Genetically detoxified tetanus toxin as a vaccine and conjugate carrier protein.

Tetanus toxoid (TTxd), developed over 100 years ago, is a clinically effective, legacy vaccine against tetanus. Due to the extreme potency of native tetanus toxin, manufacturing and regulatory efforts often focus on TTxd production, standardization, and safety, rather than product modernization. Recently, a genetically detoxified, full-length tetanus toxin protein (8MTT) was reported as a tetanus vaccine alternative to TTxd (Przedpelski et al. mBio, 2020). Here we describe the production of 8MTT in Gor/MetTM E. coli, a strain engineered to have an oxidative cytoplasm, allowing for the expression of soluble, disulfide-bonded proteins. The strain was also designed to efficiently cleave N-terminal methionine, the obligatory start amino acid for E. coli expressed proteins. 8MTT was purified as a soluble protein from the cytoplasm in a two-column protocol to > 99 % purity, yielding 0.5 g of purified 8MTT/liter of fermentation broth with low endotoxin contamination, and antigenic purity of 3500 Lf/mg protein nitrogen. Mouse immunizations showed 8MTT to be an immunogenic vaccine and effective as a carrier protein for peptide and polysaccharide conjugates. These studies validate 8MTT as commercially viable and, unlike the heterogenous tetanus toxoid, a uniform carrier protein for conjugate vaccines. The development of a recombinant, genetically detoxified toxin produced in E. coli aligns the tetanus vaccine with modern manufacturing, regulatory, standardization, and safety requirements.

Androgen-Induced Relaxation of Uterine Myocytes Is Mediated by Blockade of Both Ca(2+) Flux and MLC Phosphorylation.

CONTEXT: Uterine quiescence must be maintained until pregnancy reaches term. Premature activation of myometrial contractility leads to preterm labor and delivery. OBJECTIVE: To scrutinize the potential of androgens to relax the myometrium and the mechanism of their action. SAMPLES: A pregnancy-derived myometrial smooth muscle cell line (PHM1-41) and myometrial strips prepared from tissues obtained from pregnant women (lean, n = 9; obese, n = 6) undergoing elective cesarean section at term and from nonpregnant C57BL/6 mice (n=5) were each utilized. DESIGN: The contraction of collagen-embedded PHM1-41s and the stretch-induced contraction of human and murine myometrial strips were assessed after incubation with Testosterone (T), dihydrotestosterone (DHT), and T conjugated to BSA. Intracellular calcium ([Ca(2+)]) and phosphorylated myosin light chain concentrations were quantified in PHM1-41s using a Fluo-4 Ca(2+) assay and in-cell Westerns, respectively. SETTING: University research institute. RESULTS: DHT and T, but not T conjugated to BSA, impaired the contractile function of PHM1-41s and of human and murine myometrial strips. The response was rapid (observed within minutes), was sustainable for up to 48 hours, and was not abolished on knockdown of the androgen receptor. DHT (100 µm) reduced the amplitude of lean strip contraction to 2 ± 2% of the pretreatment value and T (100 µm) to 3.3 ± 1%. These values for obese strips were 15 ± 6.7% and 11 ± 6.7%, respectively. At the same doses, in murine strips, DHT reduced the amplitude to 4.8 ± 3% and T to 4.9 ± 3%. DHT (50 µm) pretreatment reduced the oxytocin-stimulated increase in [Ca(2+)] (P < .0001; n = 6) and phosphorylated myosin light chain (P < .05; n = 5) in PHM1-41s. CONCLUSION: Lipid-soluble androgens could be developed as tocolytic agents for the treatment of preterm labor.

Lipopolysaccharide promotes contraction of uterine myocytes via activation of Rho/ROCK signaling pathways.

Myometrial contraction is a central feature of labor. Although a link between infection and preterm labor is widely accepted, surprisingly little is known about the mechanisms coupling infection-induced inflammation to myocyte contractile machinery. This study explores the myocyte response to pathogen-derived ligands in vitro. The pregnant human myometrial cell line PHM1-41 and primary cultured uterine myocytes responded to Toll-like receptor (TLR) ligands, including the bacterial wall component LPS, which at 100 ng/ml increased contraction of cells embedded within collagen gels over 72 h compared to PBS. LPS-treated myocytes secreted inflammatory mediators, including prostaglandin F2α, the cytokines TNF-α and IL-6, and a range of chemokines. The contractile response to LPS required TLR4 signaling and was independent of prostaglandin synthesis. Neutralizing TNF-α had no effect on LPS-mediated contraction; however, the Rho-associated protein kinase (ROCK) inhibitors Y-27632 (10 µM) and GSK-269962 (50 nM) both abrogated the contractile response. The finding of LPS-mediated contraction was supported by a 1.38 ± 0.072-fold (mean±SE) increase in myosin light-chain phosphorylation 48 h post-treatment, assessed by in-cell Western blot analysis. Together, these data suggest that, in addition to modulating the local inflammatory environment, pathogen-derived ligands may directly promote myometrial contractility via Rho/ROCK signaling, thus contributing to preterm labor-mediated preterm birth.

Molecular regulators of resolution of inflammation: potential therapeutic targets in the reproductive system.

Inflammatory processes are central to reproductive events including ovulation, menstruation, implantation and labour, while inflammatory dysregulation is a feature of numerous reproductive pathologies. In recent years, there has been much research into the endogenous mechanisms by which inflammatory reactions are terminated and tissue homoeostasis is restored, a process termed resolution. The identification and characterisation of naturally occurring pro-resolution mediators including lipoxins and annexin A1 has prompted a shift in the field of anti-inflammation whereby resolution is now observed as an active process, triggered as part of a normal inflammatory response. This review will address the process of resolution, discuss available evidence for expression of pro-resolution factors in the reproductive tract and explore possible roles for resolution in physiological reproductive processes and associated pathologies.