TIP peptide inhalation in experimental acute lung injury: effect of repetitive dosage and different synthetic variants

BACKGROUND: Inhalation of TIP peptides that mimic the lectin-like domain of TNF-α is a novel approach to attenuate pulmonary oedema on the threshold to clinical application. A placebo-controlled porcine model of acute respiratory distress syndrome (ARDS) demonstrated a reduced thermodilution-derived extravascular lung water index (EVLWI) and improved gas exchange through TIP peptide inhalation within three hours. Based on these findings, the present study compares a single versus a repetitive inhalation of a TIP peptide (TIP-A) and two alternate peptide versions (TIP-A, TIP-B). METHODS: Following animal care committee approval ARDS was induced by bronchoalveolar lavage followed by injurious ventilation in 21 anaesthetized pigs. A randomised-blinded three-group setting compared the single-dosed peptide variants TIP-A and TIP-B as well as single versus repetitive inhalation of TIP-A (n = 7 per group). Over two three-hour intervals parameters of gas exchange, transpulmonary thermodilution, calculated alveolar fluid clearance, and ventilation/perfusion-distribution were assessed. Post-mortem measurements included pulmonary wet/dry ratio and haemorrhage/congestion scoring. RESULTS: The repetitive TIP-A inhalation led to a significantly lower wet/dry ratio than a single dose and a small but significantly lower EVLWI. However, EVLWI changes over time and the derived alveolar fluid clearance did not differ significantly. The comparison of TIP-A and B showed no relevant differences. Gas exchange and ventilation/perfusion-distribution significantly improved in all groups without intergroup differences. No differences were found in haemorrhage/congestion scoring. CONCLUSIONS: In comparison to a single application the repetitive inhalation of a TIP peptide in three-hour intervals may lead to a small additional reduction the lung water content. Two alternate TIP peptide versions showed interchangeable characteristics

Adenosine-producing regulatory B cells in head and neck cancer

Background Multiple mechanisms of immunosuppression have been identified in the tumor microenvironment including regulatory B cells (Breg). Recently, we have shown that Breg suppress T cell function by production of adenosine (ADO). However, the autocrine effect of ADO on B cells and the role of Breg in head and neck cancer remains unclear. Methods Blood (n = 42) and tumor tissue (n = 39) of head and neck cancer patients and healthy donors (n = 60) were analyzed by FACS. The effect of ADO on phenotype, intracellular signaling pathways, Ca2+ influx and ADO production was analyzed in Breg and effector B cells (Beff) by FACS, luminescence and mass spectrometry. The blockage of the ADO receptor A2A was analyzed in a murine head and neck cancer model. Results ADO-producing Breg were found in tumor tissue and peripheral blood. ADO inhibited the intracellular Bruton’s tyrosine kinase (BTK) and Ca2+ influx only in Beff. The inhibition of BTK by ibrutinib mimicked the effect of ADO, and ibrutinib reduced the production of ADO by downregulation of CD39 in vitro. The inhibition of ADO receptor A2A significantly reduced tumor mass and increased B cell infiltration, in vivo. Conclusion Our data demonstrate the presence of a novel ADO-producing Breg population within the tumor microenvironment in mice and humans. A new model is proposed on how ADO-producing Breg can influence the function of Beff cells in healthy donors and cancer patients. Thus, the modulation of the ADO pathway in B cells may serve as a therapeutic approach for cancer patients

Bisphosphonates: restrictions for vasculogenesis and angiogenesis: inhibition of cell function of endothelial progenitor cells and mature endothelial cells in vitro

Abstract Bisphosphonate-associated osteonecrosis of the jaws (BP-ONJ) is one of the main side effects in patients treated with bisphosphonates for metastasis to the bone or osteoporosis. BP-ONJ usually occurs in patients treated with highly potent nitrogen-containing bisphosphonates. The exact mechanism of action and etiopathology is still unknown. In addition to inhibition of bone remodelling, an anti-angiogenetic effect has become the focus of research. The aim of these study was to investigate the effect of different bisphosphonates on human umbilicord vein endothelial cells (HUVEC) and endothelial progenitor cells (EPC), which play an important role in angiogenesis. Using varying concentrations, the impact of one non-nitrogencontaining bisphosphonate (clodronate) and three nitrogencontaining bisphosphonates (ibandronate, pamidronate and zoledronate) on HUVEC and EPC was analysed. The biologic behaviour of HUVEC after incubation with different bisphosphonates was measured in a Boyden migration assay as well as in a 3D angiogenesis assay. The number of apoptotic cells was measured by Tunnel assay. To underline the importance of neoangiogenesis in the context of BP-ONJ, we measured the EPC number after incubation with different bisphosphonates in vitro. HUVEC and EPC were significantly influenced by bisphosphonates at different concentrations compared with the non-treated control groups. The nitrogen-containing bisphosphonates pamidronate and zoledronate had the greatest impact on the cells, whereas clodronate followed by ibandronate was less distinct on cell function. These results underline the hypothesis that inhibited angiogenesis induced by bisphosphonates might be of relevance in the development and maintenance of BP-ONJ. The increased impact by highly potent bisphosphonates on HUVEC and EPC may explain the high prevalence of BP-ONJ in patients undergoing this treatment

The influence of bisphosphonates on human osteoblast migration and integrin aVb3/tenascin C gene expression in vitro

<p>Abstract</p> <p>Background</p> <p>Bisphosphonates are therapeutics of bone diseases, such as Paget's disease, multiple myeloma or osteoclastic metastases. As a severe side effect the bisphosphonate induced osteonecrosis of the jaw (BONJ) often requires surgical treatment and is accompanied with a disturbed wound healing.</p> <p>Therefore, the influence on adhesion and migration of human osteoblasts (hOB) after bisphosphonate therapy has been investigated by morphologic as well as gene expression methods.</p> <p>Methods</p> <p>By a scratch wound experiment, which measures the reduction of defined cell layer gap, the morphology and migration ability of hOB was evaluated. A test group of hOB, which was stimulated by zoledronate 5 × 10^-5M, and a control group of unstimulated hOB were applied. Furthermore the gene expression of integrin aVb3 and tenascin C was quantified by Real-Time rtPCR at 5data points over an experimental period of 14 days. The bisphosphonates zoledronate, ibandronate and clodronate have been compared with an unstimulated hOB control.</p> <p>Results</p> <p>After initially identical migration and adhesion characteristics, zoledronate inhibited hOB migration after 50 h of stimulation. The integrinavb3 and tenascin C gene expression was effected by bisphosphonates in a cell line dependent manner with decreased, respectively inconsistent gene expression levels over time. The non-nitrogen containing bisphosphonates clodronate led to decreased gene expression levels.</p> <p>Conclusion</p> <p>Bisphosphonates seem to inhibit hOB adhesion and migration. The integrin aVb3 and tenascin C gene expression seem to be dependent on the cell line. BONJ could be enhanced by an inhibition of osteoblast adhesion and migration. The gene expression results, however, suggest a cell line dependent effect of bisphosphonates, which could explain the interindividual differences of BONJ incidences.</p

The impact of bisphosphonates on the osteoblast proliferation and Collagen gene expression in vitro

<p>Abstract</p> <p>Background</p> <p>Bisphosphonates are widely used in the clinical treatment of bone diseases with increased bone resorption. In terms of side effects, they are known to be associated with osteonecrosis of the jaw (BONJ).</p> <p>The objective of this study was to evaluate the effect of bisphosphonates on osteoblast proliferation by cell count and gene expression analysis of cyclin D1 <it>in vitro</it>. Furthermore, the gene expression of the extracellular matrix protein collagen type I was evaluated. Nitrogen-containing and non-nitrogen-containing bisphosphonates have been compared on gene expression levels.</p> <p>Methods</p> <p>Human osteoblast obtained from hip bone were stimulated with zoledronate, ibandronate and clodronate at concentrations of 5 × 10^-5M over the experimental periods of 1, 2, 5, 10 and 14 days. At each point in time, the cells were dissolved, the mRNA extracted, and the gene expression level of cyclin D1 and collagen type I were quantified by Real-Time RT-PCR. The gene expression was compared to an unstimulated osteoblast cell culture for control.</p> <p>Results</p> <p>The proliferation appeared to have been influenced only to a small degree by bisphosphonates. Zolendronate led to a lower cyclin D1 gene expression after 10 days. The collagen gene expression was enhanced by nitrogen containing bisphosphonates, decreased however after day 10. The non-nitrogen-containing bisphosphonate clodronate, however, did not significantly influence cyclin D1 and collagen gene expression.</p> <p>Conclusions</p> <p>The above data suggest a limited influence of bisphosphonates on osteoblast proliferation, except for zoledronate. The extracellular matrix production seems to be initially advanced and inhibited after 10 days. Interestingly, clodronate has little influence on osteoblast proliferation and extracellular matrix production in terms of cyclin D1 and collagen gene expression.</p