Comparison of PCR and clinical laboratory tests for diagnosing <Emphasis Type="Italic">H. pylori</Emphasis> infection in pediatric patients

Background

Histology and/or culture are generally considered the gold standard for the detection of H. pylori infection. Especially in children, these tests may result in a false negative outcome because of patchy distribution of the organism in the stomach mucosa. We have developed a PCR assay utilizing nested primer pairs directed against a subunit of the H. pylori urease gene (ureA). As part of a prospective evaluation of diagnostic tests to aid in detecting H. pylori infection in children, the aim of this study was to compare our PCR and Western blot assays with results obtained from histologic examination of biopsy specimens, rapid urease tests, and an FDA approved serologic assay and published PCR results to determine if we could validate the assays for diagnostic use on our patient population.

Results

Gastric biopsy specimens obtained from 101 pediatric patients were evaluated for the presence of H. pylori using histologic techniques, rapid urease (CLOtest) test and the PCR assay. Serum samples from each patient were assayed using both ELISA and Western Blot for antibodies to H. pylori. A total of 32 patients tested were positive by at least one of the methods evaluated. Thirteen patients had positive histology, 13 had a positive CLOtest, and 17 patients had positive H. pylori PCR. Out of the 13 CLO positive patients, 12 were positive by histologic analysis and all 13 were positive by PCR. Results of serologic tests on the same population did not correlate well with other assays. Twenty-eight patients showed serologic evidence of H. pylori infection, of which 9 were both CLO and histology positive and 12 were positive by PCR. Of the seropositive patients, 26 were ELISA positive, 13 were positive by Western blot, and 11 by both serologic methods.

Conclusions

The results obtained suggest that our nested PCR assay has the specificity and sensitivity necessary for clinical application when compared to standard histologic examination and rapid urease test. In addition, we found the current commercially available approved ELISA method appears unable to accurately detect H. pylori in this population. The Western blot assay yielded better concordance with CLOtest and histology, but not as good as the nested PCR assay.

     

Human defensin gene copy number polymorphisms: comprehensive analysis of independent variation in alpha- and beta-defensin regions at 8p22-p23

To investigate defensin gene copy number polymorphisms, a quantitative real-time PCR assay was developed and used to study DNA from 27 unrelated individuals of diverse ethnic and racial backgrounds. The DEFB4 and DEFB103A genes varied in tandem, with copy numbers 2 to 8, with a mode of 6 per diploid genome (PDG). The combined copy numbers of the DEFA1 and DEFA3 genes ranged from 5 to 14, with a mode of 10 copies PDG. The copy numbers of the DEFA1/3 genes varied independently of those of the DEFB4 and DEFB103A genes. The amount of HNP-1 and HNP-3 peptides expressed in neutrophils was found to be proportional to the combined copy number of DEFA1 and DEFA3. The DEFA3 allele was absent in 7/27 subjects. The highly copy-number-variable DEFA1 and DEFA3 genes are flanked by other defensin genes present uniformly at 2 copies PDG. The remarkable variability in defensin gene copy numbers could contribute to differences in individual resistance to infections.     

Contribution of endothelin to pulmonary vascular tone under normoxic and hypoxic conditions

The contribution of endothelin to resting pulmonary vascular tone and hypoxic pulmonary vasoconstriction in humans is unknown. We studied the hemodynamic effects of BQ-123, an endothelin type A receptor antagonist, on healthy volunteers exposed to normoxia and hypoxia. Hemodynamics were measured at room air and after 15 min of exposure to hypoxia (arterial PO(2) 99.8 +/- 1.8 and 49.4 +/- 0.4 mmHg, respectively). Measurements were then repeated in the presence of BQ-123. BQ-123 decreased pulmonary vascular resistance (PVR) 26% and systemic vascular resistance (SVR) 21%, whereas it increased cardiac output (CO) 22% (all P < 0.05). Hypoxia raised CO 28% and PVR 95%, whereas it reduced SVR 23% (all P < 0.01). During BQ-123 infusion, hypoxia increased CO 29% and PVR 97% and decreased SVR 22% (all P < 0.01). The pulmonary vasoconstrictive response to hypoxia was similar in the absence and presence of BQ-123 [P = not significant (NS)]. In vehicle-treated control subjects, hypoxic pulmonary vasoconstriction did not change with repeated exposure to hypoxia (P = NS). Endothelin contributes to basal pulmonary and systemic vascular tone during normoxia, but does not mediate the additional pulmonary vasoconstriction induced by acute hypoxia.     

Biological Activity of Human Granulocyte-Macrophage Colony Stimulating Factor is Maintained in a Fusion with Seed Glutelin Peptide

Human granulocyte-macrophage colony stimulating factor (GM-CSF), a cytokine with many applications in clinical medicine, was produced specifically in the seeds of transgenic tobacco plants. Two rice endosperm-specific glutelin promoters of different size and sequence, Gt1 and Gt3, were used to direct expression. Also in the Gt3 construct, the GM-CSF coding region was in fusion with the first 24 nucleotides of the mature rice glutelin sequence at its 5' end. With the Gt1 construct plants, seed extracts contained the recombinant human GM-CSF protein up to a level of 0.03% of total soluble protein. Transgenic seed extracts actively stimulated the growth of human TF-1 cells suggesting that the seed-produced GM-CSF alone and in fusion with the rice glutelin peptide was stable and biologically active. Furthermore, native tobacco seed extracts inhibited the activity of E. coli-derived GM-CSF in this cytokine-dependent cell line. The seeds of F1 generation plants retained the biological activity of human GM-CSF protein indicating that the human coding sequence was stably inherited. The feasibility of oral delivery of such stable seed-produced cytokines is discussed.     

Bacteriophage T4 Head Morphogenesis IV. Comparison of Gene 16-, 17-, and 49-Defective Head Structures2

Defective heads present in extracts of bacteriophage T4 gene 16, 17, or 49 mutant-infected cells have been characterized. All appeared as empty shells when examined by negative-stain electron microscopy and showed essentially the same polypeptide pattern on sodium dodecyl sulfate-acrylamide gels. However, when analyzed by several other methods, gene 16- and 17-defective heads were shown to differ markedly from phage heads present in gene 49-defective extracts. First, the gene 16- and 17-defective structures were found to possess a large number of attached tails (50%, rather than about 5%). Second, they contained less nuclease-resistant deoxyribonucleic acid (DNA) (3 versus 18% of a phage equivalent), had a smaller sedimentation coefficient (240 versus 315S), and a lighter density (1.31 vs. 1.34 g/ml) than gene 49-defective heads. Third, they were not attached to the intracellular DNA pool through a deoxyribonuclease-sensitive linkage. Finally, 8-nm diameter capsomers were clearly revealed on the surface of many gene 16- and 17-defective structures. There was a total of 305 ± 25 capsomers per particle, which yielded an approximate molecular weight of 84 × 10⁶ for these heads. The capsomers were presumably not seen on gene 49-defective heads because of the large amount (18%) of associated DNA.     

Spores and soil from six sides: interdisciplinarity and the environmental biology of anthrax (Bacillus anthracis)

Environmentally transmitted diseases are comparatively poorly understood and managed, and their ecology is particularly understudied. Here we identify challenges of studying environmental transmission and persistence with a six‐sided interdisciplinary review of the biology of anthrax (Bacillus anthracis). Anthrax is a zoonotic disease capable of maintaining infectious spore banks in soil for decades (or even potentially centuries), and the mechanisms of its environmental persistence have been the topic of significant research and controversy. Where anthrax is endemic, it plays an important ecological role, shaping the dynamics of entire herbivore communities. The complex eco‐epidemiology of anthrax, and the mysterious biology of Bacillus anthracis during its environmental stage, have necessitated an interdisciplinary approach to pathogen research. Here, we illustrate different disciplinary perspectives through key advances made by researchers working in Etosha National Park, a long‐term ecological research site in Namibia that has exemplified the complexities of the enzootic process of anthrax over decades of surveillance. In Etosha, the role of scavengers and alternative routes (waterborne transmission and flies) has proved unimportant relative to the long‐term persistence of anthrax spores in soil and their infection of herbivore hosts. Carcass deposition facilitates green‐ups of vegetation to attract herbivores, potentially facilitated by the role of anthrax spores in the rhizosphere. The underlying seasonal pattern of vegetation, and herbivores' immune and behavioural responses to anthrax risk, interact to produce regular ‘anthrax seasons’ that appear to be a stable feature of the Etosha ecosystem. Through the lens of microbiologists, geneticists, immunologists, ecologists, epidemiologists, and clinicians, we discuss how anthrax dynamics are shaped at the smallest scale by population genetics and interactions within the bacterial communities up to the broadest scales of ecosystem structure. We illustrate the benefits and challenges of this interdisciplinary approach to disease ecology, and suggest ways anthrax might offer insights into the biology of other important pathogens. Bacillus anthracis, and the more recently emerged Bacillus cereus biovar anthracis, share key features with other environmentally transmitted pathogens, including several zoonoses and panzootics of special interest for global health and conservation efforts. Understanding the dynamics of anthrax, and developing interdisciplinary research programs that explore environmental persistence, is a critical step forward for understanding these emerging threats.     

Dynamic complexity of visuo-motor coordination: an extension of Bernstein’s conception of the degrees-of-freedom problem

 Extending Bernstein’s spatial conception of the degrees-of-freedom problem in the human motor system, we introduce a method developed from the theory of non-linear dynamics that allows one to quantify the spatio-temporal, i.e. dynamic, complexity of visuo-motor coordination. The correlation dimension D is used to measure the effective number of dynamic degrees of freedom in the coordination that a subject uses when performing a visuo-motor tracking task. The validity of the estimator employed is demonstrated. Visuo-motor coordination had a low-dimensional (mean D±SD=6.07 ±0.82) dynamic structure, which was consistent with deterministic chaos rather than with pure stochastic noise. D correlated with tracking performance, P. Both D and P were closely related to the degree of visuo-motor compatibility that the task presented to the subject. However, for short periods of training P increased, but D did not. As these seemingly contradictory results suggest, our dynamic conception of the degrees-of-freedom problem may reveal far more intricate visuo-motor interactions than Bernstein could identify on the basis of his spatial analyses of bodily movement patterns and by the methods of evaluation that were available to him at the time. Received: 19 April 1995/Accepted in revised form: 17 June 1996     

The molecular mechanism of hepcidin-mediated ferroportin down-regulation

Ferroportin (Fpn) is the only known iron exporter in vertebrates. Hepcidin, a peptide secreted by the liver in response to iron or inflammation, binds to Fpn, inducing its internalization and degradation. We show that after binding of hepcidin, Fpn is tyrosine phosphorylated at the plasma membrane. Mutants of human Fpn that do not get internalized or that are internalized slowly show either absent or impaired phosphorylation. We identify adjacent tyrosines as the phosphorylation sites and show that mutation of both tyrosines prevents hepcidin-mediated Fpn internalization. Once internalized, Fpn is dephosphorylated and subsequently ubiquitinated. An inability to ubiquitinate Fpn does not prevent hepcidin-induced internalization, but it inhibits the degradation of Fpn. Ubiquitinated Fpn is trafficked through the multivesicular body pathway en route to degradation in the late endosome/lysosome. Depletion of proteins involved in multivesicular body trafficking (Endosome Sorting Complex Required for Transport proteins), by small-interfering RNA, reduces the trafficking of Fpn-green fluorescent to the lysosome.