Host susceptibility to the attaching and effacing bacterial pathogen Citrobacter rodentium

Many studies have shown that genetic susceptibility plays a key role in determining whether bacterial pathogens successfully infect and cause disease in potential hosts. Surprisingly, whether host genetics influence the pathogenesis of attaching and effacing (A/E) bacteria such as enteropathogenic and enterohemorrhagic Escherichia coli has not been examined. To address this issue, we infected various mouse strains with Citrobacter rodentium, a member of the A/E pathogen family. Of the strains tested, the lipopolysaccharide (LPS) nonresponder C3H/HeJ mouse strain experienced more rapid and extensive bacterial colonization than did other strains. Moreover, the high bacterial load in these mice was associated with accelerated crypt hyperplasia, mucosal ulceration, and bleeding, together with very high mortality rates. Interestingly, the basis for the increased susceptibility was not due to LPS hyporesponsiveness, as the genetically related but LPS-responsive C3H/HeOuJ and C3H/HeN mouse strains were also susceptible to infection. Analysis of the intestinal pathology in these susceptible strains revealed significant crypt epithelial cell apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end label staining) as well as bacterial translocation to the mesenteric lymph nodes. Further studies with infection of SCID (T- and B-lymphocyte-deficient) C3H/HeJ mice demonstrated that loss of lymphocytes had no effect on bacterial numbers but did reduce crypt cell apoptosis and delayed mortality. These studies thus identify the adaptive immune system, crypt cell apoptosis, and bacterial translocation but not LPS responsiveness as contributing to the tissue pathology and mortality seen during C. rodentium infection of highly susceptible mouse strains. Determining the basis for these strains' susceptibility to intestinal colonization by an A/E pathogen will be the focus of future studies.     

Cytopathic effects of Treponema denticola chymotrypsin-like proteinase on migrating and stratified epithelial cells.

The effects of Treponema denticola and its outer membrane-bound chymotrypsin-like proteinase on periodontal ligament epithelial cell cultures at different stages of maturity were studied. In sparse cultures with migrating epithelial cells, large intracellular vacuoles were formed rapidly following exposure to live T. denticola. Treponemes showing structural damage were seen occasionally inside membrane-bound vesicles. Intensive membrane blebbing occurred in infected cells and continued for up to 48 h before the cell died. Blebbing could also be induced by a purified chymotrypsin-like proteinase of T. denticola. Cortical actin and alpha-actinin of the bacterium-treated cells showed disorganization, and pericellular fibronectin was degraded by both whole T. denticola and the isolated proteinase. Epithelial cells with well-formed lateral cell contacts appeared to be more resistant to the effects of T. denticola than migrating isolated cells. In multilayer epithelial cultures, adhesion of T. denticola and membrane blebbing were observed infrequently. There was no evidence of invasion of T. denticola into epithelial multilayers. However, immunogold electron microscopy showed rapid transport of T. denticola chymotrypsin-like proteinase into newly formed large intracellular vacuoles within the epithelial layers. These vacuoles were lined by membranes studded with ribosomes. T. denticola-treated epithelial multilayers had loose cell contacts, collapsed intercellular spaces, and increased permeability. Through its capacity to cause these unique cytopathic effects, the chymotrypsin-like proteinase of T. denticola has the potential to contribute to the initiation of periodontal disease.     

Genetic study of oxygen resistance and melanization in Cryptococcus neoformans.

Genetic analysis of oxygen-sensitive mutants of Cryptococcus neoformans revealed two loci (oxy1 and oxy2) linking hyperoxia sensitivity to production of melanin, a known virulence factor. Hyperoxia-sensitive strain 562 (oxy1 oxy2) is albino and avirulent. oxy2-defective strains lacking the oxy1 defect are melanin deficient but show normal hyperoxia resistance. Mutants defective at three additional mapped melanin loci fail to show hyperoxia sensitivity in the oxy1 background. Revertants of strain 562, which regain the ability to synthesize melanin by mutation at suppressor sites unlinked to oxy2, retain the oxygen sensitivity conferred by their oxy1 and oxy2 defects. These data identify the melanin gene oxy2 as unique in its association of hyperoxia resistance and melanization.     

Human CD8+ intraepithelial lymphocytes: a unique model to study the regulation of effector cytotoxic T lymphocytes in tissue

Summary:  The epithelium of the human small intestine contains a large population of intraepithelial cytolytic αβ T-cell receptor (TCR) CD8αβ T lymphocytes (IE-CTLs), whose main role is to sustain epithelial integrity by rapidly eliminating infected and damaged cells. In mouse, the recognition of inducible/modified self-molecules, i.e. non-classical major histocompatibility complex (MHC) class I molecules, is mediated by the TCR and natural killer receptors (NKRs) co-expressed on the cell surface of a non-conventional autoreactive CD8αααβTCR cell subset. In contrast, in humans, the recognition of non-classical MHC class I molecules induced by stress and inflammation on intestinal epithelial cells (IECs) is principally mediated by NKRs expressed on conventional CD8αβαβTCR cells. By sensing microenvironmental signals of inflammation and stress through NKRs, IE-CTLs fine tune their TCR activation threshold. Furthermore, IE-CTLs under particular conditions, involving interleukin-15 upregulation, acquire the capacity to kill distressed intestinal epithelial cells in an antigen non-specific manner. Adaptive IE-CTLs appear hence to have autoreactive properties and modulate their immune response based on innate signals, reflecting the fitness of the tissue.     

Genotyping of clinical methicillin-susceptible Staphylococcus aureus isolates in a Dutch teaching hospital

Methicillin-susceptible Staphylococcus aureus isolates, recovered from 204 patients in our hospital in a 22-month period, were characterized by pulsed-field gel electrophoresis. Among the multiple S. aureus types six clonal lineages dominated, comprising isolates from 158 patients. Despite the limited genetic variation, cross-transmission was made plausible only sporadically.     

Human observer detection experiments with mammograms and power-law noise

We determined contrast thresholds for lesion detection as a function of lesion size in both mammograms and filtered noise backgrounds with the same average power spectrum, P(f)=B/f3. Experiments were done using hybrid images with digital images of tumors added to digitized normal backgrounds, displayed on a monochrome monitor. Four tumors were extracted from digitized specimen radiographs. The lesion sizes were varied by digital rescaling to cover the range from 0.5 to 16 mm. Amplitudes were varied to determine the value required for 92% correct detection in two-alternative forced-choice (2AFC) and 90% for search experiments. Three observers participated, two physicists and a radiologist. The 2AFC mammographic results demonstrated a novel contrast-detail (CD) diagram with threshold amplitudes that increased steadily (with slope of 0.3) with increasing size for lesions larger than 1 mm. The slopes for prewhitening model observers were about 0.4. Human efficiency relative to these models was as high as 90%. The CD diagram slopes for the 2AFC experiments with filtered noise were 0.44 for humans and 0.5 for models. Human efficiency relative to the ideal observer was about 40%. The difference in efficiencies for the two types of backgrounds indicates that breast structure cannot be considered to be pure random noise for 2AFC experiments. Instead, 2AFC human detection with mammographic backgrounds is limited by a combination of noise and deterministic masking effects. The search experiments also gave thresholds that increased with lesion size. However, there was no difference in human results for mammographic and filtered noise backgrounds, suggesting that breast structure can be considered to be pure random noise for this task. Our conclusion is that, in spite of the fact that mammographic backgrounds have nonstationary statistics, models based on statistical decision theory can still be applied successfully to estimate human performance.     

Extinction of the human leukocyte antigen homozygosity effect after two doses of the measles-mumps-rubella vaccine

We have reported associations between human leukocyte antigen (HLA) homozygosity and low measles antibody levels after one dose of the measles, mumps, and rubella (MMR) vaccine. Here, we examined associations between HLA homozygosity and immune responses to MMR after two doses of vaccine. We examined associations between HLA homozygosity and measles antibody levels in a group of 178 children (cohort 1) as well as associations between homozygosity and antibody levels and lymphoproliferative responses to MMR in 346 children (cohort 2). In cohort 1, HLA homozygotes and heterozygotes had similar increases in measles antibody levels after a second dose of measles vaccine. In cohort 2, HLA homozygosity was not associated with measles immune measures after two doses of vaccine. Homozygosity at the DPB locus was associated with increased rubella antibody levels, and homozygosity at the class IA alleles was associated with lower mumps lymphoproliferative response. Homozygosity at increasing numbers of loci was also associated with lower mumps antibody levels and lymphoproliferative response. Therefore, two doses of the MMR vaccine appear to induce sufficient antibody levels and lymphoproliferative responses against measles and rubella, regardless of HLA homozygosity status. However, children who are HLA homozygous may be less protected against mumps compared with children who are heterozygous.     

Tip-oriented adherence of Treponema denticola to fibronectin.

The adherence of Treponema denticola to ligands on cell surfaces or in basement membranes of periodontal tissues might play an important role in its pathogenicity. A direct microscopic assay was used to examine the binding of T. denticola to fibronectin and other protein substrates adsorbed on plastic cover slips. All strains of T. denticola that were tested adhered to fibronectin but to different degrees. The strains which bound in high numbers frequently bound by their tips. Type strain ATCC 33520 bound to fibronectin in high numbers (149 +/- 11.3 bacteria per microscopic field), with 60% bound by the tips. Strain e' bound in high numbers (140 +/- 10.2) and had the highest percentage of tip binding (98%); strain e bound in lowest numbers (39 +/- 8.2) and had the lowest percentage of tip binding (15%). Laminin supported binding at a level similar to that of fibronectin, as did fibronectin fragments which contained the cell binding domain peptides, RGDS. Type IV collagen and non-RGDS peptides did not support binding. Binding to fibronectin and laminin was inhibited by the addition of antifibronectin and antilaminin antibodies. By lowering the incubation temperature from 37 to 4 degrees C, the number of cells that attached decreased by 60% and tip binding was reduced by 50%. Pretreatment of the cells with collagen did not affect binding, whereas fibronectin pretreatment enhanced binding by 50% and laminin pretreatment resulted in a decrease of 60%. T. denticola adheres by its tips to fibronectin-coated surfaces, which suggests that fibronectin-specific adhesins cluster at the tips.     

Chemical and immunological comparison of surface fibrils of strains representing six taxonomic groups of Actinomyces viscosus and Actinomyces naeslundii.

Human isolates of Actinomyces viscosus and Actinomyces naeslundii have been divided into six clusters in a numerical taxonomy study. Surface fibrils of strains representing these clusters were isolated and purified. Chemical analyses revealed that the major component of all fibrils was protein and that although differences in percentages of specific amino acid residues were found, the relative proportions of basic, acidic, polar uncharged, and nonpolar amino acids were rather similar among clusters. All of the fibrils except those from strain B236 (cluster 2) either failed to migrate or penetrated only slightly into gels during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even after boiling, reduction, or alkylation. Immunological studies by electron microscopic examination of fibril-antibody immunocomplexes, whole bacterial cell agglutination, inhibition of hemagglutination, and immunofluorescence by using antifibril antisera and antibodies demonstrated that strains of typical A. naeslundii (cluster 5) have a specific fibril-associated antigen(s) distinct from those of strains of other clusters. Cross-reactions for atypical A. naeslundii (cluster 3) were few. The fibrils from A. viscosus clusters 1, 2, 4, and 6 demonstrated several cross-reactions. By absorbing antifibril antibodies with cross-reactive strains it was possible to obtain cluster-specific antibodies, as determined by whole cell agglutination, only for cluster 5. Absorbed antifibril antisera for both A. naeslundii clusters 3 and 5 were specific by indirect immunofluorescence, whereas anti-cluster 1 fibril antisera cross-reacted only with other A. viscosus cluster representatives. Purification of Actinomyces fibrils by methods used for appendages of other species yields preparations containing common antigens among taxonomic groups. However, absorbing antifibril antisera, gamma globulin, or both has promise for producing cluster-specific reagents useful in identification.