Association of 4p14 TLR locus with antibodies to Helicobacter pylori.

A genome-wide association study among Europeans related polymorphisms of the Toll-like receptor (TLR) locus at 4p14 and the Fcγ receptor 2a locus at 1q23.3 to Helicobacter pylori serologic status. We replicated associations of 4p14 but not 1q23.3 with anti-Helicobacter pylori antibodies in 1402 Finnish males. Importantly, our analysis clarified that the phenotype affected by 4p14 is quantitative level of these antibodies rather than association with seropositivity per se. In addition, we annotated variants at 4p14 as expression quantitative trait loci (eQTL) associated with TLR6/10 and FAM114A1. Our findings suggest that 4p14 polymorphisms are linked to host immune response to H. pylori infection but not to its acquisition.

Increased Helicobacter pylori-associated gastric cancer risk in the Andean region of Colombia is mediated by spermine oxidase.

Helicobacter pylori infection causes gastric cancer, the third leading cause of cancer death worldwide. More than half of the world's population is infected, making universal eradication impractical. Clinical trials suggest that antibiotic treatment only reduces gastric cancer risk in patients with non-atrophic gastritis (NAG), and is ineffective once preneoplastic lesions of multifocal atrophic gastritis (MAG) and intestinal metaplasia (IM) have occurred. Therefore, additional strategies for risk stratification and chemoprevention of gastric cancer are needed. We have implicated polyamines, generated by the rate-limiting enzyme ornithine decarboxylase (ODC), in gastric carcinogenesis. During H. pylori infection, the enzyme spermine oxidase (SMOX) is induced, which generates hydrogen peroxide from the catabolism of the polyamine spermine. Herein, we assessed the role of SMOX in the increased gastric cancer risk in Colombia associated with the Andean mountain region when compared with the low-risk region on the Pacific coast. When cocultured with gastric epithelial cells, clinical strains of H. pylori from the high-risk region induced more SMOX expression and oxidative DNA damage, and less apoptosis than low-risk strains. These findings were not attributable to differences in the cytotoxin-associated gene A oncoprotein. Gastric tissues from subjects from the high-risk region exhibited greater levels of SMOX and oxidative DNA damage by immunohistochemistry and flow cytometry, and this occurred in NAG, MAG and IM. In Mongolian gerbils, a prototype colonizing strain from the high-risk region induced more SMOX, DNA damage, dysplasia and adenocarcinoma than a colonizing strain from the low-risk region. Treatment of gerbils with either α-difluoromethylornithine, an inhibitor of ODC, or MDL 72527 (N(1),N(4)-Di(buta-2,3-dien-1-yl)butane-1,4-diamine dihydrochloride), an inhibitor of SMOX, reduced gastric dysplasia and carcinoma, as well as apoptosis-resistant cells with DNA damage. These data indicate that aberrant activation of polyamine-driven oxidative stress is a marker of gastric cancer risk and a target for chemoprevention.

Effect of green lentils on colonic function, nitrogen balance, and serum lipids in healthy human subjects.

Green lentils are an increasingly popular food, but their effects on human colonic function and serum lipids have been studied little. Nine healthy males aged 19-38 y consumed for 3-wk periods a controlled Western diet and the same diet supplemented with 130 g dry lentils/d [which contained 11.8 g non-starch polysaccharide (NSP)] incorporated into loaves, cakes, and soups. Protein was equilibrated with soy protein isolate and carbohydrate with soft drinks. Radioopaque markers were used to calculate mean transit time (MTT) and to correct fecal weight for infrequency of bowel movements. Feces were collected throughout the study and blood was taken on 2 d at the end of each period. Lentils increased fecal weight from 131 +/- 12 g/d (means +/- SEM) to 189 +/- 17.4 g/d (44.9%) (P < 0.005). MTT was unchanged: 46 +/- 6 h for the control diet and 43 +/- 4 h for the lentils (NS). Fecal nitrogen was increased to 2.49 +/- 0.08 g/d for lentils compared with 1.74 +/- 0.09 g/d for the control diet (P < 0.001) and urine nitrogen decreased to 15.31 +/- 0.52 g/d with the lentils compared with 15.90 +/- 0.51 g/d for the control diet (P < 0.05); nitrogen balance was unaffected. Serum lipids were unchanged by addition of lentils to the diet. Green lentils were effective in increasing fecal weight and can therefore make a valuable contribution to a healthy diet.

Intake of carbohydrate and its components--international comparisons, trends over time, and effects of changing to low-fat diets.

Carbohydrate constitutes the major source of dietary energy for all peoples of the world. However, it has been difficult to make accurate determinations of intakes of carbohydrate and its constituents because of lack of individual assessments in which carbohydrate components are included. For many countries, only food balance information is available and values for total carbohydrate are often derived by difference. Available information indicates that carbohydrate consumption decreased in many industrialized nations as prosperity led to an increased consumption of fat. Fat intakes have fallen over the past two decades and carbohydrate intakes have increased, but still do not approach the 60-70%contribution of carbohydrate to total energy in developing countries. A negative image for carbohydrate has led to a reluctance to accept it as a legitimate dietary component, particularly in North America. New evidence of the beneficial effects of starch in the diet indicates that increased consumption of carbohydrate, especially in the form of starch, should be promoted in Western countries.

Analysis of a dimethacrylate copolymer (bis-GMA and TEGDMA) network by DSC and 13C solution and solid-state NMR spectroscopy.

In this work the effect of dilution with TEGDMA on the kinetics of Bis-GMA polymerization and on the extent of polymerization or degree of conversion was studied using (a) DSC and (b) NMR. The systems with lower viscosity and lower Tg exhibited higher extent of polymerization. For Bis-GMA/TEGDMA mixtures the calculated Tg values were found to be higher than the experimental values suggesting that a dilution effect is predominant rather than intermolecular hydrogen bonding. Solid state NMR has been shown to be a convenient method for measuring the total amount of conversion in a mixed monomer system. The disappearance of the NMR solution spectrum was used to reveal overall polymerization kinetics.

Small bowel haemangioma with local lymph node involvement presenting as intussusception.

Gastrointestinal haemangiomas make up 0.05% of all intestinal neoplasms. They are sometimes multiple and usually present with pain, bleeding, and obstruction. An associated haemangiomatous change in regional lymph nodes has not been reported previously. A woman of 21 years presented with abdominal pain and vomiting. Abdominal ultrasound and computed tomography scan showed a lower abdominal mass. Laparotomy revealed a small bowel tumour causing an intussusception together with enlarged mesenteric lymph nodes. Pathological examination revealed a small bowel haemangioma with mesenteric node involvement. The pathogenesis of haemangiomatous involvement of lymph nodes is discussed. Hamartomatous change is the likely cause in this patient.

Signature patterns of DNA restriction fragments of Helicobacter pylori before and after treatment.

The genomic DNA of Helicobacter pylori was studied by electrophoretic analysis of restriction endonuclease fragments. Twenty seven isolates from eight patients in the United Kingdom, obtained before and after treatment with nitrofurantoin, and two reference strains from Australia and Peru were investigated. Digestion of DNA with HaeIII, which gave the clearest band pattern of the 20 enzymes tested, showed that each set of isolates from a single patient had a unique band pattern. The DNA signature band patterns of strains from different patients were less than or equal to 62% similar (average 43%); similarities of patterns from the same patient were generally greater than or equal to 86%. Some minor but reproducible polymorphisms (less than or equal to five bands) in the signature region were detected in most consecutive isolates. Plasmid DNA was detected in isolates from five patients, but major pattern differences were attributed to genomic variation. It is concluded that the HaeIII DNA digest signature fingerprints provide a reproducible and sensitive method of discriminating between isolates of H pylori.

Bacterial translocation and immunohistochemical measurement of gut immune function.

AIMS: The local immune response in the small bowel mucosa might play a role in bacterial translocation (BT). The aim of this study was to quantify immune cells and secretory antibodies in the small bowel mucosa, and relate this to BT as assessed by culture of a mesenteric lymph node. METHODS: Immunohistochemical techniques were used to measure the frequency of plasma cells and IgA and IgM positive cells in the lamina propria and semiquantitatively to assess mucosal surface IgA and IgM values in small bowel specimens obtained from 11 patients in whom positive evidence of BT had been identified in a mesenteric lymph node harvested at the time of laparotomy. These were compared with similar specimens obtained from 11 patients in whom a similar lymph node had yielded no growth. RESULTS: BT was associated with a significantly increased median frequency of plasma cells (p < 0.01) and IgA positive cells (p < 0.05) in the lamina propria. The frequency of IgM positive cells was also higher in these patients, although this difference was not significant. In addition, semiquantitatively scored IgA and IgM concentrations at the mucosal surface were both significantly higher in the patients in whom BT had been identified (p = 0.006 and 0.016, respectively). CONCLUSION: Higher numbers of plasma cells and higher IgA and IgM values are present in the small bowel mucosa of patients in whom BT has been shown to occur, suggesting an increased local immune response.

Evaluation of a selective enrichment technique for the isolation of Campylobacter pylori.

To cultivate Campylobacter pylori from contaminated biopsy specimens, Brucella broth was supplemented with 10% fetal calf serum, 1% Vitox, 1000 units/ml polymyxin B sulfate, 10 micrograms/ml vancomycin, and 2 micrograms/ml amphotericin B. Pseudomonas aeruginosa, Candida albicans, and Enterococcus fecalis were cocultivated with C. pylori. All four strains of C. pylori were recoverable at 24 h. When 21 C. pylori strains were studied in pure culture, 86% grew in the selective enrichment medium. In a clinical study, the selective enrichment technique resulted in isolation of C. pylori from 50% of patient samples, compared with isolation from only 36% of samples with agar cultivation. The selective enrichment technique may be more sensitive than techniques currently employed to isolate C. pylori from gastric tissue.

Biotype and macromolecular profiles of cytotoxin-producing strains of Helicobacter pylori from antral gastric mucosa.

Biotype, genome, protein and plasmid profile diversity amongst 40 epidemiologically unrelated strains of Helicobacter pylori was studied. Strains were API Zym biotypes II, III and IV but most (87%) were biotype II. Four subsets of strains were defined on a combination of motility (56% positive) and cytotoxin production (44% positive). A close association (P = 0.45) between these two features was observed for 69% of strains. Each strain of H. pylori had a unique DNA type defined by either HaeIII or HindIII total digest patterns and by ribopatterns, except for DNA of the rare strains not cut by these endonucleases. Strain diversity was confirmed by one-dimensional SDS-PAGE electrophoretic protein patterns. No consistent associations between cytotoxin activity and overall ribopattern or band subsets within a ribopattern were detected. Some strains (39%) contained a plasmid but the presence of plasmids was not consistently associated with either cytotoxin activity, biotype, motility or ribopattern. We conclude that the cytotoxin-producing strains of H. pylori were genomically as diverse as the non-cytotoxin producing strains.

Motility as a factor in the colonisation of gnotobiotic piglets by Helicobacter pylori.

Non-motile variants of Helicobacter pylori (strain 26695) occurred with a frequency of 1.6 (SD 0.4) x 10(-4) variants/cell/division cycle, and reversion to the motile form occurred with a frequency of less than 10(-7) variants/cell/division cycle. The two forms remained greater than 90% pure for up to 50 cell divisions and differed only in the presence or absence of motility and flagella. Bacteria were recovered from nine of 10 gnotobiotic piglets inoculated orally with motile H. pylori, but from only two of eight inoculated with the non-motile variant. The motile form survived for 21 days in infected piglets, but the non-motile variant survived for only 6 days. Bacteria recovered from piglets inoculated with the non-motile variant were non-motile. These data support the hypothesis that motility is a colonisation factor for H. pylori.

Cytotoxic activity in broth-culture filtrates of Campylobacter pylori.

Broth-culture filtrates of Campylobacter pylori induced non-lethal cytopathic effects in vitro in 7 of 9 mammalian cell lines tested. Transmission electronmicroscopy revealed that the response consisted of intracellular vacuolisation. Intestine 407 cells were among the most responsive and were used for routine assay. About 55% of isolates of C. pylori tested, originating from four geographic regions worldwide, produced cytotoxic activity. The activity was neutralisable by specific antisera to broth-culture filtrates or to sonicated bacteria but not by antisera to other bacterial preparations. Cytotoxic activity was heat-labile (70 degrees C for 30 min), was protease-sensitive and ammonium-sulphate precipitable. It did not pass through an ultrafiltration membrane with a nominal mol.-wt limit of 100 X 10(3). It was concluded that C. pylori can produce a factor that alters cultured cells in vitro. The relevance of this factor to the pathogenesis of gastritis associated with C. pylori remains to be determined.

Needlestick injuries: how can we teach people better about risk assessment?

At work people run some small risk of death or injury which is directly attributable to their occupation. In biomedical sciences the accidental puncture of the skin by hypodermic needles, other instruments or broken glass has long been regarded as an occupational hazard and there is increasing concern that staff could become infected with a range of micro-organisms, including hepatitis B and the Human Immunodeficiency Virus (HIV). Needlestick injuries should be preventable if staff are trained effectively and take care about disposal of used syringes and needles. Staff at risk must be offered pre-exposure vaccination for hepatitis B and resources must be provided for special training. Fundamental changes may be required in methods and equipment and a number of new ways of targeting groups of health care staff with information are discussed.

Performance characteristics of a dual-energy detector for digital scan projection radiography.

An energy discriminating x-ray detector has been developed for dual-energy, scan projection digital radiography. The detector is comprised of a pair of x-ray intensifying screen/linear photodiode arrays, aligned one behind the other. Energy discrimination is achieved by employing a low atomic number phosphor in the front screen and a high atomic number phosphor in the back screen. The x-ray response, modulation transfer function, and defective quantum efficiency of the detector are reported along with the experimental methodology utilized for the measurements. Also presented is an analysis which indicates that in a typical patient's lung field, the detector can resolve the projected density (g/cm2) of a 3-mm-thick, 1-cm2 area of bone to better than 1.5%.

Measured performance characteristics of a solid-state linear detector array.

Experimental studies of a solid-state linear detector array developed for a prototype scanning slit digital chest radiographic unit have been completed. The detector consists of a strip of scintillating material, optically coupled to a linear silicon photodiode array. Measured performance characteristics of the detector, such as sensitivity, modulation transfer function, and detective quantum efficiency, are presented for several different scintillators. Results indicate that direct x-ray absorption events in the silicon photodiode can degrade detective quantum efficiency. Results also indicate that the inexpensive preamplifier circuits used in the digital chest prototype contribute negligible noise at diagnostic x-ray photon fluence rates.

An investigation into the effect of a probiotic on gut immune function in surgical patients.

BACKGROUND: The gut-associated lymphoid tissue (GALT) is an important component of the gut barrier. We have previously demonstrated a significant increase in various parameters of gut immune function in association with bacterial translocation. Animal studies have suggested that the probiotic Lactobacillus plantarum 299v improves the immunological status of the intestinal mucosa. The aim of this study was to determine whether the same is true in humans. METHOD: This was a prospective randomised controlled study, in which immunohistochemical techniques were used to measure the concentrations of plasma cells, IgA positive cells and IgM positive cells in the lamina propria, together with the concentrations of IgA and IgM at the mucosal surface in specimens of normal small bowel obtained from patients undergoing elective abdominal surgery who had consumed an oral preparation containing the probiotic Lactobacillus plantarum 299v (ProViva) during the immediate preoperative period. These were compared with similar specimens obtained from control subjects who did not receive the probiotic. RESULTS: A total of 22 patients were studied (probiotic group n = 11, control group n = 11). The median volume of ProViva consumed was 3250 ml (range 2100-9000 ml), for a median duration of 9 days (range 5-18 days). There were no significant differences between the probiotic and control groups in terms of concentrations of plasma cells, IgA positive cells or IgM positive cells in the lamina propria. There was a significantly higher concentration of IgM at the mucosal surface in the control group (P = 0.02, Fishers Exact test mid P), but no difference in terms of IgA. CONCLUSIONS: The increase in IgA observed in the intestinal mucosa in response to probiotics in animal studies does not occur in humans. The significance of the increase in IgM at the mucosal surface in the controls is unclear.

Analysis for residual host cell proteins and DNA in process streams of a recombinant protein product expressed in Escherichia coli cells.

Analyses of crude samples from biotechnology processes are often required in order to demonstrate that residual host cell impurities are reduced or eliminated during purification. In later stages of development, as the processes are further developed and finalized, there is a tremendous volume of testing required to confirm the absence of residual host cell proteins (HCP) and DNA. Analytical tests for these components are very challenging since (1). they may be present at levels that span a million-fold range, requiring substantial dilutions; (2). are not a single component, often existing as fragments and a variety of structures; (3). require high sensitivity for final steps in process; and (4). are present in very complex matrices including other impurities, the product, buffers, salts and solvents. Due to the complex matrices and the variety of potential analytes, the methods of analysis are not truly quantitative for all species. Although these limitations are well known, the assays are still very much in demand since they are required for approval of new products. Methods for final products, described elsewhere, focus on approaches to achieve regulatory requirements. The study described herein will describe the technical rationale for measuring the clearance of HCP and DNA in the entire bioprocessing to purification from an Escherichia coli-derived expression system. Three analytical assays, namely, reversed-phase high-performance liquid chromatography (RP-HPLC), enzyme-linked immunosorbent assay (ELISA), and Threshold Total DNA Assay, were utilized to quantify the protein product, HCP and DNA, respectively. Product quantification is often required for yield estimation and is useful since DNA and HCP results are best expressed as a ratio to product for calculation of relative purification factors. The recombinant E. coli were grown to express the protein of interest as insoluble inclusion bodies (IB) within the cells. The IB were isolated by repeated homogenization and centrifugation and the inclusion body slurry (IBS) was solubilized with urea. After refolding the product, the solution was loaded on several commonly used ion exchangers (CM, SP, DEAE, and Q). Product was eluted in a salt gradient mode and fractions were collected and analyzed for product, HCP and DNA. The IBS used for this study contained about 15 mg/ml product, 38 mg/ml HCP and 1.1 mg/ml DNA. Thus, the relative amounts of HCP and DNA in the IBS was excessive, and about 10(3) times greater than typical (because the cells and IB were not processed with the normal number of washing steps during isolation). This was of interest since similar samples may be encountered when working with non-inclusion body systems, such as periplasmic expressions, or in cases where the upstream unit operations under-perform in IB cleaning. The study described herein describes the development of three robust methods that provide the essential process data needed. These findings are of general interest to other projects since applications of similar analytical technology may be used as a tool to develop processes, evaluate clearance of impurities, and produce a suitable product.

Cultures of pig macrophages.

Freshly collected blood from pigs was defibrinated with glass beads. The white cells were collected on a Ficoll-Hypaque mixture and grown in RPMI 1640 medium containing 20 per cent fetal calf serum. Monocytes adhered to the plastic or glass flasks and the erythrocytes and lymphocytes remained in suspension. The adherent cells assumed the appearance of macrophages and the mean cell size at eight days was 40 mum. Phagocytosis was demonstrated and, in the first few days, incorporation of tritiated thymidine occurred. Cells remained viable in culture for more than eight weeks. Histo-chemical studies and the appearance of the cells under the electron microscope are described.

Ultrasound-guided Tru-cut biopsy of the breast.

Ultrasound-guided automated Tru-cut needle biopsy may be used as an alternative to fine needle aspiration cytology for the assessment of discrete mass lesions of the breast. This is a retrospective study of 187 biopsies, comparing the results with a final diagnosis obtained from subsequent excision or outpatient follow-up. Biopsies were performed using a spring-loaded gun under ultrasound guidance. Invasive malignancy was demonstrated in 114 biopsies, 98 of which were subjected to surgery, with no false-positives. Twelve biopsies contained 'atypical cells', pre-invasive malignancy or risk factors for invasive carcinoma, ten of which proved to be invasive malignancy on excision. Normal or benign tissue was found in 61 biopsies, but of those that proceeded to excision biopsy, 16 were invasive or in situ carcinoma. The sensitivity of the procedure for detecting significant pathology was 88.7%, and the specificity 100%. When used as part of triple assessment, the sensitivity increases to 97.9%. Ultrasound-guided Tru-cut needle biopsy is a well-tolerated and reliable procedure for providing a tissue diagnosis of malignancy before definitive treatment, and obviating the need for formal excision biopsy of lesions for which there is a low index of suspicion.