Randall J. Mrsny

A three-session webinar series was held by the IQ Consortium in early May 2023 on the topic of in vitro release testing of long acting injectable and paren

Neutrophil migration involves a dynamic balance of chemoattractant and inhibitory signals. We previously identified hepoxilin A ₃ (HxA ₃ ) as a potent chemoattractant that directs neutrophil migration across intestinal epithelia and showed that endocannabinoids can suppress migration through the cannabinoid 2 receptor (CB ₂ R). Here, we reveal that HxA ₃ uses the transient receptor potential vanilloid type 2 (TRPV2) cation channel to promote transmigration, a process finely tuned by physical TRPV2-CB ₂ R interaction. In resting neutrophils, surface-localized CB ₂ R responding to endocannabinoids suppresses migration, while a small fraction of TRPV2 at the plasma membrane “senses” HxA ₃ . HxA ₃ drives the movement of an intracellular “storage” pool of TRPV2 to the leading edge of migration where physical interaction with CB ₂ R establishes cell surface receptor complexes to direct neutrophil migration. Postactivation, TRPV2 is shunted toward degradation, while CB ₂ R is both replaced and recycled to the plasma membrane to establish a coordinated system capable of maintaining directional fidelity to HxA ₃ gradients. These findings uncover a previously unrecognized layer of neutrophil trafficking regulation.

Mono-ADP-ribosyl transferase (mART) proteins are secreted virulence factors produced by several human pathogens, the founding member being diphtheria toxin (DT). Pseudomonas aeruginosa can also secrete a mART toxin, known as exotoxin A (PE), but with an organization of its three functional domains (receptor, translocation, and enzymatic elements) that is opposite to DT. Two additional PE-like toxins (PLTs) have been identified from Vibrio cholerae and Aeromonas hydrophila, suggesting more PLT family members may exist. Database mining discovered six additional putative homologues, considerably extending this group of PLTs across a wide range of bacterial species. Here, we examine sequence and structural information for these new family members with respect to previously identified PLTs. The X-ray crystal structures of four new homologues show the conservation of critical features responsible for structure and function. This study shows the potential of these newly described toxins for the development of novel drug delivery platforms. Additionally, genomic analysis suggests horizontal gene transfer to account for the wide distribution of PLTs across a range of eubacteria species, highlighting the need to monitor emerging pathogens and their virulence factors.

Mankind has recently had to deal a series of virus-mediated pandemics, resulting in extensive morbidity and mortality rates that have severely strained healthcare systems. While dealing with viral infections as a healthcare concern is not new, our exceptionally mobile society has added to the critical challenge of limiting pathogen spread of a highly transmissible virus prior to the generation, testing, and distribution of safe and effective vaccines. The tremendous global effort put forth to address the recent pandemic induced by SARS-CoV-2 infection has highlighted many of the strengths and weaknesses of how vaccines are identified, tested, and used to provide protection. These uncertainties are exacerbated by the lack of clear and consistent messaging that can occur when the processes of research, development, and clinical testing that normally requires years of study and consideration are compressed into a few months. In this commentary, I will provide some background on the intramuscular (IM), subcutaneous (SC), and intradermal (ID) administration routes used for injectable vaccines and some information on potential immunological outcomes. With this background, I will address the recent FDA decision to allow an approved vaccine against monkeypox virus to be administered by ID, as well as its initial approval route via SC, injection as a dose-sparing strategy to maximize immunization numbers using current stockpiles.

Cholix (Chx) is secreted by non-pandemic strains of Vibrio cholerae in the intestinal lumen. For this exotoxin to induce cell death in non-polarized cells in the intestinal lamina propria, it must traverse the epithelium in the fully intact form. We identified host cell elements in polarized enterocytes associated with Chx endocytosis and apical to basal (A→B) vesicular transcytosis. This pathway overcomes endogenous mechanisms of apical vesicle recycling and lysosomal targeting by interacting with several host cell proteins that include the 75 kDa glucose-regulated protein (GRP75). Apical endocytosis of Chx appears to involve the single membrane spanning protein TMEM132A, and interaction with furin before it engages GRP75 in apical vesicular structures. Sorting within these apical vesicles results in Chx being trafficked to the basal region of cells in association with the Lectin, Mannose Binding 1 protein LMAN1. In this location, Chx interacts with the basement membrane-specific heparan sulfate proteoglycan perlecan in recycling endosomes prior to its release from this basal vesicular compartment to enter the underlying lamina propria. While the furin and LMAN1 elements of this Chx transcytosis pathway undergo cellular redistribution that are reflective of the polarity shifts noted for coatamer complexes COPI and COPII, GRP75 and perlecan fail to show these dramatic rearrangements. Together, these data define essential steps in the A→B transcytosis pathway accessed by Chx to reach the intestinal lamina propria where it can engage and intoxicate certain non-polarized cells. The Vibrio cholerae exotoxin protein cholix interacts with a number of host cell proteins, including GRP75, to facilitate its vesicular transcytosis across polarized intestinal epithelial cells following apical endocytosis.

Background: P-glycoprotein (P-gp) plays a critical role in protection of the intestinal epithelia by mediating efflux of drugs/xenobiotics from the intestinal mucosa into the gut lumen. Recent studies bring to light that P-gp also confers a critical link in communication between intestinal mucosal barrier function and the innate immune system. Yet, despite knowledge for over 10 years that P-gp plays a central role in gastrointestinal homeostasis, the precise molecular mechanism that controls its functional expression and regulation remains unclear. Here, we assessed how the intestinal microbiome drives P-gp expression and function.Results: We have identified a “functional core” microbiome of the intestinal gut community, specifically genera within theClostridiaandBacilliclasses, that is necessary and sufficient for P-gp induction in the intestinal epithelium in mouse models. Metagenomic analysis of this core microbial community revealed that short-chain fatty acid and secondary bile acid production positively associate with P-gp expression. We have further shown these two classes of microbiota-derived metabolites synergistically upregulate P-gp expression and function in vitro and in vivo. Moreover, in patients suffering from ulcerative colitis (UC), we find diminished P-gp expression coupled to the reduction of epithelial-derived anti-inflammatory endocannabinoids and luminal content (e.g., microbes or their metabolites) with a reduced capability to induce P-gp expression.Conclusion: Overall, by means of both in vitro and in vivo studies as well as human subject sample analysis, we identify a mechanistic link between cooperative functional outputs of the complex microbial community and modulation of P-gp, an epithelial component, that functions to suppress overactive inflammation to maintain intestinal homeostasis. Hence, our data support a new cross-talk paradigm in microbiome regulation of mucosal inflammation.