Chronic unilateral arm lymphedema correlates with increased intima-media thickness in the brachial artery.

Drainage of the arterial wall via adventitial lymphatic vessels has been shown to play a pivotal role for vessel wall homeostasis. Also, retrograde cholesterol transport is ensured via this route, but no studies exist to demonstrate that lymphatic stasis would represent a mechanism to initiate atherosclerotic lesion formation in human arteries. To test this hypothesis, we embarked on a simple clinical experiment, assessing wall thickness in limb arteries with lymphedema after surgical intervention, with the contralateral limb serving as control. Using ultrasound imaging, the differential thickness was assessed separately for the three arterial wall layers. The potential of disease progression by lymphostasis was addressed by depiction of longitudinal results according to the time after lymph dissection.

Std fimbriae-fucose interaction increases Salmonella-induced intestinal inflammation and prolongs colonization.

Expression of ABO and Lewis histo-blood group antigens by the gastrointestinal epithelium is governed by an α-1,2-fucosyltransferase enzyme encoded by the Fut2 gene. Alterations in mucin glycosylation have been associated with susceptibility to various bacterial and viral infections. Salmonella enterica serovar Typhimurium is a food-borne pathogen and a major cause of gastroenteritis. In order to determine the role of Fut2-dependent glycans in Salmonella-triggered intestinal inflammation, Fut2+/+ and Fut2-/- mice were orally infected with S. Typhimurium and bacterial colonization and intestinal inflammation were analyzed. Bacterial load in the intestine of Fut2-/- mice was significantly lower compared to Fut2+/+ mice. Analysis of histopathological changes revealed significantly lower levels of intestinal inflammation in Fut2-/- mice compared to Fut2+/+ mice and measurement of lipocalin-2 level in feces corroborated histopathological findings. Salmonella express fimbriae that assist in adherence of bacteria to host cells thereby facilitating their invasion. The std fimbrial operon of S. Typhimurium encodes the π-class Std fimbriae which bind terminal α(1,2)-fucose residues. An isogenic mutant of S. Typhimurium lacking Std fimbriae colonized Fut2+/+ and Fut2-/- mice to similar levels and resulted in similar intestinal inflammation. In vitro adhesion assays revealed that bacteria possessing Std fimbriae adhered significantly more to fucosylated cell lines or primary epithelial cells in comparison to cells lacking α(1,2)-fucose. Overall, these results indicate that Salmonella-triggered intestinal inflammation and colonization are dependent on Std-fucose interaction.

Ex vivo perfusion of the isolated rat small intestine as a novel model of Salmonella enteritis.

Using an ex vivo perfused rat small intestinal model, we examined pathological changes to the tissue, inflammation induction, as well as dynamic changes to smooth muscle activity, metabolic competence, and luminal fluid accumulation during short-term infection with the enteropathogenic bacteria Salmonella enterica serovar Typhimurium and Yersinia enterocolitica. Although few effects were seen upon Yersinia infection, this system accurately modeled key aspects associated with Salmonella enteritis. Our results confirmed the importance of the Salmonella Pathogenicity Island 1 (SPI1)-encoded type 3 secretion system (T3SS) in pathology, tissue invasion, inflammation induction, and fluid secretion. Novel physiological consequences of Salmonella infection of the small intestine were also identified, namely, SPI-1-dependent vasoconstriction and SPI-1-independent reduction in the digestive and absorptive functions of the epithelium. Importantly, this is the first small animal model that allows for the study of Salmonella-induced fluid secretion. Another major advantage of this model is that one can specifically determine the contribution of resident cell populations. Accordingly, we can conclude that recruited cell populations were not involved in the pathological damage, inflammation induction, fluid accumulation, nutrient absorption deficiency, and vasoconstriction observed. Although fluid loss induced by Salmonella infection is hypothesized to be due to damage caused by recruited neutrophils, our data suggest that bacterial invasion and inflammation induction in resident cell populations are sufficient for fluid loss into the lumen. In summary, this model is a novel and useful tool that allows for detailed examination of the early physiopathological effects of Salmonella infection on the small intestine.

Cytotoxic necrotizing factor-Y boosts Yersinia effector translocation by activating Rac protein.

Pathogenic Yersinia spp. translocate the effectors YopT, YopE, and YopO/YpkA into target cells to inactivate Rho family GTP-binding proteins and block immune responses. Some Yersinia spp. also secrete the Rho protein activator cytotoxic necrotizing factor-Y (CNF-Y), but it has been unclear how the bacteria may benefit from Rho protein activation. We show here that CNF-Y increases Yop translocation in Yersinia enterocolitica-infected cells up to 5-fold. CNF-Y strongly activated RhoA and also delayed in time Rac1 and Cdc42, but when individually expressed, constitutively active mutants of Rac1, but not of RhoA, increased Yop translocation. Consistently, knock-out or knockdown of Rac1 but not of RhoA, -B, or -C inhibited Yersinia effector translocation in CNF-Y-treated and control cells. Activation or knockdown of Cdc42 also affected Yop translocation but much less efficiently than Rac. The increase in Yop translocation induced by CNF-Y was essentially independent of the presence of YopE, YopT, or YopO in the infecting Yersinia strain, indicating that none of the Yops reported to inhibit translocation could reverse the CNF-Y effect. In summary, the CNF-Y activity of Yersinia strongly enhances Yop translocation through activation of Rac.

Systematic review and meta-analysis of the effect of fecal microbiota transplantation on behavior in animals.

The bi-directional interaction between gut microbiota and the central nervous system has been coined the gut microbiota-brain axis. Fecal microbiota transplantation (FMT) is the administration of a solution of fecal matter from a donor into the intestinal tract of a recipient. Preclinical FMT experiments are essential to prove causality in the context of the gut microbiota-brain axis. In this systematic review, we assess the body of evidence related to the ability of FMT to modulate an animal's behavior. Accordingly, we provide a detailed summary of the use of FMT in behavior-related animal studies, an extensive risk of bias analysis, and a meta-analysis of the overall effect of FMT on behavioral outcome measures in 64 studies, representing 4889 animals. The resulting meta-analysis revealed FMT was effective at changing animal behavior, thereby substantiating evidence for the gut microbiota-brain axis. However, our study also highlights an urgent need for methodological safeguards within this research field to reduce the risk of bias and improve the internal validity of future studies.

Salmonella enterica Infection of Human and Mouse Colon Organoid-Derived Monolayers.

Intestinal epithelial organoids reflect the morphology and function of an in vivo epithelial barrier. The composition of epithelial cell types reflects the cellular composition of the original tissue (small or large intestine) and organoids can be grown from different species. Thus, intestinal organoids constitute an ideal model to investigate infections of different hosts with enteric pathogens. In this chapter, we will focus on Salmonella infection of human and mouse colonoids grown in a 2D monolayer on permeable filter supports.

Bacterial pathogenesis: exploiting cellular adherence.

Cell adhesion molecules, such as integrins, cadherins, the immunoglobulin superfamily of cell adhesion molecules and selectins, play important structural roles and are involved in various signal transduction processes. As an initial step in the infectious process, many bacterial pathogens adhere to cell adhesion molecules as a means of exploiting the underlying signaling pathways, entering into host cells or establishing extracellular persistence. Often, bacteria are able to bind to cell adhesion molecules by mimicking or acting in place of host cell receptors or their ligands. Recent studies have contributed to our understanding of bacterial adherence mechanisms and the consequences of receptor engagement; they have also highlighted alternative functions of cell adhesion molecules.

Measuring endogenous corticosterone in laboratory mice - a mapping review, meta-analysis, and open source database.

Evaluating stress in laboratory animals is a key principle in animal welfare. Measuring corticosterone is a common method to assess stress in laboratory mice. There are, however, numerous methods to measure glucocorticoids with differences in sample matrix (e.g., plasma, urine) and quantification techniques (e.g., enzyme immunoassay or radioimmunoassay). Here, the authors present a mapping review and a searchable database, giving a complete overview of all studies mea­suring endogenous corticosterone in mice up to February 2018. For each study, information was recorded regarding mouse strain and sex; corticosterone sample matrix and quantification technique; and whether the study covered the research theme animal welfare, neuroscience, stress, inflammation, or pain (the themes of specific interest in our con­sortium). Using all database entries for the year 2012, an exploratory meta-regression was performed to determine the effect of predictors on basal corticosterone concentrations. Seventy-five studies were included using the predictors sex, time-since-lights-on, sample matrix, quantification technique, age of the mice, and type of control. Sex, time-since-lights-on, and type of control significantly affected basal corticosterone concentrations. The resulting database can be used, inter alia, for preventing unnecessary duplication of experiments, identifying knowledge gaps, and standardizing or heterogenizing methodologies. These results will help plan more efficient and valid experiments in the future and can answer new questions in silico using meta-analyses.

Virulence is positively selected by transmission success between mammalian hosts.

Virulence, defined as damage to the host, is a trait of pathogens that evolutionary theory suggests benefits the pathogen in the "struggle for existence". Pathogens employ virulence mechanisms that contribute to disease. Central to the evolution of virulence of the infectious agents causing an array of bacterial disease is the evolutionary acquisition of type III secretion, a macromolecular complex that creates a syringe-like apparatus extending from the bacterial cytosol to the eukaryotic cytosol and delivers secreted bacterial virulence factors (effectors) into host cells. In this work, we quantify the contribution of virulence determinants to the evolutionary success of a pathogen. Using a natural pathogen of mice, we show that virulence factors provide a selective advantage by enhancing transmission between hosts. Virulence factors that have a major contribution to disease were absolutely required for transmission of the pathogen to naive hosts. Virulence-factor mutants with more subtle defects in pathogenesis had quantifiable roles in the time required to transmit the pathogen between mice. Virulence-factor mutants were also found to lose in competition with wild-type bacteria when iteratively transmitted from infected to uninfected mice. These results directly demonstrate that virulence is selected via the fitness advantage it provides to the host-to-host cycle of pathogenic species.

CD14 and ALPK1 Affect Expression of Tight Junction Components and Proinflammatory Mediators upon Bacterial Stimulation in a Colonic 3D Organoid Model.

Cd14 and Alpk1 both encode pathogen recognition receptors and are known candidate genes for affecting severity in inflammatory bowel diseases. CD14 acts as a coreceptor for bacterial lipopolysaccharide (LPS), while ALPK1 senses ADP-D-glycero-beta-D-manno-heptose, a metabolic intermediate of LPS biosynthesis. Intestinal barrier integrity can be influenced by CD14, whereas to date, the role of ALPK1 in maintaining barrier function remains unknown. We used colon-derived 3D organoids, first characterised for growth, proliferation, stem cell markers, and expression of tight junction (TJ) components using qPCR and immunohistochemistry. They showed characteristic crypt stem cells, apical shedding of dead cells, and TJ formation. Afterwards, organoids of different genotypes (WT, Il10 -/-, Cd14 -/-, and Alpk1 -/-) were then stimulated with either LPS or Escherichia coli Nissle 1917 (EcN). Gene expression and protein levels of cytokines and TJ components were analysed. WT organoids increased expression of Tnfα and tight junction components. Cd14 -/- organoids expressed significantly less Tnfα and Ocln after LPS stimulation than WT organoids but reacted similarly to WT organoids after EcN stimulation. In contrast, compared to WT, Alpk1 -/- organoids showed decreased expression of different TJ and cytokine genes in response to EcN but not LPS. However, Western blotting revealed an effect of ALPK1 on TJ protein levels. These findings demonstrate that Cd14, but not Alpk1, alters the response to LPS stimulation in colonic epithelial cells, whereas Alpk1 is involved in the response upon bacterial challenge.

Bacteriophage Therapy for Critical Infections Related to Cardiothoracic Surgery.

(1) Objective: Bacterial resistance to conventional antibiotic therapy is an increasingly significant worldwide challenge to human health. The objective is to evaluate whether bacteriophage therapy could complement or be a viable alternative to conventional antibiotic therapy in critical cases of bacterial infection related to cardiothoracic surgery. (2) Methods: Since September 2015, eight patients with multi-drug resistant or especially recalcitrant Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli infections were treated with bacteriophage preparations as a therapy of last resort according to Article 37 of the Declaration of Helsinki. Patients had infections associated with immunosuppression after organ transplantation or had infections of vascular grafts, implanted medical devices, and surgical wounds. Individualized phage preparations were administered locally, orally, or via inhalation for different durations depending on the case. All patients remained on conventional antibiotics during bacteriophage treatment. (3) Results: Patients ranged in age from 13 to 66 years old (average 48.5 ± 16.7) with seven males and one female. Eradication of target bacteria was reached in seven of eight patients. No severe adverse side effects were observed. (4) Conclusions: Phage therapy can effectively treat bacterial infections related to cardiothoracic surgery when conventional antibiotic therapy fails.

Warming and cooling device using thermoelectric Peltier elements tested on male mice.

Hypothermia is a treatment strategy for different clinical conditions and an essential part of cardiopulmonary bypass in complex cardiac procedures. Clinically, cooling patients is achieved via a mattress and heat exchanger integrated into a membrane oxygenator connected to a waterbed using a refrigerator system based on volatile and toxic liquids. Peltier elements are known as environmentally friendly thermoelectric generators that enable rapid warming and cooling. In this paper, we describe the construction of a novel device for rapid and precise control of mouse warming and cooling using thermoelectric Peltier elements. Six male BALB/c mice were subjected to deep hypothermia and were rewarmed under full physiological monitoring. After rewarming, all animals were observed for two hours, and pathology was evaluated in several organs. All animals tolerated the rapid cooling process well and remained active after rewarming. Temperature-relevant changes were seen via electrocardiography, with heart-rate patterns showing a strong linear correlation to body temperature. No myocardial ischaemia was seen. However, two animals experienced bradycardic atrial fibrillation which spontaneously converted to normal sinus rhythm during rewarming. No histological damage was seen in the heart, liver, kidney or lungs. Our device can effectively be used for heat shock and hypothermia studies in mice, and we foresee no obstacles for its application to other small rodents such as hamsters and young rats. In comparison to known experimental and clinical methods of hypothermia, our device is environmentally friendly, cost-effective and easy to handle, allowing precise control and maintenance of body temperatures ranging from 18℃ to 42℃.

Four hours of veno-venous extracorporeal membrane oxygenation using bi-caval cannulation affects kidney function and induces moderate lung damage in a mouse model.

BACKGROUND: Improvement of single site cannulation for extracorporeal membrane oxygenation (ECMO) therapy is pivotal for reduction of patient morbidity and mortality in respiratory failure. To further improve the cardiopulmonary outcomes and reduce end organ damage, we established a murine model for single site cannulation with a double lumen cannula. RESULTS: We created a hemodynamically stable double lumen cannula and successfully implanted it through the jugular vein into the upper and lower vena cava. This allowed adequate drainage of the blood. Blood gas analysis showed excellent oxygenation and CO2 reduction. There was no excessive bleeding. No signs of right heart congestion were present which was confirmed in the histological analysis of the liver. Histology demonstrated moderate lung damage and mild acute kidney injury. Neutrophil infiltration was similar in ECMO and sham kidneys. CONCLUSIONS: Veno-venous extracorporeal circulation deteriorates kidney function and promotes moderate pulmonary damage.

Fibrin glue as a local drug-delivery system for bacteriophage PA5.

Fibrin glue has been used clinically for decades in a wide variety of surgical specialties and is now being investigated as a medium for local, prolonged drug delivery. Effective local delivery of antibacterial substances is important perioperatively in patients with implanted medical devices or postoperatively for deep wounds. However, prolonged local application of antibiotics is often not possible or simply inadequate. Biofilm formation and antibiotic resistance are also major obstacles to antibacterial therapy. In this paper we test the biocompatibility of bacteriophages incorporated within fibrin glue, track the release of bacteriophages from fibrin scaffolds, and measure the antibacterial activity of released bacteriophages. Fibrin glue polymerized in the presence of the PA5 bacteriophage released high titers of bacteriophages during 11 days of incubation in liquid medium. Released PA5 bacteriophages were effective in killing Pseudomonas aeruginosa PA01. Overall, our results show that fibrin glue can be used for sustained delivery of bacteriophages and this strategy holds promise for many antibacterial applications.

Persistent Salmonella enterica Serovar Typhimurium Infection Induces Protease Expression During Intestinal Fibrosis.

BACKGROUND: Intestinal fibrosis is a common and serious complication of Crohn's disease characterized by the accumulation of fibroblasts, deposition of extracellular matrix, and formation of scar tissue. Although many factors including cytokines and proteases contribute to the development of intestinal fibrosis, the initiating mechanisms and the complex interplay between these factors remain unclear. METHODS: Chronic infection of mice with Salmonella enterica serovar Typhimurium was used to induce intestinal fibrosis. A murine protease-specific CLIP-CHIP microarray analysis was employed to assess regulation of proteases and protease inhibitors. To confirm up- or downregulation during fibrosis, we performed quantitative real-time polymerase chain reaction (PCR) and immunohistochemical stainings in mouse tissue and tissue from patients with inflammatory bowel disease. In vitro infections were used to demonstrate a direct effect of bacterial infection in the regulation of proteases. RESULTS: Mice develop severe and persistent intestinal fibrosis upon chronic infection with Salmonella enterica serovar Typhimurium, mimicking the pathology of human disease. Microarray analyses revealed 56 up- and 40 downregulated proteases and protease inhibitors in fibrotic cecal tissue. Various matrix metalloproteases, serine proteases, cysteine proteases, and protease inhibitors were regulated in the fibrotic tissue, 22 of which were confirmed by quantitative real-time PCR. Proteases demonstrated site-specific staining patterns in intestinal fibrotic tissue from mice and in tissue from human inflammatory bowel disease patients. Finally, we show in vitro that Salmonella infection directly induces protease expression in macrophages and epithelial cells but not in fibroblasts. CONCLUSIONS: In summary, we show that chronic Salmonella infection regulates proteases and protease inhibitors during tissue fibrosis in vivo and in vitro, and therefore this model is well suited to investigating the role of proteases in intestinal fibrosis.

Vasa Vasorum Angiogenesis: Key Player in the Initiation and Progression of Atherosclerosis and Potential Target for the Treatment of Cardiovascular Disease.

Plaque microvascularization and increased endothelial permeability are key players in the development of atherosclerosis, from the initial stages of plaque formation to the occurrence of acute cardiovascular events. First, endothelial dysfunction and increased permeability facilitate the entry of diverse inflammation-triggering molecules and particles such as low-density lipoproteins into the artery wall from the arterial lumen and vasa vasorum (VV). Recognition of entering particles by resident phagocytes in the vessel wall triggers a maladaptive inflammatory response that initiates the process of local plaque formation. The recruitment and accumulation of inflammatory cells and the subsequent release of several cytokines, especially from resident macrophages, stimulate the expansion of existing VV and the formation of new highly permeable microvessels. This, in turn, exacerbates the deposition of pro-inflammatory particles and results in the recruitment of even more inflammatory cells. The progressive accumulation of leukocytes in the intima, which trigger proliferation of smooth muscle cells in the media, results in vessel wall thickening and hypoxia, which further stimulates neoangiogenesis of VV. Ultimately, this highly inflammatory environment damages the fragile plaque microvasculature leading to intraplaque hemorrhage, plaque instability, and eventually, acute cardiovascular events. This review will focus on the pivotal roles of endothelial permeability, neoangiogenesis, and plaque microvascularization by VV during plaque initiation, progression, and rupture. Special emphasis will be given to the underlying molecular mechanisms and potential therapeutic strategies to selectively target these processes.

Veno-Venous Extracorporeal Membrane Oxygenation in a Mouse.

The use of extracorporeal membrane oxygenation (ECMO) has increased substantially in recent years. ECMO has become a reliable and effective therapy for acute as well as end-stage lung diseases. With the increase in clinical demand and prolonged use of ECMO, procedural optimization and prevention of multi-organ damage are of critical importance. The aim of this protocol is to present a detailed technique of veno-venous ECMO in a non-intubated, spontaneously breathing mouse. This protocol demonstrates the technical design of the ECMO and surgical steps. This murine ECMO model will facilitate the study of pathophysiology related to ECMO (e.g., inflammation,bleeding and thromboembolic events). Due to the abundance of genetically modified mice, the molecular mechanisms involved in ECMO-related complications can also be dissected.

Treatment of infected lungs by ex vivo perfusion with high dose antibiotics and autotransplantation: A pilot study in pigs.

The emergence of multi-drug resistant bacteria threatens to end the era of antibiotics. Drug resistant bacteria have evolved mechanisms to overcome antibiotics at therapeutic doses and further dose increases are not possible due to systemic toxicity. Here we present a pilot study of ex vivo lung perfusion (EVLP) with high dose antibiotic therapy followed by autotransplantation as a new therapy of last resort for otherwise incurable multidrug resistant lung infections. Severe Pseudomonas aeruginosa pneumonia was induced in the lower left lungs (LLL) of 18 Mini-Lewe pigs. Animals in the control group (n = 6) did not receive colistin. Animals in the conventional treatment group (n = 6) received intravenous application of 2 mg/kg body weight colistin daily. Animals in the EVLP group (n = 6) had their LLL explanted and perfused ex vivo with a perfusion solution containing 200 µg/ml colistin. After two hours of ex vivo treatment, autotransplantation of the LLL was performed. All animals were followed for 4 days following the initiation of treatment. In the control and conventional treatment groups, the infection-related mortality rate after five days was 66.7%. In the EVLP group, there was one infection-related mortality and one procedure-related mortality, for an overall mortality rate of 33.3%. Moreover, the clinical symptoms of infection were less severe in the EVLP group than the other groups. Ex vivo lung perfusion with very high dose antibiotics presents a new therapeutic option of last resort for otherwise incurable multidrug resistant pneumonia without toxic side effects on other organs.

Blood cytokine expression correlates with early multi-organ damage in a mouse model of moderate hypothermia with circulatory arrest using cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) with moderate hypothermic cardiac arrest (MHCA) is essential for prolonged complex procedures in cardiac surgery and is associated with postoperative complications. Although cytokine release provoked through MHCA under CPB plays a pivotal role in postoperative organ damage, the pathomechanisms are unclear. Here, we investigated the cytokine release pattern and histological organ damage after MHCA using a recently described mouse CPB model. Eight BALB/c mice underwent 60 minutes of circulatory arrest under CPB, were successively rewarmed and reperfused. Blood cytokine concentrations and liver and kidney function parameters were measured and histological changes to these organs were compared to control animals. Our results showed a marked increase in proinflammatory cytokines and histological changes in the kidney, lung, and liver after CPB. Furthermore, clinical chemistry showed signs of hemolysis and acute kidney injury. These results suggest early onset of solid organ injury which correlates with increased leukocyte infiltration. A better understanding of the interplay between pro-inflammatory cytokine activation and solid organ injury in this model of CBP with MHCA will inform strategies to reduce organ damage during cardiac surgeries in the clinic.

Novel mouse model of cardiopulmonary bypass.

OBJECTIVES: Cardiopulmonary bypass (CPB) is an essential component of many cardiac interventions, and therefore, there is an increasing critical demand to minimize organ damage resulting from prolonged extracorporeal circulation. Our goal was to develop the first clinically relevant mouse model of CPB and to examine the course of extracorporeal circulation by closely monitoring haemodynamic and oxygenation parameters. METHODS: Here, we report the optimization of device design, perfusion circuit and microsurgical techniques as well as validation of physiological functions during CPB in mice after circulatory arrest and reperfusion. Validation of the model required multiple blood gas analyses, and therefore, this initial report describes an acute model that is incompatible with survival due to the need of repetitive blood draws. RESULTS: Biochemical and histopathological assessment of organ damage revealed only mild changes in the heart and lungs and signs of the beginning of acute organ failure in the liver and kidneys. CONCLUSIONS: This new CPB mouse model will facilitate preclinical testing of therapeutic strategies in cardiovascular diseases and investigation of CPB in relation to different insults and pre-existing comorbidities. In combination with genetically modified mice, this model will be an important tool to dissect the molecular mechanisms involved in organ damage related to extracorporeal circulation.