Kevin Dhaliwal

Surface-enhanced Raman scattering in cancer detection and imaging.

Technologies that use surface-enhanced Raman scattering (SERS) have experienced significant growth in biomedical research during the past 4 years. In this review we summarize the progress in SERS for cancer diagnostics, including multiplexed detection and identification of new biomarkers, single-nucleotide polymorphisms, and circulating tumor cells. SERS is also used as a non-invasive tool for cancer imaging with immunoSERS microscopy, histological analysis of biopsies, and in vivo detection of tumors. We discuss the future of SERS probes compatible with multiple imaging modalities and their potential for clinical translation (e.g., endoscope-based and intraoperative imaging as tools for surgical guidance). Moreover, we highlight the potential of SERS agents for targeted drug delivery and photothermal therapy.

Ly6Chi Monocytes Direct Alternatively Activated Profibrotic Macrophage Regulation of Lung Fibrosis

RATIONALE Idiopathic pulmonary fibrosis (IPF) is a devastating disease. Antiinflammatory therapies, including corticosteroids, are of no benefit. The role of monocytes and macrophages is therefore controversial. OBJECTIVES To define the role of monocytes and macrophages during lung fibrogenesis and resolution, and explore the phenotype of the cells involved. METHODS We used multiple in vivo depletional strategies, backed up by adoptive transfer techniques. Further studies were performed on samples from patients with IPF. MEASUREMENTS AND MAIN RESULTS Depletion of lung macrophages during fibrogenesis reduced pulmonary fibrosis as measured by lung collagen (P = 0.0079); fibrosis score (P = 0.0051); and quantitative polymerase chain reaction for surrogate markers of fibrosis Col1 (P = 0.0083) and a-smooth muscle actin (P = 0.0349). There was an associated reduction in markers of the profibrotic alternative macrophage activation phenotype, Ym1 (P = 0.0179), and Arginase 1. The alternative macrophage marker CD163 was expressed on lung macrophages from patients with IPF. Depletion of Ly6Chi circulating monocytes reduced pulmonary fibrosis (P = 0.0052) and the number of Ym1- positive alternatively activated lung macrophages (P = 0.0310). Their adoptive transfer during fibrogenesis exacerbated fibrosis (P = 0.0304); however, adoptively transferred CD45.1 Ly6Chi cells were not found in the lungs of recipient CD45.2 mice. CONCLUSIONS We demonstrate the importance of circulating monocytes and lung macrophages during pulmonary fibrosis, and emphasize the importance of the alternatively activated macrophage phenotype. We show that Ly6Chi monocytes facilitate the progression of pulmonary fibrosis, but are not obviously engrafted into lungs thereafter. Finally, we provide empirical data to suggest that macrophages may have a resolution-promoting role during the reversible phase of bleomycin-induced pulmonary fibrosis.

Galectin-3 reduces the severity of pneumococcal pneumonia by augmenting neutrophil function

The Gram-positive Streptococcus pneumoniae is the leading cause of community-acquired pneumonia worldwide, resulting in high mortality. Our in vivo studies show that galectin-3(-/-) mice develop more severe pneumonia after infection with S. pneumoniae, as demonstrated by increased bacteremia and lung damage compared to wild-type mice and that galectin-3 reduces the severity of pneumococcal pneumonia in part by augmenting neutrophil function. Specifically, we show that 1) galectin-3 directly acts as a neutrophil-activating agent and potentiates the effect of fMLP, 2) exogenous galectin-3 augments neutrophil phagocytosis of bacteria and delays neutrophil apoptosis, 3) phagocytosis of apoptotic neutrophils by galectin-3(-/-) macrophages is less efficient compared to wild type, and 4) galectin-3 demonstrates bacteriostatic properties against S. pneumoniae in vitro. Furthermore, ad-back of recombinant galectin-3 in vivo protects galectin-3-deficient mice from developing severe pneumonia. Together, these results demonstrate that galectin-3 is a key molecule in the host defense against pneumococcal infection. Therapeutic strategies designed to augment galectin-3 activity may both enhance inflammatory cell function (by directly affecting neutrophil responsiveness and prolonging neutrophil longevity) and have direct bacteriostatic activity, improving clinical outcomes after severe pneumococcal infection.

Macrophage/monocyte depletion by clodronate, but not diphtheria toxin, improves renal ischemia/reperfusion injury in mice

The role of resident renal mononuclear phagocytes in acute kidney injury is controversial with experimental data suggesting both deleterious and protective functions. To help resolve this, we used mice transgenic for the human diphtheria toxin receptor under the control of the CD11b promoter and treated them with diphtheria toxin, or liposomal clodronate, or both to deplete monocyte/mononuclear phagocytes prior to renal ischemia/reperfusion injury. Although either system effectively depleted circulating monocytes and resident mononuclear phagocytes, depletion was most marked in diphtheria toxin-treated mice. Despite this, diphtheria toxin treatment did not protect from renal ischemia. In contrast, mice treated with clodronate exhibited reduced renal failure and acute tubular necrosis, suggesting key differences between these depletion strategies. Clodronate did not deplete CD206-positive renal macrophages and, unlike diphtheria toxin, left resident CD11c-positive cells unscathed while inducing dramatic apoptosis in hepatic and splenic mononuclear phagocyte populations. Abolition of the protected phenotype by administration of diphtheria toxin to clodronate-treated mice suggested that the protective effect of clodronate resulted from the presence of a cytoprotective intrarenal population of mononuclear phagocytes sensitive to diphtheria toxin-mediated ablation.

Monocytes Control Second-Phase Neutrophil Emigration in Established Lipopolysaccharide-induced Murine Lung Injury

RATIONALE Acute lung injury (ALI) is an important cause of morbidity and mortality, with no currently effective pharmacological therapies. Neutrophils have been specifically implicated in the pathogenesis of ALI, and there has been significant research into the mechanisms of early neutrophil recruitment, but those controlling the later phases of neutrophil emigration that characterize disease are poorly understood. OBJECTIVES To determine the influence of peripheral blood monocytes (PBMs) in established ALI. METHODS In a murine model of LPS-induced ALI, three separate models of conditional monocyte ablation were used: systemic liposomal clodronate (sLC), inducible depletion using CD11b diphtheria toxin receptor (CD11b DTR) transgenic mice, and antibody-dependent ablation of CCR2(hi) monocytes. MEASUREMENTS AND MAIN RESULTS PBMs play a critical role in regulating neutrophil emigration in established murine LPS-induced lung injury. Gr1(hi) and Gr1(lo) PBM subpopulations contribute to this process. PBM depletion is associated with a significant reduction in measures of lung injury. The specificity of PBM depletion was demonstrated by replenishment studies in which the effects were reversed by systemic PBM infusion but not by systemic or local pulmonary infusion of mature macrophages or lymphocytes. CONCLUSIONS These results suggest that PBMs, or the mechanisms by which they influence pulmonary neutrophil emigration, could represent therapeutic targets in established ALI.

C5a Mediates Peripheral Blood Neutrophil Dysfunction in Critically Ill Patients

RATIONALE Critically ill patients are highly susceptible to hospital-acquired infection. Neutrophil function in critical illness remains poorly understood. OBJECTIVES To characterize and define mechanisms of peripheral blood neutrophil (PBN) dysfunction in critically ill patients. To determine whether the inflamed lung contributes additional phagocytic impairment. METHODS Prospective collection of blood and bronchoalveolar lavage fluid from patients with suspected ventilator-associated pneumonia and from age- and sex-matched volunteers; laboratory analysis of neutrophil functions. MEASUREMENTS AND MAIN RESULTS Seventy-two patients and 21 volunteers were included. Phagocytic capacity of PBNs was 36% lower in patients than in volunteers (P < 0.0001). From several biologically plausible candidates only activated complement was significantly associated with impaired PBN phagocytosis (P < 0.0001). Phagocytosis was negatively correlated with serum C3a and positively correlated with expression of C5a receptor type 1 (CD88) on PBNs. C5a recapitulated impaired PBN phagocytosis and significantly down-regulated CD88 expression in vitro. C5a-mediated phagocytic impairment was prevented by blocking either CD88 or phosphoinositide 3-kinase, and completely reversed by granulocyte-macrophage colony-stimulating factor. C5a also impaired killing of Pseudomonas aeruginosa by, and migration of, PBNs, indicating that effects were not restricted to phagocytosis. Bronchoalveolar lavage fluid leukocytes from patients also demonstrated significantly impaired function, and lavage supernatant reduced phagocytosis in healthy neutrophils by 43% (P = 0.0001). However, lavage fluid did not affect CD88 expression and lavage-mediated impairment of phagocytosis was not blocked by anti-CD88 antibody. CONCLUSIONS Critically ill patients have significant dysfunction of PBNs, which is mediated predominantly by activated complement. Further, profound complement-independent neutrophil dysfunction occurs in the inflamed lung.

The role of formylated peptides and formyl peptide receptor 1 in governing neutrophil function during acute inflammation.

Neutrophil migration to sites of inflammation and the subsequent execution of multiple functions are designed to contain and kill invading pathogens. These highly regulated and orchestrated processes are controlled by interactions between numerous receptors and their cognate ligands. Unraveling and identifying those that are central to inflammatory processes may represent novel therapeutic targets for the treatment of neutrophil-dominant inflammatory disorders in which dysregulated neutrophil recruitment, function, and elimination serve to potentiate rather than resolve an initial inflammatory insult. The first G protein-coupled receptor to be described on human neutrophils, formyl peptide receptor 1 (FPR1), is one such receptor that plays a significant role in the execution of these functions through multiple intracellular signaling pathways. Recent work has highlighted important observations with regard to both receptor function and the importance and functional relevance of FPR1 in the pathogenesis of a range of both sterile and infective inflammatory conditions. In this review, we explore the multiple components of neutrophil migration and function in both health and disease, with a focus on the role of FPR1 in these processes. The current understanding of FPR1 structure, function, and signaling is examined, alongside discussion of the potential importance of FPR1 in inflammatory diseases suggesting that FPR1 is a key regulator of the inflammatory environment.

Diagnostic importance of pulmonary interleukin-1β and interleukin-8 in ventilator-associated pneumonia

Background Ventilator-associated pneumonia (VAP) is the most commonly fatal nosocomial infection. Clinical diagnosis of VAP remains notoriously inaccurate. The hypothesis was tested that significantly augmented inflammatory markers distinguish VAP from conditions closely mimicking VAP. Methods A prospective, observational cohort study was carried out in two university hospital intensive care units recruiting 73 patients with clinically suspected VAP, and a semi-urban primary care practice recruiting a reference group of 21 age- and sex-matched volunteers. Growth of pathogens at >104 colony-forming units (cfu)/ml of bronchoalveolar lavage fluid (BALF) distinguished VAP from “non-VAP”. Inflammatory mediators were quantified in BALF and serum. Mediators showing significant differences between patients with and without VAP were analysed for diagnostic utility by receiver operator characteristic (ROC) curves. Results Seventy-two patients had recoverable lavage—24% had VAP. BALF interleukin-1β (IL-1β), IL-8, granulocyte colony-stimulating factor and macrophage inflammatory protein-1α were significantly higher in the VAP group (all p<0.005). Using a cut-off of 10 pg/ml, BALF IL-1β generated negative likelihood ratios for VAP of 0.09. In patients with BALF IL-1β <10 pg/ml the post-test probability of VAP was 2.8%. Using a cut-off value for IL-8 of 2 ng/ml, the positive likelihood ratio was 5.03. There was no difference in cytokine levels between patients with sterile BALF and those with growth of <104 cfu/ml. Conclusions BALF IL-1β and IL-8 are amongst the strongest markers yet identified for accurately demarcating VAP within the larger population of patients with suspected VAP. These findings have potential implications for reduction in unnecessary antibiotic use but require further validation in larger populations.

Imaging inflammation: Molecular strategies to visualize key components of the inflammatory cascade, from initiation to resolution

Dysregulation of inflammation is central to the pathogenesis of innumerable human diseases. Understanding and tracking the critical events in inflammation are crucial for disease monitoring and pharmacological drug discovery and development. Recent progress in molecular imaging has provided novel insights into spatial associations, molecular events and temporal sequelae in the inflammatory process. While remaining a burgeoning field in pre-clinical research, increasing application in man affords researchers the opportunity to study disease pathogenesis in humans in situ thereby revolutionizing conventional understanding of pathophysiology and potential therapeutic targets. This review provides a description of commonly used molecular imaging modalities, including optical, radionuclide and magnetic resonance imaging, and details key advances and translational opportunities in imaging inflammation from initiation to resolution.

Highly specific, multi-branched fluorescent reporters for analysis of human neutrophil elastase

Human neutrophil elastase (HNE) is a serine protease implicated in the pathogenesis of acute and chronic inflammatory disease. Here a series of, internally quenched, single fluorophore fluorescent reporters were synthesised that allowed the rapid, highly specific and sensitive analysis of HNE activity over closely related proteases.