Activity-based Tools for Interrogating Host Biology During Infection.

Host cells sense and respond to pathogens by dynamically regulating cell signaling. The rapid modulation of signaling pathways is achieved by post-translational modifications (PTMs) that can alter protein structure, function, and/or binding interactions. By using chemical probes to broadly profile changes in enzyme function or side-chain reactivity, activity-based protein profiling (ABPP) can reveal PTMs that regulate host-microbe interactions. While ABPP has been widely utilized to uncover microbial mechanisms of pathogenesis, in this review, we focus on more recent applications of this technique to the discovery of host PTMs and enzymes that modulate signaling within infected cells. Collectively, these advances underscore the importance of ABPP as a tool for interrogating the host response to infection and identifying potential targets for host-directed therapies.

Analysis of gene expression from human breastmilk cells: A comparison between low and high producers, and the influence of anxiety and depression on milk production, gene expression and bacterial production.

BACKGROUND: Breastmilk is considered the gold standard of infant nutrition. Many mothers have difficulty with breastfeeding and over 50% of women stop due to perceived low production. AIMS AND METHODS: Our study compared gene expression in 8 samples of low and high producers of milk. All subjects were administered GAD-7 and PHQ-9 questionnaires. Low-producers were all found to have more depression and anxiety compared to high-producers. RESULTS: We did not find significant differences between gene expression between low and high milk producers. Only 5 of 8 samples contained a significant number of human cells. We did find differences in the amount of various bacterial populations. CONCLUSION: Our results indicate that gene expression in breastmilk is complicated by collection methods. We recommend that even though some women produced less than 600 ml of milk over a 24-hour period of time, due to the nature of the bacteria found in milk they try to breastfeed as much as they can for the health benefits of their infants. the rich bacterial diversity in all patients including the low producers strongly suggests that even women producing lesser quantities of milk confer their children numerous benefits by breastfeeding them.

1-(4-nitrobenzenesulfonyl)-4-penylpiperazine increases the number of Peyer's patch-associated regenerating crypts in the small intestines after radiation injury.

OBJECTIVE: Exposure to lethal doses of radiation has severe effects on normal tissues. Exposed individuals experience a plethora of symptoms in different organ systems including the gastrointestinal (GI) tract, summarized as Acute Radiation Syndrome (ARS). There are currently no approved drugs for mitigating GI-ARS. A recent high-throughput screen performed at the UCLA Center for Medical Countermeasures against Radiation identified compounds containing sulfonylpiperazine groups with radiation mitigation properties to the hematopoietic system and the gut. Among these 1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine (Compound #5) efficiently mitigated gastrointestinal ARS. However, the mechanism of action and target cells of this drug is still unknown. In this study we examined if Compound #5 affects gut-associated lymphoid tissue (GALT) with its subepithelial domes called Peyer's patches. METHODS: C3H mice were irradiated with 0 or 12 Gy total body irradiation (TBI). A single dose of Compound #5 or solvent was administered subcutaneously 24 h later. 48 h after irradiation the mice were sacrificed, and the guts examined for changes in the number of visible Peyer's patches. In some experiments the mice received 4 daily injections of treatment and were sacrificed 96 h after TBI. For immune histochemistry gut tissues were fixed in formalin and embedded in paraffin blocks. Sections were stained with H&E, anti-Ki67 or a TUNEL assay to assess the number of regenerating crypts, mitotic and apoptotic indices. Cells isolated from Peyer's patches were subjected to immune profiling using flow cytometry. RESULTS: Compound #5 significantly increased the number of visible Peyer's patches when compared to its control in non-irradiated and irradiated mice. Additionally, assessment of total cells per Peyer's patch isolated from these mice demonstrated an overall increase in the total number of Peyer's patch cells per mouse in Compound #5-treated mice. In non-irradiated animals the number of CD11bhigh in Peyer's patches increased significantly. These Compound #5-driven increases did not coincide with a decrease in apoptosis or an increase in proliferation in the germinal centers inside Peyer's patches 24 h after drug treatment. A single dose of Compound #5 significantly increased the number of CD45+ cells after 12 Gy TBI. Importantly, 96 h after 12 Gy TBI Compound #5 induced a significant rise in the number of visible Peyer's patches and the number of Peyer's patch-associated regenerating crypts. CONCLUSION: In summary, our study provides evidence that Compound #5 leads to an influx of immune cells into GALT, thereby supporting crypt regeneration preferentially in the proximity of Peyer's patches.

Nanoparticle-releasing nanofiber composites for enhanced in vivo vaginal retention.

Current approaches for topical vaginal administration of nanoparticles result in poor retention and extensive leakage. To overcome these challenges, we developed a nanoparticle-releasing nanofiber delivery platform and evaluated its ability to improve nanoparticle retention in a murine model. We individually tailored two components of this drug delivery system for optimal interaction with mucus, designing (1) mucoadhesive fibers for better retention in the vaginal tract, and (2) PEGylated nanoparticles that diffuse quickly through mucus. We hypothesized that this novel dual-functioning (mucoadhesive/mucus-penetrating) composite material would provide enhanced retention of nanoparticles in the vaginal mucosa. Equivalent doses of fluorescent nanoparticles were vaginally administered to mice in either water (aqueous suspension) or fiber composites, and fluorescent content was quantified in cervicovaginal mucus and vaginal tissue at time points from 24 h to 7d. We also fabricated composite fibers containing etravirine-loaded nanoparticles and evaluated the pharmacokinetics over 7d. We found that our composite materials provided approximately 30-fold greater retention of nanoparticles in the reproductive tract at 24 h compared to aqueous suspensions. Compared to nanoparticles in aqueous suspension, the nanoparticles in fiber composites exhibited sustained and higher etravirine concentrations after 24 h and up to 7d, demonstrating the capabilities of this new delivery platform to sustain nanoparticle release out to 3d and drug retention out to one week after a single administration. This is the first report of nanoparticle-releasing fibers for vaginal drug delivery, as well as the first study of a single delivery system that combines two components uniquely engineered for complementary interactions with mucus.

Chitosan enhances nanoparticle delivery from the reproductive tract to target draining lymphoid organs.

To prime adaptive immune responses from the female reproductive tract (FRT), particulate antigens must be transported to draining lymph nodes (dLNs) since there are no local organized lymphoid structures equivalent to those found in the respiratory or gastrointestinal tracts. However, little is known about how to safely and effectively navigate successive barriers to transport such as crossing the epithelium and gaining access to migratory cells and lymphatic drainage that provide entry into dLNs. Here, we demonstrate that intravaginal pre-treatment with chitosan significantly facilitates translocation of nanoparticles (NPs) across the multilayered vaginal epithelium to target dLNs. In addition, chitosan pre-treatment was found to enhance NP associations with immunogenic antigen presenting cells in the vaginal submucosa. These observations indicate that chitosan may have great potential as an adjuvant for both local and systemic protective immunity against viral infections in the FRT.

Engineering immunity in the mucosal niche against sexually transmitted infections.

The mucosal surfaces of the genital tract are the site of entry to over 30 different bacterial, parasitic, and viral pathogens that are the cause of sexually transmitted infections (STIs) including HIV. Women and adolescent girls are more severely impacted by STIs than men due in part to a greater biological susceptibility for acquiring infections and differences in disease sequelae. While it is widely accepted that preventative vaccines against the most commonly transmitted STIs would have a major impact on decreasing the global health burden of STIs for women worldwide, several challenges preclude their development. The female genital tract is a complex niche of microflora, hormonal influences, and immune tissues and cells that result in a mucosal immune system that is distinct from other mucosal sites and from our systemic immune system. An appreciation of these differences and their effect on shaping mucosal immunity to sexually transmitted pathogens is an important determinant for the design of effective STI vaccines. Here we describe the anatomy and mucosal immune system of the female reproductive tract, and discuss bioengineering strategies to design mucosal vaccines that overcome delivery challenges and coordinate the presentation kinetics and compartmentalization of antigens and adjuvants to relevant mucosal immune cell subsets. In particular, we describe recent progress in understanding the role of specific mucosal dendritic cell subsets in facilitating immune responses to pathogenic microbes in the genital mucosa. We also discuss the development of pathogen-mimicking materials that may be useful for engineering protective immunity in this mucosal niche.

Effect of Mucosal Cytokine Administration on Selective Expansion of Vaginal Dendritic Cells to Support Nanoparticle Transport.

PROBLEM: The capacity of antigen-carrying vaccine nanoparticles (NPs) administered vaginally to stimulate local immune responses may be limited by the relatively low numbers of antigen-presenting cells (APCs) in the genital mucosa. Because inflammation is associated with increased susceptibility to sexually transmitted infections, we sought to increase APC numbers without causing inflammation. METHOD OF STUDY: In this study, we evaluated intravaginal delivery of chemokines, growth factors, or synthetic adjuvants to expand APCs in reproductive tissues. RESULTS: We found that granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated expansion of CD11b+ dendritic cells (DCs) within 24 hr of intravaginal administration, with no effect on Langerhans cells or macrophages. Expansion of the CD11b+ DC population was not associated with increased inflammatory cytokine production, and these cells retained phagocytic function. CONCLUSION: Our data suggest that non-inflammatory expansion of mucosal APCs by intravaginal GM-CSF could be used as an adjuvanting strategy to potentiate the genital immune response to nanoparticulate mucosal vaccines.

Nanoparticulate cellular patches for cell-mediated tumoritropic delivery.

The targeted delivery of therapeutics to tumors remains an important challenge in cancer nanomedicine. Attaching nanoparticles to cells that have tumoritropic migratory properties is a promising modality to address this challenge. Here we describe a technique to create nanoparticulate cellular patches that remain attached to the membrane of cells for up to 2 days. NeutrAvidin-coated nanoparticles were anchored on cells possessing biotinylated plasma membrane. Human bone marrow derived mesenchymal stem cells with nanoparticulate patches retained their inherent tumoritropic properties as shown using a tumor model in a 3D extracellular matrix. Additionally, human umbilical vein endothelial cells with nanoparticulate patches were able to retain their functional properties and form multicellular structures as rapidly as unmodified endothelial cells. These results provide a novel strategy to actively deliver nanostructures and therapeutics to tumors utilizing stem cells as carriers and also suggest that nanoparticulate cellular patches may have applications in tissue regeneration.