Bifidobacterial lipoglycan as a new cause for false-positive platelia Aspergillus enzyme-linked immunosorbent assay reactivity

We previously hypothesized that a lipoglycan of Bifidobacterium bifidum subsp. pennsylvanicum cross-reacts with the Platelia Aspergillus (PA) enzyme-linked immunosorbent assay (ELISA) based on the presence of galactofuranosyl epitopes in the cell wall (M. A. S. H. Mennink-Kersten, R. R. Klont, A. Warris, H. J. M. Op den Camp, and P. E. Verweij, Lancet 363:325-327, 2004). We tested this hypothesis by testing bacterial suspensions of different bifidobacterial species and other gram-positive and -negative bacteria with the PA ELISA, which is used to detect circulating galactomannan for the serodiagnosis of invasive aspergillosis. Furthermore, neonatal fecal samples were enumerated for bifidobacteria by fluorescence in situ hybridization (FISH) and tested for PA ELISA reactivity. All bifidobacteria, except B. infantis and B. adolescentis, showed reactivity 6- to 600-fold higher compared to the controls (i.e., Micrococcus luteus and Propionibacterium freudenreichii, which contain a cell wall lipomannan). Eggerthella lenta showed a 25-fold-higher reactivity. ELISA reactivity was clearly shown to be associated with bacterial lipoglycans containing a beta-1,5-galactofuranosyl chain. All neonatal feces showed PA ELISA reactivity and associated numbers of bifidobacteria. Since high concentrations of bifidobacteria are present in the human gut, these bacteria or excreted lipoglycan may cause false serum PA ELISA reactivity in selected patient groups, especially neonates.     

The rational design of TAP inhibitors using peptide substrate modifications and peptidomimetics

The major histocompatibility complex (MHC)-encoded transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum. This step precedes the binding of peptides to MHC class I molecules and is essential for cell surface expression of the MHC class I/peptide complex. TAP has a broad sequence specificity and a preference for peptides of around 9 amino acids. To synthesize inhibitors for TAP, we studied various alterations of the peptide substrate. The results indicate that TAP is stereospecific and that peptide bonds engineered into isosteric structures can improve translocation of the peptide. Furthermore, TAP is able to translocate peptides with large side chains that correspond to a peptide of ～ 21 amino acids in extended conformation. Peptides with longer side chains compete for the peptide binding site of TAP but fail to be translocated. Therefore, they represent the first rationally designed inhibitors of TAP.     

Detection of Cyclospora cayetanensis oocysts in human fecal specimens by flow cytometry

A diagnosis of cyclosporiasis typically involves stool examinations for the presence of Cyclospora oocysts by means of microscopy. In recent years, flow cytometry has been gaining in popularity as a novel method of detecting pathogens in environmental and clinical samples. The present study is an evaluation of a flow cytometric method for the detection and enumeration of Cyclospora oocysts in human fecal specimens associated with food-borne outbreaks of cyclosporiasis in Ontario, Canada. Flow cytometry results were generally very comparable to the original microscopy results for these specimens, in terms of both presence or absence of oocysts and relative oocyst concentrations. Of the 34 fecal specimens confirmed positive for Cyclospora by microscopy, 32 were also found positive by flow cytometry, and 2 others were considered equivocal. Of the eight fecal specimens reported to be negative by microscopy, two were found positive by flow cytometry and five others were considered equivocal. These two flow cytometry-positive samples and one of the equivocal samples were confirmed by microscopic reexamination, suggesting that flow cytometry may be more sensitive than microscopy. While the sample preparation time for flow cytometry is similar to or slightly longer than that for microscopy, the actual analysis time is much shorter. Further, because flow cytometry is largely automated, an analyst's levels of fatigue and expertise will not influence results. Flow cytometry appears to be a useful alternative to microscopy for the screening of large numbers of stool specimens for Cyclospora oocysts, such as in an outbreak situation.     

Phylogenetic analysis of rhinovirus isolates collected during successive epidemic seasons

Human rhinoviruses (HRV) have been shown to be the major causative agent for mild respiratory infections, but also associated with more serious diseases, such as acute otitis media and pneumonia in children, and asthma. Despite the economical and medical importance of HRV, little is known about the circulation and genetic diversity of HRV during a given season. The aim of this study was to genetically characterize HRV strains causing acute respiratory infections in a cohort of small children during a 2 years follow-up time. Genetic relationships between 61 HRV field isolates were studied using partial genomic sequencing in the VP4/VP2 region (420 nt) and phylogenetic analysis of these sequences. Sequences from the clinical isolates clustered in the two previously known phylogenetic clades, the designated genetic group 2 (including HRV 14) being more predominant. The maximum genetic variation within group 1 was 32.3% and within group 2 it was 32.7%. Several distinct clusters could be observed, some of which were strictly seasonal, whereas some other variants were detected during several seasons. The results of this study show striking genetic diversity of the HRV strains circulating in a given community during a short time.     

Effects of a feed additive blend on broilers challenged with heat stress

ABSTRACT

We evaluated a blend of medium-chain fatty acids (MCFA), organic acids, and a polyphenol antioxidant on gut integrity. Eighty Ross Broilers were exposed to 20–22°C (control – normothermic) or to 35–39.5°C (heat stress) for eight hours a day for a period of 1 or 5 days. Birds were fed a standard diet, or a diet supplemented with the test blend. Thereafter, birds were euthanized, and intestinal sections were excised for morphological, morphometric and gene expression analyses. Blood samples were collected for glucose-6-phosphate dehydrogenase (G6PD), glutathione peroxidase (GSH-Px) activity and trolox equivalent antioxidant capacity (TEAC) determination. Heart and liver tissues were used to quantify the expression of heat shock proteins 60 and 70 (HSP60 and HSP70, respectively) and inhibitor of kappa light chain gene enhancer in B cells alpha (IKBA). The jejunum was the most sensitive intestinal section, where heat stress modulated the expression of HSP70, of the inflammatory markers IKBA, interleukin 8 (IL-8), interferon gamma (IFNγ), and toll-like receptor 4 (TLR4). Moreover, expression of tight junctions (CLDN1, ZO1 and ZO2) and nutrient transporters (PEPT1 and EAAT3) was modulated especially in the jejunum. In conclusion, the feed additive blend protected intestines during heat stress from the decrease in villus height and crypt depth, and from the increase in villus width. Especially in the jejunum, heat stress played an important role by modulating oxidative stress and inflammation, impairing gut integrity and nutrient transport, and such deleterious effects were alleviated by the feed additive blend.

RESEARCH HIGHLIGHTS

• Jejunum is the most sensitive intestinal segment during heat stress.

• Heat stress affects the expression of tight junctions and nutrient transporters.

• Feed management helps to alleviate the disturbances caused by heat stress.

• A blend of MCFA, organic acids and a polyphenol protects broilers under heat stress.

     

Secretin and VIP-stimulated adenylate cyclase from rat heart

The exact positions of microelectrodes used to measure thePO₂ in the cerebral cortex of the rat were determined by staining the tissue with Alcian Blue. The measurement sites were subsequently located under a light microscope and correlated with the capillary and cellular arrangement of the cortex. The microelectrodes used for thePO₂ measurements were made of gold glass fibers; the Alcian Blue was injected hydrostatically through a micropipette attached to thePO₂ microelectrode. The sites where dye had been deposited were seen under a light microscope as green blue spots about 100 m in diameter. The capillaries were visualized by silver nitrate perfusion. Differences between the localPO₂ values in the neo- and the archeocortex were found. In the neocortex the meanPO₂ was 31 mm Hg, capillary volume 1.6%, capillary surface area 980/mm², capillary length 13.5/mm; whereas in the archeocortex these values where 21 mm Hg, 0.9%, 820/mm² and 9.4/mm respectively. These data indicate a relationship between the microcirculatory transport system and the local oxygen tension and provide further evidence that the meanPO₂ level tends to decrease when moving from the surface into the archeocortex.Supported by the Deutsche ForschungsgemeinschaftReported in part at the 3rd Symposium of ISOTT, Cambridge, GB, 1977; and at the 27th International Congress of Physiological Sciences, Paris, France, 1977     

Group-specific identification of polioviruses by PCR using primers containing mixed-base or deoxyinosine residue at positions of codon degeneracy.

We have developed a method for differentiating polioviruses from nonpolio enteroviruses using PCR. A pair of panpoliovirus PCR primers were designed to match intervals encoding amino acid sequences within VP1 that are strongly conserved among polioviruses. The initiating primer hybridizes with codons of a 7-amino-acid sequence that has been found only in polioviruses; the second primer matches codons of a domain thought to interact with the cell receptor. The panpoliovirus PCR primers contain mixed-base and deoxyinosine residues to compensate for the high degeneracy of the targeted codons. All RNAs from 48 vaccine-related and 110 wild poliovirus isolates of all three serotypes served as efficient templates for amplification of 79-bp product. None of the genomic sequences of 49 nonpolio enterovirus reference strains were amplified under equivalent reaction conditions. Sensitivities of poliovirus detection were as low as 100 fg (equivalent to approximately 25,000 genomic copies or 25 to 250 PFU) when the amplified products were visualized by ethidium bromide fluorescence. These degenerate PCR primers should aid in the detection of all polioviruses, including those wild poliovirus isolates for which genotype-specific reagents are unavailable.     

cagA-Positive Helicobacter pylori Populations in China and The Netherlands Are Distinct

The aim of this research was to study whether and to what extent Chinese cagA-positive Helicobacter pylori isolates differ from those in The Netherlands. Analysis of random amplified polymorphic DNA (RAPD)-PCR-assessed DNA fingerprints of chromosomal DNA of 24 cagA-positive H. pylori isolates from Dutch (n = 12) and Chinese (n = 10) patients yielded the absence of clustering. Based on comparison of the sequence of a 243-nucleotide part of cagA, the Dutch (group I) and Chinese (group II) H. pylori isolates formed two separate branches with high confidence limits in the phylogenetic tree. These two clusters were not observed when the sequence of a 240-bp part of glmM was used in the comparison. The number of nonsynonymous substitutions was much higher in cagA than in glmM, indicating positive selection. The average levels of divergence of cagA at the nucleotide and protein levels between group I and II isolates were found to be high, 13.3 and 17.9%, respectively. Possibly, the pathogenicity island (PAI) that has been integrated into the chromosome of the ancestor of H. pylori now circulating in China contained a different cagA than the PAI that has been integrated into the chromosome of the ancestor of H. pylori now circulating in The Netherlands. We conclude that in China and The Netherlands, two distinct cagA-positive H. pylori populations are circulating.