Multiple three-column osteotomies successfully correcting cervicothoracic kyphosis in the setting of ankylosing spondylitis: illustrative case.

BACKGROUND: Ankylosing spondylitis (AS) is an autoimmune spondylarthritis often associated with rigid kyphoscoliosis. The authors describe a surgical approach that employs multilevel three-column osteotomies for the restoration of normal global alignment. OBSERVATIONS: A 48-year-old male with a past medical history of AS presented to the clinic with a stooped-over posture: his chin-brow vertical angle (CBVA) was 58.0°; T1 slope (T1S), 97.8°; thoracic kyphosis (TK; T1-12), 94.2°; proximal TK (T1-5), 50.8°; distal TK (T5-12), 43.5°; and sagittal vertical axis (SVA), 22.6 cm. A two-stage procedure was planned. During stage 1, instrumentation was placed from C5 to T10, followed by a T3 vertebral column resection. During stage 2, bilateral pedicle screws were placed from T11 to the pelvis. An L3 pedicle subtraction osteotomy (PSO) was completed and was followed by a T7 PSO. Postoperatively, the patient had significant postural improvement: CBVA was 29.3°; T1S, 57.8°; TK, 77.3°; proximal TK, 33.5°; distal TK, 43.8°; and SVA, 15 cm. At 6 years postoperatively, the patient continued to do well and was without evidence of construct breakdown. LESSONS: The authors propose that multilevel three-column osteotomies, if optimally located, successfully correct spinal malalignment associated with AS.

A fluorogenic, peptide-based probe for the detection of Cathepsin D in macrophages.

Cathepsin D is a protease that is an effector in the immune response of macrophages, yet to date, only a limited number of probes have been developed for its detection. Herein, we report a water soluble, highly sensitive, pH insensitive fluorescent probe for the detection of Cathepsin D activity that provides a strong OFF/ON signal upon activation and with bright emission at 515 nm. The probe was synthesised using a combination of solid and solution-phase chemistries, with probe optimisation to increase its water solubility and activation kinetics by addition of a long PEG chain (5 kDa) at the C-terminus. A BODIPY fluorophore allowed detection of Cathepsin D across a wide pH range, important as the protease is active both at the low pH found in lysosomes and also in higher pH phagolysosomes, and in the cytosol. The probe was successfully used to detect Cathepsin D activity in macrophages challenged by exposure to bacteria.

NRF2 Activation Reprograms Defects in Oxidative Metabolism to Restore Macrophage Function in Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is a disease characterized by persistent airway inflammation and disordered macrophage function. The extent to which alterations in macrophage bioenergetics contribute to impaired antioxidant responses and disease pathogenesis has yet to be fully delineated. Objectives: Through the study of COPD alveolar macrophages (AMs) and peripheral monocyte-derived macrophages (MDMs), we sought to establish if intrinsic defects in core metabolic processes drive macrophage dysfunction and redox imbalance. Methods: AMs and MDMs from donors with COPD and healthy donors underwent functional, metabolic, and transcriptional profiling. Measurements and Main Results: We observed that AMs and MDMs from donors with COPD display a critical depletion in glycolytic- and mitochondrial respiration-derived energy reserves and an overreliance on glycolysis as a source for ATP, resulting in reduced energy status. Defects in oxidative metabolism extend to an impaired redox balance associated with defective expression of the NADPH-generating enzyme, ME1 (malic enzyme 1), a known target of the antioxidant transcription factor NRF2 (nuclear factor erythroid 2-related factor 2). Consequently, selective activation of NRF2 resets the COPD transcriptome, resulting in increased generation of TCA cycle intermediaries, improved energetic status, favorable redox balance, and recovery of macrophage function. Conclusions: In COPD, an inherent loss of metabolic plasticity leads to metabolic exhaustion and reduced redox capacity, which can be rescued by activation of the NRF2 pathway. Targeting these defects, via NRF2 augmentation, may therefore present an attractive therapeutic strategy for the treatment of the aberrant airway inflammation described in COPD.

A mechanistic evaluation of human beta defensin 2 mediated protection of human skin barrier in vitro.

The human skin barrier, a biological imperative, is impaired in inflammatory skin diseases such as atopic dermatitis (AD). Staphylococcus aureus is associated with AD lesions and contributes to pathological inflammation and further barrier impairment. S. aureus secretes extracellular proteases, such as V8 (or 'SspA'), which cleave extracellular proteins to reduce skin barrier. Previous studies demonstrated that the host defence peptide human beta-defensin 2 (HBD2) prevented V8-mediated damage. Here, the mechanism of HBD2-mediated barrier protection in vitro is examined. Application of exogenous HBD2 provided protection against V8, irrespective of timeline of application or native peptide folding, raising the prospect of simple peptide analogues as therapeutics. HBD2 treatment, in context of V8-mediated damage, modulated the proteomic/secretomic profiles of HaCaT cells, altering levels of specific extracellular matrix proteins, potentially recovering V8 damage. However, HBD2 alone did not substantially modulate cellular proteomic/secretomics profiles in the absence of damage, suggesting possible therapeutic targeting of lesion damage sites only. HBD2 did not show any direct protease inhibition or induce expression of known antiproteases, did not alter keratinocyte migration or proliferation, or form protective nanonet structures. These data validate the barrier-protective properties of HBD2 in vitro and establish key protein datasets for further targeted mechanistic analyses.

Severe kyphoscoliosis correction utilizing sublaminar bands in the setting of a vertebral column resection: a unique technical report.

PURPOSE: Severe kyphoscoliosis produces combined coronal and sagittal imbalance, a challenging presentation of Adult Spinal Deformity (ASD). Vertebral column resection (VCR) provides three-dimensional correction typically reserved for severe and/or fixed deformities. The challenge of a VCR is to stabilize the spinal column during incremental reduction maneuvers to correct the deformity. We describe novel use of sub-laminar bands with a temporary pivoting rod construct to achieve coronal and sagittal correction in one reductive maneuver. METHODS: All available notes, imaging, and reports were summarized for inclusion. RESULTS: A 67-year-old male presented severe, progressive thoracolumbar kyphoscoliosis, subsequent back pain, and difficulty in ambulating. Given the progression, surgical intervention was undertaken. The patient underwent an L3 VCR followed by instrumentation from T2 to pelvis. Kyphoscoliosis correction was performed via a single reduction maneuver using novel pivoting-rod construct and sub-laminar bands followed by quad-rod construct placement. Fresh, frozen, femoral head allograft, and BMP were utilized. The patient was placed in a Jewett brace for 6 weeks postoperatively and recovered. His condition remains optimized at three-year follow-up. CONCLUSION: Correcting three-dimensional deformities can require vertebral column resection and pose risk during correction. We propose a sub-laminar band with a pivot-rod construct as a safe, effective technique, limiting the number of reduction maneuvers.

Change in somatosensory evoked potentials detect an acute arterial stent occlusion in lower extremity during spinal surgery: Case report.

The authors report a case in which an acute thrombosis of a pre-existing arterial stent occurs in a patient's lower extremity during a lumbar spinal fusion surgery. The event was detected by acute changes in somatosensory evoked potentials (SSEPs) which were being monitored during the procedure. The neurophysiology technologist reported a 10 % increased latency and 50 % loss of amplitude in the left posterior tibial nerve recordings. While still in the operating room, further investigation, including doppler and arteriogram, demonstrated a complete occlusion of one of the two contiguous stents within the superficial femoral artery (SFA). A vascular surgeon was then able to emergently perform trans-arterial thrombectomy and restore flow through the extremity while still in the operating room. The observed events demonstrate the ability of SSEP monitoring to potentially detect arterial occlusion early, allowing for a rapid diagnosis and expedient treatment, in this case immediate, thus avoiding significant limb threatening morbidity.

The antimicrobial peptide cathelicidin drives development of experimental autoimmune encephalomyelitis in mice by affecting Th17 differentiation.

Multiple sclerosis (MS) is a highly prevalent demyelinating autoimmune condition; the mechanisms regulating its severity and progression are unclear. The IL-17-producing Th17 subset of T cells has been widely implicated in MS and in the mouse model, experimental autoimmune encephalomyelitis (EAE). However, the differentiation and regulation of Th17 cells during EAE remain incompletely understood. Although evidence is mounting that the antimicrobial peptide cathelicidin profoundly affects early T cell differentiation, no studies have looked at its role in longer-term T cell responses. Now, we report that cathelicidin drives severe EAE disease. It is released from neutrophils, microglia, and endothelial cells throughout disease; its interaction with T cells potentiates Th17 differentiation in lymph nodes and Th17 to exTh17 plasticity and IFN-γ production in the spinal cord. As a consequence, mice lacking cathelicidin are protected from severe EAE. In addition, we show that cathelicidin is produced by the same cell types in the active brain lesions in human MS disease. We propose that cathelicidin exposure results in highly activated, cytokine-producing T cells, which drive autoimmunity; this is a mechanism through which neutrophils amplify inflammation in the central nervous system.

Galectin-3 inhibitor GB0139 protects against acute lung injury by inhibiting neutrophil recruitment and activation.

Rationale: Galectin-3 (Gal-3) drives fibrosis during chronic lung injury, however, its role in acute lung injury (ALI) remains unknown. Effective pharmacological therapies available for ALI are limited; identifying novel concepts in treatment is essential. GB0139 is a Gal-3 inhibitor currently under clinical investigation for the treatment of idiopathic pulmonary fibrosis. We investigate the role of Gal-3 in ALI and evaluate whether its inhibition with GB0139 offers a protective role. The effect of GB0139 on ALI was explored in vivo and in vitro. Methods: The pharmacokinetic profile of intra-tracheal (i.t.) GB0139 was investigated in C57BL/6 mice to support the daily dosing regimen. GB0139 (1-30 µg) was then assessed following acute i.t. lipopolysaccharide (LPS) and bleomycin administration. Histology, broncho-alveolar lavage fluid (BALf) analysis, and flow cytometric analysis of lung digests and BALf were performed. The impact of GB0139 on cell activation and apoptosis was determined in vitro using neutrophils and THP-1, A549 and Jurkat E6 cell lines. Results: GB0139 decreased inflammation severity via a reduction in neutrophil and macrophage recruitment and neutrophil activation. GB0139 reduced LPS-mediated increases in interleukin (IL)-6, tumor necrosis factor alpha (TNFα) and macrophage inflammatory protein-1-alpha. In vitro, GB0139 inhibited Gal-3-induced neutrophil activation, monocyte IL-8 secretion, T cell apoptosis and the upregulation of pro-inflammatory genes encoding for IL-8, TNFα, IL-6 in alveolar epithelial cells in response to mechanical stretch. Conclusion: These data indicate that Gal-3 adopts a pro-inflammatory role following the early stages of lung injury and supports the development of GB0139, as a potential treatment approach in ALI.

Inhibition of Cyclin-Dependent Kinase 9 Downregulates Cytokine Production Without Detrimentally Affecting Human Monocyte-Derived Macrophage Viability.

Cyclin-dependent kinase (CDK) inhibitor drugs (CDKi), such as R-roscovitine and AT7519, induce neutrophil apoptosis in vitro and enhance the resolution of inflammation in a number of in vivo models. This class of compounds are potential novel therapeutic agents that could promote the resolution of acute and chronic inflammatory conditions where neutrophil activation contributes to tissue damage and aberrant tissue repair. In this study we investigated CDKi effects on macrophage pro-inflammatory mediator production and viability. Treatment of human monocyte-derived macrophages (MDMs) with the CDKi AT7519 and R-roscovitine at concentrations that induce neutrophil apoptosis had no significant effect on control or LPS-activated MDM apoptosis and viability, and did not detrimentally affect MDM efferocytosis of apoptotic cells. In addition, enhanced efferocytosis, induced by the glucocorticoid dexamethasone, was also unaffected after a short time treatment with R-roscovitine. Macrophage cytokine responses to inflammatory stimuli are also of importance during inflammation and resolution. As a key target of CDKi, CDK9, is involved in protein transcription via the RNA polymerase II complex, we investigated the effect of CDKi drugs on cytokine production. Our data show that treatment with AT7519 significantly downregulated expression and release of key MDM cytokines IL-6, TNF, IL-10 and IL-1ß, as well as markers of pro-inflammatory macrophage polarisation. R-Roscovitine was also able to downregulate inflammatory cytokine protein secretion from MDMs. Using siRNA transfection, we demonstrate that genetic knock-down of CDK9 replicates these findings, reducing expression and release of pro-inflammatory cytokines. Furthermore, overexpression of CDK9 in THP-1 cells can promote a pro-inflammatory phenotype in these cells, suggesting that CDK9 plays an important role in the inflammatory phenotype of macrophages. Overall, this study demonstrates that pharmacological and genetic targeting of CDK9 inhibits an inflammatory phenotype in human MDMs. As such these data indicate that CDK9 may be key to therapeutically targeting pro-inflammatory macrophage functions during chronic inflammation.

Optical motion capture accuracy is task-dependent in assessing wrist motion.

Optical motion capture (OMC) systems are commonly used to capture in-vivo three-dimensional joint kinematics. However, the skin-based markers may not reflect the underlying bone movement, a source of error known as soft tissue artifact (STA). This study examined STA during wrist motion by evaluating the agreement between OMC and biplanar videoradiography (BVR). Nine subjects completed 7 different wrist motion tasks: doorknob rotation to capture supination and pronation, radial-ulnar deviation, flexion-extension, circumduction, hammering, and pitcher pouring. BVR and OMC captured the motion simultaneously. Wrist kinematics were quantified using helical motion parameters of rotation and translation, and Bland-Altman analysis quantified the mean difference (bias) and 95% limit of agreement (LOA). The rotational bias of doorknob pronation, a median bias of -4.9°, was significantly larger than the flexion-extension (0.7°, p < 0.05) and radial-ulnar deviation (1.8°, p < 0.01) tasks. The rotational LOA range was significantly smaller in the flexion-extension task (5.9°) compared to pitcher (11.6°, p < 0.05) and doorknob pronation (17.9°, p < 0.05) tasks. The translation bias did not differ between tasks. The translation LOA range was significantly larger in circumduction (9.8°) compared to the radial-ulnar deviation (6.3°, p < 0.05) and pitcher (3.4°, p < 0.05) tasks. While OMC technology has a wide-range of successful applications, we demonstrated it has relatively poor agreement with BVR in tracking wrist motion, and that the agreement depends on the nature and direction of wrist motion.

Accuracy of an electrogoniometer relative to optical motion tracking for quantifying wrist range of motion.

Methods for capturing wrist range of motion (RoM) vary in complexity, cost, and sensitivity. Measures by manual goniometer, though an inexpensive modality, provide neither dynamic nor objective motion data. Conversely, optical motion capture systems are widely used in three-dimensional scientific motion capture studies but are complex and expensive. The electrogoniometer bridges the gap between portability and objective measurement. Our study aims to evaluate the accuracy of a 2 degree of freedom electrogoniometer using optical motion capture as the reference for in vivo wrist motion. First, a mechanical system constructed from two plastic pipes and a universal joint mimicked a human wrist to assess the inherent accuracy of the electrogoniometer. Simulations of radial/ulnar deviation (R/U), flexion/extension (F/E) and circumduction were evaluated. Second, six subjects performed three RoM tasks of R/U deviation, F/E, and circumduction for evaluation of the in vivo accuracy. Bland-Altman analysis quantified the accuracy. The mechanical experiment reported greater accuracy than the in vivo study with mean difference values less than ±1°. The in vivo accuracy varied across RoM tasks, with mean differences greatest in the F/E task (7.2°). Smaller mean differences values were reported in the R/U deviation task (-0.8°) and the circumduction task (1.2°).

Dynamic Thermal Mapping of Localized Therapeutic Hypothermia in the Brain.

Although whole body cooling is used widely to provide therapeutic hypothermia for the brain, there are undesirable clinical side effects. Selective brain cooling may allow for rapid and controllable neuroprotection while mitigating these undesirable side effects. We evaluated an innovative cerebrospinal fluid (CSF) cooling platform that utilizes chilled saline pumped through surgically implanted intraventricular catheters to induce hypothermia. Magnetic resonance thermal imaging of the healthy sheep brain (n = 4) at 7.0T provided dynamic temperature measurements from the whole brain. Global brain temperature was 38.5 ± 0.8°C at baseline (body temperature of 39.2 ± 0.4°C), and decreased by 3.1 ± 0.3°C over ∼30 min of cooling (p < 0.0001). Significant cooling was achieved in all defined regions across both the ipsilateral and contralateral hemispheres relative to catheter placement. On cooling cessation, global brain temperature increased by 3.1 ± 0.2°C over ∼20 min (p < 0.0001). Rapid and synchronized temperature fall/rise on cooling onset/offset was observed reproducibly with rates ranging from 0.06-0.21°C/min, where rewarming was faster than cooling (p < 0.0001) signifying the importance of thermoregulation in the brain. Although core regions (including the subcortex, midbrain, olfactory tract, temporal lobe, occipital lobe, and parahippocampal cortex) had slightly warmer (∼0.2°C) baseline temperatures, after cooling, temperatures reached the same level as the non-core regions (35.6 ± 0.2°C), indicating the cooling effectiveness of the CSF-based cooling device. In summary, CSF-based intraventricular cooling reliably reduces temperature in all identified brain regions to levels known to be neuroprotective, while maintaining overall systemic normothermia. Dynamic thermal mapping provides high spatiotemporal temperature measurements that can aid in optimizing selective neuroprotective protocols.

Exposure to the antimicrobial peptide LL-37 produces dendritic cells optimized for immunotherapy.

Immunization of patients with autologous, ex vivo matured dendritic cell (DC) preparations, in order to prime antitumor T-cell responses, is the focus of intense research. Despite progress and approval of clinical approaches, significant enhancement of these personalized immunotherapies is urgently needed to improve efficacy. We show that immunotherapeutic murine and human DC, generated in the presence of the antimicrobial host defense peptide LL-37, have dramatically enhanced expansion and differentiation of cells with key features of the critical CD103+/CD141+ DC subsets, including enhanced cross-presentation and co-stimulatory capacity, and upregulation of CCR7 with improved migratory capacity. These LL-37-DC enhanced proliferation, activation and cytokine production by CD8+ (but not CD4+) T cells in vitro and in vivo. Critically, tumor antigen-presenting LL-37-DC increased migration of primed, activated CD8+ T cells into established squamous cell carcinomas in mice, and resulted in tumor regression. This advance therefore has the potential to dramatically enhance DC immunotherapy protocols.

Cathelicidins and the Onset of Labour.

Preterm birth, defined as delivery before 37 weeks of gestation, is the leading cause of neonatal mortality and morbidity. Infection and inflammation are frequent antecedents of spontaneous preterm birth. Cathelicidin, an antimicrobial host defence peptide, is induced by infection and inflammation and although expressed in the reproductive tract and fetal tissues, its role in the pathogenesis of spontaneous preterm birth is unknown. Here we demonstrate that cathelicidin expression is increased at RNA and protein level in the mouse uterus in a model of inflammation-induced labour, where ultrasound guided intrauterine injection of lipopolysaccharide (LPS) at E17 stimulates preterm delivery within 24 hours. Cathelicidin-deficient (Camp-/-) mice are less susceptible to preterm delivery than wild type mice following intrauterine injection of 1 µg of LPS, and this is accompanied by a decrease in circulating IL-6, an inflammatory mediator implicated in the onset of labour. We also show that the proportion of cathelicidin expressing cells in the myometrium is higher in samples obtained from women in labour at term than pre-labour. Together, these data suggest that cathelicidin has roles in mediating pro-inflammatory responses in a murine model of inflammation-induced labour, and in human term labour.

Cathelicidin is a "fire alarm", generating protective NLRP3-dependent airway epithelial cell inflammatory responses during infection with Pseudomonas aeruginosa.

Pulmonary infections are a major global cause of morbidity, exacerbated by an increasing threat from antibiotic-resistant pathogens. In this context, therapeutic interventions aimed at protectively modulating host responses, to enhance defence against infection, take on ever greater significance. Pseudomonas aeruginosa is an important multidrug-resistant, opportunistic respiratory pathogen, the clearance of which can be enhanced in vivo by the innate immune modulatory properties of antimicrobial host defence peptides from the cathelicidin family, including human LL-37. Initially described primarily as bactericidal agents, cathelicidins are now recognised as multifunctional antimicrobial immunomodulators, modifying host responses to pathogens, but the key mechanisms involved in these protective functions are not yet defined. We demonstrate that P. aeruginosa infection of airway epithelial cells promotes extensive infected cell internalisation of LL-37, in a manner that is dependent upon epithelial cell interaction with live bacteria, but does not require bacterial Type 3 Secretion System (T3SS). Internalised LL-37 acts as a second signal to induce inflammasome activation in airway epithelial cells, which, in contrast to myeloid cells, are relatively unresponsive to P. aeruginosa. We demonstrate that this is mechanistically dependent upon cathepsin B release, and NLRP3-dependent activation of caspase 1. These result in LL-37-mediated release of IL-1ß and IL-18 in a manner that is synergistic with P. aeruginosa infection, and can induce caspase 1-dependent death of infected epithelial cells, and promote neutrophil chemotaxis. We propose that cathelicidin can therefore act as a second signal, required by P. aeruginosa infected epithelial cells to promote an inflammasome-mediated altruistic cell death of infection-compromised epithelial cells and act as a "fire alarm" to enhance rapid escalation of protective inflammatory responses to an uncontrolled infection. Understanding this novel modulatory role for cathelicidins, has the potential to inform development of novel therapeutic strategies to antibiotic-resistant pathogens, harnessing innate immunity as a complementation or alternative to current interventions.

An Orally Active Galectin-3 Antagonist Inhibits Lung Adenocarcinoma Growth and Augments Response to PD-L1 Blockade.

A combination therapy approach is required to improve tumor immune infiltration and patient response to immune checkpoint inhibitors that target negative regulatory receptors. Galectin-3 is a ß-galactoside-binding lectin that is highly expressed within the tumor microenvironment of aggressive cancers and whose expression correlates with poor survival particularly in patients with non-small cell lung cancer (NSCLC). To examine the role of galectin-3 inhibition in NSCLC, we tested the effects of galectin-3 depletion using genetic and pharmacologic approaches on syngeneic mouse lung adenocarcinoma and human lung adenocarcinoma xenografts. Galectin-3-/- mice developed significantly smaller and fewer tumors and metastases than syngeneic C57/Bl6 wild-type mice. Macrophage ablation retarded tumor growth, whereas reconstitution with galectin-3-positive bone marrow restored tumor growth in galectin-3-/- mice, indicating that macrophages were a major driver of the antitumor response. Oral administration of a novel small molecule galectin-3 inhibitor GB1107 reduced human and mouse lung adenocarcinoma growth and blocked metastasis in the syngeneic model. Treatment with GB1107 increased tumor M1 macrophage polarization and CD8+ T-cell infiltration. Moreover, GB1107 potentiated the effects of a PD-L1 immune checkpoint inhibitor to increase expression of cytotoxic (IFNγ, granzyme B, perforin-1, Fas ligand) and apoptotic (cleaved caspase-3) effector molecules. In summary, galectin-3 is an important regulator of lung adenocarcinoma progression. The novel galectin-3 inhibitor presented could provide an effective, nontoxic monotherapy or be used in combination with immune checkpoint inhibitors to boost immune infiltration and responses in lung adenocarcinoma and potentially other aggressive cancers. SIGNIFICANCE: A novel and orally active galectin-3 antagonist inhibits lung adenocarcinoma growth and metastasis and augments response to PD-L1 blockade.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/7/1480/F1.large.jpg.

Blood Neutrophils Are Reprogrammed in Bronchiectasis.

RATIONALE: Excessive neutrophilic airway inflammation is the central feature of bronchiectasis, but little is known about neutrophils in bronchiectasis. OBJECTIVES: To assess blood neutrophil phenotype in patients with bronchiectasis while stable and during exacerbations. METHODS: In the clinically stable arm of this study, there were eight healthy volunteers, eight patients with mild bronchiectasis, and eight patients with severe bronchiectasis. In addition, six patients with severe bronchiectasis were compared with six patients with community-acquired pneumonia at the start and end of an exacerbation. We assessed neutrophils for spontaneous apoptosis, cell surface marker expression, degranulation, reactive oxygen species generation, phagocytosis, and killing of Pseudomonas aeruginosa (PAO1). In addition, blood neutrophil function was compared with airway neutrophil function in bronchiectasis. MEASUREMENTS AND MAIN RESULTS: In stable bronchiectasis, compared with healthy volunteers, blood neutrophils had significantly prolonged viability, delayed apoptosis, increased CD62L shedding, upregulated CD11b expression, increased myeloperoxidase release, and impaired neutrophil phagocytosis and killing of PAO1. Bronchiectatic airway neutrophils had significantly lower bacterial phagocytosis and killing than their matched autologous blood neutrophils. Both blood and airway neutrophil phagocytosis and killing were impaired at the start of an exacerbation and improved following antibiotic treatment. In pneumonia, there was a significant improvement in phagocytosis and killing after treatment with antibiotics. During infections, there was no difference in phagocytosis, but there was significantly increased bacterial killing at the start and end of infection in pneumonia compared with bronchiectasis exacerbations. CONCLUSIONS: In bronchiectasis stable state, peripheral blood neutrophils are reprogrammed and have prolonged survival. This impairs their functional ability of bacterial phagocytosis and killing, thereby perpetuating the vicious circle in bronchiectasis.

Microbial Host Interactions and Impaired Wound Healing in Mice and Humans: Defining a Role for BD14 and NOD2.

Chronic wounds cause significant patient morbidity and mortality. A key factor in their etiology is microbial infection, yet skin host-microbiota interactions during wound repair remain poorly understood. Microbiome profiles of noninfected human chronic wounds are associated with subsequent healing outcome. Furthermore, poor clinical healing outcome was associated with increased local expression of the pattern recognition receptor NOD2. To investigate NOD2 function in the context of cutaneous healing, we treated mice with the NOD2 ligand muramyl dipeptide and analyzed wound repair parameters and expression of antimicrobial peptides. Muramyl dipeptide treatment of littermate controls significantly delayed wound repair associated with reduced re-epithelialization, heightened inflammation, and up-regulation of murine ß-defensins 1, 3, and particularly 14. We postulated that although murine ß-defensin 14 might affect local skin microbial communities, it may further affect other healing parameters. Indeed, exogenously administered murine ß-defensin 14 directly delayed mouse primary keratinocyte scratch wound closure in vitro. To further explore the role of murine ß-defensin 14 in wound repair, we used Defb14-/- mice and showed they had a global delay in healing in vivo, associated with alterations in wound microbiota. Taken together, these studies suggest a key role for NOD2-mediated regulation of local skin microbiota, which in turn affects chronic wound etiology.

IL-1ß-Induced Protection of Keratinocytes against Staphylococcus aureus-Secreted Proteases Is Mediated by Human ß-Defensin 2.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease that results in significant morbidity. A hallmark of AD is disruption of the critical barrier function of upper epidermal layers, causatively linked to environmental stimuli, genetics, and infection, and a critical current target for the development of new therapeutic and prophylactic interventions. Staphylococcus aureus is an AD-associated pathogen producing virulence factors that induce skin barrier disruption in vivo and contribute to AD pathogenesis. We show, using immortalized and primary keratinocytes, that S. aureus protease SspA/V8 is the dominant secreted factor (in laboratory and AD clinical strains of S. aureus) inducing barrier integrity impairment and tight junction damage. V8-induced integrity damage was inhibited by an IL-1ß-mediated mechanism, independent of effects on claudin-1. Induction of keratinocyte expression of the antimicrobial/host defense peptide human ß-defensin 2 (hBD2) was found to be the mechanism underpinning this protective effect. Endogenous hBD2 expression was required and sufficient for protection against V8 protease-mediated integrity damage, and exogenous application of hBD2 was protective. This modulatory property of hBD2, unrelated to antibacterial effects, gives new significance to the defective induction of hBD2 in the barrier-defective skin lesions of AD and indicates therapeutic potential.