Shock due to Gram-negative bacterial sepsis is a consequence of acute inflammatory response to lipopolysaccharide (LPS) or endotoxin released from bacteria. LPS is a major constituent of the outer membrane of Gram-negative bacteria, and its terminal disaccharide phospholipid (lipid A) portion contains the key structural features responsible for toxic activity. Based on the proposed structure of nontoxic Rhodobacter capsulatus lipid A, a fully stabilized endotoxin antagonist E5531 has been synthesized. In vitro, E5531 demonstrated potent antagonism of LPS-mediated cellular activation in a variety of systems. In vivo, E5531 protected mice from LPS-induced lethality and, in cooperation with an antibiotic, protected mice from a lethal infection of viable Escherichia coli.
ER-112022 is a novel acyclic synthetic lipid (17), that are important modulators of LPS activity. Several lines of evidence suggest that TLR4 is the main signal-transducing molecule in the LPS receptor complex. First, animals that lack a functional TLR4, but not those without a functional TLR2, are markedly hyporesponsive to LPS (11, 18 -22). Second, LPS induces activation of HEK293 cells transfected with TLR4 cDNA, and this activation is blocked by a lipid A-based LPS antagonist (12). Third, TLR4 is responsible for the fine discriminatory ability of the LPS receptor. For example, the unique species-specific pharmacology associated with small alterations in lipid A structure can be attributed to differences in the primary structure of TLR4 (23, 24). In contrast, TLR2 appears to be involved in responses to a variety of bacterial compounds, such as bacterial lipoproteins, peptidoglycan, and lipoarabinomannan, as well as whole bacteria and yeast particles (21,22,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34). It is still unclear whether TLR2 can mediate lipid A signals (35), although it is possible that TLR2 represents an LPS receptor involved in the recognition of non-enteric endotoxins.An important approach to understanding how any receptor system functions is to define its pharmacology. Partial lipid A structures have been important in investigating mechanisms of LPS binding and cell activation (36). These compounds include several naturally occurring, bacterially derived LPS antagonists such as deacylated LPS, lipid IVa, and Rhodobacter sphaeroides lipid A (37-39) as well as several synthetic lipid A-like
A series of novel, synthetic compounds containing lipids linked to a phosphate-containing acyclic backbone are shown to have similar biological properties to lipopolysaccharide (LPS). These compounds showed intrinsic agonistic properties when tested for their ability to stimulate tumor necrosis factor-␣ in human whole blood and interleukin-6 in U373 human glioblastoma cells without added LPS coreceptor CD14. The presence of the LPS antagonist E5564 completely blocked responses, suggesting that the novel compounds and LPS share a common mechanism of cell activation. Stereoselectivity of the molecules was observed in vitro; compounds with an R,R,R,R-configuration were strongly agonistic, whereas compounds with an R,S,S,R-configuration were much weaker in their activity on human whole blood and U373 cells. We also tested the effect of the compounds in cells transfected with the LPS receptor Tolllike receptor 4 (TLR4), with similar results, further supporting a shared mechanism with LPS. This was confirmed in vivo where the agonists failed to elicit cytokine responses in C3H/HeJ mice lacking TLR4 signaling. Because LPS-like molecules enhance immune responses, the compounds were mixed with tetanus toxoid and administered to mice in an immunization protocol to test for adjuvant activity. They enhanced the generation of specific antibodies against tetanus toxoid. Our results indicate that these unique compounds behave as agonists of TLR4, resulting in responses similar to those elicited by LPS. They display adjuvant activity in vivo and may be useful for the development of vaccine therapies.
During the COVID-19 public health emergency, many actions have been undertaken to help ensure that patients and health care providers have timely and continued access to high-quality medical devices to respond effectively. The development and validation of new testing supplies and equipment, including collection swabs, has helped to expand the availability and capability for various diagnostic, therapeutic, and protective medical devices in high demand during the COVID-19 emergency. Here, we report the initial validation of a new injection-molded anterior nasal swab, ClearTip™, that was experimentally validated in a laboratory setting as well as in independent clinical studies in comparison to gold standard flocked swabs. We have also developed an in vitro anterior nasal tissue model which offers a novel, efficient, and clinically relevant validation tool to replicate the clinical swabbing workflow with high fidelity, while being accessible, safe, reproducible, and time- and cost-effective. ClearTip™ displayed greater inactivated virus release in the benchtop model, confirmed by its greater ability to report positive samples in a small clinical study in comparison to flocked swabs. We also quantified the detection of biological materials, as a proxy for viral material, in multi-center pre-clinical and clinical studies which showed a statistically significant difference in one study and a reduction in performance in comparison to flocked swabs. Taken together, these results emphasize the compelling benefits of non-absorbent injection-molded anterior nasal swabs for COVID-19 detection, comparable to standard flocked swabs. Injection-molded swabs, as ClearTip™, could have the potential to support future swab shortages, due to its manufacturing advantages, while offering benefits in comparison to highly absorbent swabs in terms of comfort, limited volume collection, and potential multiple usage.
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