Hemolytic and antifungal activity of liposome-entrapped amphotericin B prepared by the precipitation method.

A new method of preparing liposomes containing amphotericin B (AmB) was developed with the purpose of reducing the toxicity of AmB without causing a loss in its antifungal activity. The procedure involved the precipitation of AmB and egg phosphatidylcholine (PC) in phosphate buffered saline (PBS, pH 7.4) or tris buffered saline (TBS, pH 7.4) by evaporating methanol and chloroform, which had been previously mixed in the buffer solution, at 4 degrees C and 600 mm Hg. The in vitro toxicity of the precipitated liposomes containing 3, 6, 9, 12, and 15 wt% AmB was compared with that of the film-swollen liposomes containing the equivalent contents of the drug. The hemolytic ability of the precipitated liposomes at 37 degrees C was 50.3% at maximum of the film-swollen liposomes at a dose of 30 micrograms AmB/ml, as measured after 17-hr incubation. The significant reduction in the hemolysis effect may in fact be attributed to the reduced rate of drug release from the precipitated liposomes. The precipitated liposomes were multilayered and aggregates of AmB were embedded in the bilayers. These aggregates of AmB would be responsible for an intensive positive peak around 330 nm and reduced toxicity. Despite the decrease in toxicity, the activity of the precipitated liposomes against Candida albicans remained almost equipotent to that of the film-swollen liposomes. Therefore, liposomes prepared by the precipitation method are less toxic but equally as active.

Contact and noncontact transscleral neodymium:YAG laser cyclophotocoagulation in a rabbit model.

PURPOSE: Contact versus noncontact transscleral neodymium:YAG laser cyclophotocoagulation was compared in a rabbit model with respect to the effects on intraocular pressure and pathologic findings. METHODS: Thirty-two rabbits received comparable energy levels (2 joules) of contact and noncontact cyclophotocoagulation. Pneumotonometry was performed every other day following treatment. RESULTS: For both the 2-week and 8-week periods following treatment, intraocular pressure was significantly lower in eyes receiving contact cyclophotocoagulation than in eyes receiving noncontact cyclophotocoagulation (for the 2-week period, mean difference in intraocular pressure was 5.3 mm Hg, n = 22, p < 0.001; for the 8-week period, mean difference in intraocular pressure was 4.3 mm Hg, n = 17, p < 0.013). Pathologic findings include acute ciliary body hemorrhage and inflammation (1 day), chronic inflammation (1 week), granulation tissue within laser lesions (2 weeks), and marked ciliary body atrophy (8 weeks). CONCLUSIONS: In our rabbit model, contact cyclophotocoagulation was more effective in lowering intraocular pressure than noncontact cyclophotocoagulation. Both contact and noncontact cyclophotocoagulation produced a variety of pathologic lesions in the rabbit eye, including cataract, phthisis bulbi, and anterior synechiae.

A retrospective study of eye disease among first grade children in Los Angeles.

BACKGROUND: The prevalence of ocular disease among children in one school district in Los Angeles, California was studied to better understand the types of eye disorders among this population as well as to develop appropriate preventive programs. METHODS: A computer-assisted retrospective analysis of date was performed from charts of 2,204 first grade children examined in the UCLA Mobile Eye Clinic. RESULTS: One or more ocular disorders were observed in 22.3 percent of the subjects. Uncorrected best monocular visual acuity was 20/40 or worse in 3.4 percent of the children. Refractive errors were diagnosed in 15.7 percent of the subjects, astigmatism in 7.6 percent, hyperopia in 6.2 percent, and myopia in 6.0 percent. Color vision deficiencies (red-green) were found in 2.6 percent of boys. The prevalences of heterophorias and heterotropias were 1.2 percent and 1.3 percent, respectively. CONCLUSIONS: The variety of ocular disorders diagnosed in this demographic setting attests to the importance of performing early and effective screening eye examinations for children.

Blood-brain barrier glucose transporter mRNA is increased in experimental diabetes mellitus.

The blood-brain barrier (BBB) glucose transporter activity in vivo is known to be down-regulated in experimental diabetes mellitus. To determine whether parallel changes in BBB glucose transporter mRNA levels occur in experimental diabetes we isolated brain microvessels, which make up the BBB in vivo, from both control and experimental diabetic rats. Microvessel RNA fractions were isolated by cesium chloride density gradient centrifugation and were applied to 1.1% agarose gels for Northern blotting. The blots were probed with [32P]-labeled cDNAs corresponding to the rat brain glucose transporter and a cDNA to alpha-actin was used to monitor the transcript level of a typical housekeeping gene. The study was repeated three times and, in all cases, the BBB glucose transporter mRNA level was increased in experimental diabetes relative to control rats. These studies suggest that factors associated with experimental diabetes mellitus in rats lead to either an increased transcription or a decreased degradation of brain capillary glucose transporter mRNA.

Brain capillary 46,000 dalton protein is cytoplasmic actin and is localized to endothelial plasma membrane.

The most abundant protein of the brain capillary, which makes up the blood-brain barrier (BBB) in vivo, is a protein that migrates at a molecular weight of approximately 46 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bovine brain capillary 46 kDa protein was purified by SDS-PAGE and Sephadex G-25 gel filtration. The purified protein migrated as a single band of molecular weight of approximately 42,000 Da on subsequent SDS-PAGE followed by silver staining. The protein was digested by trypsin and tryptic peptides were analyzed by reverse phase high-performance liquid chromatography (HPLC). Two of these peptides, 11 and 18 amino acids in length, were sequenced and found to be identical to amino acid sequences corresponding to portions of cytoplasmic actin. The SDS-PAGE gel-purified 46 kDa protein was also subjected to limited proteolysis using S. aureus V8 protease, and this resulted in the formation of a prominent 31 kDa doublet as well as smaller proteolytic fragments, and these fragments were of identical molecular weight to those generated from limited proteolysis of bovine actin. Electron microscopic immunoperoxidase studies with primary cultures of bovine brain capillary endothelium showed that immunoreactive actin is intimately associated with the plasma membranes. In conclusion, the brain capillary 46 kDa protein is cytoplasmic actin and is localized to the endothelial plasma membrane. Modulations of brain capillary endothelial actin may play a role in the regulation of BBB permeability.

Transscleral and transcorneal iontophoresis of vancomycin in rabbit eyes.

We examined the ability of transscleral and transcorneal iontophoresis to deliver vancomycin into the aqueous humor, the vitreous humor, and the cornea of rabbit eyes. Control eyes receiving subconjunctival injection (25 mg) attained peak aqueous, vitreous, and corneal concentrations (mean +/- S.E.M.) of 14.73 +/- 0.35 mcg/ml (at 4 hours after injection), 1.10 +/- 0.78 mcg/ml (2 hours), and 1167 +/- 63 mcg/g (1 hour), respectively. Eyes receiving transscleral iontophoresis (3.5 mA for 10 minutes) attained significantly higher vitreal levels than controls: 6.33 +/- 0.25 mcg/ml (p less than 0.001; 1 hour), 13.43 +/- 2.32 mcg/ml (p less than 0.01; 2 hours), 11.93 +/- 0.76 mcg/ml (p less than 0.001; 4 hours), 8.40 +/- 0.60 mcg/ml (p less than 0.001; 8 hours). Eyes receiving transcorneal iontophoresis (0.5 mA for 5 minutes) attained earlier and significantly higher aqueous and corneal levels than controls. Aqueous humor levels were 16.20 +/- 3.19 mcg/ml (p less than 0.05; 1 hour) and 20.20 +/- 0.43 mcg/ml (p less than 0.001; 2 hours). Corneal levels were 10799 +/- 755 mcg/g (p less than 0.001; 0.5 hour), 4856 +/- 606 mcg/g (p less than 0.005; 1.0 hour), 2185 +/- 71 mcg/g (p less than 0.001; 2 hours), and 710 +/- 112 mcg/g (p less than 0.025; 4 hours). Corneal endothelial cell counts were decreased by 8.8% (p = 0.08) after transcorneal iontophoresis of vancomycin and 5.4% (p less than 0.02) following Balanced Salt Solution (BSS). However, corneal thickness were not significantly increased by iontophoresis of either vancomycin or BSS. These experiments show that transscleral and transcorneal iontophoresis are efficacious in delivering high concentrations of vancomycin into the aqueous and vitreous humor and the cornea.

Amyloid angiopathy of Alzheimer's disease: amino acid composition and partial sequence of a 4,200-dalton peptide isolated from cortical microvessels.

The cardinal lesions of Alzheimer's disease are neurofibrillary tangles, senile neuritic plaques, and vascular amyloid, the latter generally involving cortical arteries and small arterioles. All three lesions are composed of amyloid-like, beta-pleated sheet fibrils. Recently, a 4,200-dalton peptide has been isolated from extraparenchymal meningeal vessels, neuritic plaques, and neurofibrillary tangles. The assumption of N-terminal homogeneity in vascular amyloid has been used as an argument for a neuronal (versus blood) origin of the peptide. However, intracortical microvessels from Alzheimer's disease have not been previously isolated. The present studies describe the isolation of a microvessel fraction from Alzheimer's disease and control fresh autopsy human brain. Alzheimer's disease isolated brain microvessels that were extensively laden with amyloid and control microvessels were solubilized in 90% formic acid and analyzed by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The arteriole fraction from the Alzheimer's subject with extensive amyloid angiopathy contained a unique 4,200-dalton peptide, whereas the arterioles or capillaries isolated from two controls and two Alzheimer's disease subjects without angiopathy did not. This peptide was purified by HPLC and amino acid composition analysis showed the peptide is nearly identical to the 4,200-dalton peptide recently isolated from neuritic plaques or from neurofibrillary tangles. Sequence analysis revealed N-terminal heterogeneity. The N-terminal sequence was: Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr, which is identical to the N-terminal sequence of the 4,200-dalton peptide isolated previously from extraparenchymal meningeal vessels and neuritic plaques.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutral amino acid transport at the human blood-brain barrier.

Transport regulates nutrient availability in the brain, and many pathways of brain amino acid metabolism are influenced by precursor supply. Therefore, amino acid transport through the blood-brain barrier (BBB) plays an important rate-affecting role in brain metabolism. Information on the Km of BBB amino acid transport provides the quantitative basis for understanding the physiological importance of BBB transport competition effects. For example, the uniquely low Km values of BBB amino acid transport as compared to other organs in the rat provides the basis for the selective vulnerability of the rat brain to changes in amino acid supply caused by nutritional factors. The development of amino acid imbalances in the human brain in parallel with amino acid imbalances in blood is likely to occur if the Km of BBB neutral amino acid transport in humans is low, e.g., 25-100 microM, as is the case for the rat. A new model system of the human BBB, the isolated human brain capillary, has been developed. Recent studies with this system indicate that the Km of phenylalanine transport into human brain microvessels is approximately the same as that found during in vivo studies with laboratory rats. These results support the emerging hypothesis that the human brain, like the rat brain, is subject to acute regulation by dietary-related amino acid imbalances, and that the major site of this regulation is the amino acid transport system at the BBB.

Binding and internalization of insulin and insulin-like growth factors by isolated brain microvessels.

Isolated brain capillaries were used as a model system to test for binding and internalization of insulin and insulin-like growth factors (IGF) I and II. At 37 degrees C, the maximum specific binding of the 125I-labeled peptides was 48.0 +/- 0.8%/mg capillary protein for IGF I, 40.6 +/- 1.4% for IGF II, and 15.1 +/- 0.6% for insulin. The concentration of unlabeled peptide needed to cause a 50% decrease in the maximum binding (ID50) was 22 ng/ml (2.9 nM), 25 ng/ml (3.3 nM), and 7 ng/ml (1.2 nM) for IGF I, IGF II, and insulin, respectively. Unlabeled insulin competed poorly for the IGF I receptor, requiring 5000 ng/ml (667 nM) to cause a 50% reduction in binding, and did not compete at all for the IGF II receptor at concentrations up to 10(5) ng/ml (17.8 microM). The IGF I receptor was further characterized by reduced polyacrylamide gel electrophoresis of the disuccinimidyl suberate cross-linked 125I-labeled IGF I receptor. The gel showed a distinct band at 133,000 Mr that was abolished by 0.6 microgram/ml (80 nM) unlabeled IGF I but not by 10.0 micrograms/ml (1780 nM) unlabeled insulin. Peptide internalization was monitored by the acidwash technique. Only 22% of the bound IGF I was internalized, but 50% of the insulin and 43% of the IGF II were acid resistant. Capillaries prelabeled with internalized 125I-insulin could then export radioactivity into fresh, insulin-free media in a time- and temperature-dependent manner. However, high-performance liquid chromatography (HPLC) and trichloroacetic acid (TCA) analysis of the released material showed that it consisted mostly of degraded peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenylalanine transport at the human blood-brain barrier. Studies with isolated human brain capillaries.

The exquisite sensitivity of brain amino acid availability to changes in plasma amino acid composition arises from the uniquely high affinity (low Km) of blood-brain barrier transport sites as compared to cell membrane transport systems in nonbrain tissues. The extension of this paradigm from rats to man assumes that the Km of blood-brain barrier amino acid transport in the human is low as in the rat. This hypothesis is tested in the present studies wherein isolated human brain capillaries are used as a model system for the human blood-brain barrier. Capillaries were obtained from autopsy brain between 20 and 45 h after death and were isolated in high yield and free of adjoining brain tissue. [3H]Phenylalanine transport into the isolated human, rabbit, or rat brain capillary was characterized by two saturable transport systems and a nonsaturable component. The Km values of phenylalanine transport into brain capillaries via the two saturable systems averaged 0.26 +/- 0.08 and 22.3 +/- 7.1 microM for five human subjects. These studies provide the first evidence for a very high affinity (Km = 0.26 microM) neutral amino acid transport system at the blood-brain barrier, and it is hypothesized that this system is selectively localized to the brain side of the blood-brain barrier. The results also show that the transport Km values for phenylalanine transport are virtually identical at both the rat and human blood-brain barrier.