Fate of nanoparticles in the central nervous system after intrathecal injection in healthy mice.

Cerebrospinal fluid (CSF) is produced in the cerebral ventricles and circulates within the subarachnoid space (SAS) of the brain and spinal cord, where it exchanges with interstitial fluid of the parenchyma. The access of CSF to the entire central nervous system (CNS) makes it an attractive medium for drug delivery. However, few intrathecal (IT) therapies have reached the clinic due, in part, to limited distribution and rapid clearance. Given the success of nanoparticle (NP) carriers in prolonging circulation and improving delivery of systemically administered agents, we sought to evaluate the distribution of IT injected NPs within the CNS. We administered fluorescent, 100 nm PEGylated-NPs into the cisterna magna of healthy mice and studied their distribution along the brain and spinal cord. Our data demonstrate that NPs are capable of distributing rapidly through the SAS along the entire neuraxis with reproducible, anatomically defined patterns of delivery. NPs were well retained within the leptomeninges for over 3 weeks, showing preference for ventral surfaces and minimal penetration into the CNS parenchyma. Clearance of NPs occurred across the cribriform plate into the nasal mucosa, with a small fraction of NPs localizing with nerve roots exiting the spinal column. Larger 10 µm particles were also capable of moving through the SAS but did not achieve as widespread distribution. These studies demonstrate the ability of NPs to achieve widespread delivery along the neuraxis and highlight IT administration as a potentially significant route of administration for delivery of nanomedicine to the subarachnoid space.

Docosahexaenoic acid therapy in peroxisomal diseases: results of a double-blind, randomized trial.

OBJECTIVES: Peroxisome assembly disorders are genetic disorders characterized by biochemical abnormalities, including low docosahexaenoic acid (DHA). The objective was to assess whether treatment with DHA supplementation would improve biochemical abnormalities, visual function, and growth in affected individuals. METHODS: This was a randomized, double-blind, placebo-controlled trial conducted at a single center. Treatment groups received supplements of DHA (100 mg/kg per day). The primary outcome measures were the change from baseline in the visual function and physical growth during the 1 year follow-up period. RESULTS: Fifty individuals were enrolled and randomized. Two were subsequently excluded from study analysis when it was determined that they had a single enzyme disorder of peroxisomal beta oxidation. Thirty-four returned for follow-up. Nine patients died during the trial of their disorder, and 5 others were lost to follow-up. DHA supplementation was well tolerated. There was no difference in the outcomes between the treated and untreated groups in biochemical function, electroretinogram, or growth. Improvements were seen in both groups in certain individuals. CONCLUSIONS: DHA supplementation did not improve the visual function or growth of treated individuals with peroxisome assembly disorders. CLASSIFICATION OF EVIDENCE: This interventional study provides Class II evidence that DHA supplementation did not improve the visual function or growth of treated individuals with peroxisome assembly disorders during an average of 1 year of follow-up in patients aged 1 to 144 months.

Hepatoprotective activity of petroleum ether, diethyl ether, and methanol extract of Scoparia dulcis L. against CCl4-induced acute liver injury in mice.

OBJECTIVES: The present study was aimed at assessing the hepatoprotective activity of 1:1:1 petroleum ether, diethyl ether, and methanol (PDM) extract of Scoparia dulcis L. against carbon tetrachloride-induced acute liver injury in mice. MATERIALS AND METHODS: The PDM extract (50, 200, and 800 mg/kg, p.o.) and standard, silymarin (100 mg/kg, p.o) were tested for their antihepatotoxic activity against CCl4-induced acute liver injury in mice. The hepatoprotective activity was evaluated by measuring aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and total proteins in serum, glycogen, lipid peroxides, superoxide dismutase, and glutathione reductase levels in liver homogenate and by histopathological analysis of the liver tissue. In addition, the extract was also evaluated for its in vitro antioxidant activity using 1, 1-Diphenyl-2-picrylhydrazyl scavenging assay. RESULTS: The extract at the dose of 800 mg/kg, p.o., significantly prevented CCl4-induced changes in the serum and liver biochemistry (P < 0.05) and changes in liver histopathology. The above results are comparable to standard, silymarin (100 mg/kg, p.o.). In the in vitro 1, 1-diphenyl-2-picrylhydrazyl scavenging assay, the extract showed good free radical scavenging potential (IC 50 38.9 +/- 1.0 mug/ml). CONCLUSIONS: The results of the study indicate that the PDM extract of Scoparia dulcis L. possesses potential hepatoprotective activity, which may be attributed to its free radical scavenging potential, due to the terpenoid constituents.

A G1103R mutation in CRB1 is co-inherited with high hyperopia and Leber congenital amaurosis.

PURPOSE: To identify the genetic basis of recessive inheritance of high hyperopia and Leber congenital amaurosis (LCA) in a family of Middle Eastern origin. MATERIALS AND METHODS: The patients were examined using standard ophthalmic techniques. DNA samples were obtained and genetic linkage was carried out using polymorphic markers flanking the known genes and loci for LCA. Exons were amplified and sequenced. RESULTS: All four members of this family affected by LCA showed high to extreme hyperopia, with average spherical refractive errors ranging from +5.00 to +10.00. Linkage was obtained to 1q31.3 with a maximal LOD score of 5.20 and a mutation found in exon 9 of the CRB1 gene, causing a G1103R substitution at a highly conserved site in the protein. CRB1 is a vertebrate homolog of the Drosophila crumbs gene, which is required for photoreceptor morphogenesis, and has been associated with either retinitis pigmentosa (RP) or LCA. This sequence variant has previously been reported as a compound heterozygote in one sporadic LCA patient. CONCLUSION: Although hyperopia has been associated with LCA, it is typically moderate and variable between patients with the same mutation. In addition, some CRB1 mutations can be associated with either RP or LCA. We have shown that hyperopia and LCA are linked to the mutant CRB1 gene itself and are not dependent on unlinked modifiers.

In vitro culture of mantle tissue of the abalone Haliotis varia Linnaeus.

The study is aimed at developing a technology for the production of in vitro pearl through tissue culture of mantle of the abalone, Haliotis varia Linnaeus, as the production of free and spherical pearls in vivo is rather difficult in abalones. In the basic study, the cell yield was intensified from the explant after 24h incubation. Among the cells liberated, the granulocytes were dominant over hyalinocytes. The size of granulocytes ranged from 3 to 16 microm and of hyalinocytes from 13 to 18 microm. Fibroblast-like cells appeared in cultures after day 2. Both granulocytes and hyalinocytes developed pseudopodial-like extensions in all directions and formed organic matrix. Granulocytes contained granules in the cytoplasm. Specific granules were responsible for nucleation of crystals. Some crystals exhibited green colour resembling mother of pearl of abalone. scanning electron microscope (SEM) study revealed the oolitic amorphous state and rhombohedral state of crystals. Its analysis through energy dispersive X-ray microanalyzer (EDS) indicated the presence of calcium. The rhombohedral crystals under polarized light showed its high birefringence (0.18) and uniaxial optically negative calcite nature with high content of calcium. A mean survival of cells was found to be 102 days in T 25 flasks and 32 days in petri dishes. Growth of cells was studied. Thirty percent of cultures were found to have contaminated during the study. The study provides basic knowledge in the development of a technology for in vitro pearl production.

Mutational analysis and clinical correlation in Leber congenital amaurosis.

UNLABELLED: Leber congenital amaurosis (LCA, MIM 204001) is a clinically and genetically heterogeneous retinal disorder characterized by severe visual loss from birth, nystagmus, poor pupillary reflexes, retinal pigmentary or atrophic changes, and a markedly diminished electroretinogram (ERG). PURPOSE: To examine 100 consecutive patients with LCA in order to assess the relative burden of the three known genes involved in LCA, namely retinal guanylyl cyclase (GUCY2D), retinal pigment epithelium protein ( RPE65), and the cone-rod homeobox (CRX), and to define their clinical correlates. METHODS: Mutational analysis and detailed clinical examinations were performed in patients diagnosed with LCA at the Johns Hopkins Center for Hereditary Eye Diseases and the Montreal Children's Hospital. RESULTS: Mutations were identified in 11% of our patients: GUCY2D mutations accounted for 6%, while RPE65 and CRX gene mutations accounted for 3% and 2%, respectively. The clinical presentation was variable; however, the visual evolution in patients with mutations in GUCY2D and CRX remained stable, while individuals with mutations in the RPE65 gene showed progressive visual loss. CONCLUSIONS: This study suggests that molecular diagnosis of Leber congenital amaurosis could provide important information concerning prognosis and course of treatment.

A CRX null mutation is associated with both Leber congenital amaurosis and a normal ocular phenotype.

PURPOSE: To identify and characterize new cone rod homeobox (CRX) mutations associated with the Leber congenital amaurosis phenotype. METHODS: The human CRX gene was sequenced in 74 consecutive patients carrying the diagnosis of Leber congenital amaurosis. RESULTS: Two mutations were identified in CRX that cause frameshifts and predict severe truncations of the encoded protein. One of these, a 1-bp insertion, spares only nine N-terminal amino acids, removing the homeodomain, WSP motif, and conserved OTX domain at the C terminus. Of the CRX mutations described in the literature, this is the first that convincingly represents a null allele of the gene. Although the patient heterozygous for this null allele is affected with Leber congenital amaurosis, it was surprising that her father, who had normal vision, was heterozygous for the same mutation. CONCLUSIONS: These results strongly suggest that haploinsufficiency of CRX is not sufficient to cause a retinal disorder. Loss of function alleles of CRX appear to cause Leber congenital amaurosis through a recessive or multigenic mechanism.

Prevalence of AIPL1 mutations in inherited retinal degenerative disease.

Leber congenital amaurosis (LCA) is the most severe form of inherited retinal dystrophy and the most frequent cause of inherited blindness in children. LCA is usually inherited in an autosomal recessive fashion, although rare dominant cases have been reported. One form of LCA, LCA4, maps to chromosome 17p13 and is genetically distinct from other forms of LCA. We recently identified the gene associated with LCA4, AIPL1 (aryl-hydrocarbon interacting protein-like 1) and identified three mutations that were the cause of blindness in five families with LCA. In this study, AIPL1 was screened for mutations in 512 unrelated probands with a range of retinal degenerative diseases to determine if AIPL1 mutations cause other forms of inherited retinal degeneration and to determine the relative contribution of AIPL1 mutations to inherited retinal disorders in populations worldwide. We identified 11 LCA families whose retinal disorder is caused by homozygous or compound heterozygous AIPL1 mutations. We also identified affected individuals in two apparently dominant families, diagnosed with juvenile retinitis pigmentosa or dominant cone-rod dystrophy, respectively, who are heterozygous for a 12-bp AIPL1 deletion. Our results suggest that AIPL1 mutations cause approximately 7% of LCA worldwide and may cause dominant retinopathy.

trp RNA-binding attenuation protein-5' stem-loop RNA interaction is required for proper transcription attenuation control of the Bacillus subtilis trpEDCFBA operon.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by a novel transcription attenuation mechanism. Tryptophan-activated TRAP binds to the nascent trp leader transcript by interacting with 11 (G/U)AG repeats, 6 of which are present in an antiterminator structure. TRAP binding to these repeats prevents formation of the antiterminator, thereby promoting formation of an overlapping intrinsic terminator. A third stem-loop structure that forms at the extreme 5' end of the trp leader transcript also plays a role in the transcription attenuation mechanism. The 5' stem-loop increases the affinity of TRAP for trp leader RNA. Results from RNA structure mapping experiments demonstrate that the 5' stem-loop consists of a 3-bp lower stem, a 5-by-2 asymmetric internal loop, a 6-bp upper stem, and a hexaloop at the apex of the structure. Footprinting results indicate that TRAP interacts with the 5' stem-loop and that this interaction differs depending on the number of downstream (G/U)AG repeats present in the transcript. Expression studies with trpE'-'lacZ translational fusions demonstrate that TRAP-5' stem-loop interaction is required for proper regulation of the trp operon. 3' RNA boundary experiments indicate that the 5' structure reduces the number of (G/U)AG repeats required for stable TRAP-trp leader RNA association. Thus, TRAP-5' stem-loop interaction may increase the likelihood that TRAP will bind to the (G/U)AG repeats in time to block antiterminator formation.

Multicentric reticulohistiocytosis masquerading as lepromatous leprosy.

A 23-year old male presented for evaluation of skin coloured, non-scaly, asymptomatic papulonodules of sizes varying from 0.5 cm to 2 cm of 4 years duration distributed all over the body including the ears. The plaques present on the face gave the appearance of a 'leonine facies'. Clinically mistaken for lepromatous leprosy in reaction the patient was treated with antileprosy and anti-inflammatory drugs in 3 other centres for months with no improvement. Systemic involvement included painful swelling of both knee joints, pericardial effusion episcleritis and enlarged liver. Negative slit smears for AFB from the nodules repeatedly and the histology of one on the skin nodules clinched the diagnosis of multicentric reticulohistiocytosis. The case is reported not only for its rarity, and varying clinical lesions simulating lepromatous leprosy but also to alert the leprologists to avert unreasonable delay in diagnosis.