HIV-1 infection affects more than 40 million people worldwide and is associated with neurocognitive disorders (HAND) in approximately 25% of the cases . HAND can develop years after viral infection and immunosuppression and remain prevalent in milder forms in patients receiving combined antiretroviral therapy (cART) . The pathogenesis of HAND involves increased blood–brain barrier (BBB) permeability, inflammation, oxidative stress, neuronal dysfunction, and death . The increased BBB permeability facilitates the migration of HIV-infected and activated monocytes into the brain, with subsequent infection and activation of resident perivascular macrophages and microglia (reviewed in ). HIV neurotoxicity occurs both directly, through toxic effects of HIV proteins, and indirectly, through macrophage activation.
Activated macrophages secrete a variety of soluble factors that are potentially toxic to neurons, including cytokines, proteases, excitotoxins, and reactive oxygen species [5–7]. One macrophage-secreted factor that could promote neuronal apoptosis is cathepsin B, a lysosomal cysteine protease with known roles in inflammation, intracellular protein degradation, and cell death [8–10]. Increased cathepsin B levels were observed in perivascular macrophages from white matter in brain tissue of patients with AIDS in the pre-HAART era . We recently found increased secretion of cathepsin B from human monocyte-derived macrophages (MDM) after HIV infection in vitro. Moreover, we found that supernatants from HIV-infected MDM had greater neurotoxic activity than supernatants from uninfected MDM, and that this neurotoxic activity could be blocked by either a cathepsin B inhibitor or an antibody to cathepsin B. The increased neurotoxic activity of cathepsin B secreted by HIV-infected MDM in vitro may result from dysregulation of its normal cellular localization and protein–protein interactions: in HIV-infected MDM, the protease was no longer sequestered in lysosomes and showed reduced interactions with its endogenous inhibitors, cystatin B and cystatin C.
We have now undertaken in-vivo studies to test the hypothesis that dysregulation of the cathepsin B pathway plays a role in the pathogenesis of HAND. In a recent pilot analysis of human postmortem tissue, we found evidence of increased cathepsin B levels in postmortem brain tissue from individuals with HAND . In the current study, we analyzed the expression of cathepsin B and cystatins B and C in samples of monocytes, plasma, and cerebrospinal fluid (CSF) obtained from the repository established for our Hispanic-Latino cohort of HIV-seropositive women and examined whether alterations in these proteins were associated with the presence or progression to HAND.
Patients and methods
The study patients are from the Hispanic-Latino longitudinal cohort of HIV-seropositive women, followed since 2001 as part of the NeuroAIDS Specialized Neuroscience Research Program at the University of Puerto Rico, Medical Science Campus. The study had the approval of the Institutional Review Board (UPR-MSC; IRB #0720109) and was conducted with the informed consent of the participants. The study is a retrospective analysis of plasma, CSF, and monocyte samples from this cohort that were collected from 2003 to 2009. Inclusion criteria for HIV-seropositive patients were as follows: HIV-infected women with or without HAART with CD4 cell count less than 500 copies/μl and/or more than 1000 viral copies while on HAART, age 18–50 years, at least ninth grade education, and nondrug users defined as those with less than five exposures in a lifetime to drugs (opiates, heroin, methamphetamine, cocaine/crack, speed ball). For the purpose of this study, tobacco or nicotine and marijuana were considered as drugs. The exclusion criteria were as follows: women 18 years old or less; opportunistic infections of the central nervous system, history of seizures, history of head trauma, or any other neuropsychiatric condition that, in the judgment of the investigations, may impact the study; underlying neuropsychiatric illness such as cerebrovascular events; prior meningitis or encephalitis; any active systemic infection or systemic illness that, in the judgement of the investigators, may impact the study; pregnant or nursing mothers, or women with recent birth (<60 days); active drug use; all patients screened with urine toxicology; patients abusing alcohol, using the Michigan Alcoholism Screening Test (MAST) more than three points; patients unwilling to give informed consent; and education level less than ninth grade. The CD4 cell counts and viral load parameters were evaluated at an AIDS Clinical Trial Group (ACTG) certified LDMS Lab 053 at the Department of Pathology of the UPR-MSC.
Patient data and samples for this study were collected in our female Hispanic cohort from 2003 to 2009. At that time, we used the American Academy of Neurology HIV-associated Dementia criteria (AAN) [13,14], which categorized cognitive performance into normal, minor cognitive motor disorder (MCMD), and HIV-associated dementia (HAD). The new nosology for research called HIV-associated neurocognitive disorder (HAND) is based on both neuropsychological performance and functional assessment. However, given the difficulty of assessing the HAND criteria in all cohorts, an alternative set of criteria was established in which the AAN criteria were modified to add a stage called asymptomatic cognitive impairment (ANI), which includes HIV-positive women who presented decreased performance of 1 SD in two or more neuropsychological tests or 2 SD in one or more tests without disturbances in activities of daily living (see modified AAN criteria, or mAAN [1,15]). Thus, our staging fits with the HAND criteria as follows: normal is equivalent to normal in both sets of criteria, ANI using the mAAN criteria is similar to the ANI using the HAND criteria, and HAD in the mAAN criteria is equivalent to HAD in the HAND criteria.
The neuropsychological tests included tests of verbal memory (trial 5, delay recall, and recognition of the Rey Auditory Verbal Learning Test), frontal executive function (Stroop word/color and Trail Making B), psychomotor speed (Symbol Digit Modalities Test and visual and auditory reaction time nondominant hand), motor speed (Trail Making A and Grooved Pegboard dominant and nondominant hand), and the Beck Depression Index. Patients were classified as having normal cognition or nondemented, ANI, MCMD, or HAD [16,17]. In one of the analyses, patients were also classified as progressors or nonprogressors based on the cognitive status results from two consecutive visits registered 6 months apart. Progressors were defined as those patients whose cognitive status worsened between the first visit and the second (i.e. from normal cognition to ANI, ANI to HAD, or normal cognition to HAD). Nonprogressors were defined as those who remained in the same category from the first visit to the second (i.e. were either normal cognition or ANI at both visits). Patients who were already classified as HAD and remained stable in this category were not considered in this analysis. Different sets of patients were used for each experiment.
Blood samples from the patients were collected in four tubes containing acid citrate dextrose anticoagulant and centrifuged to obtain plasma for storage in 0.5-ml aliquots at −80°C. Peripheral blood mononuclear cells (PBMC) were isolated using Lymphosep medium (MP Biomedicals, Solon, Ohio, USA) and frozen for flow cytometry analyses as described in ACTG manuals (www.hanc.info). Briefly, for freezing, cells were initially suspended in 20% dimethyl sulfoxide (DMSO) in Roswell Park Memorial Institute media (RPMI) on ice and slowly diluted with 10% DMSO and 50% fetal bovine serum (FBS) in RPMI for a final concentration of 5 × 107 cells/ml. Cells were kept on ice and immediately aliquoted into 1-ml vials. Cells were transferred to a freezing container (Mr. Frosty, Nalgene, New York, USA), kept at −80°C for less than 2 weeks, and then transferred to liquid nitrogen.
Monocyte isolation from PBMC was done by positive selection using magnetic cell sorting columns and CD14+ microbeads (Miltenyi Biotech, Auburn, California, USA). For analyses of the expression of cathepsin and cystatins in monocytes from progressors and nonprogressors, isolated monocytes were lysed (5-mmol/l Tris–HCl, 0.1% Triton X-100, pH 8.0) and treated with protease inhibitors from Sigma–Aldrich (St Louis, Missouri, USA). These lysates came from monocytes with viability levels of over 90% and had been stored at −80°C for up to 3 years.
Expression of cathepsin B and its inhibitors in plasma and cerebrospinal fluid
Cathepsin B expression was quantified by ELISA (R&D Systems, Minnesota, Minneapolis, USA) according to the manufacturer's instructions. CSF was diluted at 1 : 5, whereas plasma was diluted at 1 : 10. Cystatin B levels were measured with an ELISA (USCN Life Science Inc., Wuhan, China) according to the manufacturer's instructions with a final dilution of 1 : 10 for plasma and 1 : 20 for CSF. Cystatin C levels were measured by ELISA (BioVendor, Candler, North Carolina, USA) following the manufacturer's instructions with final dilutions of 1 : 400 for plasma and CSF. All samples were assayed in duplicate and read at 450 nm in an ELISA Reader (Varioskan Flash; Thermo Fisher Scientific, Waltham, Massachusetts, USA).
Cathepsin B activity
Cathepsin B activity was detected using the Cathepsin B Activity Kit (BioVision; Milpitas, California, USA). This kit is a fluorescence-based assay that utilizes the preferred cathepsin B substrate sequence RR labeled with amino-4-trifluoromethyl coumarin (AFC), which is cleaved by cathepsin B to release free AFC. Samples were assayed in duplicate following the manufacturer's instructions. The free AFC was quantified using a fluorescence plate reader (Varioskan Flash; Thermo Fisher Scientific) with excitation at 400 nm and emission at 505 nm.
Intracellular expression of cathepsin B and cystatin B
We analyzed intracellular cathepsin B and cystatin B levels in monocytes of 30 patients from PBMC stored in liquid nitrogen for less than a year of cryopreservation. Immediately after removing vial from liquid nitrogen, cells were thawed by placing them in a 37°C water bath followed by ice bath for 2 min. Contents were transferred into a 50-ml centrifugation tube with slow addition of 10 ml of 20% FBS in RPMI (should be at room temperature) over 3 min while mixing gently. Cells were diluted up to 30 ml with RPMI 20% FBS and left standing for 5–10 min before centrifugation at 1100 rpm for 10 min to remove the DMSO-containing supernatants. Cells were resuspended in RPMI/10% FBS and counted using trypan blue dye to determine viability.
For determination of CD14 positive monocytes, PBMCs (1 × 106 cells) were incubated with anti-CD14-PE antibody (BD Biosciences, San Jose, California, USA) for 1 h at 4°C. For detection of intracellular cathepsin B and cystatin B levels, anti-CD14 PE-labeled PBMCs were permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences), incubated with anticystatin B (Sigma, St Louis, Missouri, USA) or anticathepsin B (Abcam, Cambridge, Massachusetts, USA) antibodies (1 : 1000) for 1 h at 4°C, and finally stained with FITC secondary antibody (1 : 500). Fluorescence levels were analyzed by flow cytometry.
Flow cytometry was carried out using a FACSCalibur cytometer (BD Biosciences). The Cell Quest software (BD Biosciences) was used for data acquisition and multivariate analysis. Monocytes were gated in forward/side scatter dot plots and FITC or PE emission was measured in the FL1 (band pass filter 525 nm) or FL2 (band pass filter 585 nm) channels. Data on scatter parameters and histograms were acquired in log mode. Ten thousand events were evaluated for each sample and the median peak channel obtained from the histograms was used to determine levels of cathepsin B and cystatin B.
Statistical analyses of age, viral load, CD4 cell counts, and therapy parameters among different cognitive groups were performed using ANOVA. Statistical significance of progression was analyzed by Mann–Whitney test. All assays were performed using commercial kits validated with their own standard curves. Samples were run in duplicates with less than 20% variability. One sample already analyzed was included in all of the assays as internal standard to test the variability of the assay, which was determined to be less than 10%. Statistical analyses of protein concentrations and activity among cognitive groups were performed by Kruskal–Wallis tests. Distributional assumptions were tested for all continuous variables using the Shapiro–Wilk test for normality. In the case that this test demonstrated nonnormal distribution, nonparametric statistics were used. Correlations were tested using the Spearman correlation test for cathepsin B activity (non-Gaussian distribution) and Pearson correlation for cathepsin B, cystatin B, and cystatin C concentration measurements (normally distributed).
This study analyzed repository samples of PBMC, monocyte lysates, plasma, and CSF from 18 HIV-seronegative controls and 63 HIV-seropositive patients from our Hispanic women cohort. Patients were stratified as 27 normal cognition, 14 ANI, and 22 HAD for analyses by cognitive group, and as 17 progressors and 14 nonprogressors for analyses of progression. Most of the patients (>80%) were on cART, with an average CSF penetration index (CPE) of 7. There were no significant differences in age, CD4 cell counts, plasma or CSF HIV RNA levels, or CPE among HIV-seropositive patients in the different cognition groups in the patient sample as a whole, or in the samples of patients used in different substudies (i.e. of PBMC, plasma and CSF). No significant differences in CD4 cell counts, plasma or CSF HIV RNA levels, or CPE were observed in samples collected from the 12 progressors versus the 17 nonprogressors at either the first or second visit. There was a significant difference in CD4 nadir (P < 0.001), with progressors having a higher CD4 nadir than nonprogressors (data not shown).
Intracellular cathepsin B and cystatin B levels are elevated in monocytes of HIV-associated dementia patients
Intracellular levels of cathepsin B and cystatin B were measured in monocytes from samples of PBMC from 16 HIV-seropositive women with normal cognition and 14 women with HAD. After thawing PBMC from liquid nitrogen storage, the cells viability was within a range of 94–98%. PBMCs were stained with antibodies against CD14 and cystatin B or cathepsin B and analyzed by flow cytometry. Intracellular levels of both proteins were significantly higher in monocytes of HIV-seropositive women with HAD than in those of HIV-seropositive women with normal cognition (Fig. 1).
Plasma cathepsin B expression and activity is increased in HIV-seropositive patients
Cathepsin B levels and activity were measured in plasma from HIV-seronegative patients (n = 18) and HIV-seropositive patients with normal cognition (n = 19), ANI (n = 12), or HAD (n = 17). Plasma levels of both cathepsin B protein and activity were significantly higher in the normal cognition and HAD groups compared with those in the HIV-seronegative control group (Fig. 2a and b). Cathepsin B activity was not significantly different in ANI patients compared with HIV-seronegative patients and lower than that in the HIV-seropositive patients with normal cognition (Fig. 2b).
Cathepsin B levels and activity were also measured in CSF of HIV-seropositive patients: normal cognition (n = 18), ANI (n = 12), and HAD (n = 17). Samples of CSF from HIV-seronegative controls were not available to be included in the experiments. There were no significant differences in CSF cathepsin B levels or activity across the three HIV-seropositive groups (Fig. 2c and d).
Cystatin B and cystatin C levels are higher in plasma of HIV-infected patients
Plasma cystatin B and cystatin C levels were significantly higher in all groups of HIV-seropositive patients than those in the HIV-seronegative group (Fig. 3a and b). There were no significant differences in cystatin B or C levels among the HIV-seropositive groups with different levels of cognitive function. Plasma cystatin C levels showed a trend toward increasing levels in ANI and HAD patients compared with the normal cognition patients. In contrast, plasma cystatin B levels were lower in HAD patients than in ANI patients. In CSF, cystatin B levels were significantly higher in HAD patients than in normal cognition and ANI patients (P < 0.05; Fig. 3c). There were no significant differences in CSF cystatin C levels among the three HIV-seropositive groups. Interestingly, in all groups, cystatin C was found at much higher levels in CSF than in plasma.
Cathepsin B expression and activity and cystatin C expression are similar in progressor and nonprogressor patients
Cathepsin B levels and activity and cystatin C levels were measured in cell lysates of monocytes collected at two consecutive visits from nonprogressors and progressors. The two groups of patients showed no significant differences in levels of any of these markers in monocyte lysates (Fig. 4a–c). We then asked whether a correlation existed between cathepsin B expression or activity relative to levels of its inhibitor, cystatin C. Cathepsin B activity was not correlated with cystatin C levels as analyzed by a Spearman correlation test (data not shown). However, a positive correlation existed between cathepsin B and cystatin C concentrations, which was stronger in progressors (r2 = 0.62, P < 0.0001) than in nonprogressors (r2 = 0.3459, P = 0.004) as determined by Pearson correlation test (Fig. 4d and e).
There is increasing evidence that lysosomal enzymes and their inhibitors play roles in the development of neurodegenerative conditions [18,19]. Cathepsin B and/or cystatins B and C have been linked to neurodegenerative diseases such as Alzheimer's disease , multiple sclerosis , and progressive myoclonus epilepsy (EPM1) . Previous in-vitro studies conducted in our laboratory demonstrated that cathepsin B and cystatins B and C are overexpressed by HIV-infected MDM and secreted into the extracellular fluid. Moreover, we demonstrated that MDM-conditioned medium induced apoptosis of cultured neuronal cells, and that this effect could be blocked by treating MDM-conditioned medium with a specific cathepsin B inhibitor, CA-074, or a monoclonal antibody to cathepsin B. In addition, preliminary immunocytochemical studies of postmortem brain tissue suggested that cathepsin B and cystatin B are upregulated in the hippocampus and basal ganglia of patients with HAND .
To further explore the potential roles of cathepsin B and its inhibitors in the development of HAND, we analyzed the expression of these proteins in repository samples of PBMC, monocytes, plasma, and CSF from a cohort of HIV-seropositive Hispanic women characterized for cognitive function . We found that intracellular levels of cathepsin B, as determined by flow cytometry of intact PBMC, were significantly increased in the CD14+ monocytes of HIV-infected women diagnosed with HAD than in those of HIV-infected women with normal cognition, as were intracellular cystatin B levels. Additional studies of cathepsin B activity and levels and cystatin C levels were performed with stored samples of cell lysates of monocytes from progressors and nonprogressors to HAD, but no difference in any of these markers was seen between the two groups. We were unable to measure cystatin B levels in that study because they were below the limit of detection with the antibody used. With regard to cathepsin B levels, the different results obtained in the flow cytometry analysis and cell lysate analysis may reflect the different methodologies used in the two studies. Flow cytometry detects intracellular proteins in living cells, whereas the lysate analyses were done on cells after separation with anti-CD14 antibodies and magnetic beads and lysis with detergents. Unfortunately, sufficient live PBMCs were not available for the studies of progressors versus nonprogressors. Currently, samples are being stored in liquid nitrogen for future analyses of progressors versus nonprogressors by flow cytometry. We did find a stronger positive correlation between cystatin C and cathepsin B concentrations in progressors relative to nonprogressors, suggesting increased secretion of cystatin C in response to cathepsin B in progressors; this possibility will require further studies. Finally, there are several distinct subpopulations of monocytes infected by HIV, which may contribute differentially to the development of HAND [23,24]. The CD14+/CD16+ monocyte subset has been characterized as a viral reservoir, with preferential susceptibility to HIV infection [25–27]. Recent work in the Hawaii Aging Group  showed that HIV DNA copy number in the CD14+/CD16+ subset correlates with HAND, and also with greater secretion of inflammatory cytokines whose expression are regulated in part by cathepsin B. Further studies of analysis of the expression of cathepsin B and cystatins B and C within different monocyte subpopulations may help to further elucidate the relationship of cystatins levels to cathepsin B activity, and how changes in monocyte levels of these proteins relate to the development of HANDs.
We next determined the concentration of cathepsin B and cystatins B and C in plasma and CSF. Plasma levels of both cathepsin B protein and activity were significantly higher in HIV-seropositive women with normal cognition and with HAD than in healthy controls, although in this sample they were not elevated in HIV-seropositive women with ANI. Cystatins B and C were higher in all three HIV-seropositive groups compared with healthy women, although their concentration exhibited a different pattern. Among the HIV-seropositive patients, there was no clear correlation between plasma levels of cathepsin B, cathepsin B activity, or cystatins B and C and cognitive status. For example, cystatin B levels were elevated in asymptomatic HIV-seropositive patients relative to those with normal cognition, whereas cathepsin B activity showed the reverse pattern. Thus, the biological significance of the changes in plasma levels of cystatin B and cathepsin B activity seen in asymptomatic patients in this study is unclear, and the relationship of these markers to HAND will require further study with larger patient sample sizes.
CSF levels of inflammatory chemokines correlate with measures of regional brain metabolism in HIV-seropositive patients , and elevated levels of certain chemokines can be detected in the CSF at early stages of HIV-1 infection, even when viral load in the CSF is undetectable . However, we found no significant differences among the HIV-seropositive groups in CSF cathepsin B or cystatin C levels, or in CSF cathepsin B activity. CSF cystatin B levels were significantly higher in HAD patients than in those with normal cognition or ANI. Measurements in CSF of healthy individuals could not be performed due to unavailability of CSF samples from uninfected donors, and this comparison remains critical. However, it was interesting to find that among HIV-seropositive groups, cathepsin B protein was present at lower levels in CSF than in plasma, whereas the reverse was true for cathepsin B activity and cystatin B and C levels.
Thus, our results show that cathepsin B is upregulated in plasma of HIV-seropositive patients as compared with HIV seronegative controls. These results are consistent with our in-vitro findings that HIV infection upregulates secretion of active cathepsin B by cultured MDM . Two recent studies have suggested roles for cathepsin B in HIV infection  and/or release from infected cells , but in our study, we found no correlation of cathepsin B levels with plasma HIV-1 RNA copy number or viral load. Previous studies showed that cystatin B expression is induced in cultured MDM after HIV infection , and this protein has been associated with HIV replication in MDM . Consistent with that observation, we saw upregulated levels of cystatin B in plasma of all HIV-seropositive groups compared with healthy controls, although cystatin B levels did not correlate with plasma HIV load. On the basis of the literature in which pro-viral DNA in activated monocytes correlates with HAND , the plasma viral load is reflective of the CD4 T-cell infection rather than the monocyte infection, which explains the lack of correlation with cystatin B. In further studies, we plan to determine the association between cystatin B in monocytes with surface activation markers and pro-viral DNA.
Cathepsin B has now been postulated to play a role in the pathogenesis of several neurodegenerative conditions. Elevated levels of cathepsin B have been reported in the plasma and CSF of individuals with Alzheimer's disease [35,36]. Cathepsin B has β-secretase activity, and in Alzheimer's disease mice model, it appears to promote neurodegeneration by increasing β-amyloid production . Luo et al. reported that cathepsin B expression is also increased after traumatic brain injury and promotes neuronal cell death via mitochondria-mediated apoptotic pathways. Cathepsin B is also upregulated in the primate hippocampus following ischemic injury, and cathepsin B inhibitors protect against neuronal cell death in that model [39,40]. Finally, cathepsin B has been reported to be elevated in the CSF of elderly individuals , suggesting that the enzyme is associated with multiple age-related neurodegenerative conditions.
In conclusion, we have demonstrated increased expression of cathepsin B levels and activity in HIV-seropositive patients, together with increased levels of cathepsin B's endogenous inhibitors, cystatins B and C. Although there was no clear correlation of any of these proteins with cognitive status among the HIV-seropositive groups, elevated levels of these proteins could contribute to the pathogenesis of HAND. For example, chronic exposure of neurons to elevated levels of cathepsin B could cause progressive neurodegeneration, even if the elevated levels of cathepsin B do not increase further during the course of infection. Moreover, we did see increased levels of cathepsin B and cystatin B in intact monocytes of HIV-seropositive patients with HAD relative to those with normal cognition. Thus, plasma levels of these proteins may not reflect monocytes levels. And, as monocytes enter the brain of HIV-infected individuals, it is monocyte levels rather than plasma levels of cathepsin B and its inhibitors that are likely more relevant to HAND pathogenesis. Previous studies of plasma, serum, and CSF samples from HIV-seropositive patients with cART in search for possible candidate biomarkers for HAND have identified promising host protein and lipid candidates, many of them monocyte-derived proteins [42–48]. However, most are also present in ANI and patients with other neurological diseases . Nevertheless, a battery of these candidate markers in plasma and CSF, especially those derived from monocytes, like sCD14 and CCL2, are being tested in longitudinal studies by the CHARTER cohort in the cART era. Studies of monocytes as targets for candidate biomarkers have demonstrated that monocyte subpopulations [49–52], proteomics profiles and antioxidants [6,53,54], and proviral DNA [28,34] have been considered as potential candidates for larger biomarker studies (reviewed by Meléndez et al.).
Again, further studies with intact monocytes, and with larger sample sizes (and including male as well as female patients), will be necessary to clarify this issue and to determine whether the levels of cathepsin B and its inhibitors in intact monocytes might serve as biomarkers for HAND.
This work was supported by grants from the National Institutes of Health R01MH083516 (L.M.M., V.W.), U54NS043011 (V.W., L.M.M.), R25GM061838 (Y.C.). Research infrastructure support was provided in part by NCRR-2G12-RR003051/NIMHHD 8G12-MD007600 Translational Proteomics Center and Flow Cytometry Facilities. Additional funding was provided by UPR Vice President and the Associate Deanship of Biomedical Sciences institutional funds at the UPR Medical Sciences Campus.
Authors contributions: manuscript writing, data analysis, and discussion was done by L.M.M. and Y.C.; ELISAs and activity experiments was done by M.P.-V.; flow cytometry was done by M.P.-V. and Y.G.; statistical analyses was done by R.L.S., M.P., and Y.C.; and human patients were provided by V.W.
Conflicts of interest
L.M.M. has a patent application approved for cystatin B, related to this manuscript (patent No. 8,143,231).
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