Utility of intraperitoneal administration as a route of AAV serotype 5 vector-mediated neonatal gene transfer

BACKGROUND: Gene transfer into a fetus or neonate can be a fundamental approach for treating genetic diseases, particularly disorders that have irreversible manifestations in adulthood. Although the potential utility of this technique has been suggested, the advantages of neonatal gene transfer have not been widely investigated. Here, we tested the usefulness of neonatal gene transfer using adeno-associated virus (AAV) vectors by comparing the administration routes and vector doses. METHODS: To determine the optimal administration route, neonates were subjected to intravenous (i.v.) or intraperitoneal (i.p.) injections of AAV5-based vectors encoding the human coagulation factor IX (hfIX) gene, and the dose response was examined. To determine the distribution of transgene expression, vectors encoding lacZ or luciferase (luc) genes were used and assessed by X-gal staining and in vivo imaging, respectively. After the observation period, the vector distribution across tissues was quantified. RESULTS: The factor IX concentration was higher in i.p.-injected mice than in i.v.-injected mice. All transgenes administered by i.p. injection were more efficiently expressed in neonates than in adults. The expression was confined to the peritoneal tissue. Interestingly, a sex-related difference was observed in transgene expression in adults, whereas this difference was not apparent in neonates. CONCLUSIONS: AAV vector administration to neonates using the i.p. route was clearly advantageous in obtaining robust transgene expression. Vector genomes and transgene expression were observed mainly in the peritoneal tissue. These findings indicate the advantages of neonatal gene therapy and would help in designing strategies for gene therapy using AAV vectors.     

Selective delivery of rifampicin incorporated into poly(DL-lactic-co-glycolic) acid microspheres after phagocytotic uptake by alveolar macrophages, and the killing effect against intracellular Mycobacterium bovis Calmette-Guérin

Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages.     

A new cottid species, Icelus sekii (Perciformes: Cottoidei), from Hokkaido, Japan

A new cottid species, Icelus sekii, is described on the basis of six specimens collected from off Rausu and Urakawa, Hokkaido Island, Japan. This species is distinguished from its congeners by the following combination of characters: supraocular and parietal spines absent; nuchal spine obscure; uppermost preopercular spine unbranched; no scales between dorsal scale row and lateral line scale row, and no scales below lateral line scale row; supraocular, parietal, and nuchal cirri present; five dark brown saddles dorsolaterally; anal fin rays 13; pectoral fin rays 15; vertebrae 12 + 24–25 = 36–37. Icelus sekii can be mature at the smallest size among the species of Icelus. As a secondary sexual character, the male holotype has unique ensiform flaps on the distal tips of the first dorsal fin.     

Construction of physical map and mapping of chromosomal virulence genes of the biovar 3 Agrobacterium (Rhizobium vitis) strain K-Ag-1

Most plant pathogenic Agrobacterium strains have been classified into three biovars, "biovar 1 (A. tumefaciens; Rhizobium radiobacter), biovar 2 (A. rhizogenes; R. rhizogenes) and biovar 3 (A. vitis; R. vitis)". The bacteria possess diverse types of genomic organization depending on the biovar. Previous genomic physical maps indicated difference in location of rDNA and chromosomally-coded virulence genes between biovar 1 and 2 genomes. In order to understand biovar 3 genome and its evolution in relation to the biovar 1, 2 and 3 genomes, we constructed physical map of a pathogenic biovar 3 strain K-Ag-1 in this study. Its genome consisted of two circular chromosomes (3.6 and 1.1 Mbp in length), and three plasmids (560, 230 and 70 kbp). Gene mapping based on the physical map showed presence of two rDNA loci in the larger chromosome and at least one rDNA locus in the smaller chromosome. Six chromosomal virulence genes, namely chvA, chvD, chvE, glgP, exoC and ros were found in the larger chromosome and not in the smaller chromosome. The location of rDNA loci is similar with that of biovar 1 genome, whereas the location of chromosomal virulence genes is similar with that of biovar 2 genome despite of the closer 16S-rRNA based phylogenetic relation of biovar 3 with biovar 1 than with biovar 2. Genomic PFGE RFLP analysis revealed that the K-Ag-1 strain, which was isolated on a kiwifruit plant in Japan, has the closest intra-species relation with two strains isolated from grapevine plants in Japan among eight biovar 3 strains examined. This datum suggests that the line of the strain is a major one in biovar 3 in Japan. Evolution of the genome of the strain is discussed based on the data.     

Importance of interspecific competition in the abundance of Aphanizomenon flos-aquae (Cyanophyceae)

The population dynamics of Aphanizomenon flos-aquae var. klebahnii Elenk. (Cyanophyceae) was studied in an artificial pond for 32 months from May 2002 to December 2004. Our previous in vitro study showed that the lower limits of water temperature and pH for its growth are within the ranges of 11°–14°C and 7.1–7.4, respectively, and it appeared that these findings are applicable to the emergence and disappearance of Ap. flos-aquae in the pond. Based on the change in the water temperature, the emergence of Ap. flos-aquae in 2004 was expected to be in late April, whereas emergence occurred after a 1-month lag period. During this period, Ankistrodesmus falcatus (Corda) Ralfs (Chlorophyceae) dominated the phytoplankton assemblage, which raised the possibility that the growth of Ap. flos-aquae was restricted by the existence of An. falcatus. We conducted mixed cultures of Ap. flos-aquae and An. falcatus at four temperatures (14°, 19°, 24°, and 29°C). After 18 days of incubation, An. falcatus dominated at 14° and 29°C whereas Ap. flos-aquae dominated at 19°C. This result indicates that a slightly higher water temperature than the growth threshold value is needed for Ap. flosaquae to outcompete An. falcatus, which agrees with the field observation. Contrary to the results of the mixed culture, the summer phytoplankton assemblage was dominated by Ap. flos-aquae, and the population of An. falcatus was less or almost absent. This variation seemed to be partly caused by the difference in nutrient conditions; concentrations of dissolved inorganic nitrogen and phosphorus in the pond were far lower than those in the culture medium. The lack of nitrogen fixation of An. falcatus seemed to be a growth disadvantage during the summer when the concentration of dissolved inorganic nitrogen was low.     

Protective roles of redox-active protein thioredoxin-1 for severe acute pancreatitis

Severe acute pancreatitis is a disease with high mortality, and infiltration of inflammatory cells and reactive oxygen species have a crucial role in the pathophysiology of this disease. Thioredoxin-1 (TRX-1) is an endogenous redox-active multifunctional protein with antioxidant and anti-inflammatory effects. TRX-1 is induced in various inflammatory conditions and shows cytoprotective effects. The aim of the present study was to clarify the protective roles of TRX-1 in the host defense mechanism against severe acute pancreatitis. Experimental acute pancreatitis was induced by intraperitoneal administration of cerulein, a CCK analog, and aggravated by lipopolysaccharide injection in transgenic mice overexpressing human TRX-1 (hTRX-1) and control C57BL/6 mice. Transgenic overexpression of hTRX-1 strikingly attenuated the severity of experimental acute pancreatitis. TRX-1 overexpression suppressed neutrophil infiltration as determined by myeloperoxidase activity, oxidative stress as determined by malondialdehyde concentration, and cytoplasmic degradation of inhibitor of kappaB-alpha, thereby suppressing proinflammatory cytokines, tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6; a neutrophil chemoattractant, keratinocyte-derived chemokine; and inducible nitric oxide synthase in the pancreas. Administration of recombinant hTRX-1 also suppressed neutrophil infiltration, reduced the inflammation of the pancreas and the lung, and improved the mortality rate. The present study suggests that TRX-1 has potent antioxidant and anti-inflammatory actions in experimental acute pancreatitis and might be a new therapeutic strategy to improve the prognosis of severe acute pancreatitis.     

Isolation and characterization of a novel small cardioactive peptide-related peptide from the brain of Octopus vulgaris

A novel small cardioactive peptide (SCP)-related peptide (oct-SCPRP: Ser-Asn-Gly-Tyr-Leu-Ala-Leu-Pro-Arg-Gln-NH2) was isolated from the brain of the octopus (Octopus vulgaris). cDNA encoding this precursor protein was cloned. Oct-SCPRP was shown to evoke contraction in the radula protractor muscle, and the precursor protein was highly homologous to the SCP family in the Mollusk but did not encode a related peptide, SCPB. The expression of oct-SCPRP mRNA was present not only in the peripheral nervous system (PNS) which is a motor center for the control of feeding, but also in the central nervous system (CNS) which is capable of complex analysis, learning, and controls behaviors.     

Regulation of food intake by acyl and des-acyl ghrelins in the goldfish

Our recent research has indicated that intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) administration of n-octanoic acid-modified ghrelin (acyl ghrelin) stimulates food intake and locomotor activity in the goldfish. The manner in which peripherally administered acyl ghrelin regulates food intake, however, remains unclear. In contrast to acyl ghrelin, non-acylated ghrelin (des-acyl ghrelin) does not exert an orexigenic action or induce hypermotility. To this extent, the biological role of des-acyl ghrelin in fish is unknown. Given the possible involvement of afferent pathways in mediating the effects of acyl ghrelin, as is known to occur in rodents, we examined the effect of capsaicin, a neurotoxin which destroys primary sensory (vagal and splanchnic) afferents, on the orexigenic activity induced by i.p.-injected acyl ghrelin. Pretreatment with i.p.-injected capsaicin (0.16 micromol/g body weight (BW)) cancelled the orexigenic action of i.p.-injected acyl ghrelin (8 pmol/g BW), although i.p.-injected capsaicin alone did not affect food intake. The effect of des-acyl ghrelin on the orexigenic action of acyl ghrelin in the goldfish was also investigated. The i.c.v. and i.p. injection of des-acyl ghrelin at doses 3-10 times higher than that of acyl ghrelin suppressed the orexigenic action of i.c.v.- and i.p.-injected acyl ghrelin (doses of 1 and 8 pmol/g BW). In contrast, injection of des-acyl ghrelin alone did not show any inhibitory effect on food intake. These results suggest that, as is seen in rodents, circulating acyl ghrelin derived from peripheral tissues acts via primary sensory afferent pathways on feeding centers in the brain. The results also show that des-acyl ghrelin inhibits acyl ghrelin-induced orexigenic activity in goldfish.