Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.
22 Related JoVE Articles!
Isolation, Processing and Analysis of Murine Gingival Cells
Institutions: Hebrew University - Hadassah Medical Center, Hebrew University - Hadassah Medical Center.
We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1
. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.
Immunology, Issue 77, Infection, Medicine, Cellular Biology, Molecular Biology, Anatomy, Physiology, Periodontology, Gingiva, Periodontitis, Flow cytometry, mice, oral mucosa, gingival cells, animal model
Chronic Salmonella Infected Mouse Model
Institutions: University of Rochester.
The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella
typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo
. Mice with Salmonella
persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella
typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella
persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.
Microbiology, Issue 39, Salmonella, intestine, colitis, chronic infection, mouse model
Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR
Institutions: Universidad Peruana Cayetano Heredia, Johns Hopkins University, University of Concepcion,Chile, University of California San Diego School of Medicine.
Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer.
Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3
. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al.
which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5
is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.
Virology, Issue 65, Medicine, Genetics, norovirus, gastroenteritis, RNA extraction, diarrhea, stool samples, PCR, RT-PCR, TaqMan, silica
Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature
Institutions: Paris Cardiovascular Research Centre, Université Paris Descartes.
causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo
models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis
adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis
infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.
Infection, Issue 86, Disease Models, Bacteria, Bacterial Infections and Mycoses, Neisseria meningitidis, purpura, vascular infection, humanized model
In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint
Institutions: University of California San Francisco, University of California San Francisco, Xradia Inc..
This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ
loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e.
from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo
conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.
Bioengineering, Issue 85, biomechanics, bone-periodontal ligament-tooth complex, concentric loads, eccentric loads, contrast agent
Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision
Institutions: University of Zürich.
Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 ± 1.1 µm for trueness and 1.6 ± 0.6 µm for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 ± 2.2 µm, precision: 12.5 ± 2.5 µm) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.
Medicine, Issue 86, Laboratories, Dental, Calibration, Technology, Dental impression, Accuracy, Trueness, Precision, Full arch scan, Abrasion
Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods
Institutions: Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8
; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10
. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers & also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13i.e.
enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 & CD44), hematopoietic/endothelial Markers (CD34, CD45 & CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) & Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE & dentin sialophosphoprotein; DSPP).14
Stem Cell Biology, Issue 69, Medicine, Developmental Biology, Cellular Biology, Bioengineering, Dental pulp tissue, Human third molar, Human dental pulp stem cells, hDPSC, Odontoblasts, Outgrown stem cells, MSC, differentiation
Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation
Institutions: University Hospital Münster, University Children's Hospital Münster.
Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo,
necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live
mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo
murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo
imaging and may significantly impact on preclinical research in various fields.
Medicine, Issue 90,
gastroenterology, in vivo imaging, murine endoscopy, diagnostic imaging, carcinogenesis, intestinal wound healing, experimental colitis
Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites
Institutions: UMR CNRS 5615, Université Lyon1, Hospices Civils de Lyon, APHP, Hôpital Rothschild.
It is generally accepted that in vitro
cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro
biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro
assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.
Medicine, Issue 93, In vitro biocompatibility, dental composites, Live/Deadstaining, 3D imaging, Confocal Microscopy, Time lapse imaging
The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms
Institutions: Bar-Ilan University, Bar-Ilan University, Bar-Ilan University, Bar-Ilan University.
Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5
. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7
. There are also reports of biocidal effect of red and near infra red8
as well as green light9
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa
. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa
is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa
strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa
. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa
was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.
Microbiology, Issue 77, Infection, Infectious Diseases, Cellular Biology, Molecular Biology, Biophysics, Chemistry, Biomedical Engineering, Bacteria, Photodynamic therapy, Medical optics, Bacterial viability, Antimicrobial treatment, Laser, Gentamycin, antibiotics, reactive oxygen species, pathogens, microorganisms, cell culture
Assessment of Vascular Function in Patients With Chronic Kidney Disease
Institutions: University of Colorado, Denver, University of Colorado, Boulder.
Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA
) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA
, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA
, aPWV, and vascular endothelial cell protein expression.
Medicine, Issue 88, chronic kidney disease, endothelial cells, flow-mediated dilation, immunofluorescence, oxidative stress, pulse-wave velocity
Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry
Institutions: Sanford-Burnham Medical Research Institute, University of California, San Diego , VA San Diego Healthcare Center, University of California, San Diego .
Although human saliva proteome and peptidome have been revealed 1-2
they were majorly identified from tryptic digests of saliva proteins. Identification of indigenous peptidome of human saliva without prior digestion with exogenous enzymes becomes imperative, since native peptides in human saliva provide potential values for diagnosing disease, predicting disease progression, and monitoring therapeutic efficacy. Appropriate sampling is a critical step for enhancement of identification of human indigenous saliva peptidome. Traditional methods of sampling human saliva involving centrifugation to remove debris 3-4
may be too time-consuming to be applicable for clinical use. Furthermore, debris removal by centrifugation may be unable to clean most of the infected pathogens and remove the high abundance proteins that often hinder the identification of low abundance peptidome.
Conventional proteomic approaches that primarily utilize two-dimensional gel electrophoresis (2-DE) gels in conjugation with in-gel digestion are capable of identifying many saliva proteins 5-6
. However, this approach is generally not sufficiently sensitive to detect low abundance peptides/proteins. Liquid chromatography-Mass spectrometry (LC-MS) based proteomics is an alternative that can identify proteins without prior 2-DE separation. Although this approach provides higher sensitivity, it generally needs prior sample pre-fractionation 7
and pre-digestion with trypsin, which makes it difficult for clinical use.
To circumvent the hindrance in mass spectrometry due to sample preparation, we have developed a technique called capillary ultrafiltration (CUF) probes 8-11
. Data from our laboratory demonstrated that the CUF probes are capable of capturing proteins in vivo
from various microenvironments in animals in a dynamic and minimally invasive manner 8-11
. No centrifugation is needed since a negative pressure is created by simply syringe withdrawing during sample collection. The CUF probes combined with LC-MS have successfully identified tryptic-digested proteins 8-11
. In this study, we upgraded the ultrafiltration sampling technique by creating a lollipop-like ultrafiltration (LLUF) probe that can easily fit in the human oral cavity. The direct analysis by LC-MS without trypsin digestion showed that human saliva indigenously contains many peptide fragments derived from various proteins. Sampling saliva with LLUF probes avoided centrifugation but effectively removed many larger and high abundance proteins. Our mass spectrometric results illustrated that many low abundance peptides became detectable after filtering out larger proteins with LLUF probes. Detection of low abundance saliva peptides was independent of multiple-step sample separation with chromatography. For clinical application, the LLUF probes incorporated with LC-MS could potentially be used in the future to monitor disease progression from saliva.
Medicine, Issue 66, Molecular Biology, Genetics, Sampling, Saliva, Peptidome, Ultrafiltration, Mass spectrometry
Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat
Institutions: University College London, University of Gothenburg.
Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli
K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli
O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.
Infection, Issue 92, Bacterial infection, neonatal bacterial meningitis, bacteremia, sepsis, animal model, K1 polysaccharide, systemic infection, gastrointestinal tract, age dependency
A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites
Institutions: Boston University School of Medicine, Boston University School of Medicine.
Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis
, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis
accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.
Immunology, Issue 90,
Pathogen-Induced Chronic Inflammation; Porphyromonas gingivalis; Oral Bone Loss; Periodontal Disease; Atherosclerosis; Chronic Inflammation; Host-Pathogen Interaction; microCT; MRI
Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90,
zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms
Institutions: The University of Michigan, Newcastle University.
There are few high-throughput in vitro
systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo
oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo
communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus
, and Porphyromonas
), a protocol will also be presented describing how to harvest the biofilm cells for further subculture or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.
Bioengineering, Issue 94, Dental plaque, biofilm, confocal laser scanning microscopy, three-dimensional structure, pyrosequencing, image analysis, image reconstruction, saliva, modeling, COMSTAT, IMARIS, IMAGEJ, multi-species biofilm communities.
Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae
Institutions: McMaster University .
Nasopharyngeal colonization by Streptococcus pneumoniae
is a prerequisite to invasion to the lungs or bloodstream1
. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2
, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae
Our mouse model of intranasal colonization is adapted from human models3
and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7
. In the first part of the model, we use a clinical isolate of S. pneumoniae
to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8
, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.
Immunology, Issue 83, Streptococcus pneumoniae, Nasal lavage, nasopharynx, murine, flow cytometry, RNA, Quantitative PCR, recruited macrophages, neutrophils, T-cells, effector cells, intranasal colonization
qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue
Institutions: Indiana University School of Medicine, Indiana University Health.
It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.
Genetics, Issue 89, qPCR, cytomegalovirus, CMV, biopsy, real-time PCR, gastrointestinal, formalin-fixed, paraffin-embedded tissue
Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy
Institutions: Sainte-Justine University Hospital Research Center, Université de Montréal.
This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells' responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.
Medicine, Issue 80, Blood Cells, Lymphocytes, Spinal Diseases, Diagnostic Techniques and Procedures, Clinical Laboratory Techniques, Dielectric Spectroscopy, Musculoskeletal Diseases, Idiopathic scoliosis, classification, prognosis, G proteins, cellular dielectric spectroscopy, PBMCs
A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia
Institutions: Universite de Montreal, Universite de Montreal, Universite de Montreal.
There are several lines of evidence supporting the role of de novo
mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo
mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo
mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1
and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo
mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2
. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo
mutations. This is the case for autism and schizophrenia3
. The male-to-female ratio of mutation rate is estimated at about 4–6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo
mutations would more frequently come from males, particularly older males4
. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo
mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo
mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.
Medicine, Issue 52, de novo mutation, complex diseases, schizophrenia, autism, rare variations, DNA sequencing
RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract
Institutions: University of Texas Southwestern Medical Center , Harvard Medical School, University of Texas Southwestern Medical Center .
(PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1
. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2
, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo
transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3
in order to identify changes in bacterial gene expression during alterations to the host’s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4
. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.
Immunology, Issue 55, Pseudomonas, RNA, murine, cecum, transcriptome, qPCR, RT-PCR, PCR
Invasion of Human Cells by a Bacterial Pathogen
Institutions: University of Bath.
Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus
. The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus
would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2
at 37°C. For S. aureus
this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 μl tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.
Infection, Issue 49, Bacterial pathogen, host cell invasion, Staphylococcus aureus, invasin