Are vancomycin dosing guidelines followed? A mixed methods study of vancomycin prescribing practices.

AIMS: Despite the availability of international consensus guidelines, vancomycin dosing and therapeutic drug monitoring (TDM) remain suboptimal. This study aimed to assess concordance of vancomycin dosing and TDM with institutional guidelines and to identify factors taken into consideration by clinicians when prescribing vancomycin. METHODS: A retrospective audit of 163 patients receiving vancomycin therapy (≥48 hours) was undertaken. Data collected included patient characteristics, dosing history and plasma vancomycin and creatinine concentrations. Concordance of dosing and TDM with institutional guidelines was evaluated. Semi-structured interviews, including simulated prescribing scenarios, were undertaken with prescribers (n = 17) and transcripts analysed. RESULTS: Plasma vancomycin concentrations (n = 1043) were collected during 179 courses of therapy. Only 24% of courses commenced with a loading dose with 72% lower than recommended. The initial maintenance dose was concordant in 42% of courses with 34% lower than recommended. Only 14% of TDM samples were trough vancomycin concentrations. Dose was not adjusted for 60% (21/35) of subtherapeutic and 43% (18/42) of supratherapeutic trough vancomycin concentrations, respectively. Interview participants reported that patient characteristics (including renal function), vancomycin concentrations, guidelines and expert advice influenced vancomycin prescribing decisions. Despite referring to guidelines when completing simulated prescribing scenarios, only 37% of prescribing decisions aligned with guideline recommendations. CONCLUSION: Poor compliance with institutional vancomycin guidelines was observed, despite prescriber awareness of available guidelines. Multifaceted strategies to support prescriber decision-making are required to improve vancomycin dosing and monitoring.

α-Synuclein accumulates in huntingtin inclusions but forms independent filaments and its deficiency attenuates early phenotype in a mouse model of Huntington's disease.

Huntington's disease (HD) is the most common of nine inherited neurological disorders caused by expanded polyglutamine (polyQ) sequences which confer propensity to self-aggregate and toxicity to their corresponding mutant proteins. It has been postulated that polyQ expression compromises the folding capacity of the cell which might affect other misfolding-prone proteins. α-Synuclein (α-syn) is a small neural-specific protein with propensity to self-aggregate that forms Parkinson's disease (PD) Lewy bodies. Point mutations in α-syn that favor self-aggregation or α-syn gene duplications lead to familial PD, thus indicating that increased α-syn aggregation or levels are sufficient to induce neurodegeneration. Since polyQ inclusions in HD and other polyQ disorders are immunopositive for α-syn, we speculated that α-syn might be recruited as an additional mediator of polyQ toxicity. Here, we confirm in HD postmortem brains and in the R6/1 mouse model of HD the accumulation of α-syn in polyQ inclusions. By isolating the characteristic filaments formed by aggregation-prone proteins, we found that N-terminal mutant huntingtin (N-mutHtt) and α-syn form independent filamentous microaggregates in R6/1 mouse brain as well as in the inducible HD94 mouse model and that N-mutHtt expression increases the load of α-syn filaments. Accordingly, α-syn knockout results in a diminished number of N-mutHtt inclusions in transfected neurons and also in vivo in the brain of HD mice. Finally, α-syn knockout attenuates body weight loss and early motor phenotype of HD mice. This study therefore demonstrates that α-syn is a modifier of polyQ toxicity in vivo and raises the possibility that potential PD-related therapies aimed to counteract α-syn toxicity might help to slow HD.

GSK3ß overexpression induces neuronal death and a depletion of the neurogenic niches in the dentate gyrus.

Overexpression of GSK3ß in transgenic mice induces learning deficits and some features associated with Alzheimer's disease (AD), including dentate gyrus (DG) atrophy. Here, we assessed whether these mice also recapitulate DG atrophy as well as impaired neurogenesis reported in AD. Ultrastructural analysis revealed that there were fewer and more disorganized neurogenic niches in these animals, coupled with an increase in the proportion of immature neurons. Indeed, the maturation of granule cells is delayed as witnessed by the alterations to the length and patterning of their dendritic trees and to the mossy fiber terminals. Together with an increase in neuronal death, these phenomena lead to a marked decrease in the number and disorganization of granule cells of the DG. Our results suggest that GSK3ß overexpression perturbs proliferation and maturation, resulting in the loss of immature neurons. In turn, the activation of microglia is stimulated in conjunction with a decrease in the birth of new functional neurons, leading to the deterioration of this structure. These data support the idea that by inducing degeneration of the DG, GSK3ß could be involved in the pathogenesis of AD.

Full motor recovery despite striatal neuron loss and formation of irreversible amyloid-like inclusions in a conditional mouse model of Huntington's disease.

The primary mechanism responsible for Huntington's disease remains unknown. Postulated early pathogenic events include the following: impaired protein folding, altered protein degradation, mitochondrial dysfunction, and transcriptional dysregulation. Although related therapies can delay disease progression in mouse models, they target downstream and probably indirect effects of mutant-huntingtin expression. Accordingly, in case they prove beneficial in humans, they might only palliate some aspects of disease. Our previous studies in the Tet/HD94 conditional model and the recently reported efficacy of RNA interference against mutant huntingtin in another mouse model support silencing mutant-huntingtin expression as a valid therapeutic approach that has the advantage of targeting toxicity at its root. Here, we address whether gene silencing can still be beneficial in the late stages of disease with detectable striatal neuron loss. Stereological analysis was applied to determine an age at which Tet/HD94 mice show a decrease in the number of striatal neurons. Then, progression of neuropathology and motor phenotype were analyzed in mice that were allowed to continue expressing mutant huntingtin and in mice that no longer expressed it. Neuronal loss did not revert in gene-off mice, but the additional loss that takes place in gene-on mice was prevented. The total number of huntingtin-containing inclusions dramatically reverted, but a small fraction of inclusions positive for the amyloid dye thioflavin-S remained. Interestingly, despite a 20% decrease in striatal neurons and the presence of amyloid-like irreversible inclusions, gene-off mice fully recover from their motor deficit, thus ruling out amyloid-like huntingtin inclusions as the main toxic species and suggesting that gene-silencing therapies might work in late stages of disease.

Biochemical, ultrastructural, and reversibility studies on huntingtin filaments isolated from mouse and human brain.

Huntington's disease (HD) and eight additional inherited neurological disorders are caused by CAG triplet-repeat expansions leading to expanded polyglutamine-sequences in their respective proteins. These triplet-CAG repeat disorders have in common the formation of aberrant intraneuronal proteinaceous inclusions containing the expanded polyglutamine sequences. These aggregates have been postulated to contribute to pathogenesis caused by conformational toxicity, sequestration of other polyglutamine-containing proteins, or by interfering with certain enzymatic activities. Testing these hypotheses has been hampered by the difficulty to isolate these aggregates from brain. Here we report that polyglutamine aggregates can be isolated from the brain of the Tet/HD94 conditional mouse model of HD, by following a method based on high salt buffer homogenization, nonionic detergent extraction, and gradient fractionation. We then verified that the method can be successfully applied to postmortem HD brains. Immunoelectron microscopy, both in human and mouse samples, revealed that the stable component of the inclusions are mutant huntingtin-containing and ubiquitin-containing fibrils. Atomic-force microscopy revealed that these fibrils have a "beads on a string" morphology. Thus, they resemble the in vitro assembled filaments made of recombinant mutant-huntingtin, as well as the Abeta and alpha-synuclein amyloid protofibrils. Finally, by shutting down transgene expression in the Tet/HD94 conditional mouse model of HD, we were able to demonstrate that these filaments, although stable in vitro, are susceptible to revert in vivo, thus demonstrating that the previously reported reversal of ubiquitin-immunoreactive inclusions does not simply reflect disassembling of the inclusions into their constituent fibrils and suggesting that any associated conformational or protein-sequestration toxicity is also likely to revert.

Neuronal induction of the immunoproteasome in Huntington's disease.

Huntington's disease (HD) inclusions are stained with anti-ubiquitin and anti-proteasome antibodies. This, together with proteasome activity studies on transfected cells, suggest that an impairment of the ubiquitin-proteasome system (UPS) may be key in HD pathogenesis. To test whether proteasome activity is impaired in vivo, we performed enzymatic assays for the three peptidase activities of the proteasome in brain extracts from the HD94 conditional mouse model of HD. We found no inhibition of any of the activities, suggesting that if UPS impairment happens in vivo, it is not at the level of the proteasome catalytic core. Intriguingly, the chymotrypsin- and trypsin-like activities increased selectively in the affected and aggregate-containing regions: cortex and striatum. Western blot analysis revealed no difference in total proteasome content whereas an increase in the interferon-inducible subunits of the immunoproteasome, LMP2 and LMP7, was observed. These subunits confer to the proteasome catalytic properties that are optimal for MHC-I peptide presentation. Immunohistochemistry in control mouse brain revealed LMP2 and LMP7 mainly in neurons. Accordingly, their increase in HD94 mice predominantly took place in neurons, and 5% of the ubiquitin-positive cortical aggregates were also LMP2-positive. Ultrastructural analysis of neurons with high level of immunoproteasome subunits revealed signs of neurodegeneration like nuclear indentation or fragmentation and dark cell appearance. The neuronal induction of LMP2 and LMP7 and the associated signs of neurodegeneration were also found in HD postmortem brains. Our results indicate that LMP2 and LMP7 participate in normal neuronal physiology and suggest a role in HD neurodegeneration.

Cooexpression of FTDP-17 tau and GSK-3beta in transgenic mice induce tau polymerization and neurodegeneration.

Here we have tested whether tau modification either by point mutation or by hyperphosphorylation can exert maximal pathogenic effects or if, on the contrary, both types of tau modifications can act synergistically to induce neuropathology. For this, we have combined transgenic mice overexpressing the enzyme GSK-3beta (Tet/GSK-3beta mice), with transgenic mice expressing Tau with a triple FTDP-17 mutation which develop prefibrillar tau-aggregates (VLW mice). Tet/GSK-3beta/VLW transgenic mice show tau hyperphosphorylation in hippocampal neurons. This is accompanied by thioflavin-S staining, and formation of filaments similar in width to those found in tauophaties. Finally, the atrophy of the hippocampal dentate gyrus observed in Tet/GSK-3beta mice develops much faster in Tet/GSK-3beta/VLW mice. All these morphological and biochemical data demonstrate that there is a synergistic contribution of both types of tau modifications and that the potential of GSK-3 inhibitors for AD therapeutics also extends to tauopathies caused by point mutations in tau gene.

Inhibition of 26S proteasome activity by huntingtin filaments but not inclusion bodies isolated from mouse and human brain.

In Huntington's disease (HD), as in the rest of CAG triplet-repeat disorders, the expanded polyglutamine (polyQ)-containing proteins form intraneuronal fibrillar aggregates that are gathered into inclusion bodies (IBs). Since IBs contain ubiquitin and proteasome subunits, it was proposed that inhibition of proteasome activity might underlie pathogenesis of polyQ disorders. Recent in vitro enzymatic studies revealed the inability of eukaryotic proteasomes to digest expanded polyQ, thus suggesting that occasional failure of polyQ to exit the proteasome may interfere with its proteolytic function. However, it has also recently been found that in vitro assembled aggregates made of synthetic polyQ fail to inhibit proteasome activity. Because synthetic polyQ aggregates lack the post-translational modifications found inside affected neurons, such as poly ubiquitylation, we decided to study the effect of mutant huntingtin (htt) aggregates isolated from the Tet/HD94 mouse model and from human HD brain tissue. Here, we show that isolated ubiquitylated filamentous htt aggregates, extracted from IBs by a previously reported method, selectively inhibited the in vitro peptidase activity of the 26S but not of the 20S proteasome in a non-competitive manner. In good agreement, immuno-electron microscopy revealed a direct interaction of htt filaments with the 19S ubiquitin-interacting regulatory caps of the 26S proteasome. Here, we also report a new method for isolation of IBs based on magnetic sorting. Interestingly, isolated IBs did not modify proteasome activity. Our results therefore show that mutant htt filamentous aggregates can inhibit proteasome activity, but only when not recruited into IBs, thus strengthening the notion that IB formation is protective by neutralizing toxicity of dispersed filamentous htt aggregates.