The Immune System Throws Its Traps: Cells and Their Extracellular Traps in Disease and Protection

The first formal description of the microbicidal activity of extracellular traps (ETs) containing DNA occurred in neutrophils in 2004. Since then, ETs have been identified in different populations of cells involved in both innate and adaptive immune responses. Much of the knowledge has been obtained from in vitro or ex vivo studies; however, in vivo evaluations in experimental models and human biological materials have corroborated some of the results obtained. Two types of ETs have been described—suicidal and vital ETs, with or without the death of the producer cell. The studies showed that the same cell type may have more than one ETs formation mechanism and that different cells may have similar ETs formation mechanisms. ETs can act by controlling or promoting the mechanisms involved in the development and evolution of various infectious and non-infectious diseases, such as autoimmune, cardiovascular, thrombotic, and neoplastic diseases, among others. This review discusses the presence of ETs in neutrophils, macrophages, mast cells, eosinophils, basophils, plasmacytoid dendritic cells, and recent evidence of the presence of ETs in B lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes. Moreover, due to recently collected information, the effect of ETs on COVID-19 is also discussed.

A trans-complementation system for SARS-CoV-2

ABSTRACTThe biosafety level-3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research and countermeasure development. Here we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing a deletion of ORF3 and envelope gene, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round, but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. The results suggest that the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.

Extracellular Metabolism Sets the Table for Microbial Cross-Feeding

SUMMARY The transfer of nutrients between cells, or cross-feeding, is a ubiquitous feature of microbial communities with emergent properties that influence our health and orchestrate global biogeochemical cycles. Cross-feeding inevitably involves the externalization of molecules. Some of these molecules directly serve as cross-fed nutrients, while others can facilitate cross-feeding. Altogether, externalized molecules that promote cross-feeding are diverse in structure, ranging from small molecules to macromolecules. The functions of these molecules are equally diverse, encompassing waste products, enzymes, toxins, signaling molecules, biofilm components, and nutrients of high value to most microbes, including the producer cell. As diverse as the externalized and transferred molecules are the cross-feeding relationships that can be derived from them. Many cross-feeding relationships can be summarized as cooperative but are also subject to exploitation. Even those relationships that appear to be cooperative exhibit some level of competition between partners. In this review, we summarize the major types of actively secreted, passively excreted, and directly transferred molecules that either form the basis of cross-feeding relationships or facilitate them. Drawing on examples from both natural and synthetic communities, we explore how the interplay between microbial physiology, environmental parameters, and the diverse functional attributes of extracellular molecules can influence cross-feeding dynamics. Though microbial cross-feeding interactions represent a burgeoning field of interest, we may have only begun to scratch the surface.

Targeted Knock-in of Transgenes into the CHO Cell Genome Using CRISPR-mediated Integration Systems

Biopharmaceutical proteins are usually produced by culturing recombinant Chinese hamster ovary (CHO) cells. High producer cell lines are screened from transfected cells with random integration of target genes. Since transgene expression is susceptible to the surrounding environment of the integrated genomic locus, producer cell lines should be selected from a large number of recombinant cells with heterogeneous transgene insertion. In contrast, targeted integration into a characterized genomic locus allows for predictable transgene expression and less clonal variability, and thus stable production of target proteins can be expected. Genome editing technology based on programmable nucleases has recently emerged as a versatile tool for precise editing of target locus in the cell genome. Here, we demonstrated targeted knock-in of transgenes into the hypoxanthine phosphoribosyltransferase (hprt) locus of CHO cells using CRISPR/Cas9 and CRISPR-mediated precise integration into target chromosome (PITCh) systems. We also generated knock-in CHO cells based on the homology-independent targeted integration (HITI) system. We evaluated the knock-in efficiency of transgenes into the hprt locus using these systems.

APPLICATION OF AMBR15 MINIBIOREACTORS IN CHARACTERIZATION OF SUSPENSION MONOCLONAL ANTIBODY CHO-PRODUCER CELL CULTURE PROCESS BASED ON QBD PRINCIPLES

A systematic approach to characterizing the cell culture process of CHO-producers of monoclonal antibodies within the framework of the concept "Quality by design" (QbD) with the use of ambr15 mini-bioreactors (TAP Biosystems) is shown.