SummaryLate infantile neuronal ceroid lipofuscinosis (LINCL), a fatal autosomal recessive neurodegenerative lysosomal storage disorder of childhood, is caused by mutations in the CLN2 gene, resulting in deficiency of the protein tripeptidyl peptidase I (TPP-I). We have previously shown that direct CNS administration of AAVrh.10hCLN2 to adult CLN2 knockout mice, a serotype rh.10 adeno-associated virus expressing the wild type CLN2 cDNA, will partially improve neurological function and survival. In this study, we explore the hypothesis that administration of AAVrh.10hCLN2 to the neonatal brain will significantly improve the results of AAVrh.10hCLN2 therapy. To assess this concept, AAVrh.10hCLN2 vector was administered directly to the CNS of CLN2 knockout mice at 2 days, 3 wk and 7 wk of age. While all treatment groups show a marked increase in total TPP-I activity over wild-type mice, neonatally treated mice displayed high levels of TPP-I activity in the CNS 1 yr after administration which was spread throughout the brain. Using behavioral markers, 2 day treated mice demonstrate marked improvement over 3 wk, 7 wk or untreated mice. Finally, neonatal administration of AAVrh.10hCLN2 was associated with markedly enhanced survival, with a median time of death 376 days for neonatal treated mice, 277 days for 3 wk treated mice, 168 days for 7 wk treated mice, and 121 days for untreated mice. These data suggest that neonatal treatment offers many unique advantages, and that early detection and treatment may be essential for maximal gene therapy for childhood lysosomal storage disorders affecting the CNS.
OBJECTIVE
To assess the risk factors for haemorrhage and renal fracture associated with renal cryoablation.
MATERIALS AND METHODS
In a porcine model, 120 cryoablations were administered in 26 pigs, with five groups of 24 ice‐balls each; in groups 1 and 2 asynchronous cryoprobe activation was evaluated for the 1.47‐ and 3.4‐mm cryoprobes (IceRods, Galil Medical, Plymouth Meeting, PA, USA), respectively; in group 3, three‐3.4 mm cryoprobes were used to examine synchronous probe activation; in group 4 the 1.47‐mm cryoprobe was used to examine single‐probe activation with premature cryoprobe extraction; and in group 5 we used a new ‘guillotine’ technique for upper‐pole renal cryoablation. Ice‐ball fractures and haemorrhage were characterized by the location, length and depth of each fracture, was well as the degree of bleeding.
RESULTS
In all, 26 domestic pigs successfully had renal cryoablation procedures. In group 1 and 4 there were no episodes of renal fracture; in group 2 renal fracture occurred in 10 (42%) trials. Group 3 had 22 (92%) renal fractures during the freeze/thaw cycle. Group 5 had 13 (54%) renal fractures during the freeze/thaw cycle, and there was an additional ice‐ball fracture during probe removal once in 24 times.
CONCLUSIONS
Renal fracture is most common with the application of larger 3.4‐mm cryoprobes in the synchronous and asynchronous setting. Under standard application, smaller (1.47‐mm) cryoprobes result in little renal fracture or bleeding. The use of the guillotine technique is associated with a greater risk of renal fracture.
The murine inhibitor of carbonic anhydrase (mICA) is a member of the superfamily related to the bilobal iron transport protein transferrin (TF), which binds a ferric ion within a cleft in each lobe. Although the gene encoding ICA in humans is classified as a pseudogene, an apparently functional ICA gene has been annotated in mice, rats, cows, pigs, and dogs. All ICAs lack one (or more) of the amino acid ligands in each lobe essential for high-affinity coordination of iron and the requisite synergistic anion, carbonate. The reason why ICA family members have lost the ability to bind iron is potentially related to acquiring a new function(s), one of which is inhibition of certain carbonic anhydrase (CA) isoforms. A recombinant mutant of the mICA (W124R/S188Y) was created with the goal of restoring the ligands required for both anion (Arg124) and iron (Tyr188) binding in the N-lobe. Absorption and fluorescence spectra definitively show that the mutant binds ferric iron in the N-lobe. Electrospray ionization mass spectrometry confirms the presence of both ferric iron and carbonate. At the putative endosomal pH of 5.6, iron is released by two slow processes indicative of high-affinity coordination. Induction of specific iron binding implies that (1) the structure of mICA resembles those of other TF family members and (2) the N-lobe can adopt a conformation in which the cleft closes when iron binds. Because the conformational change in the N-lobe indicated by metal binding does not impact the inhibitory activity of mICA, inhibition of CA was tentatively assigned to the C-lobe. Proof of this assignment is provided by limited trypsin proteolysis of porcine ICA.
Our in vitro study indicates that this radiopaque, thermosensitive polymer is able to transiently occlude the ureter without damaging the urothelium while withstanding the pressure of ureteroscopic irrigation, stone motion, and laser energy.
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