A gene from the locus of enterocyte effacement that is required for enteropathogenic Escherichia coli to increase tight-junction permeability encodes a chaperone for EspF

Disruption of the barrier properties of the enterocyte tight junction is believed to be important in the pathogenesis of diarrhea caused by enteropathogenic Escherichia coli (EPEC). This phenotype can be measured in vitro as the ability of EPEC to reduce transepithelial resistance (TER) across enterocyte monolayers and requires the products of the locus of enterocyte effacement (LEE) and, in particular, the type III secreted effector protein EspF. We report a second LEE-encoded gene that is also necessary for EPEC to fully reduce TER. rorf10 is not necessary for EPEC adherence, EspADB secretion, or formation of attaching and effacing lesions. However, rorf10 mutants have a diminished TER phenotype, reduced intracellular levels of EspF, and a reduced ability to translocate EspF into epithelial cells. The product of rorf10 is a 14-kDa intracellular protein rich in alpha-helices that specifically interacts with EspF but not with Tir or other EPEC secreted proteins. These properties are consistent with the hypothesis that rorf10 encodes a type III secretion chaperone for EspF, and we rename this protein CesF, the chaperone for EPEC secreted protein F.     

Cytotoxicity of Hemolytic, Cytotoxic Necrotizing Factor 1-Positive and -Negative Escherichia coli to Human T24 Bladder Cells

Approximately one-half of Escherichia coli isolates from patients with cystitis or pyelonephritis produce the pore-forming cytotoxin hemolysin, a molecule with the capacity to lyse erythrocytes and a range of nucleated cell types. A second toxin, cytotoxic necrotizing factor 1 (CNF1), is found in approximately 70% of hemolytic, but rarely in nonhemolytic, isolates. To evaluate the potential interplay of these two toxins, we used epidemiological and molecular biologic techniques to compare the cytotoxicity of hemolytic, CNF1⁺, and CNF1⁻ cystitis strains toward human T24 bladder epithelial cells in vitro. A total of 29 isolates from two collections of cystitis-associated E. coli were evaluated by using methylene blue staining of bladder monolayers at 1-h intervals after inoculation with each strain. Most (20 of 29) isolates damaged or destroyed the T24 monolayer (less than 50% remaining) within 4 h after inoculation. As a group, CNF1⁺ isolates from one collection (11 strains) were less cytotoxic at 4 h than the CNF1⁻ strains in that collection (P = 0.009), but this pattern was not observed among isolates from the second collection (18 strains). To directly evaluate the role of CNF1 in cytotoxicity of hemolytic E. coli without the variables present in multiple clinical isolates, we constructed mutants defective in production of CNF1. Compared to the CNF1⁺ parental isolates, no change in cytotoxicity was detected in these cnf1 mutants. Our results indicate that CNF1 does not have a detectable effect on the ability of hemolytic E. coli to damage human bladder cell monolayers in vitro.     

Anaphylactoid shock – a common cause of death in heroin addicts?

We measured mast-cell tryptase in postmortem blood from 22 heroin addicts dying suddenly after injection. In 32%, the concentration of tryptase was elevated (≥10 μg/1), and the mean value of tryptase was significantly different from a control group dying from known, nonimmunologic causes ( P <0.05). The increased tryptase concentrations indicate that death was preceded by systemic mast-cell degranulation. All victims of drug deaths had morphine in blood, most below 0.2 μg/ml. In 71% of the victims of drug-related deaths with tryptase values ≥10 μg/1, the intermediate degradation product, 6–monoacetyl-morphine, was not found in blood, whereas this was the case in only two victims with values below that cutoff point. This indicates that those with high tryptase concentrations survived longer than those with lower values. No correlation was found between the IgE levels and tryptase in either group, supporting the hypothesis that tryptase release was not mediated by an allergic reaction. The well-known property of opiates to stimulate unspecifically the liberation of histamine and other constituents of mast-cell granules offers one explanation of our observations. The results suggest that many heroin fatalities are caused by an anaphylactoid reaction.     

Treatment with aflatoxin B1 for immortalization of human fibroblasts from Li-Fraumeni patients

Immortalization of normal human fibroblasts is a very rare event. Multiple genes such as p53 and cellular senescence genes are possibly involved in immortalization of human fibroblasts, suggesting that multiple treatments with carcinogens are required for the immortalization. We describe here the procedure for immortalization of human fibroblasts (MDAH 087) from Li-Fraumeni cancer syndrome with a germ-line p53 mutation. The cells were subjected to multiple treatments with aflatoxin B₁ (AFB₁) in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant (PMS), and 3 of 9 MDAH 087 cell cultures treated 1–3 times with 0.1–1 µg/ml AFB₁ became immortal, defined as continuous growth for over 300 population doublings after the first treatment. However, cultures of human fibroblasts from a normal embryo treated under the same conditions failed to escape senescence. The results indicate that the model of human fibroblasts with a mutated p53 allele exposed to AFB₁ is potentially useful for studying mechanisms of chemically induced immortalization.     

Genotyping hepatitis C virus by heteroduplex mobility analysis using temperature gradient capillary electrophoresis

The genotype of the infecting hepatitis C virus (HCV) helps determine the patient's prognosis and the duration of treatment. Heteroduplex mobility analysis (HMA) is a rapid, inexpensive method for genotyping of HCV that does not require sequencing. We developed an HMA that uses temperature gradient capillary electrophoresis (TGCE) to differentiate HCV genotypes. A 56-bp region of the HCV 5' untranslated region (UTR) that was conserved within a genotype yet whose sequence differed between genotypes was amplified for HMA-TGCE analysis. HCV amplicons of types 1, 2a, 2b, 3a, 4, and 6a were hybridized in pairs and analyzed by TGCE. Amplicons hybridized to the same subtype yielded one homoduplex peak, while hybridization of different subtypes resulted in heteroduplexes and generated multiple TGCE peaks. Heteroduplexes contain thermodynamically unstable nucleotide mismatches that reduced their TGCE mobilities compared to those of homoduplexes. Three HCV subtypes (subtypes 1a, 3a, and 4) generated unique peak patterns when they were combined with each genotype analyzed and were chosen as the reference genotypes. A blinded study with 200 HCV-infected samples was 97% accurate compared to genotyping by 5' UTR sequence analysis. The majority of discordant results were unexpected sequence variants; however, five of nine sequence variants were correctly genotyped. The assay also detected and correctly genotyped mixed HCV infections. Compared to conventional HMA, TGCE improves the resolution, with better separation of heteroduplexes and homoduplexes. All common HCV genotypes can be detected and differentiated by this HMA-TGCE assay.     

Excitatory amino acid receptors of the electrosensory system: the NR1/NR2B N-methyl-D-aspartate receptor

The amino acid sequence of the N-methyl-D-aspartate (NMDA) receptor subunit NR2B from the brown ghost knife fish Apteronotus leptorhynchus has been determined and compared with the sequence of the murine NR2B. This comparison revealed high levels of sequence conservation throughout the ligand binding and membrane spanning segments. The functional properties of the NR1 and NR2B receptor complex were examined by coexpression in HEK cells. The recombinant AptNR1/NR2B receptors produced robust currents after stimulation with glutamate or NMDA in the presence of glycine. Measurements of the concentration dependencies for these agonists indicated that the agonist binding sites on the apteronotid receptor are highly conserved, with nearly identical agonist affinities to those of the murine NR1/NR2B receptor. The kinetic responses of the fish receptor were also highly conserved, with deactivation rates for the AptNR2B receptor matching those of the murine NR2B containing receptor. Evidently, most of the unique functional properties that reside in the NR2B receptor subunit have been well conserved in teleost NMDA receptors. On the other hand, the apteronitid receptor displayed a lowered sensitivity to voltage-dependent Mg(2+) block and a reduced affinity for the NR2B-specific noncompetitive antagonist ifenprodil. We conclude that the functional properties that result from the incorporation of the NR2B receptor in the NMDA receptor complex have been maintained since the evolutionary divergence of teleost and mammalian organisms.     

Evidence for involvement of a tumor suppressor gene on 1p in malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNST) are rare soft-tissue malignancies. The genetic basis of these tumors is still poorly understood. Cytogenetic analyses predominantly revealed complex karyotypes, precluding the identification of recurrent chromosomal changes. We report loss of 1p material in a near-diploid karyotype with few or no additional structural chromosome changes in two sporadic cases of MPNST, indicating an important role of 1p loss in MPNST development. In one of these two tumors, a distal 1p deletion (1p31.2 approximately pter) was detected suggesting involvement of a tumor suppressor gene located within this distal region of 1p. Further evidence for recurrent 1p loss in MPNST was obtained by interphase fluorescence in situ hybridization, which showed loss of 1p material in 3 out of 13 tumors. These findings together with data from the literature suggest that loss of a tumor suppressor gene located within distal 1p is implicated in the pathogenesis of MPNST.     

Sulfolipid deficiency does not affect the virulence of Mycobacterium tuberculosis H37Rv in mice and guinea pigs

Lipids that are found only in the cell envelope of pathogenic mycobacteria, such as those containing multiple methyl-branched fatty acids, have long been thought to play a role in pathogenesis. Among these complex lipids, sulfolipids have been the most extensively studied over the last 50 years. The numerous biological effects exhibited by purified sulfolipids on phagocytic cells led to the idea that these molecules are probably important virulence factors facilitating the intracellular survival of Mycobacterium tuberculosis. However, definitive evidence to support this concept has been lacking. The recent construction of an isogenic sulfolipid-deficient mutant of M. tuberculosis H37Rv (Sirakova et al., J. Biol. Chem. 276:16833-16839, 2001) has for the first time provided the opportunity to directly assess the contribution of these complex lipids to pathogenesis. In the present study, we show that against all expectations, sulfolipid deficiency does not significantly affect the replication, persistence, and pathogenicity of M. tuberculosis H37Rv in mice and guinea pigs or in cultured macrophages.     

Babesia bovis : identification of immunodominant merozoite surface proteins in soluble culture-derived exoantigen

Babesia bovis merozoite proteins presenting as exoantigens in in vitro culture supernatants have been characterized. Bovine antisera to B. bovis exoantigens were used to immunoprecipitate [³⁵S]-methionine metabolically labeled or lactoperoxidase-catalyzed radioiodinated B. bovis merozoite proteins. A total of 24 metabolically labeled proteins ranging in molecular weight from 24,000 to 225,000 Da and 9 radioiodinated proteins with molecular weights varying between 24,000 and 225,000 Da were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monoclonal antibodies to B. bovis merozoite surface proteins were also used to immunoprecipitate metabolically labeled exoantigens directly from in vitro culture supernatants. These results demonstrate epitopes from at least nine merozoite surface proteins present in the exoantigen fraction, among which are the recently characterized major surface antigens 1 and 2, rhoptry-associated protein 1, and spherical body protein 2. Received: 1 April 1997 / Accepted: 6 May 1997     

Dendritic cells transfected with cytopathic self-replicating RNA induce crosspriming of CD8+ T cells and antiviral immunity

A potential shortcoming of nonlive vaccines is their relative inefficiency in generating T cell responses, thus limiting their application in infections requiring cellular immunity. Here, we present a system to induce cellular immunity and to study the immunological implications of time-delayed dendritic cell (DC) apoptosis and antigen reprocessing in vivo. We generated a self-replicating cytopathic pestivirus RNA to enhance production and presentation of hepatitis C virus (HCV) antigens and to induce apoptosis in DC 24-48 hr after transfection. Replicon-transfected H-2(b) DCs used to immunize HLA-A2 transgenic mice induced protection upon challenge with a vaccinia virus expressing HCV antigens. Induction of cell death enhanced the immunogenicity of DC-associated antigen. Transfer of cellular material from vaccine DCs to endogenous antigen presenting cells was visualized in lymph nodes and spleen, and crossprimed CD8(+) T cells were characterized. The findings are relevant for the rational design of vaccines against noncytopathic pathogens like HCV.