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.
24 Related JoVE Articles!
Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein
Institutions: George Mason University.
Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3
. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6
. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ
PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.
Infectious Disease, Issue 43, HIV-1, Rev, GFP, lentiviral vector, RRE
Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding
Institutions: School of Medicine, New York University.
The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' surface envelope glycoprotein. We previously proposed that the crown of the V3 loop, although dynamic and sequence variable, is constrained throughout the population of HIV-1 viruses to an immunologically relevant β-hairpin tertiary structure. Importantly, there are thousands of different V3 loop crown sequences in circulating HIV-1 viruses, making 3D structural characterization of trends across the diversity of viruses difficult or impossible by crystallography or NMR. Our previous successful studies with folding of the V3 crown1, 2
used the ab initio
accessible in the ICM-Pro molecular modeling software package (Molsoft LLC, La Jolla, CA) and suggested that the crown of the V3 loop, specifically from positions 10 to 22, benefits sufficiently from the flexibility and length of its flanking stems to behave to a large degree as if it were an unconstrained peptide freely folding in solution. As such, rapid ab initio
folding of just this portion of the V3 loop of any individual strain of the 60,000+ circulating HIV-1 strains can be informative. Here, we folded the V3 loop of the R2 strain to gain insight into the structural basis of its unique properties. R2 bears a rare V3 loop sequence thought to be responsible for the exquisite sensitivity of this strain to neutralization by patient sera and monoclonal antibodies4, 5
. The strain mediates CD4-independent infection and appears to elicit broadly neutralizing antibodies. We demonstrate how evaluation of the results of the folding can be informative for associating observed structures in the folding with the immunological activities observed for R2.
Infection, Issue 43, HIV-1, structure-activity relationships, ab initio simulations, antibody-mediated neutralization, vaccine design
New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals
Institutions: Ragon Institute of MGH, MIT, and Harvard, Massachusetts General Hospital.
CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro
. Flow-sorted CD3+
Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.
Infection, Issue 75, Infectious Diseases, Medicine, Immunology, Virology, Cellular Biology, Molecular Biology, Lymphocytes, T-Lymphocytes, Regulatory, HIV, Culture Techniques, flow cytometry, cell culture, Treg expansion, regulatory T cells, CD4+ T cells, Tregs, HIV-1, virus, HIV-1 infection, AIDS, clinical techniques
Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays
Institutions: Rush University Medical Center.
Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2
. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3
. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4
. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6
. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7
. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8
called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51
Cr from within the target cell in the presence of NK cells.
Immunology, Issue 49, innate immunity, HIV-1, natural killer cell, cytolytic assay, degranulation assay, primary lymphocytes
Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test
Institutions: University of Bern, MCL Laboratories Inc..
Tuberculosis (TB) due to Mycobacterium tuberculosis
(MTB) remains a major public health issue: the infection affects up to one third of the world population1
, and almost two million people are killed by TB each year.2
Universal access to high-quality, patient-centered treatment for all TB patients is emphasized by WHO's Stop TB Strategy.3
The rapid detection of MTB in respiratory specimens and drug therapy based on reliable drug resistance testing results are a prerequisite for the successful implementation of this strategy. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods. Ineffective TB detection and the emergence and transmission of drug-resistant MTB strains increasingly jeopardize global TB control activities.2
Effective diagnosis of pulmonary TB requires the availability - on a global scale - of standardized, easy-to-use, and robust diagnostic tools that would allow the direct detection of both the MTB complex and resistance to key antibiotics, such as rifampicin (RIF). The latter result can serve as marker for multidrug-resistant MTB (MDR TB) and has been reported in > 95% of the MDR-TB isolates.4, 5
The rapid availability of reliable test results is likely to directly translate into sound patient management decisions that, ultimately, will cure the individual patient and break the chain of TB transmission in the community.2
Cepheid's (Sunnyvale, CA, U.S.A.) Xpert MTB/RIF assay6, 7
meets the demands outlined above in a remarkable manner. It is a nucleic-acids amplification test for 1) the detection of MTB complex DNA in sputum or concentrated sputum sediments; and 2) the detection of RIF resistance-associated mutations of the rpoB
It is designed for use with Cepheid's GeneXpert Dx System that integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences using real-time PCR and reverse transcriptase PCR. The system consists of an instrument, personal computer, barcode scanner, and preloaded software for running tests and viewing the results.9
It employs single-use disposable Xpert MTB/RIF cartridges that hold PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated.6
Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to universal-access point-of-care settings3
, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.10
Immunology, Issue 62, tuberculosis, drug resistance, rifampicin, rapid diagnosis, Xpert MTB/RIF test
Implantation of the Syncardia Total Artificial Heart
Institutions: Virginia Commonwealth University, Virginia Commonwealth University.
With advances in technology, the use of mechanical circulatory support devices for end stage heart failure has rapidly increased. The vast majority of such patients are generally well served by left ventricular assist devices (LVADs). However, a subset of patients with late stage biventricular failure or other significant anatomic lesions are not adequately treated by isolated left ventricular mechanical support. Examples of concomitant cardiac pathology that may be better treated by resection and TAH replacement includes: post infarction ventricular septal defect, aortic root aneurysm / dissection, cardiac allograft failure, massive ventricular thrombus, refractory malignant arrhythmias (independent of filling pressures), hypertrophic / restrictive cardiomyopathy, and complex congenital heart disease. Patients often present with cardiogenic shock and multi system organ dysfunction. Excision of both ventricles and orthotopic replacement with a total artificial heart (TAH) is an effective, albeit extreme, therapy for rapid restoration of blood flow and resuscitation. Perioperative management is focused on end organ resuscitation and physical rehabilitation. In addition to the usual concerns of infection, bleeding, and thromboembolism common to all mechanically supported patients, TAH patients face unique risks with regard to renal failure and anemia. Supplementation of the abrupt decrease in brain natriuretic peptide following ventriculectomy appears to have protective renal effects. Anemia following TAH implantation can be profound and persistent. Nonetheless, the anemia is generally well tolerated and transfusion are limited to avoid HLA sensitization. Until recently, TAH patients were confined as inpatients tethered to a 500 lb pneumatic console driver. Recent introduction of a backpack sized portable driver (currently under clinical trial) has enabled patients to be discharged home and even return to work. Despite the profound presentation of these sick patients, there is a 79-87% success in bridge to transplantation.
Medicine, Issue 89, mechanical circulatory support, total artificial heart, biventricular failure, operative techniques
Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays
Institutions: NCI-Frederick, University of Pittsburgh, Tuffts University.
Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART).
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19
and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15
,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19
. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.
Immunology, Issue 55, single genome sequencing, SGS, real-time PCR, single-copy assay, SCA, HIV-1, ultra-sensitive, RNA extraction
The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism
Institutions: University of Montréal, McGill University, University of Minnesota.
Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33
. To help improve this understanding, proton magnetic resonance spectroscopy (1
H-MRS) can be used as it allows the in vivo
quantification of brain metabolites such as γ-aminobutyric acid (GABA) and glutamate in a region-specific manner 41
. In fact, a recent study demonstrated that 1
H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34
. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1
H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31
. Methodological factors to consider and possible modifications to the protocol are also discussed.
Neuroscience, Issue 93, proton magnetic resonance spectroscopy, transcranial direct current stimulation, primary motor cortex, GABA, glutamate, stroke
Corticospinal Excitability Modulation During Action Observation
Institutions: Universita degli Studi di Padova.
This study used the transcranial magnetic stimulation/motor evoked potential (TMS/MEP) technique to pinpoint when the automatic tendency to mirror someone else's action becomes anticipatory simulation of a complementary act. TMS was delivered to the left primary motor cortex corresponding to the hand to induce the highest level of MEP activity from the abductor digiti minimi (ADM; the muscle serving little finger abduction) as well as the first dorsal interosseus (FDI; the muscle serving index finger flexion/extension) muscles. A neuronavigation system was used to maintain the position of the TMS coil, and electromyographic (EMG) activity was recorded from the right ADM and FDI muscles. Producing original data with regard to motor resonance, the combined TMS/MEP technique has taken research on the perception-action coupling mechanism a step further. Specifically, it has answered the questions of how and when observing another person's actions produces motor facilitation in an onlooker's corresponding muscles and in what way corticospinal excitability is modulated in social contexts.
Behavior, Issue 82, action observation, transcranial magnetic stimulation, motor evoked potentials, corticospinal excitability
Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing
Institutions: University Hospital Center and University of Lausanne.
Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.
Immunology, Issue 92, blood culture, bacteriology, identification, antibiotic susceptibility testing, MALDI-TOF MS.
Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies
Institutions: Flemish Institute for Technological Research (VITO), Hasselt University, Hasselt University, Leuven University.
The microcirculation consists of blood vessels with diameters less than 150 µm. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.
Medicine, Issue 92, retina, microvasculature, image analysis, Central Retinal Arteriolar Equivalent, Central Retinal Venular Equivalent, air pollution, particulate matter, black carbon
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
Institutions: University of KwaZulu-Natal, Durban, South Africa, Jembi Health Systems, University of Amsterdam, Stanford Medical School.
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
Medicine, Issue 85, Biomedical Technology, HIV-1, HIV Infections, Viremia, Nucleic Acids, genetics, antiretroviral therapy, drug resistance, genotyping, affordable
Community-based Adapted Tango Dancing for Individuals with Parkinson's Disease and Older Adults
Institutions: Emory University School of Medicine, Brigham and Woman‘s Hospital and Massachusetts General Hospital.
Adapted tango dancing improves mobility and balance in older adults and additional populations with balance impairments. It is composed of very simple step elements. Adapted tango involves movement initiation and cessation, multi-directional perturbations, varied speeds and rhythms. Focus on foot placement, whole body coordination, and attention to partner, path of movement, and aesthetics likely underlie adapted tango’s demonstrated efficacy for improving mobility and balance. In this paper, we describe the methodology to disseminate the adapted tango teaching methods to dance instructor trainees and to implement the adapted tango by the trainees in the community for older adults and individuals with Parkinson’s Disease (PD). Efficacy in improving mobility (measured with the Timed Up and Go, Tandem stance, Berg Balance Scale, Gait Speed and 30 sec chair stand), safety and fidelity of the program is maximized through targeted instructor and volunteer training and a structured detailed syllabus outlining class practices and progression.
Behavior, Issue 94, Dance, tango, balance, pedagogy, dissemination, exercise, older adults, Parkinson's Disease, mobility impairments, falls
The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo
Institutions: Australian National University.
The ability to monitor T cell responses in vivo
is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into >250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+
T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+
T cell-mediated killing of FTA target cells and CD4+
T cell-meditated help of FTA B cell target cells in real time in vivo
by flow cytometry. Since >250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.
the cumulative magnitude of the response) and a qualitative (e.g
. functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.
Immunology, Issue 88, Investigative Techniques, T cell response, Flow Cytometry, Multiparameter, CTL assay in vivo, carboxyfluorescein succinimidyl ester (CFSE), CellTrace Violet (CTV), Cell Proliferation Dye eFluor 670 (CPD)
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
Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology
Institutions: DNA Medicine Institute, Harvard Medical School, NASA Glenn Research Center, ZIN Technologies.
Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.
Cellular Biology, Issue 93, Point-of-care, prototype, diagnostics, spaceflight, reduced gravity, parabolic flight, flow cytometry, fluorescence, cell counting, micromixing, spiral-vortex, blood mixing
Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors
Institutions: National Cancer Institute.
Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.
Immunology, Issue 86, HIV, cytotoxicity, infectivity, luciferase, drug resistance, integrase, reverse transcriptase
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Confocal Imaging of Confined Quiescent and Flowing Colloid-polymer Mixtures
Institutions: University of Houston.
The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed assembly1-3
, drug delivery4
, improved hydrocarbon recovery5-7
, and flowable electrodes for energy storage8
. Suspensions containing fluorescent colloids and non-adsorbing polymers are appealing model systems, as the ratio of the polymer radius of gyration to the particle radius and concentration of polymer control the range and strength of the interparticle attraction, respectively. By tuning the polymer properties and the volume fraction of the colloids, colloid fluids, fluids of clusters, gels, crystals, and glasses can be obtained9
. Confocal microscopy, a variant of fluorescence microscopy, allows an optically transparent and fluorescent sample to be imaged with high spatial and temporal resolution in three dimensions. In this technique, a small pinhole or slit blocks the emitted fluorescent light from regions of the sample that are outside the focal volume of the microscope optical system. As a result, only a thin section of the sample in the focal plane is imaged. This technique is particularly well suited to probe the structure and dynamics in dense colloidal suspensions at the single-particle scale: the particles are large enough to be resolved using visible light and diffuse slowly enough to be captured at typical scan speeds of commercial confocal systems10
. Improvements in scan speeds and analysis algorithms have also enabled quantitative confocal imaging of flowing suspensions11-16,37
. In this paper, we demonstrate confocal microscopy experiments to probe the confined phase behavior and flow properties of colloid-polymer mixtures. We first prepare colloid-polymer mixtures that are density- and refractive-index matched. Next, we report a standard protocol for imaging quiescent dense colloid-polymer mixtures under varying confinement in thin wedge-shaped cells. Finally, we demonstrate a protocol for imaging colloid-polymer mixtures during microchannel flow.
Chemistry, Issue 87, confocal microscopy, particle tracking, colloids, suspensions, confinement, gelation, microfluidics, image correlation, dynamics, suspension flow
Molecular Evolution of the Tre Recombinase
Institutions: Max Plank Institute for Molecular Cell Biology and Genetics, Dresden.
Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells.
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.
Cell Biology, Issue 15, HIV-1, Tre recombinase, Site-specific recombination, molecular evolution
Using Visual and Narrative Methods to Achieve Fair Process in Clinical Care
Institutions: Brandeis University, Brandeis University.
The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is respect for patient's values, preferences, and expressed needs. Yet specific approaches to gaining this understanding and translating it to quality care in the clinical setting are lacking. From a patient perspective quality is not a simple concept but is best understood in terms of five dimensions: technical outcomes; decision-making efficiency; amenities and convenience; information and emotional support; and overall patient satisfaction. Failure to consider quality from this five-pronged perspective results in a focus on medical outcomes, without considering the processes central to quality from the patient's perspective and vital to achieving good outcomes. In this paper, we argue for applying the concept of fair process in clinical settings. Fair process involves using a collaborative approach to exploring diagnostic issues and treatments with patients, explaining the rationale for decisions, setting expectations about roles and responsibilities, and implementing a core plan and ongoing evaluation. Fair process opens the door to bringing patient expertise into the clinical setting and the work of developing health care goals and strategies. This paper provides a step by step illustration of an innovative visual approach, called photovoice or photo-elicitation, to achieve fair process in clinical work with acquired brain injury survivors and others living with chronic health conditions. Applying this visual tool and methodology in the clinical setting will enhance patient-provider communication; engage patients as partners in identifying challenges, strengths, goals, and strategies; and support evaluation of progress over time. Asking patients to bring visuals of their lives into the clinical interaction can help to illuminate gaps in clinical knowledge, forge better therapeutic relationships with patients living with chronic conditions such as brain injury, and identify patient-centered goals and possibilities for healing. The process illustrated here can be used by clinicians, (primary care physicians, rehabilitation therapists, neurologists, neuropsychologists, psychologists, and others) working with people living with chronic conditions such as acquired brain injury, mental illness, physical disabilities, HIV/AIDS, substance abuse, or post-traumatic stress, and by leaders of support groups for the types of patients described above and their family members or caregivers.
Medicine, Issue 48, person-centered care, participatory visual methods, photovoice, photo-elicitation, narrative medicine, acquired brain injury, disability, rehabilitation, palliative care
Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase
Institutions: Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg.
HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies primarily target virus enzymes or virus-cell fusion, suppressing the viral life cycle without eradicating the infection. Since the integrated provirus is not targeted by these approaches, new resistant strains of HIV-1 may emerge. Here, we report that the engineered recombinase Tre (see Molecular evolution of the Tre recombinase , Buchholz, F., Max Planck Institute for Cell Biology and Genetics, Dresden) efficiently excises integrated HIV-1 proviral DNA from the genome of infected cells. We produced loxLTR containing viral pseudotypes and infected HeLa cells to examine whether Tre recombinase can excise the provirus from the genome of HIV-1 infected human cells. A virus particle-releasing cell line was cloned and transfected with a plasmid expressing Tre or with a parental control vector. Recombinase activity and virus production were monitored. All assays demonstrated the efficient deletion of the provirus from infected cells without visible cytotoxic effects. These results serve as proof of principle that it is possible to evolve a recombinase to specifically target an HIV-1 LTR and that this recombinase is capable of excising the HIV-1 provirus from the genome of HIV-1-infected human cells.
Before an engineered recombinase could enter the therapeutic arena, however, significant obstacles need to be overcome. Among the most critical issues, that we face, are an efficient and safe delivery to targeted cells and the absence of side effects.
Medicine, Issue 16, HIV, Cell Biology, Recombinase, provirus, HeLa Cells
Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools
Institutions: Brigham and Women's Hospital.
Cellular Biology, Issue 8, microfluidics, diagnostics, capture, blood, HIV, bioengineering