Long-term follow-up of liver-directed, adeno-associated vector-mediated gene therapy in the canine model of hemophilia A.

Questions remain concerning the long-term efficacy, safety, and site(s) of transgene expression following adeno-associated vector (AAV) therapy. We report a long-term follow-up of 8 (male = 4, hemizygous, and female = 4, homozygous) dogs with severe hemophilia A treated with a single portal vein infusion of a B-domain-deleted (BDD)-canine FVIII (cFVIII) AAV vector (median dose = 1.25 × 1013 vg/kg, AAV2 = 4, AAV6 = 3, and AAV8 = 1). After a median follow-up of 10.8 years (8.2-12.0 years), persistent FVIII:C (median one-stage = 12.7%, chromogenic = 7.2%) was seen in all responding dogs (n = 6), with improvement in annualized bleed rates (pre = 3.9 vs post = 0.3 event per year; P = .003). Anti-AAV capsid neutralizing antibodies (nAbs) toward the dosed capsid were detected throughout the study, with limited cross-reactivity to other capsids. nAb titers for all capsid serotypes declined with time, although they remained at levels precluding redosing with the same capsid. AAV-BDD-cFVIII DNA was detected in the liver of all dogs (median = 0.15 vg per diploid genome), with lower levels in the spleen in 4 dogs (median = 0.005 vg per diploid genome). Consistent with the liver-specific promoter, BDD-cFVIII mRNA was only detected in the liver. Postmortem examination demonstrated no evidence of chronic liver disease or liver malignancy. Persistent FVIII expression and an improved bleeding phenotype was seen for more than a decade after vector delivery. This is the longest follow-up reported in a preclinical model supporting long-term efficacy and safety of AAV-mediated gene therapy.

Factors Associated with Pre-Event Hydration Status and Drinking Behavior of Middle-Aged Cyclists.

OBJECTIVES: Water is an essential nutrient for thermoregulation, metabolism, cognition, and overall physiological homeostatic function. However, aging adults display a blunted thirst mechanism and subsequently have an increased risk for dehydration or hyponatremia. Fluid consumption behaviors are modifiable and the importance of practicing adequate drinking behaviors for aging adults is amplified during exercise. Identification of aging adult's hydration beliefs and how they attain hydration advice could provide valuable information into ways to promote better drinking habits to reduce fluid imbalances. Thus, this investigation evaluated the knowledge, beliefs and behaviors of middle-aged cyclists (MA) that were associated with hydration status and drinking behavior, before and during a 164-km mass-participation event (ambient temperature, 33.3±2.8ºC(mean±SD)). DESIGN: This cross-sectional field study retrospectively grouped participants by their second urine specific gravity (Usg) measurement of the event morning prior to a mass participation cycling event. Usg was assessed via handheld refractometer. SETTING: The Hotter N' Hell Hundred 164-km cycling event in Wichita Falls, Texas during the month of August. PARTICIPANTS: 36 male recreational cyclists (age, 53±9 y(mean±SD)). MEASUREMENTS: Participants were grouped according their urine specific gravity as either slightly hyperhydrated (SH; n=12, Usg≤1.014), euhydrated (EUH; n=12, Usg, 1.015-1.020), or slightly dehydrated (SD; n=12, Usg≥1.021). Exercise histories and questionnaires were recorded 24-48 h prior to the cycling event. RESULTS: Regardless of pre-event hydration status, all groups experienced a similar body mass loss during the 164-km event and finished with statistically similar exercise times; also, drinking behavior within all groups was influenced by multiple factors. The primary factors associated with MA cyclist drinking behavior were trial and error/personal history and thirst; further, the majority of cyclists (≥65%) in SH, EUH, and SD believed that dehydration affects performance negatively. The least important factors included rehydration recommendations from scientific and sports medicine organizations, plus information from sports drink manufacturers. CONCLUSION: Considering the complexity of the present findings and the physiological changes that accompany aging such as delayed thirst perception, we recommend that MA cyclists formulate an individualized drinking plan that is based on observations during exercise.

Strategy to detect pre-existing immunity to AAV gene therapy.

Gene therapy may offer a new treatment option, particularly for patients with severe hemophilia, based on recent research. However, individuals with pre-existing immunity to adeno-associated viruses (AAVs) may be less likely to benefit from AAV vector-based therapies. To study pre-existing AAV5 immunity in humans, we validated two complementary, sensitive, and scalable in vitro assays to detect AAV5 total antibodies and transduction inhibition (TI). Using these two assays, we found that 53% of samples from 100 healthy male individuals were negative in both assays, 18% were positive in both assays, 5% were positive for total antibodies but negative for TI and, of interest, 24% were negative for total antibodies but positive for TI activity, suggesting the presence of non-antibody-based neutralizing factors in human plasma. Similar findings were obtained with 24 samples from individuals with hemophilia A. On the basis of these results, we describe the development of a dual-assay strategy to identify individuals without total AAV5 antibodies or neutralizing factors who may be more likely to respond to AAV5-directed gene therapy. These assays offer a universal, transferrable platform across laboratories to assess the global prevalence of AAV5 antibodies and neutralizing factors in large patient populations to help inform clinical development strategies.

Integrating the Forensic Sciences in Wildlife Case Investigations: A Case Report of Pentobarbital and Phenytoin Toxicosis in a Bald Eagle (Haliaeetus leucocephalus).

The application of medical knowledge to the purpose of law is the foundation of forensic pathology. A forensic postmortem examination often involves the expertise of multiple scientific disciplines to reconstruct the full story surrounding the death of an animal. Wildlife poses additional challenges in forensic investigations due to little or no associated history, and the disruptive effects of decomposition. To illustrate the multidisciplinary nature of wildlife forensic medicine, the authors outline a case of secondary pentobarbital/phenytoin toxicosis in a bald eagle (Haliaeetus leucocephalus). The eagle was the single fatality in a group of 8 birds that fed on euthanized domestic cat remains that had been improperly disposed of in a landfill. Cooperation between responding law enforcement officers, pathologists, and other forensic scientists led to the successful diagnosis and resolution of the case.

Morphological identification of animal hairs: Myths and misconceptions, possibilities and pitfalls.

The examination of hair collected from crime scenes is an important and highly informative discipline relevant to many forensic investigations. However, the forensic identification of animal (non-human) hairs requires different skill sets and competencies to those required for human hair comparisons. The aim of this is paper is not only to highlight the intrinsic differences between forensic human hair comparison and forensic animal hair identification, but also discuss the utility and reliability of the two in the context of possibilities and pitfalls. It also addresses and dispels some of the more popular myths and misconceptions surrounding the microscopical examination of animal hairs. Furthermore, future directions of this discipline are explored through the proposal of recommendations for minimum standards for the morphological identification of animal hairs and the significance of the newly developed guidelines by SWGWILD is discussed.

On the selective cleavage of nitrous oxide by metal-amide complexes.

Computational investigation of nitrous oxide cleavage by metal-amide systems has shown that a bimetallic mechanism is compatible with the remarkable experimental observation of selective N-N bond scission by a di-molybdenum system, and that the interplay between the bonding energetics of reactants and products can both provide an explanation for the observed cleavage selectivity and be utilized to modify and design chemical behaviour.

On the unprecedented level of dinitrogen activation in the calix[4]arene complex of Nb(III).

The calix[4]arene niobium(III) complex ([L]Nb-N=N-Nb[L] where [L] = p-tert-butylcalix[4]arene), reported to bind N(2) in a µ(2)-linear dimeric capacity and to activate the N(2) triple bond to 1.39 Å, corresponding to the longest N(2) bond known in the end-on coordination mode, was subjected to a computational investigation involving both density functional and wavefunction based methods to establish the basis for the unprecedented level of activation. Replacement of the calix[4]arene ligand with hydroxide or methoxide ligands reveals that the organic backbone structure of the calix[4]arene ligand exerts negligible electronic influence over the metal centre, serving only to geometrically constrain the coordinating phenoxide groups. A fragment bonding analysis shows that metal-to-dinitrogen π* backbonding is the principal Nb-N interaction, providing a strong electronic basis for analogy with other well-characterised three- and four-coordinate complexes which bind N(2) end-on. While the calculated structure of the metallacalix[4]arene unit is reproduced with high accuracy, as is also the Nb-Nb separation, the calculated equilibrium geometry of the complex under a variety of conditions consistently indicates against a 1.39 Å activation of the N(2) bond. Instead, the calculated N-N distances fall within the range 1.26-1.30 Å, a result concordant with closely related three- and four-coordinate µ(2)-N(2) complexes as well as predictions derived from trends in N-N stretching frequency for a number of crystallographically characterized linear N(2) activators. A number of potential causes for this bond length discrepancy are explored.

Responses of neurons in the rostral ventrolateral medulla to whole body rotations: comparisons in decerebrate and conscious cats.

The responses to vestibular stimulation of brain stem neurons that regulate sympathetic outflow and blood flow have been studied extensively in decerebrate preparations, but not in conscious animals. In the present study, we compared the responses of neurons in the rostral ventrolateral medulla (RVLM), a principal region of the brain stem involved in the regulation of blood pressure, to whole body rotations of conscious and decerebrate cats. In both preparations, RVLM neurons exhibited similar levels of spontaneous activity (median of ∼17 spikes/s). The firing of about half of the RVLM neurons recorded in decerebrate cats was modulated by rotations; these cells were activated by vertical tilts in a variety of directions, with response characteristics suggesting that their labyrinthine inputs originated in otolith organs. The activity of over one-third of RVLM neurons in decerebrate animals was altered by stimulation of baroreceptors; RVLM units with and without baroreceptor signals had similar responses to rotations. In contrast, only 6% of RVLM neurons studied in conscious cats exhibited cardiac-related activity, and the firing of just 1% of the cells was modulated by rotations. These data suggest that the brain stem circuitry mediating vestibulosympathetic reflexes is highly sensitive to changes in body position in space but that the responses to vestibular stimuli of neurons in the pathway are suppressed by higher brain centers in conscious animals. The findings also raise the possibility that autonomic responses to a variety of inputs, including those from the inner ear, could be gated according to behavioral context and attenuated when they are not necessary.

Mapping of neural pathways that influence diaphragm activity and project to the lumbar spinal cord in cats.

During breathing, the diaphragm and abdominal muscles contract out of phase. However, during other behaviors (including vomiting, postural adjustments, and locomotion) simultaneous contractions are required of the diaphragm and other muscle groups including abdominal muscles. Recent studies in cats using transneuronal tracing techniques showed that in addition to neurons in the respiratory groups, cells in the inferior and lateral vestibular nuclei (VN) and medial pontomedullary reticular formation (MRF) influence diaphragm activity. The goal of the present study was to determine whether neurons in these regions have collateralized projections to both diaphragm motoneurons and the lumbar spinal cord. For this purpose, the transneuronal tracer rabies virus was injected into the diaphragm, and the monosynaptic retrograde tracer Fluoro-Gold (FG) was injected into the Th13-L1 spinal segments. A large fraction of MRF and VN neurons (median of 72 and 91%, respectively) that were infected by rabies virus were dual-labeled by FG. These data show that many MRF and VN neurons that influence diaphragm activity also have a projection to the lumbar spinal cord and thus likely are involved in coordinating behaviors that require synchronized contractions of the diaphragm and other muscle groups.

Effects of postural changes and removal of vestibular inputs on blood flow to and from the hindlimb of conscious felines.

Considerable data show that the vestibular system contributes to blood pressure regulation. Prior studies reported that lesions that eliminate inputs from the inner ears attenuate the vasoconstriction that ordinarily occurs in the hindlimbs of conscious cats during head-up rotations. These data led to the hypothesis that labyrinthine-deficient animals would experience considerable lower body blood pooling during head-up postural alterations. The present study tested this hypothesis by comparing blood flow though the femoral artery and vein of conscious cats during 20-60 degrees head-up tilts from the prone position before and after removal of vestibular inputs. In vestibular-intact animals, venous return from the hindlimb dropped considerably at the onset of head-up tilts and, at 5 s after the initiation of 60 degrees rotations, was 66% lower than when the animals were prone. However, after the animals were maintained in the head-up position for another 15 s, venous return was just 33% lower than before the tilt commenced. At the same time point, arterial inflow to the limb had decreased 32% from baseline, such that the decrease in blood flow out of the limb due to the force of gravity was precisely matched by a reduction in blood reaching the limb. After vestibular lesions, the decline in femoral artery blood flow that ordinarily occurs during head-up tilts was attenuated, such that more blood flowed into the leg. Contrary to expectations, in most animals, venous return was facilitated, such that no more blood accumulated in the hindlimb than when labyrinthine signals were present. These data show that peripheral blood pooling is unlikely to account for the fluctuations in blood pressure that can occur during postural changes of animals lacking inputs from the inner ear. Instead, alterations in total peripheral resistance following vestibular dysfunction could affect the regulation of blood pressure.

Responses of thoracic spinal interneurons to vestibular stimulation.

Vestibular influences on outflow from the spinal cord are largely mediated via spinal interneurons, although few studies have recorded interneuronal activity during labyrinthine stimulation. The present study determined the responses of upper thoracic interneurons of decerebrate cats to electrical stimulation of the vestibular nerve or natural stimulation of otolith organs and the anterior and posterior semicircular canals using rotations in vertical planes. A majority of thoracic interneurons (74/102) responded to vestibular nerve stimulation at median latencies of 6.5 ms (minimum of approximately 3 ms), suggesting that labyrinthine inputs were relayed to these neurons through trisynaptic and longer pathways. Thoracic interneuronal responses to vertical rotations were similar to those of graviceptors such as otolith organs, and a wide array of tilt directions preferentially activated different cells. Such responses were distinct from those of cells in the cervical and lumbar enlargements, which are mainly elicited by ear-down tilts and are synchronous with stimulus position when low rotational frequencies are delivered, but tend to be in phase with stimulus velocity when high frequencies are employed. The dynamic properties of thoracic interneuronal responses to tilts were instead similar to those of thoracic motoneurons and sympathetic preganglionic neurons. However, the preferred tilt directions of the interneurons were more heterogeneous than thoracic spinal outputs, showing that the outputs do not simply reflect an addition of local interneuronal activity.

Physical presence during gamma stereotactic radiosurgery.

During an invited visit to the University of Pittsburgh Medical Center (UPMC) gamma knife facility, officials from the Nuclear Regulatory Commission (NRC) observed what they considered as an apparent violation of the physical presence requirements specified in 10 CFR 35.615(f)(3). This event initiated an inspection and two different but related investigations by the NRC Office of Investigations (OI). Based on the NRC inspection and investigations, the NRC identified three apparent violations that were under consideration for escalated enforcement. The University of Pittsburgh (licensee) requested an Alternative Dispute Resolution (ADR) session with the NRC to resolve issues related to whether a violation occurred, the appropriate enforcement action, and the appropriate corrective action. As a result of the ADR mediation session, the licensee and NRC agreed to final disposition of this matter by way of a single violation of the regulatory requirement in 10 CFR 35.24(b), whereby the licensee's Radiation Safety Officer failed to ensure that the physical presence requirements of 10 CFR 35.615(f)(3) were consistently met and failed to ensure that written directives were consistently signed by the Authorized User in accordance with 10 CFR 35.32. In addition to corrective actions the licensee had already taken to prevent recurrence, it also agreed to inform other licensees in the industry of this event, so that they may learn from this incident and take appropriate actions to assure that these types of violations do not occur at their institutions.

Pseudomembranous colitis in four patients with cystic fibrosis following lung transplantation.

In the present study, 4 patients with cystic fibrosis undergoing lung transplantation (from a total of 137) who developed fulminant pseudomembranous colitis are described. Initial presentation was variable and the mortality rate was 50% despite urgent colectomy. In one case the presenting abdominal distension was thought to be due to meconium ileus equivalent. It is concluded that Clostridium difficile colitis may be a difficult diagnosis in patients with cystic fibrosis and follows a fulminant course after lung transplantation.

Occupational asthma caused by Arabidopsis thaliana: a case of laboratory plant allergy.

A 36-yr-old male never-smoker with an 8-yr history of hay fever but no past history of asthma undertook a 3-yr research project involving the plant Arabidopsis thaliana. The subject was based in a small laboratory with an attached growing room. After 30 months of research, he began to develop breathlessness within 5-10 min of entering the laboratory. Initial investigations confirmed asthma with airflow obstruction (forced expiratory volume in one second (FEV(1))/forced vital capacity was 3.01/4.75 L; predicted values were 3.67/4.43 L) and increased airway responsiveness. Serial peak expiratory flow measurements showed a work-related pattern. A supervised workplace challenge test led to a fall in FEV(1) from the baseline value of 3.10 L to 1.95 L within 20 min of entering the growing room. Skin-prick solutions were prepared from Arabidopsis leaves and flower heads; positive 4-mm responses were obtained to the flower heads (i.e. to the pollen). Arabidopsis is a member of the Brassicaceae family. It is used extensively in plant biology research as its genome is small, has been fully sequenced and is easily manipulated. The present article represents the first reported case of occupational asthma due to Arabidopsis thaliana.

Responses of rostral fastigial nucleus neurons of conscious cats to rotations in vertical planes.

The rostral fastigial nucleus (RFN) of the cerebellum is thought to play an important role in postural control, and recent studies in conscious nonhuman primates suggest that this region also participates in the sensory processing required to compute body motion in space. The goal of the present study was to examine the dynamic and spatial responses to sinusoidal rotations in vertical planes of RFN neurons in conscious cats, and determine if they are similar to responses reported for monkeys. Approximately half of the RFN neurons examined were classified as graviceptive, since their firing was synchronized with stimulus position and the gain of their responses was relatively unaffected by the frequency of the tilts. The large majority (80%) of graviceptive RFN neurons were activated by pitch rotations. Most of the remaining RFN units exhibited responses to vertical oscillations that encoded stimulus velocity, and approximately 50% of these velocity units had a response vector orientation aligned near the plane of a single vertical semicircular canal. Unlike in primates, few feline RFN neurons had responses to vertical rotations that suggested integration of graviceptive (otolith) and velocity (vertical semicircular canal) signals. These data indicate that the physiological role of the RFN may differ between primates and lower mammals. The RFN in rats and cats in known to be involved in adjusting blood pressure and breathing during postural alterations in the transverse (pitch) plane. The relatively simple responses of many RFN neurons in cats are appropriate for triggering such compensatory autonomic responses.

Responses of caudal vestibular nucleus neurons of conscious cats to rotations in vertical planes, before and after a bilateral vestibular neurectomy.

Although many previous experiments have considered the responses of vestibular nucleus neurons to rotations and translations of the head, little data are available regarding cells in the caudalmost portions of the vestibular nuclei (CVN), which mediate vestibulo-autonomic responses among other functions. This study examined the responses of CVN neurons of conscious cats to rotations in vertical planes, both before and after a bilateral vestibular neurectomy. None of the units included in the data sample had eye movement-related activity. In labyrinth-intact animals, some CVN neurons (22%) exhibited graviceptive responses consistent with inputs from otolith organs, but most (55%) had dynamic responses with phases synchronized with stimulus velocity. Furthermore, the large majority of CVN neurons had response vector orientations that were aligned either near the roll or vertical canal planes, and only 18% of cells were preferentially activated by pitch rotations. Sustained head-up rotations of the body provide challenges to the cardiovascular system and breathing, and thus the response dynamics of the large majority of CVN neurons were dissimilar to those of posturally-related autonomic reflexes. These data suggest that vestibular influences on autonomic control mediated by the CVN are more complex than previously envisioned, and likely involve considerable processing and integration of signals by brainstem regions involved in cardiovascular and respiratory regulation. Following a bilateral vestibular neurectomy, CVN neurons regained spontaneous activity within 24 h, and a very few neurons (<10%) responded to vertical tilts <15 degrees in amplitude. These findings indicate that nonlabyrinthine inputs are likely important in sustaining the activity of CVN neurons; thus, these inputs may play a role in functional recovery following peripheral vestibular lesions.

Commissioning and performance of the variable line spacing plane grating monochromator beamline at the Canadian Light Source.

The variable line spacing plane grating monochromator beamline at the Canadian Light Source (CLS) employs three grazing incidence variable line spacing gratings to cover a photon energy range of 5-250 eV. It uses a 185 mm period length planar permanent magnet insertion device as the photon source, sharing a straight section with another soft x-ray beamline at the CLS. The commissioning and performance of the beamline is reported. The high resolution photoabsorption spectra of Ar and PF(5) gases are reported. A resolving power of over 40,000 for photons in the low energy region and >10,000 for a wider energy range (8-200 eV) can be achieved. A photon flux of up to 2 x 10(12) photons/s per 100 mA with slit settings of 50 microm has been measured.