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Background: Pancreatic ductal adenocarcinoma (PDAC) represents a major clinical challenge due to its tumor microenvironment, which exhibits immune-suppressive properties that facilitate cancer progression, metastasis, and therapy resistance. Interleukin 1 (IL-1) signaling has been implicated as a driver in this process. Mechanistically, both IL-1α and IL-1β bind to the IL-1 receptor type 1, forming a complex with IL-1-receptor accessory protein (IL1RAP), which triggers downstream signaling pathways. The IL1RAP blocking antibody nadunolimab is currently in clinical development, but the precise consequences of inhibiting IL-1 signaling in PDAC remains elusive., Methods: To evaluate the biological relevance of blocking IL1RAP using nadunolimab in a PDAC animal model, human PDAC cells and cancer-associated fibroblasts (CAFs) were co-transplanted into mice. To study the underlying mechanisms of IL1RAP blockade ex vivo, co-cultured PDAC cells and CAFs were treated with nadunolimab prior to RNA sequencing. Migration assays were performed to assess how nadunolimab affects interactions between CAFs and myeloid immune cells. Finally, to establish a clinical correlation between IL1RAP expression and nadunolimab treatment effects, we analyzed tumor biopsies from a clinical phase I/II study in which nadunolimab was administered to patients., Results: In the xenograft mouse model, nadunolimab exhibited antitumor effects only when human CAFs were co-transplanted with PDAC cells. IL-1 stimulation induced CAFs to secrete chemokines that recruited neutrophils and monocytes. The secretion of this chemokine and the migration of myeloid cells were inhibited by nadunolimab. Media conditioned by IL-1-stimulated CAFs sustained a neutrophil population with a tissue invasion phenotype, an effect that was reversed by nadunolimab. In a cohort of metastatic late-stage PDAC patients receiving nadunolimab as monotherapy, high IL1RAP expression in tumors was associated with extended progression-free survival., Conclusions: Our study demonstrates that targeting IL1RAP on CAFs inhibits IL-1-induced chemokine secretion and recruitment of neutrophils and monocytes, thereby counteracting the immunosuppressive microenvironment in PDAC. These findings highlight the therapeutic potential of targeting IL1RAP in PDAC., Competing Interests: Competing interests: NH, PS, CG, SM, KvW, CRM, DL, and MJ are current employees of, and/or hold stocks or stock options in Cantargia AB, Lund, Sweden. The use of nadunolimab for cancer treatment is patented within WO 2015/13602., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Identifying biomarkers in fibrotic lung disease is key for early anti-fibrotic intervention. Dynamic contrast-enhanced (DCE) MRI offers valuable perfusion-related insights in fibrosis but adapting human MRI methods to rodents poses challenges. Here, we explored these translational challenges for the inflammatory and fibrotic phase of a bleomycin lung injury model in rats. Eleven male Sprague-Dawley rats received a single intratracheal dose of bleomycin (1000iU), four control rats received saline. Imaging was performed on days 7 and 28 post-induction. Ultra-short echo time imaging was used to image the lung for 7 minutes after which Clariscan was injected intravenously. Lung signal changes were measured for an additional 21 minutes. Images were reconstructed with a sliding-window approach, providing a temporal resolution of 10 seconds per image. After imaging on day 28, animals were euthanized, and lungs were collected for histology. Bleomycin-exposed rats initially exhibited reduced body weight, recovering to control levels after 20 days. Lung volume increased in bleomycin animals from 4.4±0.9 ml in controls to 5.5±0.5 ml and 6.5±1.2 ml on day 7 and 28. DCE-MRI showed no change of initial gradient of relative enhancement in the curves between controls and bleomycin animals on day 7 and 28 post-induction. On day 7, the DCE-MRI washout phase in bleomycin animals had higher signals than the saline group and than observed at a later time point. Lung pixels were binned in 7 enhancement classes. On day 28, the size of low relative enhancement bins almost doubled in volume compared to controls and animals on day 7 post-induction. Histology on day 28 suggests that findings could be explained by changes in lung tissue density due to lung volume increase. Adapting this clinical MRI method to rodents at 9.4T remains a challenge. Future studies may benefit from lower field strength MRI combined with higher temporal resolution DCE-MRI., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 in ‘t Zandt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Drug-induced interstitial lung disease (ILD) is crucial to detect early to achieve the best treatment outcome. Optimally, non-invasive imaging biomarkers can be used for early detection of disease progression and treatment follow-up. Therefore, reliable in vivo models are warranted in new imaging biomarker development to accelerate better-targeted treatment options. Single-dose bleomycin models have, for a long time, served as a reference model in fibrosis and lung injury research. Here, we aimed to use a clinically more relevant animal model by systemic exposure to bleomycin and assessing disease progression over time by combined magnetic resonance imaging (MRI) and positron emission tomography (PET) imaging., Methods: C57BL/6 mice received bleomycin (i.p. 35iU/kg) or saline as control twice per week for 4 weeks. Mice were monitored until 2 weeks after cessation of bleomycin administration (w4 + 1 and w4 + 2), referred to as the resting period. MRI scans were performed in weeks 3 and 4 and during the resting weeks. [ 18 F]FDG-PET was performed at the last week of dosing (w4) and 2 weeks after the last dosing (w4 + 2). Lung tissue sections were stained with Masson's trichrome and evaluated by modified Ashcroft scoring. Lung volume and lesion volumes were assessed using MRI, as well as 3D mapping of the central airways., Results and Discussion: Bleomycin-challenged mice showed increased lung weights ( p  < 0.05), while total lung volume was unchanged (w4 and onward). Histology analysis demonstrated fibrotic lesions emanating from the distal parts of the lung. Fibrosis progression was visualized by MRI with significantly increased high signal in bleomycin-exposed lungs compared to controls ( p  < 0.05). In addition, a significant increase in central airway diameter ( p  < 0.01) was displayed in bleomycin-exposed animals compared to controls and further continued to dilate as the disease progressed, comparing the bleomycin groups over time ( p  < 0.05-0.001). Lung [ 18 F]FDG uptake was significantly elevated in bleomycin-exposed mice compared to controls ( p  < 0.05)., Conclusion: Non-invasive imaging displayed progressing lesions in the lungs of bleomycin-exposed mice, using two distinct MRI sequences and [ 18 F]FDG-PET. With observed fibrosis progression emanating from distal lung areas, dilation of the central airways was evident. Taken together, this chronic bleomycin-exposure model is translationally more relevant for studying lung injury in ILD and particularly in the context of DIILD., Competing Interests: Truly Labs—CRO performing in vivo model development. KW was the CEO of Truly Labs and NF, JL, and CC were employed at Truly Labs at the time when studies were conducted. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mahmutovic Persson, Fransén Petterson, Liu, in 't Zandt, Carvalho, Örbom, Olsson and von Wachenfeldt.)

Objective: Accurate imaging biomarkers that indicate disease progression at an early stage are highly important to enable timely mitigation of symptoms in progressive lung disease. In this context, reproducible experimental models and readouts are key. Here, we aim to show reproducibility of a lung injury rat model by inducing disease and assessing disease progression by multi-modal non-invasive imaging techniques at two different research sites. Furthermore, we evaluated the potential of fibroblast activating protein (FAP) as an imaging biomarker in the early stage of lung fibrosis., Methods: An initial lung injury rat model was set up at one research site (Lund University, Lund, Sweden) and repeated at a second site (Radboudumc, Nijmegen, The Netherlands). To induce lung injury, Sprague-Dawley rats received intratracheal instillation of bleomycin as one single dose (1,000 iU in 200 µL) or saline as control. Thereafter, longitudinal images were acquired to track inflammation in the lungs, at 1 and 2 weeks after the bleomycin challenge by magnetic resonance imaging (MRI) and [ 18 F]FDG-PET. After the final [ 18 F]FDG-PET scan, rats received an intravenous tracer [ 89 Zr]Zr-DFO-28H1 (anti-FAP antibody) and were imaged at day 15 to track fibrogenesis. Upon termination, bronchoalveolar lavage (BAL) was performed to assess cell and protein concentration. Subsequently, the biodistribution of [ 89 Zr]Zr-DFO-28H1 was measured ex vivo and the spatial distribution in lung tissue was studied by autoradiography. Lung sections were stained and fibrosis assessed using the modified Ashcroft score., Results: Bleomycin-challenged rats showed body weight loss and increased numbers of immune cells and protein concentrations after BAL compared with control animals. The initiation and progression of the disease were reproduced at both research sites. Lung lesions in bleomycin-exposed rats were visualized by MRI and confirmed by histology. [ 18 F]FDG uptake was higher in the lungs of bleomycin-challenged rats compared with the controls, similar to that observed in the Lund study. [ 89 Zr]Zr-DFO-28H1 tracer uptake in the lung was increased in bleomycin-challenged rats compared with control rats ( p  = 0.03)., Conclusion: Here, we demonstrate a reproducible lung injury model and monitored disease progression using conventional imaging biomarkers MRI and [ 18 F]FDG-PET. Furthermore, we showed the first proof-of-concept of FAP imaging. This reproducible and robust animal model and imaging experimental set-up allows for future research on new therapeutics or biomarkers in lung disease., Competing Interests: Truly Labs is a CRO performing in vivo model development. KvW is the CEO of Truly Labs and NFP is employed at Truly Labs. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Boswinkel, Raavé, Veltien, Scheenen, Fransén Petterson, in ‘t Zandt, Olsson, von Wachenfeldt, Heskamp and Mahmutovic Persson.)

IL-1α and IL-1β are both involved in several aspects of tumor biology, including tumor initiation, progression, metastasis, and not least in resistance to various therapies. IL-1α can function as an alarmin to signal cellular stress, and acts to induce downstream events, including production of IL-1β, to amplify the signal. Both IL-1α and IL-1β act through the same receptor complex, IL-1R1-IL1RAP, to mediate signal transduction. IL1RAP is expressed on tumor cells and in the tumor microenvironment by for example CAF, macrophages and endothelial cells. The anti-IL1RAP antibody nadunolimab (CAN04) inhibits both IL-1α and IL-1β signaling and induces ADCC of IL1RAP-expressing tumor cells. As both IL-1α and IL-1β mediate chemoresistance, the aim of this study was to explore the potential synergy between nadunolimab and chemotherapy. This was performed using the NSCLC PDX model LU2503 and the syngeneic MC38 model, in addition to in vitro cell line experiments. We show that chemotherapy induces expression and release of IL-1α from tumor cells and production of IL-1β-converting enzyme, ICE, in the tumor stroma. IL-1α is also demonstrated to act on stromal cells to further induce the secretion of IL-1β, an effect disrupted by nadunolimab. Nadunolimab, and its surrogate antibody, synergize with platinum-based as well as non-platinum-based chemotherapy to induce potent anti-tumor effects, while blockade of only IL-1β signaling by anti-IL-1β antibody does not achieve this effect. In conclusion, blockade of IL1RAP with nadunolimab reduces IL-1-induced chemoresistance of tumors., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

For drug-induced interstitial lung disease (DIILD) translational imaging biomarkers are needed to improve detection and management of lung injury and drug-toxicity. Literature was reviewed on animal models in which in vivo imaging was used to detect and assess lung lesions that resembled pathological changes found in DIILD, such as inflammation and fibrosis. A systematic search was carried out using three databases with key words "Animal models", "Imaging", "Lung disease", and "Drugs". A total of 5749 articles were found, and, based on inclusion criteria, 284 papers were selected for final data extraction, resulting in 182 out of the 284 papers, based on eligibility. Twelve different animal species occurred and nine various imaging modalities were used, with two-thirds of the studies being longitudinal. The inducing agents and exposure (dose and duration) differed from non-physiological to clinically relevant doses. The majority of studies reported other biomarkers and/or histological confirmation of the imaging results. Summary of radiotracers and examples of imaging biomarkers were summarized, and the types of animal models and the most used imaging modalities and applications are discussed in this review. Pathologies resembling DIILD, such as inflammation and fibrosis, were described in many papers, but only a few explicitly addressed drug-induced toxicity experiments.

Non-invasive imaging biomarkers (IBs) are warranted to enable improved diagnostics and follow-up monitoring of interstitial lung disease (ILD) including drug-induced ILD (DIILD). Of special interest are IB, which can characterize and differentiate acute inflammation from fibrosis. The aim of the present study was to evaluate a PET-tracer specific for Collagen-I, combined with multi-echo MRI, in a rat model of DIILD. Rats were challenged intratracheally with bleomycin, and subsequently followed by MRI and PET/CT for four weeks. PET imaging demonstrated a significantly increased uptake of the collagen tracer in the lungs of challenged rats compared to controls. This was confirmed by MRI characterization of the lesions as edema or fibrotic tissue. The uptake of tracer did not show complete spatial overlap with the lesions identified by MRI. Instead, the tracer signal appeared at the borderline between lesion and healthy tissue. Histological tissue staining, fibrosis scoring, lysyl oxidase activity measurements, and gene expression markers all confirmed establishing fibrosis over time. In conclusion, the novel PET tracer for Collagen-I combined with multi-echo MRI, were successfully able to monitor fibrotic changes in bleomycin-induced lung injury. The translational approach of using non-invasive imaging techniques show potential also from a clinical perspective.

A large number of systemically administered drugs have the potential to cause drug-induced interstitial lung disease (DIILD). We aim to characterize a model of DIILD in the rat and develop imaging biomarkers (IBs) for detection and quantification of DIILD. In this study, Sprague-Dawley rats received one single dose of intratracheal (i.t.) bleomycin and were longitudinally imaged at day 0, 3, 7, 14, 21, and 28 post dosing, applying the imaging techniques magnetic resonance imaging (MRI) and positron emission tomography (PET)/computed tomography (CT). Bronchoalveolar lavage fluid (BALF) was analyzed for total protein and inflammatory cells. Lungs were saved for further evaluation by gene analysis using quantitative-PCR and by histology. Lung sections were stained with Masson's-Trichrome staining and evaluated by modified Ashcroft score. Gene expression profiling of inflammatory and fibrotic markers was performed on lung tissue homogenates. Bleomycin induced significant increase in total protein concentration and total cell count in bronchoalveolar lavage (BAL), peaking at day 3 ( p > 0.001) and day 7 ( p > 0.001) compared to control, respectively. Lesions measured by MRI and PET signal in the lungs of bleomycin challenged rats were significantly increased during days 3-14, peaking at day 7. Two subgroups of animals were identified as low- and high-responders by their different change in total lung volume. Both groups showed signs of inflammation initially, while at later time points, the low-responder group recovered toward control, and the high-responder group showed sustained lung volume increase, and significant increase of lesion volume ( p < 0.001) compared to control. Lastly, important inflammatory and pro-fibrotic markers were assessed from lung tissue, linking observed imaging pathological changes to gene expression patterns. In conclusion, bleomycin-induced lung injury is an adequate animal model for DIILD studies and for translational lung injury assessment by MRI and PET imaging. The scenario comprised disease responses, with different fractions of inflammation and fibrosis. Thereby, this study improved the understanding of imaging and biological biomarkers in DIILD and lung injury., (Copyright © 2020 Mahmutovic Persson, Falk Håkansson, Örbom, Liu, von Wachenfeldt and Olsson.)

Background: Obesity and metabolic syndrome (MetS) are major risk factors for atherosclerotic diseases; however, a causal link remains elusive. Animal models resembling human MetS and its complications, while important, are scarce. We aimed at developing a porcine model of human MetS., Methods: Forty pigs with familial hypercholesterolemia were fed a high fat + fructose diet for 30 weeks. Metabolic assessments and subcutaneous fat biopsies were obtained at 18 and 30 weeks, and fat distribution was assessed by CT-scans. Postmortem, macrophage density, and phenotype in fat tissues were quantified along with atherosclerotic burden., Results: During the experiment, we observed a >4-fold in body weight, a significant but small increase in fasting glucose (4.1 mmol/L), insulin (3.1 mU/L), triglycerides (0.5 mmol/L), and HDL cholesterol (2.6 mmol/L). Subcutaneous fat correlated with insulin resistance, but intra-abdominal fat correlated inversely with insulin resistance and LDL cholesterol. More inflammatory macrophages were found in visceral versus subcutaneous fat, and inflammation decreased in subcutaneous fat over time., Conclusions: MetS based on human criteria was not achieved. Surprisingly, visceral fat seemed part of a healthier metabolic and inflammatory profile. These results differ from human findings, and further research is needed to understand the relationship between obesity and MetS in porcine models.

Background: Immunization with oxidized LDL (oxLDL) reduces atherosclerosis in rodents. We tested the hypothesis that treatment with a human recombinant monoclonal antibody against oxLDL will reduce the burden or composition of atherosclerotic lesions in hypercholesterolemic minipigs., Methods and Results: Thirty-eight hypercholesterolemic minipigs with defective LDL receptors were injected with an oxLDL antibody or placebo weekly for 12weeks. An 18F-fluorodeoxyglucose positron emission tomography (FDG PET) scan (n=9) was performed before inclusion and after 3months of treatment. Blood samples were obtained prior to each injection. Following the last injection all animals were sacrificed, and the heart, aorta, and iliac arteries were removed. The left anterior descending coronary artery was sectioned at 5mm intervals for quantitative and qualitative assessments of atherosclerosis, including immunohistochemical phenotyping of macrophages using a pan-macrophage marker (CD68) and markers for putative pro-atherogenic (cathepsin S) and atheroprotective (CD163) macrophages. Aorta, right coronary artery, and left iliac artery were stained en face with Sudan IV and the amount of atherosclerosis quantified. There was no effect of treatment on plasma lipid profile, vascular FDG-PET signal or the amount of atherosclerosis in any of the examined arteries. However, immunostaining of coronary lesions revealed reduced cathepsin S positivity in the treated group compared with placebo (4.8% versus 8.2% of intima area, p=0.03) with no difference in CD68 or CD163 positivity., Conclusions: In hypercholesterolemic minipigs, treatment with a human recombinant monoclonal antibody against oxLDL reduced cathepsin S in coronary lesions without any effect on the burden of atherosclerosis or aortic FDG-PET signal., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

The critical role played by IgE in allergic asthma is well-documented and clinically precedented, but some patients in whom IgE neutralization may still offer clinical benefit are excluded from treatment with the existing anti-IgE therapy, omalizumab, due to high total IgE levels or body mass. In this study, we sought to generate a novel high affinity anti-IgE antibody (MEDI4212) with potential to treat a broad severe asthma patient population. Analysis of body mass, total and allergen-specific IgE levels in a cohort of severe asthmatics was used to support the rationale for development of a high affinity IgE-targeted antibody therapeutic. Phage display technology was used to generate a human IgG1 lead antibody, MEDI4212, which was characterized in vitro using binding, signaling and functional assay systems. Protein crystallography was used to determine the details of the interaction between MEDI4212 and IgE. MEDI4212 bound human IgE with an affinity of 1.95 pM and was shown to target critical residues in the IgE Cε3 domain critical for interaction with FcεRI. MEDI4212 potently inhibited responses through FcεRI and also prevented the binding of IgE to CD23. When used ex vivo at identical concentration, MEDI4212 depleted free-IgE from human sera to levels ~1 log lower than omalizumab. Our results thus indicate that MEDI4212 is a novel, high affinity antibody that binds specifically to IgE and prevents IgE binding to its receptors. MEDI4212 effectively depleted free-IgE from human sera ex vivo to a level (1 IU/mL) anticipated to provide optimal IgE suppression in severe asthma patients.

Oxidation of LDL (oxLDL) is a crucial step in the development of cardiovascular disease. Treatment with antibodies directed against oxLDL can reduce atherosclerosis in rodent models through unknown mechanisms. We demonstrate that through a novel mechanism of immune complex formation and Fc-γ receptor (FcγR) engagement, antibodies targeting oxLDL (MLDL1278a) are anti-inflammatory on innate immune cells via modulation of Syk, p38 MAPK phosphorylation and NFκB activity. Subsequent administration of MLDL1278a in diet-induced obese (DIO) nonhuman primates (NHP) resulted in a significant decrease in pro-inflammatory cytokines and improved overall immune cell function. Importantly, MLDL1278a treatment improved insulin sensitivity independent of body weight change. This study demonstrates a novel mechanism by which an anti-oxLDL antibody improves immune function and insulin sensitivity independent of internalization of oxLDL. This identifies MLDL1278a as a potential therapy for reducing vascular inflammation in diabetic conditions.

We describe the discovery of novel inhibitors of prostaglandin D2 synthase (PGDS) through fragment-based lead generation and structure-based drug design. A library of 2500 low-molecular-weight compounds was screened using 2D nuclear magnetic resonance (NMR), leading to the identification of 24 primary hits. Structure determination of protein-ligand complexes with the hits enabled a hit optimization process, whereby we harvested increasingly more potent inhibitors out of our corporate compound collection. Two iterative cycles were carried out, comprising NMR screening, molecular modeling, X-ray crystallography, and in vitro biochemical testing. Six novel high-resolution PGDS complex structures were determined, and 300 hit analogues were tested. This rational drug design procedure culminated in the discovery of 24 compounds with an IC 50 below 1 microM in the in vitro assay. The best inhibitor (IC 50 = 21 nM) is one of the most potent inhibitors of PGDS to date. As such, it may enable new functional in vivo studies of PGDS and the prostaglandin metabolism pathway.

Background: Improved asthma control by combinations of inhaled glucocorticosteroids (GCs) and long-acting beta(2)-agonists (LABAs) includes a reduced frequency and severity of exacerbations. In view of the association of exacerbations with increased airway inflammation, the question has arisen as to whether LABAs are able to complement the known anti-inflammatory activity of GCs. To address this, we studied the effects of a LABA, formoterol (FORM), and a GC, budesonide (BUD), alone and in combination, on bronchial epithelial cell-mediated eosinophil superoxide production in vitro., Methods: We employed 2 experimental approaches. First, superoxide production by human eosinophils incubated with conditioned medium (CM) from human bronchial epithelial cells cultured for 24 h with vehicle, BUD, FORM or BUD + FORM was measured (Epi/Eos assay). Second, eosinophils were stimulated with vehicle-CM to which the drugs were added (Eos assay). Superoxide production was determined as the superoxide dismutase-inhibitable reduction of ferricytochrome C., Results: CM increased eosinophil superoxide generation (p < 0.01) and epithelial-derived granulocyte macrophage colony-stimulating factor was the mediator responsible. In both assays, FORM dose-dependently inhibited eosinophil superoxide similarly and in the same concentration range as BUD. The BUD + FORM combination was more effective than BUD alone, and it completely inhibited CM-induced superoxide production in the Epi/Eos assay, suggesting complementary effects of both drugs on bronchial epithelial cells and eosinophils., Conclusions: The cooperative, inhibitory effects of BUD and FORM on eosinophils and bronchial epithelial cells, in terms of their effects on eosinophil superoxide production, may represent a possible mechanism for the enhanced anti-inflammatory efficacy of BUD and FORM combination therapy of asthma.

The inflammation of the lungs in chronic obstructive pulmonary disease (COPD) is characterised by increased numbers of macrophages, neutrophils and T-cells. Decline in lung function in these patients has been correlated to the number of CD8+ T-cells present in the lung as well as to a decline in the ratio of CD4+/CD8+ T-cells. Although systemic components are likely to be present, circulating lymphocyte populations in COPD patients have not been well characterised. This study aimed at correlating lung function to expression of five different T-cell activation markers on peripheral blood CD4+ and CD8+ T-cells in COPD patients and matched smokers. Furthermore, proportions of lymphocyte populations and degree of systemic T-cell activation in COPD patients were compared to that in smokers and never-smokers. Peripheral blood lymphocytes from six never-smokers, eight smokers and 17 smokers with COPD were analysed using flowcytometry. The number of lymphocytes per millilitre was higher in smokers than in never-smokers. No differences were found between the three groups in regard to proportions of lymphocyte populations, but the number of CD4+ T-cells in smokers was higher than in both never-smokers and COPD patients. The degree of T-cell activation was similar in all patient groups; however, a clear correlation between CD69 expression on CD4+ T-cells and lung function (FEV(1)% of predicted) was found when examining current smokers, with or without COPD. Elevated numbers of CD69+ CD4+ T-cells in blood thus seem to be protective against airway obstruction in smokers while still exposed to cigarette smoke, the main inducer of COPD.

Chronic obstructive pulmonary disease (COPD) is a disease primarily caused by cigarette smoking, which in turn has been shown to affect the susceptibility to and progression of airway infections. The question addressed in this study was how components from cigarette smoke could affect the defence mechanisms of T-cells and epithelial cells, and thereby contribute to the development of the COPD pathology. T-cells and monocytes were isolated from buffycoats from healthy donors and T-cell responses studied in response to cigarette smoke extract (CSE). Activation level (CD25 expression), proliferation (BrdU incorporation) and intracellular expression of the cytotoxic markers granzyme-b and TIA-1 were determined using flowcytometry. Normal human bronchial epithelial cells were obtained from Cambrex and differentiated in air-liquid interface cultures. After exposure to CSE barrier function (trans-epithelial electric resistance, TEER), MUC5AC and interleukin-8 production were measured. T-cell activation, proliferation and expression of the cytotoxic proteins granzyme-b and TIA-1 were significantly reduced in response to 0.5-1% of CSE. The epithelial cells were more resistant to CSE and responded at doses 20 times higher than T-cells. The expression of interleukin-8 and MUC5AC was significantly increased after exposure to 15% and 30% CSE and TEER was largely unaffected at 30% CSE but clearly reduced at 40% CSE. This study shows that mechanisms, in both T-cells and airway epithelial cells, involved in the defence against infectious agents are modulated by CSE.

alpha1-Antitrypsin (AAT), a major endogenous inhibitor of serine proteases, plays an important role in minimizing proteolytic injury to host tissue at sites of infection and inflammation. There is now increasing evidence that AAT undergoes post-translational modifications to yield by-products with novel biological activity. One such molecule, the C-terminal fragment of AAT, corresponding to residues 359-394 (C-36 peptide) has been reported to stimulate significant pro-inflammatory activity in monocytes and neutrophils in vitro. In this study we showed that C-36 peptide is present in human lung tissue and mimics the effects of lipopolysaccharide (LPS), albeit with lower magnitude, by inducing monocyte cytokine (TNFalpha, IL-1beta) and chemokine (IL-8) release in conjunction with the activation of nuclear factor-kappaB (NF-kappaB). Using receptor blocking antibodies and protein kinase inhibitors, we further demonstrated that C-36, like LPS, utilizes CD14 and Toll-like receptor 4 (TLR4) receptors and enzymes of the mitogen-activated protein kinase (MAPK) signaling pathways to stimulate monocyte TNFalpha release. The specificity of C-36 effects were demonstrated by failure of a shorter peptide (C-20) to elicit biological activity and the failure of C-36 to inhibit CD3/CD28-stimulated IL-2 receptor expression or proliferation in T-cells which lack TLR4 and CD14. We suggest that C-36 mediates its effects though the activation of LPS signaling pathways.