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Our previous and present studies demonstrated that innocuous, mechanical stimulation of the ulnar nerve at HT7, an acupoint at the ulnar nerve, suppresses drug-seeking behaviors and drug-induced DA release in NAc11, 12, 25, 27. During such stimulation, the superficial or deep afferents of ulnar nerve are activated and the afferent signals are transmitted via A-fiber of ulnar nerve11. The afferent inputs reduce drug-induced DA release in the NAc by normalizing the decreased activity of VTA GABA neurons in addicted rats9, 12, 22. While our previous and present studies demonstrated that under drug-induced conditions, innocuous sensory inputs produced a consistent suppression of psychomotor behaviors and DA release11, 12, 25, 27, others have shown mixed results in responses of midbrain DA neurons to innocuous stimulation in drug-naïve conditions. For examples, non-noxious stimuli such as foot pressure and cervical probing elicit short latency facilitation or inhibition of midbrain DA neurons in drug-naïve rats28. Innocuous sensory stimulation by electrical stimulation or 80–100 mmHg pressure to the skin can produce a slight decrease in DA release in the dorsal striatum4, 5 or a mild increase in DA release in the NAc15. This discrepancy may be due to anatomical segregation of functional subgroups in midbrain DA neurons responding to sensory stimuli. In support of this, one previous study demonstrated that DA neurons activated by sensory stimuli are located dorsolaterally in the substantia nigra pars, while DA neurons inhibited by the stimuli are found ventromedially, some in the VTA29. In the present study, the afferent inputs, which were conveyed via DC pathway, attenuated cocaine-induced c-fos expression in NAc. It led to the speculation that afferent inputs from cutaneous innocuous stimuli would inhibit DA neurons in the VTA causing a reduced DA release in NAc, thereby suppressing cocaine-induced psychomotor behaviors.
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study
| 99.94 |
How does the activation of DC pathways by ulnar MS suppress drug-seeking behaviors? The LHb, an epithalamic structure, projects to the VTA and the RMTg, which contain GABA neurons that inhibit DAergic activity and the behavioral effects of drugs of abuse19. Electrical stimulation of LHb neurons elicits a strong inhibition of VTA DA neurons, while inhibition of the LHb is associated with the activation of DA neurons18. Activation of the habenula reduces addiction to cocaine, nicotine, morphine and ethanol19, 30, 31, while lesions of the habenula increase drug seeking behaviors32. Our findings demonstrate that ulnar MS increased both local temperature and c-fos expression in LHb neurons projecting to the VTA/RMTg regions and LHb lesions blocked ulnar inhibition of cocaine locomotion, indicating that excitation of habenula neurons by ulnar MS can suppress psychomotor responses or NAc-neuronal activity to cocaine (Figs 4–6). Taken together, these findings provide convincing evidence that the DC somatosensory pathway is functionally linked to the LHb-VTA/RMTg pathway, which may underlie somatosensory inhibition of cocaine-induced psychomotor behaviors. Although the LHb has been shown to receive somatosensory inputs16, 20, it is not known yet how somatosensory inputs enter the LHb, which receives input from structures, such as the prefrontal cortex, lateral hypothalamus and entopeduncular nucleus33. As there are no known direct pathways projecting from VPL to the LHb, linking DC somatosensory inputs to the LHb may require multi-synaptic pathways. Lesions of the VPL abolished neuronal activation of LHb by peripheral stimulation, suggesting a putative VPL-LHb link. Others have shown that the VPL projects to prefrontal cortex34, which sends excitatory inputs to the LHb35. Connections between the VPL and LHb may be linked via the prefrontal cortex, which requires further elucidation. Regardless, previous studies have demonstrated that LHb neurons are sensitive to noxious stimuli. For example, LHb neurons respond to peripheral noxious stimuli16 and the expression of the immediately early gene c-fos in LHb is induced after noxious stimuli36. Our findings suggest that innocuous stimulation of the ulnar nerve would activate LHb neurons, as measured by local temperature, c-fos expression and in vivo extracellular recordings in the LHb. Thus, there is the possibility that noxious as well as innocuous stimuli can activate LHb neurons under certain conditions and inhibit psychomotor response to cocaine.
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study
| 100.0 |
The present study revealed that stimulation of the ulnar nerve suppressed cocaine-induced hyperlocomotion and the effects were completely abolished by lesions to the DC pathway and LHb, but not to the STT. Additionally, activation of the DC pathway or the LHb was detected following ulnar MS. These findings strongly suggest that the DC somatosensory pathway conveys an inhibitory signal from the periphery to mesolimbic reward circuits and the activation of spinal DC pathway can inhibit psychomotor response to cocaine.
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study
| 100.0 |
Male Sprague-Dawley rats (270–320 g, Daehan Animal, Seoul, Korea) were used for this study. All rats had free access to food and water and were maintained on a 12 hr light-dark cycle. All procedures were approved by the Institutional Animal Care and Use Committee at the Daegu Haany University and conducted in accordance with National Institutes of Health guidelines for the care and use of laboratory animals. Each group consisted of 6–8 rats, unless stated otherwise.
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study
| 99.94 |
Locomotor activity was measured with an image analysis system (Ethovision 3.1, Noldus Information Technology BV, Netherlands) as previously described11. Briefly, each animal was placed into a square open field box (40 cm × 40 cm × 45 cm) and monitored with an overhead video camera and video tracking software. After recording baseline activity for 30 min, the animal was given an intraperitoneal (i.p.) injection of cocaine (15 mg/kg) and monitored for up to 60 min after injection. The distance travelled during each 10-min period was analyzed. The data are expressed as a percentage of baseline activity.
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study
| 100.0 |
Since our previous studies showed that mechanical stimulation of a needle inserted into the ulnar tunnel (Guyon’s Canal or HT7 acupuncture Shenmen Point) in the forelimb produces intense and reproducible inhibitory effects on various addictive behaviors caused by cocaine, morphine and ethanol9, 11, 12, 22, a peripheral stimulation was performed as described in our previous experiments11. Mechanical stimulation was performed about 1 min after cocaine injection. Briefly, while an assistant lightly restrained the rat, needles (0.10 mm in diameter, 10 mm in length of needle; Dongbang Medical Co., Korea) were inserted bilaterally 3 mm deep into the ulnar tunnel on the transverse crease of the wrist of the forepaw. By using a newly-developed mechanical instrument that consisted of a custom-made control unit and a mechanical vibrator connected to the needle, the inserted needle was mechanically stimulated for 20 sec in duration at an intensity of 1.3 m/sec2, maintained for 1 min after needle insertion and subsequently withdrawn. Following mechanical stimulation, locomotor activity was monitored for 60 min after cocaine injection. To assess the possibility that the mechanical stimulation of the wrist impaired locomotor behaviors, the radial side of wrist joint was mechanically stimulated in an identical fashion as a control condition.
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study
| 100.0 |
A small skin incision was made longitudinally on the medial part of the elbow to expose the ulnar nerve under isoflurane anesthesia. The ulnar nerve was bilaterally ligated with 4–0 silk and cut around the medial head of the triceps muscle of both forelimbs. Forty-eight to 72 hr after the nerve injury, the rats were subjected to the locomotor activity test following acute cocaine injection.
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study
| 99.94 |
Lesions of DC or STT were performed as previously described37, 38 with slight modifications. Briefly: (1) dorsal column lesions were made bilaterally to a depth of 0.4 mm into the cord using fine tipped Dumont #5 forceps (Fine Science Tools, USA) with aid of a dissecting microscope after a laminectomy at the C3 portion of the spinal cord; (2) For surgical lesions of cuneate nucleus (CN; second-order neurons of DC pathway), the animal was placed in the stereotaxic frame with the head tilted forward, the obex was exposed surgically and the cuneate nucleus was macerated with the fine forceps along its length; (3) For lesioning the STT pathway, the ventrolateral funiculus (VLF) at the C3 portion of the spinal cord was lifted with a fire polished glass rod and the tip of a 22 gauge needle was used to damage the left and right ventrolateral quadrants. Animals in the sham group in each experiment were subjected to a laminectomy or surgical exposure of the obex without damaging the nerve. The rats were allowed to recover for at least 7 days after surgery.
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| 100.0 |
For VPL lesions, ibotenic acid (0.5 μl/site) was bilaterally microinjected (third-order neurons of the DC pathway)39. The anesthetized rat (pentobarbital, 50 mg/kg, i.p.) was placed in a stereotaxic frame, and two holes were drilled in the skull to access the following coordinates: anterior, −2.5~−3 mm; lateral, ±2.75~3 mm; deep, 6 mm40. A 26-gauge Hamilton syringe (Reno, NV, USA) filled with either ibotenic acid or saline was infused at a rate of 0.5 μl/min using a microinjection pump (Pump 22, Harvard Apparatus, USA). The syringe was left in place for at least 5 min to facilitate diffusion after injection. For electrolytic lesion of bilateral LHb, tungsten electrodes insulated except at 0.5 mm tip were inserted in the following coordinates: anterior, −3.5 mm; lateral, ±0.7 mm, deep −4.9 mm. Lesions were made by passing ± 0.35 mA of DC current for 8 sec alternately. The rats were allowed to recover for at least 7 days after surgery.
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study
| 99.94 |
At the termination of the experiments, all rats were sacrificed for histological confirmation of lesions. All lesions were confirmed by toluidine blue stain. The animals were perfused with phosphate-buffered saline (PBS) and then with 4% paraformaldehyde. The brains and spinal cords were removed, post-fixed in 4% paraformaldehyde and cryoprotected in 30% sucrose. The tissue was then sectioned into 30 μm-thick sections using a cryostat (Leica CM 1850; Leica Biosystems, Germany) and stained with toluidine blue. Only those rats with histologically-verified lesions were included in the data analysis.
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study
| 99.94 |
The in vivo extracellular single unit recordings in the CN were performed according to a previously published method41. In brief, rats were deeply anesthetized with an intraperitoneal (i.p.) injection of sodium pentobarbital (60 mg/kg) for surgery, were mounted on the stereotaxic frame, and were supplemented with sodium pentobarbital (5 mg/kg/h) infused intravenously through a jugular vein catheter during the recording process to maintain anesthesia. The adequacy of anesthesia was monitored by the lack of withdrawal reflexes to noxious stimuli and the absence of corneal blink reflexes. Body temperature was maintained at 37 °C with a thermostatically controlled heating blanket with a rectal probe. Extracellular potentials of single neurons in the CN were recorded using a carbon-filament glass microelectrode (Carbostar-1, Kation Scientific, USA) at a depth of 0.0–0.8 mm ventral from the dorsal surface of the medulla, 1 mm caudal to the obex and 1–2 mm lateral from the midline40. Neurons were classified as low-threshold (LT) or wide-dynamic range (WDR) neurons based on the response to mechanical somatic stimuli. LT neurons were excited by only innocuous stimuli (light pressure), and WDR neurons responded to both innocuous (light pressure) and noxious stimuli (blunt clip). The signals were amplified (Iso80, World Precision Instruments, USA) and recorded using a micro1401 and Spike2 software (Cambridge Electronic Design, UK).
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study
| 100.0 |
Single-unit electrophysiological recordings in the LHb were performed with a slight modification as described previously42. In brief, rats (n = 7) were placed in a stereotaxic frame under pentobarbital anesthesia (60 mg/kg, i.p.). Anesthesia level was maintained by intravenous infusion of sodium pentobarbital (15 mg/kg/hr) during the recording. A single microcarbon (7 μm diameter) filament-filled glass electrode (carbon fiber electrode, impedence 0.4–1.2 MΩ, Kation Scientific, USA) was positioned into the LHb (anterior, −3.5 mm; lateral ± 0.7 mm; deep, −4.9 mm) with one-axis water hydraulic micromanipulator (Narishige, USA). Neuronal signals were amplified 10,000 times, filtered at 0.3 to 10 kHz and digitized by using an Iso80 amplifier and a microCED-1401. Single-unit activity was isolated by Spike2 software. Once stable baseline activity was achieved for at least 10 min, mechanical stimulation was applied to the ulnar site for 20 sec. Firing rates were evaluated for 20 sec before, 20 sec during and 20 sec after mechanical stimulation.
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study
| 100.0 |
Brain or spinal cord sections were removed after perfusion with 4% paraformaldehyde, postfixed and cryosectioned into 30 µm-thick sections at the level of the CN (coordinates: anterior, −14.30~−14.60 mm; lateral, ±1.0~1.4 mm; deep, −7.8~−8.2 mm), the NAc (coordinates: anterior, 1.60~1.30 mm; lateral, ±0.6~1.0 mm; deep, −6.8~−7.4 mm) or the LHb (coordinates: anterior, −3.5 mm; lateral, ±0.7 mm; deep, −4.9 mm). The sections were incubated overnight (approximately 16 hr) at 4 °C with anti-c-fos rabbit polyclonal antibodies (1:200; Sigma, USA) followed by a 2-hr incubation at room temperature with a biotinylated donkey anti-rabbit Alexa Fluor 594 (red; 1:500; Sigma, USA) and mounted onto gelatin-coated slides. The sections were photographed and quantified using confocal laser scanning microscopy (LSM700, Carl Zeiss, Germany).
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study
| 99.94 |
After placing the animal into a stereotaxic frame under pentobarbital anesthesia (50 mg/kg, i.p.), the retrograde tracer Fluorogold (0.1 µl) was injected bilaterally to diffuse into the VTA/RMTg region (coordinates: anterior, −6.7 mm; lateral, 0.6 mm; deep, 8 mm) using a 5 μl Hamilton syringe controlled by a syringe pump at a rate of 0.1 μl/3 min. At least 4 days were allowed for the dye to diffuse.
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study
| 87.94 |
The local brain temperature is monitored as an indicator of functional neural activation21. Temperature changes in the LHb following peripheral stimulation were monitored as described previously43 with slight modifications. In brief, under pentobarbital-induced anesthesia (50 mg/kg, i.p.), an access hole was drilled through the skull over the habenula (coordinates: anterior, −3.5 mm; lateral, ±0.7 mm; deep, −4.9 mm) or the control site (coordinates: anterior, −3.5 mm; lateral, ±2.0 mm; deep, −4.9 mm), and a thermocouple needle microprobe (NJ-07013, World Precision Instruments, USA) was slowly lowered to the desired target depth. The thermocouple probe was connected to a BAT-12 digital thermometer (Physitemp, USA) and a data acquisition system (Powerlab, ADinstrument, Australia). The LHb temperature was measured while body temperature was maintained at approximately 36.5 °C with a heating pad.
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study
| 100.0 |
All data are presented as the means ± standard error of the mean (SEM) and analyzed by one- or two-way repeated measurement analysis of variance (ANOVA) followed by post hoc testing using the Tukey method or t-test, where appropriate. Statistical significance was considered at p < 0.05.
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study
| 99.94 |
Despite the success of HIV-1 therapies in reducing the concentration of virus in the bloodstream , a long-lived reservoir of infectious virus persists in CD4 T cells [2–6] and perhaps other cell types . Although most of the proviral DNA within CD4 T cells is not able to replicate , replication-competent virus can persist in long-lived resting memory CD4 T cells in a quiescent state [4,5,9–11]. These latently infected cells, which are replenished through proliferation or new infection can release infectious virus when reactivated . HIV-1 can also be derived from ongoing cycles of replication of CD4 T cells in tissue compartments where antiretroviral drugs have difficulty reaching–the so called drug sanctuaries [13–15]–and viral particles produced from infected CD4 T helper follicular cells that are captured and presented on the follicular dendritic cell network .
|
review
| 98.4 |
An effective cure strategy will need to target both the latent and active viral reservoir. Thus far, strategies to eliminate the viral reservoir have focused on early initiation of antiretroviral therapy (ART) , increasing the administered amount of current antiretroviral drugs or manipulation of cellular and viral transcription factors that eliminate transcriptional or post-transcriptional blocks [19–23]. Curative strategies focused on the activation of dormant virus that would lead to its destruction via host immune or viral cytopathic effects have not led to a reduction in the number of infected cells, however [8,24–29].
|
review
| 99.9 |
The enrichment of infected cells within secondary lymphoid tissue and lymph nodes suggest a critical role for these anatomical sites in sheltering persistently infected cells during therapy . HIV-1 RNA is particularly abundant in germinal centers within lymphoid tissue [31–33]. Regulatory mechanisms that prevent self-reactivity in the germinal center keep this microenvironment relatively safe from cytotoxic CD8 T cells and natural killer (NK) cells that target and destroy infected cells [34–38], thereby giving this unique tissue compartment immune privilege. This impaired protective immunity along with natural epigenetic silencing mechanisms and poor drug penetration allows the virus continue to replicate . As neither antiretroviral drugs, nor immune cells are able to fully abate ongoing cycle of replication in lymphoid tissue, other strategies need to be sought.
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review
| 68.2 |
A novel approach is to influence how CD4 T cells traffic between lymphoid tissue and peripheral sites where antiretroviral drugs effectively penetrate. Immune cell migration plays a critical role in immunity. Trafficking is affected by the cell’s differentiation status and activation state and is orchestrated by chemokines, integrins and selectins, as well as cues from the microenvironment [40–42]. Humanized monoclonal antibodies that influence immune cell traffic are currently being explored for the treatment of inflammatory disorders and cancerous tumors . A therapeutic strategy that could influence the traffic of CD4 T cells so that HIV-1 infected cells migrate more quickly out of drug sanctuaries could also control persistent replication.
|
review
| 99.8 |
Here, we develop a mathematical framework that defines how manipulating CD4 T cell trafficking could influence HIV-1 control. By boosting the traffic of CD4 T cells harboring virus out of drug sanctuaries to regions where antiretroviral drugs effectively penetrate, the virus cannot continue to replenish the persistent viral reservoir. Our analyses show that if the rate of trafficking of infected CD4 T cells exceeds a certain critical threshold, ongoing cycles of viral replication in drug sanctuaries become unsustainable. This strategy points towards a novel adjunct strategy to the treatment of HIV-1 infection and a promising new approach to a functional cure.
|
study
| 99.94 |
The spatial and dynamic mathematical model (model 1; see Fig 1 and Eqs 1–4) has two spatial compartments that differ in size and in the effectiveness of antiretroviral therapy: the drug sanctuaries (i = 0) and the main compartment (i = 1). In the drug sanctuaries, the effectiveness of antiretroviral therapy (the proportional decrease in infection probability due to drugs) is insufficient to block ongoing cycles of HIV-1 replication. Although many drug sanctuaries could exist within the body, for simplicity we model the sum of these sanctuaries as a single compartment. Our threshold results are robust to this assumption. Notably, the drug sanctuaries could include small regions within lymphoid tissue . It is clear that these regions must be very small because HIV-1 infected cells in lymphoid tissue, as well as HIV-1 RNA in plasma, declines by many orders of magnitude on antiretroviral therapy . The main compartment represents regions of the body that include peripheral blood and the majority of the lymphoid tissue where antiretroviral therapy is sufficiently effective that sustained viral replication is not possible.
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study
| 99.94 |
The model tracks the number of susceptible and infected cells in each of two spatial compartments, which can differ in size and in the effectiveness of antiretroviral therapy. Within each spatial compartment, infected cells can only transmit infection to other cells within the same spatial compartment, but a fraction of transmission is blocked by antiretroviral drugs. The model includes trafficking of cells between the two compartments, the rate of which can be changed using a trafficking therapy. The model also includes cell turnover.
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study
| 99.9 |
Within each of the two spatial compartments there is homogenous mixing that facilitates the spread of HIV-1 between infected and uninfected CD4 T cells. Though the risk of a cell becoming infected within each compartment comes only from that compartment, there is also trafficking of CD4 T cells between compartments . Principally, we consider the impact of an envisaged therapy that increases the rate at which CD4 T cells traffic between these two compartments. We also investigate the impact of combining such a “trafficking therapy” with therapy that improves immunological control inside drug sanctuaries (“immune therapy”). These spatial dynamics are built upon a standard compartmental model of the impact of antiretroviral drug therapy on the infection of CD4 T cells .
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study
| 100.0 |
A fraction (ui) of the body’s CD4 T cells are assumed to be in each compartment (u0 + u1 = 1) and the production rate of uninfected CD4 T cells in the body is apportioned between the drug sanctuaries (Λu0 day-1) and the main compartment (Λu1 day-1). In the absence of antiretroviral therapy, susceptible cells (Xi) within each compartment become infected cells (Yi) at a rate equal to the product of the density of infected cells in the same compartment (Yi/ui day-1) and the transmission parameter β. This assumption ensures that the basic reproductive number of each compartment, if it were isolated (i.e. in the absence of traffic of cells between compartments), is not directly related to the size of the compartment. Antiretroviral therapy blocks a fraction, zi, of these infections. The parameter zi is allowed to vary between compartments, with z0 ≪ z1 ≈ 1, so that antiretroviral therapy is highly effective in the main compartment. In the absence of trafficking therapy, cells travel from compartment i to the other compartment at a per cell rate of τi day-1. The constraint, τ0u0 = τ1u1, ensures no change in compartment size in the absence of infection. The envisaged trafficking therapy that we focus on acts to increase trafficking in both directions by a factor κ. In adaptations of this model discussed later, we also model trafficking therapy that independently manipulates the rate that cells flow into or out of drugs sanctuaries. Infected cells in compartment i are cleared at a per cell rate of δi day-1. These rates are free to vary between compartments to account for the possibility that drug sanctuaries may also be subject to impaired infected cell clearance rates (δ0 < δ1).
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study
| 100.0 |
In the presence of immune therapy directed at the drug sanctuaries, the cell clearance rate in the drug sanctuaries can be increased. In both compartments, susceptible cells are cleared at a per cell rate of α day-1. The resulting set of coupled, non-linear ordinary differential equations describing the change in the number of susceptible cells (Eqs 1 and 2) and infected cells (Eqs 3 and 4) in the drug sanctuaries and main compartment over time is provided below.
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study
| 99.94 |
Our mathematical model reveals a threshold condition in which HIV-1 infection is dependent upon the rate that CD4 T cells traffic between compartments (S1 Text). Assuming that the drug sanctuaries are small in size compared to the main compartment, viral replication in the drug sanctuaries is only sustainable when the rate of trafficking of infected CD4 T cells between the drug sanctuaries and the main compartment is below a critical threshold value. Should infected CD4 T cells traffic from the drug sanctuaries into the main compartment faster than the critical threshold rate, the number of secondarily infected cells that arise and remain in the drug sanctuaries would fall below one, and ongoing replication in the sanctuaries would become unsustainable. That is, the basic reproductive number in the drug sanctuaries would be less than one and ongoing replication could not persist in the long-term.
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study
| 100.0 |
Fig 2 illustrates the threshold for ongoing replication as a function of the rate at which CD4 T cells move between compartments and the effectiveness of antiretroviral therapy in the drug sanctuaries (see S1 Text for derivation). Here, antiretroviral drug therapy is assumed to be highly effective in the main compartment (z1 = 0.97) and the drug sanctuaries are small in size compared to the main compartment (see S1 Fig for threshold analysis for larger sanctuaries). There is a negative relationship between the effectiveness of antiretroviral therapy in the drug sanctuaries and the rate of cell trafficking between compartments at the threshold for ongoing replication. Even perfect drug sanctuaries (z0 = 0) can only support ongoing replication provided the rate that CD4 T cells traffic through them is below a critical threshold. When immune control in the sanctuary is boosted, the critical threshold for trafficking is lower, that is, stopping ongoing replication in drug sanctuaries becomes easier.
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study
| 100.0 |
The grey line plots the relationship between the per cell rate that CD4 T cells traffic out of drug sanctuaries (κτ0 day-1) and the effectiveness of antiretroviral therapy in the drug sanctuaries (z0), under the assumption that the drug sanctuaries are also immune sanctuaries (δ0 = 0.5 day-1,δ1 = 1 day-1). Boosting immune control in the sanctuaries so that there are equal infected cell clearance rates in each compartment (δ0 = δ1 = 1 day-1) acts additively with boosting cell trafficking rates so that the threshold for sustainable replication is decreased (black line). This figure reveals that antiretroviral therapy, trafficking therapy and immune therapy could all work in synergy to halt ongoing replication in drug sanctuaries. Guided by clinical findings, these plots assume that antiretroviral therapy is very effective in the main compartment (z1 = 0.97) and drug sanctuaries are very small in size compared to the main compartment.
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study
| 100.0 |
In demonstrating that ongoing cycles of replication in drug sanctuaries require CD4 T cell trafficking between compartments to be below a critical threshold, our model suggests a novel approach to HIV-1 control. It predicts that therapy designed to increase the trafficking of CD4 T cells into and out of drug sanctuaries above a threshold rate could halt ongoing cycles of replication. Although a ‘trafficking therapy’ of this sort would affect infected and uninfected cells equally, the mechanism through which it would work is by decreasing the mean residence time of infected cells in drug sanctuaries, moving them into the main compartment where drug concentrations are sufficient to block new rounds of infection with HIV-1 (Fig 3).
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study
| 100.0 |
In the absence of therapy that promotes cell trafficking (top left), trafficking of CD4 T cells in and out of the drug sanctuaries is slow enough to allow ongoing cycles of replication. The sanctuaries include small regions within lymph nodes (purple region). The effectiveness of antiretroviral therapy is assumed to be high in other regions, referred to as the main compartment, including elsewhere in lymph nodes (pale region) and in the blood. In the main compartment, continuous cycles of replication are unsustainable. When trafficking therapy increases the trafficking rate above the critical threshold (bottom right), CD4 T cells move more rapidly through the lymphatic system, including between the drug sanctuaries and the main compartment. The egress of infected CD4 T cells from the drug sanctuaries lowers their density in this spatial compartment. As a result, fewer virus particles are produced in the drug sanctuaries. If trafficking is fast enough, the lower density of virus particles and infected cells in the drug sanctuaries combine to ensure that ongoing cycles of infection, either through cell-to-cell infection or free virus, is not sustainable.
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study
| 99.94 |
This can be understood by evaluating, R, the effective reproductive number for the system in the presence of therapy, that is, the average number of secondary infected cells generated by one primary infected cell in a given population of target cells. The formal derivation of R is presented in S1 Text. Under the simplifying assumption that the CD4 T cell population in the drug sanctuaries (at a given value X^0) is very small compared to that in the main compartment, the effective reproductive number (Eq 5) can be understood by focusing on just the dynamics concerning the drug sanctuaries (Fig 4).
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study
| 100.0 |
The effective reproductive number can be understood by focusing on just the dynamics concerning infected cell numbers in the drug sanctuaries (Y0) when the drug sanctuaries hold only a small fraction of the body’s CD4 T cells. Under this assumption the net traffic of infected cells out of the drug sanctuaries is approximately κτ0Y0 day-1 (see S1 Text for details). The dynamics of infected cells in the drug sanctuaries will therefore be governed by a single influx rate (new infections at rate βX^0(1−z0)/u0 day-1) and two efflux rates (cell clearance at rate δ0 Y0 day-1 and cell egress at rate κτ0Y0 day-1).
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study
| 100.0 |
Notice that the effective reproductive number is equal to the rate at which one infected cell infects others (βX^0(1−z0)/u0 day-1), multiplied by the mean residence time of infected cells in drug sanctuaries (1/(δ0 + κτ0) days). With this expression, it is clear that the effective reproductive number can be reduced by either: increasing the rate that cells traffic through drug sanctuaries (higher κ); reducing the number of susceptible cells (target cells) in drug sanctuaries (lower X^0); increasing the effectiveness of antiretroviral therapy in drug sanctuaries (higher z0); or boosting immune control and thus increasing the clearance rate of infected cells in drug sanctuaries (higher δ0).
|
study
| 99.94 |
The immune system is a highly diverse, complex system and any mathematical model must make simplifying assumptions. The result we present here is robust to any refinements that maintain the core processes drawn in Fig 4. Should the average time that any infected cell spends within the drug sanctuaries be shorter than the average time required for infection of one other uninfected cell, continuous rounds of infection in drug sanctuaries are not sustainable.
|
other
| 99.7 |
Fig 5 shows model predictions of the impact of changes to the trafficking rate or enhanced immune control upon the dynamics of susceptible and infected CD4 T cells in patients taking antiretroviral therapy. In these simulations, prior to time 0, infected cell numbers under antiretroviral therapy are assumed to have reached an equilibrium state due to continuous replication in drug sanctuaries. After time 0, trafficking therapy, immune therapy or both are applied. Fig 5A shows that if the CD4 T cell trafficking rate is increased above the defined critical threshold there is a transient increase in infected cells in the main compartment as cells are washed out of the drug sanctuaries into the main compartment. Following this, infected cell counts in both compartments decline as replication becomes unsustainable in the drug sanctuaries.
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study
| 100.0 |
These figures show model predictions of the impact of therapeutic interventions on infected cell numbers in the drug sanctuaries (red lines) and main compartment (black lines). In each figure, prior to time 0 (grey shaded area), the host is only taking antiretroviral therapy (ART). At this stage, the effectiveness of antiretroviral therapy is high in the main compartment of the body (z1 = 0.97), but lower in the drug sanctuaries (z0 = 0.6). Furthermore, the per cell rate that cells traffic between compartments (governed by parameter τ0 = 0.5 day-1) and the per cell rate that infected cells are cleared from the drug sanctuaries (δ0 = 0.5 day-1) are slow enough to allow ongoing cycles of replication to persist in the drug sanctuaries. At time 0, additional therapy that either increases the cell trafficking rate (a and b), increases the cell clearance rate of infected cells from the drug sanctuaries (c), or both (d) is applied. In a), the trafficking rate is increased sufficiently (κ = 5) that ongoing viral replication is no longer sustainable. In b), the trafficking rate is increased to a level (κ = 3.5) just below the critical threshold and infected cell numbers decline to a new, lower equilibrium in the drug sanctuaries, but in the main compartment they increase. In c), the per cell clearance rate of infected cells in the drug sanctuaries is increased to the same level as in the main compartment (δ0 = 1 day-1). Here, the assumption that the effective drug concentration is highly impaired in the drug sanctuaries results in infected cell numbers declining to a new equilibrium. In d) both the per cell trafficking rate and the per cell clearance rate are increased to levels (κ = 3.5 and δ0 = 1 day-1) which independently would not stop ongoing replication (b and c), but, in combination, do so. All parameter values used in these calculations are provided in S1 Table.
|
study
| 100.0 |
If CD4 T cell trafficking is increased, but to a rate that is still below the critical threshold, the resulting infected cell number depends upon the balance between two effects: first any increase in incidence fueled by a net influx of susceptible cells into the drug sanctuaries (see S2 Text for discussion); and second, faster removal of infected cells from drug sanctuaries. If the trafficking rate is increased to a level just below the critical threshold, infected cell numbers decline to a new, lower equilibrium in the drug sanctuaries, but in the main compartment they increase (Fig 5B). Increasing the clearance of infected cells within drug sanctuaries acts synergistically with increased trafficking of cells out of drug sanctuaries.
|
study
| 100.0 |
Immune therapy that increases the rate of infected-cell clearance in the drug sanctuaries is another approach to stopping ongoing replication and could be encouraged through enhancing the function or penetration of CD8 T cells , natural killer (NK) cells or innate immunity . But unless clearance rates in the sanctuaries can be boosted beyond the rates observed in the main compartment, this approach alone is likely to be insufficient to halt persistent replication in sanctuaries where the effectiveness of antiretroviral therapy is also very low (Fig 5C). However, trafficking therapy and immune therapy can be expected to act synergistically so that treatments that combine a sub-threshold trafficking therapy and a sub-threshold immune therapy could be sufficient to halt replication (Fig 5D). Improving the penetration of antiretroviral drugs into sanctuaries is a third approach to tackling persistent replication that could work in synergy with these approaches (Fig 2).
|
review
| 98.3 |
The human immune response is a complex system composed of multiple cell types operating in many parts of the body. This model is a major simplification of that system aimed at understanding the features most salient to the question in hand–namely how to halt ongoing replication in drug sanctuaries. We reiterate that any modelled therapy that leads to each infected cell in a drug sanctuary giving rise to less than one further infected cell will lead to a modelled cure.
|
other
| 99.9 |
To explore the implications of some of the most pertinent additional complexities we present additional models in the Supporting Information, as follows. In S2 Text (Model 2) we model trafficking therapy that does not directly affect population sizes in each compartment. In S3 Text (Model 3) we model the impact of trafficking therapy that can independently change the rate that cells flow into or out of drugs sanctuaries. In S4 Text we explore the role of free virus under homogenous mixing (Model 4) or spatial heterogeneity (Model 5). In S5 Text (Model 6) we explore the impact of including cells that carry their treatment status with them as they move from the main compartment into the drug sanctuary. Our results remain robust to all these additional complexities.
|
study
| 100.0 |
Model 5 further highlights that any intervention that increases the trafficking rate of free virus, in addition to, or instead of, CD4 T cells, is capable of halting ongoing replication in drug sanctuaries. This is one putative mechanism for how antibodies that bind α4β7 –an integrin involved in gut homing and demonstrated to integrate into the envelope of HIV-1 –have led to significantly improved control in simian immunodeficiency virus (SIV) infected monkeys (S6 Text). Furthermore, Model 3 shows that just reducing the inflow of CD4 T cells into, or increasing the outflow of CD4 T cells from drug sanctuaries, can also be sufficient to halt persistent replication. This explicitly highlights a fundamental concept of our study: that any intervention that reduces the average residency time of infected CD4 T cells in the drug sanctuaries can halt persistent replication in these regions.
|
study
| 100.0 |
We developed a mathematical model that demonstrates that even a perfect drug sanctuary can only support ongoing replication if infected CD4 T cells traffic through it slowly enough. That is, only anatomical sites that, to a certain degree, are isolated in terms of CD4 T cell mixing have the potential to act as drug sanctuaries. Because persistent replication in drug sanctuaries contributes to the maintenance of the HIV-1 reservoir, our finding opens up an entirely new approach to eliminating this source of virus through the regulation of the trafficking of CD4 T cells to lymphoid tissue.
|
study
| 100.0 |
Increased CD4 T cell trafficking could eliminate persistent replication by promoting infected cells out of drug sanctuaries and into regions with clinically effective drug concentrations where the virus they produce cannot infect uninfected cells. As the average number of secondary infections arising from each infected cell is reduced, ongoing cycles of viral replication no longer remain sustainable. Thus, a novel way to address the problem of concentrations of drugs that are low is to promote the trafficking of infected cells away from drug sanctuaries to anatomical regions where antiretroviral drugs penetrate effectively.
|
study
| 99.94 |
Here we have demonstrated the theoretical potential of using trafficking therapy to stop ongoing replication in drug sanctuaries. There are several practical challenges to the development of safe and effective drugs that can achieve this, however. While this strategy is not restricted to any particular tissue that might harbour a sanctuary, no single therapeutic agent would be expected to usefully impact trafficking to all drug sanctuary sites. By regulating a receptor required for trafficking into one particular tissue, CD4 T-cells could be redistributed to another tissue, where drug sanctuaries also exist. As we have demonstrated (see Eq 5), this could increase the effective reproductive number in these sanctuaries and add to the challenge of HIV-1 clearance from them.
|
study
| 100.0 |
Even within a particular tissue, cells may respond differently to trafficking signals owing to the expression of distinct homing receptors and their differentiation status and activation state [40–42]. Whereas effector memory and central memory T cells recirculate between blood, lymphoid tissue, and lymph, resident memory T cells do not circulate through the blood . Thus, functional changes are associated with T cell migration. Although the existence of some cell types within a drug sanctuary that are unresponsive or less responsive to trafficking signals does not preclude an effective outcome of trafficking therapy, it does make it more difficult. The outcome of trafficking therapy would depend upon the fraction of cells of each type, the degree to which each of their trafficking patterns can be manipulated and the degree to which these different cell types mix together. Ultimately, if different cells types within a sanctuary mix homogeneously, and if the average residency time of infected cells in a drug sanctuary (across all cell types) can be sufficiently reduced, replication can still be rendered unsustainable.
|
study
| 99.9 |
Despite these challenges, targeting just the most important cell types in sanctuary sites that contribute most significantly to persistent infection could still prove worthwhile. By significantly reducing the size of the reservoir, but not necessarily eliminating it, trafficking therapy could be used alongside other therapies or in pursuit of a functional cure (see S6 Text for comments). For instance, gut associated lymphoid tissue is an important site of persistent HIV-1 and the trafficking of immune cells to the gastrointestinal tract can be reduced by integrin inhibitors (vedolizumab and natalizumab) that are routinely used for the treatment of Crohn’s disease and ulcerative colitis . These drugs consist of monoclonal antibodies that selectively target the α4β7 integrin receptor, expressed on immune cells. When used to treat simian immunodeficiency virus-infected monkeys, vedolizumab reduced the amount of virus detected in the blood . Under the hypothesis presented here, the anti- α4β7 antibody likely helped to control simian immunodeficiency virus infection after antiretroviral drug cessation by encouraging CD4 T cells to traffic away from gut-associated lymphoid tissue, to other tissue sites that have high concentrations of antiretroviral drugs.
|
review
| 64.9 |
Trafficking of immune cells into and out of lymph nodes is essential for immune surveillance of foreign invaders. CD4 T cells have a preference to home to germinal centers once they are activated and follicular helper T cells only circulate amongst the lymph follicles . As immune privileged sites for the activation and selection of B cell responses, the germinal center is outside the scope of cytotoxic CD8 T cells or natural killer cells. Accordingly, germinal centers are plausible foci for persistent viral replication and offer particular challenges in regards to the use of trafficking therapy.
|
other
| 70.06 |
Because productive HIV-1 infection predominantly occurs in activated CD4 T cells , it is implicit that HIV-1 infected cells are less permissive to egress cues compared to uninfected CD4 T cells that normally travel through the germinal center. This degree of difference would significantly reduce the impact of trafficking therapy compared with our model predictions that rely on the assumption that infected and uninfected CD4 T cells respond equally to trafficking therapy. Hypothetical mechanistic approaches to orchestrating T cell trafficking to germinal centers (S7 Text) would involve manipulation of pathways that are central to the immune response; for example S1P-S1PR1-mediated egress of immune cells from lymph nodes into efferent lymphatics . In chronic infection, virus-specific CD8 T cells are largely excluded from the germinal center or are functionally impaired within it . Thus, a potentially effective adjunctive strategy for control of the viral reservoir would be to restore the ability of these cells to kill infected cells by blocking the PD-1/PD-L1 inhibitory pathway or allow entry of functional antigen-specific T cells by directly engineering CD8 T cells to express CXCR5
|
study
| 99.94 |
Whether adjunct therapy that targets ongoing replication in drug sanctuaries could ever achieve a full or functional cure will clearly depend upon the relative importance of ongoing replication compared to other sources that contribute to the HIV-1 reservoir in treated patients . It is currently unknown whether ongoing replication accounts for the majority or a minority of the persistent pool of replication competent HIV-1 and whether alternative sources, including latent infection and homeostatic proliferation of latently infected cells, would be self-sustaining in the absence of ongoing replication that continuously tops up the reservoir. If the pools of virus are self-sustaining, drugs that targets these alternative sources will need to be included in any comprehensive therapeutic strategy to control or clear the viral reservoir.
|
other
| 99.6 |
Arguably, the concept of cell trafficking as therapy is a counterintuitive one because it opposes traditional ideas about the role of mixing in increasing infection rates . Since antiretroviral therapy is highly effective throughout most of the body, including the peripheral blood, drug sanctuaries in HIV-1 infection are akin to small islands of ongoing replication in a sea of highly effective drug concentrations. In such a situation, it is intuitively appealing that increased trafficking of CD4 T cells could reduce their residency time in drug sanctuaries and contribute to a functional cure for HIV-1 infection.
|
review
| 99.5 |
A cluster heatmap is a popular graphical method for visualizing high dimensional data. In it, a table of numbers is scaled and encoded as a tiled matrix of colored cells. The rows and columns of the matrix are ordered to highlight patterns and are often accompanied by dendrograms and extra columns of categorical annotation. The ongoing development of this iconic visualization, spanning over more than a century, has provided the foundation for one of the most widely used of all bioinformatics displays (Wilkinson and Friendly, 2009). When using the R language for statistical computing (R Core Team, 2016), there are many available packages for producing static heatmaps, such as: stats, gplots, heatmap3, fheatmap, pheatmap and others. Recently released packages also allow for more complex layouts; these include gapmap, superheat and ComplexHeatmap (Gu et al., 2016). The next evolutionary step has been to create interactive cluster heatmaps, and several solutions are already available. However, these solutions, such as the idendro R package (Sieger et al., 2017), are often focused on providing an interactive output that can be explored only on the researcher's personal computer. Some solutions do exist for creating shareable interactive heatmaps. However, these are either dependent on a specific online provider, such as XCMS Online, or require JavaScript knowledge to operate, such as InCHlib. In practice, when publishing in academic journals, the reader is left with a static figure only (often in a png or pdf format).
|
review
| 99.56 |
To fill this gap, we have developed the heatmaply R package for easily creating a shareable HTML file that contains an interactive cluster heatmap. The interactivity is based on a client-side JavaScript code that is generated based on the user's data, after running the following command:
|
other
| 99.94 |
The HTML file contains a publication-ready, interactive figure that allows the user to zoom in as well as see values when hovering over the cells. This self-contained HTML file can be made available to interested readers by uploading it to the researcher's homepage or as a Supplementary Material in the journal's server. Concurrently, this interactive figure can be displayed in RStudio's viewer pane, included in a Shiny application, or embedded in a knitr/RMarkdown HTML documents.
|
other
| 99.94 |
The rest of this paper offers guidelines for creating effective cluster heatmap visualization. Figure 1 demonstrates the suggestions from this section on data from project Tycho (van Panhuis et al., 2013), while the online Supplementary Material includes the interactive version, as well as several examples of using the package on real-world biological data.
|
other
| 99.9 |
The (square root) number of people infected by Measles in 50 states, from 1928 to 2003. Vaccines were introduced in 1963, An interactive version is available in the following URL: https://cdn.rawgit.com/talgalili/heatmaplyExamples/564da09e/inst/doc/measles_heatmaply.html
|
other
| 99.94 |
The generation of cluster heatmaps is a subtle process (Gehlenborg and Wong, 2012; Weinstein, 2008), requiring the user to make many decisions along the way. The major decisions to be made deal with the data matrix and the dendrogram. The raw data often need to be transformed in order to have a meaningful and comparable scale, while an appropriate color palette should be picked. The clustering of the data requires us to decide on a distance measure between the observation, a linkage function, as well as a rotation and coloring of branches that manage to highlight interpretable clusters. Each such decision can have consequences on the patterns and interpretations that emerge. In this section, we go through some of the arguments in the function heatmaply, aiming to make it easy for the user to tune these important statistical and visual parameters. Our toy example visualizes the effect of vaccines on measles infection. The output is given in the static Figure 1, while an interactive version is available online in the Supplementary file ‘measles.html’. Both were created using:
|
other
| 99.75 |
The first argument of the function (x) accepts a matrix of the data. In the measles data, each row corresponds with a state, each column with a year (from 1928 to 2003), and each cell with the number of people infected with measles per 100 000 people. In this example, the data were scaled twice—first by not giving the raw number of cases with measles, but scaling them relatively to 100 000 people, thus making it possible to more easily compare between states. And second by taking the square root of the values. This was done since all the values in the data represent the same unit of measure, but come from a right-tailed distribution of count data with some extreme observations. Taking the square root helps with bringing extreme observations closer to one another, helping to avoid an extreme observation from masking the general pattern. Other transformations that may be considered come from Box-Cox or Yeo-Johnson family of power transformations. If each column of the data were to represent a different unit of measure, then leaving the values unchanged will often result in the entire figure being un-usable due to the column with the largest range of values taking over most of the colors in the figure. Possible per-column transformations include the scale function, suitable for data that are relatively normal. normalize, and percentize functions bring data to the comparable 0–1 scale for each column. The normalize function preserves the shape of each column’s distribution by subtracting the minimum and dividing by the maximum of all observations for each column. The percentize function is similar to ranking but with the simpler interpretation of each value being replaced by the percent of observations that have that value or below. It uses the empirical cumulative distribution function of each variable on its own values. The sparseness of the dataset can be explored using is.na10.
|
study
| 99.94 |
Once the data are adequately scaled, it is important to choose a good color palette for the data. Other than being pretty, an ideal color palette should have three (somewhat conflicting) properties: (i) Colorful, spanning as wide a palette as possible so as to make differences easy to see; (ii) Perceptually uniform, so that values close to each other have similar-appearing colors compared with values that are far away, consistently across the range of values; and (iii) Robust to colorblindness, so that the above properties hold true for people with common forms of colorblindness, as well as printing well in grey scale. The default passed to the color argument in heatmaply is viridis, which offers a sequential color palette, offering a good balance of these properties. Divergent color scale should be preferred when visualizing a correlation matrix, as it is important to make the low and high ends of the range visually distinct. A helpful divergent palette available in the package is cool_warm (other alternatives in the package include RdBu, BrBG, or RdYlBu, based on the RColorBrewer package). It is also advisable to set the limits argument to range from -1 to 1.
|
other
| 99.8 |
Passing NULL to the Colv argument, in our example, removed the column dendrogram (since we wish to keep the order of the columns, relating to the years). The row dendrogram is automatically calculated using hclust with a Euclidean distance measure and the average linkage function. The user can choose to use an alternative clustering function (hclustfun), distance measure (dist_method), or linkage function (hclust_method), or to have a dendrogram only in the rows/columns or none at all (through the dendrogram argument). Also, the users can supply their own dendrogram objects into the Rowv (or Colv) arguments. The preparation of the dendrograms can be made easier using the dendextend R package (Galili, 2015) for comparing and adjusting dendrograms. These choices are all left for the user to decide. Setting the k_col/k_row argument to NA makes the function search for the number of clusters (from 2 to 10) by which to color the branches of the dendrogram. The number picked is the one that yields the highest average silhouette coefficient (based on the find_k function from dendextend). Lastly, the heatmaply function uses the seriation package to find an ‘optimal’ ordering of rows and columns (Hahsler et al., 2008). This is controlled using the seriation argument where the default is ‘OLO’ (optimal-leaf-order)—which rotates the branches so that the sum of distances between each adjacent leaf (label) will be minimized (i.e.: optimize the Hamiltonian path length that is restricted by the dendrogram structure). The other arguments in the example were omitted since they are self-explanatory—the exact code is available in the heatmaplyExamples package.
|
other
| 99.8 |
In order to make some of the above easier, we created the shinyHeatmaply package (available on CRAN) which offers a GUI to help guide the researcher with the heatmap construction, with the functionality to export the heatmap as an html file and summaries parameter specifications to reproduce the heatmap with heatmaply. For a more detailed step-by-step demonstration of using heatmaply on biological datasets, you should explore the heatmaplyExamples package (https://github.com/talgalili//heatmaplyExamples).
|
other
| 99.94 |
Experimental evidence suggests that mechanical ventilation (MV) could cause specific lung damage (i.e., ventilator-induced lung injury [VILI]) . Moreover, MV could per se modify the efficacy of lung immunity and promote an overwhelming inflammatory state if pneumonia develops [3–6]. Actually, a link has been shown between the magnitude of pulmonary and systemic inflammatory responses and outcomes in VAP patients .
|
study
| 70.2 |
Statins are lipid-lowering agents that possess immunomodulatory properties that could be in part mediated by the down-regulation of the toll-like receptors (TLRs), involved in pathogens recognition by immune cells [8, 9]. Among TLRs, TLR-2 is likely to sense the immune response to S. aureus . Statins could be protective in pneumonia according to some concordant clinical data [11, 12]. In addition, experimental studies have demonstrated a lung-protective effect through VILI attenuation [13, 14]. Outcomes in VAP patients were improved by prior treatment with statins . This remains however a controversial issue .
|
review
| 99.9 |
We hypothesized that statins could influence the course of MRSA pneumonia in a rabbit model . We used spontaneously breathing (SB) and ventilated animals treated with linezolid (LNZ), atorvastatin or the combination of both, to assess the effect of statins in both settings (i.e. MRSA pneumonia with or without MV).
|
study
| 100.0 |
Male New Zealand White rabbits (3.0 [0.2] Kg) were bred in the University of Burgundy animal facility (Dijon, France). Animal use and handling were approved by the local veterinary committee (i.e., Comité d’Ethique de l’Expérimentation Animale Grand Campus Dijon [C2EA– 105]), and were performed according to the European laws for animal experimentation in accordance with current recommendations mentioned in the Guide for the Care and Use of Laboratory Animals, National Institutes of Health No. 92–23, revised 1985. Animals were randomized into the SB or MV group within each experiment as follows: control uninfected (SB or MV, n = 5 in each), infected (SB+MRSA [n = 11] or MV+MRSA [n = 11]), statin pretreatment infected (SB+MRSA+Statin or MV+MRSA+Statin, n = 5 in each), LNZ-treated infected group (SB+MRSA+LNZ or MV+MRSA+LNZ, n = 5 in each) and the statin pretreatment LNZ-treated infected group (SB+MRSA+Statin+LNZ or MV+MRSA+Statin+LNZ, n = 5 in each).
|
study
| 99.94 |
Under general anesthesia provided by ketamine 3.3 mg/Kg (Panpharma, France) and xylazine 1 mg/Kg (Rompun®, Bayer, Germany), a cuff tube of 2.5 mm (Mallinckrodt™, Covidien®, U.S.A.) was inserted through the mouth into the trachea under view control. The animal was connected to a volume-controlled respirator (Servo ventilator 900C, Siemens®, Germany) (tidal volume of 12 mL/Kg with zero end-expiratory pressure [ZEEP], a respiratory rate of 25 bpm and an 0.5 inspired fraction of O2). Rabbits were kept anesthetized and paralyzed throughout the experiment with midazolam (0.06 mg/Kg/h) (Hypnovel®, Roche, Switzerland) and cisatracurium besilate (0.3 mg/Kg/h) (Nimbex®, GlaxoSmithKline, U.K). Isotonic serum was infused.
|
other
| 96.1 |
The USA300 PVL- strain of S. aureus was used. At 48 hours before experimentation began, several colonies were harvested, cultured on MRSA2 agar plates (Biomérieux) and then incubated for 24 hours at 37°C. One colony was inoculated into 10 mL of BHI and was incubated for 6 hours before being cultured for 18 hours. A mean titer 9.5 Log10 colony-forming units per milliliter (CFU/mL) was used . In the SB groups, inoculum (0.5 mL) was gently flushed through a catheter briefly introduced into the trachea. The animals were then placed upright for 15s and allowed to go back to their cage before being sacrificed 24 hours later. In the MV groups, animals were subjected to MV for 24 hours before being instilled intrabronchially with MRSA. The animals were then kept under MV for 24 hours before sacrifice. Controls received 0.5 mL of saline.
|
study
| 100.0 |
Linezolid (Zyvoxid®, Pfizer, United States) was delivered (125 mg of LNZ in 10 mL of 5% α-cyclodextrin solution) over 1 day through two subcutaneous injections (50 mg/kg), 4 and 16 hours after bacterial challenge, as previously described . Blood samples were obtained 1, 2, 4, 6 and 12 hours after the first drug injection and stored at -80°C. Linezolid concentrations were then determined by high-performance liquid chromatography (limit of detection = 0.03 mg/L).
|
study
| 99.94 |
All the animals were sacrificed by an intravenous overdose of thiopental (100 mg/kg) and then exsanguinated. Autopsies were carried out so that the lungs and spleen were aseptically taken. They were harvested for ribonucleic acid (RNA) extraction and microscopic examination. The remaining tissue was homogenized to determine cytokine concentrations and for bacterial culture.
|
study
| 99.9 |
A tissue sample of 1 cm3 that focused on a macroscopic lesion was fixed in formalin and embedded in paraffin. Five-micrometer sections were obtained and colored with hematoxylin and eosin. After viewing five fields per sector in a blinded manner, each section was assigned a numerical histologic score ranging from 0 to 3 according to the degree of PMN infiltration, hemorrhage, and edema in the interstitial and alveolar spaces .
|
study
| 99.94 |
Each pulmonary lobe was isolated from the whole lung, homogenized and used for serial ten-fold dilution cultures. The mean pulmonary bacterial concentration was calculated (i.e. mean concentration = ∑ [lobar concentration x lobar weight]/ lobar weights). Spleens were processed similarly. An S. aureus-positive spleen culture was considered a marker of bacteremia.
|
study
| 99.94 |
Fresh heparinized blood from spontaneously breathing (SB) untreated rabbits (n = 10), rabbits subjected to 24-hour MV as described above (n = 5), or either SB or MV rabbits given atorvastatin (20 mg/kg once a day) 72 hours prior to venipuncture was obtained and diluted 1:2 with RPMI 1640 medium (GibcoTM Life Technologies, Saint Aubin, France). Blood was plated at 120 μL/well in a 96-well plate and incubated for 15 minutes at 37°C. Atorvastatin pills were dissolved in RPMI 1640 medium (10 nmol/mL in final volume), added to whole blood (60 μL/well) and incubated for 24 hours at 37°C. Then, heat-killed S. aureus (HKSA) were added and the blood was incubated for 24 hours at 37°C . Cell viability was checked (propidium iodide dying assay) and culture supernatants were finally removed and kept frozen at -80°C until cytokine concentrations were determined.
|
study
| 100.0 |
RNA was extracted from lung samples using the RNA GenElute kit (Sigma-Aldrich, St Louis, MO, USA). Quantitative PCR was performed using IQ Sybergreen Supermix (Bio-Rad, Hercules, CA, USA). Melting curves were performed to ensure the presence of a single amplicon. The following primers were used: rTlr2, forward 5’-TGT CTG TCA CCG AAC CGA ATC CAC-3’ and reverse 5’-TCA GGT TTT TCA GCG TCA GCA GGG-3’ ; and rGapdh, forward 5’-ATG TTT GTG ATG GGC GTG AAC C-3’ and reverse 5’-CCC AGC ATC GAA GGT AGA GGA-3’.
|
study
| 99.9 |
Continuous variables between the different treatment arms (i.e., atorvastatin alone, LNZ alone or the combination of both, in either SB or MV animals) were compared using 2-way ANOVA, the non-parametric Mann-Whitney U test or the Kruskall-Wallis test, as appropriate, and post hoc corrections for multiple comparisons were applied. The Fisher exact test was used for the comparison of categorical variables.
|
study
| 99.94 |
There was a trend toward a reduction of the pulmonary bacterial load in SB and in MV rabbits treated with LNZ, without any improvement in tissue damage (Figs 1 and 2). However, whereas no bacteremia was detected in the SB treated rabbits, LNZ failed apparently to prevent MRSA systemic spillover in MV animals since 59% of them had positive spleen cultures (Fig 1).
|
study
| 100.0 |
Pulmonary bacterial clearance expressed as the median (IQR) ratio between the amounts of bacteria recovered from the lung culture and the initial Staphylococcus aureus inoculum 24 hours after bacterial challenge in either spontaneously breathing or mechanically ventilated animals. Pulmonary-to-systemic bacterial translocation expressed as the spleen positive cultures rate 24 hours after bacteria instillation according to the experimental group in either spontaneously breathing or mechanically ventilated animals with Staphylococcus aureus pneumonia. Values are presented as percentages (right). Kruskall-Wallis test with Dunn correction for multiple comparisons or Fisher exact test was used as appropriate for intergroup comparisons. CFU: colony forming unit; MRSA: methicillin-resistant S. aureus; LNZ: linezolid; SB: spontaneously breathing; MV: mechanical ventilation; IQR: interquartile range.
|
study
| 100.0 |
Histologic score ranging from 0 to 3, based on the degree of polymorphonuklear infiltration, haemorrhage, and oedema in the interstitial and alveolar spaces, following tracheal instillation of saline (controls) or 9 log10CFU of Staphylococcus aureus in either spontaneously breathing rabbits or animals subjected to mechanical ventilation with various treatment combinations. Mean values (SD) are shown (A) as well as representative light microphotographs of rabbit lungs in various conditions fixed at the same transpulmonary pressure (hematoxylin and eosin x400) (B). Two-tailed Mann-Whitney U test was used for all intergroup comparisons. CFU: colony forming unit; MRSA: methicilline-resistant S. aureus; LNZ: linezolid; SB: spontaneously breathing; MV: mechanical ventilation; SD: standard deviation.
|
study
| 100.0 |
Inflammatory cytokines (interleukin [IL]-8 and tumor necrosis factor [TNF]-α) concentrations in lung (A) and spleen (B) homogenates 24 hours after tracheal instillation of saline (controls) 109 CFU of Staphylococcus aureus in either spontaneously breathing rabbits or animals subjected to mechanical ventilation with various treatment combinations. Values are presented as means (SD). 2-way ANOVA test was used for all intergroup comparisons with Tukey’s adjustment for multiple comparisons. CFU: colony forming unit; MRSA: methicilline-resistant S. aureus; LNZ: linezolid; SB: spontaneously breathing; MV: mechanical ventilation; SD: standard deviation.
|
study
| 100.0 |
Serum concentrations of interleukin (IL)-8 and tumor necrosis factor (TNF)-α expressed as % of the mean baseline value measured by enzyme-linked immune-sorbent assay at baseline (100%; 24 hours before bacterial airway challenge), just before intra-tracheal instillation of 109 CFU of S. aureus (H0), and then 8 (H8) and 24 hours (H24) after instillation, in the spontaneously breathing (A) and the mechanically ventilated animals (B). Concentrations measured in non-infected controls are shown in panel C. Variables were compared at each time-point with Mann-Whitney U test and p values were adjusted for multiple comparisons. (A): (left) * denotes p = 0.014 between rabbits treated with atorvastatin vs. untreated animals, and ** denotes p = 0.027 between rabbits treated with LNZ+atorvastatin vs. untreated animals; (right) * denotes p = 0.049 between rabbits treated with LNZ vs. untreated animals, and ** denotes p = 0.006 between rabbits treated with LNZ+atorvastatin vs. untreated animals. (B): (left) * denotes p = 0.004 between rabbits treated with atorvastatin vs. untreated animals, ** denotes p = 0.003 between rabbits treated with LNZ vs. untreated animals, and *** denotes p = 0.014 between rabbits treated with LNZ+atorvastatin vs. untreated animals; (right) * denotes p = 0.034 between rabbits treated with atorvastatin vs. untreated animals, ** denotes p = 0.013 between rabbits treated with LNZ vs. untreated animals, and *** denotes p = 0.002 between rabbits treated with LNZ+atorvastatin vs. untreated animals. CFU: colony forming unit; MRSA: methicillin-resistant S. aureus; LNZ: linezolid; SB: spontaneously breathing; MV: mechanical ventilation. CFU: colony forming unit; MRSA: methicilline-resistant S. aureus; LNZ: linezolid; SB: spontaneously breathing; MV: mechanical ventilation; IQR: interquartile range.
|
study
| 100.0 |
Significantly lower blood levels of IL-8 were achieved in SB rabbits treated with atorvastatin prior to pneumonia and then treated with LNZ 24 hours following the bacterial challenge than in rabbits treated with LNZ alone. The same additive anti-inflammatory effect of statin was observed in MV rabbits according to TNF-α blood levels as early as the 8th hour following the onset of infection (75% vs. 169% of the baseline value; p<0.001).
|
study
| 100.0 |
After HKSA ex vivo stimulation, levels of both IL-8 and TNF-α were higher in the blood taken from MV rabbits (Fig 5). If MV rabbits were given statin prior to MV, or if the drug was added to the medium prior to ex vivo stimulation, TNF-α levels were significantly decreased as low as baseline values.
|
study
| 100.0 |
Interleukin (IL)-8 and tumor necrosis factor (TNF)-α mean levels (SD) in the supernatant of whole blood from either spontaneously breathing rabbits or animals subjected to mechanical ventilation. Atorvastatin was added to the medium (“statin”) or given to the rabbit prior to blood sampling (“Premed”). Values are presented as means (SD). 2-way ANOVA test was used for all intergroup comparisons with Tukey’s adjustment for multiple comparisons. One missing [IL-8] and one missing [TNF-α] value in the Whole blood+MRSA and in the Whole blood+MRSA+statin groups. MRSA: methicillin-resistant S. aureus; SB: spontaneously breathing; MV: mechanical ventilation; SD: standard deviation.
|
study
| 100.0 |
TLR2 gene expression was measured in both lung (left) and spleen (right) homogenates in either spontaneously breathing rabbits (SB) or animals subjected to mechanical ventilation (MV) with or without atorvastatin pre-treatment (n = 5 in each group). All values are reported as fold increases compared with SB rabbits. Results are expressed as mean±standard deviation. Kolmogorov-Smirnov test was used for all intergroup comparisons.
|
study
| 100.0 |
We showed herein in a rabbit model of MRSA pneumonia that, although LNZ was efficient in keeping in check bacterial growth within the lung of both SB and MV animals, infection could be considered as more severe in the latter, since pulmonary-to-systemic translocation of S. aureus was apparently not prevented by LNZ in the ventilated animals. The lung inflammatory response was in part dampened by LNZ in both SB and MV rabbits, but interestingly, an additive effect was achieved by pre-treatment with atorvastatin. Similarly, we showed that LNZ alone failed to completely abrogate the systemic release of inflammatory mediators subsequent to pneumonia, whereas its combination with atorvastatin succeeded in doing so. This anti-inflammatory effect, however, could have hampered the host’s ability to control systemic diffusion of S. aureus, thereby promoting bacterial spillover into the bloodstream in MV animals.
|
study
| 100.0 |
Cumulative experimental evidence supports the notion that MV itself, especially if adverse settings including large VT are applied, could be harmful in the context of bacterial pneumonia by promoting pulmonary and systemic inflammation [17, 21, 22]. In the present study, we showed that although LNZ therapy was successful in SB rabbits, its efficacy was limited to the lung compartment in MV rabbits. Interestingly, our findings in our model of pneumococci pneumonia treated with moxifloxacin were similar . No significant histological findings were likely to account for such a difference in extra-pulmonary bacterial diffusion, but unseen lung damage is not excluded.
|
study
| 100.0 |
The pulmonary release of TNF-α was reduced by LNZ in both SB and MV rabbits, as was the bacterial burden. It remains unclear as to what extent the anti-inflammatory properties of LNZ per se contributed to such a decrease in this key mediator. Actually, experimental studies have shown that LNZ was able to reduce pulmonary inflammation in mice with MRSA pneumonia, in terms of MIP-2 concentrations, thus limiting in turn PMN recruitment and lung injury . In addition, since high levels of IL-8 were still reached despite LNZ therapy, the net effect on lung inflammation is uncertain. Thus, no positive beneficial effect on tissue injury was found.
|
study
| 99.94 |
Treatment with atorvastatin prior to the bacterial infection had anti-inflammatory effects per se in the infected lung of both SB and MV animals according to TNF-α concentrations, as reported in vitro [9, 25]. It has been shown that human alveolar macrophages incubated with simvastatin released lower amounts of inflammatory mediators in response to a TLR2 agonist, and statin pretreatment decreased lung inflammation following lipopolysaccharide inhalation in volunteers [9, 26].
|
study
| 100.0 |
However, statin therapy alone tended to decrease pulmonary bacterial clearance in MV animals, leading in turn to worse lung injury, thus underlining the need for efficient antimicrobial therapy in combination with the drug. These findings emphasize also the fact that the beneficial effect of statins on VILI is uncertain and should be balanced with their deleterious impact on bacterial clearance . Moreover, the enhanced bactericidal activity of PMNs reported in mice with S. aureus pneumonia treated with simvastatin is unlikely in our model . Appropriate antibiotics could thus be the prerequisite for any beneficial adjunctive effect of statin therapy in patients with VAP.
|
study
| 99.94 |
The magnitude and persistence of the systemic inflammation are considered key determinants of outcomes in the setting of VAP . Atorvastatin pretreatment could blunt the host inflammatory response within the blood compartment as reported in animal models of sepsis . However, it may be deleterious in our model since bacteremia is an independent predictor of death in MRSA VAP patients . Linezolid might have immuno-modulatory properties likely to dampen MRSA virulence factors, including the expression of pro-inflammatory signals and could thus be beneficial in a mouse model of pneumonia despite persisting bacteremia . Accordingly, the overall effect of the combination of atorvastatin and LNZ could protect the host against a protracted systemic inflammatory response, although delaying S. aureus clearance from the blood compartment. However, further experimental studies are necessary before any definitive conclusions can be drawn.
|
study
| 100.0 |
Statins exert various immunomodulatory effects but the underlying mechanisms remain unclear . Data from in vitro studies suggest that statins could interfere with the TLR pathway, thereby modulating the response to microbial products. However, depending on the experimental model, the net effect of statin treatment could be pro- or anti-inflammatory [25, 31]. For instance, statins could have anti-inflammatory effects through TLR down-regulation . Accordingly, we found that TLR2 gene expression down-regulation by statin within the lung could account for its anti-inflammatory effect. Interestingly, this is in line with previous findings from our group and others showing that tlr2 up-regulation within the lung of animals subjected to MV probably accounted for overreaction to its agonist including S. aureus cell wall compounds [6, 32]. In contrast, other mechanisms are probably involved within the blood, suggesting that the spleen was a surrogate for peripheral blood mononuclear cells . Moreover, the effects of statin probably depend on the molecule tested, the cell type and its environment, as well as both dose and duration of treatment prior to stimulation. Herein, we showed that despite one negative effect of atorvastatin treatment prior to pneumonia, namely the systemic release of IL-8 in MV rabbits, ex vivo experiments showed that whole blood taken from pretreated MV rabbits overreacted to HKSA stimulation in terms of IL-8 production. These findings, as well as in vitro studies, illustrate to what extent impact of statin on the inflammatory response is equivocal.
|
study
| 100.0 |
Although experimental findings support the protective impact of statins in the setting of VAP, strong clinical evidence is still lacking. Actually, the only large multicenter RCT published so far showed that atorvastatin had no effect on the outcome of patients with suspected VAP . However, we have shown that statin treatment prior to VAP was an independent predictor of ICU survival . Similarly, it has been shown by one RCT that overall mortality was reduced in patients with the most severe VAP who received statin prior to infection, as already demonstrated previously in another trial, which included patients with sepsis from various origins [35, 36]. Thus, one cannot exclude the possibility that statins could be beneficial to the host only if given prior to the infection, as was the case in our model. Several explanations could be proposed: (i) more than one administration of the drug is required before critical concentrations in the blood and lung are reached; (ii) the clinical suspicion of VAP is probably later than the actual onset of pneumonia (i.e., airway challenge with bacteria triggering the immune response); (iii) immunomodulation has to occur very early during sepsis in order to prevent any overwhelming pro-inflammatory response.
|
study
| 99.94 |
Several limitations of our study should be mentioned. First, the MV with “high” VT and without PEEP we used could be considered not clinically relevant. However, it has recently been reported that such settings were still used, especially in the operating room . In addition, it is known that lung injury is heterogeneous in ARDS patients. As a result, poorly aerated areas of the lung usually coexist with overstretched ones, even if “low-VT” is applied, as shown in human studies . It is therefore quite possible that such parts of the lung may react as described in the present study if bacterial challenge occurred in patients subjected to “protective” MV. Third, TLR2 assessment was limited to gene expression, and the corresponding protein was not measured. Fourth, our study may be underpowered given the small size of the experimental groups. In addition, we should acknowledge that the LNZ concentrations achieved in the treated animals did not reach the usually reported values in humans, thereby undermining the clinical relevance of our findings (Fig 7). However, we used a MRSA strain, for which the MIC for LNZ was low (i.e., 1 mg/L). In addition, it has recently been shown that the LNZ concentration actually obtained in critically ill patients, especially those with lung injury, were probably not as high as those in healthy volunteers . Finally, our model does not exactly mimic the pathophysiology of VAP since a large number of bacteria were directly inoculated into the airways rather than repeatedly inhaled in small numbers. In addition, there was no mortality attributable to pneumonia in our model, at least over the 48-hour observation period. As a result it remains difficult to evaluate the clinical relevance of our findings.
|
study
| 100.0 |
Atorvastatin treatment prior to pneumonia provides an anti-inflammatory effect within the lung and the systemic compartment of rabbits with MRSA pneumonia treated by LNZ. This effect may be mediated at least in part by TLR2 down-regulation. Whether such an impact is beneficial for the host remains unclear.
|
study
| 99.94 |
The European population is the most aged in the world, with 24% of the population 60 years or older. It is projected to remain the most aged population in the coming decades, with 34% of the population projected to be 60 years or older in 2050 . This overall aging of the population is accompanied by a substantial increase in prevalence of chronic conditions. Two thirds of older adults in Europe who have reached retirement age have at least two chronic conditions .
|
other
| 99.9 |
This co-existence of multiple chronic conditions, defined as “multimorbidity”, is a common phenomenon in older people, and its occurrence increases with age . Multimorbid chronic diseases are associated with increased rate of mortality and disability, reduced function levels, increased polypharmacy, poor health-related quality of life (HRQoL) and more health care utilization (costs, number of physician visits, length of hospital stay) [5–7]. In this context, health care should aim to increase the life span cost-efficiently while maintaining HRQoL and the ability to perform activities of daily life . Most studies have shown impaired HRQoL by gender with the presence of many chronic diseases or with aging in older people [10–13].
|
review
| 99.7 |
When studying the impact of morbidities on HRQoL, morbidities are usually considered separately . Most treatment plans and clinical guidelines target single diseases , but an effective intervention for one disease could be less effective or deleterious with the presence of coexisting conditions . Regarding associations between morbidities, patterns of morbidities can be established. However, few studies are identifying patterns and potential factors underlying such associations [16–20]. The interest of these patterns is to consider the interrelations or the cumulative effect between different morbidities. Methodological approaches that consider such patterns that are well-adapted to the respective study populations are needed, as is the need to understand the patterns of disease combinations and their complexity. The identification and standardization of patterns of multimorbidity might help in organizing specific treatment strategies and system-wide initiatives to improve the care of people with various types and degrees of multimorbidity. However, more evidence on multimorbidity patterns is required.
|
review
| 99.9 |
We aimed to describe the multimorbidity patterns in adults aged 55 years or older by using national French data from the Supplémentation en VItamines et Minéraux AntioXydants 2 (SU.VI.MAX 2) study. We also aimed to assess the association between multimorbidity patterns and HRQoL among older people overall and by age and gender.
|
study
| 99.94 |
Our study is based on the data from SU.VI.MAX 2 study, which is an additional observational follow-up study, organized 5 years after the end of the initial SU.VI.MAX trial. The initial SU.VI.MAX trial was a randomized, double-blind, placebo-controlled primary prevention trial assessing the efficacy of a daily antioxidant supplementation in the incidence of cardiovascular disease and cancer. Eligibility criteria of SU.VI.MAX participants were described in previous publication [21–23]. This initial trial was launched in 1994–95 and had a planned follow-up of 8 years (until 2002).
|
study
| 99.5 |
The SU.VI.MAX and SU.VI.MAX 2 studies were approved by the Ethics Committee for Studies with Human Subjects of the Paris-Cochin Hospital (CCPPRB nos. 706 and 2364, respectively) and the Commission Nationale Informatique et Liberté (CNIL nos. 334641 and 907094, respectively). Written informed consent was obtained from all participants. Clinical Trial Registration at clinicaltrial.gov (number NCT00272428).
|
other
| 99.94 |
The SU.VI.MAX 2 participants were recruited through a postal campaign organized in 2007–2009 among all SU.VI.MAX participants. From the full initial SU.VI.MAX cohort (N = 13,017), 6,850 participants were agreed to participate in the SU.VI.MAX 2 study, and 5,925 participants were older than 55 years at enrollment and received a geriatric assessment . Sociodemographic characteristics data, morbidities data and quality of life data were available for 5,647 aged 55 years or older for inclusion in the present analyses (Fig 1).
|
study
| 100.0 |
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