Clonal succession after prolonged antiretroviral therapy rejuvenates CD8+ T cell responses against HIV-1.

Human immunodeficiency virus 1 (HIV-1) infection is characterized by a dynamic and persistent state of viral replication that overwhelms the host immune system in the absence of antiretroviral therapy (ART). The impact of prolonged treatment on the antiviral efficacy of HIV-1-specific CD8+ T cells has nonetheless remained unknown. Here, we used single-cell technologies to address this issue in a cohort of aging individuals infected early during the pandemic and subsequently treated with continuous ART. Our data showed that long-term ART was associated with a process of clonal succession, which effectively rejuvenated HIV-1-specific CD8+ T cell populations in the face of immune senescence. Tracking individual transcriptomes further revealed that initially dominant CD8+ T cell clonotypes displayed signatures of exhaustion and terminal differentiation, whereas newly dominant CD8+ T cell clonotypes displayed signatures of early differentiation and stemness associated with natural control of viral replication. These findings reveal a degree of immune resilience that could inform adjunctive treatments for HIV-1.

Antigenic Restimulation of Virus-Specific Memory CD8+ T Cells Requires Days of Lytic Protein Accumulation for Maximal Cytotoxic Capacity.

In various infections or vaccinations of mice or humans, reports of the persistence and the requirements for restimulation of the cytotoxic mediators granzyme B (GrB) and perforin (PRF) in CD8+ T cells have yielded disparate results. In this study, we examined the kinetics of PRF and GrB mRNA and protein expression after stimulation and associated changes in cytotoxic capacity in virus-specific memory cells in detail. In patients with controlled HIV or cleared respiratory syncytial virus (RSV) or influenza virus infections, all virus-specific CD8+ T cells expressed low PRF levels without restimulation. Following stimulation, they displayed similarly delayed kinetics for lytic protein expression, with significant increases occurring by days 1 to 3 before peaking on days 4 to 6. These increases were strongly correlated with, but were not dependent upon, proliferation. Incremental changes in PRF and GrB percent expression and mean fluorescence intensity (MFI) were highly correlated with increases in HIV-specific cytotoxicity. mRNA levels in HIV-specific CD8+ T-cells exhibited delayed kinetics after stimulation as with protein expression, peaking on day 5. In contrast to GrB, PRF mRNA transcripts were little changed over 5 days of stimulation (94-fold versus 2.8-fold, respectively), consistent with posttranscriptional regulation. Changes in expression of some microRNAs, including miR-17, miR-150, and miR-155, suggested that microRNAs might play a significant role in regulation of PRF expression. Therefore, under conditions of extremely low or absent antigen levels, memory virus-specific CD8+ T cells require prolonged stimulation over days to achieve maximal lytic protein expression and cytotoxic capacity.IMPORTANCE Antigen-specific CD8+ T cells play a major role in controlling most virus infections, primarily by perforin (PRF)- and granzyme B (GrB)-mediated apoptosis. There is considerable controversy regarding whether PRF is constitutively expressed, rapidly increased similarly to a cytokine, or delayed in its expression with more prolonged stimulation in virus-specific memory CD8+ T cells. In this study, the degree of cytotoxic capacity of virus-specific memory CD8+ T cells was directly proportional to the content of lytic molecules, which required antigenic stimulation over several days for maximal levels. This appeared to be modulated by increases in GrB transcription and microRNA-mediated posttranscriptional regulation of PRF expression. Clarifying the requirements for maximal cytotoxic capacity is critical to understanding how viral clearance might be mediated by memory cells and what functions should be induced by vaccines and immunotherapies.

Immunologic Control of HIV-1: What Have We Learned and Can We Induce It?

PURPOSE OF REVIEW: A large amount of data now exists on the virus-specific immune response associated with spontaneous or induced immunologic control of lentiviruses. This review focuses on how the current understanding of HIV-specific immunity might be leveraged into induction of immunologic control and what further research is needed to accomplish this goal. RECENT FINDINGS: During chronic infection, the function most robustly associated with immunologic control of HIV-1 is CD8+ T cell cytotoxic capacity. This function has proven difficult to restore in HIV-specific CD8+ T cells of chronically infected progressors in vitro and in vivo. However, progress has been made in inducing an effective CD8+ T cell response prior to lentiviral infection in the macaque model and during acute lentiviral infection in non-human primates. Further study will likely accelerate the ability to induce an effective CD8+ T cell response as part of prophylactic or therapeutic strategies.

Factors Associated With Influenza in an Emergency Department Setting.

BACKGROUND: Emergency departments (EDs) become more overcrowded during peak respiratory virus season. Distinguishing influenza from other viruses is crucial to implement social distancing practices, early treatment, and prompt disposition. OBJECTIVES: We sought to determine factors associated with influenza among a prospective cohort of consecutive ED patients with acute respiratory illness (ARI). METHODS: Between December 2016 and March 2017, trained research assistants screened consecutive ED patients with ARI symptoms. ARI criteria included measured fever at home or in the ED >38°C and a cough, sore throat, or rhinorrhea with a duration of symptoms >12 hours and <1 week. After consent, research assistants collected demographics and clinical history using a standardized data form, and patients had a polymerase chain reaction-based assay that is nearly 100% sensitive for influenza. Univariate analysis was conducted on all predictor variables. Significant variables were entered into a multivariate logistic regression model to find factors that were independently associated with influenza. RESULTS: One hundred nineteen patients consented to enrollment and 31% were found to be positive for influenza. Myalgia, the absence of gastrointestinal symptoms (no diarrhea or vomiting), sore throat, chills, headache, and oxygen saturation ≥97% were significant on univariate analysis and were entered into the multivariate model. Myalgia (adjusted odds ratio [AOR] 3.9), the absence of gastrointestinal symptoms (AOR 4.7), and oxygen saturation ≥97% (AOR 2.8) were significant independent factors of influenza. CONCLUSION: The presence of myalgia, the absence of gastrointestinal symptoms, and oxygen saturation ≥97% are factors that can help distinguish influenza from other acute respiratory illnesses in the ambulatory ED population.

Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking.

Mycobacterium tuberculosis (Mtb) disrupts anti-microbial pathways of macrophages, cells that normally kill bacteria. Over 40 years ago, D'Arcy Hart showed that Mtb avoids delivery to lysosomes, but the molecular mechanisms that allow Mtb to elude lysosomal degradation are poorly understood. Specialized secretion systems are often used by bacterial pathogens to translocate effectors that target the host, and Mtb encodes type VII secretion systems (TSSSs) that enable mycobacteria to secrete proteins across their complex cell envelope; however, their cellular targets are unknown. Here, we describe a systematic strategy to identify bacterial virulence factors by looking for interactions between the Mtb secretome and host proteins using a high throughput, high stringency, yeast two-hybrid (Y2H) platform. Using this approach we identified an interaction between EsxH, which is secreted by the Esx-3 TSSS, and human hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). ESCRT has a well-described role in directing proteins destined for lysosomal degradation into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), ensuring degradation of the sorted cargo upon MVB-lysosome fusion. Here, we show that ESCRT is required to deliver Mtb to the lysosome and to restrict intracellular bacterial growth. Further, EsxH, in complex with EsxG, disrupts ESCRT function and impairs phagosome maturation. Thus, we demonstrate a role for a TSSS and the host ESCRT machinery in one of the central features of tuberculosis pathogenesis.

HIV vaccines induce CD8+ T cells with low antigen receptor sensitivity.

Current HIV vaccines designed to stimulate CD8+ T cells have failed to induce immunologic control upon infection. The functions of vaccine-induced HIV-specific CD8+ T cells were investigated here in detail. Cytotoxic capacity was significantly lower than in HIV controllers and was not a consequence of low frequency or unaccumulated functional cytotoxic proteins. Low cytotoxic capacity was attributable to impaired degranulation in response to the low antigen levels present on HIV-infected targets. The vaccine-induced T cell receptor (TCR) repertoire was polyclonal and transduction of these TCRs conferred the same reduced functions. These results define a mechanism accounting for poor antiviral activity induced by these vaccines and suggest that an effective CD8+ T cell response may require a vaccination strategy that drives further TCR clonal selection.

Using a novel rapid viral test to improve triage of emergency department patients with acute respiratory illness during flu season.

BACKGROUND: Acute respiratory illnesses (ARI) are mostly viral in etiology and cause significant morbidity and mortality. Point of care PCR (POC-PCR) is a promising new technology for rapid virus identification but utility in the Emergency Department (ED) is not yet defined. OBJECTIVES: Primarily, to investigate the value of POC-PCR in rapidly identifying RSV and influenza in the setting of ED triage. Additionally, to assess whether rapid knowledge of accurate test results would improve patient management by preventing nosocomial transmission and optimizing the prescription of antimicrobials for ARIs. STUDY DESIGN: A prospective cohort study of consecutive ED patients with ARI symptoms during peak flu season was conducted. Patient nasopharyngeal swabs were collected and tested using a POC-PCR device; physicians and patients were blinded to results. Virus positive and negative groups were compared by ED patient room placement and antimicrobial therapy ordered. Specificity and sensitivity were calculated using laboratory-PCR as the gold standard. RESULTS: Of 119 participants, 52.9% were POC-PCR positive - Influenza A (42.9%), RSV (41.3%), influenza B (15.9%). Nearly 70% of virus positive patients were placed rooms shared with non-ARI patients. Antibiotics were prescribed for 27.3% of virus positive patients, and 77.8% of oseltamivir-eligible patients did not receive therapy. POC-PCR was 100% sensitive (95% CI, 80.5-100.0%) and 95.2% specific (95% CI, 76.2-99.9%). CONCLUSIONS: Rapid POC-PCR for influenza and RSV in ED triage has excellent sensitivity and specificity and the potential to improve social distancing practices through better triage and increase appropriate prescription of antimicrobials.

Impact of Rapid Molecular Respiratory Virus Testing on Real-Time Decision Making in a Pediatric Emergency Department.

Acute respiratory illnesses (ARIs) are usually viral [influenza, respiratory syncytial virus (RSV)] and account for 25% of emergency department (ED) peak-season visits. Laboratory PCR testing is accurate albeit slow, whereas rapid antigen testing is inaccurate. We determined the impact of bedside PCR (molecular point-of-care test; mPOCT) on pediatric ARI management. This was a prospective cohort study of consecutive pediatric patients with ED-ordered respiratory PCR test, enrolled over 9 weeks during peak flu season. On ordering, ED physicians were interviewed to ascertain real-time plans if given immediate influenza/RSV PCR results for the current patient. Two groups were compared: actual management and management adjusted for mPOCT results. We compared ED length of stay (LOS), tests ordered, and antibiotic/antiviral ordering. One-hundred thirty-six respiratory PCR panels were ordered, 71 by admitting team, 61 for ED management. Of 61 ED-initiated tests, physicians indicated in 39 cases (64%) they would change patient management were bedside viral results available. Physicians would have decreased ED LOS by 33 minutes, ordered fewer tests (18%; P < 0.001) with average patient charge savings of $669, fewer antibiotics among discharged patients (17%; P = 0.043), and increased appropriate antiviral use (13%; P = 0.023). Rapid bedside ARI mPOCT PCR has the potential to decrease ED LOS, reduce diagnostic tests and patient charges, and increase appropriate use of antibiotics and antiviral agents.

Characterizing Readmissions After Bariatric Surgery.

INTRODUCTION: Readmissions are an important quality metric for surgery. Here, we compare characteristics of readmissions across laparoscopic Roux-en-Y gastric bypass (LRYGB), sleeve gastrectomy (LSG), and adjustable gastric band (LAGB). METHODS: Demographic, intraoperative, anthropometric, and laboratory data were prospectively obtained for 1775 patients at a single academic institution. All instances of readmissions within 1 year were recorded. Data were analyzed using STATA, release 12. RESULTS: For the 1775 patients, 113 (6.37 %) were readmitted. Mean time to readmission was 52.1 days. Of all the readmissions, 64.6 % were within 30 days, 22.1 % from 30 to 90 days, 1.77 % from 90 to 180 days, and 11.5 % from 180 to 365 days. Incidence of 30-day readmissions varied across surgeries (LRYGB: 7.17 %; LAGB: 3.05 %; LSG: 4.25 %, p = 0.04). Time to readmission varied as well, with 90.0 % of LSG and 80.0 % of LABG patients within the first 30 days, versus 60.8 % of LRYGB (p = 0.02). The most common causes of readmissions were gastrointestinal issues related to index procedure (34.5 %) and did not vary across surgeries. In multivariable logistic regression, index hospital length of stay (LOS) was associated with readmission (OR = 1.07, 95 % CI 1.02-1.13, p = 0.01). CONCLUSIONS: Readmissions after bariatric surgery are associated with high index hospital LOS, and a measureable proportion of procedure-related readmissions can occur up to 1 year, especially for LRYGB.

The Influence of Resected Gastric Weight upon Weight Loss after Sleeve Gastrectomy.

Bariatric surgery is an effective and enduring treatment for obesity. Sleeve gastrectomy (SG) has emerged as an increasingly prevalent surgical intervention. Further investigation is required to determine optimal standardization of SG. Data were collected prospectively for 64 patients who underwent a laparoscopic vertical SG between December 2010 and February 2013 at a single academic institution. Demographic, intraoperative, and postoperative (postop) data were collected for all patients including weighing each resected stomach. The total resected gastric weight varied widely. Preoperatively, patients in the upper tercile for resected gastric weight were more likely to be male (lower 10%, middle 23%, upper 52%, P = 0.006) and had greater initial weights (lower 255.9%, middle 245.1%, upper 280.0%, P = 0.019). The resected gastric weight (g) varied by tercile (mean of all, 131.24 ± 39.8; lower, 93.9 ± 10.9; middle, 127.4 ± 11.7; upper 172.7 ± 37.9, P = 0.000). Patients were followed for 1-year postop with follow-up data for 94 per cent (60/64) of participants. Per cent excess weight loss (EWL) was obtained at three, six, and 12 months postop. At 12 months, there was a trend toward increased per cent EWL in the upper tercile (lower 61.1%, middle 54.1%, upper 90.5%, P = 0.057). In conclusion, while the amount of gastric sleeve resected can vary, this study shows that intraoperative assessment of resected sleeve weight can help evaluate adequacy of resection. Improved 12-month per cent EWL in patients with greater resected tissue demonstrate potentially improved outcomes.

Active transport of bile acids decreases mucin 2 in neonatal ileum: implications for development of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants, but its etiology remains unclear. We have previously shown that mucin 2 (Muc2) positive goblet cells are significantly decreased in NEC. We have also shown that ileal bile acids (BAs) are significantly increased during the development of this disease. Because BAs can affect mucins, we hypothesized that elevated ileal BAs contribute to decreased Muc2 in experimental NEC. The role of Muc2 in NEC was evaluated in Winnie +/+ mice, a strain that produces aberrant Muc2. Muc2 and trefoil factor 3 (Tff3) were assessed in neonatal rats subjected to the NEC protocol when bile acids were removed, and in ileal explants from newborn and older rats cultured with and without BAs. Further, the role of active transport of BAs was determined using neonatal rats given the apical sodium dependent bile acid transporter (Asbt) inhibitor SC-435 and in neonatal Asbt knockout mice subjected to the NEC protocol. Mice with aberrant Muc2 had significantly greater incidence and severity of NEC. Using both in vivo and ex vivo techniques, we determined that BAs decrease Muc2 positive cells in neonatal but not older ileum. However, Tff3 positive cells are not decreased by BAs. In addition, active transport of BAs is required for BAs to decrease Muc2 in immature ileum. These data show that functional Muc2 plays a critical role in the prevention of NEC and BAs can potentiate the decreased Muc2 in disease development. Further, BAs have a more profound effect on Muc2 in immature versus older ileum, which may explain at least in part why NEC occurs almost exclusively in premature infants.