Noncanonical autophagy is required for type I interferon secretion in response to DNA-immune complexes.

Toll-like receptor-9 (TLR9) is largely responsible for discriminating self from pathogenic DNA. However, association of host DNA with autoantibodies activates TLR9, inducing the pathogenic secretion of type I interferons (IFNs) from plasmacytoid dendritic cells (pDCs). Here, we found that in response to DNA-containing immune complexes (DNA-IC), but not to soluble ligands, IFN-α production depended upon the convergence of the phagocytic and autophagic pathways, a process called microtubule-associated protein 1A/1B-light chain 3 (LC3)-associated phagocytosis (LAP). LAP was required for TLR9 trafficking into a specialized interferon signaling compartment by a mechanism that involved autophagy-related proteins, but not the conventional autophagic preinitiation complex, or adaptor protein-3 (AP-3). Our findings unveil a new role for nonconventional autophagy in inflammation and provide one mechanism by which anti-DNA autoantibodies, such as those found in several autoimmune disorders, bypass the controls that normally restrict the apportionment of pathogenic DNA and TLR9 to the interferon signaling compartment.

Novel methodology to measure pre-procedure antimicrobial prophylaxis: integrating text searches with structured data from the Veterans Health Administration's electronic medical record.

BACKGROUND: Antimicrobial prophylaxis is an evidence-proven strategy for reducing procedure-related infections; however, measuring this key quality metric typically requires manual review, due to the way antimicrobial prophylaxis is documented in the electronic medical record (EMR). Our objective was to electronically measure compliance with antimicrobial prophylaxis using both structured and unstructured data from the Veterans Health Administration (VA) EMR. We developed this methodology for cardiac device implantation procedures. METHODS: With clinician input and review of clinical guidelines, we developed a list of antimicrobial names recommended for the prevention of cardiac device infection. We trained the algorithm using existing fiscal year (FY) 2008-15 data from the VA Clinical Assessment Reporting and Tracking-Electrophysiology (CART-EP), which contains manually determined information about antimicrobial prophylaxis. We merged CART-EP data with EMR data and programmed statistical software to flag an antimicrobial orders or drug fills from structured data fields in the EMR and hits on text string searches of antimicrobial names documented in clinician's notes. We iteratively tested combinations of these data elements to optimize an algorithm to accurately classify antimicrobial use. The final algorithm was validated in a national cohort of VA cardiac device procedures from FY2016-2017. Discordant cases underwent expert manual review to identify reasons for algorithm misclassification. RESULTS: The CART-EP dataset included 2102 procedures at 38 VA facilities with manually identified antimicrobial prophylaxis in 2056 cases (97.8%). The final algorithm combining structured EMR fields and text note search results correctly classified 2048 of the CART-EP cases (97.4%). In the validation sample, the algorithm measured compliance with antimicrobial prophylaxis in 16,606 of 18,903 cardiac device procedures (87.8%). Misclassification was due to EMR documentation issues, such as antimicrobial prophylaxis documented only in hand-written clinician notes in a format that cannot be electronically searched. CONCLUSIONS: We developed a methodology with high accuracy to measure guideline concordant use of antimicrobial prophylaxis before cardiac device procedures using data fields present in modern EMRs. This method can replace manual review in quality measurement in the VA and other healthcare systems with EMRs; further, this method could be adapted to measure compliance in other procedural areas where antimicrobial prophylaxis is recommended.

Analysis of the Effects of the Bruton's tyrosine kinase (Btk) Inhibitor Ibrutinib on Monocyte Fcγ Receptor (FcγR) Function.

The irreversible Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fcγ receptors (FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on FcγR-mediated activities in monocytes. We found that ibrutinib did not affect monocyte FcγR-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFNγ priming because NK cells produce IFNγ in response to antibody therapy. Pretreatment of monocytes with IFNγ abrogated the effects of ibrutinib on FcγR-mediated cytokine production, suggesting that IFNγ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function.

Interferon-γ Promotes Antibody-mediated Fratricide of Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is characterized by the proliferation of immature myeloid lineage blasts. Due to its heterogeneity and to the high rate of acquired drug resistance and relapse, new treatment strategies are needed. Here, we demonstrate that IFNγ promotes AML blasts to act as effector cells within the context of antibody therapy. Treatment with IFNγ drove AML blasts toward a more differentiated state, wherein they showed increased expression of the M1-related markers HLA-DR and CD86, as well as of FcγRI, which mediates effector responses to therapeutic antibodies. Importantly, IFNγ was able to up-regulate CD38, the target of the therapeutic antibody daratumumab. Because the antigen (CD38) and effector receptor (FcγRI) were both simultaneously up-regulated on the AML blasts, we tested whether IFNγ treatment of the AML cell lines THP-1 and MV4-11 could stimulate them to target one another after the addition of daratumumab. Results showed that IFNγ significantly increased daratumumab-mediated cytotoxicity, as measured both by 51Cr release and lactate dehydrogenase release assays. We also found that the combination of IFNγ and activation of FcγR led to the release of granzyme B by AML cells. Finally, using a murine NSG model of subcutaneous AML, we found that treatment with IFNγ plus daratumumab significantly attenuated tumor growth. Taken together, these studies show a novel mechanism of daratumumab-mediated killing and a possible new therapeutic strategy for AML.

Complement C5a potentiates uric acid crystal-induced IL-1ß production.

Anaphylatoxin C5a released upon complement activation is associated with both acute and chronic inflammations such as gout. The pathogenesis of gout was identified as uric acid crystal deposition in the joints that activates inflammasome, leading to IL-1ß release. However, little is known about the interaction between complement activation and monosodium urate/uric acid (MSU) crystal-induced inflammasome activation or IL-1ß production. Here, we report that MSU crystal-induced proinflammatory cytokines/chemokines in human whole blood is predominantly regulated by C5a through its interaction with C5a receptor. C5a induces pro-IL-1ß and IL-1ß production in human primary monocytes, and potentiates MSU or cholesterol crystals in IL-1ß production. This potentiation is caspase-1 dependent and requires intracellular Ca(2+) mobilization, K(+) efflux, and cathepsin B activity. Our results provide insight into the role of C5a as an endogenous priming signal that is required for the initiation of uric acid crystal-induced IL-1ß production. C5a could potentially be a therapeutic target together with IL-1ß antagonists for the treatment of complement-dependent and inflammasome-associated diseases.

Fcγ receptor-induced soluble vascular endothelial growth factor receptor-1 (VEGFR-1) production inhibits angiogenesis and enhances efficacy of anti-tumor antibodies.

Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt-1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing the mechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-κB, we found that a microRNA, miR181a, negatively regulates FcγR-mediated sFlt-1 production and that NF-κB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy.

Activation of plasmacytoid dendritic cells promotes AML-cell fratricide.

Acute myeloid leukemia (AML) is characterized by the proliferation of immature myeloid blasts and a suppressed immune state. Interferons have been previously shown to aid in the clearance of AML cells. Type I interferons are produced primarily by plasmacytoid dendritic cells (pDCs). However, these cells exist in a quiescent state in AML. Because pDCs express TLR 7-9, we hypothesized that the TLR7/8 agonist R848 would be able to reprogram them toward a more active, IFN-producing phenotype. Consistent with this notion, we found that R848-treated pDCs from patients produced significantly elevated levels of IFNß. In addition, they showed increased expression of the immune-stimulatory receptor CD40. We next tested whether IFNß would influence antibody-mediated fratricide among AML cells, as our recent work showed that AML cells could undergo cell-to cell killing in the presence of the CD38 antibody daratumumab. We found that IFNß treatment led to a significant, IRF9-dependent increase in CD38 expression and a subsequent increase in daratumumab-mediated cytotoxicity and decreased colony formation. These findings suggest that the tolerogenic phenotype of pDCs in AML can be reversed, and also demonstrate a possible means of enhancing endogenous Type I IFN production that would promote daratumumab-mediated clearance of AML cells.

Angioleiomyoma of the Lower Lip.

Leiomyoma is a benign smooth muscle tumor that occurs most frequently in the uterine myometrium, gastrointestinal tract, skin, and lower extremities. Leiomyoma rarely affects the oral cavity. Angioleiomyoma (vascular leiomyoma) is a histological subtype of the leiomyoma. The diagnosis is commonly determined by histopathological studies. This case report shows a 57-year-old male patient with a lesion of the lower lip. After laser excision, hematoxylin and eosin and smooth muscle actin staining confirmed the diagnosis of angioleiomyoma.

Adenomatoid Odontogenic Tumor Associated with Impacted Mandibular Canine - A Case Report.

Adenomatoid odontogenic tumor (AOT) is a relatively rare, benign, hamartomatous, and cystic odontogenic neoplasm that was first described more than a century ago. It accounts for 2-7% of all odontogenic tumors. The lesion still continues to intrigue experts with its varied histomorphology and controversies regarding its development. The present article describes a case of cystic AOT with an unusual histomorphology associated with an impacted 43 in a 15-year-old male.

Development and Validation of a Semi-Automated Surveillance Algorithm for Cardiac Device Infections: Insights from the VA CART program.

Procedure-related cardiac electronic implantable device (CIED) infections have high morbidity and mortality, highlighting the urgent need for infection prevention efforts to include electrophysiology procedures. We developed and validated a semi-automated algorithm based on structured electronic health records data to reliably identify CIED infections. A sample of CIED procedures entered into the Veterans' Health Administration Clinical Assessment Reporting and Tracking program from FY 2008-2015 was reviewed for the presence of CIED infection. This sample was then randomly divided into training (2/3) validation sets (1/3). The training set was used to develop a detection algorithm containing structured variables mapped from the clinical pathways of CIED infection. Performance of this algorithm was evaluated using the validation set. 2,107 unique CIED procedures from a cohort of 5,753 underwent manual review; 97 CIED infections (4.6%) were identified. Variables strongly associated with true infections included presence of a microbiology order, billing codes for surgical site infections and post-procedural antibiotic prescriptions. The combined algorithm to detect infection demonstrated high c-statistic (0.95; 95% confidence interval: 0.92-0.98), sensitivity (87.9%) and specificity (90.3%) in the validation data. Structured variables derived from clinical pathways can guide development of a semi-automated detection tool to surveil for CIED infection.

Ectopically Positioned Tooth Piercing the Philtrum.

A variety of eruption disturbances arise during the transitional dentition period, which can be broadly classified as disturbances related to time and disturbances related to position. The occurrence of ectopic eruption is relatively common, but ectopically positioned tooth piercing the philtrum is a rare clinical presentation. This is a case report of a 70-year-old female who presented with the chief complaint of an abnormally positioned tooth piercing out from the upper lip to the Department of Oral and Maxillofacial Surgery, Karnavati School of Dentistry, Uvarsad, Gandhinagar, Gujarat, India. As per the patient's history, labially erupted tooth was piercing the philtrum for 60 years and it was visible extraorally from the philtrum and was painful.

Real-world effectiveness of infection prevention interventions for reducing procedure-related cardiac device infections: Insights from the veterans affairs clinical assessment reporting and tracking program.

OBJECTIVE: To measure the association between receipt of specific infection prevention interventions and procedure-related cardiac implantable electronic device (CIED) infections. DESIGN: Retrospective cohort with manually reviewed infection status. SETTING: Setting: National, multicenter Veterans Health Administration (VA) cohort. PARTICIPANTS: Sampling of procedures entered into the VA Clinical Assessment Reporting and Tracking-Electrophysiology (CART-EP) database from fiscal years 2008 through 2015. METHODS: A sample of procedures entered into the CART-EP database underwent manual review for occurrence of CIED infection and other clinical/procedural variables. The primary outcome was 6-month incidence of CIED infection. Measures of association were calculated using multivariable generalized estimating equations logistic regression. RESULTS: We identified 101 procedure-related CIED infections among 2,098 procedures (4.8% of reviewed sample). Factors associated with increased odds of infections included (1) wound complications (adjusted odds ratio [aOR], 8.74; 95% confidence interval [CI], 3.16-24.20), (2) revisions including generator changes (aOR, 2.4; 95% CI, 1.59-3.63), (3) an elevated international normalized ratio (INR) >1.5 (aOR, 1.56; 95% CI, 1.12-2.18), and (4) methicillin-resistant Staphylococcus colonization (aOR, 9.56; 95% CI, 1.55-27.77). Clinically effective prevention interventions included preprocedural skin cleaning with chlorhexidine versus other topical agents (aOR, 0.41; 95% CI, 0.22-0.76) and receipt of ß-lactam antimicrobial prophylaxis versus vancomycin (aOR, 0.60; 95% CI, 0.37-0.96). The use of mesh pockets and continuation of antimicrobial prophylaxis after skin closure were not associated with reduced infection risk. CONCLUSIONS: These findings regarding the real-world clinical effectiveness of different prevention strategies can be applied to the development of evidence-based protocols and infection prevention guidelines specific to the electrophysiology laboratory.

Prolonged antimicrobial prophylaxis following cardiac device procedures increases preventable harm: insights from the VA CART program.

BACKGROUND: The rate of cardiovascular implantable electronic device (CIED) infection is increasing coincident with an increase in the number of device procedures. Preprocedural antimicrobial prophylaxis reduces CIED infections; however, there is no evidence that prolonged postprocedural antimicrobials additionally reduce risk. Thus, we sought to quantify the harms associated with this approach. OBJECTIVE: To measure the association between Clostridium difficile infection (CDI), acute kidney injury (AKI) and receipt of prolonged postprocedural antimicrobials. METHODS: CIED procedures entered into the VA Clinical Assessment Reporting and Tracking Electrophysiology (CART-EP) database during fiscal years 2008-2016 were included. The primary outcome was 90-day incidence of CDI and the secondary outcome was the 7-day incidence of AKI. The primary exposure measure was duration of postprocedural antimicrobial therapy. Associations were measured using Cox-proportional hazards and binomial regression. RESULTS: Prolonged postprocedural antimicrobial therapy was identified following 3,331 of 6,497 CIED procedures (51.3%), and the median duration of prophylaxis was 5 days. Prolonged postprocedural antimicrobial use was associated with increased risk of CDI (hazard ratio [HR], 2.90; 95% confidence interval [CI], 1.54-5.46). Of the 27 patients who developed CDI, 11 subsequently died. Postprocedural antimicrobial use with ≥2 antimicrobials was associated with an increased risk of AKI (OR, 4.16; 95% CI, 2.50-6.90). The impact was particularly significant when one of the dual agents prescribed was vancomycin (adjusted OR, 8.41; 95% CI, 5.53-12.79). CONCLUSIONS: Prolonged antimicrobial prophylaxis following CIED procedures increases preventable harm; this practice should be discouraged in procedural settings such as the cardiac electrophysiology laboratory.

A bioconjugate of Pseudomonas cepacia lipase with alginate with enhanced catalytic efficiency.

A bioconjugate of Pseudomonas cepacia lipase with alginate was prepared by simple adsorption. Atomic force microscope (AFM) images showed that this bioconjugate resulted from adsorption rather than entrapment of the enzyme as enzyme molecules were visible on the gel surface. The soluble bioconjugate exhibited increased enzyme activity in terms of high effectiveness factor (effectiveness factor was 3 for the immobilized preparation) and greater Vmax/Km value (Vmax/Km increased 25 times upon immobilization). This constitutes one of the less frequently observed instances of lipase activation by lid opening as a result of binding to a predominantly hydrophilic molecule. The bioconjugate was also more stable at 55 degrees C as compared to the free enzyme and could be reused for oil hydrolysis up to 4 cycles without any loss in activity. Fluorescence emission spectroscopy showed that the immobilized enzyme had undergone definite conformational changes.

A comparative study of sheath potential profile measurements with laser-heated and current-heated emissive probes.

Emissive Langmuir probe is one of the most efficient diagnostic tools available for plasma potential measurements. Extensive studies have been carried out in designing different kinds of conventional (electrically heated) emissive probes (CEPs) to estimate the plasma potential. Laser heated emissive probe (LHEP) has been developed with certain advantages over the conventional probes such as low evaporation rate of the probe material, high lifetime, and high emission levels. Most importantly, the LHEP uses laser to heat the probe-tip and does not require electric current to heat the probe-tip like in CEP. The heating current in CEP substantially affects the plasma potential measurements, especially in the regions of plasma where high electric and magnetic field gradients are present. In this paper, we studied the plasma potential structures in sheath-presheath region using both LHEP and CEP in an unmagnetized dc-filament discharge plasma. Measurements of sheath spatial potential profile using laser heated emissive probe are compared with those obtained using conventional emissive probe.

Noncanonical autophagy: one small step for LC3, one giant leap for immunity.

Noncanonical autophagy is utilized by phagocytes to kill and digest extracellular pathogens. This process is initiated at the cell surface by receptors that recruit elements of the autophagy machinery, like LC3, to the phagosome. Also known as LC3-associated phagocytosis, the intersection of autophagy and phagocytosis was initially described as a pathway that limits the proliferation of engulfed pathogens by expediting phagosome maturation. Emerging evidences suggest that this pathway confers previously unsuspected versatility to the immune response as it regulates functions like the interferon pathway, dead cell clearance, and antigen presentation. Here we review recent advances in understanding the functional consequences of linking the autophagy machinery to phagocytosis in innate immunity.

An NF-κB-independent and Erk1/2-dependent mechanism controls CXCL8/IL-8 responses of airway epithelial cells to cadmium.

Airway epithelial cells in the lung are the first line of defense against pathogens and environmental pollutants. Inhalation of the environmental pollutant cadmium has been linked to the development of lung cancer and chronic obstructive pulmonary disease, which are diseases characterized by chronic inflammation. To address the role of airway epithelial cells in cadmium-induced lung inflammation, we investigated how cadmium regulates secretion of interleukin 8 (IL-8) by airway epithelial cells. We show that exposure of human airway epithelial cells to subtoxic doses of cadmium in vitro promotes a characteristic inflammatory cytokine response consisting of IL-8, but not IL-1ß or tumor necrosis factor-alpha. We also found that intranasal delivery of cadmium increases lung levels of the murine IL-8 homologs macrophage inflammatory protein-2 and keracinocyte-derived chemokine and results in an influx of Gr1+ cells into the lung. We determined that inhibition of the nuclear factor-κB (NF-κB) pathway had no effect on cadmium-induced IL-8 secretion by human airway epithelial cells, suggesting that IL-8 production was mediated through an NF-κB-independent pathway. Mitogen-activated protein kinases (MAPKs) are often involved in proinflammatory signaling. Cadmium could activate the main MAPKs (i.e., p38, JNK, and Erk1/2) in human airway epithelial cells. However, only pharmacological inhibition of Erk1/2 pathway or knockdown of the expression of Erk1 and Erk2 using small interfering RNAs suppressed secretion of IL-8 induced by cadmium. Our findings identify cadmium as a potent activator of the proinflammatory cytokine IL-8 in lung epithelial cells and reveal for the first time the role of an NF-κB-independent but Erk1/2-dependent pathway in cadmium-induced lung inflammation.

LyGDI, a novel SHIP-interacting protein, is a negative regulator of FcγR-mediated phagocytosis.

SHIP and SHIP-2 are inositol phosphatases that regulate FcγR-mediated phagocytosis through catalytic as well as non-catalytic mechanisms. In this study we have used two-dimensional fluorescence difference gel electrophoresis (DIGE) analysis to identify downstream signaling proteins that uniquely associate with SHIP or SHIP-2 upon FcγR clustering in human monocytes. We identified LyGDI as a binding partner of SHIP, associating inducibly with the SHIP/Grb2/Shc complex. Immunodepletion and competition experiments with recombinant SHIP domains revealed that Grb2 and the proline-rich domain of SHIP were necessary for SHIP-LyGDI association. Functional studies in primary human monocytes showed that LyGDI sequesters Rac in the cytosol, preventing it from localizing to the membrane. Consistent with this, suppression of LyGDI expression resulted in significantly enhanced FcγR-mediated phagocytosis.

Thrombospondin-1 contributes to mortality in murine sepsis through effects on innate immunity.

BACKGROUND: Thrombospondin-1 (TSP-1) is involved in many biological processes, including immune and tissue injury response, but its role in sepsis is unknown. Cell surface expression of TSP-1 on platelets is increased in sepsis and could activate the anti-inflammatory cytokine transforming growth factor beta (TGFß1) affecting outcome. Because of these observations we sought to determine the importance of TSP-1 in sepsis. METHODOLOGY/PRINCIPAL FINDINGS: We performed studies on TSP-1 null and wild type (WT) C57BL/6J mice to determine the importance of TSP-1 in sepsis. We utilized the cecal ligation puncture (CLP) and intraperitoneal E. coli injection (i.p. E. coli) models of peritoneal sepsis. Additionally, bone-marrow-derived macrophages (BMMs) were used to determine phagocytic activity. TSP-1-/- animals experienced lower mortality than WT mice after CLP. Tissue and peritoneal lavage TGFß1 levels were unchanged between animals of each genotype. In addition, there is no difference between the levels of major innate cytokines between the two groups of animals. PLF from WT mice contained a greater bacterial load than TSP-1-/- mice after CLP. The survival advantage for TSP-1-/- animals persisted when i.p. E. coli injections were performed. TSP-1-/- BMMs had increased phagocytic capacity compared to WT. CONCLUSIONS: TSP-1 deficiency was protective in two murine models of peritoneal sepsis, independent of TGFß1 activation. Our studies suggest TSP-1 expression is associated with decreased phagocytosis and possibly bacterial clearance, leading to increased peritoneal inflammation and mortality in WT mice. These data support the contention that TSP-1 should be more fully explored in the human condition.