Design of a symmetry-broken tetrahedral protein cage by a method of internal steric occlusion.

Methods in protein design have made it possible to create large and complex, self-assembling protein cages with diverse applications. These have largely been based on highly symmetric forms exemplified by the Platonic solids. Prospective applications of protein cages would be expanded by strategies for breaking the designed symmetry, for example, so that only one or a few (instead of many) copies of an exterior domain or motif might be displayed on their surfaces. Here we demonstrate a straightforward design approach for creating symmetry-broken protein cages able to display singular copies of outward-facing domains. We modify the subunit of an otherwise symmetric protein cage through fusion to a small inward-facing domain, only one copy of which can be accommodated in the cage interior. Using biochemical methods and native mass spectrometry, we show that co-expression of the original subunit and the modified subunit, which is further fused to an outward-facing anti-GFP DARPin domain, leads to self-assembly of a protein cage presenting just one copy of the DARPin protein on its exterior. This strategy of designed occlusion provides a facile route for creating new types of protein cages with unique properties.

Design of Ligand-Operable Protein-Cages That Open Upon Specific Protein Binding.

Protein nanocages have diverse applications in medicine and biotechnology, including molecular delivery. However, although numerous studies have demonstrated the ability of protein nanocages to encapsulate various molecular species, limited methods are available for subsequently opening a nanocage for cargo release under specific conditions. A modular platform with a specific protein-target-based mechanism of nanocage opening is notably lacking. To address this important technology gap, we present a new class of designed protein cages, the Ligand-Operable Cage (LOC). LOCs primarily comprise a protein nanocage core and a fused surface binding adaptor. The geometry of the LOC is designed so that binding of a target protein ligand (or multiple copies thereof) to the surface binder is sterically incompatible with retention of the assembled state of the cage. Therefore, the tight binding of a target ligand drives cage disassembly by mass action, subsequently exposing the encapsulated cargo. LOCs are modular; direct substitution of the surface binder sequence can reprogram the nanocage to open in response to any target protein ligand of interest. We demonstrate these design principles using both a natural and a designed protein cage as the core, with different proteins acting as the triggering ligand and with different reporter readouts─fluorescence unquenching and luminescence─for cage disassembly. These developments advance the critical problem of targeted molecular delivery and detection.

Design of a symmetry-broken tetrahedral protein cage by a method of internal steric occlusion.

Methods in protein design have made it possible to create large and complex, self-assembling protein cages with diverse applications. These have largely been based on highly symmetric forms exemplified by the Platonic solids. Prospective applications of protein cages would be expanded by strategies for breaking the designed symmetry, e.g., so that only one or a few (instead of many) copies of an exterior domain or motif might be displayed on their surfaces. Here we demonstrate a straightforward design approach for creating symmetry-broken protein cages able to display singular copies of outward-facing domains. We modify the subunit of an otherwise symmetric protein cage through fusion to a small inward-facing domain, only one copy of which can be accommodated in the cage interior. Using biochemical methods and native mass spectrometry, we show that co-expression of the original subunit and the modified subunit, which is further fused to an outward-facing anti-GFP DARPin domain, leads to self-assembly of a protein cage presenting just one copy of the DARPin protein on its exterior. This strategy of designed occlusion provides a facile route for creating new types of protein cages with unique properties.

Association of Tibial Tubercle-Trochlear Groove Distance With Risk of ACL Graft Failure.

Background: Limited evidence suggests a positive correlation between tibial tubercle-trochlear groove (TT-TG) distance and the risk of native anterior cruciate ligament (ACL) tear. The relationship between TT-TG distance and the risk of ACL graft failure is unknown. Hypothesis: TT-TG distance is independently associated with risk of ACL graft failure. Study Design: Cohort study; Level of evidence, 3. Methods: All patients who underwent ACL revision surgery between 2010 and 2018 at a single institution were identified. A control cohort underwent primary ACL reconstruction (ACLR) between 2006 and 2015, with no evidence of graft failure at 8.1 ± 2.5 years postoperatively. Record review included anthropometrics, graft type, and estimated Tegner activity score at ≥6 months after primary ACLR. Magnetic resonance imaging (MRI) scans after native ACL tear (controls) or graft failure (revision cohort) were assessed for (1) TT-TG distance, (2) proximal tibial slopes, (3) depth of tibial plateau concavity, and (4) tunnel position (revision cohort). Associations between ACL graft failure and MRI measurements, surgical variables, and patient characteristics were evaluated with logistic regression analyses. Sensitivity analyses, excluding patients with tunnel malposition, were performed to confirm multivariable results in patients with "ideal" tunnel placement. Results: Participants included 153 patients who underwent revisions and 144 controls. Controls were older than the patients who underwent revision (26.6 ± 8.8 vs 20.6 ± 7.3 years; P < .001). The mean TT-TG distance and lateral posterior tibial slope (PTS) were smaller for the control group than for the revision group (TT-TG: 9.3 ± 3.9 vs 11.2 ± 4.2 mm; P < .001; lateral PTS: 6.2° ± 3.3° vs 7.2° ± 3.6°; P = .01). TT-TG distance, lateral PTS, and age were associated with risk of ACL graft failure by multivariable analysis (OR, 1.15; 95% CI, 1.07-1.23; P < .001; OR, 1.13; 95% CI, 1.04-1.22; P = .004; and OR, 0.90; 95% CI, 0.87-0.94; P < .001, respectively). With sensitivity analyses, TT-TG distance, lateral PTS, and age at index surgery remained significantly and independently associated with ACL graft failure. Conclusion: Increased TT-TG distance, increased lateral PTS, and younger age are independently associated with increased odds of ACL graft failure. Patients with these characteristics may require a more comprehensive strategy to reduce the risk of ACL reinjury.

Opioid Use in Robotic-Arm Assisted Total Knee Arthroplasty: A Comparison to Conventional Manual Total Knee Arthroplasty.

INTRODUCTION: Opioids are frequently prescribed in the postoperative management of total knee arthroplasty (TKA) with multiple factors influencing postoperative opioid use. Robotic-arm-assisted TKA (raTKA) was developed with the goal of improving alignment and outcomes while decreasing soft tissue injury. The purpose of this study was to compare postoperative opioid consumption in raTKA and conventional manual TKA (mTKA) cohorts. MATERIALS AND METHODS: A consecutive series of unilateral primary TKAs performed 1/1/16 to 12/31/17 were included. Patients with major procedures requiring opioids occurring within one year of TKA were excluded. A single-surgeon raTKA cohort of 127 patients (Group 1) was compared to a same-surgeon cohort of 119 mTKAs (Group 2) using the same cemented implant design and a two-surgeon cohort of 410 mTKA (Group 3). Groups were subdivided into opioid naïve (ON) and opioid exposed (OE). Length of hospitalization and postoperative opioid utilization up to one year were compared between groups and collectively without separating raTKA and mTKA. Statistical analysis included Chi-square, Student's t-test, and Wilcoxon rank sum tests. RESULTS: For both ON and OE patients, Group 1 demonstrated reduced inpatient mean daily oral morphine milligram equivalent (MME) compared to Group 3 (ON p=0.007; OE p=0.034), a shorter hospitalization compared to Group 2 (ON p=0.02; OE p=0.012), and fewer opioids prescribed at discharge compared to Group 2 (ON p=0.005; OE p=0.081) and Group 3 (ON p<0.001; OE p=0.036). No differences in opioid prescriptions were seen at three months or after. Regardless of surgical technique OE patients had higher inpatient opioid utilization (p<0.001) as well as cumulative outpatient prescription quantity (MME 1050 ON, 2660 OE) and duration (ON 0.5%; OE 28.3%) at one year (p<0.001). CONCLUSION: Less opioids were prescribed at discharge and used during hospitalization in raTKA compared to mTKA though no differences in opioid use were seen at further time points. Preoperative opioid use remains a dominant factor in postoperative opioid utilization regardless of TKA surgical technique.

Temporal Wedge Defects in Glaucoma: Structure/Function Correlation With Threshold Automated Perimetry of the Full Visual Field.

PRECIS: We used the Open Perimetry Interface (OPI) to design a static automated perimetry test of the full visual field. About half of our glaucoma cohort had defects in the far peripheral inferotemporal visual field that correlate well with related damage to the superior nasal optic disc. AIMS: To test the hypothesis that in glaucoma patients with mild visual loss, perimetric nerve fiber bundle defects present outside 30 degrees will correlate well with areas of Cirrus ocular coherence tomography (OCT) retinal nerve fiber layer thinning. METHODS: We tested 27 consecutive glaucoma subjects with mild vision loss (mean deviation better than -4 dB) with a SITA standard test, 2 size V custom OPI tests (OPI 30-2 and OPI Peripheral) and Cirrus OCT. Two observers assigned qualitative grades to each type of visual field test based on their level of correlation with OCT retinal nerve fiber layer and ganglion cell thickness. RESULTS: Discrete temporal wedge defects were found on the OPI peripheral V test in 26% of cases whereas more extensive inferior temporal loss (including inferior temporal wedge defect region) was present in 22% of other cases. OCT data correlated best with the OPI peripheral test for 8 glaucoma subjects. The OPI central 30-2 test correlated best for 9 glaucoma subjects; the remainder of subjects had equal central/peripheral correlations. CONCLUSIONS: About half of our glaucoma cohort have defects in the far peripheral inferotemporal visual field that correlate well with related damage to the superior nasal optic disc. Adding a threshold automated perimetry test of the far periphery improves structure/function correlations and adds useful clinical information.

Threshold Automated Perimetry of the Full Visual Field in Patients With Glaucoma With Mild Visual Loss.

PRECIS: The authors used the Open Perimetry Interface to design a static automated perimetry test of the full field. Abnormal test locations in the nasal midperiphery and temporal inferior sector area best separated glaucomas from normals. PURPOSE: The peripheral visual field in glaucoma outside 30 degrees is largely unexplored with static perimetry. Their goal was to use threshold static automated perimetry to characterize the visual loss in glaucoma of the central 30 degrees and the far periphery. PATIENTS AND METHODS: The authors administered the 30-2 perimetric test to 27 patients with early stage glaucoma (with mean deviation better than -4 dB) with the Goldmann III and V stimulus sizes and a custom test from 30 to up to 87 degrees with the size V stimulus twice within a month. The authors quantified (1) the retest variability, (2) the proportion of patients flagged as abnormal (at level 0.05) on the basis of pointwise probability distributions obtained from 63 ocular healthy observers, (3) the pointwise statistical distance using the Kullback-Leibler divergence between normal and glaucoma eyes, and (4) the effect of eccentricity on visual loss. RESULTS: Size V 30-2 testing identified significantly more abnormal test locations (36%) than size III 30-2 (30%; P=0.004). Kullback-Leibler divergence between healthy and glaucoma distributions was greatest for the nasal midperipheral test locations and the inferior temporal sector area. A more pronounced decrease was found in visual sensitivity with eccentricity in the patients with glaucoma compared with the ocular healthy participants across the full visual field (P<0.001). CONCLUSIONS: Patients with glaucoma demonstrate a systematic decrease in sensitivity with eccentricity across the full visual field. Goldmann size V stimuli better detected visual loss in patients with glaucoma with mild loss than size III.

Association between Hospice Spending on Patient Care and Rates of Hospitalization and Medicare Expenditures of Hospice Enrollees.

BACKGROUND: Care at the end of life is increasingly fragmented and is characterized by multiple hospitalizations, even among patients enrolled with hospice. OBJECTIVE: To determine whether hospice spending on direct patient care (including the cost of home visits, drugs, equipment, and counseling) is associated with hospital utilization and Medicare expenditures of hospice enrollees. DESIGN: Longitudinal, observational cohort study (2008-2010). SETTING/SUBJECTS: Medicare beneficiaries (N = 101,261) enrolled in a national random sample of freestanding hospices (N = 355). MEASUREMENTS: We used Medicare Hospice Cost reports to estimate hospice spending on direct patient care and Medicare claim data to estimate rates of hospitalization and Medicare expenditures. RESULTS: Hospice mean direct patient care costs were $86 per patient day, the largest component being patient visits by hospice staff (e.g., nurse, physician, and counselor visits). After case-mix adjustment, hospices spending the most on direct patient care had patients with 5.2% fewer hospital admissions, 6.3% fewer emergency department visits, 1.6% fewer intensive care unit stays, and $1,700 less in nonhospice Medicare expenditures per patient compared with hospices spending the least on direct patient care (p < 0.01 for each comparison). Ninety percent of hospices with the lowest spending on direct patient care and highest rates of hospital use were for-profit hospices. CONCLUSIONS: Patients cared for by hospices with lower direct patient care costs had higher hospitalization rates and were overrepresented by for-profit hospices. Greater investment by hospices in direct patient care may help Centers for Medicare and Medicaid Services avoid high-cost hospital care for patients at the end of life.

TNF-α and sTNF-RII Are Associated with Pain Following Hip Fracture Surgery in Older Adults.

Objective: To explore whether plasma inflammatory mediators on postoperative day 3 (POD3) are associated with pain scores in older adults after hip fracture surgery. Design: Cross-sectional study. Setting: Mount Sinai Hospital, New York, New York. Subjects: Forty patients age 60 years or older who presented with acute hip fracture at Mount Sinai Hospital between November 2011 and April 2013. Methods: Plasma levels of six inflammatory mediators of the nuclear factor kappa B pathway were measured using blood collected on POD3. Self-reported pain scores (i.e., pain with resting, walking, and transferring) were assessed at baseline (prefracture) and on POD3. Linear regression models using log-transformed data were performed to determine associations between inflammatory mediators and postoperative pain. Results: Interleukin 18 (IL-18) was positively associated with POD3 resting pain score in the unadjusted model (ß = 0.66, P = 0.03). Tumor necrosis factor α (TNF-α) and soluble TNF receptor II (sTNF-RII) were positively associated with POD3 resting pain score in the adjusted model (ß = 0.99, P = 0.03, and ß = 0.86, P = 0.04, respectively). Moreover, TNF-α was positively associated with POD3 walking pain score in the adjusted model (ß = 1.59, P = 0.05). Pain with transferring was not associated with these inflammatory mediators. Conclusions: These findings suggest that TNF-α and its receptors may influence pain following hip fracture. Further study of the TNF-α pathway may inform future clinical applications that monitor and treat pain in the vulnerable elderly who are unable to accurately report pain.

The microtubule plus-end-tracking protein TACC3 promotes persistent axon outgrowth and mediates responses to axon guidance signals during development.

BACKGROUND: Formation of precise neuronal connections requires proper axon guidance. Microtubules (MTs) of the growth cone provide a critical driving force during navigation of the growing ends of axons. Pioneer MTs and their plus-end tracking proteins (+TIPs) are thought to play integrative roles during this navigation. TACC3 is a + TIP that we have previously implicated in regulating MT dynamics within axons. However, the role of TACC3 in axon guidance has not been previously explored. RESULTS: Here, we show that TACC3 is required to promote persistent axon outgrowth and prevent spontaneous axon retractions in embryonic Xenopus laevis neurons. We also show that overexpressing TACC3 can counteract the depolymerizing effect of low doses of nocodazole, and that TACC3 interacts with MT polymerase XMAP215 to promote axon outgrowth. Moreover, we demonstrate that manipulation of TACC3 levels interferes with the growth cone response to the axon guidance cue Slit2 ex vivo, and that ablation of TACC3 causes pathfinding defects in axons of developing spinal neurons in vivo. CONCLUSION: Together, our results suggest that by mediating MT dynamics, the + TIP TACC3 is involved in axon outgrowth and pathfinding decisions of neurons during embryonic development.

The transcriptome of HIV-1 infected intestinal CD4+ T cells exposed to enteric bacteria.

Global transcriptome studies can help pinpoint key cellular pathways exploited by viruses to replicate and cause pathogenesis. Previous data showed that laboratory-adapted HIV-1 triggers significant gene expression changes in CD4+ T cell lines and mitogen-activated CD4+ T cells from peripheral blood. However, HIV-1 primarily targets mucosal compartments during acute infection in vivo. Moreover, early HIV-1 infection causes extensive depletion of CD4+ T cells in the gastrointestinal tract that herald persistent inflammation due to the translocation of enteric microbes to the systemic circulation. Here, we profiled the transcriptome of primary intestinal CD4+ T cells infected ex vivo with transmitted/founder (TF) HIV-1. Infections were performed in the presence or absence of Prevotella stercorea, a gut microbe enriched in the mucosa of HIV-1-infected individuals that enhanced both TF HIV-1 replication and CD4+ T cell death ex vivo. In the absence of bacteria, HIV-1 triggered a cellular shutdown response involving the downregulation of HIV-1 reactome genes, while perturbing genes linked to OX40, PPAR and FOXO3 signaling. However, in the presence of bacteria, HIV-1 did not perturb these gene sets or pathways. Instead, HIV-1 enhanced granzyme expression and Th17 cell function, inhibited G1/S cell cycle checkpoint genes and triggered downstream cell death pathways in microbe-exposed gut CD4+ T cells. To gain insights on these differential effects, we profiled the gene expression landscape of HIV-1-uninfected gut CD4+ T cells exposed to bacteria. Microbial exposure upregulated genes involved in cellular proliferation, MAPK activation, Th17 cell differentiation and type I interferon signaling. Our findings reveal that microbial exposure influenced how HIV-1 altered the gut CD4+ T cell transcriptome, with potential consequences for HIV-1 susceptibility, cell survival and inflammation. The HIV-1- and microbe-altered pathways unraveled here may serve as a molecular blueprint to gain basic insights in mucosal HIV-1 pathogenesis.

Using Nurse Practitioner Co-Management to Reduce Hospitalizations and Readmissions Within a Home-Based Primary Care Program.

Nurse practitioner (NP) co-management involves an NP and physician sharing responsibility for the care of a patient. This study evaluates the impact of NP co-management for clinically complex patients in a home-based primary care program on hospitalizations, 30-day hospital readmissions, and provider satisfaction. We compared preenrollment and postenrollment hospitalization and 30-day readmission rates of home-bound patients active in the Nurse Practitioner Co-Management Program within the Mount Sinai Visiting Doctors Program (MSVD) (n = 87) between January 1, 2012, and July 1, 2013. Data were collected from electronic medical records. An anonymous online survey was administered to all physicians active in the MSVD in July 2013 (n = 13).After enrollment in co-management, patients have lower annual hospitalization rates (1.26 vs. 2.27, p = .005) and fewer patients have 30-day readmissions (5.8% vs. 17.2%, p = .004). Eight of 13 physicians feel "much" or "somewhat" less burned out by their work after implementation of co-management. The high level of provider satisfaction and reductions in annual hospitalization and readmission rates among high-risk home-bound patients associated with NP co-management may yield not only benefits for patients, caregivers, and providers but also cost savings for institutions.

Low abundance of colonic butyrate-producing bacteria in HIV infection is associated with microbial translocation and immune activation.

OBJECTIVE: Gut microbial translocation is a major driving force behind chronic immune activation during HIV-1 infection. HIV-1-related intestinal dysbiosis, including increases in mucosa-associated pathobionts, may influence microbial translocation and contribute to mucosal and systemic inflammation. Thus, it is critical to understand the mechanisms by which gut microbes and their metabolic products, such as butyrate, influence immune cell function during HIV-1 infection. DESIGN: A cross-sectional study was performed to compare the relative abundance of butyrate-producing bacterial (BPB) species in colonic biopsies and stool of untreated, chronic HIV-1-infected (n = 18) and HIV-1-uninfected (n = 14) study participants. The effect of exogenously added butyrate on gut T-cell activation and HIV-1 infection was evaluated using an ex-vivo human intestinal cell culture model. METHODS: Species were identified in 16S ribosomal RNA sequence datasets. Ex-vivo isolated lamina propria mononuclear cells were infected with C-C chemokine receptor type 5-tropic HIV-1Bal, cultured with enteric gram-negative bacteria and a range of butyrate doses, and lamina propria T-cell activation and HIV-1 infection levels measured. RESULTS: Relative abundance of total BPB and specifically of Roseburia intestinalis, were lower in colonic mucosa of HIV-1-infected versus HIV-1-uninfected individuals. In HIV-1-infected study participants, R. intestinalis relative abundance inversely correlated with systemic indicators of microbial translocation, immune activation, and vascular inflammation. Exogenous butyrate suppressed enteric gram-negative bacteria-driven lamina propria T-cell activation and HIV-1 infection levels in vitro. CONCLUSION: Reductions in mucosal butyrate from diminished colonic BPB may exacerbate pathobiont-driven gut T-cell activation and HIV replication, thereby contributing to HIV-associated mucosal pathogenesis.

Developing triggers for the surgical intensive care unit for palliative care integration.

PURPOSE: Despite the growing acceptance of palliative care as a component of high-quality care for patients with serious illness, it remains underutilized in the surgical critical care setting. This article provides insight into a model for palliative care integration into the surgical intensive care unit (SICU), using triggers. METHODS: We performed a prospective cohort study after the implementation of a new set of palliative care triggers in the SICU of an 1170-bed tertiary medical center over the course of 9 months. We aimed to determine the ability of these triggers to identify patients who would benefit from palliative care consultation. RESULTS: There were 517 SICU admissions during the period of interest. Of this cohort, patients who had not yet been discharged at the time of analysis were excluded (n=25), and the remaining underwent analysis (n=492). Factors significantly associated with hospital death or hospice discharge were repeat SICU admission, metastatic/advanced cancer, SICU physician referral, and the matching of 2 or more secondary criteria. CONCLUSIONS: A series of triggers can help identify patients who may benefit from palliative care consultation. This approach can be used in intensive care settings to facilitate palliative care integration.

Impact of Inpatient Palliative Care on Treatment Intensity for Patients with Serious Illness.

BACKGROUND: Palliative care is associated with decreased treatment intensity and improved quality for individual patients at the end of life, but little is known about how hospital-wide outcomes are affected by the diffusion of palliative care principles. OBJECTIVE: We examined the relationship between presence of palliative care programs and hospitals' average treatment intensity, as indicated by mean intensive care unit (ICU) length of stay (LOS) and days under Medicare hospice coverage, in the last six months of life among Medicare beneficiaries aged 67 and over with serious chronic illness. METHODS: We linked hospital-level data from the American Hospital Association Annual Survey, National Palliative Care Registry, and Dartmouth Atlas of Health Care to examine hospital-level treatment intensity for chronically ill Medicare beneficiaries who died in 2010. We used propensity score-adjusted linear regression to estimate the relationship between palliative care programs and hospitals' mean ICU LOS and hospice length of enrollment. RESULTS: Among 974 hospitals meeting inclusion criteria, we compared 295 hospitals with palliative care programs to 679 hospitals without. Hospitals with palliative care programs were higher volume, more likely to be teaching hospitals, and have oncology services and less likely to be located in rural areas. In propensity score weighted analyses, the mean ICU LOS in hospitals with palliative care was shorter by 0.23 days (standard error [SE] = 0.26), but this was not statistically significant (p = 0.76). In addition, the mean length of hospice enrollment among beneficiaries served by hospitals with palliative care was longer by 0.22 days (SE = 0.61), but also was not statistically significant (p = 0.76). CONCLUSIONS: Hospital-based palliative care programs alone may not be sufficient to impact ICU LOS or hospice length of enrollment for all chronically ill older adults admitted to hospitals. Future work should measure hospital-wide palliative care outcomes and effects of core palliative knowledge and skills provided by nonpalliative care specialists.

The Impact of Reported Hospice Preferred Practices on Hospital Utilization at the End of Life.

BACKGROUND: The Affordable Care Act requires hospices to report quality measures across a range of processes and practices. Yet uncertainties exist regarding the impact of hospice preferred practices on patient outcomes. OBJECTIVE: Assess the impact of 6 hospice preferred practices and hospice organizational characteristics on hospital utilization and death using the first national data on hospice preferred practices. DESIGN: Longitudinal cohort study (2008-2011) of Medicare beneficiaries (N=149,814) newly enrolled in a national random sample of hospices (N=577) from the National Hospice Survey (84% response rate) and followed until death. OUTCOME MEASURES: The proportion of patients at each hospice admitted to the hospital, emergency department (ED), and intensive care unit (ICU), and who died in the hospital after hospice enrollment. RESULTS: Hospices that reported assessing patient preferences for site of death at admission had lower odds of being in the highest quartile for hospital death (AOR=0.36; 95% CI, 0.14-0.93) and ED visits (AOR=0.27; 95% CI, 0.10-0.76). Hospices that reported more frequently monitoring symptoms had lower odds of being in the highest quartile for ICU stays (AOR=0.48; 95% CI, 0.24-0.94). In adjusted analyses, a higher proportion of patients at for-profit compared with nonprofit hospices experienced a hospital admission (15.3% vs. 10.9%, P<0.001), ED visit (21.8% vs. 15.6%, P<0.001), and ICU stay (5.1% vs. 3.0%, P<0.001). CONCLUSIONS: Hospitalization of patients following hospice enrollment varies substantially across hospices. Two of the 6 preferred practices examined were associated with hospitalization rates and for-profit hospices had persistently high hospitalization rates regardless of preferred practice implementation.

Interferon Alpha Subtype-Specific Suppression of HIV-1 Infection In Vivo.

UNLABELLED: Although all 12 subtypes of human interferon alpha (IFN-α) bind the same receptor, recent results have demonstrated that they elicit unique host responses and display distinct efficacies in the control of different viral infections. The IFN-α2 subtype is currently in HIV-1 clinical trials, but it has not consistently reduced viral loads in HIV-1 patients and is not the most effective subtype against HIV-1 in vitro We now demonstrate in humanized mice that, when delivered at the same high clinical dose, the human IFN-α14 subtype has very potent anti-HIV-1 activity whereas IFN-α2 does not. In both postexposure prophylaxis and treatment of acute infections, IFN-α14, but not IFN-α2, significantly suppressed HIV-1 replication and proviral loads. Furthermore, HIV-1-induced immune hyperactivation, which is a prognosticator of disease progression, was reduced by IFN-α14 but not IFN-α2. Whereas ineffective IFN-α2 therapy was associated with CD8(+) T cell activation, successful IFN-α14 therapy was associated with increased intrinsic and innate immunity, including significantly higher induction of tetherin and MX2, increased APOBEC3G signature mutations in HIV-1 proviral DNA, and higher frequencies of TRAIL(+) NK cells. These results identify IFN-α14 as a potent new therapeutic that operates via mechanisms distinct from those of antiretroviral drugs. The ability of IFN-α14 to reduce both viremia and proviral loads in vivo suggests that it has strong potential as a component of a cure strategy for HIV-1 infections. The broad implication of these results is that the antiviral efficacy of each individual IFN-α subtype should be evaluated against the specific virus being treated. IMPORTANCE: The naturally occurring antiviral protein IFN-α2 is used to treat hepatitis viruses but has proven rather ineffective against HIV in comparison to triple therapy with the antiretroviral (ARV) drugs. Although ARVs suppress the replication of HIV, they fail to completely clear infections. Since IFN-α acts by different mechanism than ARVs and has been shown to reduce HIV proviral loads, clinical trials are under way to test whether IFN-α2 combined with ARVs might eradicate HIV-1 infections. IFN-α is actually a family of 12 distinct proteins, and each IFN-α subtype has different efficacies toward different viruses. Here, we use mice that contain a human immune system, so they can be infected with HIV. With this model, we demonstrate that while IFN-α2 is only weakly effective against HIV, IFN-α14 is extremely potent. This discovery identifies IFN-α14 as a more powerful IFN-α subtype for use in combination therapy trials aimed toward an HIV cure.

Enhancement of HIV-1 infection and intestinal CD4+ T cell depletion ex vivo by gut microbes altered during chronic HIV-1 infection.

BACKGROUND: Early HIV-1 infection is characterized by high levels of HIV-1 replication and substantial CD4 T cell depletion in the intestinal mucosa, intestinal epithelial barrier breakdown, and microbial translocation. HIV-1-induced disruption of intestinal homeostasis has also been associated with changes in the intestinal microbiome that are linked to mucosal and systemic immune activation. In this study, we investigated the impact of representative bacterial species that were altered in the colonic mucosa of viremic HIV-1 infected individuals (HIV-altered mucosal bacteria; HAMB) on intestinal CD4 T cell function, infection by HIV-1, and survival in vitro. Lamina propria (LP) mononuclear cells were infected with CCR5-tropic HIV-1BaL or mock infected, exposed to high (3 gram-negative) or low (2 gram-positive) abundance HAMB or control gram-negative Escherichia coli and levels of productive HIV-1 infection and CD4 T cell depletion assessed. HAMB-associated changes in LP CD4 T cell activation, proliferation and HIV-1 co-receptor expression were also evaluated. RESULTS: The majority of HAMB increased HIV-1 infection and depletion of LP CD4 T cells, but gram-negative HAMB enhanced CD4 T cell infection to a greater degree than gram-positive HAMB. Most gram-negative HAMB enhanced T cell infection to levels similar to that induced by gram-negative E. coli despite lower induction of T cell activation and proliferation by HAMB. Both gram-negative HAMB and E. coli significantly increased expression of HIV-1 co-receptor CCR5 on LP CD4 T cells. Lipopolysaccharide, a gram-negative bacteria cell wall component, up-regulated CCR5 expression on LP CD4 T cells whereas gram-positive cell wall lipoteichoic acid did not. Upregulation of CCR5 by gram-negative HAMB was largely abrogated in CD4 T cell-enriched cultures suggesting an indirect mode of stimulation. CONCLUSIONS: Gram-negative commensal bacteria that are altered in abundance in the colonic mucosa of HIV-1 infected individuals have the capacity to enhance CCR5-tropic HIV-1 productive infection and depletion of LP CD4 T cells in vitro. Enhanced infection appears to be primarily mediated indirectly through increased expression of CCR5 on LP CD4 T cells without concomitant large scale T cell activation. This represents a novel mechanism potentially linking intestinal dysbiosis to HIV-1 mucosal pathogenesis.

Interferon-α Subtypes in an Ex Vivo Model of Acute HIV-1 Infection: Expression, Potency and Effector Mechanisms.

HIV-1 is transmitted primarily across mucosal surfaces and rapidly spreads within the intestinal mucosa during acute infection. The type I interferons (IFNs) likely serve as a first line of defense, but the relative expression and antiviral properties of the 12 IFNα subtypes against HIV-1 infection of mucosal tissues remain unknown. Here, we evaluated the expression of all IFNα subtypes in HIV-1-exposed plasmacytoid dendritic cells by next-generation sequencing. We then determined the relative antiviral potency of each IFNα subtype ex vivo using the human intestinal Lamina Propria Aggregate Culture model. IFNα subtype transcripts from the centromeric half of the IFNA gene complex were highly expressed in pDCs following HIV-1 exposure. There was an inverse relationship between IFNA subtype expression and potency. IFNα8, IFNα6 and IFNα14 were the most potent in restricting HIV-1 infection. IFNα2, the clinically-approved subtype, and IFNα1 were both highly expressed but exhibited relatively weak antiviral activity. The relative potencies correlated with binding affinity to the type I IFN receptor and the induction levels of HIV-1 restriction factors Mx2 and Tetherin/BST-2 but not APOBEC3G, F and D. However, despite the lack of APOBEC3 transcriptional induction, the higher relative potency of IFNα8 and IFNα14 correlated with stronger inhibition of virion infectivity, which is linked to deaminase-independent APOBEC3 restriction activity. By contrast, both potent (IFNα8) and weak (IFNα1) subtypes significantly induced HIV-1 GG-to-AG hypermutation. The results unravel non-redundant functions of the IFNα subtypes against HIV-1 infection, with strong implications for HIV-1 mucosal immunity, viral evolution and IFNα-based functional cure strategies.

Divergent Synthesis of Chondroitin Sulfate Disaccharides and Identification of Sulfate Motifs that Inhibit Triple Negative Breast Cancer.

Glycosaminoglycans (GAGs) regulate many important physiological processes. A pertinent issue to address is whether GAGs encode important functional information via introduction of position specific sulfate groups in the GAG structure. However, procurement of pure, homogenous GAG motifs to probe the "sulfation code" is a challenging task due to isolation difficulty and structural complexity. To this end, we devised a versatile synthetic strategy to obtain all the 16 theoretically possible sulfation patterns in the chondroitin sulfate (CS) repeating unit; these include rare but potentially important sulfated motifs which have not been isolated earlier. Biological evaluation indicated that CS sulfation patterns had differing effects for different breast cancer cell types, and the greatest inhibitory effect was observed for the most aggressive, triple negative breast cancer cell line MDA-MB-231.