Name: methods for rapid transfer and localization of lyme disease pathogens within the tick gut
Uploaded: 2011-02-14T20:00:00
Description: Watch this Scientific Journal Video about Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut at JoVE.com

We sincerely thank members of the Pal laboratory for assistance with the preparation of this demonstration. This study was supported by PHS grants AI076684 and AI080615 from NIH/NIAID.

1. Microinjection of Nymphal Ixodes scapularis Ticks
1. Preparing needles
<ol>
<li>Fabricate several microinjection needles by heating and pulling 1 mm glass 	capillary tubes (World Precision Instruments) in a glass micropipette puller device (Narishige). Carefully remove the fragile capillary tubes.</li>
<li>Store pulled needles (with tip facing upward) on adhesive tape in a Petri dish.</li>
</ol>
2. Preparing B. burgdorferi
<ol>
<li>Grow <e...

<ol>
	<li>Steere, A. C., Coburn, J., Glickstein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emergence+of+Lyme+disease.">The emergence of Lyme disease.</a> J Clin Invest. 113, 1093-1101 (2004).</li><li>Barthold, S. W., Diego, C., Philipp, M. T., Samuels, D. S., Radolf, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Borrelia, Molecular Biology, Host Interaction and Pathogenesis. , 353-405 (2010).</li><li>Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OspC+facilitates+Borrelia+burgdorferi+invasion+of+Ixodes+scapularis+salivary+glands.">OspC facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands.</a> J Clin Invest. 113, 220-230 (2004).</li><li>Yang, X. F., Pal, U., Alani, S. M., Fikrig, E., Norgard, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+for+OspA/B+in+the+life+cycle+of+the+Lyme+disease+spirochete.">Essential role for OspA/B in the life cycle of the Lyme disease spirochete.</a> J Exp Med. 199, 641-648 (2004).</li><li>Zhang, X., Yang, X., Kumar, M., Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BB0323+function+is+essential+for+Borrelia+burgdorferi+virulence+and+persistence+through+tick-rodent+transmission+cycle.">BB0323 function is essential for Borrelia burgdorferi virulence and persistence through tick-rodent transmission cycle.</a> J Infect Dis. 200, 1318-1330 (2009).</li><li>Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TROSPA,+an+Ixodes+scapularis+receptor+for+Borrelia+burgdorferi.">TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi.</a> Cell. 119, 457-468 (2004).</li><li>Barbour, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cultivation+of+Lyme+disease+spirochetes.">Isolation and cultivation of Lyme disease spirochetes.</a> Yale J Biol Med. 57, 521-525 (1984).</li><li>Narasimhan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Ixodes+scapularis+anticoagulation+by+using+RNA+interference.">Disruption of Ixodes scapularis anticoagulation by using RNA interference.</a> Proc Natl Acad Sci U S A. 101, 1141-1146 (2004).</li><li>Narasimhan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tick+antioxidant+facilitates+the+Lyme+disease+agent's+successful+migration+from+the+mammalian+host+to+the+arthropod+vector.">A tick antioxidant facilitates the Lyme disease agent's successful migration from the mammalian host to the arthropod vector.</a> Cell Host Microbe. 2, 7-18 (2007).</li><li>Broadwater, A. H., Sonenshine, D. E., Hynes, W. L., Ceraul, S., DeSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glass+capillary+tube+feeding:+a+method+for+infecting+nymphal+Ixodes+scapularis+(Acari:+Ixodidae)+with+the+Lyme+disease+spirochete+Borrelia+burgdorferi.">Glass capillary tube feeding: a method for infecting nymphal Ixodes scapularis (Acari: Ixodidae) with the Lyme disease spirochete Borrelia burgdorferi.</a> J Med Entomol. 39, 285-292 (2002).</li><li>Policastro, P. F., Schwan, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+infection+of+Ixodes+scapularis+larvae+(Acari:+Ixodidae)+by+immersion+in+low+passage+cultures+of+Borrelia+burgdorferi.">Experimental infection of Ixodes scapularis larvae (Acari: Ixodidae) by immersion in low passage cultures of Borrelia burgdorferi.</a> J Med Entomol. 40, 364-370 (2003).</li><li>Fuente, d. e. l. a., Kocan, J., M, K., Almazan, C., Blouin, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+for+the+study+and+genetic+manipulation+of+ticks.">RNA interference for the study and genetic manipulation of ticks.</a> Trends Parasitol. 23, 427-433 (2007).</li></ol>

Here we demonstrate a microinjection-based procedure for rapid and efficient infection of nymphal Ixodes ticks with the bacterial pathogen B. burgdorferi. We also describe a confocal immunofluorescence procedure for the detection of B. burgdorferi in the tick gut in situ. Although our demonstration involves nymphal gut, similar procedures are also applicable for other developmental stages of ticks, such as larva or adults 8,9. However, due to their smaller size, the technique may b...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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		<tbody>
		
<tr>
<td>Glass capillary tubes</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TW100F-4</td>
<td>&nbsp;</td>
<tr>
<td>Vertical glass puller</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>PC-10</td>
<td>&nbsp;</td>
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<td>Petroff-Hausser counting chamber</td>
<td>&nbsp;</td>
<td>Hausser scientific</td>
<td>3900</td>
<td>&nbsp;</td>
<tr>
<td>Microloader pipette tips</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>930001007</td>
<td>&nbsp;</td>
<tr>
<td>Femtojet microinjector</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>920010504</td>
<td>&nbsp;</td>
<tr>
<td>Foot control FemtJet</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>920005098</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate buffered saline</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP665-1</td>
<td>Filter-sterilized</td>
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methods for rapid transfer and localization of lyme disease pathogens within the tick gut

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1. A tick-borne murine model 2 has been developed to study Lyme disease in the laboratory. While na&iacute;ve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease 3,4. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks 5. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA 6. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

Watch this Scientific Journal Video about Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut at JoVE.com

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks. Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours. We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

methods-for-rapid-transfer-localization-lyme-disease-pathogens-within

Research

JoVE Journal

Immunology and Infection

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This process has notably lower efficiencies in the Lyme disease spirochete relative to other, better-characterized Gram-negative bacteria. The rate of success of transformation is highly dependent upon having concentrated amounts of high-quality DNA from specific backgrounds and is subject to significant strain-to-strain variability. Alternative means for introducing foreign DNA (i.e., shuttle vectors, fluorescent reporters, and antibiotic-resistance markers) into B. burgdorferi could be an important addition to the armamentarium of useful tools for the genetic manipulation of the Lyme disease spirochete. Bacteriophage have been well-recognized as natural mechanisms for the movement of DNA among bacteria in a process called transduction. In this study, a method has been developed for using the ubiquitous borrelial phage &#966;BB-1 to transduce DNA between B. burgdorferi cells of both the same and different genetic backgrounds. The transduced DNA includes both borrelial DNA and heterologous DNA in the form of small shuttle vectors. This demonstration suggests a potential use of phage-mediated transduction as a complement to electroporation for the genetic manipulation of the Lyme disease spirochete. This report describes methods for the induction and purification of phage &#966;BB-1 from B. burgdorferi, the use of this phage in transduction assays, and the selection and screening of potential transductants.

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

The ability of bacteriophage to move DNA between bacterial cells makes them effective tools for the genetic manipulation of their bacterial hosts. Presented here is a methodology for inducing, recovering, and using &#966;BB-1, a bacteriophage of Borrelia burgdorferi, to transduce heterologous DNA between different strains of the Lyme disease spirochete.

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This ...