Protocols in Current Issue
Protocols in Past Issues
0 Q&A 4394 Views Apr 5, 2021

Most vaccines require co-delivery of an adjuvant in order to generate the desired immune responses. However, many currently available adjuvants are non-biodegradable, have limited efficacy, and/or poor safety profile. Thus, new adjuvants, or self-adjuvanting vaccine delivery systems, are required. Here, we proposed a self-adjuvanting delivery system that is fully defined, biodegradable, and non-toxic. The system is produced by conjugation of polyleucine to peptide antigen, followed by self-assembly of the conjugate into nanoparticles. The protocol includes solid-phase peptide synthesis of the vaccine conjugate, purification, self-assembly and physicochemical characterization of the product. Overall, this protocol describes, in detail, the production of a well-defined and effective self-adjuvanting delivery system for peptide antigens, along with tips for troubleshooting.

0 Q&A 3461 Views Mar 20, 2021

The mucus layer in the gastrointestinal tract covers the apical surface of intestinal epithelial cells, protecting the mucosal tissue from enteric pathogen and commensal microorganisms. The mucus is primarily composed of glycosylated protein called mucins, which are produced by goblet cells, a type of columnar epithelial cells in the intestinal tract. Defective mucin barrier facilitates infection caused by enteric pathogen and triggers inflammation due to invasion of commensal or opportunistic pathogens into the intestinal epithelial mucosa. Several bacterial species in the gut produce enzymes that are capable of degradation of the mucus. Defective mucin production or increased abundance of mucolytic bacteria are clinically linked to inflammatory bowel disease. Measurement of mucolytic enzymes in the feces, therefore, can be implicated in clinical and experimental research on intestinal disorders. Here, we describe a step-by-step procedure for the measurement of the mucolytic enzyme activity in fecal samples.

0 Q&A 3498 Views Mar 20, 2020
Ex vivo biophysical measurements provide valuable insights into understanding both physiological and pathogenic processes. One critical physiological mechanism that is regulated by these biophysical properties is cilia-generated flow that mediates mucociliary clearance, which is known to provide protection against foreign particles and pathogens in the upper airway. To measure ciliary clearance, several techniques have been implemented, including the use of radiolabeled particles and imaging with single-photon emission computerized tomography (SPECT) methods. Although non-invasive, these tests require the use of specialized equipment, limiting widespread use. Here we describe a method of ex vivo imaging of cilia-generated flow, adapted from previously reported methods, to make it more accessible and higher throughput for researchers. We excise trachea from mice quickly after euthanasia, cut it longitudinally and place it in an inhouse made slide. We apply fluorescent particles to measure particle movement under a fluorescent microscope, followed by analysis with ImageJ, allowing calculation of fluid flow generated by cilia under different conditions. This method enables ex vivo measurements in tissue with minimal investment or special equipment, giving opportunity to investigate and discover important biophysical properties associated with ciliary movement of the trachea in physiology and disease.
0 Q&A 4068 Views Apr 20, 2019
Intranasal administration of vaccine adjuvants directly deliver therapeutic agents to the lungs to induce potent lung mucosal immune responses. Cyclic di-GMP (CDG) is a promising mucosal vaccine adjuvant candidate capable of inducing protective immunity. This protocol describes an in vivo approach to induce and detect mucosal (lung) and systemic (blood and spleen) vaccine adjuvant responses of CDG. This protocol also includes the methods to detect both humoral and cellular immune responses of CDG adjuvant. Last, this protocol can be used to study other cyclic dinucleotides as mucosal vaccine adjuvants.
0 Q&A 4720 Views Dec 20, 2018
Eimeria vermiformis is a tissue specific, intracellular protozoan that infects the murine small intestinal epithelia, which has been widely used as a coccidian model to study mucosal immunology. This mouse infection model is valuable to investigate the mechanisms of host protection against primary and secondary infection in the small intestine. Here, we describe the generation of an E. vermiformis stock solution, preparation of sporulated E. vermiformis to infect mice and determination of oocysts burden. This protocol should help to establish a highly reproducible natural infection challenge model to study immunity in the small intestine. The information obtained from using this mouse model can reveal fundamental mechanisms of interaction between the pathogen and the immune response, e.g., provided by intraepithelial lymphocytes (IEL) at the basolateral site of epithelial cells but also a variety of other immune cell populations present in the gut.
0 Q&A 3720 Views Nov 5, 2018
Activation of inflammasomes in peritoneal macrophages and intestinal epithelial cells (IEC) leads to the release of eicosanoids. To assess the amount of eicosanoids released by IEC, lipids need to be isolated from whole tissue previous to analysis by lipid mass spectrometry or ELISA. This protocol describes how to isolate lipids from intestinal tissue for analysis by PGE2-ELISA and normalize to tissue protein content.
0 Q&A 7260 Views May 5, 2018
We have developed a protocol to purify RNA from DSS (Dextran Sulfate Sodium)-treated mouse tissues. This method, which prevents downstream inhibition of q-RT-PCR observed in DSS-treated tissues, relies on successive precipitations with lithium chloride.
0 Q&A 5500 Views Apr 20, 2018
Enteroaggregative Escherichia coli (EAEC) is a recognized cause of acute diarrhea among both children and adults worldwide. EAEC strains are characterized by the presence of aggregative adherence fimbriae (AAF), which play a key role in pathogenesis by mediating attachment to the intestinal mucosa and by triggering host inflammatory responses. The aggregative adherence fimbria II (AAF/II) is the most important adherence factor of EAEC prototype strain 042 (EAEC042) to intestinal cells. Multiple receptors for AAF/II on epithelial cells have been identified including the transmembrane signaling mucin Muc1. This protocol describes a method to measure adherence of EAEC strains to HEK293 cells expressing the Muc1 glycoprotein.
0 Q&A 6282 Views Feb 5, 2018
Colonization and penetration of the epithelium is the infection-initiating route of mucosal pathogens. The epithelium counteracts infection by eliciting host cell responses while maintaining the mucosal barrier function. The obligate human sexually transmitted bacterium Neisseria gonorrhoeae, or gonococcus (GC) infects the female reproductive tract primarily from the endocervical epithelium. Due to lack of an infection model that mimics all aspects of human infections in the female reproductive tract, GC pathogenesis is poorly understood. This protocol takes advantage of the viability and functional integrity of human cervical tissues propagated in culture to generate an ex vivo infection model. This tissue model maintains the nature of the infection target and environment without any manipulation such as immortalization of epithelial cells by viruses. Using immunofluorescence microscopy, the interaction of GC with the endocervical epithelium was analyzed.
0 Q&A 8330 Views Dec 20, 2017
The oral microbiome has been implicated as a trigger for immune responsiveness in the oral cavity, particularly in the setting of the inflammatory disease periodontitis. The protocol presented here is aimed at characterizing the oral microbiome in murine models at steady state and during perturbations of immunity or physiology. Herein, we describe murine oral microbiome sampling procedures, processing of low biomass samples and subsequent microbiome characterization based on 16S rRNA gene sequencing.

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