[Show abstract][Hide abstract]ABSTRACT: Listeriosis is one of the most lethal bacterial diseases for fetuses and infants. However, pregnant women who get infected with Listeria may experience only mild symptoms, making the diagnosis difficult, even when the fetus is fatally infected.
To reveal features of this infection, we conducted a multimodality imaging study of Listeria-induced miscarriage, using a pregnant mouse model. In this model, fetal morbidity and mortality can be observed in utero, noninvasively, and the timing and extent of infection can be carefully controlled. By employing in vivo bioluminescence imaging (BLI), perinatal infections were localized over time such that a correlation of infection to outcome could be determined without the need to kill the animal subject. The morbidity and viability of fetuses were assessed with ultrasound, and fetal morphology was imaged using magnetic resonance imaging (MRI).
The ultrasound revealed sustained fetal bradycardia, the slowing of the fetal heartbeat, in infected fetuses, with an association between slowed fetal heart rate and strong bioluminescent signal.
Uninfected fetuses showing no bioluminescent signal in the same uterine horn exhibited normal heartbeats. Thus, fetal bradycardia during infection was localized to the infected fetus and was not systemic or disseminated.
Full-text available · Article · Feb 2012 · Pediatric Research
[Show abstract][Hide abstract]ABSTRACT: We used mast cell-engrafted genetically mast cell-deficient C57BL/6-Kit(W-sh/W-sh) mice to investigate the roles of mast cells and mast cell-derived tumor necrosis factor in two models of severe bacterial infection. In these mice, we confirmed findings derived from studies of mast cell-deficient WBB6F(1)-Kit(W/W-v) mice indicating that mast cells can promote survival in cecal ligation and puncture (CLP) of moderate severity. However, we found that the beneficial role of mast cells in this setting can occur independently of mast cell-derived tumor necrosis factor. By contrast, using mast cell-engrafted C57BL/6-Kit(W-sh/W-sh) mice, we found that mast cell-derived tumor necrosis factor can increase mortality during severe CLP and can also enhance bacterial growth and hasten death after intraperitoneal inoculation of Salmonella typhimurium. In WBB6F(1)-Kit(W-sh/W-sh) mice, mast cells enhanced survival during moderately severe CLP but did not significantly change the survival observed in severe CLP. Our findings in three types of genetically mast cell-deficient mice thus support the hypothesis that, depending on the circumstances (including mouse strain background, the nature of the mutation resulting in a mast cell deficiency, and type and severity of infection), mast cells can have either no detectable effect or opposite effects on survival during bacterial infections, eg, promoting survival during moderately severe CLP associated with low mortality but, in C57BL/6-Kit(W-sh/W-sh) mice, increasing mortality during severe CLP or infection with S. typhimurium.
Full-text available · Article · Feb 2010 · American Journal Of Pathology
[Show abstract][Hide abstract]ABSTRACT: Murine listeriosis is one of the most comprehensive and well-studied models of infection, and Listeria monocytogenes has provided seminal information regarding bacterial pathogenesis. However, many aspects of the mouse model remain poorly understood, including carrier states and chronic colonization which represent important features of the spectrum of host-pathogen interaction. Bone marrow has recently been shown to harbor L. monocytogenes, which spreads from this location to the central nervous system. Bone could, therefore, be an important chronic reservoir, but this infection is difficult to study because it involves only a few bacteria and the extent of infection cannot be assessed until after the animal is sacrificed. We employed in vivo bioluminescence imaging to localize L. monocytogenes bone infections over time in live mice, revealing that the bacteria grow in discrete foci. These lesions can persist in many locations in the legs of mice and are not accompanied by a histological indication such as granuloma or a neutrophil infiltratate. We demonstrate that highly attenuated hly mutants, which have defective intracellular replication, are capable of prolonged focal infection of the bone marrow for periods of up to several weeks. These results support the recently proposed hypothesis that the bone marrow is a unique niche for L. monocytogenes.
Article · Feb 2009 · Disease Models and Mechanisms
[Show abstract][Hide abstract]ABSTRACT: We present a handheld dual-axes confocal microscope that is based on a two-dimensional microelectromechanical systems (MEMS) scanner. It performs reflectance and fluorescence imaging at 488 nm wavelength, with three-dimensional imaging capability. The fully packaged microscope has a diameter of 10 mm and acquires images at 4 Hz frame rate with a maximum field of view of 400 microm x 260 microm. The transverse and axial resolutions of the handheld probe are 1.7 microm and 5.8 microm, respectively. Capability to perform real time small animal imaging is demonstrated in vivo in transgenic mice.
Full-text available · Article · Jun 2008 · Optics Express
[Show abstract][Hide abstract]ABSTRACT: A combination of targeted probes and new imaging technologies provides a powerful set of tools with the potential to improve the early detection of cancer. To develop a probe for detecting colon cancer, we screened phage display peptide libraries against fresh human colonic adenomas for high-affinity ligands with preferential binding to premalignant tissue. We identified a specific heptapeptide sequence, VRPMPLQ, which we synthesized, conjugated with fluorescein and tested in patients undergoing colonoscopy. We imaged topically administered peptide using a fluorescence confocal microendoscope delivered through the instrument channel of a standard colonoscope. In vivo images were acquired at 12 frames per second with 50-lm working distance and 2.5-lm (transverse) and 20-lm (axial) resolution. The fluorescein-conjugated peptide bound more strongly to dysplastic colonocytes than to adjacent normal cells with 81% sensitivity and 82% specificity. This
Full-text available · Article · May 2008 · Nature Medicine
[Show abstract][Hide abstract]ABSTRACT: A combination of targeted probes and new imaging technologies provides a powerful set of tools with the potential to improve the early detection of cancer. To develop a probe for detecting colon cancer, we screened phage display peptide libraries against fresh human colonic adenomas for high-affinity ligands with preferential binding to premalignant tissue. We identified a specific heptapeptide sequence, VRPMPLQ, which we synthesized, conjugated with fluorescein and tested in patients undergoing colonoscopy. We imaged topically administered peptide using a fluorescence confocal microendoscope delivered through the instrument channel of a standard colonoscope. In vivo images were acquired at 12 frames per second with 50-microm working distance and 2.5-microm (transverse) and 20-microm (axial) resolution. The fluorescein-conjugated peptide bound more strongly to dysplastic colonocytes than to adjacent normal cells with 81% sensitivity and 82% specificity. This methodology represents a promising diagnostic imaging approach for the early detection of colorectal cancer and potentially of other epithelial malignancies.
Full-text available · Article · May 2008 · Nature medicine
[Show abstract][Hide abstract]ABSTRACT: The process by which bacteria regulate flagellar expression is known as phase variation and in Salmonella enterica this process permits the expression of one of two flagellin genes, fliC or fljB, at any one time. Salmonella Typhi (S. Typhi) is normally not capable of phase variation of flagellar antigen expression as isolates only harbour the fliC gene (H:d) and lacks an equivalent fljB locus. However, some S. Typhi isolates, exclusively from Indonesia, harbour an fljB equivalent encoded on linear plasmid, pBSSB1 that drives the expression of a novel flagellin named H:z66. H:z66+S. Typhi isolates were stimulated to change flagellar phase and genetically analysed for the mechanism of variation. The phase change was demonstrated to be unidirectional, reverting to expression from the resident chromosomal fliC gene. DNA sequencing demonstrated that pBSSB1 linear DNA was still detectable but that these derivatives had undergone deletion and were lacking fljA(z66) (encoding a flagellar repressor) and fljB(z66). The deletion end-point was found to involve one of the plasmid termini and a palindromic repeat sequence within fljB(z66), distinct to that found at the terminus of pBSSB1. These data demonstrate that, like some Streptomyces linear elements, at least one of the terminal inverted repeats of pBSSB1 is non-essential, but that a palindromic repeat sequence may be necessary for replication.
Full-text available · Article · Jan 2008 · Molecular Microbiology
[Show abstract][Hide abstract]ABSTRACT: Author Summary
Flagella are whip-like structures found on the surface of bacterial cells that mediate swimming. Flagella contain a protein called flagellin, which is recognised as a danger signal by the immune system. Salmonella Typhi, the bacteria that causes typhoid fever, normally have flagella called H:d, but some strains only from Indonesia express distinct flagella, called H:z66. In this study we have located and sequenced the genes responsible for expressing these alternative flagella. Remarkably, these genes are located on a linear plasmid, an extra-chromosomal element that we have named pBSSB1. The significance of this finding is that linear plasmids are relatively common in bacterial species such as Streptomyces and Borrelia. However, such a linear element has never previously been described in enteric bacteria such as Escherichia coli and Salmonella. The identification of this novel linear plasmid in genetically tractable bacteria will facilitate future studies on the biology of linear plasmids and the pathogenicity of both flagella and Salmonella Typhi.
Full-text available · Article · Jun 2007 · PLoS Pathogens
[Show abstract][Hide abstract]ABSTRACT: The Annotation of pBSSB1
Table S1 includes the sequential CDS numbers, the strand of the CDSs, the location of the CDSs, and the size (amino acids) of the putative protein encoded by the CDSs. Any features or hypothetical function of the putative proteins are also given. For putative proteins with significant BLASTP hits against the GenBank database, the organism, locus tag designation, and E-value of the best hit is provided.
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[Show abstract][Hide abstract]ABSTRACT: The tirs of pBSSB1
(A) Map of the terminal (3 kbp) at either end of pBSSB1 showing the tirs and the adjacent genes. The locations of the primer sites for the lambda red recombinase kanamycin insertion are highlighted by pink arrows marked 1, 2, 3, and 4; these correspond to primers z66_red_1 to z66_red_4, respectively (Table S1). The locations of the primers for PCR probing of the tirs are shown by yellow arrows a, b, c, d, and e, which correspond to primers tir_a to tir_e, respectively (Table S1).
(B) Agarose gel of PCR amplicons produced within the tirs. Upper bands were amplified using DNA from S. Typhi In20 as a template; lower bands were amplified using DNA from E. coli SGB33 as a template. Sizes are estimated in comparison to Hyperladder I (Hp). Lanes correspond to the combination of primers used in the PCR reaction; these are designated in (A). Amplicons produced by a+d and b+d using DNA from E. coli SGB33 as the template are approximately 1.5 kbp larger than those of S. Typhi In20; this is due to the kanamycin cassette insertion at position 1,295.
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[Show abstract][Hide abstract]ABSTRACT: Listeria monocytogenes is a ubiquitous gram-positive bacterium that can cause systemic and often life-threatening disease in immunocompromised hosts.
This organism is largely an intracellular pathogen; however, we have determined that it can also grow extracellularly in animals,
in the lumen of the gallbladder. The significance of growth in the gallbladder with respect to the pathogenesis and spread
of listeriosis depends on the ability of the bacterium to leave this organ and be disseminated to other tissues and into the
environment. Should this process be highly inefficient, growth in the gallbladder would have no impact on pathogenesis or
spread, but if it occurs efficiently, bacterial growth in this organ may contribute to listeriosis and dissemination of this
organism. Here, we use whole-body imaging to determine the efficacy and kinetics of food- and hormone-induced biliary excretion
of L. monocytogenes from the murine gallbladder, demonstrating that transit through the bile duct into the intestine can occur within 5 min of
induction of gallbladder contraction by food or cholecystokinin and that movement of bacteria through the intestinal lumen
can occur very rapidly in the absence of fecal material. These studies demonstrate that L. monocytogenes bacteria replicating in the gallbladder can be expelled from the organ efficiently and that the released bacteria move into
the intestinal tract, where they pass into the environment and may possibly reinfect the animal.
Full-text available · Article · Apr 2006 · Infection and Immunity
[Show abstract][Hide abstract]ABSTRACT: The bacterium Listeria monocytogenes can cause a life-threatening systemic illness in humans. Despite decades of progress in animal models of listeriosis, much
remains unknown about the processes of infection and colonization. Here, we report that L. monocytogenes can replicate in the murine gall bladder and provide evidence that its replication there is extracellular and intraluminal.
In vivo bioluminescence imaging was employed to determine the location of the infection over time in live animals, revealing
strong signals from the gall bladder over a period of several days, in diseased as well as asymptomatic animals. The data
suggest that L. monocytogenes may be carried in the human gall bladder.
Full-text available · Article · Mar 2004 · Science
[Show abstract][Hide abstract]ABSTRACT: Heme oxygenase (HO), a key catabolic enzyme in the conversion of heme to bilirubin, is an ideal target for reducing bilirubin production and preventing pathological jaundice in newborn infants. Metalloporphyrins (Mps) have been well characterized as competitive inhibitors of HO and have been evaluated as potential chemopreventive agents for neonatal jaundice. However, in addition to reducing HO activity, many Mps have been shown to increase HO-1 transcription, which would likely reduce their potential therapeutic utility. The differential effects of Mps on the transcription of HO-1 were therefore evaluated in living transgenic (Tg) reporter mice. Of the compounds evaluated, we observed that zinc bis-glycol porphyrin (ZnBG), a potent inhibitor of HO enzyme activity, did not alter HO-1 transcription patterns in Tg mice. Whole body images of HO-1 transcription patterns did, however; reveal increases in HO-1 transcription in Tg mice after treatment with other Mps, heme and cadmium chloride (CdCl(2)). Intravenous injections of CdCl(2) resulted in expression patterns that differed in tempo and location from those observed in Tg mice treated with intraperitoneal injections. Spatiotemporal analyses of transcriptional regulation in living animals accelerated the assessment of an adverse effect of Mps by revealing different patterns of HO-1 transcription. Among the known inhibitors of HO enzyme activity that were evaluated in this study, ZnBG did not significantly affect HO-1 transcription and therefore may be well suited for the prevention of neonatal jaundice.
Full-text available · Article · Nov 2002 · Journal of Molecular Medicine
[Show abstract][Hide abstract]ABSTRACT: Lymphocytes are highly mobile cells that travel throughout the body in response to a tremendous variety of stimuli. Revealing lymphocyte trafficking patterns in vivo is necessary for a complete understanding of immune function, as well as cell-cell and cell-tissue interactions in immune development and in response to insult. Although the location of cell populations in various tissues at any given point in time may be revealed by techniques such as flow cytometry and immunofluorescence, these methods are not readily amenable to the assessment of dynamic cell migration patterns in vivo. In the past 5 years, technologies for imaging molecular and cellular changes in living animals have advanced to a point where it is possible to reveal the migratory paths of these vitally important cells. Here, we review one advancement in cellular imaging, in vivo bioluminescence imaging, which addresses the problem of lymphocyte tracking. This imaging strategy has the potential to elucidate the temporal patterns of immune responses and the spatial distribution of lymphocytes within the body.
Full-text available · Article · Jan 2002 · Experimental Hematology