Perioperative arterial catheterization: A prospective evaluation of ultrasound, infection, and patient-focused outcomes.

BACKGROUND: There is little information regarding complications of arterial catheterization in modern clinical care. We aimed to determine the incidence of abnormal duplex vascular ultrasound and catheter related infections following perioperative arterial catheterization. METHODS: Patients requiring arterial catheterization for elective surgery were included and insertion details collected prospectively. Duplex ultrasound evaluation was performed 24 h after catheter removal. Symptomatic patients were identified by self-reported questionnaire. On Day 7, patients answered questions by telephone, related to the insertion site, pain, and function. Results of catheter tip and blood culture analyses were sought. Univariate associations of patient and surgical characteristics with abnormal ultrasound were assessed with p < 0.05 considered significant. RESULTS: Of 339 catheterizations, 105 (40%) had ultrasound evaluation. Catheters were indwelling for median (IQR, range) duration of 6.0 h (4.4-8.2, 1.8-28) with no catheter-related infections. There were 16 (15.2%, 95% CI 9.0%-23.6%) abnormal results, including 14 radial artery thromboses, one radial artery dissection, and one radial vein thrombosis. Those with abnormal ultrasound results were more likely to have had Arrow catheters inserted (68.8% vs 27%, p = 0.023) and more than one skin puncture (37.5% vs 26.8%, p = 0.031). Two of the 16 (12.5%) patients with abnormal ultrasound results reported new symptoms related to the hand compared with nine of the 88 (10.2%) with normal results (p = 0.1). No patients required urgent referral for management. CONCLUSIONS: Thrombosis was the most common abnormality and was usually asymptomatic. There were no infections, few post-operative symptoms, and minimal functional impairment following arterial catheterization.

The role of dermis resident macrophages and their interaction with neutrophils in the early establishment of Leishmania major infection transmitted by sand fly bite.

There is substantial experimental evidence to indicate that Leishmania infections that are transmitted naturally by the bites of infected sand flies differ in fundamental ways from those initiated by needle inocula. We have used flow cytometry and intravital microscopy (IVM) to reveal the heterogeneity of sand fly transmission sites with respect to the subsets of phagocytes in the skin that harbor L. major within the first hours and days after infection. By flow cytometry analysis, dermis resident macrophages (TRMs) were on average the predominant infected cell type at 1 hr and 24 hr. By confocal IVM, the co-localization of L. major and neutrophils varied depending on the proximity of deposited parasites to the presumed site of vascular damage, defined by the highly localized swarming of neutrophils. Some of the dermal TRMs could be visualized acquiring their infections via transfer from or efferocytosis of parasitized neutrophils, providing direct evidence for the "Trojan Horse" model. The role of neutrophil engulfment by dermal TRMs and the involvement of the Tyro3/Axl/Mertk family of receptor tyrosine kinases in these interactions and in sustaining the anti-inflammatory program of dermal TRMs was supported by the effects observed in neutrophil depleted and in Axl-/-Mertk-/- mice. The Axl-/-Mertk-/- mice also displayed reduced parasite burdens but more severe pathology following L. major infection transmitted by sand fly bite.

Th1-Th2 Cross-Regulation Controls Early Leishmania Infection in the Skin by Modulating the Size of the Permissive Monocytic Host Cell Reservoir.

The impact of T helper (Th) 1 versus Th2 immunity on intracellular infections is attributed to classical versus alternative activation of macrophages leading to resistance or susceptibility. However, observations in multiple infectious settings demonstrate deficiencies in mediators of Th1-Th2 immunity, which have paradoxical or no impact. We report that prior to influencing activation, Th1/Th2 immunity first controls the size of the permissive host cell reservoir. During early Leishmania infection of the skin, IFN-γ- or STAT6-mediated changes in phagocyte activation were counteracted by changes in IFN-γ-mediated recruitment of permissive CCR2+ monocytes. Monocytes were required for early parasite expansion and acquired an alternatively activated phenotype despite the Th1 dermal environment required for their recruitment. Surprisingly, STAT6 did not enhance intracellular parasite proliferation, but rather modulated the size and permissiveness of the monocytic host cell reservoir via regulation of IFN-γ and IL-10. These observations expand our understanding of the Th1-Th2 paradigm during infection.

Investigating Socioeconomic Disparities in the Potential Healthy Eating and Physical Activity Environments of Churches.

Faith-based settings have the potential to improve health in underresourced communities, but little research has quantified and compared health-promoting elements in church environments. This study examines the number of potential indoor and outdoor physical activity opportunities, healthy eating opportunities, healthy living media, and total environmental resources present in churches (n = 54) in a rural, southeastern US county and the relationship between these resources and neighborhood income. In our sample, most churches offered potential indoor and outdoor opportunities for physical activity and healthy eating opportunities, with more variability in the number of healthy living media items on display compared to other environmental components. Common potential opportunities present in churches for physical activity included a fellowship hall and green/open space, while potential opportunities for healthy eating frequently included a refrigerator and sink. Compared to those in medium- and high-income neighborhoods, churches in low-income neighborhoods scored higher on measures of potential outdoor physical activity opportunities and lower on measures of total potential environment resources, healthy eating opportunities, healthy living media, and indoor physical activity opportunities, though only indoor physical activity opportunities reached statistical significance. Potential opportunities for using existing resources in and around churches for health promotion should be investigated further, particularly in rural areas.

Loop-mediated isothermal amplification (LAMP): An advanced molecular point-of-care technique for the detection of Leishmania infection.

Leishmaniasis, caused by protozoan parasites of the Leishmania genus, represents an important health problem in many regions of the world. Lack of effective point-of-care (POC) diagnostic tests applicable in resources-limited endemic areas is a critical barrier to effective treatment and control of leishmaniasis. The development of the loop-mediated isothermal amplification (LAMP) assay has provided a new tool towards the development of a POC diagnostic test based on the amplification of pathogen DNA. LAMP does not require a thermocycler, is relatively inexpensive, and is simple to perform with high amplification sensitivity and specificity. In this review, we discuss the current technical developments, applications, diagnostic performance, challenges, and future of LAMP for molecular diagnosis and surveillance of Leishmania parasites. Studies employing the LAMP assay to diagnose human leishmaniasis have reported sensitivities of 80% to 100% and specificities of 94% to 100%. These observations suggest that LAMP offers a good molecular POC technique for the diagnosis of leishmaniasis and is also readily applicable to screening at-risk populations and vector sand flies for Leishmania infection in endemic areas.

ER-stress mobilization of death-associated protein kinase-1-dependent xenophagy counteracts mitochondria stress-induced epithelial barrier dysfunction.

The gut microbiome contributes to inflammatory bowel disease (IBD), in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD, and dysfunctional epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here, we treated human colonic biopsies, epithelial colonoids, and epithelial cells with an uncoupler of oxidative phosphorylation, dinitrophenol (DNP), with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6, an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis, induction of ER stress (i.e. the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather, specific mobilization of the ATF6 arm of ER stress and recruitment of DAPK1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note, epithelia with a Crohn's disease-susceptibility mutation in the autophagy gene ATG16L1 exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress-ATF6-DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria, thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation.

NOX2-Derived Reactive Oxygen Species Control Inflammation during Leishmania amazonensis Infection by Mediating Infection-Induced Neutrophil Apoptosis.

Reactive oxygen species (ROS) produced by NADPH phagocyte oxidase isoform (NOX2) are critical for the elimination of intracellular pathogens in many infections. Despite their importance, the role of ROS following infection with the eukaryotic pathogen Leishmania has not been fully elucidated. We addressed the role of ROS in C57BL/6 mice following intradermal infection with Leishmania amazonensis. Despite equivalent parasite loads compared with wild-type (WT) mice, mice deficient in ROS production by NOX2 due to the absence of the gp91 subunit (gp91phox-/-) had significantly more severe pathology in the later stages of infection. Pathology in gp91phox-/- mice was not associated with alterations in CD4+ T cell-mediated immunity but was preceded by enhanced neutrophil accumulation at the dermal infection site. Ex vivo analysis of infected versus uninfected neutrophils revealed a deficiency in infection-driven apoptosis in gp91phox-/- mice versus WT mice. gp91phox-/- mice presented with higher percentages of healthy or necrotic neutrophils but lower percentages of apoptotic neutrophils at early and chronic time points. In vitro infection of gp91phox-/- versus WT neutrophils also revealed reduced apoptosis and CD95 expression but increased necrosis in infected cells at 10 h postinfection. Provision of exogenous ROS in the form of H2O2 reversed the necrotic phenotype and restored CD95 expression on infected gp91phox-/- neutrophils. Although ROS production is typically viewed as a proinflammatory event, our observations identify the importance of ROS in mediating appropriate neutrophil apoptosis and the importance of apoptosis in inflammation and pathology during chronic infection.

Evaluation of recombinant Leishmania polyprotein plus glucopyranosyl lipid A stable emulsion vaccines against sand fly-transmitted Leishmania major in C57BL/6 mice.

Numerous experimental Leishmania vaccines have been developed to prevent the visceral and cutaneous forms of Leishmaniasis, which occur after exposure to the bite of an infected sand fly, yet only one is under evaluation in humans. KSAC and L110f, recombinant Leishmania polyproteins delivered in a stable emulsion (SE) with the TLR4 agonists monophosphoryl lipid A or glucopyranosyl lipid A (GLA) have shown protection in animal models. KSAC+GLA-SE protected against cutaneous disease following sand fly transmission of Leishmania major in susceptible BALB/c mice. Similar polyprotein adjuvant combinations are the vaccine candidates most likely to see clinical evaluation. We assessed immunity generated by KSAC or L110f vaccination with GLA-SE following challenge with L. major by needle or infected sand fly bite in resistant C57BL/6 mice. Polyprotein-vaccinated mice had a 60-fold increase in CD4(+)IFN-γ(+) T cell numbers versus control animals at 2 wk post-needle inoculation of L. major, and this correlated with a 100-fold reduction in parasite load. Immunity did not, however, reach levels observed in mice with a healed primary infection. Following challenge by infected sand fly bite, polyprotein-vaccinated animals had comparable parasite loads, greater numbers of neutrophils at the challenge site, and reduced CD4(+)IFN-γ(+)/IL-17(+) ratios versus nonvaccinated controls. In contrast, healed animals had significantly reduced parasite loads and higher CD4(+)IFN-γ(+)/IL-17(+) ratios. These observations demonstrate that vaccine-induced protection against needle challenge does not necessarily translate to protection following challenge by infected sand fly bite.

Direct demonstration of CD4 T cell cooperation in the primary in vivo generation of CD4 effector T cells.

Many observations bear upon the cellular and molecular requirements for CD4 T cell activation. The interaction of CD4 T cells with dendritic cells (DC), central to the induction of most immune responses, is the most studied. However, leukocytes other than DC can dramatically affect the induction and differentiation of CD4 T cells into effector cells. We recently provided indirect evidence that in vivo CD4 T cooperation facilitates the activation of CD4 T cells. Here, we demonstrate that the activation of CD4 T cells, specific for the hen egg lysozyme (HEL)(105) (-120) peptide, is optimally achieved when BALB/c mice are immunized with additional MHC class II-binding HEL peptides in incomplete Freund's adjuvant. This cooperation cannot be mimicked by the coadministration of LPS or of an agonistic antibody to CD40, at the time of immunization. In contrast, OX40-OX40L interactions are necessary for CD4 T cell cooperation in that an OX40 agonistic antibody can replace, and an OX40L-blocking antibody can abrogate, CD4 T cell cooperation in situations where such cooperation would otherwise enhance the activation of CD4 T cells.

CD4 T cell cooperation is required for the in vivo activation of CD4 T cells.

We address here the role of CD4 T cell cooperation in the activation of CD4 T cells. Administration of aggregated hen egg lysozyme (HEL) without microbial adjuvant to BALB/c mice normally generates cytokine-producing CD4 T cells specific for the HEL major peptide, HEL(105-120), as well as CD4 T cells specific for HEL non-major peptides. The prior administration of HEL(105-120) ablates the generation of cytokine-secreting CD4 T cells specific for HEL(105-120), as well as the CD4 T cells specific for HEL non-major peptides, normally generated upon HEL challenge. Thus, the activation of HEL non-major peptide-specific CD4 T cells appears to depend upon the HEL(105-120)-specific CD4 T cell population. In contrast, when HEL(105-120) and saline-treated mice are challenged with HEL coupled to ovalbumin (OVA), CD4 T cell responses to HEL non-major peptides and to OVA are the same, whereas treated mice still do not generate cytokine-secreting cells specific for HEL(105-120). We infer that the administration of HEL(105-120) does not generate regulatory cells capable of down-regulating CD4 T cell responses to HEL and OVA peptides. OVA-specific CD4 T cells restore the generation of HEL non-major peptide-specific T cells in the absence of HEL major peptide-specific T cells. We conclude that the generation of CD4 T cells producing IL-2, IFN-gamma and IL-4 requires CD4 T cell cooperation and that this cooperation is not mediated simply by CD40-CD40L interactions. We also conclude from these observations that there is no requirement for a microbial or danger signal for CD4 T cell activation.

In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies.

Infection with the obligate intracellular protozoan Leishmania is thought to be initiated by direct parasitization of macrophages, but the early events following transmission to the skin by vector sand flies have been difficult to examine directly. Using dynamic intravital microscopy and flow cytometry, we observed a rapid and sustained neutrophilic infiltrate at localized sand fly bite sites. Invading neutrophils efficiently captured Leishmania major (L.m.) parasites early after sand fly transmission or needle inoculation, but phagocytosed L.m. remained viable and infected neutrophils efficiently initiated infection. Furthermore, neutrophil depletion reduced, rather than enhanced, the ability of parasites to establish productive infections. Thus, L.m. appears to have evolved to both evade and exploit the innate host response to sand fly bite in order to establish and promote disease.

Analysis of cytokine-producing Th cells from hen egg lysozyme-immunized mice reveals large numbers specific for "cryptic" peptides and different repertoires among different Th populations.

We employed an optimized ex vivo enzyme-linked immunospot assay for enumerating and defining the peptide specificity of all the hen egg lysozyme (HEL)-specific Th cells producing IL-2, IFN-gamma, or IL-4, in different lymphoid organs of HEL-immunized BALB/c and CBA mice. Previous studies, employing T cell proliferation assays, demonstrated that lymph node cells from BALB/c mice immunized with HEL emulsified in complete Freund's adjuvant (CFA) are specific for HEL(105-120). In contrast, we found that the spleens of BALB/c mice immunized with HEL/CFA, or with heat-aggregated HEL on aluminum hydroxide adjuvant, contain IL-4-producing T cells specific for other HEL peptides, previously characterized as "cryptic", with consistent responses to HEL(11-25). The Th repertoire expressed in different lymphoid organs of the same immunized mouse can be different, as can the repertoire of Th cells producing different cytokines and present in one lymphoid organ. In addition, we found that the repertoire of Th cells generated depends upon the adjuvant employed. Lastly, the summation of responses elicited by a panel of non-overlapping HEL peptides is equal to that elicited by HEL. This high-resolution study thus illustrates that the Th repertoire generated upon HEL immunization depends upon diverse parameters, and that the natural processing of HEL gives rise to more diverse peptides then previously evident from studies employing T cell proliferation assays.