Self-expanding metal stents in the palliation of malignant dysphagia: outcome analysis in 100 consecutive patients.

SUMMARY. Patients with inoperable esophageal malignancy often undergo palliative self-expanding metal stent insertion. This analysis of cases shows that although such stents provide good palliation of dysphagia, complications frequently occur. Complications reported were pain after insertion, bleeding, food bolus impaction, stent migration and increased gastroesophageal reflux. Furthermore, in patients with esophageal adenocarcinoma, survival was less if the distal end of the stent entered the stomach, rather than lying entirely within the esophagus. Reduced survival, in this group with gastroesophageal junction tumors, may be a result of increased gastroesophageal reflux leading to pulmonary aspiration. Stents incorporating an antireflux valve have been shown to reduce symptomatic gastroesophageal reflux. It may be that such valves offer a survival advantage where stent insertion ablates the function of the lower esophageal sphincter. Further studies are needed to assess the role of antireflux stents on survival in patients with gastroesophageal junction tumors.

Detection of Mycobacterium tuberculosis group organisms in human and mouse joint tissue by reverse transcriptase PCR: prevalence in diseased synovial tissue suggests lack of specific association with rheumatoid arthritis.

Infection with mycobacterial species, including Mycobacterium tuberculosis, has long been implicated in the etiopathology of rheumatoid arthritis (RA) on the basis of clinical and pathological similarities between tuberculosis and RA. Despite evidence of immune responses to mycobacterial antigens in RA patient synovial fluid, cross-reactivity between these and host joint antigens, and the presence of M. tuberculosis protein antigen in RA synovial fluid, a definite causal association with RA has not been shown. Previous studies from our laboratory using reverse transcriptase PCR (RT-PCR) of bacterial rRNAs have shown RA synovium to be colonized by a diverse range of bacteria, most of commensal origin. However, M. tuberculosis group organism (MTG) RNA sequences were found in one RA patient tissue. Since this was considered of sufficient interest to warrant further investigation, we devised a M. tuberculosis-specific nested RT-PCR test which could be used for detection of MTG in a mixed pool of bacterial crDNAs. This test was used to investigate the distribution of MTG in RA synovial tissue and also non-RA arthritis and healthy control tissues and was also used to examine the tissue distribution of MTG in an acute and chronic model of M. tuberculosis infection in the BALB/c mouse. MTG sequences were found in a high proportion of RA patient synovial tissues but also in non-RA arthritis control tissues at lower frequency. This likely reflects trafficking of persistent M. bovis BCG to inflamed joint tissue, irrespective of cause. MTG were not found in healthy synovial tissue or the tissue of patients with undifferentiated arthritis. In both the acute and chronic models of infection in BALB/c mice, M. tuberculosis was also found to have trafficked to joint tissues, however, no signs of inflammation, arthritis, or pathology associated with M. tuberculosis infection was seen. These combined results would argue against a specific causal role of MTG in RA-like arthritis; however, their role as adjuvant in immune dysfunction in an innately susceptible host cannot be excluded.

Iron acquisition and virulence in Helicobacter pylori: a major role for FeoB, a high-affinity ferrous iron transporter.

The genome sequence of Helicobacter pylori suggests that this bacterium possesses several Fe acquisition systems, including both Fe2+- and Fe3+-citrate transporters. The role of these transporters was investigated by generating insertion mutants in feoB, tonB, fecA1 and fecDE. Fe transport in the feoB mutant was approximately 10-fold lower than in the wild type (with 0.5 microM Fe), irrespective of whether Fe was supplied in the Fe2+ or Fe3+ form. In contrast, transport rates were unaffected by the other mutations. Complementation of the feoB mutation fully restored both Fe2+ and Fe3+ transport. The growth inhibition exhibited by the feoB mutant in Fe-deficient media was relieved by human holo-transferrin, holo-lactoferrin and Fe3+-dicitrate, but not by FeSO4. The feoB mutant had less cellular Fe and was more sensitive to growth inhibition by transition metals in comparison with the wild type. Biphasic kinetics of Fe2+ transport in the wild type suggested the presence of high- and low-affinity uptake systems. The high-affinity system (apparent Ks = 0.54 microM) is absent in the feoB mutant. Transport via FeoB is highly specific for Fe2+ and was inhibited by FCCP, DCCD and vanadate, indicating an active process energized by ATP. Ferrozine inhibition of Fe2+ and Fe3+ uptake implied the concerted involvement of both an Fe3+ reductase and FeoB in the uptake of Fe supplied as Fe3+. Taken together, the results are consistent with FeoB-mediated Fe2+ uptake being a major pathway for H. pylori Fe acquisition. feoB mutants were unable to colonize the gastric mucosa of mice, indicating that FeoB makes an important contribution to Fe acquisition by H. pylori in the low-pH, low-O2 environment of the stomach.

Genotype-phenotype analysis in X-linked Emery-Dreifuss muscular dystrophy and identification of a missense mutation associated with a milder phenotype.

Direct sequencing of the emerin gene in 22 families with Emery-Dreifuss muscular dystrophy (EMD) revealed mutations in 21 (95%), confirming that emerin mutations can be identified in the majority of families with X-linked EMD. Most emerin mutations result in absence of the protein. In this study three mutations (a missense mutation Pro183Thr and two in-frame deletions removing residues 95-99 and 236-241, respectively) were unusual in being associated with expression of mutant protein. The phenotype in these families was compared in detail with the clinical features in cases with typical null mutations. For the in-frame deletions there were no significant differences. In the family with the missense mutation the phenotype was milder. Age at onset was later for first symptoms and for development of ankle contractures and muscle weakness. These findings have diagnostic implications as well as pointing to functionally important regions of the emerin protein.

A novel satellite/microsatellite combination in the genome of the marine shrimp, Penaeus vannamei.

In our studies of repeated sequences in the genome of the marine shrimp, Penaeus vannamei (Pv), we have discovered a novel combination of sequence elements. We inserted restriction fragments of genomic DNA into a plasmid vector and screened for recombinant plasmids containing repeated sequences. Ten of the resulting isolates contained representatives of the same repeated element, a satellite sequence present in one or more blocks of tandemly repeated units. The cloned repeat units range in size from 139 to 188 bp. Embedded within each cloned repeat unit are 6-15 copies of a tandemly repeated pentanucleotide microsatellite. The genome of Pv contains approx. 1,000,000 copies of this satellite/microsatellite unit. Sequences that cross-hybridize strongly with this structure were found in the genomes of lobster and crayfish, but not in other species of the genus Penaeus.

Isolation of a retrovirus from two fish cell lines developed from chinook salmon (Oncorhynchus tshawytscha) with plasmacytoid leukaemia.

Two new cell lines developed from chinook salmon with plasmacytoid leukaemia have been found to be producing a virus. The virus has been identified as a retrovirus based on: type of c.p.e. induced in culture; morphology and density of the particle; presence of Mn(2+)-dependent, poly(rA)-directed reverse transcriptase activity which was associated with a density of 1.16 to 1.18 g/ml in sucrose; electrophoretic pattern of the polypeptides from purified virions; elevated [3H]UTP labelling of RNA in the cell cultures occurring at a density of 1.16 to 1.18 g/ml in sucrose. This report describes the first isolation of a retrovirus from a salmonid cell line.

Unusual eosinophilic granule cell proliferation in coho salmon (Oncorhynchus kisutch).

Whitish, opaque, coalescing masses were observed coating the visceral organs of numerous adult coho salmon when they were dissected for artificial spawning in the autumn of 1983 at the Puntledge River hatchery, British Columbia. One affected fish was observed in November 1991. Histological examination of 10 of these fish (nine in 1983 and one in 1991) revealed that the lesions consisted of unusual proliferations of eosinophilic granule cells (EGCs) with some characteristics of neoplasia. The proliferative lesions extended throughout the gastrointestinal tract and infiltrated through all layers of the gut. Pancreatic tissue and adipose tissues associated with the pyloric caeca were also infiltrated and replaced by the proliferating EGCs. A mild diffuse infiltration was also observed in the spleen, liver and kidney. Histochemical analysis and ultrastructural examination showed the proliferating cells to be essentially identical with the EGCs which are usually concentrated in the stratum granulosum of the intestine in normal salmonid fishes.

Development of a 14C-urea breath test in ferrets colonised with Helicobacter mustelae: effects of treatment with bismuth, antibiotics, and urease inhibitors.

A 14C-urea breath test analogous to its clinical counterpart is described for use in ferrets naturally or experimentally infected with Helicobacter mustelae. The test is performed within a sealed glass metabolism chamber through which air is drawn at a constant rate and expired breath collected into sodium hydroxide. Peak 14CO2 production occurred approximately 1 hour after substrate administration. Both inter- and intra-animal responses were highly reproducible, with mean coefficients of variation less than 10%. Other than enhancing peak 14CO2 levels very slightly, fasting had little influence on the response. In infant animals challenged with H mustelae, breath test activity increased linearly with the total count of culturable bacteria isolated from the antrum. Treatment of established infections with colloidal bismuth subcitrate (DeNol) for 4 weeks resulted in clearance of all detectable bacteria but retention of some breath test activity. Subsequent regrowth of bacteria was parallelled by an increase in the breath test response. Inclusion of amoxycillin and metronidazole in the treatment regimen, however, eradicated all the bacteria and almost totally eliminated 14CO2 production. This response parallels the clinically observed suppressive effect on H pylori achieved with bismuth alone relative to the total eradication seen with triple therapy. A single oral dose of the urease inhibitor, flurofamide, inhibited over 90% of the response for at least 24 hours. Acetohydroxamic acid was less effective. These findings suggest that in the ferret H mustelae model, breath test analysis can be a useful, non-invasive alternative to endoscopy for evaluation of agents affecting either growth of the organism or urease activity.

Tubulin isoforms in the brine shrimp, Artemia: primary gene products and their posttranslational modification.

The brine shrimp, Artemia, contains 3 alpha- and 2 beta-tubulins as shown by Coomassie Blue staining of two-dimensional gels. In order to study the biosynthetic origins of the isotubulins, we hybridized cloned Drosophila tubulin genes, under stringent conditions, to blots of Artemia DNA and RNA. Southern blot analyses indicate a tubulin gene family of limited complexity. One size class of alpha- and beta-tubulin mRNA at 1800 bases was observed on Northern blots. Fluorograms of Artemia tubulin synthesized in vitro, revealed one alpha- and one beta-tubulin on two-dimensional gels, indicating that each mRNA is translated into one polypeptide and that additional tubulin spots observed on Coomassie-stained two-dimensional gels may arise posttranslationally. Artemia tubulin, which was either purified to homogeneity, or in crude cell-free extracts, was analyzed with a panel of tubulin-specific antibodies. The presence of acetylated tubulin, restricted to one of the three major alpha-tubulin spots on two-dimensional gels, demonstrated that Artemia tubulin diversity is partially generated by posttranslational mechanisms. Artemia tubulin reacted very well with an antibody to tyrosinated tubulin, but there was no, or very little, detectable detyrosinated tubulin unless the purified Artemia tubulin was exposed to carboxypeptidase. The results suggest that all microtubule-dependent events in Artemia, a complex metazoan animal, are accomplished with microtubules composed from a limited repertoire of tubulins and that none of these events require appreciable amounts of detyrosinated tubulin.

Interspersion of histone and 5S RNA genes in Artemia.

Four recombinant lambda phage containing histone genes were selected from a library of Artemia genomic DNA fragments. The histone gene organization of Artemia resembles that of other invertebrates in that all five genes are clustered and repeated in tandem with approximate repeat lengths of 8.5 kb and 9.3 kb. Each recombinant lambda phage isolate hybridizes with five histone mRNAs and unexpectedly also with 5S ribosomal RNA. Hybridization kinetics have shown the number of histone genes to be about 95-100 copies per haploid genome. An identical number of copies was determined for a hybridization probe containing the 5S gene but no histone genes. We have not found any evidence for a separate set of repeated 5S genes outside this histone + 5S block.

Absence of detectable 5-methylcytosine in DNA of embryos of the brine shrimp, Artemia.

DNA from three developmental stages of the brine shrimp, Artemia was found to be undermethylated compared to DNA from calf thymus and salmon sperm. Using high-performance liquid chromatography we found that DNA hydrolysates from both dormant cysts and prefeeding nauplii contain less than 1 residue of 5-methylcytosine per 59 kilobases, placing Artemia DNA in the "insect type" category. The absence of detectable 5-methylcytosine in DNA of developing Artemia supports the view that methylation status alone cannot account for regulation of transcription in protostomes, and that DNA methylation may be more common among deuterostomes.

Molecular cloning and characterization of ribosomal RNA genes from the brine shrimp.

A library of genomic DNA from the brine shrimp, Artemia, has been constructed with the Charon 4A phage vector, utilizing EcoRI passenger fragments. Screening this library with purified Xenopus laevis cloned rDNA genes has resulted in the identification and plaque purification of a recombinant containing a complete Artemia (18 S + 26 S) rDNA repeat unit. A physical map derived from the analysis of restriction endonuclease digests of the repeat unit, which measures 13.9 kilobase pairs, is similar to the map derived from genomic DNA. In common with several other species, the 26 S rRNA gene terminates with a HindIII recognition site.

Isolation and partial purification of phthalate ester hydrolyzing enzyme(s) from the brine shrimp, Artemia.

Isolation and 30-fold purification of a di-n-butyl phthalate (DNBP) hydrolyzing enzyme from synchronously hatched larvae of the brine shrimp, Artemia, are reported. The activity of the enzyme increases during larval development simultaneously with the acute toxic action of DNBP on the larvae. The enzyme is not stimulated by 10 mM sodium cholate, is stable for days to weeks in solution at 4 degrees C, indefinitely stable at -70 degrees C and is distinct from the previously reported trypsin- and chymotrypsin-like proteases [1].

CV-1 cells release proteins that facilitate infection by simian virus 40 and echovirus 6.

Uninfected simian cells (CV-1) produce two different proteins, one of which enhances the infectivity of echovirus 6 and the other enhances the infectivity of SV40 in less susceptible cells. The enhancers are released by metabolizing cells into the culture medium. The SV40 enhancer protein is larger and less acidic than the echovirus enhancer. The SV40 enhancer protein can be dissociated into 2 subunits with apparent molecular weights of 42,000 and 24,000. The echovirus enhancer protein consists of 2 subunits with apparent molecular weights of 28,000 and 31,000. Electrophoretically purified enhancer proteins interact with virions and retain their biological activities and viral specificities. The SV40 enhancer stimulates expression of SV40-T antigen under conditions in which untreated virus does not initiate expression of T antigen. This result and the observation that the enhancers do not increase the infectivity of the nucleic acids of their respective viruses suggest that the enhancers act either at the stage of penetration or uncoating.