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.

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.

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.

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.

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.

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.

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.

Toll-like receptor 3 plays a role in myocardial infarction and ischemia/reperfusion injury.

Innate immune and inflammatory responses mediated by Toll like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. This study examined the role of TLR3 in myocardial injury induced by two models, namely, myocardial infarction (MI) and I/R. First, we examined the role of TLR3 in MI. TLR3 deficient (TLR3(-/-)) and wild type (WT) mice were subjected to MI induced by permanent ligation of the left anterior descending (LAD) coronary artery for 21days. Cardiac function was measured by echocardiography. Next, we examined whether TLR3 contributes to myocardial I/R injury. TLR3(-/-) and WT mice were subjected to myocardial ischemia (45min) followed by reperfusion for up to 3days. Cardiac function and myocardial infarct size were examined. We also examined the effect of TLR3 deficiency on I/R-induced myocardial apoptosis and inflammatory cytokine production. TLR3(-/-) mice showed significant attenuation of cardiac dysfunction after MI or I/R. Myocardial infarct size and myocardial apoptosis induced by I/R injury were significantly attenuated in TLR3(-/-) mice. TLR3 deficiency increases B-cell lymphoma 2 (BCL2) levels and attenuates I/R-increased Fas, Fas ligand or CD95L (FasL), Fas-Associated protein with Death Domain (FADD), Bax and Bak levels in the myocardium. TLR3 deficiency also attenuates I/R-induced myocardial nuclear factor KappaB (NF-κB) binding activity, Tumor necrosis factor alpha (TNF-α) and Interleukin-1 beta (IL-1ß) production as well as I/R-induced infiltration of neutrophils and macrophages into the myocardium. TLR3 plays an important role in myocardial injury induced by MI or I/R. The mechanisms involve activation of apoptotic signaling and NF-κB binding activity. Modulation of TLR3 may be an effective approach for ameliorating heart injury in heart attack patients.

Microbial exposure alters HIV-1-induced mucosal CD4+ T cell death pathways Ex vivo.

BACKGROUND: Early HIV-1 infection causes massive CD4+ T cell death in the gut and translocation of bacteria into the circulation. However, the programmed cell death (PCD) pathways used by HIV-1 to kill CD4+ T cells in the gut, and the impact of microbial exposure on T cell loss, remain unclear. Understanding mucosal HIV-1 triggered PCD could be advanced by an ex vivo system involving lamina propria mononuclear cells (LPMCs). We therefore modeled the interactions of gut LPMCs, CCR5-tropic HIV-1 and a commensal gut bacterial species, Escherichia coli. In this Lamina Propria Aggregate Culture (LPAC) model, LPMCs were infected with HIV-1BaL by spinoculation and cultured in the presence or absence of heat killed E.coli. CD4+ T cell numbers derived from flow cytometry and viable cell counts were reported relative to mock infection. Viable cells were identified by viability dye exclusion (AqVi), and intracellular HIV-1 Gag p24 protein was used to identify infected cells. Annexin V and AqVi were used to identify apoptotic versus necrotic cells. Caspase-1 and Caspase-3 activities were blocked using specific inhibitors YVAD and DEVD, respectively. RESULTS: CD4+ T cell depletion following HIV-1 infection was reproducibly observed by 6 days post infection (dpi). Depletion at 6 dpi strongly correlated with infection frequency at 4 dpi, was significantly blocked by Efavirenz treatment, and was primarily driven by p24-negative cells that were predominantly necrotic. HIV-1 infection significantly induced CD4+ T-cell intrinsic Caspase-1 activity, whereas Caspase-1 inhibition, but not Caspase-3 inhibition, significantly blocked CD4+ T cell depletion. Exposure to E.coli enhanced HIV-1 infection and CD4+ T depletion, and significantly increased the number of apoptotic p24+ cells. Notably, CD4+ T cell depletion in the presence of E.coli was partially blocked by Caspase-3, but not by Caspase-1 inhibition. CONCLUSIONS: In the LPAC model, HIV-1 induced Caspase-1 mediated pyroptosis in bystander CD4+ T cells, but microbial exposure shifted the PCD mechanism toward apoptosis of productively infected T cells. These results suggest that mucosal CD4+ T cell death pathways may be altered in HIV-infected individuals after gut barrier function is compromised, with potential consequences for mucosal inflammation, viral dissemination and systemic immune activation.

Increased Escherichia coli-induced interleukin-23 production by CD16+ monocytes correlates with systemic immune activation in untreated HIV-1-infected individuals.

The level of microbial translocation from the intestine is increased in HIV-1 infection. Proinflammatory cytokine production by peripheral antigen-presenting cells in response to translocated microbes or microbial products may contribute to systemic immune activation, a hallmark of HIV-1 infection. We investigated the cytokine responses of peripheral blood myeloid dendritic cells (mDCs) and monocytes to in vitro stimulation with commensal enteric Escherichia coli in peripheral blood mononuclear cells (PBMC) from untreated HIV-1-infected subjects and from uninfected controls. Levels of interleukin 23 (IL-23) produced by PBMC from HIV-1-infected subjects in response to E. coli stimulation were significantly higher than those produced by PBMC from uninfected subjects. IL-23 was produced primarily by CD16(+) monocytes. This subset of monocytes was increased in frequency and expressed higher levels of Toll-like receptor 4 (TLR4) in HIV-1-infected individuals than in controls. Blocking TLR4 on total CD14(+) monocytes reduced IL-23 production in response to E. coli stimulation. Levels of soluble CD27, an indicator of systemic immune activation, were elevated in HIV-1-infected subjects and were associated with the percentage of CD16(+) monocytes and the induction of IL-23 by E. coli, providing a link between these parameters and systemic inflammation. Taken together, these results suggest that IL-23 produced by CD16(+) monocytes in response to microbial stimulation may contribute to systemic immune activation in HIV-1-infected individuals.

HIV-1 infection of human intestinal lamina propria CD4+ T cells in vitro is enhanced by exposure to commensal Escherichia coli.

Microbial translocation has been linked to systemic immune activation in HIV-1 disease, yet mechanisms by which microbes may contribute to HIV-associated intestinal pathogenesis are poorly understood. Importantly, our understanding of the impact of translocating commensal intestinal bacteria on mucosal-associated T cell responses in the context of ongoing viral replication that occurs early in HIV-1 infection is limited. We previously identified commensal Escherichia coli-reactive Th1 and Th17 cells in normal human intestinal lamina propria (LP). In this article, we established an ex vivo assay to investigate the interactions between Th cell subsets in primary human LP mononuclear cells (LPMCs), commensal E. coli, and CCR5-tropic HIV-1(Bal). Addition of heat-killed E. coli to HIV-1-exposed LPMCs resulted in increases in HIV-1 replication, CD4 T cell activation and infection, and IL-17 and IFN-γ production. Conversely, purified LPS derived from commensal E. coli did not enhance CD4 T cell infection. E. coli exposure induced greater proliferation of LPMC Th17 than Th1 cells. Th17 cells were more permissive to infection than Th1 cells in HIV-1-exposed LPMC cultures, and Th17 cell infection frequencies significantly increased in the presence of E. coli. The E. coli-associated enhancement of infection was dependent on the presence of CD11c(+) LP dendritic cells and, in part, on MHC class II-restricted Ag presentation. These results highlight a potential role for translocating microbes in impacting mucosal HIV-1 pathogenesis during early infection by increasing HIV-1 replication and infection of intestinal Th1 and Th17 cells.

Mental health disparities between Roma and non-Roma children in Romania and Bulgaria.

BACKGROUND: The Roma population, one of the largest minority groups in Europe, experience discrimination and stigma associated with marginalized social position. Few studies have examined mental illnesses in the Roma, and none have examined the Roma children. The present study estimates mental health and behavioral disorders among Roma children in comparison to non-Roma children in educational institutions. METHODS: Data were drawn from the School Children Mental Health Study in Europe (SCHME) study in Romania (Roma children identified by parent report, N = 70; non-Roma, N = 925) and Bulgaria (Roma children identified by exclusively-Roma schools, N = 65; non-Roma, N = 1312). The Strengths and Difficulties Questionnaire was given to the parents and teachers to measure child mental health; children reported on their mental health through the Dominique Interactive. Control covariates included child sex and age, and parental characteristics when parent reports were available. RESULTS: Based on the child's own report, Roma children had a higher odds of any internalizing disorder (OR = 2.99, 95% C.I. 2.07-4.30), phobias (OR = 4.84, 95% C.I. 3.19-7.35), separation anxiety disorder (OR = 2.54, 95% C.I. 1.72-3.76), generalized anxiety disorder (OR = 2.95, 95% C.I. 1.75-4.96), and major depressive disorder (OR = 3.86, 95% C.I. 2.31-6.37). Further Roma children had a higher odds of any externalizing disorder (OR = 2.84, 95% C.I. 1.78-4.54), oppositional defiant disorder (OR = 3.35, 95% C.I. 1.93-5.82), ADHD (OR = 2.37, 95% C.I. 1.26-4.46), and conduct disorder (OR = 3.63, 95% C.I. 2.04-6.46). Based on the report of teachers, Roma children had higher odds of emotional problems (OR = 2.03, 95% C.I. 1.20-3.44), peer-relational problems (OR = 2.76, 95% C.I. 1.73-4.41) and prosocial behavior (OR = 2.75, 95% C.I. 1.75-4.33). CONCLUSION: Roma children experience a higher burden of mental health problems compared with their non-Roma counterparts. Attention to child health and mental health among the Roma is urgently needed, as these children experience a constellation of health problems associated with poverty as well as experiences of stigma and discrimination.

Association of functional impairment with inflammation and immune activation in HIV type 1-infected adults receiving effective antiretroviral therapy.

BACKGROUND: The relationships of inflammation, immune activation, and immunosenescence markers with functional impairment in aging human immunodeficiency virus type 1 (HIV-1)-infected persons are unknown. METHODS: HIV-infected persons who were aged 45-65 years, had a plasma HIV-1 RNA load of <48 copies/mL, and were receiving antiretroviral therapy underwent standardized functional testing. In a nested case-control analysis, low-functioning cases were matched (1:1-2) by age, sex, and HIV-1 diagnosis date to high-functioning controls. Markers of inflammation, T-cell activation, microbial translocation, immunosenescence, and immune recovery were used to estimate functional status in conditional logistic regression. Primary analyses were adjusted for CD4(+) T-cell count, smoking, and hepatitis. RESULTS: Thirty-one low-functioning cases were compared to 49 high-functioning controls. After statistical adjustment, lower proportions of CD4(+) T cells and higher proportion of CD8(+) T cells, higher CD38/HLA-DR expression on CD8(+) T cells, and higher interleukin-6 were associated with a significantly greater odds of low functional status (odds ratio, ≥ 1.1 for all analyses; P ≤ .03). Other inflammatory, senescence, and microbial translocation markers were not significantly different (P ≥ .11 for all analyses) between low-functioning and high-functioning groups. CONCLUSIONS: Functional impairment during successful antiretroviral therapy was associated with higher CD8(+) T-cell activation and interleukin 6 levels. Interventions to decrease immune activation and inflammation should be evaluated for their effects on physical function and frailty.

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, 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.

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.

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.

Impaired neutrophil activity and increased susceptibility to bacterial infection in mice lacking glucose-6-phosphatase-beta.

Neutropenia and neutrophil dysfunction are common in many diseases, although their etiology is often unclear. Previous views held that there was a single ER enzyme, glucose-6-phosphatase-alpha (G6Pase-alpha), whose activity--limited to the liver, kidney, and intestine--was solely responsible for the final stages of gluconeogenesis and glycogenolysis, in which glucose-6-phosphate (G6P) is hydrolyzed to glucose for release to the blood. Recently, we characterized a second G6Pase activity, that of G6Pase-beta (also known as G6PC), which is also capable of hydrolyzing G6P to glucose but is ubiquitously expressed and not implicated in interprandial blood glucose homeostasis. We now report that the absence of G6Pase-beta led to neutropenia; defects in neutrophil respiratory burst, chemotaxis, and calcium flux; and increased susceptibility to bacterial infection. Consistent with this, G6Pase-beta-deficient (G6pc3-/-) mice with experimental peritonitis exhibited increased expression of the glucose-regulated proteins upregulated during ER stress in their neutrophils and bone marrow, and the G6pc3-/- neutrophils exhibited an enhanced rate of apoptosis. Our results define a molecular pathway to neutropenia and neutrophil dysfunction of previously unknown etiology, providing a potential model for the treatment of these conditions.

Localized immunosuppressive environment in the foreign body response to implanted biomaterials.

The implantation of synthetic biomaterials initiates the foreign body response (FBR), which is characterized by macrophage infiltration, foreign body giant cell formation, and fibrotic encapsulation of the implant. The FBR is orchestrated by a complex network of immune modulators, including diverse cell types, soluble mediators, and unique cell surface interactions. The specific tissue locations, expression patterns, and spatial distribution of these immune modulators around the site of implantation are not clear. This study describes a model for studying the FBR in vivo and specifically evaluates the spatial relationship of immune modulators. We modified a biomaterials implantation in vivo model that allowed for cross-sectional in situ analysis of the FBR. Immunohistochemical techniques were used to determine the localization of soluble mediators, ie, interleukin (IL)-4, IL-13, IL-10, IL-6, transforming growth factor-beta, tumor necrosis factor-alpha, interferon-gamma, and MCP-1; specific cell types, ie, macrophages, neutrophils, fibroblasts, and lymphocytes; and cell surface markers, ie, F4/80, CD11b, CD11c, and Ly-6C, at early, middle, and late stages of the FBR in subcutaneous implant sites. The cytokines IL-4, IL-13, IL-10, and transforming growth factor-beta were localized to implant-adherent cells that included macrophages and foreign body giant cells. A better understanding of the FBR in vivo will allow the development of novel strategies to enhance biomaterial implant design to achieve better performance and safety of biomedical devices at the site of implant.

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.