Endogenous Rab38 regulates LRRK2's membrane recruitment and substrate Rab phosphorylation in melanocytes.

Point mutations in leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease and augment LRRK2's kinase activity. However, cellular pathways that endogenously enhance LRRK2 kinase function have not been identified. While overexpressed Rab29 draws LRRK2 to Golgi membranes to increase LRRK2 kinase activity, there is little evidence that endogenous Rab29 performs this function under physiological conditions. Here, we identify Rab38 as a novel physiologic regulator of LRRK2 in melanocytes. In mouse melanocytes, which express high levels of Rab38, Rab32, and Rab29, knockdown (or CRISPR knockout) of Rab38, but not Rab32 or Rab29, decreases phosphorylation of multiple LRRK2 substrates, including Rab10 and Rab12, by both endogenous LRRK2 and exogenous Parkinson's disease-mutant LRRK2. In B16-F10 mouse melanoma cells, Rab38 drives LRRK2 membrane association and overexpressed kinase-active LRRK2 shows striking pericentriolar recruitment, which is dependent on the presence of endogenous Rab38 but not Rab32 or Rab29. Consistently, knockdown or mutation of BLOC-3, the guanine nucleotide exchange factor for Rab38 and Rab32, inhibits Rab38's regulation of LRRK2. Deletion or mutation of LRRK2's Rab38-binding site in the N-terminal armadillo domain decreases LRRK2 membrane association, pericentriolar recruitment, and ability to phosphorylate Rab10. In sum, our data identify Rab38 as a physiologic regulator of LRRK2 function and lend support to a model in which LRRK2 plays a central role in Rab GTPase coordination of vesicular trafficking.

Angiotensin II receptor inhibition ameliorates liver fibrosis and enhances hepatocellular carcinoma infiltration by effector T cells.

Although viral hepatocellular carcinoma (HCC) is declining, nonviral HCC, which often is the end stage of nonalcoholic or alcoholic steatohepatitis (NASH, ASH), is on an upward trajectory. Immune checkpoint inhibitors (ICIs) that block the T cell inhibitory receptor PD-1 were approved for treatment of all HCC types. However, only a minority of HCC patients show a robust and sustained response to PD-1 blockade, calling for improved understanding of factors that negatively impact response rate and duration and the discovery of new adjuvant treatments that enhance ICI responsiveness. Using a mouse model of NASH-driven HCC, we identified peritumoral fibrosis as a potential obstacle to T cell-mediated tumor regression and postulated that antifibrotic medications may increase ICI responsiveness. We now show that the angiotensin II receptor inhibitor losartan, a commonly prescribed and safe antihypertensive drug, reduced liver and peritumoral fibrosis and substantially enhanced anti-PD-1-induced tumor regression. Although losartan did not potentiate T cell reinvigoration, it substantially enhanced HCC infiltration by effector CD8+ T cells compared to PD-1 blockade alone. The beneficial effects of losartan correlated with blunted TGF-ß receptor signaling, reduced collagen deposition, and depletion of immunosuppressive fibroblasts.

Angiotensin II receptor inhibition ameliorates liver fibrosis and enhances hepatocellular carcinoma infiltration by effector T cells.

Although viral hepatocellular carcinoma (HCC) is declining, non-viral HCC, which often is the end-stage of non-alcoholic or alcoholic steatohepatitis (NASH, ASH), is on an upward trajectory. Immune checkpoint inhibitors (ICI) that block the T cell inhibitory receptor PD-1 were approved for treatment of all HCC types. However, only a small portion of HCC patients show a robust and sustained response to PD-1 blockade, calling for improved understanding of factors that negatively impact response rate and duration and the discovery of new adjuvant treatments that enhance ICI responsiveness. Using a mouse model of NASH-driven HCC, we identified peritumoral fibrosis as a potential obstacle to T cell mediated tumor regression and postulated that anti-fibrotic medications may increase ICI responsiveness. We now show that the angiotensin II receptor inhibitor losartan, a commonly prescribed and safe antihypertensive drug, reduced liver and peritumoral fibrosis and substantially enhanced anti-PD-1 induced tumor regression. Although losartan did not potentiate T cell reinvigoration, it substantially enhanced HCC infiltration by effector CD8 + T cells compared to PD-1 blockade alone. The beneficial effects of losartan correlated with inhibition of TGF-ß receptor signaling, collagen deposition and depletion of immunosuppressive fibroblasts. Significance: Immune checkpoint inhibitors are used in HCC treatment but overall response rates for single agent PD-1/PD-L1 blockers have remained stubbornly low. Using a mouse model of NASH-driven HCC, we show that co-treatment with the safe and inexpensive angiotensin II receptor inhibitor losartan substantially enhanced anti-PD-1 triggered HCC regression. Although losartan did not influence the reinvigoration of exhausted CD8 + T cells it considerably enhanced their intratumoral invasion, which we postulated to be compromised by peritumoral fibrosis. Indeed, the beneficial effect of losartan correlated with inhibition of TGF-ß signaling and collagen deposition, and depletion of immunosuppressive fibroblasts. Losartan should be evaluated for its adjuvant activity in HCC patients undergoing PD-1/PD-L1 blocking therapy.

Epigenomic Blood-Based Early Detection of Pancreatic Cancer Employing Cell-Free DNA.

BACKGROUND & AIMS: Early detection of pancreatic cancer (PaC) can drastically improve survival rates. Approximately 25% of subjects with PaC have type 2 diabetes diagnosed within 3 years prior to the PaC diagnosis, suggesting that subjects with type 2 diabetes are at high risk of occult PaC. We have developed an early-detection PaC test, based on changes in 5-hydroxymethylcytosine (5hmC) signals in cell-free DNA from plasma. METHODS: Blood was collected from 132 subjects with PaC and 528 noncancer subjects to generate epigenomic and genomic feature sets yielding a predictive PaC signal algorithm. The algorithm was validated in a blinded cohort composed of 102 subjects with PaC, 2048 noncancer subjects, and 1524 subjects with non-PaCs. RESULTS: 5hmC differential profiling and additional genomic features enabled the development of a machine learning algorithm capable of distinguishing subjects with PaC from noncancer subjects with high specificity and sensitivity. The algorithm was validated with a sensitivity for early-stage (stage I/II) PaC of 68.3% (95% confidence interval [CI], 51.9%-81.9%) and an overall specificity of 96.9% (95% CI, 96.1%-97.7%). CONCLUSIONS: The PaC detection test showed robust early-stage detection of PaC signal in the studied cohorts with varying type 2 diabetes status. This assay merits further clinical validation for the early detection of PaC in high-risk individuals.

Benefits of Early Thoracic Surgery in the Elderly During the COVID-19 Pandemic: Lessons Learned From Performing a Video-Assisted Thoracotomy.

Elderly patients are often considered poor surgical candidates for intra-thoracic operations due to the number of comorbidities, increased risks associated with general anesthesia, decreased cardiopulmonary reserve, and overall increased frailty. In addition, coronavirus disease 2019 (COVID-19) is a critical psychosocial factor that, through secondary effects, can prevent patients from receiving optimal care. Patients are reduced to having limited contact with family, often a vital support system, which can contribute to feelings of hopelessness, loneliness, and depression. We report the case of a 95-year-old female who presented to the emergency department with increasing supplemental oxygen requirements two weeks after a ground-level fall. She was found to have multiple rib fractures and a left-sided hemothorax. Initial management included aggressive respiratory therapy, multiple pigtail chest tubes, and thrombolytics; however, these measures failed to drain the intrathoracic hematoma. Her care was complicated by the psychosocial and isolation factors of COVID-19 which led to the patient exhibiting symptoms of hopelessness, grief, lack of appetite, and loneliness. As conservative management did not improve her clinical care the patient required a video-assisted thoracoscopic surgery (VATS) to manage the retained hemothorax and facilitate re-expansion of her atelectatic lung. Once the patient was removed from COVID-19 precautions, she was taken to surgery and postoperatively the patient reported minimal pain, participated more in physical therapy, and increased her oral intake. In this unique case, a 95-year-old patient with a hemothorax that was successfully treated with a VATS had her clinical care complicated by the psychosocial implications of COVID-19.

A Randomized Pilot Study Assessing if SEDLine Monitoring During Induction of Surgical Patients is Associated With Reduced Dosage of Administered Induction Agents.

Background. Intubations, especially in emergent settings, carry a high risk of hemodynamic instability with potentially catastrophic outcomes. Weight-based dosing of induction drugs can be inappropriately high for elective or emergent intubations and lead to hemodynamic instability. We hypothesized that monitoring the patient state index of SEDLine monitors (Masimo, Irvine, CA) would decrease the dose of induction drugs in the operating room during elective intubations.Methods. In this randomized study, SEDLine monitoring was provided to the intervention group but not to the control group during the induction of anesthesia in the operating room. Anesthesia providers in the intervention group were advised to titrate induction drugs to a Patient State Index of <50 before proceeding with intubation. The primary outcome was the induction dose of propofol and etomidate per kilogram normalized to propofol dose equivalents. Secondary outcomes included supplemental doses of ketamine, midazolam, fentanyl, phenylephrine, and ephedrine per kg, time from induction to intubation, administration of additional propofol or vasopressors after induction, mean arterial pressure ≥ or <65 mmHg, and lowest mean arterial pressure post-induction.Results. We found no significant difference in propofol equivalents between groups (P = .41). Using a SEDLine decreased the odds that a patient would require vasopressors during induction (odds ratio of .39 [95% confidence interval, .15-.98]).Conclusion. SEDLine monitoring during induction did not decrease dosing of the induction drugs etomidate and propofol but decreased the odds of receiving vasopressors. Further studies are warranted to assess the utility of processed electroencephalography in emergent intubations outside of the operating room.

Surgical Site Infection in Thoracic Surgery Is Not Associated With Perioperative Hypothermia.

INTRODUCTION: The Surgical Care Improvement Project (SCIP) added the SCIP-Inf-10 measure to mandate that all surgical patients have perioperative temperature management to reduce surgical site infection. While the basis of this measure originated in colorectal surgery, we hypothesized that this would also apply to thoracic surgery patients. METHODS: This was a retrospective single-center pilot study reviewing two years of thoracic surgery cases for the incidence and duration of hypothermia during the operation and surgical site infection occurring within 30 days. Hypothermia was defined as a core temperature of < 36° C.  Results: A total of 317 patients were included in the study. Sixty-two percent of patients were identified as hypothermic. The average intraoperative temperature was 35.4°C ± 0.8°C in the hypothermic group and 36.4°C ± 0.3°C in the normothermic group. There were four surgical site infections in the study with three cases from the <36°C group (p = 1). There was no difference in average post-anesthesia care unit length of stay between the groups. The average hospital length of stay was 5.5 ± 5.2 days for the hypothermic group and 8.6 ± 12.8 days for the normothermic group (p=0.0024). CONCLUSION: Perioperative hypothermia was common in thoracic surgery and did not have a negative impact on surgical site infection.

Early-life programming of mesenteric lymph node stromal cell identity by the lymphotoxin pathway regulates adult mucosal immunity.

Redundant mechanisms support immunoglobulin A (IgA) responses to intestinal antigens. These include multiple priming sites [mesenteric lymph nodes (MLNs), Peyer's patches, and isolated lymphoid follicles] and various cytokines that promote class switch to IgA, even in the absence of T cells. Despite these backup mechanisms, vaccination against enteric pathogens such as rotavirus has limited success in some populations. Genetic and environmental signals experienced during early life are known to influence mucosal immunity, yet the mechanisms for how these exposures operate remain unclear. Here, we used rotavirus infection to follow antigen-specific IgA responses through time and in different gut compartments. Using genetic and pharmacological approaches, we tested the role of the lymphotoxin (LT) pathway-known to support IgA responses-at different developmental stages. We found that LT-ß receptor (LTßR) signaling in early life programs intestinal IgA responses in adulthood by affecting antibody class switch recombination to IgA and subsequent generation of IgA antibody-secreting cells within an intact MLN. In addition, early-life LTßR signaling dictates the phenotype and function of MLN stromal cells to support IgA responses in the adult. Collectively, our studies uncover new mechanistic insights into how early-life LTßR signaling affects mucosal immune responses during adulthood.

Reduction of plasma angiopoietin-like 2 after cardiac surgery is related to tissue inflammation and senescence status of patients.

OBJECTIVES: A strong relationship between high circulating angiopoietin-like 2 (ANGPTL2) levels, a proinflammatory adipokine, and cardiovascular diseases has been reported. Our objective was to determine whether plasma ANGPTL2 and high-sensitivity C-reactive protein (hs-CRP) levels change postoperatively in patients who underwent heart valve surgery and/or coronary artery bypass grafting. We hypothesized that a corrective cardiac surgery would decrease ANGPTL2 levels. METHODS: In 47 prospectively recruited patients who underwent coronary artery bypass grafting (n = 16), valve replacement (n = 16), or both (n = 15), we measured plasma ANGPTL2 and hs-CRP levels preoperatively, at 24 hours, at 3 to 5 days (hospital discharge), and at 30 to 90 days (follow-up) after surgery. Mediastinal adipose tissue and distal fragments of the left internal mammary artery (IMA) were harvested during surgery and mRNA expression of inflammatory and senescence markers was assessed using real-time quantitative polymerase chain reaction. RESULTS: ANGPTL2 and hs-CRP levels were elevated 24 hours after surgery and then returned to baseline levels. We noted, however, a dichotomy among patients: compared with baseline, plasma ANGPTL2 levels either significantly decreased (n = 21/47) or increased (n = 26/47) after surgery. In contrast, hs-CRP levels were identical between groups (P = .997). Patients in the increased group were older (P = .002) with a higher systolic blood pressure (P = .038) at baseline. Moreover, changes in ANGPTL2 levels (ΔANGPTL2 = final minus initial levels) positively correlated with mRNA expression of tumor necrosis factor α and interleukin 8 in mediastinal adipose tissue and IMA (P < .05) and with the senescence-associated marker cyclin-dependent kinase inhibitor 1 in IMA (P = .009). CONCLUSIONS: In younger patients with lower levels of tissue inflammation and arterial senescence load, ANGPTL2, but not hs-CRP levels decreased after cardiac surgery, suggesting that circulating ANGPTL2 reflects tissue inflammation and senescence.

Nucleic acid delivery to mesenchymal stem cells: a review of nonviral methods and applications.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stem cells that can be isolated and expanded from many tissues, and are being investigated for use in cell therapies. Though MSC therapies have demonstrated some success, none have been FDA approved for clinical use. MSCs lose stemness ex vivo, decreasing therapeutic potential, and face additional barriers in vivo, decreasing therapeutic efficacy. Culture optimization and genetic modification of MSCs can overcome these barriers. Viral transduction is efficient, but limited by safety concerns related to mutagenicity of integrating viral vectors and potential immunogenicity of viral antigens. Nonviral delivery methods are safer, though limited by inefficiency and toxicity, and are flexible and scalable, making them attractive for engineering MSC therapies. MAIN TEXT: Transfection method and nucleic acid determine efficiency and expression profile in transfection of MSCs. Transfection methods include microinjection, electroporation, and nanocarrier delivery. Microinjection and electroporation are efficient, but are limited by throughput and toxicity. In contrast, a variety of nanocarriers have been demonstrated to transfer nucleic acids into cells, however nanocarrier delivery to MSCs has traditionally been inefficient. To improve efficiency, plasmid sequences can be optimized by choice of promoter, inclusion of DNA targeting sequences, and removal of bacterial elements. Instead of DNA, RNA can be delivered for rapid protein expression or regulation of endogenous gene expression. Beyond choice of nanocarrier and nucleic acid, transfection can be optimized by priming cells with media additives and cell culture surface modifications to modulate barriers of transfection. Media additives known to enhance MSC transfection include glucocorticoids and histone deacetylase inhibitors. Culture surface properties known to modulate MSC transfection include substrate stiffness and specific protein coating. If nonviral gene delivery to MSCs can be sufficiently improved, MSC therapies could be enhanced by transfection for guided differentiation and reprogramming, transplantation survival and directed homing, and secretion of therapeutics. We discuss utilized delivery methods and nucleic acids, and resulting efficiency and outcomes, in transfection of MSCs reported for such applications. CONCLUSION: Recent developments in transfection methods, including nanocarrier and nucleic acid technologies, combined with chemical and physical priming of MSCs, may sufficiently improve transfection efficiency, enabling scalable genetic engineering of MSCs, potentially bringing effective MSC therapies to patients.

Mechanisms of unprimed and dexamethasone-primed nonviral gene delivery to human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are under intense study for applications of cell and gene therapeutics because of their unique immunomodulatory and regenerative properties. Safe and efficient genetic modification of hMSCs could increase their clinical potential by allowing functional expression of therapeutic transgenes or control over behavior and differentiation. Viral gene delivery is efficient, but suffers from safety issues, while nonviral methods are safe, but highly inefficient, especially in hMSCs. Our lab previously demonstrated that priming cells before delivery of DNA complexes with dexamethasone (DEX), an anti-inflammatory glucocorticoid drug, significantly increases hMSC transfection success. This work systematically investigates the mechanisms of hMSC transfection and DEX-mediated enhancement of transfection. Our results show that hMSC transfection and its enhancement by DEX are decreased by inhibiting classical intracellular transport and nuclear import pathways, but DEX transfection priming does not increase cellular or nuclear internalization of plasmid DNA (pDNA). We also show that hMSC transgene expression is largely affected by pDNA promoter and enhancer sequence changes, but DEX-mediated enhancement of transfection is unaffected by any pDNA sequence changes. Furthermore, DEX-mediated transfection enhancement is not the result of increased transgene messenger RNA transcription or stability. However, DEX-priming increases total protein synthesis by preventing hMSC apoptosis induced by transfection, resulting in increased translation of transgenic protein. DEX may also promote further enhancement of transgenic reporter enzyme activity by other downstream mechanisms. Mechanistic studies of nonviral gene delivery will inform future rationally designed technologies for safe and efficient genetic modification of clinically relevant cell types.

The Association of Preoperative Hematocrit and Transfusion with Mortality in Patients Undergoing Elective Non-cardiac Surgery.

BACKGROUND: Patients with anemia frequently undergo surgery, as it is unclear at what threshold clinicians should consider delaying surgery for preoperative anemia optimization. The primary objective of this study was to determine whether there is an association of varying degrees of anemia and transfusion with 30-day mortality. METHODS: This is a retrospective cohort study using the American College of Surgeons National Surgical Quality Improvement Program database from 2011 to 2013. Cohorts were analyzed based on preoperative hematocrit range-patients with: (1) no anemia, (2) hematocrit ≥33% and <36% in females or <39% in males, (3) hematocrit ≥30% and <33%, (4) hematocrit ≥27% and <30%, (5) hematocrit ≥24% and <27%, and (6) hematocrit ≥21% and less than 24%. Multivariable logistic regression was used to analyze the association of anemia and transfusion with 30-day in-hospital mortality. RESULTS: The odds for 30-day mortality increased incrementally as the hematocrit ranges decreased, in which preoperative hematocrit between 21 and 24% had the highest odds for this outcome (odds ratio [OR] 6.50, p < 0.0001) compared to the reference group (no anemia). The use of transfusion increased the odds of mortality even further (OR 5.57, p < 0.0001). Among patients that received an intra-/postoperative transfusion, preoperative anemia was not predictive of mortality. CONCLUSIONS: Healthcare providers making preoperative clinical decisions for patients undergoing elective surgery should consider the degree of preoperative anemia and likelihood of perioperative transfusion.

Preoperative Functional Status Is Associated With Unplanned Intubations Following Thyroidectomies.

BACKGROUND: Unplanned postoperative intubation is an important event that may influence the outcome of thyroid- and parathyroidectomies. We performed a focused study on the association of preoperative functional status with unplanned intubation outcomes in these relatively common surgeries. METHODS: Utilizing data from the National Surgical Quality Improvement Program database from 2007 to 2013, a propensity score-matched retrospective cohort study was performed assessing this outcome in the functionally independent versus dependent groups. Kaplan-Meier survival analysis and a Cox proportional hazards model were performed to assess the difference. RESULTS: There were a total of 98,035 thyroid- and parathyroidectomies identified from the National Surgical Quality Improvement Program from 2007 to 2013. After propensity score matching, there were 1,862 and 931 cases in the independent and dependent group, respectively. There were 11 versus 33 per 1,000 persons in the independent and dependent group, respectively, who experienced an unplanned intubation within 30 d following surgery (P < .001). The dependent group showed worse intubation-free survival over 30 d (P < .001). There were no differences in this outcome during postoperative days 0-1 (P = .17). Dependent functional status was statistically significantly associated with unplanned intubations up to 30 d postoperatively (hazard ratio 2.4, 95% CI 1.4-4.18, P = .002). CONCLUSIONS: Preoperative functional status is a good marker for identifying patients at risk for re-intubation after thyroid- and parathyroidectomy.

Severity of Acute Kidney Injury in the Post-Lung Transplant Patient Is Associated With Higher Healthcare Resources and Cost.

OBJECTIVE: Perioperative risk factors and the clinical impact of acute kidney injury (AKI) and failure after lung transplantation are not well described. The incidences of AKI and acute renal failure (ARF), potential perioperative contributors to their development, and postdischarge healthcare needs were evaluated. DESIGN: Retrospective. SETTING: University hospital. PARTICIPANTS: Patients undergoing lung transplantation between January 1, 2011 and December 31, 2015. MEASURED DATA: The incidences of AKI and ARF, as defined using the Risk, Injury, Failure, Loss, End-Stage Renal Disease criteria, were measured. Perioperative events were analyzed to identify risk factors for renal compromise. A comparison of ventilator days, intensive care unit (ICU) and hospital lengths of stay (LOS), 1-year readmissions, and emergency department visits was performed among AKI, ARF, and uninjured patients. MEASUREMENTS AND MAIN RESULTS: Ninety-seven patients underwent lung transplantation; 22 patients developed AKI and 35 patients developed ARF. Patients with ARF had significantly longer ICU LOS (12 days v 4 days, p < 0.001); ventilator days (4.5 days v 1 day, p < 0.001); and hospital LOS (22.5 days v 14 days, p < 0.001) compared with uninjured patients. Patients with AKI also had significantly longer ICU and hospital LOS. Patients with ARF had significantly more emergency department visits and hospital readmissions (2 v 1 readmissions, p = 0.002) compared with uninjured patients. A univariable analysis suggested that prolonged surgical time, intraoperative vasopressor use, and cardiopulmonary bypass use were associated with the highest increased risk for AKI. Intraoperative vasopressor use and cardiopulmonary bypass mean arterial pressure <60 mmHg were identified as independent risk factors by multivariable analysis for AKI. CONCLUSION: The severity of AKI was associated with an increase in the use of healthcare resources after surgery and discharge. Certain risk factors appeared modifiable and may reduce the incidence of AKI and ARF.

Hemodynamic Consequence of Hand Ventilation Versus Machine Ventilation During Transport After Cardiac Surgery.

OBJECTIVES: The hemodynamic consequences of ventilation of intubated patients during transport either by hand or using a transport ventilator have not been reported in patients after cardiac surgery. The authors hypothesized that bag-mask ventilation would alter end-tidal CO2 during transport and hemodynamic parameters in patients post-cardiac surgery. DESIGN: A prospective, randomized trial. SETTING: A university-affiliated tertiary care hospital. PARTICIPANTS: Cardiac surgery patients. INTERVENTIONS: Thirty-six patients were randomized to hand ventilation or machine ventilation. Hemodynamic variables including blood pressure, heart rate, peripheral saturation of oxygen, and end-tidal carbon dioxide (ETCO2) were measured in these patients prior to transport, every 2 minutes during transport and upon arrival in the intensive care unit (ICU). Pulmonary artery pressure (PA) pressures were measured at origin and at destination. MEASUREMENTS AND MAIN RESULTS: Outcomes were changes from baseline in end-tidal CO2, hemodynamic changes from baseline and pulmonary artery pressure changes from origin to destination. The average transport time between the 2 groups was not different: 5 minutes for patients ventilated by hand and 5.47 minutes for patients ventilated with a transport ventilator (p = 0.369 by 2-sided t-test). The difference in all measured changes in ETCO2 between hand-ventilated and machine-ventilated patients during transport was 2.74 mmHg (p = 0.013). The difference between operating room and ICU ETCO2 from each cohort was 1.31 mmHg (p = 0.067). The difference in PAmean measured at origin and destination was 0.783 mmHg (p = 0.622). All other hemodynamic variables were not different during transport. CONCLUSIONS: Hand ventilation during transport was associated with greater change from baseline of ETCO2 compared to machine ventilation during transport after cardiac surgery, but this did not translate into any difference in hemodynamic changes upon arrival in ICU. A hemodynamic benefit of machine transport ventilation to cardiac patients was not demonstrated.

Rhesus θ-defensin-1 (RTD-1) exhibits in vitro and in vivo activity against cystic fibrosis strains of Pseudomonas aeruginosa.

OBJECTIVES: Chronic endobronchial infections with Pseudomonas aeruginosa contribute to bronchiectasis and progressive loss of lung function in patients with cystic fibrosis. This study aimed to evaluate the therapeutic potential of a novel macrocyclic peptide, rhesus θ-defensin-1 (RTD-1), by characterizing its in vitro antipseudomonal activity and in vivo efficacy in a murine model of chronic Pseudomonas lung infection. METHODS: Antibacterial testing of RTD-1 was performed on 41 clinical isolates of P. aeruginosa obtained from cystic fibrosis patients. MIC, MBC, time-kill and post-antibiotic effects were evaluated following CLSI-recommended methodology, but using anion-depleted Mueller-Hinton broth. RTD-1 was nebulized daily for 7 days to cystic fibrosis transmembrane conductance regulator (CFTR) F508del-homozygous mice infected using the agar bead model of chronic P. aeruginosa lung infection. In vivo activity was evaluated by change in lung bacterial burden, airway leucocytes and body weight. RESULTS: RTD-1 exhibited potent in vitro bactericidal activity against mucoid and non-mucoid strains of P. aeruginosa (MIC90 = 8 mg/L). Cross-resistance was not observed when tested against MDR and colistin-resistant isolates. Time-kill studies indicated very rapid, concentration-dependent bactericidal activity of RTD-1 with ≥3 log10 cfu/mL reductions at concentrations ≥4× MIC. No post-antibiotic effect was observed. In vivo, nebulized treatment with RTD-1 significantly decreased lung P. aeruginosa burden (mean difference of -1.30 log10 cfu; P = 0.0061), airway leucocytes (mean difference of -0.37 log10; P = 0.0012) and weight loss (mean difference of -12.62% at day 7; P < 0.05) when compared with controls. CONCLUSIONS: This study suggests that RTD-1 is a promising potential therapeutic agent for cystic fibrosis airway disease.

Postnatal exposure to voluntary exercise but not the antioxidant catechin protects the vasculature after a switch to an atherogenic environment in middle-age mice.

We aimed to evaluate the lasting functional imprinting of exercise (EX) and catechin (CAT) on the vascular function of middle-age mice switched to a proatherogenic environment. C57BL/6J mice (n = 10-15 in each group) fed a regular diet (RD) were exposed from the age of 1 to 9 months either to EX (voluntary running; 2.7 ± 0.2 km/day), to the polyphenol CAT (30 mg/kg/day in drinking water), or to physical inactivity (PI). At 9 months of age, EX and CAT were stopped and mice either remained on the RD or were fed a Western diet (WD) for an additional 3 months. At 12 months of age, mice from all groups fed a WD had similar body mass, systolic blood pressure, and plasma total cholesterol, glucose, insulin, and isoprostane. Compared to the RD, the WD induced an indomethacin-sensitive aortic endothelium-dependent and independent dysfunction in PI mice (p < 0.05) that was prevented by both EX and CAT; this benefit was associated with a higher (p < 0.05) non-nitric oxide/non-prostacyclin endothelium-dependent relaxation. While EX, but not PI or CAT, prevented vascular dysfunction induced by the WD in cerebral arteries, it had no effect in femoral arteries. The profiles of activity of antioxidant enzymes and of proinflammatory gene expression in the aorta suggest a better adaptation of EX > CAT > PI mice to stress. In conclusion, our data suggest that a postnatal exposure to EX, but not to CAT, imprints an adaptive defense capacity in the vasculature against a deleterious change in lifestyle.

Doxycycline exhibits anti-inflammatory activity in CF bronchial epithelial cells.

A hallmark of cystic fibrosis is the massive recruitment of neutrophils into the lung compartment in response to chronic Pseudomonas aeruginosa infection. The overexuberant neutrophilic response results in release of proteases (e.g. neutrophil elastase and matrix metalloproteinase-9) leading to matrix breakdown, airway remodeling, and progressive loss of lung function. Doxycycline is used clinically for the management of periodontitis due to its potent direct inhibition of matrix metalloproteinases; however, little is known regarding its potential anti-inflammatory properties and clinical utility in the context of cystic fibrosis airway disease. CF (IB3-1) and corrected (S9) bronchial epithelial cell lines were used to determine the cytotoxicity and anti-inflammatory effects of doxycycline in-vitro. Exposure to doxycycline, at low concentrations, resulted in minimal cell death and dose dependent reductions in release of CXCL-8 and MMP-9 protein. To confirm these findings, mechanistic analysis revealed ERK 1/2, p38, and JNK, but not NF-κB p65 dependent cell signaling inhibition with doxycycline treatment. These findings indicate that doxycycline exhibits anti-inflammatory activity in CF lung epithelial cells at concentrations below the cytotoxic potential. These data are encouraging and indicate in-vivo studies are warranted.

Pharmacokinetics of doxycycline in adults with cystic fibrosis.

Cystic fibrosis (CF) is characterized by a chronic neutrophilic inflammatory response resulting in airway remodeling and progressive loss of lung function. Doxycycline is a tetracycline antibiotic that inhibits matrix metalloproteinase 9, a protease known to be associated with the severity of lung disease in CF. The pharmacokinetics of doxycycline was investigated during the course of a clinical trial to evaluate the short-term efficacy and safety in adults with CF. Plasma samples were obtained from 14 patients following a single intravenous dose and after 2 and 4 weeks of oral administration of doses ranging from 40 to 200 mg daily. The data were analyzed using noncompartmental and compartmental pharmacokinetics. The maximum concentration of drug in serum (C(max)) and area under the concentration-time curve from 0 h to infinity (AUC(0-∞)) values ranged from 1.0 to 3.16 mg/liter and 15.2 to 47.8 mg/liter × h, respectively, following single intravenous doses of 40 to 200 mg. C(max) and time to maximum concentration of drug in serum (T(max)) values following multiple-dose oral administration ranged from 1.15 to 3.04 mg/liter and 1.50 to 2.33 h, respectively, on day 14 and 1.48 to 3.57 mg/liter and 1.00 to 2.17 on day 28. Predose sputum/plasma concentration ratios on days 14 and 28 ranged from 0.33 to 1.1 (mean, 0.71 ± 0.33), indicating moderate pulmonary penetration. A 2-compartment model best described the combined intravenous and oral data. Absorption was slow and delayed (absorption rate constant [K(a)], 0.414 h(-1); lag time, 0.484 h) but complete (bioavailability [F], 1.16). The distribution and elimination half-lives were 0.557 and 18.1 h, respectively. Based on these data, the plasma concentrations at the highest dose, 200 mg/day, are in the range reported to produce anti-inflammatory effects in vivo and should be evaluated in clinical trials.

Up-regulation of thromboxane A2 impairs cerebrovascular eNOS function in aging atherosclerotic mice.

We previously reported that in healthy mouse cerebral arteries, endothelial nitric oxide synthase (eNOS) produces H2O2, leading to endothelium-dependent dilation. In contrast, thromboxane A2 (TXA2), a potent pro-oxidant and pro-inflammatory endogenous vasoconstrictor, is associated with eNOS dysfunction. Our objectives were to elucidate whether (1) the cerebrovascular eNOS-H2O2 pathway was sensitive to oxidative stress associated with aging and dyslipidemia and (2) TXA2 contributed to cerebral eNOS dysfunction. Atherosclerotic (ATX = LDLR(-/-); hApoB(+/+)) and wild-type (WT) control mice were used at 3 and 12 months old (m/o). Three-m/o ATX mice were treated with the cardio-protective polyphenol catechin for 9 months. Dilations to ACh and the simultaneous eNOS-derived H2O2 production were recorded in isolated pressurized cerebral arteries. The age-associated decrease in cerebral eNOS-H2O2 pathway observed in WT was premature in ATX mice, decreasing at 3 m/o and abolished at 12 m/o. Thromboxane synthase inhibition by furegrelate increased dilations at 12 months in WT and at 3 and 12 months in ATX mice, suggesting an anti-dilatory role of TXA2 with age hastened by dyslipidemia. In addition, the non-selective NADP(H) oxidase inhibitor apocynin improved the eNOS-H2O2 pathway only in 12-m/o ATX mice. Catechin normalized the function of this pathway, which became sensitive to L-NNA and insensitive to furegrelate or apocynin; catechin also prevented the rise in TXA2 synthase expression. In conclusion, the age-dependent cerebral endothelial dysfunction is precocious in dyslipidemia and involves TXA2 production that limits eNOS activity. Preventive catechin treatment reduced the impact of endogenous TXA2 on the control of cerebral tone and maintained eNOS function.