Mechanistic studies on the degradation and protein release characteristics of poly(lactic-co-glycolic-co-hydroxymethylglycolic acid) nanospheres.

The purpose of this study was to gain mechanistic insights into the effect of different formulation parameters on the degradation and release behavior of protein-loaded nanoparticulate carrier systems based on an aliphatic polyester with pendant hydroxyl groups, poly(lactic-co-glycolic-hydroxymethyl glycolic acid) (pLGHMGA). Bovine serum albumin (BSA) was used as a model protein. BSA-loaded pLGHMGA nanospheres of 400-700 nm were prepared using a solvent evaporation method using pLGHMGA of different molecular weights and different compositions. Also, the concentration of pLGHMGA in the organic phase was varied. The nanospheres showed a continuous mass loss accompanied by continuous decrease in number average molecular weight, which indicates that the degradation of the nanospheres is by bulk degradation with a rapid release of water-soluble low molecular weight fragments. On the basis of NMR analysis, it is concluded that intramolecular transesterification precedes extensive hydrolysis of the polymer and degradation of the nanospheres. BSA-loaded freeze-dried nanospheres showed a significant burst release of 40-50% of the BSA loading. In contrast, nonfreeze-dried samples showed a small burst of around 10-20%, indicating that freeze-drying induced pore formation. Nonlyophilized nanospheres prepared from pLGHMGA with 64/18/18 lactic/glycolic/hydroxymethylglycolic acid (L/G/HMG) ratio showed a relatively fast release of BSA for the next 30 days. Nanospheres prepared from a more hydrophobic pLGHMGA (74/13/13, L/G/HMG) showed a two-phase release. Circular dichroism analysis showed that the secondary structure of the released protein was preserved. This study shows a correlation between release behavior and particle erosion rate, which can be modulated by the copolymer composition.

Venous systolic thrill and murmur in the neck: a consequence of severe tricuspid insufficiency.

A palpable venous systolic thrill and murmur at the base of the neck are described as new physical findings in five patients with severe tricuspid regurgitation. In two of these patients, the tricuspid valve had been resected as treatment for infective endocarditis related to intravenous drug abuse. The third patient had severe chronic pulmonary disease with right heart failure. The fourth patient had a complex congenital defect in which the mitral valve served as the venous atrioventricular valve and was severely incompetent. The fifth patient suffered from long-standing rheumatic mitral and tricuspid disease with pulmonary hypertension 10 years after placement of a mitral prosthesis. From these observations, it is apparent that pulsatile retrograde flow in the cervical veins resulting from severe right-sided atrioventricular valve incompetence can produce a palpable systolic thrill and murmur at the base of the neck.

Increased nitrovasodilator sensitivity in endothelial nitric oxide synthase knockout mice: role of soluble guanylyl cyclase.

Endogenously produced nitric oxide (NO) modulates nitrovasodilator-induced relaxation. We investigated the underlying mechanism in wild-type (WT) mice and endothelial NO synthase knockout (eNOS(-/-)) mice to determine whether a chronic lack of endothelial NO alters the soluble guanylyl cyclase (sGC) pathway. In aortic segments from eNOS(-/-) mice, the vasodilator sensitivity to sodium nitroprusside (SNP) was significantly greater than that in WT mice. There was no difference in sensitivity to the G-kinase I activator 8-para-chlorophenylthio-cGMP or to cromakalim. N(omega)-Nitro-L-arginine had no effect on the SNP-induced relaxation in eNOS(-/-) but increased the sensitivity in WT mice so it was no longer different than that of eNOS(-/-). Basal cGMP levels in aortic rings were significantly lower in eNOS(-/-) mice than in WT mice. SNP (300 nmol/L) induced a significantly greater cGMP accumulation in eNOS(-/-) mice than in WT mice. The maximal SNP-induced (10 micromol/L) increase in cGMP was similar in both strains. SNP-stimulated sGC activity was significantly greater in eNOS(-/-) mice than in WT mice. Incubation of aortic segments from WT mice with N(omega)-nitro-L-arginine increased sGC activity, an effect prevented by coincubation with SNP (10 micromol/L). The aortic expressions of the sGC alpha1 and beta1 subunits in WT and eNOS(-/-) mice were identical as determined with Western blot analysis. These data suggest that chronic exposure to endothelium-derived NO, as well as acute exposure to nitrovasodilator-derived NO, desensitizes sGC to activation by NO but does not alter sGC expression. Both the acute cessation of endothelial NO formation in WT mice and the chronic deficiency of NO in eNOS(-/-) mice restore the NO sensitivity of sGC and enhance vascular smooth muscle relaxation in response to nitrovasodilator agents.

Left ventricular diastolic filling in valvular aortic stenosis.

The pattern of left ventricular diastolic filling in patients with valvular aortic stenosis (AS) as assessed by gated blood pool scintigraphy has received little attention. Twenty-one normal persons (group 1), 24 patients with significant AS and ejection fractions of 50% or more (group 2) and 15 patients with significant AS and ejection fractions less than 50% (group 3) were studied. From the time-activity curve, the peak filling rate and mean filling rate (as end-diastolic volumes [EDV]/s) and percent stroke volume filled at first third of diastole and at the end of the rapid filling period were determined. Group 2 had a reduced peak filling rate (2.58 +/- 0.65 EDV/s, p less than 0.05) compared with group 1 (3.11 +/- 0.65 EDV/s). Similarly, the percent stroke volume filled at the end of the rapid filling period was reduced in group 2. Group 3 patients had a lower peak filling rate and mean filling rate than group 1 patients. However, percent stroke volume filled at first third of diastole and percent stroke volume filled at the end of the rapid filling period were greater in group 3 than in group 1. Two distinct patterns of diastolic filling were noted in patients with AS. Group 2 patients had reduced peak filling rates with less diastolic filling during the rapid filling period. Although the peak filling rate was reduced in group 3, more complete filling occurred during the rapid filling period.

Early manifestation of noncalcific aortic stenosis after porcine valve replacement.

Symptoms of noncalcific aortic stenosis developed in a 57-year-old man 3 months after implantation of a Carpentier-Edwards porcine heterograft. The glutaraldehyde-processed bioprosthesis was removed 7 months after implantation and replaced with a No. 3 Medtronic Hall valve.

Left ventricular diastolic filling in patients with left ventricular dysfunction.

The pattern of abnormal left ventricular diastolic filling and its specificity in coronary disease patients with severe left ventricular dysfunction has received little attention. We evaluated the left ventricular diastolic filling curve derived from gated blood pool scans in 21 normals, 61 coronary disease patients with ejection fractions less than or equal to 30%, and 51 congestive cardiomyopathy patients with ejection fraction less than or equal to 30%. The peak filling rate (PFR), peak ejection rate (PER), PFR/PER and the % stroke volume filled at 1/3 of diastole (%SV-1/3 DT) and at the end of the rapid filling period (%SV-RFP) were determined for each group. The PFR and PER were reduced in both coronary disease and congestive cardiomyopathy groups. The PFR/PER was increased in the coronary disease group (1.19 +/- 0.28) and congestive cardiomyopathy group (1.21 +/- 0.32) as compared to normals (0.93 +/- 0.20, P less than 0.001). A greater %SV-1/3 DT and %SV-RFP were noted in both coronary disease and congestive cardiomyopathy groups. Coronary disease and congestive cardiomyopathy patients with a mean pulmonary capillary pressure (PCP) greater than or equal to 18 mm Hg had a greater PFR/PER, %SV-1/3 DT, and %SV-RFP than patients with a PCP less than 18 mm Hg. An abnormal and nonspecific pattern of left ventricular diastolic filling is present in both coronary disease and congestive cardiomyopathy patients and is characterized by an increased PFR/PER, a greater %SV-1/3 DT, and a greater %SV-RFP. This pattern may be related to elevated PCPs.

Changes in serum CPK, LDH, and their isoenzymes in the perioperative period in patients undergoing craniotomy.

The perioperative changes in the serum concentration of creatine phosphokinase (CPK) and its isoenzymes MM, MB, and BB and of lactic dehydrogenase (LDH) and its isoenzymes LDH1 to LDH5 were determined during craniotomy in order to distinguish operation-induced changes in these enzymes from those due to acute myocardial infarction and malignant hyperthermia. Twenty-eight male patients, 29 to 76 years of age (mean +/- SD = 58 +/- 13.2 years), undergoing craniotomy for tumor reseaction (n = 26) or cerebral artery aneurysm clipping (n = 2) were included in this study. Ten serial blood samples were obtained from each patient: one sample before and another after induction of anesthesia, and eight samples after the incision, over a period of 70 h. The preinduction serum CPK level of 97 +/- 32 U/L (mean +/- SD) increased gradually and significantly and reached the peak level of 542 +/- 116 U/L 34 h after incision (p <0.05). Whereas all of the CPK isoenzymes increased in terms of U/L after incision, only the MM fraction (expressed as percent of total CPK) increased, and the MB and BB fractions (expressed as percent of total CPK) decreased. The preinduction serum LDH level of 150 +/- 42 U/L (mean +/- SD) increased gradually after incision and reached the peak level of 210 +/- 32 U/L 58 h after incision (p <0.05). LDH2 as a percent of total LDH decreased significantly, but the LDH1/LDH2 ratio did not change. LDH4 and LDH5, as percents of total LDH, increased significantly. The large increases in total serum CPK and the concomitant decrease in MB percent after craniotomy may minimize and/or mask the percentage increase in the MB level following acute myocardial infarction. The perioperative serum CPK level as a marker in the diagnosis of malignant hyperthermia should be interpreted in light of the present results and in conjunction with clinical symptomatology.

Subepicardial aneurysm of the left ventricle: report of a case and review of the literature.

A case of subepicardial aneurysm is reported and 16 other cases found in the literature are reviewed. Although the subepicardial aneurysms are distinct from true and false aneurysms, their propensity for rupture is similar to false aneurysm. The incidence of this aneurysm is increasing due to the use of thrombolytic therapy in myocardial infarction. Early invasive diagnostic test is leading to its more frequent discovery.

Simplified count-based estimates of left ventricular volume.

Accurate count-based radionuclide estimates of left ventricular volume without the use of a blood sample have not been well described. Resting gated blood pool scans were obtained within 24 hours of catheterization in 31 patients (group 1), and simultaneously with thermodilution cardiac outputs in 29 other patients (group 2) at rest and during an induced-volume change (intervention). End-diastolic and end-systolic volumes were calculated from the single-plane angiogram in Group 1 and from the combination of thermodilution stroke volume and radionuclide ejection fraction in group 2. Excellent correlations were noted between scintigraphic counts and angiographic volumes (r = 0.964), between scintigraphic counts and thermodilution-derived volumes (r = 0.979), and between interventional scintigraphic counts and interventional thermodilution-derived volumes (r = 0.941). Thermodilution and scintigraphic volume changes during the intervention were well correlated (r = 0.85). Accurate count based estimates of left ventricular volume without a blood sample are feasible at rest and during an intervention.

Serum creatine phosphokinase, lactic dehydrogenase, and their isoenzymes in the perioperative period.

The purpose of the present investigation was to determine the normal perioperative variations in the serum concentration of creatine phosphokinase (CPK) and its isoenzymes MM, MB, and BB, and of lactic dehydrogenase (LDH) and its isoenzymes LDH1 to LDH5 to distinguish operation-induced changes in these enzymes from those due to acute myocardial infarction or malignant hyperthermia. In 30 patients, 52 to 75 years of age undergoing elective orthopedic operations, 10 serial blood samples were obtained in the perioperative period: two samples before skin incision and eight samples after the incision over a time span of 70 hours. The preinduction mean serum CPK level of 141 U/L increased gradually and significantly and reached a maximum mean concentration of 809 U/L 34 hours after incision (p less than 0.01). The CPK-MM percent increased after incision, whereas that of CPK-MB and CPK-BB decreased, although their absolute values in terms of U/L rose. The preinduction mean serum LDH value of 173 U/L increased gradually after incision and achieved peak levels at 34 hours (203 U/L) and 58 hours (210 U/L) after incision (p less than 0.05). The LDH1:LDH2 ratio did not change. The LDH5 percent increased and peaked 10 hours after incision (p less than 0.05). There was a significant correlation between severity of operation-induced tissue damage and the serum CPK concentration (p less than 0.001). The large increase in total CPK (primarily MM fraction) occurring after surgery may minimize the percentile effects caused by an increase in MB level due to myocardial infarction.

The effect of lauryl capping group on protein release and degradation of poly(D,L-lactic-co-glycolic acid) particles.

The aim of this study was to investigate the effect of a specific and frequently used end group (lauryl alcohol) on the protein release and degradation kinetics of poly(DL-lactic-co-glycolic acid) particles of different sizes. Lauryl-capped PLGA and uncapped PLGA (referred to as PLGA-capped and PLGA-COOH, respectively) particles (0.3, 1 and 20 µm) were prepared by a double emulsion solvent evaporation technique. Bovine serum albumin (BSA) was used as a model protein for release studies. During degradation (PBS buffer, pH7.4 at 37°C), a slower dry mass loss was observed for 0.3 µm particles than for particles of 1 and 20 µm. It was further shown that PLGA-capped particles showed slower mass loss likely due to its more hydrophobic nature. It was found that the ester bond hydrolysis rate was substantially slower for PLGA-capped particles and that the rate increased with particle size. Particles showed enrichment in lactic acid content (and thus a decrease in glycolic acid content) in time, and interestingly PLGA-capped particles showed also an enrichment of the lauryl alcohol content. No difference was observed in degradation kinetics between BSA loaded and blank particles. Independent of size, PLGA-COOH based particles showed, after a small burst, a sustained and nearly complete release of BSA during 60-80 days. On the other hand, particles based on PLGA-capped showed a much slower release and exhibited incomplete release, accompanied by the presence of an insoluble residue remaining even after 180 days. FTIR analysis of this residue showed that it contained both polymer and protein. Considering the polymer enrichment in lauryl alcohol, the incomplete release observed for PLGA-capped is likely attributed to interactions between the protein and the lauryl end group. In conclusion, since PLGA-COOH, in contrast to the capped derivative, shows complete degradation as well as quantitative release of an entrapped protein, this polymer is preferred for the design of protein formulations.

Effect of chronic anemia on left ventricular ejection time and myocardial performance in man.

The duration of systolic time interval in ten anemic, (mean Hb 3.46) and otherwise healthy individuals (mean age 15) was measured from simultaneous recording of electrocardiogram, phonocardiogram and carotid pulsation. The preejection period (PEP); left ventricular ejection time (LVET) and ratio of PEP/LVET from 20 consecutive beats were calculated and averaged. Anemia was corrected slowly and study was repeated (mean 3 1/2 weeks) later and a similar data for comparison was obtained; heart rate decreased, LVET lengthened and the ratio of PEP/LVET remained unchanged. The lengthening of the LVET was due to slower heart rate. LVET before and after correction of anemia could be predicted from regression formula reported for normals. The unchanged PEP/LVET before and after the anemia indicates no significant alteration in myocardial performance at rest.

Time course alterations of QTC interval due to hypaque 76.

Sequential measurement of QT interval during left ventricular angiography was made 30 seconds and one, three, five and ten minutes after injection of hypaque 76. The subjects were ten patients found to have normal left ventricles and coronary arteries. Significant QTC prolongation occurred in 30 seconds to one minute in association with marked hypotension and elevation of cardiac output.

Primary congestive cardiomyopathy. Hemodynamic and autopsy findings.

Eight patients with congestive cardiomyopathy were examined at autopsy. All showed significant mural thrombi. The majority had repeated pulmonary and peripheral emboli. Cardiac catheterization was considered helpful in the diagnosis. Anticogaulation may be worthwhile in addition to the routine therapy for cardiomyopathic heart failure.

A gp91phox containing NADPH oxidase selectively expressed in endothelial cells is a major source of oxygen radical generation in the arterial wall.

Reactive oxygen species (ROS) play an important role in regulating vascular tone and intracellular signaling; the enzymes producing ROS in the vascular wall are, however, poorly characterized. We investigated whether a functionally active NADPH oxidase similar to the leukocyte enzyme, ie, containing the subunits p22phox and gp91phox, is expressed in endothelial cells (ECs) and smooth muscle cells (SMCs). Phorbol 12-myristate 13-acetate (PMA), a stimulus for leukocyte NADPH oxidase, increased ROS generation in cultured ECs and endothelium-intact rat aortic segments, but not in SMCs or endothelium-denuded arteries. NADPH enhanced chemiluminescence in all preparations. p22phox mRNA and protein was detected in ECs and SMCs, whereas the expression of gp91phox was confined to ECs. Endothelial gp91phox was identical to the leukocyte form as determined by sequence analysis. In contrast, mitogenic oxidase-1 (mox1) was expressed in SMCs, but not in ECs. To determine the functional relevance of gp91phox expression, experiments were performed in aortic segments from wild-type, gp91phox(-/-), and endothelial NO synthase (eNOS)(-/-) mice. PMA-induced ROS generation was comparable in aortae from wild-type and eNOS(-/-) mice, but was attenuated in segments from gp91phox(-/-) mice. Endothelium-dependent relaxation was greater in aortae from gp91phox(-/-) than from wild-type mice. The ROS scavenger tiron increased endothelium-dependent relaxation in segments from wild-type, but not from gp91phox(-/-) mice. These data demonstrate that ECs, in contrast to SMCs, express a gp91phox-containing leukocyte-type NADPH oxidase. This enzyme is a major source for arterial ROS generation and affects the bioavailability of endothelium-derived NO.

Candida prosthetic valve endocarditis: prospective study of six cases and review of the literature.

Candida prosthetic valve endocarditis (PVE) is a rare entity; however, its incidence is expected to increase given the recent increase in incidence of nosocomial bloodstream candida infection. This report reviews six cases of candida PVE studied prospectively plus 12 cases previously reported in the literature. Transesophageal echocardiography was more sensitive than transthoracic echocardiography in detecting vegetations. Valvular replacement combined with antifungal therapy has been the standard treatment. However, successful therapy with long-term administration of oral fluconazole has been reported for five patients. The mortality due to candida PVE was high, especially when PVE was complicated by congestive heart failure and persistent fungemia. For uncomplicated PVE, the mortality rate for patients receiving antifungal therapy alone (40%) was no worse than for those receiving combined medical and surgical therapy (33%).

The iron (Fe) and atherosclerosis study (FeAST): a pilot study of reduction of body iron stores in atherosclerotic peripheral vascular disease.

BACKGROUND: Levels of body iron stores, represented by the serum ferritin concentration, rise with age after adolescence in men and menopause in women. This rise has been implicated mechanistically and epidemiologically in the pathogenesis of atherosclerosis through iron-induced oxygen free radical-mediated lipid oxidation. However, the precise contribution of iron stores to atherosclerosis and its complications are unknown because prospective randomized trials designed to test effects of reduction of iron stores on clinical outcomes in this disease have not been performed. METHODS AND RESULTS: In preparation for a prospective randomized trial, a randomized pilot study was conducted to evaluate the feasibility, safety, and methodologic accuracy of calibrated reduction in iron stores by phlebotomy in a cohort of patients with advanced peripheral vascular disease. Phlebotomy resulted in a significant reduction in serum ferritin concentration to near targeted levels. Thus the formula for calculating the volume of blood to be removed to achieve a predetermined decrement in serum ferritin concentration was accurate and phlebotomy was not associated with any adverse laboratory or clinical effects. CONCLUSIONS: Reduction of body iron stores to a predetermined level is feasible and can be achieved in a timely manner with excellent patient compliance. Prospective randomized trials of calibrated reduction of body iron stores may be undertaken to define their pathophysiologic significance in atherosclerosis and other diseases in which excessive iron-induced oxidative stress has been implicated.