Tumour angiogenesis in Epstein-Barr virus-associated post-transplant smooth muscle tumours

Epstein-Barr virus (EBV)-associated post-transplant smooth muscle tumours (PTSMT), are rare complications following organ/stem cell transplantation. Despite the mainly benign behaviour of PTSMT, alternative therapies are needed for those patients with progressive tumours. In tumours not approachable by surgery or reduction of immunosuppression, the angiogenic microenvironment might be a potential target of therapy, an approach that is well utilised in other soft tissue neoplasms. In a previous study, we evaluated the expression of EBV-related genes and the microRNA profile in PTSMT, but so far the characteristics of angiogenesis in PTSMT are not known. Therefore, the aim of this study was to evaluate the expression pattern of angiogenesis-related genes in PTSMT, in order to identify potential target molecules for anti-angiogenic therapy. PTSMT (n = 5 tumours) were compared with uterine leiomyomas (n = 7). Analyses included real-time PCR of 45 angiogenesis-associated genes, immunohistochemistry (CD31, prostaglandin endoperoxide synthase 1/PTGS1) and assessment of tumour vascularisation by conventional histopathology. PTSMT showed similar or fewer vessels than leiomyomas. Of the genes under investigation, 23 were down-deregulated (pro-angiogenic and some anti-angiogenic factors) and five were up-regulated (e.g. PTGS1 which is expressed at very low levels in leiomyomas but moderately higher levels in PTSMT). In summary, no particular target molecule could be identified, because tumour angiogenesis in PTSMT is characterised by low levels of major pro-angiogenic factors and there is no prominent increase in tumour vascularisation. EBV can induce angiogenesis via its viral late membrane protein 1 (LMP1) but PTSMT frequently do not express LMP1, which could be an explanation why, despite EBV infection, PTSMT show no exaggerated tumour angiogenesis

A combined method for correlative 3D imaging of biological samples from macro to nano scale

Correlative analysis requires examination of a specimen from macro to nano scale as well as applicability of analytical methods ranging from morphological to molecular. Accomplishing this with one and the same sample is laborious at best, due to deformation and biodegradation during measurements or intermediary preparation steps. Furthermore, data alignment using differing imaging techniques turns out to be a complex task, which considerably complicates the interconnection of results. We present correlative imaging of the accessory rat lung lobe by combining a modified Scanning Laser Optical Tomography (SLOT) setup with a specially developed sample preparation method (CRISTAL). CRISTAL is a resin-based embedding method that optically clears the specimen while allowing sectioning and preventing degradation. We applied and correlated SLOT with Multi Photon Microscopy, histological and immunofluorescence analysis as well as Transmission Electron Microscopy, all in the same sample. Thus, combining CRISTAL with SLOT enables the correlative utilization of a vast variety of imaging techniques

Anti-inflammatory and immunomodulatory properties of alpha1-antitrypsin without inhibition of elastase

The rationale of alpha1-antitrypsin (AAT) augmentation therapy to treat progressive emphysema in AAT-deficient patients is based on inhibition of neutrophil elastase; however, the benefit of this treatment remains unclear. Here we show that clinical grade AAT (with elastase inhibitory activity) and a recombinant form of AAT (rAAT) without anti-elastase activity reduces lung inflammatory responses to LPS in elastase-deficient mice. WT and elastase-deficient mice treated with either native AAT or rAAT exhibited significant reductions in infiltrating neutrophils (23% and 68%), lavage fluid levels of TNF-alpha (70% and 80%), and the neutrophil chemokine KC (CXCL1) (64% and 90%), respectively. Lung parenchyma TNF-alpha, DNA damage-inducible transcript 3 and X-box binding protein-1 mRNA levels were reduced in both mouse strains treated with AAT; significantly lower levels of these genes, as well as IL-1beta gene expression, were observed in lungs of AAT-deficient patients treated with AAT thera py compared with untreated patients. In vitro, LPS-induced cytokines from WT and elastase-deficient mouse neutrophils, as well as neutrophils of healthy humans, were similarly reduced by AAT or rAAT; human neutrophils adhering to endothelial cells were decreased by 60-80% (P < 0.001) with either AAT or rAAT. In mouse pancreatic islet macrophages, LPS-induced surface expression of MHC II, Toll-like receptor-2 and -4 were markedly lower (80%, P < 0.001) when exposed to either AAT or rAAT. Consistently, in vivo and in vitro, rAAT reduced inflammatory responses at concentrations 40- to 100-fold lower than native plasma-derived AAT. These data provide evidence that the anti-inflammatory and immunomodulatory properties of AAT can be independent of elastase inhibition

Streptococcus pneumoniae triggers progression of pulmonary fibrosis through pneumolysin

Rationale: Respiratory tract infections are common in patients suffering from pulmonary fibrosis. The interplay between bacterial infection and fibrosis is characterised poorly. Objectives: To assess the effect of Gram-positive bacterial infection on fibrosis exacerbation in mice. Methods Fibrosis progression in response to Streptococcus pneumoniae was examined in two different mouse models of pulmonary fibrosis. Measurements and main results: We demonstrate that wild-type mice exposed to adenoviral vector delivery of active transforming growth factor-beta 1 (TGF beta 1) or diphteria toxin (DT) treatment of transgenic mice expressing the DT receptor (DTR) under control of the surfactant protein C (SPC) promoter (SPC-DTR) to induce pulmonary fibrosis developed progressive fibrosis following infection with Spn, without exhibiting impaired lung protective immunity against Spn. Antibiotic treatment abolished infection-induced fibrosis progression. The cytotoxin pneumolysin (Ply) of Spn caused this phenomenon in a TLR4-independent manner, as Spn lacking Ply (Spn Delta ply) failed to trigger progressive fibrogenesis, whereas purified recombinant Ply did. Progressive fibrogenesis was also observed in AdTGF beta 1-exposed Ply-challenged TLR4 KO mice. Increased apoptotic cell death of alveolar epithelial cells along with an attenuated intrapulmonary release of antifibrogenic prostaglandin E2 was found to underlie progressive fibrogenesis in Ply-challenged AdTGF beta 1-exposed mice. Importantly, vaccination of mice with the non-cytotoxic Ply derivative B (PdB) substantially attenuated Ply-induced progression of lung fibrosis in AdTGF beta 1-exposed mice. Conclusions: Our data unravel a novel mechanism by which infection with Spn through Ply release induces progression of established lung fibrosis, which can be attenuated by protein-based vaccination of mice