A graphical tool for locating inconsistency in network meta-analyses

BACKGROUND: In network meta-analyses, several treatments can be compared by connecting evidence from clinical trials that have investigated two or more treatments. The resulting trial network allows estimating the relative effects of all pairs of treatments taking indirect evidence into account. For a valid analysis of the network, consistent information from different pathways is assumed. Consistency can be checked by contrasting effect estimates from direct comparisons with the evidence of the remaining network. Unfortunately, one deviating direct comparison may have side effects on the network estimates of others, thus producing hot spots of inconsistency. METHODS: We provide a tool, the net heat plot, to render transparent which direct comparisons drive each network estimate and to display hot spots of inconsistency: this permits singling out which of the suspicious direct comparisons are sufficient to explain the presence of inconsistency. We base our methods on fixed-effects models. For disclosure of potential drivers, the plot comprises the contribution of each direct estimate to network estimates resulting from regression diagnostics. In combination, we show heat colors corresponding to the change in agreement between direct and indirect estimate when relaxing the assumption of consistency for one direct comparison. A clustering procedure is applied to the heat matrix in order to find hot spots of inconsistency. RESULTS: The method is shown to work with several examples, which are constructed by perturbing the effect of single study designs, and with two published network meta-analyses. Once the possible sources of inconsistencies are identified, our method also reveals which network estimates they affect. CONCLUSION: Our proposal is seen to be useful for identifying sources of inconsistencies in the network together with the interrelatedness of effect estimates. It opens the way for a further analysis based on subject matter considerations

The Aspergillus giganteus antifungal protein AFPNN5353 activates the cell wall integrity pathway and perturbs calcium homeostasis

Background The antifungal protein AFPNN5353 is a defensin-like protein of Aspergillus giganteus. It belongs to a group of secretory proteins with low molecular mass, cationic character and a high content of cysteine residues. The protein inhibits the germination and growth of filamentous ascomycetes, including important human and plant pathogens and the model organsims Aspergillus nidulans and Aspergillus niger. Results We determined an AFPNN5353 hypersensitive phenotype of non-functional A. nidulans mutants in the protein kinase C (Pkc)/mitogen-activated protein kinase (Mpk) signalling pathway and the induction of the α-glucan synthase A (agsA) promoter in a transgenic A. niger strain which point at the activation of the cell wall integrity pathway (CWIP) and the remodelling of the cell wall in response to AFPNN5353. The activation of the CWIP by AFPNN5353, however, operates independently from RhoA which is the central regulator of CWIP signal transduction in fungi. Furthermore, we provide evidence that calcium (Ca2+) signalling plays an important role in the mechanistic function of this antifungal protein. AFPNN5353 increased about 2-fold the cytosolic free Ca2+ ([Ca2+]c) of a transgenic A. niger strain expressing codon optimized aequorin. Supplementation of the growth medium with CaCl2 counteracted AFPNN5353 toxicity, ameliorated the perturbation of the [Ca2+]c resting level and prevented protein uptake into Aspergillus sp. cells. Conclusions The present study contributes new insights into the molecular mechanisms of action of the A. giganteus antifungal protein AFPNN5353. We identified its antifungal activity, initiated the investigation of pathways that determine protein toxicity, namely the CWIP and the Ca2+ signalling cascade, and studied in detail the cellular uptake mechanism in sensitive target fungi. This knowledge contributes to define new potential targets for the development of novel antifungal strategies to prevent and combat infections of filamentous fungi which have severe negative impact in medicine and agriculture.FWF, P19970-B11, Characterization of the toxicity of PA

Sterol Composition of Clinically Relevant Mucorales and Changes Resulting from Posaconazole Treatment

Mucorales are fungi with increasing importance in the clinics. Infections take a rapidly progressive course resulting in high mortality rates. The ergosterol biosynthesis pathway and sterol composition are of interest, since they are targeted by currently applied antifungal drugs. Nevertheless, Mucorales often exhibit resistance to these drugs, resulting in therapeutic failure. Here, sterol patterns of six clinically relevant Mucorales (Lichtheimia corymbifera, Lichtheimia ramosa, Mucor circinelloides, Rhizomucor pusillus, Rhizopus arrhizus, and Rhizopus microsporus) were analysed in a targeted metabolomics fashion after derivatization by gas chromatography-mass spectrometry. Additionally, the effect of posaconazole (POS) treatment on the sterol pattern of R. arrhizus was evaluated. Overall, fifteen different sterols were detected with species dependent variations in the total and relative sterol amount. Sterol analysis from R. arrhizus hyphae confronted with sublethal concentrations of posaconazole revealed the accumulation of 14-methylergosta-8,24-diene-3,6-diol, which is a toxic sterol that was previously only detected in yeasts. Sterol content and composition were further compared to the well-characterized pathogenic mold Aspergillus fumigatus. This work contributes to a better understanding of the ergosterol biosynthesis pathway of Mucorales, which is essential to improve antifungal efficacy, the identification of targets for novel drug design, and to investigate the combinatorial effects of drugs targeting this pathway

The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes

Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on Mucor and Rhizopus. This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of Lichtheimia corymbifera, one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in Galleria mellonella during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1-2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence

Safety and feasibility of intranasal heroin-assisted treatment: 4-week preliminary findings from a Swiss multicentre observational study

Background: Heroin-assisted treatment (HAT) is effective for individuals with severe opioid use disorder (OUD) who do not respond sufficiently to other opioid agonist treatments. It is mostly offered with injectable diacetylmorphine (DAM) or DAM tablets creating a barrier for individuals who need the rapid onset of action but are either unable or unwilling to inject, or primarily snort opioids. To explore another route of administration, we evaluated the safety and feasibility of intranasal (IN) DAM. Methods: This is a multicentre observational cohort study among patients in Swiss HAT. All patients planning to receive IN DAM within the treatment centres were eligible to participate. Participants were either completely switched to IN DAM or received IN DAM in addition to other DAM formulations or opioid agonists. Patients were followed up for four weeks. Sociodemographic characteristics, current HAT regimen, reasons for starting IN DAM, IN DAM doses, number of injection events in the sample, IN DAM continuation rate, and appearance of adverse events and nose-related problems were evaluated. Results: Participants (n = 52) reported vein damage, preference for nasal route of administration, and desire of a stronger effect or for a less harmful route of administration as primary reasons for switching to IN DAM. After four weeks, 90.4% of participants (n = 47) still received IN DAM. Weekly average realised injection events decreased by 44.4% from the month before IN DAM initiation to the month following. No severe adverse events were reported. Conclusions: After four weeks, IN DAM was a feasible and safe alternative to other routes of administration for patients with severe OUD in HAT. It addressed the needs of individuals with OUD and reduced injection behaviour. More long-term research efforts are needed to systematically assess efficacy of and patient satisfaction with IN DAM

The Role of lncRNAs TAPIR-1 and -2 as Diagnostic Markers and Potential Therapeutic Targets in Prostate Cancer

In search of new biomarkers suitable for the diagnosis and treatment of prostate cancer, genome-wide transcriptome sequencing was carried out with tissue specimens from 40 prostate cancer (PCa) and 8 benign prostate hyperplasia patients. We identified two intergenic long non-coding transcripts, located in close genomic proximity, which are highly expressed in PCa. Microarray studies on a larger cohort comprising 155 patients showed a profound diagnostic potential of these transcripts (AUC~0.94), which we designated as tumor associated prostate cancer increased lncRNA (TAPIR-1 and -2). To test their therapeutic potential, knockdown experiments with siRNA were carried out. The knockdown caused an increase in the p53/TP53 tumor suppressor protein level followed by downregulation of a large number of cell cycle- and DNA-damage repair key regulators. Furthermore, in radiation therapy resistant tumor cells, the knockdown leads to a renewed sensitization of these cells to radiation treatment. Accordingly, in a preclinical PCa xenograft model in mice, the systemic application of nanoparticles loaded with siRNA targeting TAPIR-1 significantly reduced tumor growth. These findings point to a crucial role of TAPIR-1 and -2 in PCa

Candida albicans Factor H Binding Molecule Hgt1p – A Low Glucose-Induced Transmembrane Protein Is Trafficked to the Cell Wall and Impairs Phagocytosis and Killing by Human Neutrophils

Complement is a tightly controlled arm of the innate immune system, facilitating phagocytosis and killing of invading pathogens. Factor H (FH) is the main fluid-phase inhibitor of the alternative pathway. Many pathogens can hijack FH from the host and protect themselves from complement-dependent killing. Candida albicans is a clinically important opportunistic yeast, expressing different FH binding molecules on its cell surface, which allow complement evasion. One such FH binding molecule is the transmembrane protein “High affinity glucose transporter 1” (Hgt1p), involved in glucose metabolism. This study demonstrated that Hgt1p transcription and expression is induced and highest at the low, but physiological glucose concentration of 0.1%. Thus, this concentration was used throughout the study. We also demonstrated the transport of Hgt1p to the fungal cell wall surface by vesicle trafficking and its release by exosomes containing Hgt1p integrated in the vesicular membrane. We corroborated Hgt1p as FH binding molecule. A polyclonal anti-Hgt1p antibody was created which interfered with the binding of FH, present in normal human serum to the fungal cell wall. A chimeric molecule consisting of FH domains 6 and 7 fused to human IgG1 Fc (FH6.7/Fc) even more comprehensively blocked FH binding, likely because FH6.7/Fc diverted FH away from fungal FH ligands other than Hgt1p. Reduced FH binding to the yeast was associated with a concomitant increase in C3b/iC3b deposition and resulted in significantly increased in vitro phagocytosis and killing by human neutrophils. In conclusion, Hgt1p also exhibits non-canonical functions such as binding FH after its export to the cell wall. Blocking Hgt1p-FH interactions may represent a tool to enhance complement activation on the fungal surface to promote phagocytosis and killing of C. albicans