Preexisting memory CD4+ T cells contribute to the primary response in an HIV-1 vaccine trial.

Naive and memory CD4+ T cells reactive with human immunodeficiency virus type 1 (HIV-1) are detectable in unexposed, unimmunized individuals. The contribution of preexisting CD4+ T cells to a primary immune response was investigated in 20 HIV-1-seronegative volunteers vaccinated with an HIV-1 envelope (Env) plasmid DNA prime and recombinant modified vaccinia virus Ankara (MVA) boost in the HVTN 106 vaccine trial (clinicaltrials.gov NCT02296541). Prevaccination naive or memory CD4+ T cell responses directed against peptide epitopes in Env were identified in 14 individuals. After priming with DNA, 40% (8/20) of the elicited responses matched epitopes detected in the corresponding preimmunization memory repertoires, and clonotypes were shared before and after vaccination in 2 representative volunteers. In contrast, there were no shared epitope specificities between the preimmunization memory compartment and responses detected after boosting with recombinant MVA expressing a heterologous Env. Preexisting memory CD4+ T cells therefore shape the early immune response to vaccination with a previously unencountered HIV-1 antigen.

Peculiar Phenotypic and Cytotoxic Features of Pulmonary Mucosal CD8 T Cells in People Living with HIV Receiving Long-Term Antiretroviral Therapy.

People living with HIV have high burdens of chronic lung disease, lung cancers, and pulmonary infections despite antiretroviral therapy (ART). The rates of tobacco smoking by people living with HIV vastly exceed that of the general population. Furthermore, we showed that HIV can persist within the lung mucosa despite long-term ART. As CD8 T cell cytotoxicity is pivotal for controlling viral infections and eliminating defective cells, we explored the phenotypic and functional features of pulmonary versus peripheral blood CD8 T cells in ART-treated HIV+ and uninfected controls. Bronchoalveolar lavage fluid and matched blood were obtained from asymptomatic ART-treated HIV+ smokers (n = 11) and nonsmokers (n = 15) and uninfected smokers (n = 7) and nonsmokers (n = 10). CD8 T cell subsets and phenotypes were assessed by flow cytometry. Perforin/granzyme B content, degranulation (CD107a expression), and cytotoxicity against autologous Gag peptide-pulsed CD4 T cells (Annexin V+) following in vitro stimulation were assessed. In all groups, pulmonary CD8 T cells were enriched in effector memory subsets compared with blood and displayed higher levels of activation (HLA-DR+) and exhaustion (PD1+) markers. Significant reductions in proportions of senescent pulmonary CD28-CD57+ CD8 T cells were observed only in HIV+ smokers. Pulmonary CD8 T cells showed lower perforin expression ex vivo compared with blood CD8 T cells, with reduced granzyme B expression only in HIV+ nonsmokers. Bronchoalveolar lavage CD8 T cells showed significantly less in vitro degranulation and CD4 killing capacity than blood CD8 T cells. Therefore, pulmonary mucosal CD8 T cells are more differentiated, activated, and exhausted, with reduced killing capacity in vitro than blood CD8 T cells, potentially contributing to a suboptimal anti-HIV immune response within the lungs.

HIV Infection and Persistence in Pulmonary Mucosal Double Negative T Cells In Vivo.

The lungs are relatively unexplored anatomical human immunodeficiency virus (HIV) reservoirs in the antiretroviral therapy (ART) era. Double negative (DN) T cells are a subset of T cells that lack expression of CD4 and CD8 (CD4- CD8-) and may have both regulatory and effector functions during HIV infection. Notably, circulating DN T cells were previously described as cellular HIV reservoirs. Here, we undertook a thorough analysis of pulmonary versus blood DN T cells of people living with HIV (PLWH) under ART. Bronchoalveolar lavage (BAL) fluid and matched peripheral blood were collected from 35 PLWH on ART and 16 uninfected volunteers without respiratory symptoms. Both PLWH and HIV-negative (HIV-) adults displayed higher frequencies of DN T cells in BAL versus blood, and these cells mostly exhibited an effector memory phenotype. In PLWH, pulmonary mucosal DN T cells expressed higher levels of HLA-DR and several cellular markers associated with HIV persistence (CCR6, CXCR3, and PD-1) than blood. We also observed that DN T cells were less senescent (CD28- CD57+) and expressed less immunosuppressive ectonucleotidase (CD73/CD39), granzyme B, and perforin in the BAL fluid than in the blood of PLWH. Importantly, fluorescence-activated cell sorter (FACS)-sorted DN T cells from the BAL fluid of PLWH under suppressive ART harbored HIV DNA. Using the humanized bone marrow-liver-thymus (hu-BLT) mouse model of HIV infection, we observed higher infection frequencies of lung DN T cells than those of the blood and spleen in both early and late HIV infection. Overall, our findings show that HIV is seeded in pulmonary mucosal DN T cells early following infection and persists in these potential cellular HIV reservoirs even during long-term ART.IMPORTANCE Reservoirs of HIV during ART are the primary reasons why HIV/AIDS remains an incurable disease. Indeed, HIV remains latent and unreachable by antiretrovirals in cellular and anatomical sanctuaries, preventing its eradication. The lungs have received very little attention compared to other anatomical reservoirs despite being immunological effector sites exhibiting characteristics ideal for HIV persistence. Furthermore, PLWH suffer from a high burden of pulmonary non-opportunistic infections, suggesting impaired pulmonary immunity despite ART. Meanwhile, various immune cell populations have been proposed to be cellular reservoirs in blood, including CD4- CD8- DN T cells, a subset that may originate from CD4 downregulation by HIV proteins. The present study aims to describe DN T cells in human and humanized mice lungs in relation to intrapulmonary HIV burden. The characterization of DN T cells as cellular HIV reservoirs and the lungs as an anatomical HIV reservoir will contribute to the development of targeted HIV eradication strategies.

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment.

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing bronchoalveolar lavage (BAL) fluid. The BAL fluid is rich in cells and can thus provide a 'snapshot' of the pulmonary immune milieu. CD4 T cells are the best characterized HIV reservoirs, while there is strong evidence to suggest that tissue macrophages, including alveolar macrophages (AMs), also serve as viral reservoirs. However, much is still unknown about the role of AMs in the context of HIV reservoir establishment and maintenance. Therefore, developing a protocol for processing BAL fluid to obtain cells that may be used in virological and immunological assays to characterize and evaluate the cell populations and subsets within the lung is relevant for understanding the role of the lungs as HIV reservoirs. Herein, we describe such a protocol, employing standard techniques such as simple centrifugation and flow cytometry. The CD4 T cells and AMs may then be used for subsequent applications, including immunophenotyping and HIV DNA and RNA quantification.

Variation in Acute Medicine Units: Measuring it, understanding it, and reducing it.

Although there are national recommendations on the function of Acute Medicine Units (AMUs), there is no single agreed best model of care. Additionally, robust data is not always available to determine whether system changes have resulted in improvement. We designed an Excel file to interface with the hospital patient management system to provide real-time data on a number of metrics including AMU length of stay (AMULOS), mortality and readmissions. This demonstrated that improving consultant continuity of care was associated with a reduction in AMULOS and reduced variation in AMULOS. Additionally, the Excel file provides timely access to consultant and individual patient-level data. These data are clinically owned, and critical for both unit governance and quality improvement work. We would encourage all AMUs to develop a similar dataset to allow standardised comparisons between units, and better understanding of the association between models of care and patient outcomes.

Reducing cardiac arrests in the acute admissions unit: a quality improvement journey.

BACKGROUND: In 2010, the acute admissions unit (AAU) at Stirling Royal Infirmary had the highest number of cardiac arrests of any ward. A quality improvement project was undertaken to reduce this to <1/1000 admissions by December 2011. METHODS: In January 2011, based on initial needs assessment, we selected three initiatives to improve cardiac arrest rate: (1) structured response to deteriorating patients; (2) analysis of adverse events; and (3) improved end-of-life decision-making. We performed a failure modes effects analysis to identify reasons for the failure of early recognition and response. Ward staff conducted weekly safety meetings to engage unit staff and promote a safety culture of continuous improvement. Additionally, in July 2011 the unit adopted a ward-based clinical team structure with twice daily consultant ward rounds. Our primary outcome measure, cardiac arrests per 1000 admissions, was measured from January 2011 to August 2012. RESULTS: Over 17 months, the number of cardiac arrests per 1000 admissions fell from a baseline of 2.8/1000 admissions to 0.8/1000 admissions (71% reduction), referrals to palliative care increased by 22 to 37/1000 admissions per month (68% increase) and the 30-day mortality of patients admitted to the AAU fell from 6.3% to 4.8% (24% relative reduction). CONCLUSIONS: Through adoption of a shared goal, application of improvement methodology including the model for improvement to test new innovations, and promotion of a safety culture in the AAU, cardiac arrests were successfully reduced to <1/1000 admissions per month with an associated significant fall in mortality. This was achieved with negligible cost.

Late neurologic and cognitive sequelae of inflicted traumatic brain injury in infancy.

OBJECTIVE: There is limited information regarding the long-term outcome of inflicted traumatic brain injury (TBI), including shaken infant syndrome. The purpose of this study was to describe the long-term neurologic, behavioral, and cognitive sequelae seen in this population. METHODS: A cross-sectional and prospective longitudinal study was conducted of 25 children with inflicted TBI in Scotland between 1980 and 1999. After consent was obtained, neurologic and cognitive examinations were performed on all participants and sequentially in the prospective cohort. Two global outcome measures were used: Glasgow Outcome Score (GOS) and Seshia's outcome score. Cognitive outcome was assessed using the Bayley Scales of Infant Development, British Ability Scales, and the Vineland Adaptive Behavior Scales. RESULTS: The mean length of follow-up was 59 months. A total of 68% of survivors were abnormal on follow-up, 36% had severe difficulties and were totally dependant, 16% had moderate difficulties, and 16% had mild difficulties on follow-up. A wide range of neurologic sequelae were seen, including motor deficits (60%), visual deficits (48%), epilepsy (20%), speech and language abnormalities (64%), and behavioral problems (52%). There was a wide range of cognitive abilities: the mean psychomotor index, 69.9 (SD: +/-25.73); and mean mental development index, 74.53 (SD: +/-28.55). Adaptive functioning showed a wide range of difficulties across all domains: communication domain (mean: 76.1; SD: +/-25.4), Daily living skills domain (mean: 76.9; SD: +/-24.3), and socialization domain (mean: 79.1; SD: +/-23.1). Outcome was found to correlate with the Pediatric Trauma Score and the Glasgow Coma Score but did not correlate with age at injury or mechanism of injury. CONCLUSIONS: Inflicted TBI has a very poor prognosis and correlates with severity of injury. Extended follow-up is necessary so as not to underestimate problems such as specific learning difficulties and attentional and memory problems that may become apparent only once the child is in school. Behavioral problems are present in 52% and begin to manifest clinically between the second and third years of life, although the consequences of frontal lobe injury may be underestimated unless follow-up is extended into adolescence and early adulthood.