Canine scent detection in the diagnosis of lung cancer: revisiting a puzzling phenomenon.

Patient prognosis in lung cancer largely depends on early diagnosis. The exhaled breath of patients may represent the ideal specimen for future lung cancer screening. However, the clinical applicability of current diagnostic sensor technologies based on signal pattern analysis remains incalculable due to their inability to identify a clear target. To test the robustness of the presence of a so far unknown volatile organic compound in the breath of patients with lung cancer, sniffer dogs were applied. Exhalation samples of 220 volunteers (healthy individuals, confirmed lung cancer or chronic obstructive pulmonary disease (COPD)) were presented to sniffer dogs following a rigid scientific protocol. Patient history, drug administration and clinicopathological data were analysed to identify potential bias or confounders. Lung cancer was identified with an overall sensitivity of 71% and a specificity of 93%. Lung cancer detection was independent from COPD and the presence of tobacco smoke and food odours. Logistic regression identified two drugs as potential confounders. It must be assumed that a robust and specific volatile organic compound (or pattern) is present in the breath of patients with lung cancer. Additional research efforts are required to overcome the current technical limitations of electronic sensor technologies to engineer a clinically applicable screening tool.

Protection against Shiga toxin-producing Escherichia coli infection by transcutaneous immunization with Shiga toxin subunit B.

Enterohemorrhagic Escherichia coli (EHEC) strains are important human food-borne pathogens. EHEC strains elaborate potent Shiga toxins (Stx1, and/or Stx2) implicated in the development of hemorrhagic colitis (HC) or hemolytic-uremic syndrome (HUS). In this report, we evaluated the immunogenicity and protective efficacy of Stx1 subunit B (StxB1) administered by transcutaneous immunization (TCI). Three groups of Dutch Belted rabbits received patches containing StxB1, StxB1 in combination with Escherichia coli heat-labile enterotoxin (LT), or LT alone. An additional group of naïve rabbits served as controls. The protective efficacy following TCI with StxB1 was assessed by challenging rabbits with a virulent Stx1-producing strain, RDEC-H19A, capable of inducing HC and HUS in rabbits. Antibodies specific to StxB1 from serum and bile samples were determined by enzyme-linked immunosorbent assay and toxin neutralization test. Rabbits immunized with StxB1 demonstrated improved weight gain and reduced Stx-induced histopathology. Rabbits receiving StxB or StxB1/LT showed a significant increase in serum immunoglobulin G titers specific to StxB1 as well as toxin neutralization titers. These data demonstrated that the StxB delivered by TCI could induce significant systemic immune responses. Thus, Stx subunit B vaccine delivered by a patch for a high-risk population may be a practical approach to prevent (and/or reduce) Stx-induced pathology.

LEE-encoded regulator (Ler) mutants elicit serotype-specific protection, but not cross protection, against attaching and effacing E. coli strains.

We previously showed that single dose orogastric immunization with an attenuated regulatory Lee-encoded regulator (ler) mutant of the rabbit enteropathogenic Escherichia coli (REPEC) strain E22 (O103:H2) protected rabbits from fatal infection with the highly virulent parent strain. In the current study we assessed the degree of homologous (serotype-specific) and heterologous (cross-serotype) protection induced by immunization with REPEC ler mutant strains of differing serotypes, or with a prototype strain RDEC-1 (O15:H-) which expresses a full array of ler up-regulated proteins. We constructed an additional ler mutant using RDEC-1 thus, permitting immunization with a ler mutant of either serotype, O15 or O103, followed by challenge with a virulent REPEC strain of the same or different serotypes. Consistent with our previous data, the current study demonstrated that rabbits immunized with a RDEC-1 ler mutant were protected from challenge with virulent RDEC-H19A (RDEC-1 transduced with Shiga toxin-producing phage H19A) of the same serotype. Rabbits immunized with RDEC-1 or E22 derivative ler mutants demonstrated significant increase in serum antibody titers to the respective whole bacterial cells expressing O antigen but not to the LEE-encoded proteins. However, immunization with the ler mutants of either E22 or RDEC-1 failed to protect rabbits from infections with virulent organisms belonging to different serotypes. In contrast, rabbits immunized with the prototype RDEC-1 were cross protected against challenge with the heterologous E22 strain as shown by normal weight gain, and the absence of clinical signs of disease or characteristic attaching and effacing (A/E) lesions. Immunization with RDEC-1 induced significantly elevated serum IgG titers to LEE-encoded proteins. We thus, demonstrated homologous protection induced by the REPEC ler mutants and heterologous protection by RDEC-1. The observed correlation between elevated immune responses to the LEE-encoded proteins and the protection against challenge with heterologous virulent REPEC strain suggests that serotype-non-specific cross protection requires the expression of, and induction of antibody to, LEE-encoded virulence factors.

Enteropathogenic Escherichia coli strain RDEC-1 produces a novel electrogenic factor active on rabbit ileum in vitro.

BACKGROUND: Attaching and effacing Escherichia coli demonstrate marked species specificity in inducing diarrhea, although its mechanism remains largely unclear. The purpose of this study was to investigate the existence of a soluble, species-specific factor that induces diarrhea in an in vitro model. METHODS: Stripped rabbit ileum was mounted in Ussing chambers, and changes in potential difference and short-circuit current were monitored after the addition of bacterial culture supernatant. RESULTS: The culture supernatant from rabbit-specific strain RDEC-1, but not from human-specific enteropathogenic Escherichia coli strain E2348/69, induced an increase in potential difference and short-circuit current in rabbit ileum mounted in Ussing chambers. This electrical signal was related to chloride ion secretion, was absent in colonic tissue, and was retained in the 30 to 100-KDa fraction of the supernatant. Preliminary experiments failed to show an involvement of calcium or cyclic nucleotides as intracellular messengers. RDEC-1 cured of a 42-MDa plasmid lost the enterotoxicity whereas conjugation of the plasmid into the negative E. coli recipient HB101 resulted in the expression of toxicity. CONCLUSIONS: The authors describe a novel, species-specific factor that helps to explain RDEC-1 diarrhea, which may be relevant to the pathogenesis of enteropathogenic Escherichia coli infection.

Complete nucleotide sequence and analysis of the locus of enterocyte Effacement from rabbit diarrheagenic Escherichia coli RDEC-1.

The pathogenicity island termed the locus of enterocyte effacement (LEE) is found in diverse attaching and effacing pathogens associated with diarrhea in humans and other animal species. To explore the relation of variation in LEE sequences to host specificity and genetic lineage, we determined the nucleotide sequence of the LEE region from a rabbit diarrheagenic Escherichia coli strain RDEC-1 (O15:H-) and compared it with those from human enteropathogenic E. coli (EPEC, O127:H6) and enterohemorrhagic E. coli (EHEC, O157:H7) strains. Differing from EPEC and EHEC LEEs, the RDEC-1 LEE is not inserted at selC and is flanked by an IS2 element and the lifA toxin gene. The RDEC-1 LEE contains a core region of 40 open reading frames, all of which are shared with the LEE of EPEC and EHEC. orf3 and the ERIC (enteric repetitive intergenic consensus) sequence present in the LEEs of EHEC and EPEC are absent from the RDEC-1 LEE. The predicted promoters of LEE1, LEE2, LEE3, tir, and LEE4 operons are highly conserved among the LEEs, although the upstream regions varied considerably for tir and the crucial LEE1 promoter, suggesting differences in regulation. Among the shared genes, high homology (>95% identity) between the RDEC-1 and the EPEC and EHEC LEEs at the predicted amino acid level was observed for the components of the type III secretion apparatus, the Ces chaperones, and the Ler regulator. In contrast, more divergence (66 to 88% identity) was observed in genes encoding proteins involved in host interaction, such as intimin (Eae) and the secreted proteins (Tir and Esps). A comparison of the highly variable genes from RDEC-1 with those from a number of attaching and effacing pathogens infecting different species and of different evolutionary lineages was performed. Although RDEC-1 diverges from some human-infecting EPEC and EHEC, most of the variation observed appeared to be due to evolutionary lineage rather than host specificity. Therefore, much of the observed hypervariability in genes involved in pathogenesis may not represent specific adaptation to different host species.

A Vibrio cholerae pathogenicity island associated with epidemic and pandemic strains.

The bacterial species Vibrio cholerae includes harmless aquatic strains as well as strains capable of causing epidemics and global pandemics of cholera. While investigating the relationship between pathogenic and nonpathogenic strains, we identified a chromosomal pathogenicity island (PAI) that is present in epidemic and pandemic strains but absent from nonpathogenic strains. Initially, two ToxR-regulated genes (aldA and tagA) were studied and were found to be associated with epidemic and pandemic strains but absent in nontoxigenic strains. The region containing aldA and tagA comprises 13 kb of previously unidentified DNA and is part of a PAI that contains a regulator of virulence genes (ToxT) and a gene cluster encoding an essential colonization factor and the cholera toxin phage receptor (toxin-coregulated pilus; TCP). The PAI is 39.5 kb in size, has low %G+C (35%), contains putative integrase and transposase genes, is flanked by att sites, and inserts near a 10Sa RNA gene (ssrA), suggesting it may be of bacteriophage origin. We found this PAI in two clinical non-O1/non-O139 cholera toxin-positive strains, suggesting that it can be transferred within V. cholerae. The sequence within this PAI includes an ORF with homology to a gene associated with the type IV pilus gene cluster of enteropathogenic Escherichia coli, a transposase from Vibrio anguillarum, and several ORFs with no known homology. As the PAI contains the CTXPhi receptor, it may represent the initial genetic factor required for the emergence of epidemic and pandemic cholera. We propose to call this island VPI (V. cholerae pathogenicity island).

The CS6 colonization factor of human enterotoxigenic Escherichia coli contains two heterologous major subunits.

The genes encoding the CS6 colonization factor were cloned from two human enterotoxigenic Escherichia coli strains of different serotypes. The DNA sequences from both clones were nearly identical and contained four open reading frames. Two of them have homology to genes encoding molecular chaperones and ushers found in many other operons encoding colonization factors. The two remaining open reading frames encode two heterologous major subunit proteins which makes CS6 unique because other colonization factors have only one major subunit. Upstream and downstream of the CS6 operon the DNA sequences of the clones diverged abruptly.

Enterohemorrhagic Escherichia coli: a family of emerging pathogens.

Enterohemorrhagic Escherichia coli (EHEC) have emerged over the last decade as important enteric pathogens because of their potential to induce both hemorrhagic colitis and fatal hemolytic uremic syndrome (HUS). HUS following EHEC colitis has become the leading cause of pediatric renal failure requiring kidney transplant in North America. The ability for EHEC to induce disease is dependent upon their ability to adhere to the intestinal mucosa in an intimate fashion, and to produce potent cytotoxins. These virulence factors (toxin production and enteroadherence) have been implicated in the pathogenesis of EHEC-induced disease. In this review we will discuss the symptomatology, epidemiology, laboratory diagnosis, pathogenesis, complications, treatment, and prevention of EHEC disease. We will review HUS with emphasis on treatment and prevention. Finally we will review animal models for EHEC infection in order to discuss their role in developing new strategies for the treatment and prevention of EHEC-associated diseases.

Cloning of the RDEC-1 locus of enterocyte effacement (LEE) and functional analysis of the phenotype on HEp-2 cells.

EPEC Escherichia coli are an important cause of epidemic diarrhea in infants. The disease is characterized by attaching and effacing (A/E) lesions where the bacteria attach intimately to the enterocyte surface resulting in localized destruction of microvilli. A 35-kb chromosomal locus termed LEE (locus of enterocyte effacement) in an EPEC strain (E2348/69) has recently been found and is thought to contain all the necessary genes for A/E lesions. RDEC-1 is a strain of E. coli that causes diarrhea in rabbits by a similar mechanism and serves as a model for human EPEC disease. We report 1) the cloning of the RDEC-1 LEE, 2) show that the RDEC-1 LEE is similar in size to the LEE in E2348/69, 3) the RDEC-1 LEE possesses all four regions of the E2348/69 LEE, 4) there are restriction site polymorphisms between the RDEC-1 LEE and that of E2348/69, and 5) the RDEC-1 LEE clone is functionally similar to E2348/69 in its fluorescent actin staining test and suggests that the LEE may be sufficient for the production of A/E lesions by these strains.

Characterization of the eaeA gene from rabbit enteropathogenic Escherichia coli strain RDEC-1 and comparison to other eaeA genes from bacteria that cause attaching-effacing lesions.

A number of enteric pathogens, including enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli, Hafnia alvei, a strain of Citrobacter freundii, and rabbit EPEC strain RDEC-1 cause attaching-effacing (AE) lesions in the gut mucosa. These bacteria have a pathogenicity cassette (locus of enterocyte effacement or LEE) containing the eaeA gene. This gene encodes intimin, an outer membrane protein required for production of AE lesions. RDEC-1, a non-invasive enteropathogen in young rabbits, produces AE lesions morphologically indistinguishable from lesions caused by human AE bacterial strains. The RDEC-1 example of E. coli diarrhea in rabbits is an important model for studying the pathogenesis of AE bacteria in a natural infection and for analyzing specific roles of the components of LEE. In order to better understand the role of intimin in the development of AE lesions, a portion of DNA within RDEC-1 LEE, containing the eaeA gene and an upstream open reading frame (ORF), was sequenced. The RDEC-1 eaeA gene shared 87%, 92%, and 93% DNA sequence identity and > 80% amino acid sequence identity with the eaeA genes of C. freundii biotype 4280, EHEC O157:H7, and EPEC O127:116, respectively. The carboxy-terminal 280 amino acid residues of intimin has 80%, 56%, and 54% identity with C. freundii, EHEC O157:H7, and EPEC O127:H6 intimins, respectively. The predicted protein encoded by the upstream ORF (156 amino acids) shares 95%, 97%, and 99% amino acid identity with predicted proteins from C. freundii. EHEC O157:H7, and EPEC O127:H6, respectively. The high degree of sequence homology of the ORF and the eueA gene of RDEC-1 with those of other AE bacteria suggests an evolutionary relationship of LEE and supports and facilitates the use of the RDEC-1 model for studying the role of LEE in pathogenesis.

Mucosal immune responses to intestinal bacterial pathogens.

Current advances in the study of gut mucosal immunology and molecular biology have enhanced our ability to understand the pathogenesis of enteric bacterial infections as well as the role of the immune system in mediating both tissue injury and protection. In this article, we review the immunopathogenesis and the protective immune response to three enteric pathogens, Vibrio cholerae, Shigella, and Salmonella. Each of these pathogens has a distinctive mechanism by which it causes disease, ie, epithelial attachment, epithelial invasion, and epithelial invasion with systemic dissemination. Pathogenicity and immune response can be conceptualized in terms of the interaction of these enteric pathogens with the gut epithelial compartment, immune inductive sites (Peyer's patch of the small intestine and lymphoid follicles of the colon), and a common immune effector compartment in the laimina propria where protective antibody is secreted. V cholerae, the representative noninvasive pathogen, has fimbrial adhesins that mediate attachment and colonization of the luminal surface of epithelial cells where organisms secrete cholera toxin (CT), a potent enterotoxin that induces a voluminous diarrhea via adenylate cyclase-dependent chloride secretion. Protective immunity is based on secretory (s) immunoglobulin A directed against whole-cell components that prevent attachment to gut epithelial cells and is enhanced by CT, an immunogen with potent adjuvant activity. Shigella, an enteric pathogen that locally invades gut epithelium, subverts the usual mechanism of immune sampling by initially invading via M cells overlying inductive sites. Subsequent macrophage invasion induces apoptosis and the release of interleukin-1, a proinflammatory cytokine. This seems to be a critical initiating event in immune-mediated tissue injury. Protective immunity is serotype specific. Infection caused by Salmonella is characterized by mucosal invasion and systemic spread mediated by the organisms ability to survive within macrophages. Both antibody and cell-mediated immunity are important for protection against Salmonella.

Assessment of attenuated Salmonella typhimurium strains designed as potential carriers of foreign antigens for mucosal localization and immuno-genicity in a rabbit model.

Neither wild type nor attenuated S. typhimurium strains induced diarrheal illness in rabbits. All strains localized to the Peyer's patch at higher concentrations than in lumenal contents or adjacent ileum. Wild type S. typhimurium C5 induced a typhoid-like illness in rabbits with severe weight loss, bacteremia, persistent splenic colonization, and serum IgG response. Both attenuated strains were disseminated to spleen (day 3) but produced minimal systemic illness. They induced biliary IgA responses greater than the wild type (day 7), but minimal serum IgG responses. Both mutants of S. typhimurium are suitable for further development as live enteric vaccines to carry foreign antigens since they localize to Peyer's patch after oral inoculation, induce biliary antibody, and produce minimal systemic disease. The attenuated strains tested are systemically disseminated. It remains to be determined whether dissemination (determined by a large virulence plasmid) is necessary for the desired mucosal immune response or acceptable for an oral vaccine strain.

Enteral immunization and challenge of volunteers given enterotoxigenic E. coli CFA/II encapsulated in biodegradable microspheres.

The development of a safe and effective vaccine against enterotoxigenic Escherichia coli (ETEC) would be useful for travellers and for young children in endemic areas. A feasibility study of an enteral ETEC vaccine prototype consisting of colonization factor antigen II (CFA/II), containing two component antigens CS1 and CS3, encapsulated in biodegradable polymer microspheres (BPM) was conducted in healthy volunteers. Ten adult volunteers swallowed intestinal tubes on days 0, 7, 14 and 28; after collection of jejunal fluid samples, 1 mg of CFA/II in BPM was administered via the tube. Volunteers kept a diary of symptoms after each dose. Secretory IgA in jejunal fluids, serum responses and circulating antibody-secreting cells (ASC) were measured before and after vaccination. The vaccine was well tolerated. Five of ten volunteers developed IgA anti-CFA/II ASC by 7 days after the last dose of vaccine; these same five vaccinees had IgA anti-CS3 ASC, and three of these five vaccinees had IgA anti-CS1 ASC. Five of ten vaccinees developed rises in jejunal fluid sIgA anti-CFA/II with peak GMT of 1:42. About 8 weeks after the first dose of vaccine, ten vaccinees and ten unvaccinated control volunteers underwent challenge with 10(9) c.f.u. ETEC E24377A (O139:H28 LT+ST+CS1+CS3+). Ten of ten controls and seven of ten vaccinees developed diarrhoea (p = 0.11, 30% vaccine efficacy). Two of the three protected vaccinees had the highest numbers of ASC and highest sIgA titres during the course of immunization, suggesting that these responses were protective and that this vaccine development strategy has merit. Future studies with higher dosages and a different dosing schedule are planned.

Role of Shiga-like toxin I in bacterial enteritis: comparison between isogenic Escherichia coli strains induced in rabbits.

BACKGROUND/AIMS: Enteroadherent Escherichia coli that produce Shiga-like toxins are important causes of human disease, including enterohemorrhagic E. coli-induced colitis (EHEC). The role of Shiga-like toxins in these illnesses is unclear. The aim of this study was to establish an animal model for human EHEC and to determine the role of Shiga-like toxin I (SLT-I) in this model. METHODS: E. coli strain RDEC-1 is an enteroadherent rabbit diarrheal pathogen. An isogenic variant of RDEC-1 (termed RDEC-H19A) producing high levels of SLT-I was obtained by infecting RDEC-1 with an SLT-I-converting bacteriophage. The effects of in vivo enteric infection produced in rabbits by RDEC-H19A were compared with those in uninfected and RDEC-1-infected animals. RESULTS: SLT-I-producing RDEC-H19A induced a severe, noninvasive, enteroadherent infection in rabbits. Clinically, infection with RDEC-H19A was more severe than infection with RDEC-1 and caused more serious histological lesions including vascular changes, edema, and more severe inflammation. Interleukin 1 and platelet-activating factor appear to be important inflammatory mediators to this infection. CONCLUSIONS: The illness induced by RDEC-H19A in rabbits resembled enterohemorrhagic E. coli-induced colitis of humans. SLT-I is an important virulence factor in the pathogenesis of EHEC.

Characterization of enterotoxigenic Escherichia coli isolated from U.S. troops deployed to the Middle East.

Enterotoxigenic Escherichia coli (ETEC) was a common cause of traveler's diarrhea in U.S. soldiers in the Middle East in 1989 and 1990. To determine which bacterial components would be useful in a vaccine, potential protective antigens (toxin, colonization factor antigen [CFA], and serotype) from 189 ETEC isolates were examined. Nearly half of the isolates expressed both ETEC toxins, 39% had only heat-stable enterotoxin (ST), and 17% had heat-labile enterotoxin (LT). CFA/I was the least common colonization factor antigen (11%), CFA/II was common (34%), as was CFA/IV (31%), and 24% expressed none of these CFAs. Fifty-seven O:H serotypes were found. Serotype O6:H16 was the most common, occurring in 29% of the ETEC isolates, usually with LT-ST and CFA/II. Generally, CFA/II was associated with expression of both toxins, CFA/IV was associated with expression of ST, and none of the CFAs was routinely found with LT. We conclude that ETEC from soldiers in the Middle East expressed a variety of antigens and that an effective vaccine will require multiple protective antigens.