Selective BCL-XL Antagonists Eliminate Infected Cells from a Primary-Cell Model of HIV Latency but Not from Ex Vivo Reservoirs.

HIV persists, despite immune responses and antiretroviral therapy, in viral reservoirs that seed rebound viremia if therapy is interrupted. Previously, we showed that the BCL-2 protein contributes to HIV persistence by conferring a survival advantage to reservoir-harboring cells. Here, we demonstrate that many of the BCL-2 family members are overexpressed in HIV-infected CD4+ T cells, indicating increased tension between proapoptotic and prosurvival family members-and suggesting that inhibition of prosurvival members may disproportionately affect the survival of HIV-infected cells. Based on these results, we chose to study BCL-XL due to its consistent overexpression and the availability of selective antagonists. Infection of primary CD4+ T cells with HIV resulted in increased BCL-XL protein expression, and treatment with two selective BCL-XL antagonists, A-1155463 and A-1551852, led to selective death of productively infected CD4+ T cells. In a primary cell model of latency, both BCL-XL antagonists drove reductions in HIV DNA and in infectious cell frequencies both alone and in combination with the latency reversing agent bryostatin-1, with little off-target cytotoxicity. However, these antagonists, with or without bryostatin-1 or in combination with the highly potent latency reversing agent combination phorbol myristate acetate (PMA) + ionomycin, failed to reduce total HIV DNA and infectious reservoirs in ex vivo CD4+ T cells from antiretroviral therapy (ART)-suppressed donors. Our results add to growing evidence that bona fide reservoir-harboring cells are resistant to multiple "kick and kill" modalities-relative to latency models. We also interpret our results as encouraging further exploration of BCL-XL antagonists for cure, where combination approaches, including with immune effectors, may unlock the ability to eliminate ex vivo reservoirs. IMPORTANCE Although antiretroviral therapy (ART) has transformed HIV infection into a manageable chronic condition, there is no safe or scalable cure. HIV persists in "reservoirs" of infected cells that reinitiate disease progression if ART is interrupted. Whereas most efforts to eliminate this reservoir have focused on exposing these cells to immune-mediated clearance by reversing viral latency, recent work shows that these cells also resist being killed. Here, we identify a "prosurvival" factor, BCL-XL, that is overexpressed in HIV-infected cells, and demonstrate selective toxicity to these cells by BCL-XL antagonists. These antagonists also reduced reservoirs in a primary-cell latency model but were insufficient to reduce "natural" reservoirs in ex vivo CD4+ T cells-adding to growing evidence that the latter are resilient in a way that is not reflected in models. We nonetheless suggest that the selective toxicity of BCL-XL antagonists to HIV-infected cells supports their prioritization for testing in combinations aimed at reducing ex vivo reservoirs.

Susceptibility to Neutralization by Broadly Neutralizing Antibodies Generally Correlates with Infected Cell Binding for a Panel of Clade B HIV Reactivated from Latent Reservoirs.

Efforts to cure human immunodeficiency virus (HIV) infection are obstructed by reservoirs of latently infected CD4+ T cells that can reestablish viremia. HIV-specific broadly neutralizing antibodies (bNAbs), defined by unusually wide neutralization breadths against globally diverse viruses, may contribute to the elimination of these reservoirs by binding to reactivated cells, thus targeting them for immune clearance. However, the relationship between neutralization of reservoir isolates and binding to corresponding infected primary CD4+ T cells has not been determined. Thus, the extent to which neutralization breadths and potencies can be used to infer the corresponding parameters of infected cell binding is currently unknown. We assessed the breadths and potencies of bNAbs against 36 viruses reactivated from peripheral blood CD4+ T cells from antiretroviral (ARV)-treated HIV-infected individuals by using paired neutralization and infected cell binding assays. Single-antibody breadths ranged from 0 to 64% for neutralization (80% inhibitory concentration [IC80] of ≤10 µg/ml) and from 0 to 89% for binding, with two-antibody combinations (results for antibody combinations are theoretical/predicted) reaching levels of 0 to 83% and 50 to 100%, respectively. Infected cell binding correlated with virus neutralization for 10 of 14 antibodies (e.g., for 3BNC117, r = 0.82 and P < 0.0001). Heterogeneity was observed, however, with a lack of significant correlation for 2G12, CAP256.VRC26.25, 2F5, and 4E10. Our results provide guidance on the selection of bNAbs for interventional cure studies, both by providing a direct assessment of intra- and interindividual variabilities in neutralization and infected cell binding in a novel cohort and by defining the relationships between these parameters for a panel of bNAbs.IMPORTANCE Although antiretroviral therapies have improved the lives of people who are living with HIV, they do not cure infection. Efforts are being directed towards harnessing the immune system to eliminate the virus that persists, potentially resulting in virus-free remission without medication. HIV-specific antibodies hold promise for such therapies owing to their ability to both prevent the infection of new cells (neutralization) and direct the killing of infected cells. We isolated 36 HIV strains from individuals whose virus was suppressed by medication and tested 14 different antibodies for neutralization of these viruses and for binding to cells infected with the same viruses (critical for engaging natural killer cells). For both neutralization and infected cell binding, we observed variation both between individuals and amongst different viruses within an individual. For most antibodies, neutralization activity correlated with infected cell binding. These data provide guidance on the selection of antibodies for clinical trials.

T-cell responses targeting HIV Nef uniquely correlate with infected cell frequencies after long-term antiretroviral therapy.

HIV-specific CD8+ T-cell responses limit viral replication in untreated infection. After the initiation of antiretroviral therapy (ART), these responses decay and the infected cell population that remains is commonly considered to be invisible to T-cells. We hypothesized that HIV antigen recognition may persist in ART-treated individuals due to low-level or episodic protein expression. We posited that if persistent recognition were occurring it would be preferentially directed against the early HIV gene products Nef, Tat, and Rev as compared to late gene products, such as Gag, Pol, and Env, which have higher barriers to expression. Using a primary cell model of latency, we observed that a Nef-specific CD8+ T-cell clone exhibited low-level recognition of infected cells prior to reactivation and robust recognition shortly thereafter. A Gag-specific CD8+ T-cell clone failed to recognized infected cells under these conditions, corresponding with a lack of detectable Gag expression. We measured HIV-specific T-cell responses in 96 individuals who had been suppressed on ART for a median of 7 years, and observed a significant, direct correlation between cell-associated HIV DNA levels and magnitudes of IFN-γ-producing Nef/Tat/Rev-specific T-cell responses. This correlation was confirmed in an independent cohort (n = 18). Correlations were not detected between measures of HIV persistence and T-cell responses to other HIV antigens. The correlation with Nef/Tat/Rev-specific T-cells was attributable to Nef-specific responses, the breadth of which also correlated with HIV DNA levels. These results suggest that ongoing Nef expression in ART-treated individuals drives preferential maintenance and/or expansion of T-cells reactive to this protein, implying sensing of infected cells by the immune system. The direct correlation, however, suggests that recognition does not result in efficient elimination of infected cells. These results raise the possibility that enhancing the cytolytic activity of Nef-specific T-cells may lead to reductions in infected cell frequencies, even in the absence of therapeutic latency reversal.

NKT cells inhibit antigen-specific effector CD8 T cell induction to skin viral proteins.

We recently demonstrated that CD1d-restricted NKT cells resident in skin can inhibit CD8 T cell-mediated graft rejection of human papillomavirus E7-expressing skin through an IFN-γ-dependent mechanism. In this study, we examined the role of systemically derived NKT cells in regulating the rejection of skin grafts expressing viral proteins. In lymph nodes draining transplanted skin, Ag-specific CD8 T cell proliferation, cytokine production, and cytotoxic activity were impaired by NKT cells. NKT cell suppression was mediated via CD11c(+) dendritic cells. Inhibition of CD8 T cell function did not require Foxp3(+) regulatory T cells or NKT cell-secreted IFN-γ, IL-10, or IL-17. Thus, following skin grafting or immunization with human papillomavirus-E7 oncoprotein, NKT cells reduce the capacity of draining lymph node-resident APCs to cross-present Ag to CD8 T cell precursors, as evidenced by impaired expansion and differentiation to Ag-specific CD8 T effector cells. Therefore, in the context of viral Ag challenge in the skin, systemic NKT cells limit the capacity for effective priming of adaptive immunity.

Combined effects of sensory cueing and limb activation on unilateral neglect in subacute left hemiplegic stroke patients: a randomized controlled pilot study.

OBJECTIVE: To compare the effects of contralesional sensory cueing and limb activation with that of sham control in the treatment of unilateral neglect after stroke. DESIGN: A randomized, single-blinded, sham-controlled pilot study. SETTING: Two rehabilitation hospitals. SUBJECTS: Forty subacute left hemiplegic stroke inpatients with unilateral neglect. INTERVENTIONS: Participants were assigned randomly to 1 of 2 groups. The experimental group wore a wristwatch cueing device over the hemiplegic arm for three hours a day, five days per week, for three weeks, and also underwent conventional rehabilitation. Patients were encouraged to move their hemiplegic arm five consecutive times after each prompt. The sham group underwent the same rehabilitation process, except they wore a sham device. MAIN MEASURES: Neglect, arm motor performance, and overall functioning were assessed pre- and posttraining, and at follow-up. RESULTS: There were no significant differences between groups in outcome measures except the neglect drawing tasks (p = 0.034) (the mean gain score from baseline to follow-up assessment was 5.2 (3.7) in the experimental group and 1.9 (3.5) in the sham group), across three time intervals. The experimental group showed greater improvement in arm motor performance than did the sham group. CONCLUSION: The results did not confirm that sensory cueing and limb activation treatment is effective when compared with those receiving placebo to reduce unilateral neglect, but it might be useful for promoting hemiplegic arm performance in stroke patients.

Factors predicting clinically significant functional gain and discharge to home in stroke in-patients after rehabilitation - A retrospective cohort study.

OBJECTIVE: This study explored factors which predict stroke survivors who could achieve "clinically significant functional gain" and return home when being discharged from a local hospital after in-patient stroke rehabilitation programme. METHODS: This study included 562 inpatients with stroke who were residing at community dwellings before onset of stroke, and transferred to a convalescent hospital for rehabilitation from four acute hospitals over one year. The main outcome variables of prediction were (a) achieving "clinically significant functional gain" as measured by (a1) achievement of "minimal clinically important difference" (MCID) of improvement in Functional Independence Measure Motor Measure (FIM-MM)", (a2) one or more level(s) of improvement in function group according to the patients' FIM-MM, and (b) discharge to home. Sixteen predictor variables were identified and studied firstly with univariate binary logistic regression and those significant variables were then put into multivariate binary logistic regression. RESULTS: Based on multivariate regression, the significant predictors for "clinically significant functional gain" were: younger age <75 years old, higher Glasgow Coma Scale score at admission, with haemorrhagic stroke, intermediate FIM-MM function group. Those significant predictors for "discharge to home" were: living with family/caregivers before stroke, higher FIM score at admission, and one or more level(s) of improvement in FIM-MM function group. CONCLUSIONS: This study identified findings consistent with overseas studies in additional to some new interesting findings. Early prediction of stroke discharge outcomes helps rehabilitation professionals and occupational therapists to focus on the use of appropriate intervention strategies and pre-discharge preparation.

BCL-2 antagonism sensitizes cytotoxic T cell-resistant HIV reservoirs to elimination ex vivo.

Curing HIV infection will require the elimination of a reservoir of infected CD4+ T cells that persists despite HIV-specific cytotoxic T cell (CTL) responses. Although viral latency is a critical factor in this persistence, recent evidence also suggests a role for intrinsic resistance of reservoir-harboring cells to CTL killing. This resistance may have contributed to negative outcomes of clinical trials, where pharmacologic latency reversal has thus far failed to drive reductions in HIV reservoirs. Through transcriptional profiling, we herein identified overexpression of the prosurvival factor B cell lymphoma 2 (BCL-2) as a distinguishing feature of CD4+ T cells that survived CTL killing. We show that the inducible HIV reservoir was disproportionately present in BCL-2hi subsets in ex vivo CD4+ T cells. Treatment with the BCL-2 antagonist ABT-199 was not sufficient to drive reductions in ex vivo viral reservoirs when tested either alone or with a latency-reversing agent (LRA). However, the triple combination of strong LRAs, HIV-specific T cells, and a BCL-2 antagonist uniquely enabled the depletion of ex vivo viral reservoirs. Our results provide rationale for novel therapeutic approaches targeting HIV cure and, more generally, suggest consideration of BCL-2 antagonism as a means of enhancing CTL immunotherapy in other settings, such as cancer.

Latent HIV reservoirs exhibit inherent resistance to elimination by CD8+ T cells.

The presence of persistent, latent HIV reservoirs in CD4+ T cells obstructs current efforts to cure infection. The so-called kick-and-kill paradigm proposes to purge these reservoirs by combining latency-reversing agents with immune effectors such as cytotoxic T lymphocytes. Support for this approach is largely based on success in latency models, which do not fully reflect the makeup of latent reservoirs in individuals on long-term antiretroviral therapy (ART). Recent studies have shown that CD8+ T cells have the potential to recognize defective proviruses, which comprise the vast majority of all infected cells, and that the proviral landscape can be shaped over time due to in vivo clonal expansion of infected CD4+ T cells. Here, we have shown that treating CD4+ T cells from ART-treated individuals with combinations of potent latency-reversing agents and autologous CD8+ T cells consistently reduced cell-associated HIV DNA, but failed to deplete replication-competent virus. These CD8+ T cells recognized and potently eliminated CD4+ T cells that were newly infected with autologous reservoir virus, ruling out a role for both immune escape and CD8+ T cell dysfunction. Thus, our results suggest that cells harboring replication-competent HIV possess an inherent resistance to CD8+ T cells that may need to be addressed to cure infection.

Effects of Arm Weight Support Training to Promote Recovery of Upper Limb Function for Subacute Patients after Stroke with Different Levels of Arm Impairments.

Purpose. The goal of this study was to investigate the effects of arm weight support training using the ArmeoSpring for subacute patients after stroke with different levels of hemiplegic arm impairments. Methods. 48 inpatients with subacute stroke, stratified into 3 groups from mild to severe upper extremity impairment, were engaged in ArmeoSpring training for 45 minutes daily, 5 days per week for 3 weeks, in addition to conventional rehabilitation. Evaluations were conducted at three measurement occasions: immediately before training (T1); immediately after training (T2); and at a 3-week follow-up (T3) by a blind rater. Results. Shoulder flexion active range of motion, Upper Extremity Scores in the Fugl-Meyer Assessment (FMA), and Vertical Catch had the greatest differences in gain scores for patients between severe and moderate impairments, whereas FMA Hand Scores had significant differences in gain scores between moderate and mild impairments. There was no significant change in muscle tone or hand-path ratios between T1, T2, and T3 within the groups. Conclusion. Arm weight support training is beneficial for subacute stroke patients with moderate to severe arm impairments, especially to improve vertical control such as shoulder flexion, and there were no adverse effects in muscle tone.

Management of simple finger injuries: the splinting regime.

Mallet finger injury, ligamentous sprain and dislocation of proximal interphalangeal (PIP) joint of fingers are very common types of simple hand injuries. Immediate correction of alignment and protection of the injured area will facilitate early joint movement while maximising functional recovery. This article is to introduce the fabrication of three simple finger splints to tackle these injuries for quick and effective conservative treatment. They are the mallet finger splint, buddy splint and dorsal finger block splint. The indications and functions of the three types of splints are discussed. The fabrication process will be illustrated; including materials needed, pattern drafting and steps of molding. Wearing regime and precautions will be highlighted to ensure effective patient compliance to splinting programme for the finger injuries.

Prevalence of visual problems among stroke survivors in Hong Kong Chinese.

BACKGROUND: Stroke, a common cerebrovascular accident, usually results in various extents of functional disability. Extensive studies have shown that ocular and visual problems are common in patients with stroke. Unfortunately, current stroke rehabilitation programs rarely address stroke-related ocular and visual problems in Hong Kong. METHODS: To examine how visual impairment (for example, deterioration in visual acuity and restriction in visual field) affects the stroke population in Hong Kong, vision screening was conducted for post-stroke patients attending in-patient and out-patient stroke clinics at two hospitals. RESULTS: One hundred and thirteen stroke patients were recruited. The percentage of various aspects of visual problems in Hong Kong post-stroke patients was generally lower than that reported in Western countries; however, a high percentage of patients had deficits in oculomotor (53.1 per cent) and vergence functions (11.5 per cent), restrictions in binocular visual field (11.5 per cent) and impairment in visual acuity (worse than 0.30 logMAR, 29.8 per cent). Conversely, only a small proportion of patients noticed problems with their vision (for example, diplopia and blurry vision) through subjective reports. This revealed that many post-stroke patients had undetected or undiagnosed ocular and visual problems. Appropriate referral was given to patients with visual problems for further evaluation and treatment. CONCLUSION: Neglecting visual problems may impose deteriorating effect on patients' stroke rehabilitation and functional independence and lead to increased incidents of injury. To address this potential hindrance in rehabilitation, formal screening for visual problems in stroke patients in a rehabilitation setting is essential.

The effect of voluntary trunk rotation and half-field eye-patching for patients with unilateral neglect in stroke: a randomized controlled trial.

OBJECTIVE: To investigate the effectiveness of voluntary trunk rotation and half-field eye-patching to treat patients with unilateral neglect in stroke. DESIGN: Pre-post, day 60 follow-up, single-blinded randomized controlled trial. SETTING: Single-centre inpatient rehabilitation hospital. SUBJECTS: Sixty subacute patients with right hemisphere stroke having unilateral neglect within eight weeks post stroke consented to participate between November 2003 and July 2005. They were randomly assigned to three comparison groups. INTERVENTIONS: Nineteen patients received daily experimental training in voluntary trunk rotation (TR) for 1 hour five times a week for 30 days. Twenty patients received the same kind of treatment together with half-field eye-patching (TR + EP). Fifteen patients in the control group received conventional training with the same contact time. MAIN OUTCOME MEASURES: Patients were assessed on days 0, 30 and 60 using the Behavioural Inattention Test, the Clock Drawing Test, and the Functional Independence Measure. RESULTS: No significant differences between voluntary trunk rotation (TR), voluntary trunk rotation and half-field eye-patching (TR + EP) and controls were found in functional performance and neglect measures at day 30 (P = 0.042-0.994) and follow-up (P = 0.052-0.911) at P = 0.005 using Bonferroni correction. CONCLUSIONS: The results of this study do not support the use of voluntary trunk rotation alone or with half-field eye-patching to improve functional performance or reduce unilateral neglect in subacute patients with stroke.

Motor relearning programme for stroke patients: a randomized controlled trial.

OBJECTIVE: To study the efficacy of the motor relearning approach in promoting physical function and task performance for patients after a stroke. DESIGN: Matched-pair randomized controlled trial. SETTING: An outpatient rehabilitation centre in Hong Kong. PARTICIPANTS: Fifty-two outpatients with either a thrombotic or haemorrhagic stroke who completed either the study or control group. INTERVENTIONS: The patients received 18 2-h sessions in six weeks of either the motor relearning programme or a conventional therapy programme. MAIN OUTCOME MEASURES: The Berg Balance Scale, the Timed Up and Go Test, the Functional Independence Measure (FIM), the modified Lawton Instrumental Activities of Daily Living (IADL) test, and the Community Integration Questionnaire. RESULTS: Patients in the motor relearning group showed significantly better performance on all but the Timed Up and Go Test when compared with the control group (F(1,150) = 6.34-41.86, P < or = 0.015). The interactions between group and occasion were significant on all five outcome measures, indicating that the rates of change across time between the motor relearning and control groups differed (F(3,150) = 3.60-33.58, P < 0.015). CONCLUSION: The motor relearning programme was found to be effective for enhancing functional recovery of patients who had a stroke. Both 'sequential' and 'function-based' concepts are important in applying the motor relearning approach to the rehabilitation of stroke patients.