Rescue of severe infantile hypophosphatasia mice by AAV-mediated sustained expression of soluble alkaline phosphatase.

Hypophosphatasia (HPP) is an inherited disease caused by a deficiency of tissue-nonspecific alkaline phosphatase (TNALP). The major symptom of human HPP is hypomineralization, rickets, or osteomalacia, although the clinical severity is highly variable. The phenotypes of TNALP knockout (Akp2(-/-)) mice mimic those of the severe infantile form of HPP. Akp2(-/-) mice appear normal at birth, but they develop growth failure, epileptic seizures, and hypomineralization and die by 20 days of age. Previously, we have shown that the phenotype of Akp2(-/-) mice can be prevented by enzyme replacement of bone-targeted TNALP in which deca-aspartates are linked to the C-terminus of soluble TNALP (TNALP-D10). In the present study, we evaluated the therapeutic effects of adeno-associated virus serotype 8 (AAV8) vectors that express various forms of TNALP, including TNALP-D10, soluble TNALP tagged with the Flag epitopes (TNALP-F), and native glycosylphosphatidylinositol-anchored TNALP (TNALP-N). A single intravenous injection of 5×10(10) vector genomes of AAV8-TNALP-D10 into Akp2(-/-) mice at day 1 resulted in prolonged survival and phenotypic correction. When AAV8-TNALP-F was injected into neonatal Akp2(-/-) mice, they also survived without epileptic seizures. Interestingly, survival effects were observed in some animals treated with AAV8-TNALP-N. All surviving Akp2(-/-) mice showed a healthy appearance and a normal activity with mature bone mineralization on X-rays. These results suggest that sustained alkaline phosphatase activity in plasma is essential and sufficient for the rescue of Akp2(-/-) mice. AAV8-mediated systemic gene therapy appears to be an effective treatment for the infantile form of human HPP.

Prolonged survival and phenotypic correction of Akp2(-/-) hypophosphatasia mice by lentiviral gene therapy.

Hypophosphatasia (HPP) is an inherited systemic skeletal disease caused by mutations in the gene encoding the tissue-nonspecific alkaline phosphatase (TNALP) isozyme. The clinical severity of HPP varies widely, with symptoms including rickets and osteomalacia. TNALP knockout (Akp2(-/-)) mice phenotypically mimic the severe infantile form of HPP; that is, TNALP-deficient mice are born with a normal appearance but die by 20 days of age owing to growth failure, hypomineralization, and epileptic seizures. In this study, a lentiviral vector expressing a bone-targeted form of TNALP was injected into the jugular vein of newborn Akp2(-/-) mice. We found that alkaline phosphatase activity in the plasma of treated Akp2(-/-) mice increased and remained at high levels throughout the life of the animals. The treated Akp2(-/-) mice survived for more than 10 months and demonstrated normal physical activity and a healthy appearance. Epileptic seizures were completely inhibited in the treated Akp2(-/-) mice, and X-ray examination of the skeleton showed that mineralization was significantly improved by the gene therapy. These results show that severe infantile HPP in TNALP knockout mice can be treated with a single injection of lentiviral vector during the neonatal period.

Leptosins isolated from marine fungus Leptoshaeria species inhibit DNA topoisomerases I and/or II and induce apoptosis by inactivation of Akt/protein kinase B.

DNA topoisomerases (topo) I and II are molecular targets of several potent anticancer agents. Thus, inhibitors of these enzymes are potential candidates or model compounds for anticancer drugs. Leptosins (Leps) F and C, indole derivatives, were isolated from a marine fungus, Leptoshaeria sp. as cytotoxic substances. In vitro cytotoxic effects of Lep were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based viability assay. Lep F inhibited the activity of topos I and II, whereas Lep C inhibited topo I in vitro. Interestingly both of the compounds were found to be catalytic inhibitors of topo I, as evidenced by the lack of stabilization of reaction intermediate cleavable complex (CC), as camptothecin (CPT) does stabilize. Furthermore, Lep C inhibited the CC stabilization induced by CPT in vitro. In vivo band depletion analysis demonstrated that Lep C likewise appeared not to stabilize CC, and inhibited CC formation by CPT, indicating that Lep C is also a catalytic inhibitor of topo I in vivo. Cell cycle analysis of Lep C-treated cells showed that Lep C appeared to inhibit the progress of cells from G(1) to S phase. Lep C induced apoptosis in RPMI8402 cells, as revealed by the accumulation of cells in sub-G(1) phase, activation of caspase-3 and the nucleosomal degradation of chromosomal DNA. Furthermore, Leps F and C inhibited the Akt pathway, as demonstrated by dose-dependent and time-dependent dephosphorylation of Akt (Ser473). Our study shows that Leps are a group of anticancer chemotherapeutic agents with single or dual catalytic inhibitory activities against topos I and II.

[A case of herpes simplex virus encephalitis characterized by prolonged psychotic symptoms in convalescence--a case report].

A 14-year-old girl was diagnosed as having herpes simplex encephalitis by a polymerase chain reaction examination of the cerebrospinal fluid. MRI showed diffuse high signal areas at the bilateral temporal lobes and right frontal lobe. She was treated with a total of 350 mg/kg of acyclovir for 23 days, and discharged without abnormal neurological findings. On the 90th day after the onset, however, she became excitable and aggressive to her family. She excessively seeked relations with several boy friends. MRI revealed dilation of the bilateral ventricles and atrophy of the temporal lobes. These pathologic changes including the atrophy of hippocampus may be responsible for the character changes in this case.