Activation of Deoxyribonuclease I by Nicotinamide as a New Strategy to Attenuate Tetracycline-Resistant Biofilms of Cutibacterium acnes.

Biofilms of Cutibacterium (C.) acnes (formerly Propionibacterium acnes) are responsible for the persistence and antibiotic resistance of acne vulgaris. In addition to the standard treatments for acne vulgaris, a common adjunctive treatment is the topical administration of nicotinamide (NAM). However, the effects of NAM on biofilms of C. acnes have never been explored. This study comprehensively investigates the effects of NAM against biofilms of C. acnes using in vitro and in vivo approaches. The results showed that NAM potentiated the efficacy of suboptimal dosing of tetracycline against C. acnes. Moreover, NAM alone decreased the formation and increased the degradation of biofilms in C. acnes. The antibiofilm effect of NAM against C. acnes was further enhanced in combination with deoxyribonuclease (DNase) I, an enzyme with known antibiofilm properties. The computational molecular docking, surface plasmon resonance analysis, and enzymatic kinetic assay demonstrated that NAM binds to DNase I and accelerated its reaction. In conclusion, NAM activates DNase I to attenuate biofilms of C. acnes. This offers valuable insights into the strategies against biofilms that are worth elaborating on in other biofilm-related chronic cutaneous infections in the future.

Ultra-microangiography in evaluating the disease activity of rheumatoid arthritis and enhancing the efficacy of ultrasonography: A preliminary study.

OBJECTIVE: Ultra-microangiography (UMA) is a novel Doppler technique that has high sensitivity for low-velocity blood flows. In this study, a distinctive imaging feature, penetrating blood vessels on the surface of eroded bones within the inflamed joints, was observed on UMA. We aimed to investigate the feasibility of UMA in assessing disease activity and identify the clinical value of the UMA feature for evaluating rheumatoid arthritis (RA). METHODS AND MATERIALS: Power-Doppler ultrasound (PDUS) and UMA were performed on small joints of RA patients. The semiquantitative scores of PDUS and UMA of the joints were assessed and compared. The UMA imaging feature of penetrating vessels on the surface of eroded bones was evaluated, and the patients were divided into three groups based on imaging features. (Group 1: no inflammatory signs; Group 2: inflammatory US features but no UMA features; and Group 3: detected UMA features). The correlations between the groups and clinical parameters were assessed. RESULTS: A total of 52 patients with RA were recruited, with 364 joints (MCP, PIP, MTP and wrist) scanned. Synovial blood vessel signals were detected for 68 by PDUS and for 93 by UMA (display rate of blood vessel signals: 18.68 % VS 25.55 %). UMA presented better display capability of blood vessels within the inflamed regions than that of PDUS in overall. Significant differences were detected in clinical scores (P < 0.0001 for DAS28 [ESR], DAS28 [CRP], SDAI, CDAI, CRP P = 0.0303, SJC P = 0.0059, and TJC P = 0.0423) between Group 2 and 3. Significant correlations between the groups and clinical parameters were also observed (DAS28 [ESR] ρ=0.750; DAS28 [CRP] ρ=0.762; SDAI ρ=0.778; CDAI ρ=0.773; CRP ρ= 0.524; SJC ρ=0.742; TJC ρ=0.693, P < 0.0001), indicating that the UMA feature was related to high disease activity. CONCLUSIONS: UMA can help enhance the detection rate of micro-vascularization. The UMA feature of the penetrating vessels on the surface of eroded bones is likely to be associated with severe disease activity.

The α9 Nicotinic Acetylcholine Receptor Mediates Nicotine-Induced PD-L1 Expression and Regulates Melanoma Cell Proliferation and Migration.

Cigarette smoking is associated with an increased risk of melanoma metastasis. Smokers show higher PD-L1 expression and better responses to PD-1/PD-L1 inhibitors than nonsmokers. Here, we investigate whether nicotine, a primary constituent of tobacco, induces PD-L1 expression and promotes melanoma cell proliferation and migration, which is mediated by the α9 nicotinic acetylcholine receptor (α9-nAChR). α9-nAChR overexpression in melanoma using melanoma cell lines, human melanoma tissues, and assessment of publicly available databases. α9-nAChR expression was significantly correlated with PD-L1 expression, clinical stage, lymph node status, and overall survival (OS). Overexpressing or knocking down α9-nAChR in melanoma cells up- or downregulated PD-L1 expression, respectively, and affected melanoma cell proliferation and migration. Nicotine-induced α9-nAChR activity promoted melanoma cell proliferation through stimulation of the α9-nAChR-mediated AKT and ERK signaling pathways. In addition, nicotine-induced α9-nAchR activity promoted melanoma cell migration via activation of epithelial-mesenchymal transition (EMT). Moreover, PD-L1 expression was upregulated in melanoma cells after nicotine treatment via the transcription factor STAT3 binding to the PD-L1 promoter. These results highlight that nicotine-induced α9-nAChR activity promotes melanoma cell proliferation, migration, and PD-L1 upregulation. This study may reveal important insights into the mechanisms underlying nicotine-induced melanoma growth and metastasis through α9-nAChR-mediated carcinogenic signals and PD-L1 expression.

Comparative effectiveness of low-level laser therapy for adult androgenic alopecia: a system review and meta-analysis of randomized controlled trials.

The purpose of this review is to explore the effectiveness of low-level laser therapy (LLLT) in the treatment of adult androgenic alopecia (AGA). A systematic search of studies on LLLT for AGA was conducted mainly in Pubmed, Embase, and Cochrane Systematic Reviews. The standardized mean difference (SMD) in the changes of hair density treated by LLLT versus sham devices was analyzed. The meta-analysis included 8 studies comprising a total of 11 double-blinded randomized controlled trials. The quantitative analysis showed a significant increase in hair density for those treated by LLLT versus sham group (SMD 1.316, 95% confidence interval, CI 0.993 to 1.639). The subgroup analysis demonstrated that LLLT increases hair growth in both genders, in both comb- and helmet-type devices, and in short- and long-term treatment course. The subgroup analysis also showed a more significant increase of hair growth for the LLLT versus sham in the low-frequency treatment group (SMD 1.555, 95% CI 1.132 to 1.978) than in the high-frequency group (SMD 0.949, 95% CI 0.644 to 1.253). The review was limited by the heterogeneity of included trials. LLLT significantly increased hair density in AGA. The meta-analysis suggests that low treatment frequency by LLLT have a better hair growth effect than high treatment frequency. LLLT represents a potentially effective treatment for AGA in both male and female. The types of LLLT devices and LLLT treatment course duration did not affect the effectiveness in hair growth.

Utility of Gram staining for diagnosis of Malassezia folliculitis.

Malassezia folliculitis (MalF) mimics acne vulgaris and bacterial folliculitis in clinical presentations. The role of Gram staining in rapid diagnosis of MalF has not been well studied. In our study, 32 patients were included to investigate the utility of Gram staining for MalF diagnosis. The final diagnoses of MalF were determined according to clinical presentation, pathological result and treatment response to antifungal agents. Our results show that the sensitivity and specificity of Gram staining are 84.6% and 100%, respectively. In conclusion, Gram staining is a rapid, non-invasive, sensitive and specific method for MalF diagnosis.

Disseminated cutaneous Mycobacterium kansasii infection presenting with Rosai-Dorfman disease-like histological features in a patient carrying anti-interferon-γ autoantibodies.

Typical cutaneous non-tuberculous mycobacteria (NTM) infections show a histopathology pattern of granulomas with admixed Langhans giant cells, and abscesses may be observed in acute lesions. Herein, we describe a patient carrying a high titer of autoantibodies to interferon (IFN)-γ with disseminated Mycobacterium kansasii infection presenting with emperipolesis and Rosai-Dorfman disease (RDD)-like histopathological features characterized by remarkable, large, pale-staining "RD cells", which were CD68 and S100 positive and CD1a negative. The patient was misdiagnosed with RDD initially, but exhibited a poor response to all interventions. A re-biopsy revealed Langhans-type multinucleated giant cells; multiple definite acid-fast bacilli were also found. M. kansasii was isolated from cultured tissues. Anti-NTM treatment was initiated. After treatment, all lesions resolved almost completely within the following month. High-titer anti-IFN-γ autoantibodies were detected during follow up, leading to the diagnosis of adult-onset immunodeficiency syndrome. In conclusion, patients carrying high-titer autoantibodies to IFN-γ who also have a disseminated cutaneous M. kansasii infection may present with RDD-like histopathological features, which may be a pitfall in the diagnosis of disseminated cutaneous NTM infections.

UVA irradiation of human skin vasodilates arterial vasculature and lowers blood pressure independently of nitric oxide synthase.

The incidence of hypertension and cardiovascular disease (CVD) correlates with latitude and rises in winter. The molecular basis for this remains obscure. As nitric oxide (NO) metabolites are abundant in human skin, we hypothesized that exposure to UVA may mobilize NO bioactivity into the circulation to exert beneficial cardiovascular effects independently of vitamin D. In 24 healthy volunteers, irradiation of the skin with two standard erythemal doses of UVA lowered blood pressure (BP), with concomitant decreases in circulating nitrate and rises in nitrite concentrations. Unexpectedly, acute dietary intervention aimed at modulating systemic nitrate availability had no effect on UV-induced hemodynamic changes, indicating that cardiovascular effects were not mediated via direct utilization of circulating nitrate. UVA irradiation of the forearm caused increased blood flow independently of NO synthase (NOS) activity, suggesting involvement of pre-formed cutaneous NO stores. Confocal fluorescence microscopy studies of human skin pre-labeled with the NO-imaging probe diaminofluorescein 2 diacetate revealed that UVA-induced NO release occurs in a NOS-independent, dose-dependent manner, with the majority of the light-sensitive NO pool in the upper epidermis. Collectively, our data provide mechanistic insights into an important function of the skin in modulating systemic NO bioavailability, which may account for the latitudinal and seasonal variations of BP and CVD.

Median arcuate ligament syndrome in the pediatric population.

OBJECTIVES: Median arcuate ligament syndrome (MALS) is a vascular compression syndrome with symptoms that overlap chronic functional abdominal pain (CFAP). We report our experience treating MALS in a pediatric cohort previously diagnosed with CFAP. PATIENTS AND METHODS: We prospectively evaluated 46 pediatric (<21years of age) patients diagnosed with MALS at a tertiary care referral center from 2008 to 2012. All patients had previously been diagnosed with CFAP. Patients were evaluated for celiac artery compression by duplex ultrasound and diagnosis was confirmed by computed tomography. Quality of life (QOL) was determined by pre- and postsurgical administration of PedsQL™ questionnaire. The patients underwent laparoscopic release of the median arcuate ligament overlying the celiac artery which included surgical neurolysis. We examined the hemodynamic changes in parameters of the celiac artery and perioperative QOL outcomes to determine correlation. RESULTS: All patients had studies suggestive of MALS on duplex and computed tomography; 91% (n=42) positive for MALS were females. All patients underwent a technically satisfactory laparoscopic surgical release resulting in a significant improvement in blood flow through the celiac artery. There were no deaths and a total of 9 complications, 8 requiring a secondary procedure; 33 patients were administered QOL surveys. 18 patients completed the survey with 15 (83%) patients reporting overall improvement in the QOL. Overall, 31/46 patients (67%) reported improvement of symptoms since the time of surgery. CONCLUSIONS: MALS was found to be more common in pediatric females than males. Laparoscopic release of the celiac artery can be performed safely in the pediatric population. Surgical release of the artery and resultant neurolysis resulted in significant improvement in the blood flow, symptoms, and overall QOL in this cohort. The overall improvement in QOL outcome measures after surgery leads us to conclude that MALS might be earlier diagnosed and possibly treated in patients with CFAP. We recommend a multidisciplinary team approach to care for these complex patients.

Murine gut microbiota and transcriptome are diet dependent.

OBJECTIVE: Here, we determine how formula feeding impacts the gut microbiota and host transcriptome. BACKGROUND: Formula-fed (FF) infants are at risk for diseases that involve complex interactions between microbes and host immune elements such as necrotizing enterocolitis. The aims of this study were to simultaneously examine the microbiota and host transcriptional profiles of FF and maternal-fed (MF) mice to evaluate how diet impacts gut colonization and host genes. METHODS: After 72 hours of FF or MF, colonic tissue was collected. 16S ribosomal RNA was sequenced with Roche GS-FLX (Genome Sequencer-FLX) pyrosequencing. Operational taxonomical unit clustering, diversity analysis, and principal coordinate analysis (PCA) were performed. Complementary DNA libraries were sequenced by Solexa. Reads were annotated by BLAST (Basic Local Alignment Search Tool) search against mouse RNA database [National Center for Biotechnology Information (NCBI) build-37] and functionally classified using the KOG (Eukaryotic Orthologous Groups) database (NCBI). RESULTS: Firmicutes (P < 0.001) was the dominant phylum in MF pups, whereas Proteobacteria (P < 0.001) and Bacteroidetes (P < 0.05) were dominant in FF mice. On the genus level, FF mice had increased Serratia (P < 0.001) and Lactococcus (P < 0.05) whereas MF mice had increased Lactobacillus (P < 0.001). PCA confirmed clustering by diet. Solexa sequencing demonstrated different (P < 0.05) messenger RNA transcript levels in 148 genes. Heme oxygenase 1 (P < 0.01), an oxidative stress marker, was increased 25-fold in FF mice. In addition, decreased vinculin (P < 0.05), a cytoskeletal protein associated with adherens junctions in FF pups suggested impaired gut structural integrity. Diet also impacted immune regulation, cell cycle control/gene expression, cell motility, and vascular function genes. CONCLUSIONS: FF shifted gut microbiota and structural integrity, oxidative stress, and immune function genes, presumably increasing vulnerability to disease in FF mice. Interrogation of microbial and host gene expression in FF neonates may offer new insight on how diet affects disease pathogenesis.

Primary hyperparathyroidism in adolescents: the same but different.

PURPOSE: Primary hyperparathyroidism has been studied more extensively in adults than in adolescents. The objective of this study is to define the similarities and differences that exist between these groups. METHODS: A retrospective review of 1,000 primary hyperparathyroidism patients undergoing parathyroidectomy at a single tertiary-care university teaching hospital between 1990 and 2004. All patients 20 years of age or younger comprised our study cohort, and were compared to two historical adult groups. RESULTS: Of 1,000 parathyroidectomies, 21 (2.1 %) were 20 years of age or younger (adolescent). The adolescents presented with higher serum calcium levels (p < 0.01) more severe symptoms (p = 0.02), more renal stones (p = 0.048), and a higher incidence of hypercalcemic crisis (p = 0.02), when compared with adults. We found that 67 % suffered from a triad of tiredness, weakness, and depression versus 39 % of adults (p = 0.02). Sestamibi scans were less helpful in the adolescents than in adults. Similar to the adults, 86 % of adolescent patients had single gland disease, and 95 % were cured at the first operation. CONCLUSION: Adolescents with primary hyperparathyroidism typically have more severe disease than adults. Contrary to popular belief, most adolescents have single gland disease and not hyperplasia associated with a genetic disorder.

Spleno-adrenal shunt: a novel alternative for portosystemic decompression in children with portal vein cavernous transformation.

PURPOSE: Children with portal vein cavernous transformation (PVCT) can develop life-threatening variceal hemorrhage from progressive portal hypertension. While spleno-renal shunt ± splenectomy is the most common portosystemic decompression surgery performed in children, we have adopted a modified spleno-adrenal (SA) shunt for complicated PVCT. We describe our 10 year experience focusing on technique evolution and treatment efficacy. METHODS: Between 2001 and 2011, 15 children (9 girls and 6 boys, ages 3-11 years, median: 6 years) with PVCT, portal hypertension, and hypersplenism were treated with SA shunt with splenectomy in Shanghai Children's Medical Center. All children in the study had endoscopy proven active esophageal variceal bleeding requiring multiple transfusions (mean: 4.2 units) with failed sclerotherapy (mean: 2.6 times). Greater omental vein pressure (GVP) approximating portal venous pressure was measured pre- and post-SA shunt. Pre- and post-operative ammonia levels were obtained. Follow-up ranged from 6 months to 10 years (mean: 4.2 ± 2 years). RESULTS: Intra-operative adrenal vein diameter and length ranged from 0.7 to 1.8 cm and 2 to 3 cm, respectively. Intra-operative GVPs pre-and post-SA shunt were (30 ± 11) and (22 ± 7) mmHg, respectively (p<0.01). On follow-up, there have been no recurrences of GI bleeding. Liver function tests remained normal in all children with the exception of elevated post-operative mean blood ammonia levels [Pre (18 ± 7) mmol/L, post (60 ± 17) mmol/L (p<0.05)] in all children. Ammonia levels normalized in all cases on outpatient follow-up. There have been no cases of hepatic encephalopathy, and all have normal age appropriate neurodevelopment (Bayley's assessment). Barium swallow and/or upper endoscopy showed interval resolution of esophageal varices in all children, and vascular ultrasound showed patent shunt anastomosis without stricture in 14 (93%). CONCLUSIONS: The left adrenal vein is a viable conduit for effective selective portosystemic decompression. Similar to the more traditional spleno-renal shunt, SA appears also to have the advantage of preventing hepatic encephalopathy preserving neurodevelopment, although the rise in post-operative ammonia levels was unexpected. Longer follow-up is needed to look for late signs of encephalopathy assessing neurodevelopment long term.

Lymphotoxin regulates commensal responses to enable diet-induced obesity.

Microbiota are essential for weight gain in mouse models of diet-induced obesity (DIO), but the pathways that cause the microbiota to induce weight gain are unknown. We report that mice deficient in lymphotoxin, a key molecule in gut immunity, were resistant to DIO. Ltbr(-/-) mice had different microbial community composition compared to their heterozygous littermates, including an overgrowth of segmented filamentous bacteria (SFB). Furthermore, cecal transplantation conferred leanness to germ-free recipients. Housing Ltbr(-/-) mice with their obese siblings rescued weight gain in Ltbr(-/-) mice, demonstrating the communicability of the obese phenotype. Ltbr(-/-) mice lacked interleukin 23 (IL-23) and IL-22, which can regulate SFB. Mice deficient in these pathways also resisted DIO, demonstrating that intact mucosal immunity guides diet-induced changes to the microbiota to enable obesity.

Novel model to study combined effects of microorganisms and oxidants on development of intestinal necrosis.

BACKGROUND: Although the cause of intestinal necrosis in diseases such as necrotizing enterocolitis (NEC) is multi-factorial, oxidants and bacteria likely are key factors. Many animal models of NEC generate histologic necrosis, but it is rare to see the gross necrosis that is observed in infants. Here, we present a novel model that produces full-thickness intestinal necrosis when chloramine-T, an oxidizing agent, is introduced directly into isolated intestinal loops. The aim of this study was to determine the role of bacteria in this model. METHODS: Three-week-old mice underwent isolated ileal loop construction by suture ligation, and either chloramine-T or saline was injected into the loop. Intestines were then returned to the abdominal cavity, and the incision was closed for 24 h, after which the intestinal loops were analyzed histologically and microbiologically. To determine if bacteria home to the site of injury, some mice had intracecal injection of luminescent Pseudomonas aeruginosa (PA). These mice were sacrificed 2, 6, 8, and 24 h later, and luminescent bacteria were localized via photon camera imaging. RESULTS: No gross necrosis was observed in the saline-treated group, but 59% of the animals in the chloramine-T-treated group had necrosis (p<0.001). Relative bacterial species numbers were calculated for untreated control animals and those with saline-treated loops and chloramine-T-treated loops without and with necrosis. Lactobacillus was detected in 60% of the control animals but was absent in all animals that underwent surgery. Methicillin-sensitive Staphylococcus aureus, PA, and Enterococcus faecium were present only in animals that underwent loop construction; however, bacterial communities did not differ according to loop treatment or the presence or absence of necrosis. After intracecal injection of PA, bacteria homed to the loop and proximal bowel even though the loop was discontinuous with the remaining bowel. CONCLUSIONS: Intraluminal chloramine-T causes full-thickness necrosis in three-week-old mice and is characterized by a loss of probiotic bacteria such as Lactobacillus. Necrotic loops of bowel become colonized rapidly with pathogenic bacteria by unconventional mechanisms. Oxidant stress and colonization by pathogenic bacteria may play important roles in intestinal necrosis across a wide spectrum of pathologic states, including NEC.

Pseudomonas aeruginosa overrides the virulence inducing effect of opioids when it senses an abundance of phosphate.

The gut during critical illness represents a complex ecology dominated by the presence of healthcare associated pathogens, nutrient scarce conditions, and compensatory host stress signals. We have previously identified key environmental cues, opioids and phosphate depletion that independently activate the virulence of Pseudomonas aeruginosa. Opioids induce quinolone signal production (PQS), whereas phosphate depletion leads to a triangulated response between MvfR-PQS, pyoverdin, and phosphosensory/phosphoregulatory systems (PstS-PhoB). Yet how P. aeruginosa manages its response to opioids during nutrient scarce conditions when growth is limited and a quorum is unlikely to be achieved is important in the context of pathogenesis in gut during stress. To mimic this environment, we created nutrient poor conditions and exposed P. aeruginosa PAO1 to the specific k-opioid receptor agonist U-50,488. Bacterial cells exposed to the k-opioid expressed a striking increase in virulence- and multi-drug resistance-related genes that correlated to a lethal phenotype in C. elegans killing assays. Under these conditions, HHQ, a precursor of PQS, rather than PQS itself, became the main inducer for pqsABCDE operon expression. P. aeruginosa virulence expression in response to k-opioids required PqsE since ΔPqsE was attenuated in its ability to activate virulence- and efflux pumps-related genes. Extracellular inorganic phosphate completely changed the transcriptional response of PAO1 to the k- opioid preventing pqsABCDE expression, the activation of multiple virulence- and efflux pumps-related genes, and the ability of P. aeruginosa to kill C. elegans. These results indicate that when P. aeruginosa senses resource abundance in the form of phosphate, it overrides its response to compensatory host signals such as opioids to express a virulent and lethal phenotype. These studies confirm a central role for phosphate in P. aeruginosa virulence that might be exploited to design novel anti- virulence strategies.

Immature oxidative stress management as a unifying principle in the pathogenesis of necrotizing enterocolitis: insights from an agent-based model.

BACKGROUND: Necrotizing enterocolitis (NEC) is a complex disease involving prematurity, enteral feeding, and bacterial effects. We propose that the underlying initial condition in its pathogenesis is reduced ability of the neonatal gut epithelial cells (NGECs) to clear oxidative stress (OS), and that when such a NGEC population is exposed to enteral feeding, the increased metabolic OS tips the population toward apoptosis, inflammation, bacterial activation, and eventual necrosis. The multi-factorial complexity of NEC requires characterization with computational modeling, and herein, we used an agent-based model (ABM) to instantiate and examine our unifying hypothesis of the pathogenesis of NEC. METHODS: An ABM of the neonatal gut was created with NGEC computational agents incorporating rules for pathways for OS, p53, tight junctions, Toll-like receptor (TLR)-4, nitric oxide, and nuclear factor-kappa beta (NF-κB). The modeled bacteria activated TLR-4 on contact with NGECs. Simulations included parameter sweeps of OS response, response to feeding, addition of bacteria, and alterations in gut mucus production. RESULTS: The ABM reproduced baseline cellular respiration and clearance of OS. Reduction in OS clearance consistent with clinical NEC led to senescence, apoptosis, or inflammation, with disruption of tight junctions, but rarely to NGEC necrosis. An additional "hit" of bacteria activating TLR-4 potentiated a shift to NGEC necrosis across the entire population. The mucus layer was modeled to limit bacterial-NGEC interactions and reduce this effect, but concomitant apoptosis in the goblet cell population reduced the efficacy of the mucus layer and limited its protective effect in simulated experiments. This finding suggests a means by which increased apoptosis at the cellular population level can lead to a transition to the necrosis outcome. CONCLUSIONS: Our ABM incorporates known components of NEC and demonstrates that impaired OS management can lead to apoptosis and inflammation of NGECs, rendering the system susceptible to an additional insult involving regionalized mucus barrier failure and TLR-4 activation, which potentiates the necrosis outcome. This type of integrative dynamic knowledge representation can be a useful adjunct to help guide and contextualize research.

Pseudomonas aeruginosa virulence expression is directly activated by morphine and is capable of causing lethal gut-derived sepsis in mice during chronic morphine administration.

OBJECTIVE: This study was designed to examine the effect of morphine administration on the intestinal mucus barrier and determine its direct effect on the virulence and lethality of Pseudomonas aeruginosa, one of the most frequent pathogens to colonize the gut of critically ill patients. BACKGROUND DATA: Surgical injury is associated with significant exposure of host tissues to morphine from both endogenous release and its use as a potent analgesic agent. Morphine use in surgical patients exposed to extreme physiologic stress is well established to result in increased infection risk. Although morphine is a known immunosuppressant, whether it directly induces virulence expression and lethality in microbes that colonize the human gut remains unknown. METHODS: Mice were implanted with a slow release morphine or placebo pellet with and without intestinal inoculation of P. aeruginosa created by direct cecal injection. Mucus production and epithelial integrity was assessed in cecal tissue via Alcian blue staining and histologic analysis. In vivo and in vitro P. aeruginosa virulence expression was examined using reporter strains tagged to the epithelial barrier disrupting protein PA-I lectin. P. aeruginosa chemotaxis toward morphine was also assayed in vitro. Finally, the direct effect of morphine to induce PA-I lectin expression was determined in the absence and presence of methylnaltrexone, a µ opioid receptor antagonist. RESULTS: Mice intestinally inoculated with P. aeruginosa and implanted with a morphine pellet demonstrated significant suppression of intestinal mucus, disrupted intestinal epithelium, and enhanced mortality; whereas exposure of mice to either systemic morphine or intestinal P. aeruginosa alone enhanced intestinal mucus without mortality, suggesting a shift in P. aeruginosa during morphine exposure to a mucus suppressing, barrier disrupting, and lethal phenotype. Direct exposure of P. aeruginosa to morphine in vitro confirmed that morphine can transform P. aeruginosa to a more virulent phenotype that is attenuated in part by methylnaltrexone. CONCLUSIONS: Morphine administration shifts intestinal P. aeruginosa to express a virulent phenotype and may play a role in its ability to causes lethal gut-derived sepsis in a susceptible host.

Targeting pediatric pedestrian injury prevention efforts: teasing the information through spatial analysis.

BACKGROUND: Pediatric pedestrian injuries remain a major cause of childhood death, hospitalization, and disability. To target injury prevention efforts, it is imperative to identify those children at risk. Racial disparities have been noted in the rates of pediatric pedestrian injury and death. Children from low-income families living in dense, urban residential neighborhoods have a higher risk of sustaining pedestrian injury. Geographic information systems (GIS) analysis of associated community factors such as child population density and median income may offer insights into prevention. METHODS: Using trauma registry E-codes for pedestrian motor vehicle crashes, children younger than 16 years were identified, who received acute care and were hospitalized at the University of Chicago Medical Center, a Level I pediatric trauma center, after being struck by a motor vehicle from 2002 to 2009. By retrospective chart review and review of the Emergency Medical Services run sheets, demographic data and details of the crash site were collected. Crash sites were aggregated on a block by block basis. A "hot spot" analysis was performed to localize clusters of injury events. Using Gi* statistical method, spatial clusters were identified at different confidence intervals using a fixed distance band of 400 m (≈ » mile). Maps were generated using GIS with 2000 census data to evaluate race, employment, income, density of public and private schools, and density of children living in the neighborhoods surrounding our medical center where crash sites were identified. Spatial correlation is used to identify statistically significant locations. RESULTS: There were 3,521 children admitted to the University of Chicago Medical Center for traumatic injuries from 2002 to 2009; 27.7% (974) of these children sustained injuries in pedestrian motor vehicle injuries. From 2002 to 2009, there were a total of 106 traumatic deaths, of which 29 (27.4%) were due to pedestrian motor vehicle crashes. Pediatric pedestrian motor vehicle crash sites occurred predominantly within low-income, predominantly African-American neighborhoods. A lower prevalence of crash sites was observed in the predominantly higher income, non-African-American neighborhoods. CONCLUSIONS: Spatial analysis using GIS identified associations between pediatric pedestrian motor vehicle crash sites and the neighborhoods served by our pediatric trauma center. Pediatric pedestrian motor vehicle crash sites occurred predominantly within low-income, African-American neighborhoods. The disparity in prevalence of crash sites is somewhat attributable to the lower density of children living in the predominantly higher income, non-African-American neighborhoods, including the community immediately around our hospital. Traffic volume patterns, as a denominator of these injury events, remain to be studied.

Pseudomonas aeruginosa potentiates the lethal effect of intestinal ischemia-reperfusion injury: the role of in vivo virulence activation.

BACKGROUND: Experimental models of intestinal ischemia-reperfusion (IIR) injury are invariably performed in mice harboring their normal commensal flora, even though multiple IIR events occur in humans during prolonged intensive care confinement when they are colonized by a highly pathogenic hospital flora. The aims of this study were to determine whether the presence of the human pathogen Pseudomonas aeruginosa in the distal intestine potentiates the lethality of mice exposed to IIR and to determine what role any in vivo virulence activation plays in the observed mortality. METHODS: Seven- to 9-week-old C57/BL6 mice were exposed to 15 minutes of superior mesenteric artery occlusion (SMAO) followed by direct intestinal inoculation of 1.0 × 10(6) colony-forming unit of P. aeruginosa PAO1 into the ileum and observed for mortality. Reiterative studies were performed in separate groups of mice to evaluate both the migration/dissemination pattern and in vivo virulence activation of intestinally inoculated strains using live photon camera imaging of both a constitutive bioluminescent P. aeruginosa PAO1 derivative XEN41 and an inducible reporter derivative of PAO1, the PAO1/lecA:luxCDABE that conditionally expresses the quorum sensing-dependent epithelial disrupting virulence protein PA 1 Lectin (PA-IL). RESULTS: Mice exposed to 15 minutes of SMAO and reperfusion with intestinal inoculation of P. aeruginosa had a significantly increased mortality rate (p < 0.001) of 100% compared with <10% for sham-operated mice intestinally inoculated with P. aeruginosa without SMAO and IIR alone (<50%). Migration/dissemination patterns of P. aeruginosa in mice subjected to IIR demonstrated proximal migration of distally injected strains and translocation to mesenteric lymph nodes, liver, spleen, lung, and kidney. A key role for in vivo virulence expression of the barrier disrupting adhesin PA-IL during IIR was established since its expression was enhanced during IR and mutant strains lacking PA-IL displayed attenuated mortality. CONCLUSIONS: The presence of intestinal P. aeruginosa potentiates the lethal effect of IIR in mice in part due to in vivo virulence activation of its epithelial barrier disrupting protein PA-IL.

Prevention of siderophore- mediated gut-derived sepsis due to P. aeruginosa can be achieved without iron provision by maintaining local phosphate abundance: role of pH.

BACKGROUND: During extreme physiological stress, the intestinal tract can be transformed into a harsh environment characterized by regio- spatial alterations in oxygen, pH, and phosphate concentration. When the human intestine is exposed to extreme medical interventions, the normal flora becomes replaced by pathogenic species whose virulence can be triggered by various physico-chemical cues leading to lethal sepsis. We previously demonstrated that phosphate depletion develops in the mouse intestine following surgical injury and triggers intestinal P. aeruginosa to express a lethal phenotype that can be prevented by oral phosphate ([Pi]) supplementation. RESULTS: In this study we examined the role of pH in the protective effect of [Pi] supplementation as it has been shown to be increased in the distal gut following surgical injury. Surgically injured mice drinking 25 mM [Pi] at pH 7.5 and intestinally inoculated with P. aeruginosa had increased mortality compared to mice drinking 25 mM [Pi] at pH 6.0 (p < 0.05). This finding was confirmed in C. elegans. Transcriptional analysis of P. aeruginosa demonstrated enhanced expression of various genes involved in media alkalization at pH 6.0 and a global increase in the expression of all iron-related genes at pH 7.5. Maintaining the pH at 6.0 via phosphate supplementation led to significant attenuation of iron-related genes as demonstrated by microarray and confirmed by QRT-PCR analyses. CONCLUSION: Taken together, these data demonstrate that increase in pH in distal intestine of physiologically stressed host colonized by P. aeruginosa can lead to the expression of siderophore-related virulence in bacteria that can be prevented without providing iron by maintaining local phosphate abundance at pH 6.0. This finding is particularly important as provision of exogenous iron has been shown to have untoward effects when administered to critically ill and septic patients. Given that phosphate, pH, and iron are near universal cues that dictate the virulence status of a broad range of microorganisms relevant to serious gut origin infection and sepsis in critically ill patients, the maintenance of phosphate and pH at appropriate physiologic levels to prevent virulence activation in a site specific manner can be considered as a novel anti-infective therapy in at risk patients.