Assessment of immunomodulatory effects of five commonly used parabens on human THP-1 derived macrophages: Implications for ecological and human health impacts.

Parabens are widely used as broad-spectrum anti-microbials and preservatives in food, cosmetics, pharmaceuticals, and personal care products. Studies suggest that the utilization of parabens has substantially increased over the past years, particularly during the global pandemic of coronavirus disease 2019 (COVID-19). Although parabens are generally recognized as safe by the U.S. FDA, some concerns have been raised regarding the potential health effects of parabens associated with immunotoxicity. Herein, we comprehensively investigated several key characteristics of immunotoxicants of five commonly used parabens (methyl-, ethyl-, propyl-, butyl-, and benzyl parabens) in human THP-1 derived macrophages, which are effector cells serving as a first line of host defense against pathogens and tumor immunosurveillance. The results indicate parabens, at concentrations found in humans and biota, significantly dampened macrophage chemotaxis and secretion of major pro-inflammatory cytokines (TNF-α and IL-6) and anti-inflammatory cytokine (IL-10), corroborating the mRNA expression profile. Furthermore, some parabens were found to markedly alter macrophage adhesion and cell surface expression of costimulatory molecules, CD80+ and CD86+, and significantly increase macrophage phagocytosis. Collectively, these findings heighten awareness of potential immunotoxicity posed by paraben exposure at biologically relevant concentrations, providing implications for human health and ecological risks associated with immune dysfunctions.

Cytotoxic stress caused by azalamellarin D (AzaD) interferes with cellular protein translation by targeting the nutrient-sensing kinase mTOR.

Analogs of pyrrole alkaloid lamellarins exhibit anticancer activity by modulating multiple cellular events. Lethal doses of several lamellarins were found to enhance autophagy flux in HeLa cells, suggesting that lamellarins may modulate protein homeostasis through the interference of proteins or kinases controlling energy and nutrient metabolism. To further delineate molecular mechanisms and their targets, our results herein show that azalamellarin D (AzaD) cytotoxicity could cause translational attenuation, as indicated by a change in eIF2α phosphorylation. Intriguingly, acute AzaD treatment promoted the phosphorylation of GCN2, a kinase that transduces the integrated stress response (ISR), and prolonged exposure to AzaD could increase the levels of the phosphorylated forms of eIF2α and the other ISR kinase protein kinase R (PKR). However, the effects of AzaD on ISR signalling were marginally abrogated in cells with genetic deletion of GCN2 and PKR, and evaluation of protein target engagement by cellular thermal shift assay (CETSA) revealed no significant interaction between AzaD and ISR kinases. Further investigation revealed that acute AzaD treatment negatively affected mechanistic target of rapamycin (mTOR) phosphorylation and signalling. The analyses by CETSA and computational modelling indicated that mTOR may be a possible protein target for AzaD. These findings indicate the potential for developing lamellarins as novel agents for cancer treatment.

Intra-articular injection of flavopiridol-loaded microparticles for treatment of post-traumatic osteoarthritis.

Rapid joint clearance of small molecule drugs is the major limitation of current clinical approaches to osteoarthritis and its subtypes, including post-traumatic osteoarthritis (PTOA). Particulate systems such as nano/microtechnology could provide a potential avenue for improved joint retention of small molecule drugs. One drug of interest for PTOA treatment is flavopiridol, which inhibits cyclin-dependent kinase 9 (CDK9). Herein, polylactide-co-glycolide microparticles encapsulating flavopiridol were formulated, characterized, and evaluated as a strategy to mitigate PTOA-associated inflammation through the inhibition of CDK9. Characterization of the microparticles, including the drug loading, hydrodynamic diameter, stability, and release profile was performed. The mean hydrodynamic diameter of flavopiridol particles was ∼15 µm, indicating good syringeability and low potential for phagocytosis. The microparticles showed no cytotoxicity in-vitro, and drug activity was maintained after encapsulation, even after prolonged exposure to high temperatures (60 °C). Flavopiridol-loaded microparticles or blank (unloaded) microparticles were administered by intraarticular injection in a rat knee injury model of PTOA. We observed significant joint retention of flavopiridol microparticles compared to the soluble flavopiridol, confirming the sustained release behavior of the particles. Matrix metalloprotease (MMP) activity, an indicator of joint inflammation, was significantly reduced by flavopiridol microparticles 3 days post-injury. Histopathological analysis showed that flavopiridol microparticles reduced PTOA severity 28 days post-injury. Taken altogether, this work demonstrates a promising biomaterial platform for sustained small molecule drug delivery to the joint space as a therapeutic measure for post-traumatic osteoarthritis. STATEMENT OF SIGNIFICANCE: Post-traumatic osteoarthritis (PTOA) begins with the deterioration of subchondral bone and cartilage after acute injuries. In spite of the prevalence of PTOA and its associated financial and psychological burdens, therapeutic measures remain elusive. A number of small molecule drugs are now under investigation to replace FDA-approved palliative measures, including cyclin-dependent kinase 9 (CDK9) inhibitors which work by targeting early inflammatory programming after injury. However, the short half-life of these drugs is a major hurdle to their success. Here, we show that biomaterial encapsulation of Flavopiridol (CDK9 inhibitor) in poly (lactic-co-glycolic acid) microparticles is a promising route for direct delivery and improved drug retention time in the knee joint. Moreover, administration of the flavopiridol microparticles reduced the severity of PTOA.

Lactate Exposure Promotes Immunosuppressive Phenotypes in Innate Immune Cells.

INTRODUCTION: Lactate secreted by tumors is not just a byproduct, but rather an active modulator of immune cells. There are few studies aimed at investigating the true effect of lactate, which is normally confounded by pH. Such a knowledge gap needs to be addressed. Herein, we studied the immunomodulatory effects of lactate on dendritic cells (DCs) and macrophages (MΦs). METHODS: Bone marrow-derived innate immune cells were treated with 50 mM sodium lactate (sLA) and incubated for 2 days or 5 days at 37 °C. Controls included media, lipopolysaccharide (LPS), MCT inhibitors (α-cyano-4-hydroxycinnamic acid and AR-C15585). Flow cytometric analysis of immune phenotypes were performed by incubating cells with specific marker antibodies and viability dye. Differential expression analyses were conducted on R using limma-voom and adjusted p-values were generated using the Bejamini-Hochberg Procedure. RESULTS: Lactate exposure attenuated DC maturation through the downregulation of CD80 and MHCII expression under LPS stimulation. For MΦs, lactate exposure resulted in M2 polarization as evidenced by the reduction of M1 markers (CD38 and iNOS), and the increase in expression of CD163 and Arg1. We also revealed the role of monocarboxylate transporters (MCTs) in mediating lactate effect in MΦs. MCT4 inhibition significantly boosted lactate M2 polarization, while blocking of MCT1/2 failed to reverse the immunosuppressive effect of lactate, correlating with the result of gene expression that lactate increased MCT4 expression, but downregulated the expression of MCT1/2. CONCLUSIONS: This research provides valuable insight on the influence of metabolic products on tumor immunity and will help to identify novel metabolic targets for augmenting cancer immunotherapies.

Polymer-loaded hydrogels serve as depots for lactate and mimic "cold" tumor microenvironments.

The burgeoning field of biomaterials for immunotherapy has aided in the understanding of foundational mechanisms of cancer immunology. In particular, implantable biomaterials can be engineered to investigate specific aspects of the tumor microenvironment either singularly or in combination. Of note, the metabolite - lactate, a byproduct of anaerobic glycolysis, is known to reprogram immune cells, resulting in increased tumor survival. An adequate model that can recapitulate intratumoral lactate concentrations does not exist. In this study, we demonstrate that a simple biomaterial platform could be developed as an instructive tool to decipher the effects of lactate in vivo. Briefly, we demonstrate that a peptide hydrogel loaded with granulocyte-macrophage colony stimulating factor and poly-(lactic-co-glycolic acid)/(lactic acid) microparticles can generate the localized lactate concentrations (∼2-22 mM) and cellular makeup of the tumor microenvironment, following subcutaneous implantation in mice. Furthermore, infiltrating immune cells adopt phenotypes similar to those seen in other in vitro and in vivo cancer models, including immunosupressive dendritic cells. This hydrogel system is a framework to interrogate immune cell modulation in cancer-like environments using safe and degradable biomaterials. Moreover, this system can be multifaceted, as incorporation of other cancer tumor environmental factors or chemotherapeutic drugs is facile and could be insightful in developing or improving immunotherapies.

Cytosolic Delivery of Proteins Using Amphiphilic Polymers with 2-Pyridinecarboxaldehyde Groups for Site-Selective Attachment.

Protein-based drugs are a promising class of therapeutics, but poor membrane permeability typically limits their application to extracellular receptors. Delivery strategies that can transport functional proteins to reach intracellular targets are needed, but with many current approaches, biomolecules become entrapped in the endosomes. This greatly reduces the effective concentrations of therapeutic agents at the target sites. Herein, we report a bioconjugation-based approach for intracellular protein delivery by site-selectively attaching amphiphilic polymers to the N-terminal positions of proteins using 2-pyridinecarboxaldehyde groups. The reaction is simple and features mild, aqueous conditions with no required genetic engineering of the proteins. Imaging studies demonstrate that the polymer-protein conjugates are successfully delivered into the cytosol of various cancer cell lines, likely through a membrane fusion mechanism. When conjugated to the delivery polymers, the activity of modified RNase A was retained and notably promoted cytotoxicity in cancer cells upon delivery to the cytosol. This work therefore provides a promising platform for protein-based material delivery for therapeutic applications.

Assessment of the Endocrine-Disrupting Effects of Trichloroethylene and Its Metabolites Using in Vitro and in Silico Approaches.

Trichloroethylene (TCE) is a ubiquitous environmental contaminant, which may have effects on both ecosystem and human health. TCE has been reported to cause several toxic effects, but little effort has been made to assess the ecological risks of TCE or its major metabolites: trichloroethanol (TCOH), trichloroacetic acid, and oxalic acid (OA). In this study, the endocrine-disrupting potential of TCE and its metabolites were investigated using in vitro and in silico approaches. We examined alterations in the steroidogenesis pathway using the NCI-H295R cell line and utilized receptor-mediated luciferase reporter cell lines to identify effects on estrogen and androgen receptors. Molecular docking was also used to explore chemical interactions with these receptors. All test chemicals except OA significantly increased 17ß-estradiol production which can be attributed to an up-regulation of 17ß-hydroxysteroid dehydrogenase. Moreover, TCOH exhibited significant antiestrogenic activity with a RIC20 (20% relative inhibitory concentration) of 3.7 × 10-7 M. Molecular docking simulation supported this finding with lower docking scores for TCOH, indicating that hydrogen bonds may stabilize the interaction between TCOH and the estrogen receptor binding pocket. These findings suggest that TCE contamination poses an endocrine-disrupting threat, which has implications for both ecological and human health.

Direct detection of nitrotyrosine-containing proteins using an aniline-based oxidative coupling strategy.

A convenient two-step method is described for the detection of nitrotyrosine-containing proteins. First, nitrotyrosines are reduced to aminophenols using sodium dithionite. Following this, an oxidative coupling reaction is used to attach anilines bearing fluorescence reporters or affinity probes. Features of this approach include fast reaction times, pmol-level sensitivity, and excellent chemoselectivity.