ATXN2 with intermediate-length CAG/CAA repeats does not seem to be a risk factor in hereditary spastic paraplegia

Hereditary spastic paraplegia (HSP) confines a group of heterogeneous neurodegenerative disorders characterized by progressive spasticity and lower limb weakness. Age of onset is highly variable even in familial cases with known mutations suggesting that the disease is modulated by other yet unknown parameters. Although progressive gait disturbances, lower limb spasticity and extensor plantar responses are hallmarks of HSP these characteristics are also found in other neurodegenerative disorders, e.g. amytrophic lateral sclerosis (ALS). HSP has been linked to ALS and frontotemporal degeneration with motor neuron disease (FTD-MND), since TDP-43 positive inclusions have recently been found in an HSP subtype, and TDP-43 are found in abundance in pathological inclusions of both ALS and FTD-MND. Furthermore, ataxin-2 (encoded by the gene ATXN2), a polyglutamine containing protein elongated in spinocerebellar ataxia type 2, has been shown to be a modulator of TDP-43 induced toxicity in ALS animal and cell models. Finally, it has been shown that ATXN2 with non-pathogenic intermediate-length CAG/CAA repeat elongations (encoding the polyglutamine tract) is a genetic risk factor of ALS. Considering the similarities in the disease phenotype and the neuropathological link between ALS and HSP we hypothesized that intermediate-length CAG/CAA repeats in ATXN2 could be a modulator of HSP. We show that in a cohort of 181 HSP patients 4.9 % of the patients had intermediate-length CAG/CAA repeats in ATXN2 which was not significantly different from the frequencies in a Danish control cohort or in American and European control populations. However, the mean age of onset was significantly lower in HSP patients with intermediate-length CAG/CAA repeats in ATXN2 compared to patients with normal length repeats. Based on these results we conclude that ATXN2 is most likely not a risk factor of HSP, whereas it might serve as a modulator of age of onset.     

Feasibility study of cavitation-induced liposomal doxorubicin release in an AT2 Dunning rat tumor model

Background: Targeted and triggered release of liposomal drug using heat or ultrasound represents a promising treatment modality able to increase the therapeutic-totoxicity ratio of encapsulated drugs. Purpose: To study the ability for high-intensity focused ultrasound to induce liposomal drug release mainly by focused inertial cavitation in vitro and in an animal model. Methods: A 1 MHz ultrasound setup has been developed for in vitro and in vivo drug release from a specific liposomal doxorubicin formulation at a target cavitation dose. Results: Controlled cavitation at 1 MHz was applied within the tumors 48 hours after liposome injection according to preliminary pharmacokinetic study. A small non-significant therapeutic effect of US-liposomal treatment was observed compared to liposomes alone suggesting no beneficial effect of ultrasound in the current setup. Conclusion: The in vitro study provided a suitable ultrasound setup for delivering a cavitation dose appropriate for safe liposomal drug release. However, when converting to an in vivo model, no therapeutic benefit was observed. This may be due to a number of reasons, one of which may be the difficulty in converting in vitro findings to an in vivo model. In light of these findings, we discuss important design features for future studies.     

Effect of ultrasound parameters on the release of liposomal calcein

The ultrasound exposure parameters that maximize drug release from dierucoyl-phosphatidylcholine (DEPC)-based liposomes were studied using two transducers operating at 300 kHz and 1 MHz. Fluorescent calcein was used as a model drug, and the release from liposomes in solution was measured using a spectrophotometer. The release of calcein was more efficient at 300 kHz than at 1 MHz, with thresholds of peak negative pressures of 0.9 MPa and 1.9 MPa, respectively. Above this threshold, the release increased with increasing peak negative pressure, mechanical index (MI), and duty cycle. The amount of drug released followed first-order kinetics and increased with exposure time to a maximal release. To increase the release further, the MI had to be increased. The results demonstrate that the MI and the overall exposure time are the major parameters that determine the drug's release. The drug's release is probably due to mechanical (cavitation) rather than thermal effects, and that was also confirmed by the detection of hydroxide radicals.     

Ultrasound-mediated destabilization and drug release from liposomes comprising dioleoylphosphatidylethanolamine

Novel sonosensitive doxorubicin-containing liposomes comprising dioleoylphosphatidylethanolamine (DOPE) as the main lipid constituent were developed and characterized in terms of ultrasound-mediated drug release in vitro. The liposome formulation showed high sonosensitivity; where approximately 95% doxorubicin was released from liposomes after 6 min of 40 kHz US exposure in buffered sucrose solution. This represented a 30% increase in release extent in absolute terms compared to liposomes comprising the saturated lipid analogue distearoylphosphatidylethanolamine (DSPE), and a 9-fold improvement in release extent when compared to standard pegylated liposomal doxorubicin, respectively. Ultrasound release experiments in the presence of serum showed a significantly reduction in sonosensitivity of DSPE-based liposomes, whilst the release properties of DOPE-based liposomes were essentially maintained. Dynamic light scattering measurements and cryo-transmission electron microscopy of DOPE-based liposomes after ultrasound treatment indicated liposome disruption and formation of various lipid structures, corroborating the high release extent. The results point to the potential of DOPE-based liposomes as a new class of drug carriers for ultrasound-mediated drug delivery.     

Role of phosphate groups in inhibition of calcium oxalate crystal growth by osteopontin

Osteopontin (OPN) inhibits the growth of calcium oxalate monohydrate (COM) and other crystal phases in a phosphorylation-dependent manner. In the present study, the role of OPN phosphate groups in adsorption to, incorporation into and inhibition of COM crystals was studied by comparing OPN isoforms differing in phosphorylation. OPN isoforms purified from rat bone (bOPN), which contains 10 phosphates, and cow milk (mOPN), which contains 25 phosphates, were compared with rat recombinant OPN (rOPN), which is not phosphorylated. Using fluorescence-labeled proteins and confocal microscopy, we show that mOPN and rOPN, like bOPN, adsorb preferentially to the edges between {100} and {121} faces of preformed COM crystals, and to a lesser extent to the {100} and {121} faces. Using scanning electron microscopy, we show that growth of COM in the presence of bOPN or mOPN results in a 'dumbbell' morphology, whereas crystals grown with rOPN are only slightly affected. COM crystals grown in the presence of low concentrations of fluorescence-labeled bOPN incorporate the protein into the crystal lattice. In crystals imaged in the {010} plane, incorporation of bOPN results in a cross-shaped pattern of fluorescence, consistent with preferential adsorption to {100}/{121} edges throughout the growth process.     

Cerebral palsy in eastern Denmark: Declining birth prevalence but increasing numbers of unilateral cerebral palsy in birth year period 1986–1998

The Cerebral Palsy Registry in eastern Denmark has been collecting cases using a uniform data sampling procedure since birth year 1979. Children are included by two child neurologists and an obstetrician. Information on pregnancy, birth, neonatal period, impairments and demographic data are registered.The total cerebral palsy birth prevalence has been significantly decreasing since the birth period 1983–1986 with 3.0 per 1000 live births until the period 1995–1998 with 2.1 per 1000 live births. The overall decrease was seen in preterm infants (<31 weeks) as well as in term infants and despite a simultaneous fall in perinatal and early neonatal mortality in the preterm group.Analysing the subtypes of CP we found a significant increase in the numbers as well as the rate of unilateral CP with a simultaneous fall in the numbers as well as the rate of bilateral CP. The explanation of this rise is not obvious. A change from bilateral periventricular lesions to unilateral is a possibility, but no major change in the neonatal handling could be documented.Regarding associated impairments, developmental delay/learning disabilities as well as motor function assessed by ability to walk (unassisted/assisted), both have changed toward higher percentage of children with unassisted walking and in need of special education.     

Impaired insulin action despite upregulation of proximal insulin signaling: novel insights into skeletal muscle insulin resistance in liver cirrhosis

BACKGROUND/AIMS: Disturbance in glucose metabolism is a common feature in liver diseases and this is associated with skeletal muscle insulin resistance. However, the underlying molecular mechanisms are unclear. To characterize skeletal muscle insulin resistance associated with liver disease, we examined muscles from animals after an acute, 5 weeks perturbation of the common bile duct. Clinical findings, elevated plasma levels of liver enzymes and histological examinations confirmed cirrhosis. METHODS/RESULTS:: Cirrhotic animals were insulin resistant and this was associated with reduced glucose transport into muscles. Interestingly, activity in the proximal part of the insulin signaling cascade was not decreased, as evinced by increased activity of key enzymes in the signal to glucose transport. Expression of the glucose transporter, GLUT4, was normal. So together these results indicate that signaling downstream of PKB/Akt and/or the translocation of GLUT4 is impaired in skeletal muscle from cirrhotic animals. CONCLUSIONS: In conclusion, in an animal model of liver cirrhosis whole body insulin resistance is associated with insulin resistance in skeletal muscles. Unlike other common forms of insulin resistance, muscles from cirrhotic animals have increased activity in the proximal insulin signaling cascade. This emphasizes the fact that skeletal muscle insulin resistance associated with liver cirrhosis is a unique entity.     

Serum Concentrations of Antibodies Against Vaccine Toxoids in Children Exposed Perinatally to Immunotoxicants

Background

Polychlorinated biphenyls (PCBs) may cause immunotoxic effects, but the detailed dose–response relationship and possible vulnerable time windows of exposure are uncertain. In this study we applied serum concentrations of specific antibodies against childhood vaccines as sentinels of immunotoxicity.

Objectives

The main objective was to assess the possible dependence of antibody concentrations against diphtheria and tetanus toxoids in children with regard to prenatal and postnatal PCB exposures.

Methods

From a cohort of 656 singleton births formed in the Faroe Islands during 1999–2001, children were invited for examination with assessment of serum antibody concentrations at 5 years (before and after a booster vaccination) and at 7 years of age. Total PCB concentrations were determined in serum from ages 5 and 7 years, and data were also available on PCB concentrations in maternal pregnancy serum, maternal milk, and, for a subgroup, the child’s serum at 18 months of age.

Results

A total of 587 children participated in the examinations at ages 5 and/or 7 years. At age 5 years, before the booster vaccination, the antidiphtheria antibody concentration was inversely associated with PCB concentrations in milk and 18-month serum. Results obtained 2 years later showed an inverse association of concentrations of antibodies against both toxoids with PCB concentrations at 18 months of age. The strongest associations suggested a decrease in the antibody concentration by about 20% for each doubling in PCB exposure. At age 5 years, the odds of an antidiphtheria antibody concentration below a clinically protective level of 0.1 IU/L increased by about 30% for a doubling in PCB in milk and 18-month serum.

Conclusions

Developmental PCB exposure is associated with immunotoxic effects on serum concentrations of specific antibodies against diphtheria and tetanus vaccinations. The immune system development during the first years of life appears to be particularly vulnerable to this exposure.