IL4I1 Is a Metabolic Immune Checkpoint that Activates the AHR and Promotes Tumor Progression.

Aryl hydrocarbon receptor (AHR) activation by tryptophan (Trp) catabolites enhances tumor malignancy and suppresses anti-tumor immunity. The context specificity of AHR target genes has so far impeded systematic investigation of AHR activity and its upstream enzymes across human cancers. A pan-tissue AHR signature, derived by natural language processing, revealed that across 32 tumor entities, interleukin-4-induced-1 (IL4I1) associates more frequently with AHR activity than IDO1 or TDO2, hitherto recognized as the main Trp-catabolic enzymes. IL4I1 activates the AHR through the generation of indole metabolites and kynurenic acid. It associates with reduced survival in glioma patients, promotes cancer cell motility, and suppresses adaptive immunity, thereby enhancing the progression of chronic lymphocytic leukemia (CLL) in mice. Immune checkpoint blockade (ICB) induces IDO1 and IL4I1. As IDO1 inhibitors do not block IL4I1, IL4I1 may explain the failure of clinical studies combining ICB with IDO1 inhibition. Taken together, IL4I1 blockade opens new avenues for cancer therapy.

Mortality Prediction by Kinetic Parameters of Lactate and S-Adenosylhomocysteine in a Cohort of Critically Ill Patients.

Tissue hypoxia is associated with the development of organ dysfunction and death in critically ill patients commonly captured using blood lactate. The kinetic parameters of serial lactate evaluations are superior at predicting mortality compared with single values. S-adenosylhomocysteine (SAH), which is also associated with hypoxia, was recently established as a useful predictor of septic organ dysfunction and death. We evaluated the performance of kinetic SAH parameters for mortality prediction compared with lactate parameters in a cohort of critically ill patients. For lactate and SAH, maxima and means as well as the normalized area scores were calculated for two periods: the first 24 h and the total study period of up to five days following ICU admission. Their performance in predicting in-hospital mortality were compared in 99 patients. All evaluated parameters of lactate and SAH were significantly higher in non-survivors compared with survivors. In univariate analysis, the predictive power for mortality of SAH was higher compared with lactate in all forms of application. Multivariable models containing SAH parameters demonstrated higher predictive values for mortality than models based on lactate parameters. The optimal models for mortality prediction incorporated both lactate and SAH parameters. Compared with lactate, SAH displayed stronger predictive power for mortality in static and dynamic application in critically ill patients.

S-Adenosylhomocysteine Is a Useful Metabolic Factor in the Early Prediction of Septic Disease Progression and Death in Critically Ill Patients: A Prospective Cohort Study.

A common final pathway of pathogenetic mechanisms in septic organ dysfunction and death is a lack or non-utilization of oxygen. Plasma concentrations of lactate serve as surrogates for the oxygen-deficiency-induced imbalance between energy supply and demand. As S-adenosylhomocysteine (SAH) was shown to reflect tissue hypoxia, we compared the ability of SAH versus lactate to predict the progression of inflammatory and septic disease to septic organ dysfunction and death. Using univariate and multiple logistic regression, we found that SAH but not lactate, taken upon patients' inclusion in the study close to ICU admission, significantly and independently contributed to the prediction of disease progression and death. Due to the stronger increase in SAH in relation to S-adenosylmethionine (SAM), the ratio of SAM to SAH, representing methylation potential, was significantly decreased in patients with septic organ dysfunction and non-survivors compared with SIRS/sepsis patients (2.8 (IQR 2.3-3.9) vs. 8.8 (4.9-13.8); p = 0.003) or survivors (4.9 (2.8-9.5) vs. 8.9 (5.1-14.3); p = 0.026), respectively. Thus, SAH appears to be a better contributor to the prediction of septic organ dysfunction and death than lactate in critically ill patients. As SAH is a potent inhibitor of SAM-dependent methyltransferases involved in numerous vital biochemical processes, the impairment of the SAM-to-SAH ratio in severely critically ill septic patients and non-survivors warrants further studies on the pathogenetic role of SAH in septic multiple organ failure.

The Use of an Inspiration-Synchronized Vibrating Mesh Nebulizer for Prolonged Inhalative Iloprost Administration in Mechanically Ventilated Patients-An In Vitro Model.

Mechanically ventilated patients suffering from acute respiratory distress syndrome (ARDS) frequently receive aerosolized iloprost. Because of prostacyclin's short half-life, prolonged inhalative administration might improve its clinical efficacy. But, this is technically challenging. A solution might be the use of inspiration-synchronized vibrating mesh nebulizers (VMNsyn), which achieve high drug deposition rates while showing prolonged nebulization times. However, there are no data comparing prolonged to bolus iloprost nebulization using a continuous vibrating mesh nebulizer (VMNcont) and investigating the effects of different ventilation modes on inspiration-synchronized nebulization. Therefore, in an in vitro model of mechanically ventilated adults, a VMNsyn and a VMNcont were compared in volume-controlled (VC-CMV) and pressure-controlled continuous mandatory ventilation (PC-CMV) regarding iloprost deposition rate and nebulization time. During VC-CMV, the deposition rate of the VMNsyn was comparable to the rate obtained with the VMNcont, but 10.9% lower during PC-CMV. The aerosol output of the VMNsyn during both ventilation modes was significantly lower compared to the VMNcont, leading to a 7.5 times longer nebulization time during VC-CMV and only to a 4.2 times longer nebulization time during PC-CMV. Inspiration-synchronized nebulization during VC-CMV mode therefore seems to be the most suitable for prolonged inhalative iloprost administration in mechanically ventilated patients.

Inhibition of lysyl oxidases synergizes with 5-azacytidine to restore erythropoiesis in myelodysplastic and myeloid malignancies.

Limited response rates and frequent relapses during standard of care with hypomethylating agents in myelodysplastic neoplasms (MN) require urgent improvement of this treatment indication. Here, by combining 5-azacytidine (5-AZA) with the pan-lysyl oxidase inhibitor PXS-5505, we demonstrate superior restoration of erythroid differentiation in hematopoietic stem and progenitor cells (HSPCs) of MN patients in 20/31 cases (65%) versus 9/31 cases (29%) treated with 5-AZA alone. This effect requires direct contact of HSPCs with bone marrow stroma components and is dependent on integrin signaling. We further confirm these results in vivo using a bone marrow niche-dependent MN xenograft model in female NSG mice, in which we additionally demonstrate an enforced reduction of dominant clones as well as significant attenuation of disease expansion and normalization of spleen sizes. Overall, these results lay out a strong pre-clinical rationale for efficacy of combination treatment of 5-AZA with PXS-5505 especially for anemic MN.

Pre-analytical processing of plasma and serum samples for combined proteome and metabolome analysis.

Metabolomic and proteomic analyses of human plasma and serum samples harbor the power to advance our understanding of disease biology. Pre-analytical factors may contribute to variability and bias in the detection of analytes, especially when multiple labs are involved, caused by sample handling, processing time, and differing operating procedures. To better understand the impact of pre-analytical factors that are relevant to implementing a unified proteomic and metabolomic approach in a clinical setting, we assessed the influence of temperature, sitting times, and centrifugation speed on the plasma and serum metabolomes and proteomes from six healthy volunteers. We used targeted metabolic profiling (497 metabolites) and data-independent acquisition (DIA) proteomics (572 proteins) on the same samples generated with well-defined pre-analytical conditions to evaluate criteria for pre-analytical SOPs for plasma and serum samples. Time and temperature showed the strongest influence on the integrity of plasma and serum proteome and metabolome. While rapid handling and low temperatures (4°C) are imperative for metabolic profiling, the analyzed proteomics data set showed variability when exposed to temperatures of 4°C for more than 2 h, highlighting the need for compromises in a combined analysis. We formalized a quality control scoring system to objectively rate sample stability and tested this score using external data sets from other pre-analytical studies. Stringent and harmonized standard operating procedures (SOPs) are required for pre-analytical sample handling when combining proteomics and metabolomics of clinical samples to yield robust and interpretable data on a longitudinal scale and across different clinics. To ensure an adequate level of practicability in a clinical routine for metabolomics and proteomics studies, we suggest keeping blood samples up to 2 h on ice (4°C) prior to snap-freezing as a compromise between stability and operability. Finally, we provide the methodology as an open-source R package allowing the systematic scoring of proteomics and metabolomics data sets to assess the stability of plasma and serum samples.

Validation of a novel, fully integrated and flexible microarray benchtop facility for gene expression profiling.

Here we describe a novel microarray platform that integrates all functions needed to perform any array-based experiment in a compact instrument on the researcher's laboratory benchtop. Oligonucle otide probes are synthesized in situ via a light- activated process within the channels of a three-dimensional microfluidic reaction carrier. Arrays can be designed and produced within hours according to the user's requirements. They are processed in a fully automatic workflow. We have characterized this new platform with regard to dynamic range, discrimination power, reproducibility and accuracy of biological results. The instrument detects sample RNAs present at a frequency of 1:100 000. Detection is quantitative over more than two orders of magnitude. Experiments on four identical arrays with 6398 features each revealed a mean coefficient of variation (CV) value of 0.09 for the 6398 unprocessed raw intensities indicating high reproducibility. In a more elaborate experiment targeting 1125 yeast genes from an unbiased selection, a mean CV of 0.11 on the fold change level was found. Analyzing the transcriptional response of yeast to osmotic shock, we found that biological data acquired on our platform are in good agreement with data from Affymetrix GeneChips, quantitative real-time PCR and--albeit somewhat less clearly--to data from spotted cDNA arrays obtained from the literature.

Intraperitoneal use of doxorubicin drug-eluting beads in sheep: a pilot safety study.

BACKGROUND: Intraperitoneal carcinmatosis and intra-abdominal tumour cell dissemination may occur with several tumour entities. At this final stage of the tumour disease, systemic chemotherapy remains only palliative and may be accompanied by severe side-effects. Alternatively, compartment-based therapy by intraperitoneal (i.p.) instillation of doxorubicin drug-eluting beads (dox-DEB) has been proposed. Here we investigated the safety, pharmacokinetics and side-effects of i.p. dox-DEB in a large animal trial. MATERIALS AND METHODS: Ten black-headed meat-sheep received i.p. polyvinyl-alcohol-encapsulated doxorubicin. The plasma concentration of doxorubicin, blood count and laboratory findings were recorded and histological examination of the organs was performed. RESULTS: After i.p. instillation, elevated serum levels of doxorubicin were obtained. After this initial phase, serum levels remained constant. Upon autopsy, no signs of systemic toxicity were detected. Beads remained within the peritoneal cavity and were not systemically distributed. However, we found significant local toxicity in five sheep, three of which died due to severe peritonitis. CONCLUSION: Doxorubicin can be successfully targeted to the peritoneal cavity by bead encapsulation. Local toxic effects must be controlled in order to facilitate clinical utility.

Development of sustained-release lipophilic calcium stearate pellets via hot melt extrusion.

The objective of this study was the development of retarded release pellets using vegetable calcium stearate (CaSt) as a thermoplastic excipient. The matrix carrier was hot melt extruded and pelletized with a hot-strand cutter in a one step continuous process. Vegetable CaSt was extruded at temperatures between 100 and 130°C, since at these temperatures cutable extrudates with a suitable melt viscosity may be obtained. Pellets with a drug loading of 20% paracetamol released 11.54% of the drug after 8h due to the great densification of the pellets. As expected, the drug release was influenced by the pellet size and the drug loading. To increase the release rate, functional additives were necessary. Therefore, two plasticizers including glyceryl monostearate (GMS) and tributyl citrate (TBC) were investigated for plasticization efficiency and impact on the in vitro drug release. GMS increased the release rate due to the formation of pores at the surface (after dissolution) and showed no influence on the process parameters. The addition of TBC increased the drug release to a higher extent. After dissolving, the pellets exhibited pores at the surface and in the inner layer. Small- and Wide-Angle X-ray Scattering (SWAXS) revealed no major change in crystalline peaks. The results demonstrated that (nearly) spherical CaSt pellets could be successfully prepared by hot melt extrusion using a hot-strand cutter as downstreaming system. Paracetamol did not melt during the process indicating a solid suspension. Due to the addition of plasticizers, the in vitro release rate could be tailored as desired.