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1.  Vaccination with gag, vif, and nef Gene Fragments Affords Partial Control of Viral Replication after Mucosal Challenge with SIVmac239 
Journal of Virology  2014;88(13):7493-7516.
Broadly targeted cellular immune responses are thought to be important for controlling replication of human and simian immunodeficiency viruses (HIV and SIV). However, eliciting such responses by vaccination is complicated by immunodominance, the preferential targeting of only a few of the many possible epitopes of a given antigen. This phenomenon may be due to the coexpression of dominant and subdominant epitopes by the same antigen-presenting cell and may be overcome by distributing these sequences among several different vaccine constructs. Accordingly, we tested whether vaccinating rhesus macaques with “minigenes” encoding fragments of Gag, Vif, and Nef resulted in broadened cellular responses capable of controlling SIV replication. We delivered these minigenes through combinations of recombinant Mycobacterium bovis BCG (rBCG), electroporated recombinant DNA (rDNA) along with an interleukin-12 (IL-12)-expressing plasmid (EP rDNA plus pIL-12), yellow fever vaccine virus 17D (rYF17D), and recombinant adenovirus serotype 5 (rAd5). Although priming with EP rDNA plus pIL-12 increased the breadth of vaccine-induced T-cell responses, this effect was likely due to the improved antigen delivery afforded by electroporation rather than modulation of immunodominance. Indeed, Mamu-A*01+ vaccinees mounted CD8+ T cells directed against only one subdominant epitope, regardless of the vaccination regimen. After challenge with SIVmac239, vaccine efficacy was limited to a modest reduction in set point in some of the groups and did not correlate with standard T-cell measurements. These findings suggest that broad T-cell responses elicited by conventional vectors may not be sufficient to substantially contain AIDS virus replication.
IMPORTANCE Immunodominance poses a major obstacle to the generation of broadly targeted, HIV-specific cellular responses by vaccination. Here we attempted to circumvent this phenomenon and thereby broaden the repertoire of SIV-specific cellular responses by vaccinating rhesus macaques with minigenes encoding fragments of Gag, Vif, and Nef. In contrast to previous mouse studies, this strategy appeared to minimally affect monkey CD8+ T-cell immundominance hierarchies, as seen by the detection of only one subdominant epitope in Mamu-A*01+ vaccinees. This finding underscores the difficulty of inducing subdominant CD8+ T cells by vaccination and demonstrates that strategies other than gene fragmentation may be required to significantly alter immunodominance in primates. Although some of the regimens tested here were extremely immunogenic, vaccine efficacy was limited to a modest reduction in set point viremia after challenge with SIVmac239. No correlates of protection were identified. These results reinforce the notion that vaccine immunogenicity does not predict control of AIDS virus replication.
PMCID: PMC4054456  PMID: 24741098
2.  Psychological Treatment of Hypnotic-Dependent Insomnia in a Primarily Older Adult Sample 
Behaviour research and therapy  2013;51(12):10.1016/j.brat.2013.09.006.
This study tested cognitive behavior therapy (CBT) in hypnotic-dependent, late middle-age and older adults with insomnia.
Seventy volunteers age 50 and older were randomized to CBT plus drug withdrawal, placebo biofeedback (PL) plus drug withdrawal, or drug withdrawal (MED) only. The CBT and PL groups received eight, 45 minute weekly treatment sessions. The drug withdrawal protocol comprised slow tapering monitored with about six biweekly, 30 minute sessions. Assessment including polysomnography (PSG), sleep diaries, hypnotic consumption, daytime functioning questionnaires, and drug screens collected at baseline, posttreatment, and 1-year follow-up.
Only the CBT group showed significant sleep diary improvement, sleep onset latency significantly decreased at posttreatment. For all sleep diary measures for all groups, including MED, sleep trended to improvement from baseline to follow-up. Most PSG sleep variables did not significantly change. There were no significant between group differences in medication reduction. Compared to baseline, the three groups decreased hypnotic use at posttreatment, down 84%, and follow-up, down 66%. There was no evidence of withdrawal side-effects. Daytime functioning, including anxiety and depression, improved by posttreatment. Rigorous methodological features, including documentation of strong treatment implementation and the presence of a credible placebo, elevated the confidence due these findings.
Gradual drug withdrawal was associated with substantial hypnotic reduction at posttreatment and follow-up, and withdrawal side-effects were absent. When supplemented with CBT, participants accrued incremental self-reported, but not PSG, sleep benefits.
PMCID: PMC3838504  PMID: 24121096
hypnotic dependence; drug withdrawal; insomnia; cognitive behavior therapy
3.  The Peaceful Mind Manual: A Protocol for Treating Anxiety in Persons with Dementia 
Behavior modification  2013;37(5):10.1177/0145445513477420.
Anxiety disorders are highly prevalent among individuals with dementia and have a significant negative impact on their lives. Peaceful Mind is a form of Cognitive-Behavioral Therapy for anxiety in persons with dementia. The Peaceful Mind manual was developed, piloted and modified over 2 years. In an open trial and a small randomized, controlled trial, it decreased anxiety and caregiver distress. The treatment meets the unique needs of individuals with dementia by emphasizing behavioral rather than cognitive interventions, slowing the pace, limiting the material to be learned, increasing repetition and practice, using cues to stimulate memory, including a friend or family member in treatment as a coach, and providing sessions in the home. The manual presented here includes modules that teach specific skills, including awareness, breathing, calming self-statements, increasing activity, and sleep management, as well as general suggestions for treatment delivery.
PMCID: PMC3872091  PMID: 23447103
Depression and anxiety  2014;31(8):653-661.
Despite their high rates of depression, homebound older adults have limited access to evidence-based psychotherapy. The purpose of this paper was to report both depression and disability outcomes of telehealth problem-solving therapy (tele-PST via Skype video call) for low-income homebound older adults over 6 months postintervention.
A 3-arm randomized controlled trial compared the efficacy of tele-PST to in-person PST and telephone care calls with 158 homebound individuals who were aged 50+ and scored 15+ on the 24-item Hamilton Rating Scale for Depression (HAMD). Treatment effects on depression severity (HAMD score) and disability (score on the WHO Disability Assessment Schedule [WHODAS]) were analyzed using mixed-effects regression with random intercept models. Possible reciprocal relationships between depression and disability were examined with a parallel-process latent growth curve model.
Both tele-PST and in-person PST were efficacious treatments for low-income homebound older adults; however the effects of tele-PST on both depression and disability outcomes were sustained significantly longer than those of in-person PST. Effect sizes (dGMA-raw) for HAMD score changes at 36 weeks were 0.68 for tele-PST and 0.20 for in-person PST. Effect sizes for WHODAS score changes at 36 weeks were 0.47 for tele-PST and 0.25 for in-person PST. The results also supported reciprocal and indirect effects between depression and disability outcomes.
The efficacy and potential low cost of tele-delivered psychotherapy show its potential for easy replication and sustainability to reach a large number of underserved older adults and improve their access to mental health services.
PMCID: PMC4122624  PMID: 24501015
depression; disability; tele-psychotherapy; homebound older adults
5.  The Peaceful Mind Program: A Pilot Test of a CBT-Based Intervention for Anxious Patients with Dementia 
To assess feasibility and to conduct a preliminary evaluation of outcomes following Peaceful Mind, a CBT-based intervention for anxiety in dementia, relative to usual care (UC).
Pilot randomized controlled trial including assessments at baseline, 3 and 6 months
Houston, TX
32 outpatients diagnosed with mild (47%) or moderate (53%) dementia receiving care through outpatient clinics at the Veterans Affairs medical center, Baylor College of Medicine, Harris County Hospital District and community day centers for dementia, and their collaterals, who spent at least 8 hours a week with them.
Peaceful Mind included up to 12 weekly in-home sessions (mean = 8.7, SD = 2.27) during the initial 3 months and up to eight brief telephone sessions (mean = 5.4, SD = 3.17) during months 3 to 6, involving self-monitoring for anxiety, deep breathing, and optional skills (coping self-statements, behavioral activation and sleep management). Patients learned skills, and collaterals served as coaches. In UC, patients received diagnostic feedback; and providers were informed of inclusion status.
Neuropsychiatric Inventory-Anxiety subscale, Rating Anxiety in Dementia scale, Penn State Worry Questionnaire-Abbreviated, Geriatric Anxiety Inventory, Geriatric Depression Scale, Quality of Life in Alzheimer’s disease, Patient Health Questionnaire, Client Satisfaction Questionnaire
Feasibility was demonstrated with regard to recruitment, attrition, and treatment characteristics. At 3 months, clinicians rated patients receiving Peaceful Mind as less anxious, and patients rated themselves as having higher quality of life; collaterals reported less distress related to loved ones’ anxiety. Although significant positive effects were not noted in other outcomes or at 6-month follow-up, the pilot nature of the trial prohibits conclusions about efficacy.
Results support that Peaceful Mind is ready for future comparative clinical trials.
PMCID: PMC3411894  PMID: 23567399
anxiety; dementia; cognitive behavioral therapy; self-ratings; proxy ratings
6.  Predictors of Inpatient Utilization among Veterans with Dementia 
Dementia is prevalent and costly, yet the predictors of inpatient hospitalization are not well understood. Logistic and negative binomial regressions were used to identify predictors of inpatient hospital utilization and the frequency of inpatient hospital utilization, respectively, among veterans. Variables significant at the P < 0.15 level were subsequently analyzed in a multivariate regression. This study of veterans with a diagnosis of dementia (n = 296) and their caregivers found marital status to predict hospitalization in the multivariate logistic model (B = 0.493, P = 0.029) and personal-care dependency to predict hospitalization and readmission in the multivariate logistic model and the multivariate negative binomial model (B = 1.048, P = 0.007, B = 0.040, and P = 0.035, resp.). Persons with dementia with personal-care dependency and spousal caregivers have more inpatient admissions; appropriate care environments should receive special care to reduce hospitalization. This study was part of a larger clinical trial; this trial is registered with NCT00291161.
PMCID: PMC4058849  PMID: 24982674
7.  Treating late-life GAD in primary care: An effectiveness pilot study 
To increase sustainability of Cognitive Behavior Therapy (CBT) in primary care for late-life anxiety, we incorporated non-expert counselors, options for telephone meetings, and integration with primary care clinicians.
This open trial examines the feasibility, satisfaction and clinical outcomes of CBT delivered by experienced and non-experienced counselors for older adults with generalized anxiety disorder (GAD). Clinical outcomes assessed worry (Penn State Worry Questionnaire), GAD (Generalized Anxiety Disorder Severity Scale), and anxiety (Beck Anxiety Inventory and Structured Interview Guide for Hamilton Anxiety Scale).
Following 3 months of treatment, Cohen’s d effect sizes for worry and anxiety ranged from .48 to .78. Patients treated by experienced and non-experienced counselors had similar reductions in worry and anxiety, although treatment outcomes were more improved on the Beck Anxiety Inventory for experienced therapists.
Preliminary results suggest adapted CBT can effectively reduce worry. The piloted modifications can provide acceptable and feasible evidence-based care.
PMCID: PMC3644354  PMID: 23588228
cognitive behavioral therapy; generalized anxiety disorder; primary care; older adults; mental health
8.  Mental Health Service Use Among Depressed, Low-Income Homebound Middle-Aged and Older Adults 
Journal of aging and health  2013;25(4):638-655.
This study examined previous mental health service use among low-income homebound middle-aged and older adults who participated in a study testing the feasibility and efficacy of telehealth problem-solving therapy for depression.
The sample consisted of 188 homebound adults aged 50 years or older. Data on mental health service use were collected at baseline. We used multivariable logistic regression analysis to examine correlates of different types of outpatient service use within the preceding 12 months.
Of the subjects, 56% reported mental health service use. Of the users, 80% had made at least one primary care mental health visit, 21% had visited a psychiatrist, and 25% had received counseling. Higher depressive symptom severity scores were positively associated with a psychiatrist visit only.
The need to improve low-income homebound older adults’ access to psychotherapy was clearly evident.
PMCID: PMC3971526  PMID: 23584592
mental health; frailty; geriatrics
9.  Exposures of 129 Preschool Children to Organochlorines, Organophosphates, Pyrethroids, and Acid Herbicides at Their Homes and Daycares in North Carolina 
Few data exist on the concurrent exposures of young children to past-use and current-use pesticides in their everyday environments. In this further analysis of study data, we quantified the potential exposures and intake doses of 129 preschool children, ages 20 to 66 months, to 16 pesticides (eight organochlorines, two organophosphates, three pyrethroids, and three acid herbicides). Environmental samples (soil, dust, outdoor air, and indoor air) and personal samples (hand wipes, solid food, and liquid food) were collected at 129 homes and 13 daycare centers in six counties in North Carolina between 2000 and 2001. α-Chlordane, γ-chlordane, heptachlor, chlorpyrifos, diazinon, cis-permethrin, trans-permethrin, and 2,4-dichlorophenoxyacetic acid (2,4-D) were detected ≥50% in two or more media in both settings. Of these pesticides, the children’s estimated median potential intake doses through dietary ingestion, nondietary ingestion, and inhalation routes were the highest for 2,4-D and cis/trans-permethrin (both 4.84 ng/kg/day), cis/trans-permethrin (2.39 ng/kg/day), and heptachlor (1.71 ng/kg/day), respectively. The children’s estimated median potential aggregate intake doses by all three routes were quantifiable for chlorpyrifos (4.6 ng/kg/day), cis/trans-permethrin (12.5 ng/kg/day), and 2,4-D (4.9 ng/kg/day). In conclusion, these children were likely exposed daily to several pesticides from several sources and routes at their homes and daycares.
PMCID: PMC4025031  PMID: 24705361
children; pesticides; exposure; intake dose; residences; daycare centers
10.  Vaccination against Endogenous Retrotransposable Element Consensus Sequences Does Not Protect Rhesus Macaques from SIVsmE660 Infection and Replication 
PLoS ONE  2014;9(3):e92012.
The enormous sequence diversity of HIV remains a major roadblock to the development of a prophylactic vaccine and new approaches to induce protective immunity are needed. Endogenous retrotransposable elements (ERE) such as endogenous retrovirus K (ERV)-K and long interspersed nuclear element-1 (LINE-1) are activated during HIV-1-infection and could represent stable, surrogate targets to eliminate HIV-1-infected cells. Here, we explored the hypothesis that vaccination against ERE would protect macaques from acquisition and replication of simian immunodeficiency virus (SIV). Following vaccination with antigens derived from LINE-1 and ERV-K consensus sequences, animals mounted immune responses that failed to delay acquisition of SIVsmE660. We observed no differences in acute or set point viral loads between ERE-vaccinated and control animals suggesting that ERE-specific responses were not protective. Indeed, ERE-specific T cells failed to expand anamnestically in vivo following infection with SIVsmE660 and did not recognize SIV-infected targets in vitro, in agreement with no significant induction of targeted ERE mRNA by SIV in macaque CD4+ T cells. Instead, lower infection rates and viral loads correlated significantly to protective TRIM5α alleles. Cumulatively, these data demonstrate that vaccination against the selected ERE consensus sequences in macaques did not lead to immune-mediated recognition and killing of SIV-infected cells, as has been shown for HIV-infected human cells using patient-derived HERV-K-specific T cells. Thus, further research is required to identify the specific nonhuman primate EREs and retroviruses that recapitulate the activity of HIV-1 in human cells. These results also highlight the complexity in translating observations of the interplay between HIV-1 and human EREs to animal models.
PMCID: PMC3961289  PMID: 24651676
11.  A controlled trial of Partners in Dementia Care: veteran outcomes after six and twelve months 
“Partners in Dementia Care” (PDC) tested the effectiveness of a care-coordination program integrating healthcare and community services and supporting veterans with dementia and their caregivers. Delivered via partnerships between Veterans Affairs medical centers and Alzheimer’s Association chapters, PDC targeted both patients and caregivers, distinguishing it from many non-pharmacological interventions. Hypotheses posited PDC would improve five veteran self-reported outcomes: 1) unmet need, 2) embarrassment about memory problems, 3) isolation, 4) relationship strain and 5) depression. Greater impact was expected for more impaired veterans. A unique feature was self-reported research data collected from veterans with dementia.
Methods and Findings
Five matched communities were study sites. Two randomly selected sites received PDC for 12 months; comparison sites received usual care. Three structured telephone interviews were completed every 6 months with veterans who could participate.
Of 508 consenting veterans, 333 (65.6%) completed baseline interviews. Among those who completed baseline interviews, 263 (79.0%) completed 6-month follow-ups and 194 (58.3%) completed 12-month follow-ups. Regression analyses showed PDC veterans had significantly less adverse outcomes than those receiving usual care, particularly for more impaired veterans after 6 months, including reduced relationship strain (B = −0.09; p = 0.05), depression (B = −0.10; p = 0.03), and unmet need (B = −0.28; p = 0.02; and B = −0.52; p = 0.08). PDC veterans also had less embarrassment about memory problems (B = −0.24; p = 0.08). At 12 months, more impaired veterans had further reductions in unmet need (B = −0.96; p < 0.01) and embarrassment (B = −0.05; p = 0.02). Limitations included use of matched comparison sites rather than within-site randomization and lack of consideration for variation within the PDC group in amounts and types of assistance provided.
Partnerships between community and health organizations have the potential to meet the dementia-related needs and improve the psychosocial functioning of persons with dementia.
Trial Registry
PMCID: PMC3978714  PMID: 24764496
12.  Vaccine-induced CD8+ T cells control AIDS virus replication 
Nature  2012;491(7422):10.1038/nature11443.
Developing a vaccine for HIV may be aided by a complete understanding of those rare cases where some HIV-infected individuals control replication of the virus1–3. The majority of these elite controllers (ECs) express HLA-B*57 or HLA-B*273. These alleles remain by far the most robust associations with low concentrations of plasma virus4,5, yet the mechanism of control in these individuals is not entirely clear. Here we vaccinated Indian rhesus macaques that express Mamu-B*08, an animal model for HLA-B*27-mediated elite control6, with three Mamu-B*08-restricted CD8+ T cell epitopes and demonstrate that these vaccinated animals controlled replication of the highly pathogenic SIVmac239 clonal virus. High frequencies of CD8+ T cells against these Vif and Nef epitopes in the blood, lymph nodes and colon, were associated with viral control. Moreover, the frequency of the Nef RL10-specific response correlated significantly with reduced acute phase viremia. Finally, two of the eight vaccinees lost control of viral replication in the chronic phase, concomitant with escape in all three targeted epitopes, further implicating these three CD8+ T cell responses in control of viral replication. Our findings indicate that narrowly targeted vaccine-induced virus-specific CD8+ T cell responses can control replication of the AIDS virus.
PMCID: PMC3883109  PMID: 23023123
13.  Escape from CD8+ T Cell Responses in Mamu-B*00801+ Macaques Differentiates Progressors from Elite Controllers 
Journal of immunology (Baltimore, Md. : 1950)  2012;188(7):10.4049/jimmunol.1102470.
A small number of HIV-infected individuals known as elite controllers experience low levels of chronic phase viral replication and delayed progression to AIDS. Specific HLA class I alleles are associated with elite control, implicating CD8+ T lymphocytes in the establishment of these low levels of viral replication. Most HIV-infected individuals that express protective HLA class I alleles, however, do not control viral replication. Approximately 50% of Mamu-B*00801+ Indian rhesus macaques control SIVmac239 replication in the chronic phase in a manner that resembles elite control in humans. We followed both the immune response and viral evolution in SIV-infected Mamu-B*00801+ animals to better understand the role of CD8+ T lymphocytes during the acute phase of viral infection, when viral control status is determined. The virus escaped from immunodominant Vif and Nef Mamu-B*00801–restricted CD8+ T lymphocyte responses during the critical early weeks of acute infection only in progressor animals that did not control viral replication. Thus, early CD8+ T lymphocyte escape is a hallmark of Mamu-B*00801+ macaques who do not control viral replication. By contrast, virus in elite controller macaques showed little evidence of variation in epitopes recognized by immunodominant CD8+ T lymphocytes, implying that these cells play a role in viral control.
PMCID: PMC3883130  PMID: 22387557
14.  Concordance of Self- and Proxy-Rated Worry and Anxiety Symptoms in Older Adults with Dementia 
Journal of anxiety disorders  2012;27(1):125-130.
We compared the psychometric performance of two validated self-report anxiety- symptom measures when rated by people with dementia versus collaterals (as proxies). Forty-one participants with mild-to-moderate dementia and their respective collaterals completed the Geriatric Anxiety Inventory, the Penn State Worry Questionnaire-Abbreviated, and a structured diagnostic interview. We used descriptive and nonparametric statistics to compare scores according to respondent characteristics. Receiver operating characteristic (ROC) curves were calculated to establish the predictive validity of each instrument by rater type against a clinical diagnosis of an anxiety disorder. Participant and collateral ratings performed comparably for both instruments. However, collaterals tended to give more severe symptom ratings, and the best-performing cut-off scores were higher for collaterals. Our findings suggest that people with mild-to-moderate dementia can give reliable self-reports of anxiety symptoms, with validity comparable to reports obtained from collaterals. Scores obtained from multiple informants should be interpreted in context.
PMCID: PMC3578141  PMID: 23270995
worry; anxiety; elders; dementia; self-ratings; proxy ratings
15.  The Pittsburgh Sleep Quality Index in Older Primary Care Patients with Generalized Anxiety Disorder: Pyschometrics and Outcomes Following Cognitive Behavioral Therapy 
Psychiatry research  2012;199(1):24-30.
The Pittsburgh Sleep Quality Index (PSQI) is a widely used, comprehensive self-report measure of sleep quality and impairment, which has demonstrated good psychometric properties within various populations, including older adults. However, the psychometric properties of the PSQI and its component scores have not been evaluated for older adults with generalized anxiety disorder (GAD). Additionally, changes in PSQI global or component scores have not been reported following cognitive-behavioral treatment (CBT) of late-life GAD. This study examined (1) the psychometric properties of the PSQI within a sample of 216 elderly primary care patients age 60 or older with GAD who were referred for treatment of worry and/or anxiety; as well as (2) response to CBT, relative to usual care, for 134 patients with principal or coprincipal GAD. The PSQI demonstrated good internal consistency reliability and adequate evidence of construct validity. Those receiving CBT experienced greater reductions in PSQI global scores at post-treatment, relative to those receiving usual care. Further, PSQI global and domain scores pertaining to sleep quality and difficulties falling asleep (i.e., sleep latency and sleep disturbances) demonstrated response to treatment over a 12-month follow-up period. Overall, results highlight the usefulness of the PSQI global and component scores for use in older adults with GAD.
PMCID: PMC3401329  PMID: 22503380
Pittsburgh Sleep Quality Index; psychometrics; generalized anxiety disorder; elderly; cognitive behavioral therapy
16.  Nox2 is a mediator of chronic CsA nephrotoxicity 
We hypothesized that Nox2, the classical phagocytic NADPH oxidase, plays an important role in calcineurin inhibitor (CNI)-induced renal fibrosis. We tested this hypothesis in vitro, in animal and in human studies. Cyclosporine A (CsA) and tacrolimus (TAC) were associated with greater levels of Nox2 mRNA and epithelial to mesenchymal transition (EMT) in NRK52E cells. CsA increased Nox2, α-SMA and phosphorylated-p38MAPK, Smad3, and NFκB proteins. Nox2 upregulation and EMT were inhibited in TGF-β1 knockout cells suggesting that TGF-β1 is required for Nox2 activation. Fisher344 rats treated with high dose CsA showed increased Nox2 in the tubulointerstitium and greater Nox2, α-SMA, phosphorylated Smad3 and nitrotyrosine by immunoblot analyses. Inhibition of Nox2 by coadministration of apocynin or diphenyleneiodonium was associated with reduced fibrogenesis. We validated these findings by treating wild type and Nox2 null (B6.129S-CybbTm1Din/J) mice with high dose CsA. Western blot analyses confirmed the absence of Nox2 and significantly lower levels of α-SMA and 4-hydroxynonenal in CsA-treated knockout mice. These findings were clinically relevant since Nox2 and α-SMA were increased in the tubulointerstitium of kidneys from 15 liver transplant recipients with biopsy-confirmed chronic CsA or TAC nephrotoxicity. In conclusion, specific Nox2 inhibition strategies may improve chronic CNI nephrotoxicity in solid organ transplantation.
PMCID: PMC3409317  PMID: 22568654
CsA; Nox2; Fibrosis; EMT; Oxidative Stress
17.  The Role of MHC Class I Allele Mamu-*07 During SIVmac239 Infection 
Immunogenetics  2011;63(12):789-807.
Virus-specific CD8+ T cells play an important role in controlling HIV/SIV replication. These T cells recognize intracellular pathogen-derived peptides displayed on the cell surface by individual MHC class I molecules. In the SIV-infected rhesus macaque model, five Mamu class I alleles have been thoroughly characterized with regard to peptide binding, and a sixth was shown to be uninvolved. In this study, we describe the peptide binding of Mamu-A1*007:01 (formerly Mamu-A*07), an allele present in roughly 5.08% of Indian-origin rhesus macaques (n=63 of 1240). We determined a preliminary binding motif by eluting and sequencing endogenously bound ligands. Subsequently, we used a positional scanning combinatorial library and panels of single amino acid substitution analogs to further characterize peptide binding of this allele and derive a quantitative motif. Using this motif, we selected and tested 200 peptides derived from SIVmac239 for their capacity to bind Mamu-A1*007:01, 33 were found to bind with an affinity of 500nM or better. We then used PBMC from SIV-infected or vaccinated but uninfected, A1*007:01-positive rhesus macaques in IFN-γ Elispot assays to screen the peptides for T cell reactivity. In all, eleven of the peptides elicited IFN-γ+ T cell responses. Six represent novel A1*007:01-restricted epitopes. Furthermore, both Sanger and ultra-deep pyrosequencing demonstrated the accumulation of amino acid substitutions within four of these six regions, suggestive of selective pressure on the virus by antigen-specific CD8+ T cells. Thus, it appears that Mamu-A1*007:01 presents SIV-derived peptides to antigen-specific CD8+ T cells and is part of the immune response to SIVmac239.
PMCID: PMC3706270  PMID: 21732180
SIV; MHC; Macaque; Epitope; Escape
18.  Psychometric Properties of a Structured Interview Guide for the Rating for Anxiety in Dementia (RAID-SI) 
Aging & mental health  2012;16(5):592-602.
The Rating Anxiety in Dementia (RAID; Shankar et al, 1999)is a clinical rating scale developed to evaluate anxiety in persons with dementia. This report explores the psychometric properties and clinical utility of a new structured interview format of the RAID (RAID-SI), developed to standardize administration and scoring based on information obtained from the patient, an identified collateral, and rater observation.
The RAID-SI was administered by trained master’s level raters. Participants were 32 persons with dementia who qualified for an anxiety treatment outcome study. Self-report anxiety, depression, and quality of life measures were administered to both the person with dementia and a collateral.
The RAID-SI exhibited adequate internal consistency reliability and inter-rater reliability. There was also some evidence of construct validity as indicated by significant correlations with other measures of patient-reported and collateral-reported anxiety, and non-significant correlations with collateral reports of patient depression and quality of life. Further, RAID-SI scores were significantly higher in persons with an anxiety diagnosis compared to those without an anxiety diagnosis.
There is evidence that the RAID-SI exhibits good reliability and validity in older adults with dementia. The advantage of the structured interview format is increased standardization in administration and scoring, which may be particularly important when RAID raters are not experienced clinicians.
PMCID: PMC3371288  PMID: 22372475
dementia; anxiety; clinical interview; assessment; Rating for Anxiety in Dementia
19.  Tubular expression of heat shock protein 27 inhibits fibrogenesis in obstructive nephropathy 
Kidney international  2012;83(1):84-92.
Morphological changes that occur during kidney injury involve actin skeleton remodeling. Here we tested whether heat shock protein 27 (HSP27), a small stress response protein involved in cytoskeletal remodeling, protects the kidney from tubulointerstitial fibrosis in obstructive nephropathy. Tubular cell HSP27 immunostaining was significantly increased in human kidneys with ureteropelvic junction obstruction; supporting the clinical relevance of our studies. To develop an animal model for mechanistic studies we generated transgenic mice that specifically overexpress human HSP27 in renal tubules, under the kidney androgen-regulated protein promoter, and determined the effects of HSP27 overexpression on epithelial-to-mesenchymal transition and tubulointerstitial fibrosis following unilateral ureteral obstruction. This was associated with decreased fibrogenesis as evidenced by significant declines in phosphorylated p38MAPK, collagen III, α-smooth muscle actin, 4-hydroxynonenal, and reduced trichrome staining following obstruction. Notably, E-cadherin and β-catenin remained at the cell membrane of tubular cells in transgenic mice with an obstructed ureter. Monocyte/macrophage infiltration, however, was not significantly affected in these transgenic mice. Thus, tubular HSP27 inhibits fibrogenesis in obstructive nephropathy. Further studies are needed to determine pathways regulating the interactions between HSP27 and the E-cadherin-β-catenin complex.
PMCID: PMC3525804  PMID: 22971995
20.  A trivalent recombinant Ad5 gag/pol/nef vaccine fails to protect rhesus macaques from infection or control virus replication after a limiting-dose heterologous SIV challenge 
Vaccine  2012;30(30):4465-4475.
It has been suggested that poor immunogenicity may explain the lack of vaccine efficacy in preventing or controlling HIV infection in the Step trial. To investigate this issue we vaccinated eight Indian rhesus macaques with a trivalent replication-incompetent adenovirus serotype 5 vaccine expressing SIV Gag, Pol, and Nef using a regimen similar to that employed in the Step trial. We detected broad vaccine-induced CD8+ (2–7 pool-specific responses) and CD4+ (5–19 pool-specific responses) T-cell responses in IFN-γ ELISPOT assays at one week post-boost using fresh PBMC. However, using cryopreserved cells at one and four weeks post-boost we observed a reduction in both the number and magnitude of most vaccine-induced responses. This demonstrates that the time points and conditions chosen to perform immune assays may influence the observed breadth and frequency of vaccine-induced T-cell responses. To evaluate protective efficacy, we challenged the immunized macaques, along with naïve controls, with repeated, limiting doses of the heterologous swarm isolate SIVsmE660. Vaccination did not significantly affect acquisition or control of virus replication in vaccinees compared to naïve controls. Post-infection we observed an average of only two anamnestic CD8+ T-cell responses per animal, which may not have been sufficiently broad to control heterologous virus replication. While the trivalent vaccine regimen induced relatively broad T-cell responses in rhesus macaques, it failed to protect against infection or control viral replication. Our results are consistent with those observed in the Step trial and indicate that SIV immunization and challenge studies in macaque models of HIV infection can be informative in assessing pre-clinical HIV vaccines.
PMCID: PMC3372643  PMID: 22569124
HIV vaccine; Adenovirus serotype 5; Simian Immunodeficiency Virus; CD8+ T cells; CD4+ T cells; Step trial
21.  T Cells Target APOBEC3 Proteins in Human Immunodeficiency Virus Type 1-Infected Humans and Simian Immunodeficiency Virus-Infected Indian Rhesus Macaques 
Journal of Virology  2013;87(11):6073-6080.
APOBEC3 proteins mediate potent antiretroviral activity by hypermutating the retroviral genome during reverse transcription. To counteract APOBEC3 and gain a replicative advantage, lentiviruses such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) have evolved the Vif protein, which targets APOBEC3 proteins for proteasomal degradation. However, the proteasome plays a critical role in the generation of T cell peptide epitopes. Whether Vif-mediated destruction of APOBEC3 proteins leads to the generation and presentation of APOBEC3-derived T cell epitopes on the surfaces of lentivirus-infected cells remains unknown. Here, using peptides derived from multiple Vif-sensitive APOBEC3 proteins, we identified APOBEC3-specific T cell responses in both HIV-1-infected patients and SIV-infected rhesus macaques. These results raise the possibility that these T cell responses may be part of the larger antiretroviral immune response.
PMCID: PMC3648095  PMID: 23536679
22.  Acute Phase CD8+ T Lymphocytes against Alternate Reading Frame Epitopes Select for Rapid Viral Escape during SIV Infection 
PLoS ONE  2013;8(5):e61383.
CD8+ T Lymphocytes (CTL) can control AIDS virus replication. However, natural selection favoring viral variants that escape CTL recognition is a common feature of both simian immunodeficiency virus (SIV) infection of macaques and HIV infection of humans. Emerging data indicate that CTL directed against alternate reading frame (ARF)-derived epitopes (a.k.a. cryptic epitopes) are important components of the total virus-specific response in SIV and HIV infection but the contributions of these responses during the critical first several weeks of infection have not been determined. We used a focused deep sequencing approach to examine acute phase viral evolution in response to CTL targeting two polypeptides encoded by ARFs of SIVmac239 in SIV-infected rhesus macaques. We report high magnitude CTL responses as early as three weeks post-infection against epitopes within both ARFs, which both overlap the 5′ end of the env gene. Further, mutations accumulated in the epitopes by three to four weeks post infection consistent with viral escape. Interestingly, these mutations largely maintained the primary amino acid sequence of the overlapping Envelope protein. Our data show that high frequency CTL target cryptic epitopes and exert selective pressure on SIV during the acute phase, underscoring the importance of these unique immune responses.
PMCID: PMC3645990  PMID: 23671565
23.  Immunogenicity of Seven New Recombinant Yellow Fever Viruses 17D Expressing Fragments of SIVmac239 Gag, Nef, and Vif in Indian Rhesus Macaques 
PLoS ONE  2013;8(1):e54434.
An effective vaccine remains the best solution to stop the spread of human immunodeficiency virus (HIV). Cellular immune responses have been repeatedly associated with control of viral replication and thus may be an important element of the immune response that must be evoked by an efficacious vaccine. Recombinant viral vectors can induce potent T-cell responses. Although several viral vectors have been developed to deliver HIV genes, only a few have been advanced for clinical trials. The live-attenuated yellow fever vaccine virus 17D (YF17D) has many properties that make it an attractive vector for AIDS vaccine regimens. YF17D is well tolerated in humans and vaccination induces robust T-cell responses that persist for years. Additionally, methods to manipulate the YF17D genome have been established, enabling the generation of recombinant (r)YF17D vectors carrying genes from unrelated pathogens. Here, we report the generation of seven new rYF17D viruses expressing fragments of simian immunodeficiency virus (SIV)mac239 Gag, Nef, and Vif. Studies in Indian rhesus macaques demonstrated that these live-attenuated vectors replicated in vivo, but only elicited low levels of SIV-specific cellular responses. Boosting with recombinant Adenovirus type-5 (rAd5) vectors resulted in robust expansion of SIV-specific CD8+ T-cell responses, particularly those targeting Vif. Priming with rYF17D also increased the frequency of CD4+ cellular responses in rYF17D/rAd5-immunized macaques compared to animals that received rAd5 only. The effect of the rYF17D prime on the breadth of SIV-specific T-cell responses was limited and we also found evidence that some rYF17D vectors were more effective than others at priming SIV-specific T-cell responses. Together, our data suggest that YF17D – a clinically relevant vaccine vector – can be used to prime AIDS virus-specific T-cell responses in heterologous prime boost regimens. However, it will be important to optimize rYF17D-based vaccine regimens to ensure maximum delivery of all immunogens in a multivalent vaccine.
PMCID: PMC3545953  PMID: 23336000
24.  Characteristics of Generalized Anxiety Disorder in Patients With Dementia 
Overlap of cognitive and anxiety symptoms (i.e., difficulty concentrating, fatigue, restlessness) contributes to inconsistent, complicated assessment of generalized anxiety disorder (GAD)in persons with dementia.
Anxious dementia patients completed a psychiatric interview, the Penn State Worry Questionnaire-Abbreviated, and the Rating for Anxiety in Dementia scale. Analyses to describe the 43 patients with and without GAD included the Wilcoxon Mann-Whitney two-sample test, Fisher’s exact test. Predictors of GAD diagnosis were identified using logistic regression.
Those with GAD were more likely to be male, have less severe dementia and endorsed more worry, and anxiety compared to patients without GAD. Gender, muscle tension and fatigue differentiated those with GAD from those without GAD.
Although this study is limited by a small sample, it describes clinical characteristics of GAD in dementia, highlighting the importance of muscle tension and fatigue in recognizing GAD in persons with dementia.
PMCID: PMC3252749  PMID: 22062223
Generalized anxiety disorder; dementia; anxiety symptoms; dementia symptoms; differential diagnosis for generalized anxiety disorder/dementia
25.  DNA/Ad5 vaccination with SIV epitopes induced epitope-specific CD4+ T cells, but few subdominant epitope-specific CD8+ T cells 
Vaccine  2011;29(43):7483-7490.
The goals of a T cell-based vaccine for HIV are to reduce viral peak and setpoint and prevent transmission. While it has been relatively straightforward to induce CD8+ T cell responses against immunodominant T cell epitopes, it has been more difficult to broaden the vaccine-induced CD8+ T cell response against subdominant T cell epitopes. Additionally, vaccine regimens to induce CD4+ T cell responses have been studied only in limited settings. In this study, we sought to elicit CD8+ T cells against subdominant epitopes and CD4+ T cells using various novel and well-established vaccine strategies. We vaccinated three Mamu-A*01+ animals with five Mamu-A*01-restricted subdominant SIV-specific CD8+ T cell epitopes. All three vaccinated animals made high frequency responses against the Mamu-A*01-restricted Env TL9 epitope with one animal making a low frequency CD8+ T cell response against the Pol LV10 epitope. We also induced SIV-specific CD4+ T cells against several MHC class II DRBw*606-restricted epitopes. Electroporated DNA with pIL-12 followed by a rAd5 boost was the most immunogenic vaccine strategy. We induced responses against all three Mamu-DRB*w606-restricted CD4 epitopes in the vaccine after the DNA prime. Ad5 vaccination further boosted these responses. Although we successfully elicited several robust epitope-specific CD4+ T cell responses, vaccination with subdominant MHC class epitopes elicited few detectable CD8+ T cell responses. Broadening the CD8+ T cell response against subdominant MHC class I epitopes was, therefore, more difficult than we initially anticipated.
PMCID: PMC3186839  PMID: 21839132

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