CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells.

Dendritic cell (DC) migration from peripheral tissues via afferent lymphatic vessels to draining lymph nodes (dLNs) is important for the organism's immune regulation and immune protection. Several lymphatic endothelial cell (LEC)-expressed adhesion molecules have thus far been found to support transmigration and movement within the lymphatic vasculature. In this study, we investigated the contribution of CD112, an adhesion molecule that we recently found to be highly expressed in murine LECs, to this process. Performing in vitro assays in the murine system, we found that transmigration of bone marrow-derived dendritic cells (BM-DCs) across or adhesion to murine LEC monolayers was reduced when CD112 was absent on LECs, DCs, or both cell types, suggesting the involvement of homophilic CD112-CD112 interactions. While CD112 was highly expressed in murine dermal LECs, CD112 levels were low in endogenous murine dermal DCs and BM-DCs. This might explain why we observed no defect in the in vivo lymphatic migration of adoptively transferred BM-DCs or endogenous DCs from the skin to dLNs. Compared to murine DCs, human monocyte-derived DCs expressed higher CD112 levels, and their migration across human CD112-expressing LECs was significantly reduced upon CD112 blockade. CD112 expression was also readily detected in endogenous human dermal DCs and LECs by flow cytometry and immunofluorescence. Upon incubating human skin punch biopsies in the presence of CD112-blocking antibodies, DC emigration from the tissue into the culture medium was significantly reduced, indicating impaired lymphatic migration. Overall, our data reveal a contribution of CD112 to human DC migration.

A Rapid Sterility Method Using Solid Phase Cytometry for Cell-Based Preparations and Culture Media and Buffers.

In this article, we demonstrate a rapid sterility testing method for non-filterable cell-based preparations and its in-process control media/buffers. The selected rapid sterility test (RST) in this work is based on the ScanRDI® system, which detects fluorescently labeled microorganisms with solid-phase cytometry. ScanRDI® has been chosen due to its sensitivity for detecting viable microorganisms down to one microbial cell with a shorter time to detection compared with the compendial sterility test (CST) method. The RST was validated for a CAR-T cell-therapy product with 4 days of time to detection (TTD) and evaluated for in-process control of media/buffers with real-time detection method success according to USP <1223>, Ph. Eur. 5.1.6, and PDA Technical Report No. 33. The validation parameters included limit of detection and equivalence in routine operations, specificity, robustness, ruggedness, and repeatability. For the validation, a combination of pharmacopoeial ATCC strains as well as in-house isolates were used. In addition, the evaluation study of this RST for in-process control of media/buffers was assessed by performing the limit of detection and equivalence with four representative microorganisms. Where applicable, results were statistically evaluated to demonstrate equivalence and no significant difference of the rapid method as compared with the CST method have been detected. All acceptance criteria have been met, and the solid-phase cytometry technology was successfully validated as an alternative sterility test for cell-based preparations and for its in-process control of media/buffer.

Optimization and Characterization of Novel ALCAM-Targeting Antibody Fragments for Transepithelial Delivery.

Activated leukocyte cell adhesion molecule (ALCAM) is a cell adhesion molecule that supports T cell activation, leukocyte migration, and (lymph)angiogenesis and has been shown to contribute to the pathology of various immune-mediated disorders, including asthma and corneal graft rejection. In contrast to monoclonal antibodies (mAbs) targeting ALCAM's T cell expressed binding partner CD6, no ALCAM-targeting mAbs have thus far entered clinical development. This is likely linked with the broad expression of ALCAM on many different cell types, which increases the risk of eliciting unwanted treatment-induced side effects upon systemic mAb application. Targeting ALCAM in surface-exposed tissues, such as the lungs or the cornea, by a topical application could circumvent this issue. Here, we report the development of various stability- and affinity-improved anti-ALCAM mAb fragments with cross-species reactivity towards mouse, rat, monkey, and human ALCAM. Fragments generated in either mono- or bivalent formats potently blocked ALCAM-CD6 interactions in a competition ELISA, but only bivalent fragments efficiently inhibited ALCAM-ALCAM interactions in a leukocyte transmigration assay. The different fragments displayed a clear size-dependence in their ability to penetrate the human corneal epithelium. Furthermore, intranasal delivery of anti-ALCAM fragments reduced leukocyte infiltration in a mouse model of asthma, confirming ALCAM as a target for topical application in the lungs.

Reassessing the adrenomedullin scavenging function of ACKR3 in lymphatic endothelial cells.

Atypical chemokine receptor 3 (ACKR3) is a scavenger of the chemokines CXCL11 and CXCL12 and of several opioid peptides. Additional evidence indicates that ACKR3 binds two other non-chemokine ligands, namely the peptide hormone adrenomedullin (AM) and derivatives of the proadrenomedullin N-terminal 20 peptide (PAMP). AM exhibits multiple functions in the cardiovascular system and is essential for embryonic lymphangiogenesis in mice. Interestingly, AM-overexpressing and ACKR3-deficient mouse embryos both display lymphatic hyperplasia. Moreover, in vitro evidence suggested that lymphatic endothelial cells (LECs), which express ACKR3, scavenge AM and thereby reduce AM-induced lymphangiogenic responses. Together, these observations have led to the conclusion that ACKR3-mediated AM scavenging by LECs serves to prevent overshooting AM-induced lymphangiogenesis and lymphatic hyperplasia. Here, we further investigated AM scavenging by ACKR3 in HEK293 cells and in human primary dermal LECs obtained from three different sources in vitro. LECs efficiently bound and scavenged fluorescent CXCL12 or a CXCL11/12 chimeric chemokine in an ACKR3-dependent manner. Conversely, addition of AM induced LEC proliferation but AM internalization was found to be independent of ACKR3. Similarly, ectopic expression of ACKR3 in HEK293 cells did not result in AM internalization, but the latter was avidly induced upon co-transfecting HEK293 cells with the canonical AM receptors, consisting of calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying protein (RAMP)2 or RAMP3. Together, these findings indicate that ACKR3-dependent scavenging of AM by human LECs does not occur at ligand concentrations sufficient to trigger AM-induced responses mediated by canonical AM receptors.

Leukocyte Trafficking in Lymphatic Vessels.

To ensure proper immune function, most leukocytes constantly move within tissues or between them using the blood and lymphatic vessels as transport routes. While afferent lymphatic vessels transfer leukocytes from peripheral tissues to draining lymph nodes (dLNs), efferent lymphatics return lymphocytes from LNs back into the blood vascular circulation. Over the last decades, great progress has been made in our understanding of leukocyte migration into and within the lymphatic compartment, leading to the approval of new drugs targeting this process. In this review, we first introduce the anatomy of the lymphatic vasculature and the main cell types migrating through lymphatics. We primarily focus on dendritic cells (DCs) and T cells, the most prominent lymph-borne cell types, and discuss the functional significance as well as the main molecules and steps involved in their migration. Additionally, we provide an overview of the different techniques used to study lymphatic trafficking.

Anaerobic co-digestion of waste yeast biomass from citric acid production and waste frying fat.

The application of spent yeast for biogas production has been studied only in the context of breweries so far. This study is focused on the anaerobic digestion of concentrated yeast biomass (CYB), being a by-product of citric acid biosynthesis. Two experimental set-ups were used in order to test CYB as a mono-substrate and co-substrate for closing the loop in accordance with the 'bioeconomy' approach. The results show that CYB allows for obtaining a high biogas yield, with a maximum of 1.45 m3 N/kgVS produced when CYB was used as a mono-substrate. The average methane concentration was 66 ± 4%. However, anaerobic digestion of CYB alone was difficult to perform because of a tendency for over-acidification, meaning that the maximum possible organic loading rate was 1 kg/(m3*d). Repeated clogging of tubes with coagulated biomass also disturbed continuous feeding. In contrast, the co-digestion of CYB with waste frying fat at a ratio of 1:20 showed stable operation during a 70-day fermentation period. The biogas yield using the substrate mixture was 1.42 m3/kgVS at an organic loading rate of 2 kg/(m3*d). The methane concentration reached 67 ± 4% and the acetate concentration did not exceed 30 mg/L during the entire fermentation.

Biomass potential analysis of aquatic biomass and challenges for its use as a nonconventional substrate in anaerobic digestion plants.

Aquatic macrophytes are important components of aquatic habitats. However, the overgrowth of aquatic plants can cause severe problems for the management of bodies of water. As a result, these plants must be removed and disposed of as waste. However, the usage of this biomass as a substrate in biogas plants would appear to be more beneficial. The present study shows the advantages and barriers to the use of harvested aquatic biomass for energy production in Germany. The results cover several aspects of this issue, such as the question of biomass potential, the quality of the harvested aquatic plants, and ensiling for the purpose of conservation for anaerobic digestion. In addition, the social aspects of the de-weeding in bodies of water are discussed.

Hydrogel substrate stress-relaxation regulates the spreading and proliferation of mouse myoblasts.

Mechanical properties of the extracellular microenvironment are known to alter cellular behavior, such as spreading, proliferation or differentiation. Previous studies have primarily focused on studying the effect of matrix stiffness on cells using hydrogel substrates that exhibit purely elastic behavior. However, these studies have neglected a key property exhibited by the extracellular matrix (ECM) and various tissues; viscoelasticity and subsequent stress-relaxation. As muscle exhibits viscoelasticity, stress-relaxation could regulate myoblast behavior such as spreading and proliferation, but this has not been previously studied. In order to test the impact of stress relaxation on myoblasts, we created a set of two-dimensional RGD-modified alginate hydrogel substrates with varying initial elastic moduli and rates of relaxation. The spreading of myoblasts cultured on soft stress-relaxing substrates was found to be greater than cells on purely elastic substrates of the same initial elastic modulus. Additionally, the proliferation of myoblasts was greater on hydrogels that exhibited stress-relaxation, as compared to cells on elastic hydrogels of the same modulus. These findings highlight stress-relaxation as an important mechanical property in the design of a biomaterial system for the culture of myoblasts. STATEMENT OF SIGNIFICANCE: This article investigates the effect of matrix stress-relaxation on spreading and proliferation of myoblasts by using tunable elastic and stress-relaxing alginate hydrogels substrates with different initial elastic moduli. Many past studies investigating the effect of mechanical properties on cell fate have neglected the viscoelastic behavior of extracellular matrices and various tissues and used hydrogels exhibiting purely elastic behavior. Muscle tissue is viscoelastic and exhibits stress-relaxation. Therefore, stress-relaxation could regulate myoblast behavior if it were to be incorporated into the design of hydrogel substrates. Altogether, we showed that stress-relaxation impacts myoblasts spreading and proliferation. These findings enable a better understanding of myoblast behavior on viscoelastic substrates and could lead to the design of more suitable substrates for myoblast expansion in vitro.