Transient depletion of macrophages alters local inflammatory response at the site of disc herniation in a transgenic mouse model.

OBJECTIVE: Macrophages are abundantly detected at sites of disc herniation, however, their function in the disease progression is unclear. We aim to investigate the functions of macrophages in acute disc herniation using a macrophage Fas-induced apoptosis (MaFIA) transgenic mouse strain. METHOD: To transiently deplete macrophages, a dimerizer, AP20187, or vehicle solution was administered via intraperitoneal injection to MaFIA mice immediately, day 1 and 2 after annular puncture induced disc herniation. Local infiltrated tissues at disc hernia and DRGs at corresponding levels were harvested to analyze immune cells and neuroinflammation on postoperative day (POD) 6 by flow cytometry and/or immunostaining. Mouse spines were harvested to analyze structures of degenerated discs and adjacent vertebrae and to assess osteoclast activity by histology and tartrate-resistant acid phosphatase (TRAP) staining on POD 6, 13, and 20, respectively. RESULTS: On POD 6, abundant macrophages were confirmed at disc hernia sites. Compared to vehicle control, AP20187 significantly reduced GFP+ cells in blood, spleen, and local inflammatory tissue. At disc hernia sites, AP20187 markedly reduced macrophages (CD11b+, F4/80+, GFP+CD11b+, CD11b+F4/80+) while increasing neutrophils and B cells. Transient macrophage depletion decreased ectopic bone formation and osteoclast activity in herniated discs and adjacent cortical bones for up to 20 days post herniation. Disc herniation elevated expressions of TNF-α, IL-6, substance P, calcitonin gene-related peptide, accompanied by increasing GFP+, CD11b+ and F4/80+ macrophages. Macrophage depletion did not attenuate these markers of neuroinflammation. CONCLUSIONS: Transient depletion of macrophages altered local inflammatory response at the site of disc herniation.

Reconnaissance of tumor immune microenvironment spatial heterogeneity in metastatic renal cell carcinoma and correlation with immunotherapy response.

A clearer understanding of the tumor immune microenvironment (TIME) in metastatic clear cell renal cell carcinoma (ccRCC) may help to inform precision treatment strategies. We sought to identify clinically meaningful TIME signatures in ccRCC. We studied tumors from 39 patients with metastatic ccRCC using quantitative multiplexed immunofluorescence and relevant immune marker panels. Cell densities were analyzed in three regions of interest (ROIs): tumor core, tumor-stroma interface and stroma. Patients were stratified into low- and high-marker density groups using median values as thresholds. Log-rank and Cox regression analyses while controlling for clinical variables were used to compare survival outcomes to patterns of immune cell distributions. There were significant associations with increased macrophage (CD68+ CD163+ CD206+ ) density and poor outcomes across multiple ROIs in primary and metastatic tumors. In primary tumors, T-bet+ T helper type 1 (Th1) cell density was highest at the tumor-stromal interface (P = 0·0021), and increased co-expression of CD3 and T-bet was associated with improved overall survival (P = 0·015) and survival after immunotherapy (P = 0·014). In metastatic tumor samples, decreased forkhead box protein 3 (FoxP3)+ T regulatory cell density correlated with improved survival after immunotherapy (P = 0·016). Increased macrophage markers and decreased Th1 T cell markers within the TIME correlated with poor overall survival and treatment outcomes. Immune markers such as FoxP3 showed consistent levels across the TIME, whereas others, such as T-bet, demonstrated significant variance across the distinct ROIs. These findings suggest that TIME profiling outside the tumor core may identify clinically relevant associations for patients with metastatic ccRCC.

Consensus approach to nasal high-flow therapy in neonates.

OBJECTIVE: Nasal high-flow therapy (nHFT) is commonly used for noninvasive respiratory support in the neonatal intensive care unit. Our objective was to determine which aspects of neonatal nHFT have achieved adequate evidence base to support consensus among experienced clinical investigators, and to document areas lacking consensus to promote future investigations. STUDY DESIGN: Prospective, modified Delphi collation of tabular queries related to specific aspects of neonatal nHFT. Seven international nHFT clinical researchers were queried regarding approaches to initiation, escalation, weaning and discontinuing nHFT. Completed tables were reviewed independently by each investigator, results clarified and discussed and areas of consensus determined. RESULTS: Consensus agreement was reached for many aspects of nHFT including: need for adequate heating and humidification, need to prevent nares occlusion, maximum flow rate of 8 l min-1, assessment of fraction of inspired oxygen (FiO2) and work of breathing for either flow escalation or weaning, equivalence of nHFT to nasal continuous positive airway pressure (nCPAP) for noninvasive support of infants of ⩾28 weeks with resolving respiratory distress and use of nHFT for noninvasive support of stable infants on nCPAP. There was general agreement for initial gas flow rates in the range of 4 to 6 l min-1 and for nHFT as primary therapy for mild respiratory distress. There was no consensus on the approach to discontinuing nHFT. CONCLUSIONS: Among an experienced group of nHFT clinical researchers, there was general consensus in the approach to neonatal nHFT. Additional randomized studies are indicated to provide better evidence related to several aspects of nHFT, as well as to identify other clinical conditions where nHFT may provide safe, effective noninvasive support.

Genomic Alterations and Outcomes with VEGF-Targeted Therapy in Patients with Clear Cell Renal Cell Carcinoma.

Background: Mutations in VHL, PBRM1, SETD2, BAP1, and KDM5C are common in clear cell renal cell carcinoma (ccRCC), and presence of certain mutations has been associated with outcomes in patients with non-metastatic disease. Limited information is available regarding the correlation between genomic alterations and outcomes in patients with metastatic disease, including response to VEGF-targeted therapy. Objective: To explore correlations between mutational profiles and cancer-specific outcomes, including response to standard VEGF-targeted agents, in patients with metastatic cc RCC. Methods: A retrospective review of 105 patients with metastatic ccRCC who had received systemic therapy and had targeted next-generation sequencing of tumors was conducted. Genomic alterations were correlated to outcomes, including overall survival and time to treatment failure to VEGF-targeted therapy. Results: The most frequent mutations were detected in VHL (83%), PBRM1 (51%), SETD2 (35%), BAP1 (24%), KDM5C (16%), and TERT (14%). Time to treatment failure with VEGF-targeted therapy differed significantly by PBRM1 mutation status (p = 0.01, median 12.0 months for MT versus 6.9 months for WT) and BAP1 mutation status (p = 0.01, median 6.4 months for MT versus 11.0 months for WT). Shorter overall survival was associated with TERT mutations (p = 0.03, median 29.6 months for MT versus 52.6 months for WT) or BAP1 mutations (p = 0.02, median 28.7 months for MT versus not reached for WT). Conclusions: Genomic alterations in ccRCC tumors have prognostic implications in patients with metastatic disease. BAP1 and TERT promoter mutations may be present in higher frequency than previously thought, and based on this data, deserve further study for their association with poor prognosis.

High-flow support in very preterm infants in Australia and New Zealand.

BACKGROUND: Randomised trials suggest that high-flow (HF) therapy is comparable with continuous positive airway pressure (CPAP) for postextubation respiratory support in neonates, and HF has been widely adopted in neonatal intensive care. METHODS: We conducted a population-based study of very preterm infants born <32 weeks' gestation within the Australian and New Zealand Neonatal Network (ANZNN) data set from 2009 to 2012, who received respiratory support with HF. RESULTS: 3372 very preterm infants were treated with HF. HF use in this population increased significantly from 15% in 2009 to 35% in 2012. In 2012, 53% (542/1029) of extremely preterm infants born <28 weeks' gestation received HF. 98% (3308/3372) of infants had received endotracheal ventilation or CPAP prior to receiving HF. The maximum HF gas flow was ≤8 L/min in almost all infants. CONCLUSIONS: HF use in extremely preterm and very preterm infants increased significantly within the ANZNN from 2009 to 2012.

Effects of elevated extracellular potassium concentrations on the class III antiarrhythmic action of sotalol.

The electrophysiological and mechanical effects of sotalol, a beta-adrenergic blocker with Class III antiarrhythmic properties, were compared with those of atenolol in rabbit right ventricular papillary muscles studied in vitro using intracellular microelectrodes. Sotalol produced a dose-dependent increase in action potential duration and effective refractory period without any effect on parameters of rapid inward current. Atenolol had no Class I or Class III effect. The actions of equipotent beta-blocking concentrations of sotalol (10(-4) mol X litre-1) and of atenolol (10(-5) mol X litre-1) in elevated extracellular potassium concentrations of 8 and 12 mmol X litre-1 were investigated. There were reductions in membrane potential, action potential amplitude and upstroke velocity in elevated potassium which were not influenced by sotalol and atenolol, apart from a small additional depression of action potential amplitude and membrane potential in 12 mmol X litre-1 potassium. Hyperkalaemia caused shortening in action potential duration but lengthening in effective refractory period (post-repolarisation refractoriness). The increase in effective refractory period over control produced by sotalol in normal potassium was preserved in elevated potassium. This effect was attributable to lengthening of action potential duration rather than alteration in the duration of post-repolarisation refractoriness. The Class III effects of sotalol are preserved even in partially depolarised fibres where rapid inward current is depressed.

The influence of acute myocardial ischaemia on the class III antiarrhythmic action of sotalol.

The effects of acute myocardial ischaemia on the Class III antiarrhythmic properties of dl-sotalol 10(-4) mol . litre-1 were studied in the isolated arterially perfused interventricular septum of the rabbit heart. Before ischaemia, sotalol increased mean action potential duration (APD90) from 267 +/- 6 to 406 +/- 19 ms (p less than 0.001 mean +/- SEM, n = 7 septa), and mean effective refractory period (ERP) from 222 +/- 7 to 291 +/- 10 ms. During 30 min zero flow global ischaemia, mean APD90 in the control group fell from 264 +/- 7 to 128 +/- 13 ms (n = 7). APD90 fell more rapidly in the sotalol group, so that the initial difference between the sotalol and control groups was abolished after 24 min ischaemia. In the sotalol group, ERP shortened rapidly during ischaemia, and was significantly less than in the control group after 30 min. These changes were associated with the development of substantial post-repolarisation refractoriness in the control but not in the sotalol group. There was a greater fall in action potential upstroke velocity in the controls than in the sotalol group. The results indicate that the Class III effect of sotalol gradually disappears during ischaemia. The effects on post-repolarisation refractoriness and upstroke velocity are compatible with the hypothesis that sotalol slows the rate of rise of extracellular potassium concentration in acute ischaemia. This is a potentially antiarrhythmic property.

Interaction of the effects of hypoxia, substrate depletion, acidosis and hyperkalaemia on the class III antiarrhythmic properties of sotalol.

The effects of hypoxia on the actions of dl-sotalol 10(-4) mol . litre-1 were studied in rabbit papillary muscles at 32 degrees C. Superfusion for 30 min with a hypoxic solution (95% N2, 5% CO2) in the presence of 5 mmol . litre-1 glucose caused moderate shortening of control action potential duration from 208 +/- 3 to 138 +/- 9 ms (mean +/- SEM). In the presence of sotalol, hypoxia caused shortening of APD90 from 399 +/- 9 ms to 249 +/- ms, but the value after 30 min was still significantly greater than in controls (p less than 0.001). Superfusion with a hypoxic, glucose-free solution, however, caused profound shortening of APD90 in controls to 80 +/- 7 ms at 30 min. The highly significant lengthening of APD90 produced by sotalol in control conditions was abolished after 5 min hypoxia. The effects of hypoxia on the effective refractory period (ERP) paralleled those on APD90. Exposure to a hypoxic, acidotic, hyperkalaemic solution (80% N2, 20% CO2, pH 6.8; K+ 12 mmol . litre-1) produced moderate shortening of APD90 with convergence of the two groups. There was an increase in ERP, with the development of an equal degree of post-repolarisation refractoriness in the control and sotalol groups. The Class III effect of sotalol is preserved under mild but lost under severely hypoxic conditions. Using "simulated ischaemic" conditions, with controlled extracellular potassium concentrations, there was no difference in the relationship between APD90 and ERP in the control and sotalol groups.

Metoprolol and ventricular repolarisation and refractoriness: lack of chronic adaptational class III effects in rabbit.

STUDY OBJECTIVE: The aim was to examine the right ventricular electrophysiological changes during metoprolol treatment and in conditions of stress in the adult rabbit. DESIGN: Metoprolol (6 mg.kg-1) was given twice daily for four weeks. Stress was induced by constant infusion of adrenaline (15.2 micrograms.kg-1.h-1) via osmotic pumps implanted in the left femoral vein. Control rabbits were treated with saline. Electrophysiological recordings were made weekly in conscious animals using bipolar pacing electrodes implanted in the right ventricular apex. SUBJECTS: Adult male New Zealand white rabbits, 2.8-3.3 kg, were used (n = 7 adrenaline/metoprolol treated, n = 7 adrenaline/saline treated, n = 7 saline/metoprolol treated, and n = 7 saline/saline treated). MEASUREMENTS AND MAIN RESULTS: Recordings were made of the effective refractory period and of the stimulus-T interval of the paced evoked response. Stimulus-T is used as an index of ventricular repolarisation time. No significant electrophysiological changes in these variables were observed throughout the study. CONCLUSIONS: These results indicate that chronic metoprolol therapy does not result in a class III antiarrhythmic effect in this in vivo rabbit model.

Subsidiary class III effects of beta blockers? A comparison of atenolol, metoprolol, nadolol, oxprenolol and sotalol.

The electrophysiological effects of various concentrations of atenolol, metoprolol, nadolol, D-oxprenolol, L-oxprenolol, and D, L-oxprenolol and D-sotalol, L-sotalol, and D, L-sotalol were compared in rabbit right ventricular papillary muscle studied in vitro using intracellular microelectrodes. An assessment of the relative beta blocking effects of D-sotalol and D, L-sotalol against the inotropic action of isoprenaline was made in the same preparation. Of the drugs tested, only oxprenolol and sotalol showed prolongation of action potential duration and effective refractory period (class III action), with oxprenolol showing, in addition, depression of maximal upstroke velocity and the presence of post-repolarisation refractoriness (class I action). When contrasted at clinically relevant concentrations, only sotalol retained a class III effect, without any actions on variables related to fast inward current. The effects of oxprenolol and sotalol were not found to be stereospecific. The mechanical experiments indicate that, in this preparation, D-sotalol has approximately one-fourteenth of the beta blocking potency of the racemic compound. It therefore merits further consideration as a useful alternative class III antiarrhythmic agent, which would be free from the side effects of beta receptor blocking treatment.

An animal model for the chronic study of ventricular repolarisation and refractory period.

We have developed a simple model permitting stable, chronic measurements of ventricular repolarisation and refractory period to be made in conscious, unsedated, unrestrained animals. The model utilises the ventricular paced evoked response, recorded from permanent pacemaker electrodes implanted into the right ventricle of New Zealand White rabbits. After a "bedding in" period of 18-21 d, measured variables are stable for long periods; the stimulus to T wave interval of the evoked response remains stable up to at least 150 d after electrode insertion. The principal advantage of the model lies in the control of heart rate by pacing, eliminating the requirement for unreliable methods of correction of repolarisation data for sinus rate. Surgical techniques are straightforward. The model can also be used for studies involving recordings of the intrinsic (non-paced) intracardiac electrogram, as the quality of the signal obtained is consistently superior to standard methods of recording the electrocardiogram in animals. The stimulus-T interval of the paced evoked response has been found to correlate significantly (r = 0.96) with action potential duration measured by transmembrane microelectrode recordings in the isolated, arterially perfused interventricular septum.

Calcium sensitivity and cardiac performance in genetic and renal models of hypertension.

We have compared cardiac performance, hypertrophy and sensitivity to calcium and verapamil of hearts of six to nine-month-old spontaneously hypertensive rats (SHR), two-kidney, one clip renal hypertensive rats (RHR) and age-matched controls. Cardiac output and heart rate were measured using an isolated perfused heart preparation. Mean blood pressure (BP) and heart weight were equally increased in SHR and RHR as was optimal left atrial filling pressure. Cardiac output was increased in both SHR and RHR at any given work load; this improvement was seen especially in SHR and at high aortic pressure (160 cm H2O) was significant in SHR but not RHR. In low [Ca2+], 0.6 mM, cardiac output of RHR and controls fell markedly, but changed little in SHR, whereas in high [Ca2+], 5.1 mM, cardiac performance deteriorated in SHR but was improved in RHR and controls. Verapamil 2 X 10(-7) M reduced Ca2+ responsiveness of RHR and controls threefold but had no effect in SHR. The results suggest there may be an abnormality of cardiac calcium utilization in inherited but not in acquired hypertension.

Are high flow nasal cannulae noisier than bubble CPAP for preterm infants?

BACKGROUND: Noise exposure in the neonatal intensive care unit is believed to be a risk factor for hearing loss in preterm neonates. Continuous positive airway pressure (CPAP) devices exceed recommended noise levels. High flow nasal cannulae (HFNC) are an increasingly popular alternative to CPAP for treating preterm infants, but there are no in vivo studies assessing noise production by HFNC. OBJECTIVE: To study whether HFNC are noisier than bubble CPAP (BCPAP) for preterm infants. METHODS: An observational study of preterm infants receiving HFNC or BCPAP. Noise levels within the external auditory meatus (EAM) were measured using a microphone probe tube connected to a calibrated digital dosimeter. Noise was measured across a range of frequencies and reported as decibels A-weighted (dBA). RESULTS: A total of 21 HFNC and 13 BCPAP noise measurements were performed in 21 infants. HFNC gas flows were 2-5 L/min, and BCPAP gas flows were 6-10 L/min with set pressures of 5-7 cm of water. There was no evidence of a difference in average noise levels measured at the EAM: mean difference (95% CI) of -1.6 (-4.0 to 0.9) dBA for HFNC compared to BCPAP. At low frequency (500 Hz), HFNC was mean (95% CI) 3.0 (0.3 to 5.7) dBA quieter than BCPAP. Noise increased with increasing BCPAP gas flow (p=0.007), but not with increasing set pressure. There was a trend to noise increasing with increasing HFNC gas flows. CONCLUSIONS: At the gas flows studied, HFNC are not noisier than BCPAP for preterm infants.

The influence of ischaemia on the electrophysiological properties of amiodarone in chronically treated rabbit hearts.

The effects of 30 min zero-flow ischaemia and reperfusion on the electrophysiological properties of amiodarone were studied in 11 rabbits treated with oral amiodarone (mean 117 mg kg-1, day-1) for 2-3 months, and 11 controls. Experiments were performed in vitro in the isolated perfused interventricular septum, and preischaemic values were compared with those obtained in right ventricular papillary muscles from the same hearts. Prior to ischaemia, mean values of action potential duration (APD90) and effective refractory period (ERP) were prolonged by 13% in the amiodarone-treated septa. Action potential upstroke velocity (Vmax) was reduced by 14% in the septa, but by 42% in papillary muscles. Ischaemia resulted in shortening of APD90 in both control and amiodarone-treated septa, with a loss of the ability of amiodarone to prolong APD90. In contrast, ischaemia resulted in a greater fall in Vmax, gross lengthening in conduction time and increase in stimulation threshold in the amiodarone-treated septa compared with controls. Reperfusion resulted in a restoration of the action of amiodarone on repolarization, and resolution of the marked effects on excitability and conduction. The electrophysiological properties of amiodarone are considerably altered in ischaemic myocardium, with a reversible loss of its ability to prolong repolarization, but evidence suggestive of a marked enhancement of its effect on the inward sodium current.

A Family Focus approach to child abuse prevention.

Family Focus is a child abuse prevention program established to assist families in the task of raising their children from birth through 3 years. Its goal is to prevent child abuse by reducing isolation, improving emotional support systems and increasing parental knowledge of child behavior and effective child management skills. It strives to be viewed by the community as a positive family resource center with its services available to any interested parent, not just those identified as being at high risk for child abuse. Family Focus provides playgroups, parent education classes and other resources. The "Infant Program" was added to the program to provide outreach and follow-up services to parents and their high risk infants birth to 2 years.

Differences in alcohol risk profiles between college students and college-age non-students presenting for care in the emergency department.

PURPOSE: Determine whether college students and non-students seeking emergency medical care experience different lifestyle choices, alcohol-use patterns, and risk profiles.METHODS: Patients aged 18-25 seen in a university-affiliated ED between August 1998 and June 1999 who had >/=1 drinks within the past year were eligible. Patients scoring >5 out of a possible 40 on the Alcohol Use Disorder Identification Test (AUDIT) were considered at elevated risk for alcohol problems and were interviewed further to determine alcohol-use patterns and history.RESULTS: Of 1,436 consenting patients, students were more likely to screen positive for alcohol problems than non-students (55% vs 43%; 478/870 vs 245/566). Furthermore, among screen-positive patients, students were more likely than non-students to binge drink in the two weeks prior to screening (82% vs 65%) and to drink illegally (60% vs 38%). Students needed fewer drinks to feel the effects of alcohol (3.9 drinks vs 5.1) and were less likely to request a referral for alcohol treatment (2% vs 8%). However, students had lower baseline risk for alcohol problems than non-students (AUDIT = 11.2 vs 13.2). Students were younger than non-students (20.2 years vs 21.4), older at their first drunken experience (15.3 vs 14.7), and less likely to smoke (51% vs 76%) or have a family history of heavy drinking (5% vs 23%). (p <.01 for all comparisons).CONCLUSIONS: Students exhibit a higher risk than non-students for behaviors associated with acute alcohol-related problems. However, they may be at lower risk for chronic alcohol problems. This university-based ED seems an appropriate venue for early identification and brief, on-site intervention for students with alcohol problems.