Background: For patients with end-stage lung disease, lung transplantation (LTx) remains the only life-saving therapy, but it is limited by the shortage of donor organs. Ex vivo perfusion (EVLP) is a new technique to expand the donor pool and significantly improve outcomes after LTx. In addition to LTx, it can also be used for surgical, regenerative and immunomodulatory approaches, as well as tumor and infection therapy. To further analyze this broad spectrum of EVLP applications, we established EVLP for the small animal model.

Ex situ lung perfusion (ESLP) is used for organ reconditioning, repair, and re-evaluation prior to transplantation. Since valid preclinical animal models are required for translationally relevant studies, we developed a 17 mL low-volume ESLP for double- and single-lung application that enables cost-effective optimal compliance “reduction” of the 3R principles of animal research. In single-lung mode, ten Fischer344 and Lewis rat lungs were subjected to ESLP and static cold storage using STEEN or PerfadexPlus. Key perfusion parameters, thermal lung imaging, blood gas analysis (BGA), colloid oncotic pressure (COP), lung weight gain, histological work-up, and cytokine analysis were performed. Significant differences between perfusion solutions but not between the rat strains were detected. Most relevant perfusion parameters confirmed valid ESLP with homogeneous lung perfusion, evidenced by uniform lung surface temperature. BGA showed temperature-dependent metabolic activities with differences depending on perfusion solution composition. COP is not decisive for pulmonary oedema and associated weight gain, but possibly rather observed chemokine profile and dextran sensitivity of rats. Histological examination confirmed intact lung architecture without infarcts or hemorrhages due to optimal organ procurement and single-lung application protocol using our in-house-designed ESLP system.

Summary Overall asthmatic symptoms can be controlled with diverse therapeutic agents. However, certain symptomatic individuals remain at risk for serious morbidity and mortality, which prompts the identification of novel therapeutic targets and treatment strategies. Thus, using an adjuvant-free T helper type 2 (Th2) murine model, we have deciphered the role of interleukin (IL)-1 signalling during allergic airway inflammation (AAI). Because functional IL-1β depends on inflammasome activation we first studied asthmatic manifestations in specific inflammasome-deficient [NACHT, LRR and PYD domains-containing protein 3 (NLRP3−/−) and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC−/−)] and IL-1 receptor type 1−/− (IL-1R1−/−) mice on the BALB/c background. To verify the onset of disease we assessed cellular infiltration in the bronchial regions, lung pathology, airway hyperresponsiveness and ovalbumin (OVA)-specific immune responses. In the absence of NLRP3 inflammasome-mediated IL-1β release all symptoms of AAI were reduced, except OVA-specific immunoglobulin levels. To address whether manipulating IL-1 signalling reduced asthmatic development, we administered the IL-1R antagonist anakinra (Kineret®) during critical immunological time-points: sensitization or challenge. Amelioration of asthmatic symptoms was only observed when anakinra was administered during OVA challenge. Our findings indicate that blocking IL-1 signalling could be a potential complementary therapy for allergic airway inflammation.

BACKGROUND:Farm-derived dust samples have been screened for bacteria with potential allergo-protective properties. Among those was Staphylococcus sciuri W620 (S. sciuri W620), which we tested with regard to its protective capacities in murine models of allergic airway inflammation.METHODS:We employed two protocols of acute airway inflammation in mice administering either ovalbumin (OVA) or house dust mite extract (HDM) for sensitization. Mechanistic studies on the activation of innate immune responses to S. sciuri W620 were carried out using human primary monocytic dendritic cells (moDC) and co-culture with autologous T cells.RESULTS:The allergo-protective properties of S. sciuri W620 were proven in a T(H)2-driven OVA model as well as in a mixed T(H)1/T(H)2 phenotype HDM model as demonstrated by abrogation of eosinophils and neutrophils in the airways after intranasal treatment. In the HDM model, lymph node cell T(H)1/T(H)2 signature cytokines were decreased in parallel. Studies on human moDC revealed an activation of TLR2 and NOD2 receptors and initiation of DC maturation following incubation with S. sciuri W620. Cytokine expression analyses after exposure to S. sciuri W620 showed a lack of IL-12 production in moDC due to missing transcription of the IL-12p35 mRNA. However, such DC selectively supported T(H)1 cytokine release by co-cultured T cells.CONCLUSION AND CLINICAL RELEVANCE:Our proof-of-concept experiments verify the screening system of farm-derived dust samples as suitable to elucidate new candidates for allergo-protection. S. sciuri W620 was shown to possess preventive properties on airway inflammation providing the basis for further mechanistic studies and potential clinical implication.

BACKGROUND:The anti-inflammatory peptide, adrenomedullin (AM), and its cognate receptor are expressed in lung tissue, but its pathophysiological significance in airway inflammation is unknown.OBJECTIVES:This study investigated whether allergen-induced airway inflammation involves an impaired local AM response.METHODS:Airway AM expression was measured in acute and chronically sensitized mice following allergen inhalation and in airway epithelial cells of asthmatic and nonasthmatic patients. The effects of AM on experimental allergen-induced airway inflammation and of AM on lung epithelial repair in vitro were investigated.RESULTS:Adrenomedullin mRNA levels were significantly (P < 0.05) reduced in acute ovalbumin (OVA)-sensitized mice after OVA challenge, by over 60% at 24 h and for up to 6 days. Similarly, reduced AM expression was observed in two models of chronic allergen-induced inflammation, OVA- and house dust mite-sensitized mice. The reduced AM expression was restricted to airway epithelial and endothelial cells, while AM expression in alveolar macrophages was unaltered. Intranasal AM completely attenuated the OVA-induced airway hyperresponsiveness and mucosal plasma leakage but had no effect on inflammatory cells or cytokines. The effects of inhaled AM were reversed by pre-inhalation of the putative AM receptor antagonist, AM ((22-52)) . AM mRNA levels were significantly (P < 0.05) lower in human asthmatic airway epithelial samples than in nonasthmatic controls. In vitro, AM dose-dependently (10(-11) -10(-7) M) accelerated experimental wound healing in human and mouse lung epithelial cell monolayers and stimulated epithelial cell migration.CONCLUSION:Adrenomedullin suppression in T(H) 2-related inflammation is of pathophysiological significance and represents loss of a factor that maintains tissue integrity during inflammation.

Background: The pathophysiological significance in airway inflammation of the anti-inflammatory peptide, adrenomedullin (AM) and its cognate receptor, which are present in the lung, is unknown.

OBJECTIVE:CRLR (calcitonin receptor-like receptor) and CD 117, the gene product of c-kit have been shown to be expressed in cells of glial tumors, especially in those with higher malignancy. Here we report the distribution of these peptides in various cellular compartments within those tumors.MATERIAL:Both receptor proteins have been investigated in 95 glial tumor biopsies of different grades.METHODS:Both proteins were visualized by immunohistochemistry with antibodies either commercially available or raised for this purpose.RESULTS:Both receptor peptides can be identified in or around tumor blood vessels. CRLR occurs in some endothelial cells, especially in the microvascular proliferations of glioblastoma multiforme, whereas CD 117 preferentially occurs in cells of the thickened vascular wall within cells of pericyte or fibroblast morphology. Both antigens are found in addition in few neoplastic cells of overt astrocyte morphology.CONCLUSIONS:The occurrence of identical antigens in glial tumor blood vessels and in neighboring tumor cells underlines the common origin of "mesenchymal" and "neuroepithelial" components of such (malignant) glial neoplasms.

Background:Vascular tumours such as Kaposi’s sarcoma and capillary haemangioma are characterised by abnormal vascularisation and proliferation of endothelial cells or neoplastic cells. Adrenomedullin, a potent vasodilative peptide, and its receptor, calcitonin receptor-like receptor (CRLR), play an important part in angiogenesis.Aim:To establish whether this system also plays a part in vascular diseases, showing abnormal proliferation such as vascular tumours.Methods:CRLR expression was investigated in several specimens of Kaposi’s sarcoma and other vascular tumours, using immunohistochemical analysis with a previously described CRLR-specific polyclonal antibody and reverse transcriptase-polymerase chain reaction.Results:Intense and specific CRLR-immunoreactive staining of neoplastic cells was observed in all specimens, which was of greater intensity than similar staining of adjacent normal endothelium.Conclusions:CRLR is expressed in vascular tumours and, with adrenomedullin, may have a role in neoplastic vascular growth.

BACKGROUND:Gliomas are the most common primary tumours of the central nervous system and exhibit rapid growth that is associated with neovascularisation. Adrenomedullin is an important tumour survival factor in human carcinogenesis. It has growth promoting effects on gliomas, and blockade of its actions has been experimentally shown to reduce the growth of glioma tissues and cell lines. There is some evidence that the calcitonin receptor-like receptor (CRLR) mediates the tumorigenic actions of adrenomedullin.AIM:To determine whether CRLR is expressed in human gliomas and the probable cellular targets of adrenomedullin.METHODS:Biopsies from 95 human gliomas of varying grade were processed for immunohistochemical analysis using a previously developed and characterised antibody to CRLR.RESULTS:All tumour specimens were positive for CRLR. As previously found in normal peripheral tissues, CRLR immunostaining was particularly intense in the endothelial cells. This was evident in all the various vascular conformations that were observed, and which are typical of gliomas. In addition, clear immunostaining of tumour cells with astrocyte morphology was observed. These were preferentially localised around vessels.CONCLUSIONS:This study has shown for the first time that the CRLR protein is present in human glioma tissue. The expression of the receptor in endothelial cells and in astrocytic tumour cells is consistent with the evidence that its endogenous ligand, adrenomedullin, may influence glioma growth by means of both direct mitogenic and indirect angiogenic effects. CRLR may be a valuable target for effective therapeutic intervention in these malignant tumours.

Calcitonin-gene-related peptide and adrenomedullin have similar and potent vascular effects, which appear to be mediated by the G protein-coupled calcitonin receptor-like (CRL) receptor. Using immunohistochemical and Western blot analyses, we have obtained novel evidence that CRL receptor is expressed in the rat vascular endothelium using an antibody to rat CRL receptor that we have raised and fully characterised. These results are an important basis for further studies aimed at determining the so far ill-defined functional significance of the extensive distribution of CRL receptor in the vascular endothelium.

During ontogenesis, adrenomedullin is essential for normal cardiovascular development. Most adrenomedullin effects are conveyed via the calcitonin receptor-like receptor in combination with appropriate receptor activity-modifying proteins. Here, we investigated the presence of this receptor during development of the rat heart by means of immunohistochemistry. Intense specific labeling was observed at endothelial cells of the large vessels and at the endocardial cushions on E14. On E16, immature endothelial cells showing CRLR-immunoreactivity, formed a subepicardiac plexus of tubes penetrating radially towards the trabecular network. They reach it at E20. Endocardial cells lining the ventricular lumen were CRLR-negative. These data suggest CRLR as highly probable candidate involved in the development of the coronary vasculature.

Background/Aims: Pharmacological and morphological studies suggest that the gut mucosal immune system and local neuropeptide-containing neurones interact. We aimed to determine whether gut immune cells are targets for calcitonin gene-related peptide (CGRP), which has potent immune regulatory properties. Methods: Using density gradient centrifugation, rat lamina propria mononuclear cells (LP-MNCs) and intra-epithelial lymphocytes (IELs) were isolated. RT-PCR was employed for the detection of mRNA of rat calcitonin receptor-like receptor (CRLR), which is considered to represent the pharmacologically defined CGRP receptor-1 subtype, as well as mRNA of the receptor activity-modifying proteins, which are essential for CRLR function and determine ligand specificity. A radioreceptor assay was employed for the detection of specific CGRP binding sites. Results: RT-PCR and DNA sequencing showed that LP-MNCs and IELs express CRLR. Incubation of isolated LP-MNCs with radiolabelled αCGRP revealed the existence of specific binding sites for CGRP. Conclusion: These novel data indicate that mucosal immune cells of the rat gut are a target for CGRP and provide significant evidence that CGRP functions as an immune regulator in the gut mucosa.