Toll-like receptor 2 orchestrates a tumor suppressor response in non-small cell lung cancer.

Targeting early-stage lung cancer is vital to improve survival. However, the mechanisms and components of the early tumor suppressor response in lung cancer are not well understood. In this report, we study the role of Toll-like receptor 2 (TLR2), a regulator of oncogene-induced senescence, which is a key tumor suppressor response in premalignancy. Using human lung cancer samples and genetically engineered mouse models, we show that TLR2 is active early in lung tumorigenesis, where it correlates with improved survival and clinical regression. Mechanistically, TLR2 impairs early lung cancer progression via activation of cell intrinsic cell cycle arrest pathways and the proinflammatory senescence-associated secretory phenotype (SASP). The SASP regulates non-cell autonomous anti-tumor responses, such as immune surveillance of premalignant cells, and we observe impaired myeloid cell recruitment to lung tumors after Tlr2 loss. Last, we show that administration of a TLR2 agonist reduces lung tumor growth, highlighting TLR2 as a possible therapeutic target.

Intrapulmonary Autoantibodies to HSP72 Are Associated with Improved Outcomes in IPF.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic interstitial lung disease, with high mortality. Currently, the aetiology and the pathology of IPF are poorly understood, with both innate and adaptive responses previously being implicated in the disease pathogenesis. Heat shock proteins (Hsp) and antibodies to Hsp in patients with IPF have been suggested as therapeutic targets and prognostic biomarkers, respectively. We aimed to study the relationship between the expression of Hsp72 and anti-Hsp72 antibodies in the BAL fluid and serum Aw disease progression in patients with IPF. METHODS: A novel indirect ELISA to measure anti-Hsp72 IgG was developed and together with commercially available ELISAs used to detect Hsp72 IgG, Hsp72 IgGAM, and Hsp72 antigen, in the serum and BALf of a cohort of IPF (n = 107) and other interstitial lung disease (ILD) patients (n = 66). Immunohistochemistry was used to detect Hsp72 in lung tissue. The cytokine expression from monocyte-derived macrophages was measured by ELISA. RESULTS: Anti-Hsp72 IgG was detectable in the serum and BALf of IPF (n = 107) and other ILDs (n = 66). Total immunoglobulin concentrations in the BALf showed an excessive adaptive response in IPF compared to other ILDs and healthy controls (p = 0.026). Immunohistochemistry detection of C4d and Hsp72 showed that these antibodies may be targeting high expressing Hsp72 type II alveolar epithelial cells. However, detection of anti-Hsp72 antibodies in the BALf revealed that increasing concentrations were associated with improved patient survival (adjusted HR 0.62, 95% CI 0.45-0.85; p = 0.003). In vitro experiments demonstrate that anti-Hsp72 complexes stimulate macrophages to secrete CXCL8 and CCL18. CONCLUSION: Our results indicate that intrapulmonary anti-Hsp72 antibodies are associated with improved outcomes in IPF. These may represent natural autoantibodies, and anti-Hsp72 IgM and IgA may provide a beneficial role in disease pathogenesis, though the mechanism of action for this has yet to be determined.

Ovine pulmonary adenocarcinoma: a large animal model for human lung cancer.

Lung cancer is the leading cause of cancer deaths worldwide. Recent progress in understanding the molecular pathogenesis of this disease has resulted in novel therapeutic strategies targeting specific groups of patients. Further studies are required to provide additional advances in diagnosis and treatment. Animal models are valuable tools for studying oncogenesis in lung cancer, particularly during the early stages of disease where tissues are rarely available from human cases. Mice have traditionally been used for studying lung cancer in vivo, and a variety of spontaneous and transgenic models are available. However, it is recognized that other species may also be informative for studies of cancer. Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring lung cancer of sheep caused by retrovirus infection and has several features in common with adenocarcinoma of humans, including a similar histological appearance and activation of common cell signaling pathways. Additionally, the size and organization of human lungs are much closer to those of sheep lungs than to those of mice, which facilitates experimental approaches in sheep that are not available in mice. Thus OPA presents opportunities for studying lung tumor development that can complement conventional murine models. Here we describe the potential applications of OPA as a model for human lung adenocarcinoma with an emphasis on the various in vivo and in vitro experimental systems available.

A randomized controlled trial of peripheral blood mononuclear cell depletion in experimental human lung inflammation.

RATIONALE: Depletion of monocytes reduces LPS-induced lung inflammation in mice, suggesting monocytes as potential therapeutic targets in acute lung injury. OBJECTIVES: To investigate whether depletion of circulating blood monocytes has beneficial effects on markers of systemic and pulmonary inflammation in a human model of acute lung inflammation. METHODS: A total of 30 healthy volunteers were enrolled in a randomized controlled trial. Volunteers inhaled LPS at baseline, and were randomized to receive active mononuclear cell depletion by leukapheresis, or sham leukapheresis, in a double-blind fashion (15 volunteers per group). Serial blood counts were measured, bronchoalveolar lavage (BAL) was performed at 9 hours, and [(18)F]fluorodeoxyglucose positron emission tomography at 24 hours. The primary endpoint was the increment in circulating neutrophils at 8 hours. MEASUREMENTS AND MAIN RESULTS: As expected, inhalation of LPS induced neutrophilia and an up-regulation of inflammatory mediators in the blood and lungs of all volunteers. There was no significant difference between the depletion and sham groups in the mean increment in blood neutrophil count at 8 hours (6.16 × 10(9)/L and 6.15 × 10(9)/L, respectively; P = 1.00). Furthermore, there were no significant differences in BAL neutrophils or protein, positron emission tomography-derived measures of global lung inflammation, or cytokine levels in plasma or BAL supernatant between the study groups. No serious adverse events occurred, and no symptoms were significantly different between the groups. CONCLUSIONS: These findings do not support a role for circulating human monocytes in the early recruitment of neutrophils during LPS-mediated acute lung inflammation in humans.

Histologic assessment of tumor-associated CD45(+) cell numbers is an independent predictor of prognosis in small cell lung cancer.

BACKGROUND: Small cell lung carcinoma (SCLC) continues to have a poor prognosis, with a 2-year survival of < 20%. Studies have suggested that SCLC may affect the immune system to allow it to evade immunologic responses. We hypothesized that any such effect would be characterized by a decrease in the lymphoid cells associated with the tumor in biopsy specimens and that this might relate to patient outcome. METHODS: Sixty-four SCLC biopsy specimens were immunohistochemically stained with anti-CD45 antibody to identify immune cells associated with the tumor. A mean CD45 count per high-power field for each case was obtained, and the results were correlated with age, sex, stage, performance status (PS), treatment with chemotherapy/radiotherapy, and overall survival. RESULTS: The median CD45 count for all cases was taken as 40 (CD45(40)). Kaplan-Meier plots demonstrated better survival for patients with a CD45(40) > 40 ( P < .009). No relationship between CD45 40 and age, sex, stage, or treatment by chemotherapy or radiotherapy was identified. Although PS was a significant predictor of survival ( P = .014), it did not correlate with CD45 40. In patients with better Eastern Cooperative Oncology Group PS (≤ 2), the CD45(40) demonstrated a highly significant survival advantage for those with CD45(40) > 40 ( P < .0001). CONCLUSIONS: The data indicate that (1) simple immunohistochemical assessment of immune cell infiltrates in routinely processed and stained biopsy specimens of primary tumors can provide prognostic information in SCLC and (2) tumor-associated CD45(+) cells in SCLC biopsy specimens may be a good clinical marker to identify patients with poor prognosis despite good PS.

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.

The role of macrophages in healing the wounded lung.

Acute tissue injury is often considered in the context of a wound. The host response to wounding is an orchestrated series of events, the fundamentals of which are preserved across all multicellular organisms. In the human lung, there are a myriad of causes of injury, but only a limited number of consequences: complete resolution, persistent and/or overwhelming inflammation, a combination of resolution/remodelling with fibrosis or progressive fibrosis. In all cases where complete resolution does not occur, there is the potential for significant ongoing morbidity and ultimately death through respiratory failure. In this review, we consider the elements of injury, resolution and repair as they occur in the lung. We specifically focus on the role of the macrophage, long considered to have a pivotal role in regulating the host response to injury and tissue repair.

Progressive severe lung injury by zinc oxide nanoparticles; the role of Zn2+ dissolution inside lysosomes.

BACKGROUND: Large production volumes of zinc oxide nanoparticles (ZnONP) might be anticipated to pose risks, of accidental inhalation in occupational and even in consumer settings. Herein, we further investigated the pathological changes induced by ZnONP and their possible mechanism of action. METHODS: Two doses of ZnONP (50 and 150 cm2/rat) were intratracheally instilled into the lungs of rats with assessments made at 24 h, 1 wk, and 4 wks after instillation to evaluate dose- and time-course responses. Assessments included bronchoalveolar lavage (BAL) fluid analysis, histological analysis, transmission electron microscopy, and IgE and IgA measurement in the serum and BAL fluid. To evaluate the mechanism, alternative ZnONP, ZnONP-free bronchoalveolar lavage exudate, and dissolved Zn2+ (92.5 µg/rat) were also instilled to rats. Acridine orange staining was utilized in macrophages in culture to evaluate the lysosomal membrane destabilization by NP. RESULTS: ZnONP induced eosinophilia, proliferation of airway epithelial cells, goblet cell hyperplasia, and pulmonary fibrosis. Bronchocentric interstitial pulmonary fibrosis at the chronic phase was associated with increased myofibroblast accumulation and transforming growth factor-ß positivity. Serum IgE levels were up-regulated by ZnONP along with the eosinophilia whilst serum IgA levels were down-regulated by ZnONP. ZnONP are rapidly dissolved under acidic conditions (pH 4.5) whilst they remained intact around neutrality (pH 7.4). The instillation of dissolved Zn2+ into rat lungs showed similar pathologies (eg., eosinophilia, bronchocentric interstitial fibrosis) as were elicited by ZnONP. Lysosomal stability was decreased and cell death resulted following treatment of macrophages with ZnONP in vitro. CONCLUSIONS: We hypothesise that rapid, pH-dependent dissolution of ZnONP inside of phagosomes is the main cause of ZnONP-induced diverse progressive severe lung injuries.

Routinely obtained diagnostic material as a source of RNA for personalized medicine in lung cancer patients.

INTRODUCTION: Therapeutic approaches to lung cancer are evolving, with personalized therapy, based on "molecular analysis" of tumors being developed. Given that approximately 90% of patients will not undergo surgery for their disease, an ability to apply these tests to small samples obtained at the time of initial pathological diagnosis is desirable. Studies in this area have produced variable results, and the minimum area of tumor tissue required for analysis has not been defined. Furthermore, such assays have not been widely applied to cytology specimens, which may be the only source of diagnostic material in many cases. METHODS: Routinely processed biopsy and cytology specimens were microdissected to enrich for tumor cells, followed by RNA extraction using QIAGEN RNeasy kit and cDNA synthesis/reverse-transcriptase polymerase chain reaction for genes including beta-actin, ERCC-1, and RRM-1, according to standard laboratory protocols. Paired biopsy and resection specimens were similarly analyzed. RESULTS: As little as 1 mm² of tumor tissue, from a 10-µm-thick section, may be used to produce RNA suitable for analysis. RNA of adequate quality and quantity for analysis may be obtained from residual, routinely processed biopsy and cytology specimens. There is good correlation between the result obtained on the tumor biopsy specimen and paired blocks from the surgical resection with respect to clinical decision making. CONCLUSION: Routinely processed clinical diagnostic samples provide a suitable source of RNA for polymerase chain reaction-based molecular analyses, potentially providing personalized medicine to all lung cancer patients.

Oxidative damage and TGF-ß differentially induce lung epithelial cell sonic hedgehog and tenascin-C expression: implications for the regulation of lung remodelling in idiopathic interstitial lung disease.

Idiopathic interstitial lung diseases (iILDs) are characterized by inflammation, hyperplasia of Type-II alveolar epithelial cells (AECs) and lung remodelling often with progressive fibrosis. It remains unclear which signals initiate iILD and/or maintain the disease processes. Using real-time RT-PCR and immunohistochemistry on archival biopsies of three patterns of iILD (usual interstitial pneumonitis/UIP, non-specific interstitial pneumonitis/NSIP and cryptogenic organizing pneumonia/COP) we investigated whether hedgehog signalling (previously associated with lung damage and repair) was functional and whether the damage associated extracellular matrix protein tenascin-C was present in activated Type-II AECs in all three iILDs. Using tissue culture, protein and mRNA detection we also determined how two signals (oxidative damage and TGF-ß) associated with iILD pathogenesis affected Sonic hedgehog (SHH) and tenascin-C production by a Type-II AEC cell line. We report that SHH pathway and tenascin-C mRNA and proteins were found in UIP, NSIP and COP. SHH signalling was most active at sites of immature organizing fibrous tissue (fibroblastic foci) in UIP. In vitro Type-II AECs constitutively secrete SHH but not tenascin-C. Oxidative injury stimulated SHH release whereas TGF-ß inhibited it. TGF-ß and oxidative damage both upregulated tenascin-C mRNA but only TGF-ß induced synthesis and release of a distinct protein isoform. SHH signalling is active in Type-II AECs from three types of ILD and all three express tenascin-C.

Absence of markers of betaretrovirus infection in human pulmonary adenocarcinoma.

A proportion of human pulmonary adenocarcinomas has been shown previously to express an antigen related to the Gag protein of a betaretrovirus, Jaagsiekte sheep retrovirus, that causes ovine pulmonary adenocarcinoma. To investigate further the hypothesis that a retrovirus might be present in human lung adenocarcinoma, we examined specimens from patients with lung cancer for evidence of retroviral infection by immunohistochemistry, reverse transcriptase-polymerase chain reaction, immunoblotting and cDNA library screening. Thirty-eight percent of the tumor samples analyzed were positive by immunohistochemistry for Gag-related antigen of Jaagsiekte sheep retrovirus. However, this antigen was not detected by immunoblotting using the same antiserum. In addition, plasma samples from the patients did not contain antibodies reacting with Gag proteins from Jaagsiekte sheep retrovirus or other betaretroviruses on immunoblots. Reverse transcriptase-polymerase chain reaction identified the expression of endogenous betaretroviruses in tumor tissue and in normal lung tissue, but no specific provirus was associated with tumor. Expression library screening did not identify the Gag-reactive antigen. This study has confirmed the expression of a Jaagsiekte sheep retrovirus Gag-related antigen in some human lung tumors but additional evidence of betaretroviral infection was not obtained. While these data do not rule out a role for a retrovirus in human pulmonary adenocarcinomas, they suggest that, if such a virus is present, it is unrelated to known betaretroviruses.

The challenge of classifying poorly differentiated tumours in the lung.

The lung is a common site for primary carcinomas as well as a wide range of metastatic carcinomas and other rarer primary and secondary tumours that clinically and radiologically may mimic lung cancer. The World Health Organization classification of lung tumours is based on the morphological appearances in resected specimens. This provides a widely applicable system for classification, which, although complex, can be applied universally. The vast majority of patients with lung cancer, however, do not have their tumours resected, and the application of the same classification system to small biopsy specimens is problematic due in large part to sampling. Recognition of this has led to the widespread use of the term 'non-small cell carcinoma'. Future developments in our understanding of lung cancer and the development of novel targeted therapies may mean that in the future classification will become based more on the molecular features of tumours that predict response to therapies and prognosis rather than simple morphology. The predictive validity of such an approach using small biopsy or cytology specimens, however, still requires to be established. The problem of tumour heterogeneity, which is problematic for morphological classification, may also pose similar challenges for molecular approaches.

Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study.

Carbon nanotubes have distinctive characteristics, but their needle-like fibre shape has been compared to asbestos, raising concerns that widespread use of carbon nanotubes may lead to mesothelioma, cancer of the lining of the lungs caused by exposure to asbestos. Here we show that exposing the mesothelial lining of the body cavity of mice, as a surrogate for the mesothelial lining of the chest cavity, to long multiwalled carbon nanotubes results in asbestos-like, length-dependent, pathogenic behaviour. This includes inflammation and the formation of lesions known as granulomas. This is of considerable importance, because research and business communities continue to invest heavily in carbon nanotubes for a wide range of products under the assumption that they are no more hazardous than graphite. Our results suggest the need for further research and great caution before introducing such products into the market if long-term harm is to be avoided.

Mutationally activated K-ras 4A and 4B both mediate lung carcinogenesis.

To examine the roles of endogenous K-ras 4A and K-ras 4B splice variants in tumorigenesis, murine lung carcinogenesis was induced by N-methyl-N-nitrosourea (MNU), which causes a K-ras mutation (G12D) that jointly affects both isoforms. Compared with age-matched K-ras(tmDelta4A/-) mice (where tumours can express mutationally activated K-ras 4B only), tumour number and size were significantly higher in K-ras(+/-) mice (where tumours can also express mutationally activated K-ras 4A), and significantly lower in K-ras(tmDelta4A/tmDelta4A) mice (where tumours can express both wild-type and activated K-ras 4B). MNU induced significantly more, and larger, tumours in wild-type than K-ras(tmDelta4A/tmDelta4A) mice which differ in that only tumours in wild-type mice can express wild-type and activated K-ras 4A. Lung tumours in all genotypes were predominantly papillary adenomas, and tumours from K-ras(+/-) and K-ras(tmDelta4A/-) mice exhibited phospho-Erk1/2 and phospho-Akt staining. Hence (1) mutationally activated K-ras 4B is sufficient to activate the Raf/MEK/ERK(MAPK) and PI3-K/Akt pathways, and initiate lung tumorigenesis, (2) when expressed with activated K-ras 4B, mutationally activated K-ras 4A further promotes lung tumour formation and growth (both in the presence and absence of its wild-type isoform) but does not affect either tumour pathology or progression, and (3) wild-type K-ras 4B, either directly or indirectly, reduces tumour number and size.

Acute myocarditis after massive phenelzine overdose.

BACKGROUND: Monoamine oxidase inhibitors (MAOIs) are uncommonly used due to their high frequency of adverse effects, including tachycardia and hypertension. Recently, there has been renewed interest in the role of this class of drugs in treating a variety of psychiatric disorders. The clinical features of MAOI overdose are poorly characterised. This paper describes a novel cardiac complication of phenelzine toxicity in a previously healthy young adult with no history of cardiovascular disease. METHODS: A 23-year-old woman presented to hospital after massive phenelzine overdose, and the clinical features and pathological findings are discussed in light of existing literature. RESULTS: Clinical features of phenelzine toxicity included reduced consciousness level, seizures, and tachycardia, in keeping with previous reports. Unexpectedly, the patient developed severe and unexplained hypotension and impaired left ventricular function, and died 3 days after initial presentation. Post-mortem examination confirmed high serum phenelzine concentrations (4.1 mg/L) and histopathological features that were consistent with drug-induced acute myocarditis. CONCLUSION: Acute myocarditis was attributed to phenelzine in the absence of any plausible alternative explanation. This possible complication should be considered in patients who develop unexplained hypotension after phenelzine overdose.

Hypersensitivity pneumonitis associated with leflunomide therapy.

Disease progression in rheumatoid arthritis is controlled with disease modifying drugs, many of which have toxic side effects. Pulmonary side effects are common and this has resulted in the development of newer medications with less pulmonary toxicity. We observed that even these newer drugs can be associated with potentially very serious pulmonary toxicity, with hypersensitivity pneumonitis developing after the initiation of leflunomide therapy. We urge caution in the use of leflunomide in patients with pulmonary side effects from other drugs or who have underlying pulmonary disease.

Inflammation-associated remodelling and fibrosis in the lung - a process and an end point.

Fibrosis by common usage in the pathological and clinical literature is the end result of a healing process and synonymous with scarring. We would argue that its use to describe a dynamic series of events which may be reversible is unhelpful and that the term 'lung remodelling' is a better description for this process as it reflects changes in tissue organization that may or may not progress to 'fibrosis' as a final fixed point. Resolution, through reversal of active lung remodelling, by therapeutic intervention is possible providing the alveolar architecture remains intact. If the lung architecture is lost then healing by permanent fibrosis with loss of organ function is inevitable.

Predicting non-small cell lung cancer expression of epidermal growth factor receptor and matrix metalloproteinase 9 from immunohistochemical staining of diagnostic biopsy samples.

Epidermal growth factor receptor (EGFR) and matrix metalloproteinase 9 (MMP9) expression in resected non-small cell lung cancer (NSCLC) has been associated with a poor prognosis. Specific inhibitors have been developed to these molecules and have entered into clinical practice. We performed immunohistochemical staining on a series of 36 resected cases of NSCLC in parallel with the associated preoperative diagnostic biopsies in order to determine whether expression of these markers in the tumour could reliably be predicted from the result obtained with the small diagnostic biopsy. The results demonstrated considerable intratumour heterogeneity of expression for both markers in the resected tumours, and this was associated with a poor negative predictive value for the result obtained with the diagnostic biopsy. We conclude that, staining small diagnostic biopsies for EGFR and MMP9 is unlikely to be helpful in defining tumour status for these markers and allowing targeted therapy.