Low-molecular weight branched polyethylenimine reduces cytokine secretion from human immune system monocytes stimulated with bacterial and fungal PAMPs.

Adaptive immunity recruits T-cells and specific antibodies against antigens, innate immune cells express pathogen recognition receptors (PRRs) that can detect various pathogen-associated molecular patterns (PAMPs) released by invading pathogens. Microbial molecular patterns, such as lipopolysaccharide (LPS) from Gram-negative bacteria, trigger signaling cascades in the host that result in the production of pro-inflammatory cytokines. LPS stimulation produces a strong immune response and excessive LPS signaling leads to dysregulation of the immune response. However, dysregulated inflammatory response during wound healing often results in chronic non-healing wounds that are difficult to control. In this work, we present data demonstrating partial neutralization of anionic LPS molecules using cationic branched polyethylenimine (BPEI). The anionic sites on the LPS molecules from Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are the lipid A moiety and BPEI binding create steric factors that hinder the binding of PRR signaling co-factors. This reduces the production of pro-inflammatory TNF-α cytokines. However, the anionic sites of Pseudomonas aeruginosa (P. aeruginosa) LPS are in the O-antigen region and subsequent BPEI binding slightly reduces TNF-α cytokine production. Fortunately, BPEI can reduce TNF-α cytokine expression in response to stimulation by intact P. aeruginosa bacterial cells and fungal zymosan PAMPs.

Acquisition of Resistance to PEGylated branched polyethylenimine Increases Pseudomonas aeruginosa Susceptibility to Aminoglycosides.

PEGylated branched polyethylenimine (PEG-BPEI) has antibacterial and antibiofilm properties. Exposure to PEG-BPEI through serial passage leads to resistant P. aeruginosa strains. The minimum inhibitory concentration (MIC) of 600 Da BPEI and PEGylated 600 Da BPEI (PEG-BPEI) in the wild-type PAO1 strain is 16 µg/ml while, after 15 serial passages, the MIC increased to 1024 µg/mL. An additional 15 rounds of passage in the absence of BPEI or PEG-BPEI did not change the 1024 µg/mL MIC. Gentamicin, Neomycin, and Tobramycin, cationic antibiotics that inhibit protein synthesis, have a 16-32 fold reduction of MIC values in PEG350-BPEI resistant strains, suggesting increased permeation. The influx of these antibiotics occurs using a self-mediated uptake mechanism, perhaps due to changes in the outer membrane Data show that resistance causes changes in genes related to outer membrane lipopolysaccharide (LPS) assembly. Mutations were noted in the gene coding for the polymerase Wzy that participates in the assembly of the O-antigen region. Other mutations were noted with wbpE and wbpI of the Wbp pathway responsible for the enzymatic synthesis of ManNAc(3NAc)A in the LPS of P. aeruginosa. These changes suggest that PEG-BPEI resistance can be countered with antibiotics to prevent the emergence of PEG-BPEI resistant bacterial populations.

Neutralizing Staphylococcus aureus PAMPs that Trigger Cytokine Release from THP-1 Monocytes.

Innate immunity has considerable specificity and can discriminate between individual species of microbes. In this regard, pathogens are "seen" as dangerous to the host and elicit an inflammatory response capable of destroying the microbes. This immune discrimination is achieved by toll-like receptors on host cells recognizing pathogens, such as Staphylococcus aureus, and microbe-specific pathogen-associated molecular pattern (PAMP) molecules, such as lipoteichoic acid (LTA). PAMPs impede wound healing by lengthening the inflammatory phase of healing and contributing to the development of chronic wounds. Preventing PAMPs from triggering the release of inflammatory cytokines will counteract the dysregulation of inflammation. Here, we use ELISA to evaluate the use of cationic molecules branched polyethylenimine (BPEI), PEGylated BPEI (PEG-BPEI), and polymyxin-B to neutralize anionic LTA and lower levels of TNF-α cytokine release from human THP-1 monocytes in a concentration-dependent manner. Additional data collected with qPCR shows that BPEI and PEG-BPEI reduce the expression profile of the TNF-α gene. Similar effects are observed for the neutralization of whole-cell S. aureus bacteria. In vitro cytotoxicity data demonstrate that PEGylation lowers the toxicity of PEG-BPEI (IC50 = 2661 µm) compared to BPEI (IC50 = 853 µM) and that both compounds are orders of magnitude less toxic than the cationic antibiotic polymyxin-B (IC50 = 79 µM). Additionally, the LTA neutralization ability of polymyxin-B is less effective than BPEI or PEG-BPEI. These properties of BPEI and PEG-BPEI expand their utility beyond disabling antibiotic resistance mechanisms and disrupting S. aureus biofilms, providing additional justification for developing these agents as wound healing therapeutics. The multiple mechanisms of action for BPEI and PEG-BPEI are superior to current wound treatment strategies that have a single modality.

Antibiofilm Activity of PEGylated Branched Polyethylenimine.

Biofilm formation is an adaptive resistance mechanism that pathogens employ to survive in the presence of antimicrobials. Pseudomonas aeruginosa is an infectious Gram-negative bacterium whose biofilm allows it to withstand antimicrobial attack and threaten human health. Chronic wound healing is often impeded by P. aeruginosa infections and the associated biofilms. Previous findings demonstrate that 600 Da branched polyethylenimine (BPEI) can restore ß-lactam potency against P. aeruginosa and disrupt its biofilms. Toxicity concerns of 600 Da BPEI are mitigated by covalent linkage with low-molecular-weight polyethylene glycol (PEG), and, in this study, PEGylated BPEI (PEG350-BPEI) was found exhibit superior antibiofilm activity against P. aeruginosa. The antibiofilm activity of both 600 Da BPEI and its PEG derivative was characterized with fluorescence studies and microscopy imaging. We also describe a variation of the colony biofilm model that was employed to evaluate the biofilm disruption activity of BPEI and PEG-BPEI.

Beyond Program Objectives.

Graduate students in eight diverse health care professional programs participated together in a pilot course entitled, "An Interprofessional Approach to Patient Care." Through postcourse discussions with the clinical nurse leader students, faculty discovered that beyond meeting program objectives, the nursing learners gained numerous unexpected insights about interprofessional collaborative practice. J Contin Educ Nurs. 2016;47(6):248-249.

Associations of retinal oximetry in persons with diabetes.

BACKGROUND: There are limited data available on the variables that might affect retinal vessel oxygen saturation (SO2) in diabetes. Therefore, the aim of this study is to assess factors associated with retinal oximetry values in persons with diabetes. DESIGN: Clinic-based cross-sectional study. PARTICIPANTS: Fifty-eight persons with diabetes aged 18+ years, recruited from the University of Melbourne, the Royal Victorian Eye and Ear Hospital, and St. Vincent's Hospital (Melbourne), Australia. METHODS: Retinal oximetry was performed using the oximetry module of the Vesselmap system (Imedos UG, Jena, Germany) in 92 diabetic eyes. Generalized estimating equation models were used to estimate the associations between candidate variables (age; gender; retinal capillary flow; duration of diabetes; hypertension; smoking status; presence of diabetic retinopathy [DR]; glycated haemoglobin; triglyceride; total cholesterol; finger SO2 and ocular perfusion pressure) with retinal oximetry measures. MAIN OUTCOME MEASURE: Arteriolar SO2, venular SO2 and the arterio-venous (A-V) difference. RESULTS: Of the candidate factors assessed, only the presence of DR was significantly associated with increased venular SO2 and decreased A-V difference in unadjusted analyses. In models adjusting for age and gender and significant variables from unadjusted analyses, compared with no DR, the presence of DR was significantly associated with greater retinal venular SO2 values (ß = 3.65%, 95% confidence interval: 0.67-6.63%) and decreased A-V difference (ß = -2.00%, 95% confidence interval: -3.46 to -0.53%). CONCLUSION: In patients with diabetes, eyes with DR were associated with increased venular SO2 and decreased A-V difference compared with eyes without DR, suggesting an altered metabolic state in DR.

Flicker light-induced retinal vasodilation is unaffected by inhibition of epoxyeicosatrienoic acids and prostaglandins in humans.

PURPOSE: To investigate the role of epoxyeicosatrienoic acids (EETs) and prostaglandins (PGs) in retinal blood vessel calibers and vasodilation during flicker light stimulation in humans. METHODS: Twelve healthy nonsmokers participated in a balanced crossover study. Oral fluconazole 400 mg and dispersible aspirin 600 mg were used to inhibit production of EETs and PGs, respectively. Retinal imaging was performed 1 hour after drug ingestion with the Dynamic Vessel Analyzer. Resting calibers of selected vessel segments were recorded in measurement units (MU). Maximum percentage dilations during flicker stimulation were calculated from baseline calibers. We then studied six participants each after fluconazole and aspirin ingestions at 30-minute intervals for 2 hours. Within-subject differences were assessed by ANOVA and Dunnett-adjusted pairwise comparisons with significance taken at P < 0.05. RESULTS: In crossover study participants, mean (SD) arteriole and venule dilations without drug administration were 4.4% (2.0%) and 4.6% (1.7%), respectively. Neither drug affected vasodilation during flicker stimulation. Mean (SD) resting arteriole and venule calibers on no-drug visits were 119.6 (10.6) MU and 145.7 (17.0) MU, respectively. Fluconazole reduced mean (±95% CI) resting venule calibers by 5.1 (4.3) MU. In repeated measures participants, neither drug affected vasodilations, but fluconazole reduced resting venule calibers over 2 hours (P < 0.001). CONCLUSIONS: Epoxyeicosatrienoic acids and prostaglandins are unlikely to be primary mediators of flicker light-induced retinal vasodilation in humans. However, EETs may play a role in the regulation of retinal vascular tone and blood flow under resting physiological conditions.

Decreased retinal capillary flow is not a mediator of the protective myopia-diabetic retinopathy relationship.

PURPOSE: The mechanisms supporting the protective relationship between a longer axial length (AL) and a decreased risk of diabetic retinopathy (DR) remain unclear. Previous studies have demonstrated reduced retinal blood flow in axial myopia, and it has been suggested that the compromised retinal capillaries in diabetes are less likely to leak and rupture as a result of this decreased flow. In this study, we therefore investigated if reduced retinal capillary flow (RCF) is a potential mechanism underpinning this protective relationship. METHODS: Retinal capillary flow was assessed using the Heidelberg Retinal Flowmeter in 150 eyes of 85 patients with diabetes aged 18+ years from the Royal Victorian Eye and Ear Hospital and St. Vincent's Hospital (Melbourne), Australia. Axial length was measured using the Intraocular Lens Master. Diabetic retinopathy was graded from two-field retinal photographs into none, mild, moderate, and severe DR using the modified Airlie House classification system. RESULTS: A total of 74 out of 150 eyes (49.3%) had DR. A longer AL was associated with decreased odds of DR presence (per mm increase in AL, odds ratio [OR] 0.61, 95% confidence interval [CI] 0.41-0.91) and DR severity (OR: 0.65; 95% CI: 0.44-0.95). However, no association was found between AL and RCF (per mm increase in AL, regression coefficient [ß] -1.80, 95% CI -13.50 to 9.50) or between RCF and DR (per unit increase in RCF, OR 1.00; 95% CI 0.99-1.00). CONCLUSIONS: Our finding suggests that diminished RCF may not be a major factor underlying the protective association between axial elongation and DR.

Flicker-induced retinal arteriole dilation is reduced by ambient lighting.

PURPOSE: To investigate the impact of ambient room lighting on the magnitude of flicker light-induced retinal vasodilations in healthy individuals. METHODS: Twenty healthy nonsmokers participated in a balanced 2 × 2 crossover study. Retinal vascular imaging was performed with the dynamic vessel analyzer under reduced or normal ambient lighting, then again after 20 minutes under the alternate condition. Baseline calibers of selected arteriole and venule segments were recorded in measurement units. Maximum percentage dilations from baseline during 20 seconds of luminance flicker were calculated from the mean of three measurement cycles. Within-subject differences were assessed by repeated measures analysis of variance with the assumption of no carryover effects and pairwise comparisons from the fitted model. RESULTS: Mean (SD) maximum arteriole dilations during flicker stimulation under reduced and normal ambient lighting were 4.8% (2.3%) and 4.1% (1.9%), respectively (P = 0.019). Maximum arteriole dilations were (mean ± 95% confidence interval) 0.7% ± 0.6% lower under normal ambient lighting compared with reduced lighting. Ambient lighting had no significant effect on maximum venular dilations during flicker stimulation or on the baseline calibers of arterioles or venules. CONCLUSIONS: Retinal arteriole dilation in response to luminance flicker stimulation is reduced under higher ambient lighting conditions. Reduced responses with higher ambient lighting may reflect reduced contrast between the ON and OFF flicker phases. Although it may not always be feasible to conduct studies under reduced lighting conditions, ambient lighting levels should be consistent to ensure that comparisons are valid.

Axial length, retinal function, and oxygen consumption: a potential mechanism for a lower risk of diabetic retinopathy in longer eyes.

PURPOSE: To determine the relationship between axial length (AL), retinal function, and relative oxygen (O2) consumption to better understand the protective effect of axial elongation on diabetic retinopathy development. METHODS: Measurements of AL, multifocal electroretinogram (mfERG), and relative O2 consumption (difference between arteriolar and venular O2 saturation levels or A-V difference) were performed on 50 healthy individuals. The relationships between AL, mfERG amplitude, and A-V difference were analyzed using linear regression models. Path analysis was performed to determine the direct and indirect effects (via mfERG amplitude) of AL on A-V difference. RESULTS: mfERG P1 amplitude was positively associated with A-V difference (ß = 0.33; 95% confidence interval [CI]: 0.23-0.42). Increased AL was significantly associated with a decrease in A-V difference (ß = -1.08; 95% CI: -1.52 to -0.65) as well as a decrease in retinal function (ß = -3.14, 95% CI: -4.07 to -2.20). Path analysis models including AL (study factor), retinal function (intermediate variable), and A-V difference (outcome variable) showed that AL had little direct association with A-V difference (ß(p) = -0.002), while the indirect effect of AL on A-V difference via changes in retinal function were substantial (ß(p) = -0.51). CONCLUSIONS: In eyes with longer AL, the reduction in A-V difference is explained by the parallel reduction in retinal function. These findings suggest that longer eyes have decreased retinal function and O2 consumption, and thus are relatively less hypoxic in the presence of diabetes, which may partly explain the reduced risk of DR in these eyes.

Retinal arteriolar dilation to flicker light is reduced on short-term retesting.

PURPOSE: To investigate the impact of retesting frequency over a short period on flicker light-induced retinal vasodilation. METHODS: Twenty healthy participants were included. The retinal vascular response to flicker light stimulation was assessed three times (at baseline and after 5 and 30 minutes of rest [tests 1, 2, and 3, respectively]) in each participant using the Dynamic Vessel Analyzer. Relative dilations of selected arteriole and venule segments during flicker stimulation and resting diameters were measured automatically. The mean vessel dilations and resting diameters were compared using repeated-measures analysis of variance. RESULTS: Participants were young (mean [SD] age, 33.1 [5.7] years) and mostly female (70%). The mean (SD) maximum arteriolar dilations during flicker stimulation were 3.23% (2.06%), 2.44% (1.62%), and 3.36% (2.11%) in tests 1, 2, and 3, respectively. The mean (SD) venular dilations were 4.26% (1.28%), 3.81% (1.61%), and 4.43% (1.73%) in tests 1, 2, and 3, respectively. The mean arteriolar dilations were significantly different across the three tests (P < 0.001). Compared with test 1, arteriolar dilations were significantly reduced after 5 minutes (P = 0.008) but not 30 minutes (P = 0.437) of rest. No significant differences were found over time for the mean venular dilations (P = 0.128). Resting diameters of selected vessels were not significantly different between tests. CONCLUSIONS: Retinal arteriolar dilation during flicker stimulation is reduced on short-term retesting, without a significant change in baseline vessel diameter, indicating decreased responsiveness to the flicker stimulus. Researchers should allow at least 30 minutes between consecutive tests to minimize suppression of the flicker response.

Decadal Cycles in the Human Cardiovascular System.

Seven of the eight authors of this report each performed physiologic self-surveillance, some around the clock for decades. We here document the presence of long cycles (decadals, including circaundecennians) in the time structure of systolic (S) and diastolic (D) blood pressure (BP) and heart rate (HR). Because of the non-stationary nature in time and space of these and other physiologic and environmental periodic components that, like the wind, can appear and disappear in a given or other geographic location at one or another time, they have been called "Aeolian". The nonlinear estimation of the uncertainties of the periods (τs) of two or more variables being compared has been used to determine whether these components are congruent or not, depending on whether their CIs (95% confidence intervals) overlap or not. Among others, congruence has been found for components with τs clustering around 10 years in us and around us. There is a selective assortment among individuals, variables and cycle characteristics (mean and circadian amplitude and acrophase). Apart from basic interest, like other nonphotic solar signatures such as transyears with periods slightly longer than one year or about 33-year Brückner-Egeson-Lockyer (BEL) cycles, about 10-year and longer cycles present in 7 of 7 self-monitoring individuals are of interest in the diagnosis of Vascular Variability Anomalies (VVAs), including MESOR-hypertension, and others. Some of the other VVAs, such as a circadian overswing, i.e., CHAT (Circadian Hyper-Aplitude-Tension), or an excessive pulse pressure, based on repeated 7-day around-the-clock records, can represent a risk of severe cardiovascular events, greater than that of a high BP. The differential diagnosis of physiologic cycles, infradians (components with a τ longer than 28 hours) as well as circadians awaits the collection of reference values for the infradian parameters of the cycles described herein. Just as in stroke-prone spontaneously hypertensive rats during the weeks after weaning CHAT precedes an elevation of the BP MESOR, a decadal overswing seems to precede the occurrence of high BP in two of the subjects here examined. Only around-the-clock monitoring in health for the collection of reference values will allow on their basis the differential diagnosis of the onsets of a circadian versus a circadecadal overswing in BP and the specification whether, and if so, when to initiate hypotensive non-drug or drug treatment.

Predicting the Crohn's disease activity index from the Harvey-Bradshaw Index.

BACKGROUND: The Crohn's Disease Activity Index (CDAI) was developed in the 1970s to assess the degree of illness in individuals with Crohn's disease and has since been used widely in clinical trials of the condition. The Harvey-Bradshaw Index (HBI) is a simplification of the CDAI, designed to make data collection and computation easier. It is purported, on the basis of a 0.93 correlation coefficient, to give "essentially the same information." However, correlation is an incomplete way to assess sameness, and this study aimed to develop a method for predicting CDAI from HBI values, including relevant prediction limits. MATERIALS AND METHODS: Data used in developing both indexes were combined. Single visits of 224 patients with Crohn's disease were plotted on a scattergram. HBI values seen were integers from 0 through 19. Mean and standard deviation of CDAI were determined for each HBI value that included a sufficient number of patients. Standard deviation of CDAI showed a linear increase with increasing HBI. Therefore, regression of CDAI on HBI was weighted on the inverse of the estimated CDAI standard deviation. RESULTS: Regression predicted a 27-CDAI-unit increase for each HBI unit. Calculated 95% prediction limits were almost straight, diverging lines, bracketing 95% of observations. A table gives central tendency and 95% prediction limits of CDAI for any HBI, as well as key clinical benchmarks. CONCLUSIONS: There is a good but far from perfect relationship between CDAI and HBI. CDAI is preferred for clinical trials; HBI is easier to use.

Identifying patient preoperative risk factors and postoperative adverse events in administrative databases: results from the Department of Veterans Affairs National Surgical Quality Improvement Program.

BACKGROUND: The Department of Veterans Affairs (DVA) National Surgical Quality Improvement Program (NSQIP) employs trained nurse data collectors to prospectively gather preoperative patient characteristics and 30-day postoperative outcomes for most major operations in 123 DVA hospitals to provide risk-adjusted outcomes to centers as quality indicators. It has been suggested that routine hospital discharge abstracts contain the same information and would provide accurate and complete data at much lower cost. STUDY DESIGN: With preoperative risks and 30-day outcomes recorded by trained data collectors as criteria standards, ICD-9-CM hospital discharge diagnosis codes in the Patient Treatment File (PTF) were tested for sensitivity and positive predictive value. ICD-9-CM codes for 61 preoperative patient characteristics and 21 postoperative adverse events were identified. RESULTS: Moderately good ICD-9-CM matches of descriptions were found for 37 NSQIP preoperative patient characteristics (61%); good data were available from other automated sources for another 15 (25%). ICD-9-CM coding was available for only 13 (45%) of the top 29 predictor variables. In only three (23%) was sensitivity and in only four (31%) was positive predictive value greater than 0.500. There were ICD-9-CM matches for all 21 NSQIP postoperative adverse events; multiple matches were appropriate for most. Postoperative occurrence was implied in only 41%; same breadth of clinical description in only 23%. In only four (7%) was sensitivity and only two (4%) was positive predictive value greater than 0.500. CONCLUSION: Sensitivity and positive predictive value of administrative data in comparison to NSQIP data were poor. We cannot recommend substitution of administrative data for NSQIP data methods.