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Iron doping of InP and GaInAsP(λ[sub g]=1.05 μm) layers grown by metalorganic molecular beam epitaxy was studied using elemental source material in combination with a conventional effusion cell. This study was aimed at the creation of semi-insulating optical waveguides under growth conditions compatible with selective area growth. Secondary ion mass spectroscopy measurements revealed a reproducible and homogeneous incorporation behavior of the iron dopant in the materials investigated. Resistivities in excess of 10[sup 9] Ω cm were obtained for both compositions at medium doping levels. GaInAsP/InP waveguide structures grown at 485 °C—the minimum temperature necessary for selective deposition—exhibited averaged resistivities of 5×10[sup 7] Ω cm in combination with optical losses of 2.5±0.5 dB/cm. © 1998 American Institute of Physics. [ABSTRACT FROM AUTHOR]

For the optimization of ventricular assist devices (VADs), flow simulations are crucial. Typically, these simulations assume single-phase flow to represent blood flow. However, blood consists of plasma and blood cells, making it a multiphase flow. Cell migration in such flows leads to a heterogeneous cell distribution, significantly impacting flow dynamics, especially in narrow gaps of less than 300 μm found in VADs. In these areas, cells migrate away from the walls, forming a cell-free layer, a phenomenon not usually considered in current VAD simulations. This paper addresses this gap by introducing a viscosity model that accounts for cell migration in microchannels under VAD-relevant conditions. The model is based on local particle distributions measured in a microchannels with a blood analog fluid. We developed a local viscosity distribution for flows with particles/cells and a cell-free layer, applicable to both blood and analog fluids, with particle volume fractions of up to 5%, gap heights of 150 μm, and Reynolds numbers around 100. The model was validated by comparing simulation results with experimental data of blood and blood analog fluid flow on wall shear stresses and pressure losses, showing strong agreement. This model improves the accuracy of simulations by considering local viscosity changes rather than assuming a single-phase fluid. Future developments will extend the model to physiological volume fractions up to 40%.

Exposure to air pollution fine particulate matter (PM 2.5 ) aggravates respiratory and cardiovascular diseases. It has been proposed that PM 2.5 uptake by alveolar macrophages promotes local inflammation that ignites a systemic response, but precise underlying mechanisms remain unclear. Here, we demonstrate that PM 2.5 phagocytosis leads to NLRP3 inflammasome activation and subsequent release of the pro-inflammatory master cytokine IL-1β. Inflammasome priming and assembly was time- and dose-dependent in inflammasome-reporter THP-1-ASC-GFP cells, and consistent across PM 2.5 samples of variable chemical composition. While inflammasome activation was promoted by different PM 2.5 surrogates, significant IL-1β release could only be observed after stimulation with transition-metal rich Residual Oil Fly Ash (ROFA) particles. This effect was confirmed in primary human monocyte-derived macrophages and murine bone marrow-derived macrophages (BMDMs), and by confocal imaging of inflammasome-reporter ASC-Citrine BMDMs. IL-1β release by ROFA was dependent on the NLRP3 inflammasome, as indicated by lack of IL-1β production in ROFA-exposed NLRP3-deficient (Nlrp3 -/- ) BMDMs, and by specific NLRP3 inhibition with the pharmacological compound MCC950. In addition, while ROFA promoted the upregulation of pro-inflammatory gene expression and cytokines release, MCC950 reduced TNF-α, IL-6, and CCL2 production. Furthermore, inhibition of TNF-α with a neutralizing antibody decreased IL-1β release in ROFA-exposed BMDMs. Using electron tomography, ROFA particles were observed inside intracellular vesicles and mitochondria, which showed signs of ultrastructural damage. Mechanistically, we identified lysosomal rupture, K + efflux, and impaired mitochondrial function as important prerequisites for ROFA-mediated IL-1β release. Interestingly, specific inhibition of superoxide anion production (O 2 •- ) from mitochondrial respiratory Complex I, but not III, blunted IL-1β release in ROFA-exposed BMDMs. Our findings unravel the mechanism by which PM 2.5 promotes IL-1β release in macrophages and provide a novel link between innate immune response and exposure to air pollution PM 2.5 ., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Rationale: Atherosclerosis is a chronic inflammatory disease of large arteries that involves an autoimmune response with autoreactive T cells and auto-antibodies recognizing Apolipoprotein B (ApoB), the core protein of low-density lipoprotein (LDL). Here, we aimed to establish a clinical association between circulating human ApoB auto-antibodies with atherosclerosis and its clinical risk factors using a novel assay to detect auto-antibodies against a pool of highly immunogenic ApoB-peptides., Methods and Results: To detect polyclonal IgM- and IgG-antibodies recognizing ApoB, we developed a chemiluminescent sandwich ELISA with 30 ApoB peptides selected by an in silico assay for a high binding affinity to MHC-II, which cover more than 80% of known MHC-II variants in a Caucasian population. This pre-selection of immunogenic self-peptides accounted for the high variability of human MHC-II, which is fundamental to allow T cell dependent generation of IgG antibodies. We quantified levels of ApoB-autoantibodies in a clinical cohort of 307 patients that underwent coronary angiography. Plasma anti-ApoB IgG and IgM concentrations showed no differences across healthy individuals ( n = 67), patients with coronary artery disease ( n = 179), and patients with an acute coronary syndrome ( n = 61). However, plasma levels of anti-ApoB IgG, which are considered pro-inflammatory, were significantly increased in patients with obesity ( p = 0.044) and arterial hypertension ( p < 0.0001). In addition, patients diagnosed with the metabolic syndrome showed significantly elevated Anti-ApoB IgG ( p = 0.002). Even when normalized for total plasma IgG, anti-ApoB IgG remained highly upregulated in hypertensive patients ( p < 0.0001). We observed no association with triglycerides, total cholesterol, VLDL, or LDL plasma levels. However, total and normalized anti-ApoB IgG levels negatively correlated with HDL. In contrast, total and normalized anti-ApoB IgM, that have been suggested as anti-inflammatory, were significantly lower in diabetic patients ( p = 0.012) and in patients with the metabolic syndrome ( p = 0.005)., Conclusion: Using a novel ELISA method to detect auto-antibodies against ApoB in humans, we show that anti-ApoB IgG associate with cardiovascular risk factors but not with the clinical appearance of atherosclerosis, suggesting that humoral immune responses against ApoB are shaped by cardiovascular risk factors but not disease status itself. This novel tool will be helpful to develop immune-based risk stratification for clinical atherosclerosis in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Marchini, Malchow, Caceres, El Rabih, Hansen, Mwinyella, Spiga, Piepenburg, Horstmann, Olawale, Li, Mitre, Gissler, Bugger, Zirlik, Heidt, Hilgendorf, Stachon, von zur Muehlen, Bode and Wolf.)

Hematopoietic progenitor kinase 1 (HPK1) is an MAP4K family member within the Ste20-like serine/threonine branch of the kinome. HPK1 expression is limited to hematopoietic cells and has a predominant role as a negative regulator of T cell function. Because of the central/dominant role in negatively regulating T cell function, HPK1 has long been in the center of interest as a potential pharmacological target for immune therapy. The development of a small molecule HPK1 inhibitor remains challenging because of the need for high specificity relative to other kinases, including additional MAP4K family members, that are required for efficient immune cell activation. Here, we report the identification of the selective and potent HPK1 chemical probe, A-745. In unbiased cellular kinase-binding assays, A-745 demonstrates an excellent cellular selectivity binding profile within pharmacologically relevant concentrations. This HPK1 selectivity translates to an in vitro immune cell activation phenotype reminiscent of Hpk1-deficient and Hpk1-kinase-dead T cells, including augmented proliferation and cytokine production. The results from this work give a path forward for further developmental efforts to generate additional selective and potent small molecule HPK1 inhibitors with the pharmacological properties for immunotherapy.

Many cellular events are driven by changes in protein expression, measurable by mass spectrometry or antibody-based assays. However, using conventional technology, the analysis of transcription factor or membrane receptor expression is often limited by an insufficient sensitivity and specificity. To overcome this limitation, we have developed a high-resolution targeted proteomics strategy, which allows quantification down to the lower attomol range in a straightforward way without any prior enrichment or fractionation approaches. The method applies isotope-labeled peptide standards for quantification of the protein of interest. As proof of principle, we applied the improved workflow to proteins of the unfolded protein response (UPR), a signaling pathway of great clinical importance, and could for the first time detect and quantify all major UPR receptors, transducers and effectors that are not readily detectable via antibody-based-, SRM- or conventional PRM assays. As transcription and translation is central to the regulation of UPR, quantification and determination of protein copy numbers in the cell is important for our understanding of the signaling process as well as how pharmacologic modulation of these pathways impacts on the signaling. These questions can be answered using our newly established workflow as exemplified in an experiment using UPR perturbation in a glioblastoma cell lines.

Schwann cells (SCs) are essential in the production of the axon-wrapping myelin sheath and provide trophic function and repair mechanisms in the peripheral nerves. Consequently, well-characterized SC in vitro models are needed to perform preclinical studies including the investigation of the complex biochemical adaptations occurring in the peripheral nervous system (PNS) under different (patho)physiological conditions. MSC80 cells represent a murine SC line used as an in vitro system for neuropathological studies. Here, we introduce the most abundant 9532 proteins identified via mass spectrometry-based protein analytics, and thus provide the most comprehensive SC protein catalogue published thus far. We cover proteins causative for inherited neuropathies and demonstrate that in addition to cytoplasmic, nuclear and mitochondrial proteins and others belonging to the protein processing machinery are very well covered. Moreover, we address the suitability of MSC80 to examine the molecular effect of a drug-treatment by analyzing the proteomic signature of Vitamin C-treated cells. Proteomic findings, immunocytochemistry, immunoblotting and functional experiments support the concept of a beneficial role of Vitamin C on oxidative stress and identified TMX1 as an oxidative stress protective factor, which might represent a promising avenue for therapeutic intervention of PNS-disorders with oxidative stress burden such as diabetic neuropathy.

Platelets are known to be key players in thrombosis and hemostasis, contributing to the genesis and progression of cardiovascular diseases. Due to their pivotal role in human physiology and pathology, platelet function is regulated tightly by numerous factors which have either stimulatory or inhibitory effects. A variety of factors, e.g., collagen, fibrinogen, ADP, vWF, thrombin, and thromboxane promote platelet adhesion and aggregation by utilizing multiple intracellular signal cascades. To quantify platelet proteins for this work, a targeted proteomics workflow was applied. In detail, platelets are isolated and lyzed, followed by a tryptic protein digest. Subsequently, a mix of stable isotope-labeled peptides of interesting biomarker proteins in concentrations ranging from 0.1 to 100 fmol is added to 3 μg digest. These peptides are used as an internal calibration curve to accurately quantify endogenous peptides and corresponding proteins in a pooled platelet reference sample by nanoLC-MS/MS with parallel reaction monitoring. In order to assure a valid quantification, limit of detection (LOD) and limit of quantification (LOQ), as well as linear range, were determined. This quantification of platelet activation and proteins by targeted mass spectrometry may enable novel diagnostic strategies in the detection and prevention of cardiovascular diseases., Competing Interests: The authors declare no conflict of interest.

Complex mass spectrometry based proteomics data sets are mostly analyzed by protein database searches. While this approach performs considerably well for sequenced organisms, direct inference of peptide sequences from tandem mass spectra, i.e., de novo peptide sequencing, oftentimes is the only way to obtain information when protein databases are absent. However, available algorithms suffer from drawbacks such as lack of validation and often high rates of false positive hits (FP). Here we present a simple method of combining results from commonly available de novo peptide sequencing algorithms, which in conjunction with minor tweaks in data acquisition ensues lower empirical FDR compared to the analysis using single algorithms. Results were validated using state-of-the art database search algorithms as well specifically synthesized reference peptides. Thus, we could increase the number of PSMs meeting a stringent FDR of 5% more than 3-fold compared to the single best de novo sequencing algorithm alone, accounting for an average of 11 120 PSMs (combined) instead of 3476 PSMs (alone) in triplicate 2 h LC-MS runs of tryptic HeLa digestion.

The promiscuous expression of tissue-restricted antigens in the thymus, driven in part by autoimmune regulator (Aire), is critical for the protection of peripheral tissues from autoimmune attack. Aire-dependent processes are thought to promote both clonal deletion and the development of Foxp3(+) regulatory T (Treg) cells, suggesting that autoimmunity associated with Aire deficiency results from two failed tolerance mechanisms. Here, examination of autoimmune lesions in Aire(-/-) mice revealed an unexpected third possibility. We found that the predominant conventional T cell clonotypes infiltrating target lesions express antigen receptors that were preferentially expressed by Foxp3(+) Treg cells in Aire(+/+) mice. Thus, Aire enforces immune tolerance by ensuring that distinct autoreactive T cell specificities differentiate into the Treg cell lineage; dysregulation of this process results in the diversion of Treg cell-biased clonotypes into pathogenic conventional T cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Although antigen recognition mediated by the T cell receptor (TCR) influences many facets of Foxp3(+) regulatory T (Treg) cell biology, including development and function, the cell types that present antigen to Treg cells in vivo remain largely undefined. By tracking a clonal population of Aire-dependent, prostate-specific Treg cells in mice, we demonstrated an essential role for dendritic cells (DCs) in regulating organ-specific Treg cell biology. We have shown that the thymic development of prostate-specific Treg cells required antigen presentation by DCs. Moreover, Batf3-dependent CD8α(+) DCs were dispensable for the development of this clonotype and had negligible impact on the polyclonal Treg cell repertoire. In the periphery, CCR7-dependent migratory DCs coordinated the activation of organ-specific Treg cells in the prostate-draining lymph nodes. Our results demonstrate that the development and peripheral regulation of organ-specific Treg cells are dependent on antigen presentation by DCs, implicating DCs as key mediators of organ-specific immune tolerance., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Many tumors express antigens that can be specifically or selectively recognized by T lymphocytes, suggesting that T-cell-mediated immunity may be harnessed for the immunotherapy of cancer. However, since tumors originate from normal cells and evolve within the context of self-tissues, the immune mechanisms that prevent the autoimmune attack of normal tissues function in parallel to restrict anti-tumor immunity. In particular, the purging of autoreactive T cells and the development of immune-suppressive regulatory T cells (Tregs) are thought to be major barriers impeding anti-tumor immune responses. Here, we discuss current understanding regarding the antigens recognized by tumor-infiltrating T-cell populations, the mechanisms that shape the repertoire of these cells, and the role of the transcription factor autoimmune regulator (Aire) in these processes. Further elucidation of these principles is likely to be critical for optimizing emerging cancer immunotherapies, and for the rational design of novel therapies exhibiting robust anti-tumor activity with limited toxicity., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Little is known about the relative contributions of self-specific regulatory T cells (Tregs) of thymic origin and induced Tregs generated extrathymically to the pool of tumor-infiltrating Tregs. We have recently demonstrated that thymic-derived Tregs reactive to a prostate-associated self antigen are highly and recurrently enriched within oncogene-driven murine prostate cancers.

Despite considerable interest in the modulation of tumor-associated Foxp3(+) regulatory T cells (T(regs)) for therapeutic benefit, little is known about the developmental origins of these cells and the nature of the antigens that they recognize. We identified an endogenous population of antigen-specific T(regs) (termed MJ23 T(regs)) found recurrently enriched in the tumors of mice with oncogene-driven prostate cancer. MJ23 T(regs) were not reactive to a tumor-specific antigen but instead recognized a prostate-associated antigen that was present in tumor-free mice. MJ23 T(regs) underwent autoimmune regulator (Aire)-dependent thymic development in both male and female mice. Thus, Aire-mediated expression of peripheral tissue antigens drives the thymic development of a subset of organ-specific T(regs), which are likely coopted by tumors developing within the associated organ.