Listeria adhesion protein orchestrates caveolae-mediated apical junctional remodeling of epithelial barrier for Listeria monocytogenes translocation.

The cellular junctional architecture remodeling by Listeria adhesion protein-heat shock protein 60 (LAP-Hsp60) interaction for Listeria monocytogenes (Lm) passage through the epithelial barrier is incompletely understood. Here, using the gerbil model, permissive to internalin (Inl) A/B-mediated pathways like in humans, we demonstrate that Lm crosses the intestinal villi at 48 h post-infection. In contrast, the single isogenic (lap- or ΔinlA) or double (lap-ΔinlA) mutant strains show significant defects. LAP promotes Lm translocation via endocytosis of cell-cell junctional complex in enterocytes that do not display luminal E-cadherin. In comparison, InlA facilitates Lm translocation at cells displaying apical E-cadherin during cell extrusion and mucus expulsion from goblet cells. LAP hijacks caveolar endocytosis to traffic integral junctional proteins to the early and recycling endosomes. Pharmacological inhibition in a cell line and genetic knockout of caveolin-1 in mice prevents LAP-induced intestinal permeability, junctional endocytosis, and Lm translocation. Furthermore, LAP-Hsp60-dependent tight junction remodeling is also necessary for InlA access to E-cadherin for Lm intestinal barrier crossing in InlA-permissive hosts. IMPORTANCE: Listeria monocytogenes (Lm) is a foodborne pathogen with high mortality (20%-30%) and hospitalization rates (94%), particularly affecting vulnerable groups such as pregnant women, fetuses, newborns, seniors, and immunocompromised individuals. Invasive listeriosis involves Lm's internalin (InlA) protein binding to E-cadherin to breach the intestinal barrier. However, non-functional InlA variants have been identified in Lm isolates, suggesting InlA-independent pathways for translocation. Our study reveals that Listeria adhesion protein (LAP) and InlA cooperatively assist Lm entry into the gut lamina propria in a gerbil model, mimicking human listeriosis in early infection stages. LAP triggers caveolin-1-mediated endocytosis of critical junctional proteins, transporting them to early and recycling endosomes, facilitating Lm passage through enterocytes. Furthermore, LAP-Hsp60-mediated junctional protein endocytosis precedes InlA's interaction with basolateral E-cadherin, emphasizing LAP and InlA's cooperation in enhancing Lm intestinal translocation. This understanding is vital in combating the severe consequences of Lm infection, including sepsis, meningitis, encephalitis, and brain abscess.

Aortic dissection detection and thrombus structure quantification using volumetric ultrasound, histology, and scanning electron microscopy.

Aortic dissection occurs when a weakened portion of the intima tears, and a separation of layers propagates along the aortic wall to form a false lumen filled with active blood flow or intramural thrombus. The unpredictable nature of aortic dissection formation and need for immediate intervention leaves limited serial human image data to study the formation and morphological changes that follow dissection. We used volumetric ultrasound examination, histology, and scanning electron microscopy (SEM) to examine intramural thrombi at well-defined timepoints after dissection occurs in apolipoprotein E-deficient mice infused with angiotensin II (n = 71). Stratification of red blood cell (RBC) morphologies (biconcave, intermediate biconcave, intermediate polyhedrocyte, and polyhedrocyte) in the thrombi with scanning electron microscopy (n = 5) was used to determine degree of thrombus deposition/contraction. Very few biconcave RBCs (1.2 ± 0.6%) were in the thrombi, and greater amounts of intermediate biconcave RBCs (25.8 ± 6.7%) were located in the descending thoracic portion of the dissection while more polyhedrocytes (14.6 ± 5.1%) and fibrin (42.3 ± 4.5%; P < .05) were found in the distal suprarenal aorta. Thrombus deposition likely plays some role in patient outcomes, and this multimodality technique can help investigate thrombus deposition and characteristics in experimental animal models and human tissue samples.

Maize near-isogenic lines with enhanced flavonoids alleviated dextran sodium sulfate-induced murine colitis via modulation of the gut microbiota.

The rising incidence of inflammatory bowel disease (IBD) has necessitated the search for safe and effective novel therapeutic strategies. Dietary flavonoids exhibited antioxidant, antiproliferative, and anticarcinogenic activities in several model systems with proven abilities to reduce inflammation and oxidative stress, thus they could be promising therapeutic agents for IBD prevention/treatment. However, understanding the role of a specific class of compounds in foods that promote health is difficult because of the chemically complex food matrices. This study aimed to utilize four maize near-isogenic lines to determine the anti-colitis effects of specific classes of flavonoids, anthocyanins and/or phlobaphenes, in a whole-food matrix. Results showed that the intake of anthocyanin and phlobaphene-enriched maize diets effectively alleviated dextran sodium sulfate (DSS)-induced colitis in mice via reducing the intestinal permeability and restoring the barrier function. Anthocyanin diets were more effective in maintaining the crypt structure and muc2 protein levels and reducing inflammation. Bacterial communities of mice consuming diets enriched with anthocyanins and phlobaphenes were more similar to the healthy control compared to the DSS control group, suggesting the role of flavonoids in modulating the gut microbiota to retrieve intestinal homeostasis. Microbiota depletion rendered these compounds ineffective against colitis. Lower serum concentrations of several phenolic acids were detected in the microbiota-depleted mice, indicating that gut microbiota plays a role in flavonoid metabolism and bioavailability.

Sea squirt-inspired bio-derived tissue sealants.

Sea squirts' or tunicates' bodies are composed of cellulose nanofibers and gallol- functionalized proteins. These sea creatures are known to heal their injuries under seawater by forming crosslinks between gallols and functional groups from other proteins in their bodies. Inspired by their wound healing mechanism, herein, we have developed a tissue sealant using zein (a plant-based protein) and tannic acid (gallol-containing polyphenol). Except for fibrin- based sealants, most commercial surgical adhesives, and sealants available today are derived from petroleum products that compromise their biodegradability. They often have complicated and multi-step synthesis processes that ultimately affect their affordability. To overcome this challenge, we ensured that these sea squirt-inspired tissue sealants are bio-based, easily synthesized, and low-cost. The sealants were studied on their own and with a food-grade enzyme transglutaminase. The adhesion performances of the sealants were found to be higher than physiological pressures in seven out of nine different tissue substrates studied here. Their performance was also better than or on par with the FDA-approved fibrin sealant Tisseel. Ex vivo models demonstrate instant sealing of leaking wounds in less than a minute. The sealants were not only cytocompatible but also showed complete wound healing on par with sutures and Tisseel when applied in vivo on skin incisions in rats. Overall, these sea squirt-inspired bio-based sealants show great potential to replace currently available wound closure methods.

Biocompatibility and Safety Assessment of Combined Topical Ozone and Antibiotics for Treatment of Infected Wounds.

Wound infections with antibiotic-resistant bacteria, particularly the Gram-negative strains, pose a substantial health risk for patients with limited treatment options. Recently topical administration of gaseous ozone and its combination with antibiotics through portable systems has been demonstrated to be a promising approach to eradicate commonly found Gram-negative strains of bacteria in wound infections. However, despite the significant impact of ozone in treating the growing number of antibiotic-resistant infections, uncontrolled and high concentrations of ozone can cause damage to the surrounding tissue. Hence, before such treatments could advance into clinical usage, it is paramount to identify appropriate levels of topical ozone that are effective in treating bacterial infections and safe for use in topical administration. To address this concern, we have conducted a series of in vivo studies to evaluate the efficacy and safety of a portable and wearable adjunct ozone and antibiotic wound therapy system. The concurrent ozone and antibiotics are applied through a wound interfaced gas permeable dressing coated with water-soluble nanofibers containing vancomycin and linezolid (traditionally used to treat Gram-positive infections) and connected to a portable ozone delivery system. The bactericidal properties of the combination therapy were evaluated on an ex vivo wound model infected with Pseudomonas aeruginosa, a common Gram-negative strain of bacteria found in many skin infections with high resistance to a wide range of currently available antibiotics. The results indicated that the optimized combination delivery of ozone (4 mg h-1) and topical antibiotic (200 µg cm-2) provided complete bacteria eradication after 6 h of treatment while having minimum cytotoxicity to human fibroblast cells. Furthermore, in vivo local and systemic toxicity studies (e.g., skin monitoring, skin histopathology, and blood analysis) on pig models showed no signs of adverse effects of ozone and antibiotic combination therapy even after 5 days of continuous administration. The confirmed efficacy and biosafety profile of the adjunct ozone and antibiotic therapy places it as a strong candidate for treating wound infection with antimicrobial-resistant bacteria and further pursuing human clinical trials.

Canary Seed (Phalaris canariensis L.) Peptides Prevent Obesity and Glucose Intolerance in Mice Fed a Western Diet.

Previous research showed that canary seed (Phalaris canariensis L.) peptides (CSP) possess robust in vitro antiobesity properties via inhibition of pancreatic lipase (PL). Nevertheless, no studies have yet explored their antiobesity properties in vivo. Consequently, we investigated the effects of CSP in C57BL/6J mice under a Western diet (WD). Mice were assigned into groups and fed a normal diet (ND) or a WD accompanied by an oral dose of CSP (250 or 500 mg/kg/day), orlistat (40 mg/kg/day), or distilled water. The results showed that consuming CSP can provide metabolic benefits, including preventing weight gain by up to 20%, increasing glucose tolerance, and reducing insulin, leptin, and LDL/VLDL levels in plasma. Conversely, total ghrelin was unaffected by CSP-500, but decreased by CSP-250, and amplified by orlistat. Surprisingly, CSP-250 was more effective in preventing weight gain and promoting satiety than CSP-500. Parallel to this, protein absorption in CSP-500 was decreased, supported by a rise in fecal crude protein (+3.5%). Similarly, fecal fat was increased by orlistat (38%) and was unaffected by CSP-250 (3.0%) and CSP (3.0%), comparatively to WD (2.5%). Despite this, both CSP treatments were equally effective in decreasing hepatic steatosis and avoiding hyperlipidemia. Furthermore, the enzymatic analysis showed that CSP-PL complexes dissociated faster (15 min) than orlistat-PL complexes (41 min). Lastly, CSP did not affect expression of hepatic lipid oxidation genes ACO and PPAR-α, but reduced the expression of the hydrolase gene LPL, and lipogenesis related genes FAS and ACC. Taken together, these results suggest that CSP antiobesity mechanism relies on lipid metabolism retardation to increase fat transit time and subsequently suppress hunger.

Experimental aortic aneurysm severity and growth depend on topical elastase concentration and lysyl oxidase inhibition.

Abdominal aortic aneurysm (AAA) formation and expansion is highly complex and multifactorial, and the improvement of animal models is an important step to enhance our understanding of AAA pathophysiology. In this study, we explore our ability to influence aneurysm growth in a topical elastase plus ß-Aminopropionitrile (BAPN) mouse model by varying elastase concentration and by altering the cross-linking capability of the tissue. To do so, we assess both chronic and acute effects of elastase concentration using volumetric ultrasound. Our results suggest that the applied elastase concentration affects initial elastin degradation, as well as long-term vessel expansion. Additionally, we assessed the effects of BAPN by (1) removing it to restore the cross-linking capability of tissue after aneurysm formation and (2) adding it to animals with stable aneurysms to interrupt cross-linking. These results demonstrate that, even after aneurysm formation, lysyl oxidase inhibition remains necessary for continued expansion. Removing BAPN reduces the aneurysm growth rate to near zero, resulting in a stable aneurysm. In contrast, adding BAPN causes a stable aneurysm to expand. Altogether, these results demonstrate the ability of elastase concentration and BAPN to modulate aneurysm growth rate and severity. The findings open several new areas of investigation in a murine model that mimics many aspects of human AAA.

Quantification of murine myocardial infarct size using 2-D and 4-D high-frequency ultrasound.

Ischemic heart disease is the leading cause of death in the United States, Canada, and worldwide. Severe disease is characterized by coronary artery occlusion, loss of blood flow to the myocardium, and necrosis of tissue, with subsequent remodeling of the heart wall, including fibrotic scarring. The current study aims to demonstrate the efficacy of quantitating infarct size via two-dimensional (2-D) echocardiographic akinetic length and four-dimensional (4-D) echocardiographic infarct volume and surface area as in vivo analysis techniques. We further describe and evaluate a new surface area strain analysis technique for estimating myocardial infarction (MI) size after ischemic injury. Experimental MI was induced in mice via left coronary artery ligation. Ejection fraction and infarct size were measured through 2-D and 4-D echocardiography. Infarct size established via histology was compared with ultrasound-based metrics via linear regression analysis. Two-dimensional echocardiographic akinetic length (r = 0.76, P = 0.03), 4-D echocardiographic infarct volume (r = 0.85, P = 0.008), and surface area (r = 0.90, P = 0.002) correlate well with histology. Although both 2-D and 4-D echocardiography were reliable measurement techniques to assess infarct, 4-D analysis is superior in assessing asymmetry of the left ventricle and the infarct. Strain analysis performed on 4-D data also provides additional infarct sizing techniques, which correlate with histology (surface strain: r = 0.94, P < 0.001, transmural thickness: r = 0.76, P = 0.001). Two-dimensional echocardiographic akinetic length, 4-D echocardiography ultrasound, and strain provide effective in vivo methods for measuring fibrotic scarring after MI.NEW & NOTEWORTHY Our study supports that both 2-D and 4-D echocardiographic analysis techniques are reliable in quantifying infarct size though 4-D ultrasound provides a more holistic image of LV function and structure, especially after myocardial infarction. Furthermore, 4-D strain analysis correctly identifies infarct size and regional LV dysfunction after MI. Therefore, these techniques can improve functional insight into the impact of pharmacological interventions on the pathophysiology of cardiac disease.

Intermittent antibiotic treatment accelerated the development of colitis in IL-10 knockout mice.

BACKGROUND AND AIMS: Many epidemiological studies suggest an association between antibiotic exposure and the development of inflammatory bowel disease [IBD]. However, the majority of these studies are observational and still the question remains, "Does the specific antibiotic administration regimen play a role in the development of colitis?" This study aimed to compare the possible effects of continuous and intermittent antibiotic exposure on the development of colitis using a colitis-susceptible IL-10 knockout [IL-10-/-] mouse model. METHODS: IL-10-/- mice [C57BL/6] were randomly assigned to a non-antibiotic group, continuous antibiotic group and intermittent antibiotic group, and observed for 30 weeks. The antibiotic cocktail was given via the drinking water. The differential response to antibiotics was assessed. RESULTS: Intermittent antibiotic treatment resulted in severe colitis with early disease onset in IL-10-/- mice. Higher unit colon weight and spleen weight were observed in intermittent antibiotic-treated mice but not in the continuous antibiotic group. Moreover, intermittent antibiotic treatment aggravated epithelial damage and colonic inflammation, mucosal barrier dysfunction and colonic allergic sensitization in IL-10-/- mice, whereas continuous antibiotic treatment ameliorated these symptoms. Male IL-10-/- mice with intermittent antibiotic exposure were more susceptible to colonic inflammation and allergic response than females. CONCLUSIONS: In summary, intermittent antibiotic exposure accelerated the development of severe colitis more than continuous antibiotic exposure in IL-10-/- male mice. In addition to the colonic damage and impaired barrier function, stimulation of allergic response may play a role in accelerating the development of colitis in genetically susceptible mice.

Role of Gut Microbiota in the Anti-Colitic Effects of Anthocyanin-Containing Potatoes.

SCOPE: Anthocyanin-containing potatoes exert anti-inflammatory activity in colitic mice. Gut bacterial dysbiosis plays a critical role in ulcerative colitis. This study examined the extent to which the anti-colitic activity of anthocyanin-containing red/purple-fleshed potatoes depends on the gut bacteria using a chemically-induced rodent model of colitis with the intact and antibiotic-ablated microbiome. METHODS AND RESULTS: Four-week-old male mice (C57BL6) are randomly assigned to the control diet or 20% purple-/red-fleshed potatoes supplemented diet group. The microbiota-ablated group received an antibiotic cocktail in drinking water. At week nine, colitis is induced by 2% dextran sulfate sodium (DSS) in drinking water for five days. Administration of antibiotics resulted in a 95% reduction in gut bacterial load and fecal SCFAs. DSS-induced elevated gut permeability and body weight loss are more pronounced in antibiotic mice compared to non-antibiotic mice. Purple- or red-fleshed potato supplementation (20% w/w) ameliorated DSS-induced reduction in colon length and mucin 2 expression levels, and increase in permeability, spleen weight, myeloperoxidase (MPO) activity, and inflammatory cytokines (IL-6, IL-17, and IL1-ß) expression levels in non-antibiotic mice, but not in gut microbiota ablated mice. CONCLUSIONS: Anthocyanin-containing potatoes are potent in alleviating colitis, and the gut microbiome is critical for the anti-colitic activity of anthocyanin-containing potatoes.

Biofilm-isolated Listeria monocytogenes exhibits reduced systemic dissemination at the early (12-24 h) stage of infection in a mouse model.

Environmental cues promote microbial biofilm formation and physiological and genetic heterogeneity. In food production facilities, biofilms produced by pathogens are a major source for food contamination; however, the pathogenesis of biofilm-isolated sessile cells is not well understood. We investigated the pathogenesis of sessile Listeria monocytogenes (Lm) using cell culture and mouse models. Lm sessile cells express reduced levels of the lap, inlA, hly, prfA, and sigB and show reduced adhesion, invasion, translocation, and cytotoxicity in the cell culture model than the planktonic cells. Oral challenge of C57BL/6 mice with food, clinical, or murinized-InlA (InlAm) strains reveals that at 12 and 24 h post-infection (hpi), Lm burdens are lower in tissues of mice infected with sessile cells than those infected with planktonic cells. However, these differences are negligible at 48 hpi. Besides, the expressions of inlA and lap mRNA in sessile Lm from intestinal content are about 6.0- and 280-fold higher than the sessle inoculum, respectively, suggesting sessile Lm can still upregulate virulence genes shortly after ingestion (12 h). Similarly, exposure to simulated gastric fluid (SGF, pH 3) and intestinal fluid (SIF, pH 7) for 13 h shows equal reduction in sessile and planktonic cell counts, but induces LAP and InlA expression and pathogenic phenotypes. Our data show that the virulence of biofilm-isolated Lm is temporarily attenuated and can be upregulated in mice during the early stage (12-24 hpi) but fully restored at a later stage (48 hpi) of infection. Our study further demonstrates that in vitro cell culture assay is unreliable; therefore, an animal model is essential for studying the pathogenesis of biofilm-isolated bacteria.

Listeria adhesion protein-expressing bioengineered probiotics prevent fetoplacental transmission of Listeria monocytogenes in a pregnant Guinea pig model.

Pregnancy is a high-risk factor for foodborne pathogen Listeria monocytogenes (Lm), which causes abortion, premature birth, or stillbirth. The primary route of Lm transmission is oral hence intestinal epithelial barrier crossing is a prerequisite for systemic spread. Intestinal barrier crossing, in part, is attributed to the interaction of Listeria adhesion protein (LAP) with its cognate receptor, Hsp60. In a recent study, we showed that oral-dosing of bioengineered Lactobacillus caseiprobiotic (BLP) expressing the LAP protected nonpregnant mice from lethal infection; however, its ability to prevent listeriosis during pregnancy is not known. Therefore, we investigated whether BLP could prevent fetoplacental transmission of Lm in a pregnant guinea pig model. After 14 consecutive days on probiotic (~109 CFU/ml in drinking water), pregnant guinea pigs (gestational days 24-28) were orally challenged with Lm (9 × 108-2.5 × 109 CFU/animal) and were euthanized 72 h post-infection. Maternal mesenteric lymph node (MLN), liver, spleen, lungs, blood, and placenta, and fetal liver were analyzed for the presence/absence of Lm. All tissues/organs from Lm-challenged naïve dams and fetuses were Lm positive. Similar tissue distribution was also seen in guinea pigs that received wild-type Lactobacillus casei (LbcWT). Remarkably, Lm was absent in the maternal blood, kidney, lungs, and placenta, and fetal liver from the BLP-fed group even though the Lm was present in the maternal liver, spleen, and MLN. BLP feeding also suppressed Lm-induced inflammatory response in mothers. These data highlight the potential for the prevention of fetoplacental transmission of Lm by LAP-expressing BLP during pregnancy.

Receptor-targeted engineered probiotics mitigate lethal Listeria infection.

Probiotic bacteria reduce the intestinal colonization of pathogens. Yet, their use in preventing fatal infection caused by foodborne Listeria monocytogenes (Lm), is inconsistent. Here, we bioengineered Lactobacillus probiotics (BLP) to express the Listeria adhesion protein (LAP) from a non-pathogenic Listeria (L. innocua) and a pathogenic Listeria (Lm) on the surface of Lactobacillus casei. The BLP strains colonize the intestine, reduce Lm mucosal colonization and systemic dissemination, and protect mice from lethal infection. The BLP competitively excludes Lm by occupying the surface presented LAP receptor, heat shock protein 60 and ameliorates the Lm-induced intestinal barrier dysfunction by blocking the nuclear factor-κB and myosin light chain kinase-mediated redistribution of the major epithelial junctional proteins. Additionally, the BLP increases intestinal immunomodulatory functions by recruiting FOXP3+T cells, CD11c+ dendritic cells and natural killer cells. Engineering a probiotic strain with an adhesion protein from a non-pathogenic bacterium provides a new paradigm to exclude pathogens and amplify their inherent health benefits.

Toward Automation of the Supine Pressor Test for Preeclampsia.

Preeclampsia leads to increased risk of morbidity and mortality for both mother and fetus. Most previous studies have largely neglected mechanical compression of the left renal vein by the gravid uterus as a potential mechanism. In this study, we first used a murine model to investigate the pathophysiology of left renal vein constriction. The results indicate that prolonged renal vein stenosis after 14 days can cause renal necrosis and an increase in blood pressure (BP) of roughly 30 mmHg. The second part of this study aimed to automate a diagnostic tool, known as the supine pressor test (SPT), to enable pregnant women to assess their preeclampsia development risk. A positive SPT has been previously defined as an increase of at least 20 mmHg in diastolic BP when switching between left lateral recumbent and supine positions. The results from this study established a baseline BP increase between the two body positions in nonpregnant women and demonstrated the feasibility of an autonomous SPT in pregnant women. Our results demonstrate that there is a baseline increase in BP of roughly 10-14 mmHg and that pregnant women can autonomously perform the SPT. Overall, this work in both rodents and humans suggests that (1) stenosis of the left renal vein in mice leads to elevation in BP and acute renal failure, (2) nonpregnant women experience a baseline increase in BP when they shift from left lateral recumbent to supine position, and (3) the SPT can be automated and used autonomously.