Targeting liposomes with protein drugs to the blood-brain barrier in vitro.

In this study, we aim to target pegylated liposomes loaded with horseradish peroxidase (HRP) and tagged with transferrin (Tf) to the BBB in vitro. Liposomes were prepared with the post-insertion technique: micelles of polyethylene glycol (PEG) and PEG-Tf were inserted into pre-formed liposomes containing HRP. Tf was measured indirectly by measuring iron via atomic absorption spectroscopy. All liposomes were around 100 nm in diameter, contained 5-13 microg HRP per mumol phospholipid and 63-74 Tf molecules per liposome (lipo Tf) or no Tf (lipo C). Brain capillary endothelial cells (BCEC) were incubated with liposomes at 4 degrees C (to determine binding) or at 37 degrees C (to determine association, i.e. binding+endocytosis) and the HRP activity, rather than the HRP amount was determined in cell lysates. Association of lipo Tf was two- to three-fold higher than association of lipo C. Surprisingly, the binding of lipo Tf at 4 degrees C was four-fold higher than the association of at 37 degrees C. Most likely this high binding and low endocytosis is explained by intracellular degradation of endocytosed HRP. In conclusion, we have shown targeting of liposomes loaded with protein or peptide drugs to the BCEC and more specifically to the lysosomes. This is an advantage for the treatment of lysosomal storage disease. However, drug targeting to other intracellular targets also results in intracellular degradation of the drug. Our experiments suggest that liposomes release some of their content within the BBB, making targeting of liposomes to the TfR on BCEC an attractive approach for brain drug delivery.     

The expression of SFRP1, SFRP3, DVL1, and DVL2 proteins in testicular germ cell tumors

Germ cell tumors of the testis are a heterogeneous group of neoplasms that affect male adolescents and young adults. Wnt signaling pathway components have been shown to be actively involved in normal and malignant germ cell differentiation and progression. In this study, we aimed to explore the expression patterns of the secreted frizzled‐related protein (SFRP) and Disheveled protein family (DVL) in a subset of testicular germ cell tumors. Eighty‐five formalin‐fixed, paraffin‐embedded tissue samples of the primary germ cell tumors of the testis were stained against SFRP1, SFRP3, DVL1, and DVL2 proteins using immunohistochemistry. SFRP1 and SFRP3 exhibited lower expression in both seminomas and mixed/non‐seminomatous tumors, compared with atrophic/benign tissue (p < 0.001). SFRP3 expression was lower than SFRP1 expression within the seminoma group (p = 0.004), but not within the mixed/non‐seminomatous group (p = 0.409). The majority of the tested cases (27/28, 96%) exhibited low DVL1 protein expression (median 0%, range 0–90%). In contrast, 20 out of 22 tested cases (91%) exhibited strong expression of DVL2 protein (median 80%, range 0–100%). No significant difference in DVL1 and DVL2 protein expression was observed between seminomas and mixed/non‐seminomatous tumors (p = 0.68 and 0.29). The secreted frizzled‐related protein and disheveled protein family members appear to be actively involved in the pathogenesis of primary testicular germ cell tumors.     

Successful use of sirolimus for refractory atrial ectopic tachycardia in a child with cardiac rhabdomyoma

Cardiac rhabdomyomas are common in tuberous sclerosis. We report a child who developed rhabdomyoma related arrhythmia refractory to antiarrhythmic drug therapy. Reversion of the atrial ectopic tachycardia was achieved with mammalian target of rapamycin pathway (mTOR) inhibitor sirolimus. As per our knowledge, this is the first time that sirolimus has been successfully used in this setting.     

Diagnostic and therapeutic challenges in patients with coexistent chronic obstructive pulmonary disease and chronic heart failure.

Chronic obstructive pulmonary disease (COPD) and heart failure (CHF) are common conditions. The prevalence of COPD ranges from 20% to 30% in patients with CHF. The diagnosis of CHF can remain unsuspected in patients with COPD, because shortness of breath is attributed to COPD. Measurement of plasma B-type natriuretic peptide (BNP) levels helps to uncover unsuspected CHF in patients with COPD and clinical deterioration. Noninvasive assessment of cardiac function may be preferable to BNP to uncover unsuspected left ventricular (LV) systolic dysfunction in patients with stable COPD. Patients with COPD or CHF develop skeletal muscle alterations that are strikingly similar. Functional intolerance correlates with severity of skeletal muscle alterations but not with severity of pulmonary or cardiac impairment in COPD and CHF, respectively. Improvement of pulmonary or cardiac function does not translate into relief of functional intolerance in patients with COPD or CHF unless skeletal muscle alterations concomitantly regress. The mechanisms responsible for skeletal muscle alterations are incompletely understood in COPD and in CHF. Disuse and low-level systemic inflammation leading to protein synthesis/degradation imbalance are likely to contribute. The presence of COPD impacts on the treatment of CHF, as COPD is still viewed as a contraindication to beta-blockade. Therefore, COPD often deprives patients with CHF due to LV systolic dysfunction of the most beneficial pharmacologic intervention. A large body of data indicates that patients with COPD tolerate well selective beta-blockade that should not be denied to CHF patients with concomitant COPD.     

High-frequency modulation of heart rate variability during exercise in patients with COPD

STUDY OBJECTIVES: To evaluate cardiac autonomic modulation in patients with COPD during peak exercise. METHODS: Fifty-three patients with COPD (mean FEV(1), 35% predicted [SD, 11% predicted]; mean PaO(2), 68 mm Hg [SD, 11 mm Hg]; mean PaCO(2), 40 mm Hg [SD, 7 mm Hg]; mean age, 61 years [SD, 10 years]; 26 women and 27 men) and 14 healthy control subjects aged 60 years (SD, 8 years) [seven women and seven men] were studied at rest and during ramped bicycle ergometry to their volitional peak. Patients were not receiving autonomic medications other than inhaled beta-agonist agents and/or anticholinergic agents. Control subjects were not receiving any medications. Cardiac autonomic modulation was assessed via time-frequency analysis (Wigner-Ville) of ECG-derived heart rate variability as the power in the low-frequency (LF) band (ie, 0.04 to 0.15 Hz) and the high-frequency (HF) band (ie, > 0.15 to 0.4 Hz) averaged from > 3 min at rest and minutes 2 through 5 of their exercise period. RESULTS: Patients with COPD had a significantly increased mean, ln-transformed HF band from rest to peak exercise (9.9 ms(2) [SD, 1.4 ms(2)] vs 10.7 ms(2) [SD, 1.4 ms(2)], respectively; p < 0.01), while the HF band was unchanged for the control group (10.7 ms(2) [SD, 1.5 ms(2)] vs 10.4 ms(2) [1.3 ms(2)], respectively; difference not significant). The mean ln-transformed LF band was significantly increased from rest to peak exercise in patients with COPD (10.9 ms(2) [SD, 1.5 ms(2)] vs 11.5 ms(2) [SD, 1.4 ms(2)], respectively; p < 0.01) and in control subjects (10.9 ms(2) [SD, 1.5 ms(2)] vs 11.5 ms(2) [SD, 1.3 ms(2)], respectively; p < 0.01). The mean LF/HF ratio was significantly decreased from rest to peak exercise in patients with COPD (3.1 [SD, 1.5] vs 2.5 [SD, 1.0], respectively; p < 0.01) and was increased in control subjects (1.9 [SD, 0.8] vs 2.4 [1.0], respectively; p < 0.01). When expressed in normalized units ([absolute power of the components]/[total power - very low frequency power] x 100), the HF band was again significantly greater during peak exercise than at rest in the patients with COPD and was unchanged during peak exercise for the control group. Autonomic changes were not significantly correlated with age, gender, body mass index, spirometry, lung volumes, resting gas exchange, or oxygen saturation during exercise. CONCLUSION: These data suggest that, in contrast to control subjects, the balance of sympathetic to parasympathetic cardiac modulation decreases in patients with COPD during maximal volitional exercise.     

Diagnostic and therapeutic approach to coexistent chronic obstructive pulmonary disease and obstructive sleep apnea

The high prevalence of both obstructive sleep apnea (OSA) and chronic obstructive pulmonary disease (COPD) in Western societies is well documented. However, OSA frequently remains unrecognized and untreated among patients with COPD. Patients with both conditions have a greater risk for fatal and nonfatal cardiovascular events compared with patients with COPD or OSA alone. Efficacious treatment with continuous positive airway pressure reduces the risk of cardiovascular complications in patients with OSA. The aim of the present review is to discuss the diagnostic approach to patients with both conditions and to delineate the benefits of timely recognition and treatment of OSA in patients with COPD.     

Glucoregulation during and after intense exercise: effects of alpha-adrenergic blockade

In intense exercise (>80% maximal oxygen consumption [VO2 max]), the 7- to 8-fold increase in glucose production (Ra) is tightly correlated with the greater than 14-fold increase in plasma norepinephrine (NE) and epinephrine (EPI). To distinguish the relative roles of alpha- and beta-adrenergic receptors, the responses of 12 control (C) lean, healthy, fit young male subjects to 87% VO2 max cycle ergometer exercise were compared with those of 7 subjects (at 83% VO2max) receiving intravenous phentolamine (Ph). The Ph group received a 70-microg/kg bolus and then 7 microg/kg/min from -30 minutes, during exercise and for 60 minutes of recovery. The data were analyzed by comparing exercise responses to exhaustion in Ph subjects (11.4 +/- 0.6 min) with those at both 12 minutes and at exhaustion in C subjects (14.6 +/- 0.3 min) and during recovery. There were no significant differences between groups in the plasma glucose response during exercise, but values were higher in C versus Ph subjects during the first 40 minutes of postexercise "recovery." The Ra response during the first 12 minutes of exercise was not different by repeated-measures ANOVA, reaching 10.6 +/- 1.3 mg/kg/min in C and 9.6 +/- 1.5 in Ph subjects at 12 minutes. However, in C subjects, Ra increased significantly to 14.1 +/- 1.2 mg/kg/min by exhaustion, and remained higher versus Ph subjects until 15 minutes of recovery. The Rd during recovery was not different between groups; thus, the higher Ra in C subjects in early recovery was responsible for the greater hyperglycemia observed in C subjects. Ph subjects showed a more rapid, marked increment (P = .002) in both plasma NE (to 64 v38 nmol/L) and EPI at exhaustion, and catecholamine concentrations remained higher in Ph versus C subjects during recovery. Whereas plasma insulin (IRI) declined in the C group, it increased 3-fold (P = .001) in the Ph group during exercise and until 15 minutes of recovery. Ph had no effect on glucagon (IRG). Thus, the glucagon to insulin ratio decreased in Ph subjects from baseline levels during exercise and early recovery, but increased in C subjects. The increase in Ra among Ph subjects despite the decrease in the glucagon to insulin ratio supports our earlier evidence that these hormones are not principal regulators of the Ra in intense exercise. The shorter time to exhaustion and markedly higher catecholamine levels in Ph subjects limited our ability to isolate the effects of alpha-adrenergic receptors on the Ra.alpha-Adrenergic receptors appear to have little influence on the Rd.