Update on the pathophysiology of transfusion-related acute lung injury.

PURPOSE OF REVIEW: The aim of this study was to discuss recent advances regarding the pathogenesis of transfusion-related acute lung injury (TRALI), which highlight the pathogenic role of macrophages. RECENT FINDINGS: TRALI remains a leading cause of transfusion-related fatalities, despite the success of the mitigation strategy, and therapeutic approaches are unavailable. Neutrophils (PMNs) are recognized pathogenic cells in TRALI. Macrophages have previously also been suggested to be pathogenic in mice via binding of C5a to their C5a-receptor, producing reactive oxygen species (ROS), which damages the pulmonary endothelium. Recent work has further highlighted the role of macrophages in the TRALI-pathogenesis. It has been shown that the protein osteopontin (OPN) released by macrophages is critical for pulmonary PMN recruitment in mice suffering from TRALI and that targeting OPN prevents the occurrence of TRALI. Another recent study demonstrated the importance of M1-polarized alveolar macrophages in murine TRALI induction by showing that α1-antitrypsin (AAT) overexpression prevented TRALI in mice through decreasing the polarization of alveolar macrophages towards the M1 phenotype. SUMMARY: Apart from PMNs, macrophages also appear to be important in the pathogenesis of TRALI. Targeting the pathogenic functions of macrophages may be a promising therapeutic strategy to explore in TRALI.

Impact of hypoxia on chemoresistance of mesothelioma mediated by the proton-coupled folate transporter, and preclinical activity of new anti-LDH-A compounds.

BACKGROUND: Expression of proton-coupled folate transporter (PCFT) is associated with survival of mesothelioma patients treated with pemetrexed, and is reduced by hypoxia, prompting studies to elucidate their correlation. METHODS: Modulation of glycolytic gene expression was evaluated by PCR arrays in tumour cells and primary cultures growing under hypoxia, in spheroids and after PCFT silencing. Inhibitors of lactate dehydrogenase (LDH-A) were tested in vitro and in vivo. LDH-A expression was determined in tissue microarrays of radically resected malignant pleural mesothelioma (MPM, N = 33) and diffuse peritoneal mesothelioma (DMPM, N = 56) patients. RESULTS: Overexpression of hypoxia marker CAIX was associated with low PCFT expression and decreased MPM cell growth inhibition by pemetrexed. Through integration of PCR arrays in hypoxic cells and spheroids and following PCFT silencing, we identified the upregulation of LDH-A, which correlated with shorter survival of MPM and DMPM patients. Novel LDH-A inhibitors enhanced spheroid disintegration and displayed synergistic effects with pemetrexed in MPM and gemcitabine in DMPM cells. Studies with bioluminescent hypoxic orthotopic and subcutaneous DMPM athymic-mice models revealed the marked antitumour activity of the LDH-A inhibitor NHI-Glc-2, alone or combined with gemcitabine. CONCLUSIONS: This study provides novel insights into hypoxia/PCFT-dependent chemoresistance, unravelling the potential prognostic value of LDH-A, and demonstrating the preclinical activity of LDH-A inhibitors.

Evaluation of Platelet Responses in Transfusion-Related Acute Lung Injury (TRALI).

Platelets are versatile cells which are capable of eliciting nonhemostatic immune functions, especially under inflammatory conditions. Depending on the specific setting, platelets may be either protective or pathogenic in acute lung injury and acute respiratory distress syndrome (ARDS). Their role in transfusion-related acute lung injury (TRALI) is less well defined; however, it has been hypothesized that recipient platelets and transfused platelets both play a pathogenic role in TRALI. Overall, despite conflicting findings, it appears that recipient platelets may play a pathogenic role in antibody-mediated TRALI; however, their contribution appears to be limited. It is imperative to first validate the involvement of recipient platelets by standardizing the animal models, methods, reagents, and readouts for lung injury and taking the animal housing environment into consideration. For the involvement of transfused platelets in TRALI, it appears that predominantly lipids such as ceramide in stored platelets are able to induce TRALI in animal models. These studies will also need to be validated, and moreover, the platelet-derived lipid-mediated mechanisms leading to TRALI will need to be investigated.

Biological and structural characterization of murine TRALI antibody reveals increased Fc-mediated complement activation.

Transfusion-related acute lung injury (TRALI) remains a leading cause of transfusion-related deaths. In most cases, anti-leukocyte antibodies in the transfusion product trigger TRALI, but not all anti-leukocyte antibodies cause TRALI. It has been shown that the anti-major histocompatibility complex (MHC) class I antibody 34-1-2S (anti-H-2Kd) causes TRALI in BALB/c mice (MHC class I haplotype H-2Kd), whereas SF1.1.10 (anti-H-2Kd) does not. In C57BL/6 mice (MHC class I haplotype H-2Kb), TRALI only occurs when anti-MHC class I antibody AF6-88.5.5.3 (anti-H-2Kb) is administered together with a high dose of 34-1-2S. It remains unknown which specific antibody characteristics are responsible for eliciting TRALI. We therefore investigated several biological and structural features of 34-1-2S compared with other anti-MHC class I antibodies, which on their own do not cause TRALI: SF1.1.10 and AF6-88.5.5.3. No substantial differences were observed between the TRALI-causing 34-1-2S and the TRALI-resistant SF1.1.10 regarding binding affinity to H-2Kd. Regarding binding affinity to H-2Kb, only AF6-88.5.5.3 potently bound to H-2Kb, whereas 34-1-2S exhibited weak but significant cross-reactivity. Furthermore, the binding affinity to FcγRs as well as the Fc glycan composition seemed to be similar for all antibodies. Similar Fc glycosylation profiles were also observed for human TRALI-causing donor anti-HLA antibodies compared with human anti-HLA antibodies from control donors. 34-1-2S, however, displayed superior complement activation capacity, which was fully Fc dependent and not significantly dependent on Fc glycosylation. We conclude that TRALI induction is not correlated with Fab- and Fc-binding affinities for antigen and FcγRs, respectively, nor with the composition of Fc glycans; but increased Fc-mediated complement activation is correlated with TRALI induction.

Splicing modulation as novel therapeutic strategy against diffuse malignant peritoneal mesothelioma.

INTRODUCTION: Therapeutic options for diffuse malignant peritoneal mesothelioma (DMPM) are limited to surgery and locoregional chemotherapy. Despite improvements in survival rates, patients eventually succumb to disease progression. We investigated splicing deregulation both as molecular prognostic factor and potential novel target in DMPM, while we tested modulators of SF3b complex for antitumor activity. METHODS: Tissue-microarrays of 64 DMPM specimens were subjected to immunohistochemical assessment of SF3B1 expression and correlation to clinical outcome. Two primary cell cultures were used for gene expression profiling and in vitro screening of SF3b modulators. Drug-induced splicing alterations affecting downstream cellular pathways were detected through RNA sequencing. Ultimately, we established bioluminescent orthotopic mouse models to test the efficacy of splicing modulation in vivo. RESULTS: Spliceosomal genes are differentially upregulated in DMPM cells compared to normal tissues and high expression of SF3B1 correlated with poor clinical outcome in univariate and multivariate analysis. SF3b modulators (Pladienolide-B, E7107, Meayamycin-B) showed potent cytotoxic activity in vitro with IC50 values in the low nanomolar range. Differential splicing analysis of Pladienolide-B-treated cells revealed abundant alterations of transcripts involved in cell cycle, apoptosis and other oncogenic pathways. This was validated by RT-PCR and functional assays. E7107 demonstrated remarkable in vivo antitumor efficacy, with significant improvement of survival rates compared to vehicle-treated controls. CONCLUSIONS: SF3B1 emerged as a novel potential prognostic factor in DMPM. Splicing modulators markedly impair cancer cell viability, resulting also in potent antitumor activity in vivo. Our data designate splicing as a promising therapeutic target in DMPM.