the luminance-brightness mapping. Here it is argued that additional compression mechanisms are required for scenes with large luminance ranges. To trace these, a series of experiments was conducted which involved expansion of the luminance range. We used a simple spatial configuration consisting of a disk on a contourless homogeneous surround (Ganzfeld). The contrast between the disk and Ganzfeld acted as the independent variable, while the size of the disk was parametrically varied. Sequential dichoptical brightness matches with a reference were carried out for both the disk and the surround. The results reveal a compression mechanism which we term brightness indention. This indention, which has not previously been reported in the literature, only occurs if the Ganzfeld is less luminous than the disk, and it takes the form of a brightness decrease of the immediate surroundings of the disk.

Induction of differentiation-regulated transcription factor Oct-6 specifically accompanies major histocompatibility complex class I down-regulation by E1A of oncogenic adenovirus type 12.

The E1A genes from adenovirus (Ad) types 5 and 12 share the capacity to cooperate with a second oncogene to transform primary rodent cells in vitro. However, only Ad12-transformed cells are oncogenic in immunocompetent rodents, an event that requires conserved region 3 (CR3) of E1A to be intact. Ad12-induced tumorigenicity correlates with the E1A-CR3-dependent down-modulation of MHC class I transcription, contributing to escape from CTL-mediated immune surveillance. Expression of MHC class I antigens is also lacking in undifferentiated embryonal carcinoma cells. In these cells, MHC class I expression increases during differentiation in a process possibly involving octamer-binding proteins. We found that both nononcogenic and oncogenic Ad-transformed cells contained the ubiquitously expressed factor Oct-1. In contrast, only oncogenic Ad12-transformed cells that are derived from primary cell cultures expressed an additional octamer-binding factor, which we identified as Oct-6. The induction of Oct-6 expression was at the RNA level and was found to require an intact CR3 domain in Ad12 E1A. Like MHC class I expression, Oct-6 expression was not affected in already established cell lines expressing Ad12 E1A. The presence of Oct-6 in Ad12-transformed cells correlated with an increase in octamer-dependent transcription of a reporter gene, relative to Ad5-transformed cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Downregulation of MHC class I expression due to interference with p105-NF kappa B1 processing by Ad12E1A.

We have previously demonstrated that expression of major histocompatibility complex (MHC) class I genes is repressed in baby rat kidney cells transformed by early region 1 of oncogenic adenovirus type 12 (Ad12E1). Reduced expression of MHC class I antigens contributes to the escape of Ad12-transformed cells from T-cell-mediated immune surveillance and to tumour induction. In this study, we show that repression of MHC class I expression by Ad12E1A is mediated via the H2TF1 element of the MHC class I promoter. This element binds NF kappa B and KBF1, two factors which play a major role in the regulation of MHC class I expression in vivo. In extracts from Ad12E1-transformed cells, binding of KBF1 and NF kappa B to the H2TF1 element is decreased. This is caused by reduced production of p50-NF kappa B1, the 50 kDa subunit shared by KBF1 and NF kappa B, due to interference with p105-NF kappa B1 processing by Ad12-13S-E1A protein. Overexpression of the p105-NF kappa B1 cDNA, or of a truncated p105-NF kappa B1 cDNA that codes for p50-NF kappa B1, restores MHC class I expression in Ad12E1-transformed cells. These data demonstrate that downregulation of MHC class I expression in Ad12E1-transformed cells is due to interference with processing of p105-NF kappa B1 by the Ad12-13S-E1A protein.

Induction of the mitogen-activated p70 S6 kinase by adenovirus E1A.

Adenovirus E1A proteins can transform primary cells in culture in conjunction with other oncogenes, such as E1B or activated ras. The modulation of various cell cycle regulators by E1A is thought to be involved in this transformation process. In this paper we show that E1A enhances the expression of the mitogen-inducible p70 S6 kinase (p70s6k), a kinase which is essential for G1 progression. p70s6k mRNA and protein levels are enhanced 3-4-fold in various E1A-expressing cell lines. Similarly, the activity of p70s6k is enhanced in E1A-expressing cells in a manner partially independent of enhanced expression of p70s6k. The induction of p70s6k correlates with the presence of conserved region 1 (CR1) of E1A and with morphological transformation by E1A. These results suggest that induction of p70s6k by E1A might be involved in transformation by E1A.

Maturational gonadotropin from the African catfish, Clarias gariepinus: purification, characterization, localization, and biological activity.

A gonadotropic hormone of the African catfish, Clarias gariepinus, was was purified and chemically characterized. Its biological activity was tested and its localization in the gonadotropic cells of the pituitary demonstrated. An ethanolic extract of 500 pituitaries of adult male and female African catfish was subjected to ion-exchange chromatography on DE-52. The 31- to 38-kDa fraction was further purified on Sephadex G-75. On rpHPLC over an ODS 120T column two major components appeared as single bands after SDS-PAGE. From the amino acid composition and sequence analysis of these fractions, compared with those of salmon and carp GTH II-alpha and salmon GTH II-beta it was concluded that they represent catfish GTH alpha- and II-beta-subunits. The biological activity of the complete hormone (the 31- to 38-kDa fraction from the G-75 column) was tested on the production of 11 beta-hydroxyandrostenedione and 17 alpha-hydroxy-20 beta-dihydroprogesterone by catfish testis in vitro. Polyclonal antibodies were raised against the purified beta-subunit. Immunocytochemical study using these showed them to bind specifically to hypophysial gonadotropic cells. To date only one form of GTH has been demonstrated in the African catfish.

Identification of the cDNA clone which encodes the 58-kDa protein containing the amino acid sequence of rat liver non-specific lipid-transfer protein (sterol-carrier protein 2). Homology with rat peroxisomal and mitochondrial 3-oxoacyl-CoA thiolases.

The relationship between the rat liver non-specific lipid-transfer protein (nsLTP) and the 58-kDa protein cross-reactive with anti-nsLTP antibodies, was investigated by cDNA analysis. A 1945-bp cDNA clone was isolated which encodes a 58.7-kDa protein. This protein is identical to the 58-kDa immunoreactive protein determined by N-terminal sequence analysis of the purified 58-kDa protein. It consists of 546 amino acid residues, of which the 123 C-terminal residues are identical to the sequence of nsLTP. The N-terminal 400 amino acid residues of the 58.7-kDa protein were found to have 23.5% identity with the sequence of both mitochondrial and peroxisomal rat 3-oxoacyl-CoA thiolases, including a hypothetical substrate-binding site. The cDNA insert hybridizes with 1.1-kb, 1.7-kb, 2.4-kb and 3.0-kb mRNA species in RNA isolated from various rat tissues and from Chinese hamster ovary (CHO) cells. Southern blot analysis suggests that these mRNA species are generated from a single gene. Mutant CHO cells, deficient in peroxisomes, lack nsLTP. We have found that the mRNA encoding nsLTP is still present in these cells, which suggests that the absence of this protein is related to the lack of peroxisomes.