Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.
20 Related JoVE Articles!
Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats
Institutions: Massachusetts General Hospital, Harvard Medical School.
Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that neural mechanisms for certain behavioral tasks and response behaviors are different in females and males. Using females in research studies can represent a challenge because of the variation of gonadal hormones during their estrous cycle. This protocol describes well-established procedures that are useful in investigating the role of estrogen in fear extinction memory consolidation in female rats. Phase of the estrous cycle and exogenous estrogen administration prior to extinction training can influence extinction recall 24 hr later. The vaginal swabbing technique for estrous phase identification described here aids the examination and manipulation of naturally cycling gonadal hormones. The use of this basic rodent model may further delineate the mechanisms by which estrogen can modulate fear extinction memory in females.
Behavior, Issue 96, estrogen, fear extinction, sex differences, estradiol, proestrus, metestrus, female, PTSD, anxiety
Preparation and Pathogen Inactivation of Double Dose Buffy Coat Platelet Products using the INTERCEPT Blood System
Institutions: Örebro University Hospital.
Blood centers are faced with many challenges including maximizing production yield from the blood product donations they receive as well as ensuring the highest possible level of safety for transfusion patients, including protection from transfusion transmitted diseases. This must be accomplished in a fiscally responsible manner which minimizes operating expenses including consumables, equipment, waste, and personnel costs, among others.
Several methods are available to produce platelet concentrates for transfusion. One of the most common is the buffy coat method in which a single therapeutic platelet unit (≥ 2.0 x1011
platelets per unit or per local regulations) is prepared by pooling the buffy coat layer from up to six whole blood donations. A procedure for producing "double dose" whole blood derived platelets has only recently been developed.
Presented here is a novel method for preparing double dose whole blood derived platelet concentrates from pools of 7 buffy coats and subsequently treating the double dose units with the INTERCEPT Blood System for pathogen inactivation. INTERCEPT was developed to inactivate viruses, bacteria, parasites, and contaminating donor white cells which may be present in donated blood. Pairing INTERCEPT with the double dose buffy coat method by utilizing the INTERCEPT Processing Set with Dual Storage Containers (the "DS set"), allows blood centers to treat each of their double dose units in a single pathogen inactivation processing set, thereby maximizing patient safety while minimizing costs. The double dose buffy coat method requires fewer buffy coats and reduces the use of consumables by up to 50% (e.g.
pooling sets, filter sets, platelet additive solution, and sterile connection wafers) compared to preparation and treatment of single dose buffy coat platelet units. Other cost savings include less waste, less equipment maintenance, lower power requirements, reduced personnel time, and lower collection cost compared to the apheresis technique.
Medicine, Issue 70, Immunology, Hematology, Infectious Disease, Pathology, pathogen inactivation, pathogen reduction, double-dose platelets, INTERCEPT Blood System, amotosalen, UVA, platelet, blood processing, buffy coat, IBS, transfusion
Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats
Institutions: University of California, Irvine (UCI).
Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.
Immunology, Issue 8, Rodent, Hypersensitivity, Mouse, Skin, Immune Reaction, Blood Draw, Serum, Video Protocol, Vaccination, Adjuvant
Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus
Institutions: Marine Biological Laboratory, University of California San Francisco, Vrije Universiteit Amsterdam, Max Planck Institute of Molecular Cell Biology and Genetics, University of Illinois Urbana-Champaign.
Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus
, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor
cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor
cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a "double decker" microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.
Cellular Biology, Issue 82, intracellular wound healing, cytoplasm, rheology, protists, ciliates, regeneration, microscopy, Stentor coeruleus
Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities
Institutions: Université de Strasbourg.
Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols.
We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.
Neuroscience, Issue 89, mice, nociception, tail immersion test, tail pressure test, morphine, analgesia, opioid-induced hyperalgesia, tolerance
Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples
Institutions: University of Manitoba, University of Manitoba.
Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo
flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.
Medicine, Issue 89, mucosal, immunology, FGT, lavage, cervical, CMC
Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research
Institutions: University of California, San Francisco, Veterans Affairs Medical Center, San Francisco, Veterans Affairs Medical Center, San Francisco.
The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone.
Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia.
The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction.
There exists a non-invasive, in vivo
method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia.
This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.
Medicine, Issue 92, endothelial function, endothelial dysfunction, brachial artery, peripheral artery disease, ultrasound, vascular, endothelium, cardiovascular disease.
A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology
Institutions: Bangor University, Russells Hall Hospital, University of Manchester.
The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.
Medicine, Issue 96, Endothelium, Cardiovascular, Flow-mediated dilatation, Carotid intima-media thickness, Atherosclerosis, Nitric oxide, Microvasculature, Laser Doppler Imaging
Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations
Institutions: Wake Forest University Health Sciences, R.J. Reynolds Tobacco Company.
Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo
suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo
assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo
assays. We show that secreted cytokines IFN-γ, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-γ, TNF-α, and MIP-1α were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo
assays, and these methods could be readily adapted for testing other products of interest.
Immunology, Issue 95, Tobacco product preparation, whole smoke-conditioned medium, human peripheral blood mononuclear cells, PBMC, lipopolysaccharide, cell death, secreted cytokines, intracellular cytokines, K562 killing assay.
Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways
Institutions: University of Colorado School of Medicine, Oregon Health & Science University, University of Colorado School of Medicine.
Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.
Neuroscience, Issue 82, male, female, sex, neuronal culture, ischemia, cell death, neuroprotection
Infinium Assay for Large-scale SNP Genotyping Applications
Institutions: Oklahoma Medical Research Foundation.
Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina's panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.
Basic Protocol, Issue 81, genomics, SNP, Genotyping, Infinium, iScan, HiScan, Illumina
Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty
Institutions: University of Arizona, University of Arizona.
A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2
. In HIV infection the syndrome occurs at a younger age.
HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal.
The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.
Medicine, Issue 77, Infection, Virology, Infectious Diseases, Anatomy, Physiology, Molecular Biology, Biomedical Engineering, Retroviridae Infections, Body Weight Changes, Diagnostic Techniques and Procedures, Physical Examination, Muscle Strength, Behavior, Virus Diseases, Pathological Conditions, Signs and Symptoms, Diagnosis, Musculoskeletal and Neural Physiological Phenomena, HIV, HIV-1, AIDS, Frailty, Depression, Weight Loss, Weakness, Slowness, Exhaustion, Aging, clinical techniques
Peptide-based Identification of Functional Motifs and their Binding Partners
Institutions: Morehouse School of Medicine, Institute for Systems Biology, Universiti Sains Malaysia.
Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.
Virology, Issue 76, Biochemistry, Immunology, Infection, Infectious Diseases, Molecular Biology, Medicine, Genetics, Microbiology, Genomics, Proteins, Exosomes, HIV, Peptides, Exocytosis, protein trafficking, secretion, HIV-1, Nef, Secretion Modification Region, SMR, peptide, AIDS, assay
Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols
Institutions: University of British Columbia , University of British Columbia , University of British Columbia , University of British Columbia .
Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3
. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4
), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5
. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3
. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7
, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9
and mask RBC surface antigens10, 11
Immunology, Issue 71, Bioengineering, Pathology, Chemistry, Biochemistry, Hematology, polymers, Blood transfusion, surface antigens, antigen camouflage, RBC modification, hyperbranched polyglycerol, HPG, red blood cells, RBC, whole blood, flow cytometry
Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood
Institutions: Yale University School of Medicine .
Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2
. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3
. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4
and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5
. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-κB, which initiates transcription of numerous genes that are critical for immune responses8
. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-κB pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9
Immunology, Issue 62, monocyte, dendritic cells, Toll-like receptors, fluorescent imaging, signaling, FACS, aging
Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil
Institutions: Oxitec Ltd, Universidade de São Paulo, Universidade de São Paulo, Moscamed Brasil, University of Oxford, Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM).
New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)1-3
. A control method known as RIDL (Release of Insects carrying a Dominant Lethal)4
, is based around SIT, but uses genetic methods to remove the need for radiation-sterilization5-8
. A RIDL strain of Ae. aegypti
was successfully tested in the field in Grand Cayman9,10
; further field use is planned or in progress in other countries around the world.
Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL control program, released as either pupae or adults11,12
To suppress a mosquito population using RIDL a large number of high quality male adults need to be reared13,14
. The following describes the methods for the mass rearing of OX513A, a RIDL strain of Ae. aegypti 8,
for release and covers the techniques required for the production of eggs and mass rearing RIDL males for a control program.
Basic Protocol, Issue 83, Aedes aegypti, mass rearing, population suppression, transgenic, insect, mosquito, dengue
Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites
Institutions: National Institutes of Health, Xiamen University.
Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3
. The malaria parasite Plasmodium yoelii
is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9
. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum
. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii
that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10
Genetic crosses in P. yoelii
and other rodent malaria parasites are conducted by infecting mice Mus musculus
with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11
. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.
Infectious Disease, Issue 47, Genetic cross, genetic mapping, malaria, rodent
Dissection of Saccharomyces Cerevisiae Asci
Institutions: Concordia University.
Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae
exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.
Cellular Biology, Issue 27, asci, ascospores, diploid, zygote, sporulation, yeast dissection, micromanipulator
Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies
Institutions: University of California, Irvine (UCI).
Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (>80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.
Immunology, issue 8, blood, cell isolation, natural killer, lymphocyte, primary cells, negative selection, PBMC, Ficoll gradient, cell separation
Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques
Institutions: University of Duisburg-Essen.
The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since then, DBS testing for decades has remained predominantly focused on the diagnosis of infectious diseases especially in resource-limited settings or the systematic screening of newborns for inherited metabolic disorders and only recently have a variety of new and innovative DBS applications begun to emerge. For many years, pre-analytical variables were only inappropriately considered in the field of DBS testing and even today, with the exception of newborn screening, the entire pre-analytical phase, which comprises the preparation and processing of DBS for their final analysis has not been standardized. Given this background, a comprehensive step-by-step protocol, which covers al the essential phases, is proposed, i.e.,
collection of blood; preparation of blood spots; drying of blood spots; storage and transportation of DBS; elution of DBS, and finally analyses of DBS eluates. The effectiveness of this protocol was first evaluated with 1,762 coupled serum/DBS pairs for detecting markers of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus infections on an automated analytical platform. In a second step, the protocol was utilized during a pilot study, which was conducted on active drug users in the German cities of Berlin and Essen.
Molecular Biology, Issue 97, Dried blood spots, filter paper cards, specimen storage, infectious diseases, hepatitis B virus, hepatitis C virus, human immunodeficiency virus