Cathepsin B and cystat

in B in HIV-seropositivewomen are associated with infection andHIV-1-associated neurocognitive disorders

Yisel Cantres-Rosarioa, Marines Plaud-Valentına,d, Yamil Gerenac,

Richard L. Skolaskye, Valerie Wojnab,d and Loyda M. Melendeza,d

Copyright © L

aDepartment of MSpecialized Neuroof Orthopedic Sur

Correspondence tSpecialized Neuro

Tel: +787 777 00Received: 24 July

DOI:10.1097/QAD

ISS

Objective: HIV-1-associated neurocognitive disorders (HAND) is triggered by immuneactivation of brain cells and remain prevalent during progressive viral infectiondespite antiretroviral therapy. Cathepsins and cystatins are lysosomal proteins secretedby macrophages and microglia, and may play important roles in neuroregulatoryresponses. Our laboratory has shown increased secretion and neurotoxicity of cathepsinB from in-vitro HIV-infected monocyte-derived macrophages, and increased expressionin postmortem brain tissue with HIV encephalitis and HAND. We hypothesized thatcystatin B and cathepsin B could represent potential biomarkers for HAND.

Methods: Monocytes, plasma, and cerebrospinal fluid (CSF) from retrospectivesamples from 63 HIV-seropositive Hispanic women were selected for this study.These were stratified as 27 normal, 14 asymptomatic, and 22 HIV dementia, and as14 progressors and 17 nonprogressors. Samples were evaluated for cystatins B and Cand cathepsin B expression and activity.

Results: Increased cathepsin B and cystatins B and C were found in plasma of HIV-seropositive women. Higher intracellular expression of cathepsin B and cystatin B werefound in monocytes from women with HIV-associated dementia (P<0.05). Significantincrease in cystatin B concentration in CSF was found in women with dementiacompared with HIV-seropositive asymptomatic women.

Conclusion: These results demonstrate that dysregulation of cystatin B–cathepsin Bsystem is operative in HIV-associated neurocognitive impairment and suggests thatintracellular expression of cystatin B and cathepsin B in monocytes could be potentialcandidate biomarkers for HIV dementia, whereas increased cathepsin B and cystatins Band C in plasma are potential candidate markers of chronic HIV-1 activation.

� 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins

AIDS 2013, 27:347–356

Keywords: cathepsin B, cerebrospinal fluid, cystatin B, cystatin C,HIV-1-associated neurocognitive disorders, HIV-1, monocytes, plasma

Introduction

HIV-1 infection affects more than 40 million peopleworldwide and is associated with neurocognitivedisorders (HAND) in approximately 25% of the cases

ippincott Williams & Wilkins. Unaut

icrobiology, bNeurology Division, Departmentscience Program, University of Puerto Rico, Medigery, John Hopkins University, Baltimore, Mary

o Loyda M. Melendez, PhD, Professor, Departmscience Program, Medical Sciences Campus, Sa

79; fax: +787 777 0078; e-mail: loyda.melende2012; revised: 2 October 2012; accepted: 9 Oc

.0b013e32835b3e47

N 0269-9370 Q 2013 Wolters Kluwer He

[1]. HAND can develop years after viral infectionand immunosuppression and remain prevalent inmilder forms in patients receiving combined antiretroviraltherapy (cART) [2]. The pathogenesis of HAND involvesincreased blood–brain barrier (BBB) permeability,

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of Internal Medicine, cSchool of Pharmacy, dNeuroAIDScal Sciences Campus, San Juan, Puerto Rico, and eDepartmentland, USA.

ent of Microbiology, School of Medicine, and NeuroAIDSn Juan 00935, Puerto Rico.

z@upr.edutober 2012.

alth | Lippincott Williams & Wilkins 347

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inflammation, oxidative stress, neuronal dysfunction, anddeath [3]. The increased BBB permeability facilitates themigration of HIV-infected and activated monocytes intothe brain, with subsequent infection and activationof resident perivascular macrophages and microglia(reviewed in [4]). HIV neurotoxicity occurs both directly,through toxic effects of HIV proteins, and indirectly,through macrophage activation.

Activated macrophages secrete a variety of soluble factorsthat are potentially toxic to neurons, including cytokines,proteases, excitotoxins, and reactive oxygen species[5–7]. One macrophage-secreted factor that couldpromote neuronal apoptosis is cathepsin B, a lysosomalcysteine protease with known roles in inflammation,intracellular protein degradation, and cell death [8–10].Increased cathepsin B levels were observed in perivascularmacrophages from white matter in brain tissue of patientswith AIDS in the pre-HAART era [11]. We recentlyfound increased secretion of cathepsin B from humanmonocyte-derived macrophages (MDM) after HIVinfection in vitro [12]. Moreover, we found thatsupernatants from HIV-infected MDM had greaterneurotoxic activity than supernatants from uninfectedMDM, and that this neurotoxic activity could be blockedby either a cathepsin B inhibitor or an antibody tocathepsin B. The increased neurotoxic activity ofcathepsin B secreted by HIV-infected MDM in vitromay result from dysregulation of its normal cellularlocalization and protein–protein interactions: in HIV-infected MDM, the protease was no longer sequesteredin lysosomes and showed reduced interactions with itsendogenous inhibitors, cystatin B and cystatin C.

We have now undertaken in-vivo studies to test thehypothesis that dysregulation of the cathepsin B pathwayplays a role in the pathogenesis of HAND. In a recent pilotanalysis of human postmortem tissue, we found evidenceof increased cathepsin B levels in postmortem brain tissuefrom individuals with HAND [12]. In the current study,we analyzed the expression of cathepsin B and cystatins Band C in samples of monocytes, plasma, and cerebrospinalfluid (CSF) obtained from the repository established forour Hispanic-Latino cohort of HIV-seropositive womenand examined whether alterations in these proteinswere associated with the presence or progression toHAND.

Patients and methods

Study patientsThe study patients are from the Hispanic-Latinolongitudinal cohort of HIV-seropositive women,followed since 2001 as part of the NeuroAIDS SpecializedNeuroscience Research Program at the University ofPuerto Rico, Medical Science Campus. The study had

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the approval of the Institutional Review Board (UPR-MSC; IRB #0720109) and was conducted with theinformed consent of the participants. The study is aretrospective analysis of plasma, CSF, and monocytesamples from this cohort that were collected from 2003 to2009. Inclusion criteria for HIV-seropositive patientswere as follows: HIV-infected women with or withoutHAART with CD4 cell count less than 500 copies/mland/or more than 1000 viral copies while onHAART, age 18–50 years, at least ninth grade education,and nondrug users defined as those with less than fiveexposures in a lifetime to drugs (opiates, heroin,methamphetamine, cocaine/crack, speed ball). For thepurpose of this study, tobacco or nicotine and marijuanawere considered as drugs. The exclusion criteria wereas follows: women 18 years old or less; opportunisticinfections of the central nervous system, history ofseizures, history of head trauma, or any other neuro-psychiatric condition that, in the judgment of theinvestigations, may impact the study; underlying neuro-psychiatric illness such as cerebrovascular events; priormeningitis or encephalitis; any active systemic infectionor systemic illness that, in the judgement of theinvestigators, may impact the study; pregnant or nursingmothers, or women with recent birth (<60 days); activedrug use; all patients screened with urine toxicology;patients abusing alcohol, using the Michigan AlcoholismScreening Test (MAST) more than three points; patientsunwilling to give informed consent; and educationlevel less than ninth grade. The CD4 cell counts and viralload parameters were evaluated at an AIDS ClinicalTrial Group (ACTG) certified LDMS Lab 053 at theDepartment of Pathology of the UPR-MSC.

Patient data and samples for this study were collected inour female Hispanic cohort from 2003 to 2009. At thattime, we used the American Academy of NeurologyHIV-associated Dementia criteria (AAN) [13,14], whichcategorized cognitive performance into normal, minorcognitive motor disorder (MCMD), and HIV-associateddementia (HAD). The new nosology for researchcalled HIV-associated neurocognitive disorder (HAND)is based on both neuropsychological performance andfunctional assessment. However, given the difficulty ofassessing the HAND criteria in all cohorts, an alternativeset of criteria was established in which the AAN criteriawere modified to add a stage called asymptomaticcognitive impairment (ANI), which includes HIV-positive women who presented decreased performanceof 1 SD in two or more neuropsychological tests or 2 SDin one or more tests without disturbances in activitiesof daily living (see modified AAN criteria, or mAAN[1,15]). Thus, our staging fits with the HAND criteriaas follows: normal is equivalent to normal in bothsets of criteria, ANI using the mAAN criteria is similarto the ANI using the HAND criteria, and HAD inthe mAAN criteria is equivalent to HAD in the HANDcriteria.

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Cathepsin B and cystatins B and C in HAND Cantres-Rosario et al. 349

The neuropsychological tests included tests of verbalmemory (trial 5, delay recall, and recognition of the ReyAuditory Verbal Learning Test), frontal executive func-tion (Stroop word/color and Trail Making B), psycho-motor speed (Symbol Digit Modalities Test and visual andauditory reaction time nondominant hand), motor speed(Trail Making A and Grooved Pegboard dominant andnondominant hand), and the Beck Depression Index.Patients were classified as having normal cognition ornondemented, ANI, MCMD, or HAD [16,17]. In one ofthe analyses, patients were also classified as progressors ornonprogressors based on the cognitive status results fromtwo consecutive visits registered 6 months apart.Progressors were defined as those patients whosecognitive status worsened between the first visit andthe second (i.e. from normal cognition to ANI, ANI toHAD, or normal cognition to HAD). Nonprogressorswere defined as those who remained in the same categoryfrom the first visit to the second (i.e. were either normalcognition or ANI at both visits). Patients who werealready classified as HAD and remained stable in thiscategory were not considered in this analysis. Differentsets of patients were used for each experiment.

Patient samplesBlood samples from the patients were collected in fourtubes containing acid citrate dextrose anticoagulant andcentrifuged to obtain plasma for storage in 0.5-ml aliquotsat �808C. Peripheral blood mononuclear cells (PBMC)were isolated using Lymphosep medium (MP Biomedi-cals, Solon, Ohio, USA) and frozen for flow cytometryanalyses as described in ACTG manuals (www.hanc.info).Briefly, for freezing, cells were initially suspended in 20%dimethyl sulfoxide (DMSO) in Roswell Park MemorialInstitute media (RPMI) on ice and slowly diluted with10% DMSO and 50% fetal bovine serum (FBS) in RPMIfor a final concentration of 5� 107 cells/ml. Cells werekept on ice and immediately aliquoted into 1-ml vials.Cells were transferred to a freezing container (Mr. Frosty,Nalgene, New York, USA), kept at �808C for less than2 weeks, and then transferred to liquid nitrogen.

Monocyte isolation from PBMC was done by positiveselection using magnetic cell sorting columns and CD14þ

microbeads (Miltenyi Biotech, Auburn, California,USA). For analyses of the expression of cathepsin andcystatins in monocytes from progressors and nonpro-gressors, isolated monocytes were lysed (5-mmol/l Tris–HCl, 0.1% Triton X-100, pH 8.0) and treated withprotease inhibitors from Sigma–Aldrich (St Louis,Missouri, USA). These lysates came from monocyteswith viability levels of over 90% and had been stored at�808C for up to 3 years.

Expression of cathepsin B and its inhibitors inplasma and cerebrospinal fluidCathepsin B expression was quantified by ELISA (R&DSystems, Minnesota, Minneapolis, USA) according to the

Copyright © Lippincott Williams & Wilkins. Unaut

manufacturer’s instructions. CSF was diluted at 1 : 5,whereas plasma was diluted at 1 : 10. Cystatin B levelswere measured with an ELISA (USCN Life Science Inc.,Wuhan, China) according to the manufacturer’s instruc-tions with a final dilution of 1 : 10 for plasma and 1 : 20 forCSF. Cystatin C levels were measured by ELISA(BioVendor, Candler, North Carolina, USA) followingthe manufacturer’s instructions with final dilutions of 1 : 400for plasma and CSF. All samples were assayed in duplicateand read at 450 nm in an ELISA Reader (Varioskan Flash;Thermo Fisher Scientific, Waltham, Massachusetts, USA).

Cathepsin B activityCathepsin B activity was detected using the Cathepsin BActivity Kit (BioVision; Milpitas, California, USA). Thiskit is a fluorescence-based assay that utilizes the preferredcathepsin B substrate sequence RR labeled with amino-4-trifluoromethyl coumarin (AFC), which is cleaved bycathepsin B to release free AFC. Samples were assayedin duplicate following the manufacturer’s instructions.The free AFC was quantified using a fluorescence platereader (Varioskan Flash; Thermo Fisher Scientific) withexcitation at 400 nm and emission at 505 nm.

Intracellular expression of cathepsin B andcystatin BWe analyzed intracellular cathepsin B and cystatin B levelsin monocytes of 30 patients from PBMC stored in liquidnitrogen for less than a year of cryopreservation.Immediately after removing vial from liquid nitrogen,cells were thawed by placing them in a 378C water bathfollowed by ice bath for 2 min. Contents were transferredinto a 50-ml centrifugation tube with slow addition of10 ml of 20% FBS in RPMI (should be at roomtemperature) over 3 min while mixing gently. Cells werediluted up to 30 ml with RPMI 20% FBS and left standingfor 5–10 min before centrifugation at 1100 rpm for10 min to remove the DMSO-containing supernatants.Cells were resuspended in RPMI/10% FBS and countedusing trypan blue dye to determine viability.

For determination of CD14 positive monocytes, PBMCs(1� 106 cells) were incubated with anti-CD14-PEantibody (BD Biosciences, San Jose, California, USA)for 1 h at 48C. For detection of intracellular cathepsin Band cystatin B levels, anti-CD14 PE-labeled PBMCswere 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) for1 h at 48C, and finally stained with FITC secondaryantibody (1 : 500). Fluorescence levels were analyzed byflow cytometry.

Flow cytometryFlow cytometry was carried out using a FACSCaliburcytometer (BD Biosciences). The Cell Quest software(BD Biosciences) was used for data acquisition and

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350 AIDS 2013, Vol 27 No 3

multivariate analysis. Monocytes were gated in forward/side scatter dot plots and FITC or PE emission wasmeasured in the FL1 (band pass filter 525 nm) or FL2(band pass filter 585 nm) channels. Data on scatterparameters and histograms were acquired in log mode.Ten thousand events were evaluated for each sample andthe median peak channel obtained from the histogramswas used to determine levels of cathepsin B and cystatin B.

Statistical analysesStatistical analyses of age, viral load, CD4 cell counts, andtherapy parameters among different cognitive groupswere performed using ANOVA. Statistical significanceof progression was analyzed by Mann–Whitney test.All assays were performed using commercial kitsvalidated with their own standard curves. Samples wererun in duplicates with less than 20% variability. Onesample already analyzed was included in all of the assays asinternal standard to test the variability of the assay, which

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Fig. 1. Intracellular expression of cystatin B and cathepsin B in modisorders. Monocytes from PBMC of 30 HIV-1-seropositive womeversus forward scatter dot plots (a) and identified using a CD14 momonoclonal antibodies (FL1 channel) (b). Representative histogrfluorescence intensities from HIV-seropositive normal cognition (dacystatin B and cathepsin B compared with the nonfluorescent mrepresentation of these differences in normal cognition (dark bars)dementia; PBMC, peripheral blood mononuclear cells.

was determined to be less than 10%. Statistical analyses ofprotein concentrations and activity among cognitive groupswere performed by Kruskal–Wallis tests. Distributionalassumptions were tested for all continuous variables usingthe Shapiro–Wilk test for normality. In the case that thistest demonstrated nonnormal distribution, nonparametricstatistics were used. Correlations were tested usingthe Spearman correlation test for cathepsin B activity(non-Gaussian distribution) and Pearson correlation forcathepsin B, cystatin B, and cystatin C concentrationmeasurements (normally distributed).

Results

Study patientsThis study analyzed repository samples of PBMC, mono-cyte lysates, plasma, and CSF from 18 HIV-seronegative

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ams of intracellular staining show significant differences inrk histogram) and HAD (grey histogram) on the expression ofonocytes (unshaded histograms to the left) (c). A graphicand HAD (grey bars) is shown in (d). HAD, HIV-associated

Cathepsin B and cystatins B and C in HAND Cantres-Rosario et al. 351

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Fig. 2. Cathepsin B expression and activity in plasma and cerebrospinal fluid. Cathepsin B concentration was measured in theplasma of individuals from different cognitive groups: normal cognition or NC (n¼19), ANI (n¼12), and HAD (n¼17) and HIV-seronegative controls. (n¼18), (a). Activity was measured in the plasma of control (n¼18), NC (n¼ 20), ANI (n¼12), and HAD(n¼17) individuals (b). Concentration of the protein was measured by ELISA in the CSF of NC (n¼18), ANI (n¼12), and HAD(n¼17) individuals (c). Activity was measured in the CSF of NC (n¼20), ANI (n¼12), and HAD (n¼ 17) individuals (d). CSF,cerebrospinal fluid; NC, normal cognition; ANI, asymptomatic neurocognitive impairment; HAD, HIV-associated dementia.

controls and 63 HIV-seropositive patients from ourHispanic women cohort. Patients were stratified as27 normal cognition, 14 ANI, and 22 HAD for analyses bycognitive group, and as 17 progressors and 14 nonpro-gressors for analyses of progression. Most of the patients(>80%) were on cART, with an average CSF penetrationindex (CPE) of 7. There were no significant differences inage, CD4 cell counts, plasma or CSF HIV RNA levels, orCPE among HIV-seropositive patients in the differentcognition groups in the patient sample as a whole, or in thesamples of patients used in different substudies (i.e. ofPBMC, plasma and CSF). No significant differences inCD4 cell counts, plasma or CSF HIV RNA levels, or CPEwere observed in samples collected from the 12 progressorsversus the 17 nonprogressors at either the first or secondvisit. There was a significant difference in CD4 nadir(P< 0.001), with progressors having a higher CD4 nadirthan nonprogressors (data not shown).

Intracellular cathepsin B and cystatin B levels areelevated in monocytes of HIV-associateddementia patientsIntracellular levels of cathepsin B and cystatin B weremeasured in monocytes from samples of PBMC from

Copyright © Lippincott Williams & Wilkins. Unaut

16 HIV-seropositive women with normal cognitionand 14 women with HAD. After thawing PBMCfrom liquid nitrogen storage, the cells viability waswithin a range of 94–98%. PBMCs were stained withantibodies against CD14 and cystatin B or cathepsin Band analyzed by flow cytometry. Intracellular levelsof both proteins were significantly higher in mono-cytes of HIV-seropositive women with HAD than inthose of HIV-seropositive women with normal cognition(Fig. 1).

Plasma cathepsin B expression and activity isincreased in HIV-seropositive patientsCathepsin B levels and activity were measured in plasmafrom HIV-seronegative patients (n¼ 18) and HIV-seropositive patients with normal cognition (n¼ 19),ANI (n¼ 12), or HAD (n¼ 17). Plasma levels of bothcathepsin B protein and activity were significantly higherin the normal cognition and HAD groups comparedwith those in the HIV-seronegative control group (Fig. 2aand b). Cathepsin B activity was not significantly differentin ANI patients compared with HIV-seronegativepatients and lower than that in the HIV-seropositivepatients with normal cognition (Fig. 2b).

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Fig. 3. Concentration of cystatins B and C in plasma and cerebrospinal fluid measured by ELISA. Cystatin B concentration wasmeasured in the plasma of HIV-seronegative controls (n¼ 18), normal cognition or NC (n¼19), ANI (n¼12), and HAD (n¼ 15)patients (a). Cystatin C concentration was measured in the plasma of control (n¼18), normal cognition (NC) (n¼18), ANI (n¼11),and HAD (n¼17) patients (b). In CSF, cystatin B was measured from NC (n¼20), ANI (n¼ 13), and HAD (n¼18) individuals (c),whereas cystatin C was measured in NC (n¼18), ANI (n¼12) and HAD (n¼ 14) individuals (d). CSF, cerebrospinal fluid; NC,normal cognition; ANI, asymptomatic neurocognitive impairment; HAD, HIV-associated dementia.

Cathepsin B levels and activity were also measured in CSFof HIV-seropositive patients: normal cognition (n¼ 18),ANI (n¼ 12), and HAD (n¼ 17). Samples of CSFfrom HIV-seronegative controls were not available to beincluded in the experiments. There were no significantdifferences in CSF cathepsin B levels or activity across thethree HIV-seropositive groups (Fig. 2c and d).

Cystatin B and cystatin C levels are higher inplasma of HIV-infected patientsPlasma cystatin B and cystatin C levels were significantlyhigher in all groups of HIV-seropositive patients thanthose in the HIV-seronegative group (Fig. 3a and b).There were no significant differences in cystatin B or Clevels among the HIV-seropositive groups with differentlevels of cognitive function. Plasma cystatin C levelsshowed a trend toward increasing levels in ANI and HADpatients compared with the normal cognition patients. Incontrast, plasma cystatin B levels were lower in HADpatients than in ANI patients. In CSF, cystatin B levelswere significantly higher in HAD patients than in normalcognition and ANI patients (P< 0.05; Fig. 3c). Therewere no significant differences in CSF cystatin C levels

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among the three HIV-seropositive groups. Interestingly,in all groups, cystatin C was found at much higher levelsin CSF than in plasma.

Cathepsin B expression and activity and cystatinC expression are similar in progressor andnonprogressor patientsCathepsin B levels and activity and cystatin C levelswere measured in cell lysates of monocytes collectedat two consecutive visits from nonprogressors andprogressors. The two groups of patients showed nosignificant differences in levels of any of these markersin monocyte lysates (Fig. 4a–c). We then asked whether acorrelation existed between cathepsin B expression oractivity relative to levels of its inhibitor, cystatin C.Cathepsin B activity was not correlated with cystatin Clevels as analyzed by a Spearman correlation test (data notshown). However, a positive correlation existed betweencathepsin B and cystatin C concentrations, which wasstronger in progressors (r2¼ 0.62, P< 0.0001) than innonprogressors (r2¼ 0.3459, P¼ 0.004) as determined byPearson correlation test (Fig. 4d and e).

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Cathepsin B and cystatins B and C in HAND Cantres-Rosario et al. 353

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Fig. 4. Cystatin C and cathepsin B in monocytes of patients categorized by progression of HIV-1-associated neurocognitivedisorders. Progressors and nonprogressors were stratified according to change in cognitive status from two consecutive visits.Cathepsin B and cystatin C concentrations and cathepsin B activity was measured in monocytes from each patient in the two visitsand the change in concentration or activity was plotted for each group for statistical analysis. Cathepsin B concentration wasmeasured in monocytes from nonprogressors (n¼11) and progressors (n¼ 16) (a). Cathepsin B activity (b) and cystatin Cconcentration (c) was measured in monocytes from nonprogressors (n¼12) and progressors (n¼ 17). Statistical significance wascalculated by Mann–Whitney test and Pearson correlation.

Discussion

There is increasing evidence that lysosomal enzymes andtheir inhibitors play roles in the development ofneurodegenerative conditions [18,19]. Cathepsin Band/or cystatins B and C have been linked toneurodegenerative diseases such as Alzheimer’s disease[20], multiple sclerosis [21], and progressive myoclonusepilepsy (EPM1) [22]. Previous in-vitro studies con-ducted in our laboratory demonstrated that cathepsin Band cystatins B and C are overexpressed by HIV-infectedMDM and secreted into the extracellular fluid. Moreover,we demonstrated that MDM-conditioned mediuminduced apoptosis of cultured neuronal cells, and thatthis effect could be blocked by treating MDM-conditioned medium with a specific cathepsin Binhibitor, CA-074, or a monoclonal antibody tocathepsin B. In addition, preliminary immunocytochemi-cal studies of postmortem brain tissue suggested thatcathepsin B and cystatin B are upregulated in thehippocampus and basal ganglia of patients with HAND[12].

Copyright © Lippincott Williams & Wilkins. Unaut

To further explore the potential roles of cathepsin B andits inhibitors in the development of HAND, we analyzedthe expression of these proteins in repository samples ofPBMC, monocytes, plasma, and CSF from a cohort ofHIV-seropositive Hispanic women characterized forcognitive function [15]. We found that intracellular levelsof cathepsin B, as determined by flow cytometry of intactPBMC, were significantly increased in the CD14þ

monocytes of HIV-infected women diagnosed withHAD than in those of HIV-infected women with normalcognition, as were intracellular cystatin B levels.Additional studies of cathepsin B activity and levels andcystatin C levels were performed with stored samples ofcell lysates of monocytes from progressors and nonpro-gressors to HAD, but no difference in any of these markerswas seen between the two groups. We were unable tomeasure cystatin B levels in that study because they werebelow the limit of detection with the antibody used. Withregard to cathepsin B levels, the different results obtainedin the flow cytometry analysis and cell lysate analysis mayreflect the different methodologies used in the twostudies. Flow cytometry detects intracellular proteins in

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living cells, whereas the lysate analyses were done on cellsafter separation with anti-CD14 antibodies and magneticbeads and lysis with detergents. Unfortunately, sufficientlive PBMCs were not available for the studies ofprogressors versus nonprogressors. Currently, samplesare being stored in liquid nitrogen for future analyses ofprogressors versus nonprogressors by flow cytometry. Wedid find a stronger positive correlation between cystatin Cand cathepsin B concentrations in progressors relative tononprogressors, suggesting increased secretion of cystatinC in response to cathepsin B in progressors; this possibilitywill require further studies. Finally, there are severaldistinct subpopulations of monocytes infected by HIV,which may contribute differentially to the developmentof HAND [23,24]. The CD14þ/CD16þ monocytesubset has been characterized as a viral reservoir, withpreferential susceptibility to HIV infection [25–27].Recent work in the Hawaii Aging Group [28] showedthat HIV DNA copy number in the CD14þ/CD16þ

subset correlates with HAND, and also with greatersecretion of inflammatory cytokines whose expression areregulated in part by cathepsin B. Further studies ofanalysis of the expression of cathepsin B and cystatins Band C within different monocyte subpopulations mayhelp to further elucidate the relationship of cystatins levelsto cathepsin B activity, and how changes in monocytelevels of these proteins relate to the developmentof HANDs.

We next determined the concentration of cathepsin B andcystatins B and C in plasma and CSF. Plasma levels of bothcathepsin B protein and activity were significantly higherin HIV-seropositive women with normal cognition andwith HAD than in healthy controls, although in thissample they were not elevated in HIV-seropositivewomen with ANI. Cystatins B and C were higher inall three HIV-seropositive groups compared with healthywomen, although their concentration exhibited adifferent pattern. Among the HIV-seropositive patients,there was no clear correlation between plasma levels ofcathepsin B, cathepsin B activity, or cystatins B and Cand cognitive status. For example, cystatin B levelswere elevated in asymptomatic HIV-seropositivepatients relative to those with normal cognition, whereascathepsin B activity showed the reverse pattern. Thus, thebiological significance of the changes in plasma levels ofcystatin B and cathepsin B activity seen in asymptomaticpatients in this study is unclear, and the relationship ofthese markers to HAND will require further study withlarger patient sample sizes.

CSF levels of inflammatory chemokines correlate withmeasures of regional brain metabolism in HIV-seropositive patients [29], and elevated levels of certainchemokines can be detected in the CSF at early stages ofHIV-1 infection, even when viral load in the CSF isundetectable [30]. However, we found no significantdifferences among the HIV-seropositive groups in CSF

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cathepsin B or cystatin C levels, or in CSF cathepsin Bactivity. CSF cystatin B levels were significantly higher inHAD patients than in those with normal cognition orANI. Measurements in CSF of healthy individuals couldnot be performed due to unavailability of CSF samplesfrom uninfected donors, and this comparison remainscritical. However, it was interesting to find that amongHIV-seropositive groups, cathepsin B protein was presentat lower levels in CSF than in plasma, whereas thereverse was true for cathepsin B activity and cystatin B andC levels.

Thus, our results show that cathepsin B is upregulated inplasma of HIV-seropositive patients as compared withHIV seronegative controls. These results are consistentwith our in-vitro findings that HIV infection upregulatessecretion of active cathepsin B by cultured MDM [12].Two recent studies have suggested roles for cathepsin B inHIV infection [31] and/or release from infected cells [32],but in our study, we found no correlation of cathepsin Blevels with plasma HIV-1 RNA copy number or viralload. Previous studies showed that cystatin B expression isinduced in cultured MDM after HIV infection [17], andthis protein has been associated with HIV replicationin MDM [33]. Consistent with that observation, we sawupregulated levels of cystatin B in plasma of all HIV-seropositive groups compared with healthy controls,although cystatin B levels did not correlate with plasmaHIV load. On the basis of the literature in which pro-viralDNA in activated monocytes correlates with HAND[34], the plasma viral load is reflective of the CD4 T-cellinfection rather than the monocyte infection, whichexplains the lack of correlation with cystatin B. In furtherstudies, we plan to determine the association betweencystatin B in monocytes with surface activation markersand pro-viral DNA.

Cathepsin B has now been postulated to play a role in thepathogenesis of several neurodegenerative conditions.Elevated levels of cathepsin B have been reported in theplasma and CSF of individuals with Alzheimer’s disease[35,36]. Cathepsin B has b-secretase activity, and inAlzheimer’s disease mice model, it appears to promoteneurodegeneration by increasing b-amyloid production[37]. Luo et al. [38] reported that cathepsin B expression isalso increased after traumatic brain injury and promotesneuronal cell death via mitochondria-mediated apoptoticpathways. Cathepsin B is also upregulated in the primatehippocampus following ischemic injury, and cathepsin Binhibitors protect against neuronal cell death in thatmodel [39,40]. Finally, cathepsin B has been reported tobe elevated in the CSF of elderly individuals [41],suggesting that the enzyme is associated with multipleage-related neurodegenerative conditions.

In conclusion, we have demonstrated increased expres-sion of cathepsin B levels and activity in HIV-seropositivepatients, together with increased levels of cathepsin B’s

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Cathepsin B and cystatins B and C in HAND Cantres-Rosario et al. 355

endogenous inhibitors, cystatins B and C. Although therewas no clear correlation of any of these proteins withcognitive status among the HIV-seropositive groups,elevated levels of these proteins could contribute to thepathogenesis of HAND. For example, chronic exposureof neurons to elevated levels of cathepsin B could causeprogressive neurodegeneration, even if the elevated levelsof cathepsin B do not increase further during the courseof infection. Moreover, we did see increased levels ofcathepsin B and cystatin B in intact monocytes of HIV-seropositive patients with HAD relative to those withnormal cognition. Thus, plasma levels of these proteinsmay not reflect monocytes levels. And, as monocytesenter the brain of HIV-infected individuals, it ismonocyte levels rather than plasma levels of cathepsinB and its inhibitors that are likely more relevant toHAND pathogenesis. Previous studies of plasma, serum,and CSF samples from HIV-seropositive patients withcART in search for possible candidate biomarkers forHAND have identified promising host protein and lipidcandidates, many of them monocyte-derived proteins[42–48]. However, most are also present in ANI andpatients with other neurological diseases [47]. Never-theless, a battery of these candidate markers in plasma andCSF, especially those derived from monocytes, likesCD14 and CCL2, are being tested in longitudinal studiesby the CHARTER cohort in the cART era. Studies ofmonocytes as targets for candidate biomarkers havedemonstrated that monocyte subpopulations [49–52],proteomics profiles and antioxidants [6,53,54], andproviral DNA [28,34] have been considered as potentialcandidates for larger biomarker studies (reviewed byMelendez et al. [55]).

Again, further studies with intact monocytes, and withlarger sample sizes (and including male as well as femalepatients), will be necessary to clarify this issue and todetermine whether the levels of cathepsin B and itsinhibitors in intact monocytes might serve as biomarkersfor HAND.

Acknowledgements

This work was supported by grants from the NationalInstitutes of Health R01MH083516 (L.M.M., V.W.),U54NS043011 (V.W., L.M.M.), R25GM061838 (Y.C.).Research infrastructure support was provided inpart by NCRR-2G12-RR003051/NIMHHD 8G12-MD007600 Translational Proteomics Center and FlowCytometry Facilities. Additional funding was provided byUPR Vice President and the Associate Deanship ofBiomedical Sciences institutional funds at the UPRMedical Sciences Campus.

Authors contributions: manuscript writing, data analysis,and discussion was done by L.M.M. and Y.C.; ELISAs

Copyright © Lippincott Williams & Wilkins. Unaut

and activity experiments was done by M.P.-V.; flowcytometry was done by M.P.-V. and Y.G.; statisticalanalyses was done by R.L.S., M.P., and Y.C.; and humanpatients were provided by V.W.

Conflicts of interestL.M.M. has a patent application approved for cystatin B,related to this manuscript (patent No. 8,143,231).

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