A novel cold-chain free VCG-based subunit vaccine protects against Chlamydia abortus-induced neonatal mortality in a pregnant mouse model.

Chlamydia abortus (Cab) causes spontaneous abortion and neonatal mortality in infected ruminants and pregnant women. Most Cab infections are asymptomatic, although they can be treated with antibiotics, signifying that control of these infections may require alternative strategies, including the use of effective vaccines. However, the limitations imposed by live attenuated and inactivated vaccines further suggest that employment of subunit vaccines may need to be considered. The efficacy of a newly generated Vibrio cholerae ghost (rVCG)-based subunit vaccine harboring the N-terminal portion of the Cab Pmp18D protein (rVCG-Pmp18.3) in preventing Cab-induced abortion or neonatal mortality was evaluated in pregnant mice. Mice were intranasally (IN) immunized and boosted twice, 2 weeks apart with the vaccine, and immunized and unimmunized mice were caged with males 4 weeks postimmunization. The mice were then infected either IN or transcervically (TC) 10 days after pregnancy, and the fertility rate was determined 7 days postpartum. Eight days after delivery, the mice were sacrificed, and Cab infectivity in the lungs and spleens was evaluated by culturing tissue homogenates in tissue culture. Our results demonstrated that the vaccine induced immune effectors that mediated complete clearance of infection in the lungs and significantly reduced Cab infectivity in the spleen following IN immunization. Vaccine immunization also afforded protection against Cab-induced upper genital tract pathology (uterine dilation). Furthermore, while there was no incidence of abortion in both immunized and unimmunized mice, immunized mice were completely protected against neonatal mortality compared to unimmunized infected controls, which lost a significant percentage of their litter 7 days postpartum. Our results establish the capability of the rVCG-Pmp18.3 vaccine to prevent infection in the lungs (mucosal) and spleen (systemic) and protect mice from Cab-induced tubal pathologies and neonatal mortality, a hallmark of Cab infection in ruminants. To advance the commercial potential of this vaccine, future studies will optimize the antigen dose and the number of vaccine doses required for protection of ruminants.

Cocaine-Specific Effects on Exosome Biogenesis in Microglial Cells.

Cocaine is a highly addictive stimulant and a well-known drug, with multiple effects on physiology. Cocaine can have direct effects on all cell types in the brain, including microglia. Microglia can be activated by other conditions, such as infection, inflammation, or injury. However, how cocaine regulates microglia and the influence of cocaine on microglial-derived exosomes remains unknown. Exosomes are nanovesicles that are responsible for intercellular communications, signaling, and trafficking necessary cargo for cell homeostasis. In this study, we hypothesized that cocaine affects exosome biogenesis and composition in BV2 microglial cells. BV2 microglial cells were cultured in exosome-depleted RPMI-1640 media and were treated according to the experimental designs. We observed that cell viability decreased by 11% at 100 µM cocaine treatment but was unaffected at other concentrations. After treatments, the exosomes were isolated from the condition media. Purified exosomes were characterized and quantified using transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA). By NTA, there was a significant decrease in particles/mL after cocaine treatment. There was a 39.5%, 58.1%, 32.3% and 28.1% decrease in particles/mL at 100 nM, 1 µM, 10 µM and 100 µM cocaine, respectively. The characterization of exosomes and exosomal protein was performed by western/dot blot analyses. Tetraspanins CD11b, CD18 and CD63 were relatively unchanged after cocaine treatment. The heat shock proteins (Hsps), Hsp70 and Hsp90, were both significantly increased at 10 µM and 100 µM, but only hsp70 was significantly increased at 10 nM. The Rab proteins were assessed to investigate their role in cocaine-mediated exosomal decrease. Rab11 was significantly decreased at 10 nM, 100 nM, 1 µM, 10 µM and 100 µM by 15%, 28%, 25%, 38% and 22%, respectively. Rab27 was decreased at all concentrations but only significantly decreased at 100 nM, 1 µM and 100 µM cocaine by 21%, 24% and 23%, respectively. Rab35 had no significant changes noted when compared to control. Rab7 increased at all cocaine concentrations but only a significant increase in expression at 100 nM and 10 µM by 1.32-fold and 1.4-fold increase. Cocaine was found to alter exosome biogenesis and composition in BV2 microglial cells. Western and dot blot analyses verified the identities of purified exosomes, and the specific protein compositions of exosomes were found to change in the presence of cocaine. Furthermore, cocaine exposure modulated the expression of exosomal proteins, such as Hsps and Rab GTPases, suggesting the protein composition and formation of microglial-derived exosomes were regulated by cocaine.

Lipopolysaccharide Administration Alters Extracellular Vesicles in Cell Lines and Mice.

Extracellular vesicles (EVs) play a fundamental role in cell and infection biology and have the potential to act as biomarkers for novel diagnostic tools. In this study, we explored the in vitro impact of bacterial lipopolysaccharide administration on cell lines that represents a target for bacterial infection in the host. Administration of lipopolysaccharide at varying concentrations to A549 and BV-2 cell lines caused only modest changes in cell death, but EV numbers were significantly changed. After treatment with the highest concentration of lipopolysaccharide, EVs derived from A549 cells packaged significantly less interleukin-6 and lysosomal-associated membrane protein 1. EVs derived from BV-2 cells packaged significantly less tumor necrosis factor after administration of lipopolysaccharide concentrations of 0.1 µg/mL and 1 µg/mL. We also examined the impact of lipopolysaccharide administration on exosome biogenesis and cargo composition in BALB/c mice. Serum-isolated EVs from lipopolysaccharide-treated mice showed significantly increased lysosomal-associated membrane protein 1 and toll-like receptor 4 levels compared with EVs from control mice. In summary, this study demonstrated that EV numbers and cargo were altered using these in vitro and in vivo models of bacterial infection.

Effects of Pseudomonas aeruginosa on Microglial-Derived Extracellular Vesicle Biogenesis and Composition.

The packaging of molecular constituents inside extracellular vesicles (EVs) allows them to participate in intercellular communication and the transfer of biological molecules, however the role of EVs during bacterial infection is poorly understood. The goal of this study was to examine the effects of Pseudomonas aeruginosa (P. aeruginosa) infection on the biogenesis and composition of EVs derived from the mouse microglia cell line, BV-2. BV-2 cells were cultured in exosome-free media and infected with 0, 1.3 × 104, or 2.6 × 104 colony forming units per milliliter P. aeruginosa for 72 h. The results indicated that compared with the control group, BV-2 cell viability significantly decreased after P. aeruginosa infection and BV-2-derived EVs concentration decreased significantly in the P. aeruginosa-infected group. P. aeruginosa infection significantly decreased chemokine ligand 4 messenger RNA in BV-2-derived infected EVs, compared with the control group (p ≤ 0.05). This study also revealed that heat shock protein 70 (p ≤ 0.05) and heat shock protein 90ß (p ≤ 0.001) levels of expression within EVs increased after P. aeruginosa infection. EV treatment with EVs derived from P. aeruginosa infection reduced cell viability of BV-2 cells. P. aeruginosa infection alters the expression of specific proteins and mRNA in EVs. Our study suggests that P. aeruginosa infection modulates EV biogenesis and composition, which may influence bacterial pathogenesis and infection.

Alcohol Exposure Impacts the Composition of HeLa-Derived Extracellular Vesicles.

Extracellular vesicles are nanosized vesicles that are under intense investigation for their role in intercellular communication. Extracellular vesicles have begun to be examined for their role in disease protection and their role as disease biomarkers and/or vaccine agents. The goal of this study was to investigate the effects of alcohol exposure on the biogenesis and composition of extracellular vesicles derived from the cervical cancer line, HeLa. The HeLa cells were cultured in exosome-free media and were either mock-treated (control) or treated with 50 mM or 100 mM of alcohol for 24 h and 48 h. Our results demonstrated that alcohol significantly impacts HeLa cell viability and exosome biogenesis/composition. Importantly, our studies demonstrate the critical role of alcohol on HeLa cells, as well as HeLa-derived extracellular vesicle biogenesis and composition. Specifically, these results indicate that alcohol alters extracellular vesicles' packaging of heat shock proteins and apoptotic proteins. Extracellular vesicles serve as communicators for HeLa cells, as well as biomarkers for the initiation and progression of disease.

The Role of Lipopolysaccharide-Induced Extracellular Vesicles in Cardiac Cell Death.

Exosomes play a crucial role in the progression of infectious diseases, as exosome release and biogenesis are affected by external factors, such as pathogenic infections. Pyrogens may aide in the progression of diseases by triggering inflammation, endothelial cell injury, and arterial plaque rupture, all of which can lead to acute coronary disease, resulting in cardiac tissue death and the onset of a cardiac event (CE). To better understand the effects of Gram-negative bacterial infections on exosome composition and biogenesis, we examined exosome characteristics after treatment of AC16 human cardiomyocytes with lipopolysaccharide (LPS), which served as a model system for Gram-negative bacterial infection. Using increasing doses (0, 0.1, 1, or 10 µg) of LPS, we showed that treatment with LPS substantially altered the composition of AC16-derived exosomes. Both the relative size and the quantity (particles/mL) of exosomes were decreased significantly at all tested concentrations of LPS treatment compared to the untreated group. In addition, LPS administration reduced the expression of exosomal proteins that are related to exosomal biogenesis. Conversely, we observed an increase in immunomodulators present after LPS administration. This evaluation of the impact of LPS on cardiac cell death and exosome composition will yield new insight into the importance of exosomes in a variety of physiological and pathological processes as it relates to disease progression, diagnosis, and treatment.

Perspective on Adenoviruses: Epidemiology, Pathogenicity, and Gene Therapy.

Human adenoviruses are large (150 MDa) doubled-stranded DNA viruses that cause respiratory infections. These viruses are particularly pathogenic in healthy and immune-compromised individuals, and currently, no adenovirus vaccine is available for the general public. The purpose of this review is to describe (i) the epidemiology and pathogenicity of human adenoviruses, (ii) the biological role of adenovirus vectors in gene therapy applications, and (iii) the potential role of exosomes in adenoviral infections.

Alcohol Modulates the Biogenesis and Composition of Microglia-Derived Exosomes.

Exosomes are small extracellular vesicles that have emerged as an important tool for intercellular communication. In the central nervous system, exosomes can mediate glia and neuronal communication. Once released from the donor cell, exosomes can act as discrete vesicles and travel to distant and proximal recipient cells to alter cellular function. Microglia cells secrete exosomes due to stress stimuli of alcohol abuse. The goal of this study was to investigate the effects of alcohol exposure on the biogenesis and composition of exosomes derived from microglia cell line BV-2. The BV-2 cells were cultured in exosome-free media and were either mock treated (control) or treated with 50 mM or 100 mM of alcohol for 48 and 72 h. Our results demonstrated that alcohol significantly impacted BV-2 cell morphology, viability, and protein content. Most importantly, our studies revealed that exosome biogenesis and composition was affected by alcohol treatment.

Pathogens and Their Effect on Exosome Biogenesis and Composition.

Exosomes are nanosized membrane microvesicles (30⁻100 nm) that have the capability to communicate intercellularly and transport cell components (i.e., miRNA, mRNA, proteins and DNA). Exosomes are found in nearly every cell type (i.e., mast cells, dendritic, tumor, and macrophages). There have been many studies that have shown the importance of exosome function as well as their unique packaging and targeting abilities. These characteristics make exosomes ideal candidates to act as biomarkers and therapeutics for disease. We will discuss the biogenesis, composition, and relationship of exosomes with non-viral microbial infections including gram-negative bacteria, gram-positive bacteria, Leishmania and Trypanosoma cruzi.