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Intestinal permeability and neutrophil activity are closely linked to inflammatory bowel disease (IBD) pathophysiology. Here we discuss two techniques for assessing permeability and neutrophil activity in mouse IBD models using near infrared (NIR) detection. To address the limitation of visible light readouts—namely high background—IRDye 800CW was used to enable rapid, non-terminal measurements of intestinal permeability. The increased sensitivity of NIR readouts for colon permeability is shown using dextran sulfate sodium (DSS) and anti-CD40 murine colitis models in response to interleukin-22 immunoglobulin Fc (IL22Fc) fusion protein and anti-p40 monoclonal antibody treatments, respectively. In addition to enhanced permeability, elevated levels of neutrophil elastase (NE) have been reported in inflamed colonic mucosal tissue. Activatable NIR fluorescent probes have been extensively used for disease activity evaluation in oncologic animal models, and we demonstrate their translatability using a NE-activatable reagent to evaluate inflammation in DSS mice. Confocal laser endomicroscopy (CLE) and tissue imaging allow visualization of spatial NE activity throughout diseased colon as well as changes in disease severity from IL22Fc treatment. Our findings with the 800CW dye and the NE probe highlight the ease of their implementation in preclinical IBD research.

Bovine mastitis results in billion dollar losses annually in the USA alone. Streptococci are among the most relevant causative agents of this disease. Conventional antibiotic therapy is often unsuccessful and contributes to development of antibiotic resistance. Bacteriophage endolysins represent a new class of antimicrobials against these bacteria. In this work, we characterized the endolysins (lysins) of the streptococcal phages λSA2 and B30 and evaluated their potential as anti-mastitis agents. When tested in vitro against live streptococci, both enzymes exhibited near-optimum lytic activities at ionic strengths, pH, and Ca²⁺ concentrations consistent with cow milk. When tested in combination in a checkerboard assay, the lysins were found to exhibit strong synergy. The λSA2 lysin displayed high activity in milk against Streptococcus dysgalactiae (reduction of CFU/ml by 3.5 log units at 100 μg/ml), Streptococcus agalactiae (2 log), and Streptococcus uberis (4 log), whereas the B30 lysin was less effective. In a mouse model of bovine mastitis, both enzymes significantly reduced intramammary concentrations of all three streptococcal species (except for B30 vs. S. dysgalactiae), and the effects on mammary gland wet weights and TNFα concentrations were consistent with these findings. Unexpectedly, the synergistic effect determined for the two enzymes in vitro was not observed in the mouse model. Overall, our results illustrate the potential of endolysins for treatment of Streptococcus-induced bovine mastitis.

Background Biological sex plays a prominent role in the prevalence and severity of a number of important stress-related gastrointestinal and immune-related diseases including IBS and allergy/anaphylaxis. Despite the establishment of sex differences in these diseases, the underlying mechanisms contributing to sex differences remain poorly understood. The objective of this study was to define the role of biological sex on mast cells (MCs), an innate immune cell central to the pathophysiology of many GI and allergic disorders. Methods Twelve-week-old C57BL/6 male and female mice were exposed to immunological stress (2 h of IgE-mediated passive systemic anaphylaxis (PSA)) or psychological stress (1 h of restraint stress (RS)) and temperature, clinical scores, serum histamine, and intestinal permeability (for RS) were measured. Primary bone marrow-derived MCs (BMMCs) were harvested from male and female mice and analyzed for MC degranulation, signaling pathways, mediator content, and RNA transcriptome analysis. Results Sexually dimorphic responses were observed in both models of PSA and RS and in primary MCs. Compared with male mice, female mice exhibited increased clinical scores, hypothermia, and serum histamine levels in response to PSA and had greater intestinal permeability and serum histamine responses to RS. Primary BMMCs from female mice exhibited increased release of β-hexosaminidase, histamine, tryptase, and TNF-α upon stimulation with IgE/DNP and A23187. Increased mediator release in female BMMCs was not associated with increased upstream phospho-tyrosine signaling pathways or downstream Ca 2+ mobilization. Instead, increased mediator release in female MCs was associated with markedly increased capacity for synthesis and storage of MC granule-associated immune mediators as determined by MC mediator content and RNA transcriptome analysis. Conclusions These results provide a new understanding of sexual dimorphic responses in MCs and have direct implications for stress-related diseases associated with a female predominance and MC hyperactivity including irritable bowel syndrome, allergy, and anaphylaxis.

Bovine mastitis results in billion dollar losses annually in the USA alone. Streptococci are among the most relevant causative agents of this disease. Conventional antibiotic therapy is often unsuccessful and contributes to development of antibiotic resistance. Bacteriophage endolysins represent a new class of antimicrobials against these bacteria. In this work, we characterized the endolysins (lysins) of the streptococcal phages [lambda]SA2 and B30 and evaluated their potential as anti-mastitis agents. When tested in vitro against live streptococci, both enzymes exhibited near-optimum lytic activities at ionic strengths, pH, and Ca.sup.2+ concentrations consistent with cow milk. When tested in combination in a checkerboard assay, the lysins were found to exhibit strong synergy. The [lambda]SA2 lysin displayed high activity in milk against Streptococcus dysgalactiae (reduction of CFU/ml by 3.5 log units at 100 [mu]g/ml), Streptococcus agalactiae (2 log), and Streptococcus uberis (4 log), whereas the B30 lysin was less effective. In a mouse model of bovine mastitis, both enzymes significantly reduced intramammary concentrations of all three streptococcal species (except for B30 vs. S. dysgalactiae), and the effects on mammary gland wet weights and TNF[alpha] concentrations were consistent with these findings. Unexpectedly, the synergistic effect determined for the two enzymes in vitro was not observed in the mouse model. Overall, our results illustrate the potential of endolysins for treatment of Streptococcus-induced bovine mastitis.

Stressful, traumatic events are a well-recognized trigger leading to acute and chronic gastrointestinal (GI) disease like Irritable Bowel Syndrome (IBS) or Inflammatory Bowel Disease (IBD). Two major types of stress which are pervasive within the world population are early life adversity and depression, both of which are strongly associated with GI disease throughout life. Though much of the underlying pathology in IBS and IBD has been described, the underlying mechanisms explain how stress may trigger onset or increased symptom severity in these chronic disease is poorly understood. The enteric cholinergic nervous system is a major regulator of GI homeostasis with broad regulatory roles over epithelial barrier permeability, epithelial cell secretion, smooth muscle contraction and motility, and immune activation. Though a role of the enteric nervous system has been describe in acute stress induced GI dysfunction, little is known about the role of this system in chronic stress, early life adversity, or in infectious models. The objective of this dissertation was to determine if the enteric cholinergic nervous system contributed to GI disease under different types of environmental challenges including early life adversity, chronic stress and pathogen challenge. To answer these questions, we utilized several different small and large animal models in combination with pharmacological agonists and antagonist of the cholinergic system. The results presented here demonstrate that different types of stressors differentially impact the enteric cholinergic system. Following early life adversity, we observed a persistent upregulation of the enteric cholinergic system, which predisposes individuals to increased intestinal secretion, permeability, motility, and upregulation of stress related genes. In a pathogen challenge model, we observed an upregulation of a non-neuronal component of the enteric cholinergic system which correlated positively with disease severity. Finally we observed that chronic stress in adulthood results in a strong down regulation of the enteric cholinergic nervous system with reduced cholinergic mediated functional secretion. Combined these findings demonstrated that different modes of stress have dichotomous impacts the enteric cholinergic system, which differentially impact GI function. Future work should focus on the precise factors impacting the function and expression of the cholinergic system in order to develop better therapies to cope with stress induced GI disease.