Cytotoxic lymphocytes rapidly respond and destroy both malignant cells and cells infected with intracellular pathogens. One mechanism, known as granule exocytosis, employs the secretory granules of these lymphocytes. These include the pore-forming protein perforin (pfp) and a family of serine proteases known as granzymes that cleave and activate effector molecules within the target cell. Over the past two decades, the study of granzymes has largely focused on the ability of these serine proteases to induce cell death. More recently, sophisticated mouse models of disease coupled with gene-targeted mice have allowed investigators to ask why granzyme subfamilies are encoded on different chromosomal loci and what broader role these enzymes might play in inflammation and immune response. Here, we provide a brief overview of the granzyme superfamily, their relationship to pfp, and their reported functions in apoptosis. This overview is followed by a comprehensive analysis of the less characterized and developing field regarding the non-apoptotic functions of granzymes.

Histone deacetylase inhibitors (HDACi) and agents such as recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL receptor (TRAIL-R) antibodies are anticancer agents that have shown promise in preclinical settings and in early phase clinical trials as monotherapies. Although HDACi and activators of the TRAIL pathway have different molecular targets and mechanisms of action, they share the ability to induce tumor cell-selective apoptosis. The ability of HDACi to induce expression of TRAIL-R death receptors 4 and 5 (DR4/DR5), and induce tumor cell death via the intrinsic apoptotic pathway provides a molecular rationale to combine these agents with activators of the TRAIL pathway that activate the alternative (death receptor) apoptotic pathway. Herein, we demonstrate that the HDACi vorinostat synergizes with the mouse DR5-specific monoclonal antibody MD5-1 to induce rapid and robust tumor cell apoptosis in vitro and in vivo. Importantly, using a preclinical mouse breast cancer model, we show that the combination of vorinostat and MD5-1 is safe and induces regression of established tumors, whereas single agent treatment had little or no effect. Functional analyses revealed that rather than mediating enhanced tumor cell apoptosis via the simultaneous activation of the intrinsic and extrinsic apoptotic pathways, vorinostat augmented MD5-1-induced apoptosis concomitant with down-regulation of the intracellular apoptosis inhibitor cellular-FLIP (c-FLIP). These data demonstrate that combination therapies involving HDACi and activators of the TRAIL pathway can be efficacious for the treatment of cancer in experimental mouse models.

The ␤ delayed deuteron decay of 6 He has been measured at the TISOL facility at TRIUMF. For the ␤-particle measurement a silicon-germanium telescope array has been used. To separate deuterons from ␣ particles and to obtain the spectral form of the deuteron decay, a d-␣ coincidence technique has been employed using two opposite silicon detectors. The ␤ branch has been determined from the geometry of the experiment and also by comparison with the well-known ␤-␣ decay modes of 16 N and 8 Li. A branching ratio of (1.8 Ϯ0.9)ϫ10 Ϫ6 above the ␤ cutoff ͑350-keV deuteron laboratory energy͒ and (2.6Ϯ1.3)ϫ10 Ϫ6 for the entire spectrum has been determined for this decay. In the course of the measurement the half-life of 6 He has been remeasured to be T 1/2 ϭ0.810Ϯ0.008 s. High statistical accuracy in the deuteron spectrum has been obtained that allowed a comparison of the shape of the deuteron spectrum to theoretical predictions.

Material

Cigarette smoking has reached epidemic proportions within many regions of the world and remains the highest risk factor for chronic obstructive pulmonary disease (COPD) and lung cancer. Squamous cell lung cancer is commonly detected in heavy smokers, where the risk of developing lung cancer is not solely defined by tobacco consumption. Although therapies that target common driver mutations in adenocarcinomas are showing some promise, they are proving ineffective in smoking-related squamous cell lung cancer. Since COPD is characterized by an excessive inflammatory and oxidative stress response, this review details how aberrant innate, adaptive and systemic inflammatory processes can contribute to lung cancer susceptibility in COPD. Activated leukocytes release increasing levels of proteases and free radicals as COPD progresses and tertiary lymphoid aggregates accumulate with increasing severity. Reactive oxygen species promote formation of reactive carbonyls that are not only tumourigenic through initiating DNA damage, but can directly alter the function of regulatory proteins involved in host immunity and tumour suppressor functions. Systemic inflammation is also markedly increased during infective exacerbations in COPD and the interplay between tumour-promoting serum amyloid A (SAA) and IL-17A is discussed. SAA is also an endogenous allosteric modifier of FPR2 expressed on immune and epithelial cells, and the therapeutic potential of targeting this receptor is proposed as a novel strategy for COPD-lung cancer overlap. AbbreviationsCOPD, chronic obstructive pulmonary disease; FGFR1, fibroblast growth factor-1; FPR2, formyl peptide receptor 2; LOH, loss of heterozygosity; LXA4, lipoxinA4; NSCLC, non-small cell lung cancer; NFE2L2, nuclear factor, erythroid 2-like 2; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha; PTEN, phosphatase and tensin homologue; SAA, serum amyloid A; SCC, squamous cell cancer; RvD1, resolvinD1

Rationale: Neutrophilic inflammation is an important pathologic feature of chronic obstructive pulmonary disease (COPD) and infectious exacerbations of COPD. Serum amyloid A (SAA) promotes neutrophilic inflammation by its interaction with lung mucosal ALX/ FPR2 receptors. However, little is known about how this endogenous mediator regulates IL-17A immunity. Objectives: To determine whether SAA causes neutrophilic inflammation by IL-17A-dependent mechanisms. Methods: The relationship between SAA and neutrophils was investigated in lung sections from patients with COPD and a chronic mouse model of SAA exposure. A neutralizing antibody to IL-17A was used to block SAA responses in vivo, and a cell-sorting strategy was used to identify cellular sources. Measurements and Main Results: SAA mRNA expression was positively associated with tissue neutrophils in COPD (P , 0.05). SAA predominately promoted expression of the T H 17 polarizing cytokine IL-6, which was opposed by 15-epi-lipoxin A 4 , a counter-regulatory mediator, and ALX/FPR2 ligand. SAA-induced inflammation was markedly reduced by a neutralizing antibody to IL-17A in vivo. Chronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and severe asthma, are characterized by an exaggerated inflammatory profile involving accumulation of neutrophils with disease progression (1). Neutrophilic inflammation increases with COPD severity despite escalating use of glucocorticosteroids (2, 3), which contributes to excessive proteinase release and host tissue damage (4). Respiratory infections also trigger acute exacerbations of COPD (AECOPD), where airway neutrophilia increases with severity (5). AECOPDs have a major impact because they lead to impaired health-related quality of life (6) and a more rapid decline in lung function (7). We have previously shown that circulating serum amyloid A (SAA) levels acutely rise during AECOPD, where its levels were predictive of event severity (8). Furthermore, we have demonstrated elevated SAA immunoreactivity in the submucosa of COPD lung sections in close proximity to the basal epithelium and show a positive correlation between secreted SAA and the neutrophil activation marker, neutrophil elastase (9). SAA promotes expression of inflammatory mediators and neutrophil chemotaxis and survival by the ALX-FPR2 receptor in a manner that is opposed by the endogenous proresolving lipid mediator lipoxin A 4 (LXA 4 ) (9-12).SAA is also a potent endogenous ligand that stimulates expression of T H 17 polarizing mediators (13,14), and influences in vitro T H 17 differentiation of CD4 1 T cells (15). IL-17A promotes inflammation by coordinating granulopoiesis and neutrophil mobilization through its regulation of leukocyte growth factors and cytokines. IL-17A is particularly central to lung immunity because innate host defenses to respiratory pathogens are compromised in mice lacking this cytokine or its receptor (IL-17RA), leading to reduced neutrophil recruitment and increased bacterial burden (16,17). An increase in IL-17A 1 im...

Formyl peptide receptor 2/lipoxin A 4 (LXA 4 ) receptor (Fpr2/ALX) co-ordinates the transition from inflammation to resolution during acute infection by binding to distinct ligands including serum amyloid A (SAA) and Resolvin D1 (RvD1). Here, we evaluated the proresolving actions of aspirin-triggered RvD1 (AT-RvD1) in an acute coinfection pneumonia model. Coinfection with Streptococcus pneumoniae and influenza A virus (IAV) markedly increased pneumococcal lung load and neutrophilic inflammation during the resolution phase. Fpr2/ALX transcript levels were increased in the lungs of coinfected mice, and immunohistochemistry identified prominent Fpr2/ALX immunoreactivity in bronchial epithelial cells and macrophages. Levels of circulating and lung SAA were also highly increased in coinfected mice. Therapeutic treatment with exogenous AT-RvD1 during the acute phase of infection (day 4-6 post-pneumococcal inoculation) significantly reduced the pneumococcal load. AT-RvD1 also significantly reduced neutrophil elastase (NE) activity and restored total antimicrobial activity in bronchoalveolar lavage (BAL) fluid (BALF) of coinfected mice. Pneumonia severity, as measured by quantitating parenchymal inflammation or alveolitis was significantly reduced with AT-RvD1 treatment, which also reduced the number of infiltrating lung neutrophils and monocytes/macrophages as assessed by flow cytometry. The reduction in distal lung inflammation in AT-RvD1-treated mice was not associated with a significant reduction in inflammatory and chemokine mediators. In summary, we demonstrate that in the coinfection setting, SAA levels were persistently increased and exogenous AT-RvD1 facilitated more rapid clearance of pneumococci in the lungs, while concurrently reducing the severity of pneumonia by limiting excessive leukocyte chemotaxis from the infected bronchioles to distal areas of the lungs.

Aims: Reactive oxygen species (ROS) are highly reactive molecules generated in different subcellular sites or compartments, including endosomes via the NOX2-containing nicotinamide adenine dinucleotide phosphate oxidase during an immune response and in mitochondria during cellular respiration. However, while endosomal NOX2 oxidase promotes innate inflammation to influenza A virus (IAV) infection, the role of mitochondrial ROS (mtROS) has not been comprehensively investigated in the context of viral infections in vivo. Results: In this study, we show that pharmacological inhibition of mtROS, with intranasal delivery of Mito-TEMPO, resulted in a reduction in airway/lung inflammation, neutrophil infiltration, viral titers, as well as overall morbidity and mortality in mice infected with IAV (Hkx31, H3N2). MitoTEMPO treatment also attenuated apoptotic and necrotic neutrophils and macrophages in airway and lung tissue. At an early phase of influenza infection, that is, day 3 there were significantly lower amounts of IL-1b protein in the airways, but substantially higher amounts of type I IFN-b following MitoTEMPO treatment. Importantly, blocking mtROS did not appear to alter the initiation of an adaptive immune response by lung dendritic cells, nor did it affect lung B and T cell populations that participate in humoral and cellular immunity. Innovation/Conclusion: Influenza virus infection promotes mtROS production, which drives innate immune inflammation and this exacerbates viral pathogenesis. This pathogenic cascade highlights the therapeutic potential of local mtROS antioxidant delivery to alleviate influenza virus pathology. Antioxid. Redox Signal. 32, 929-942.