Alemtuzumab for refractory primary systemic vasculitis-a randomised controlled dose ranging clinical trial of efficacy and safety (ALEVIATE).

BACKGROUND: Primary systemic vasculitis (PSV) is a heterogeneous group of autoimmune conditions. There is an unmet need for alternative therapies that lead to sustained remission in patients with refractory disease. Alemtuzumab, an anti-CD52 antibody, depletes lymphocytes for prolonged periods and, in retrospective studies, has induced sustained, treatment-free remissions in patients with refractory/relapsing vasculitis but has raised safety concerns of infection and secondary autoimmunity. This phase IIb clinical trial aimed to assess the efficacy and safety of alemtuzumab, at two different doses, in inducing remission in refractory vasculitis patients. METHODS: The ALEVIATE trial was a randomised, prospective, open-label, dose ranging clinical trial. Patients with refractory ANCA-associated vasculitis (AAV) or Behçet's disease (BD) were randomised to receive either 60 mg or 30 mg alemtuzumab. Treatments were administered at baseline and 6 months or earlier where clinically appropriate. A maximum of three treatments were allowed within the 12-month study period. RESULTS: Twenty-three patients received at least one dose of alemtuzumab. Twelve had AAV, and 11 a diagnosis of BD. The median age was 40 years (range 28-44), with a prior disease duration of 61 months (42-103). Sixteen (70%) achieved either complete (6/23, 26%) or partial (10/23, 44%) response at 6 months. Eight (35%) maintained remission to the end of the trial without relapse. Ten severe adverse events were observed in 7 (30%) patients; 4 were related to alemtuzumab. There were no differences in clinical endpoints between the 60 and 30 mg alemtuzumab treatment groups. CONCLUSION: In a selected group of refractory vasculitis patients, alemtuzumab led to remission in two thirds of patients at 6 months. Remission was maintained to 12 months in a third of the patients, and the safety profile was acceptable. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01405807, EudraCT Number: 2009-017087-17. Registered on April 07, 2011.

Identification of Astrotactin2 as a Genetic Modifier That Regulates the Global Orientation of Mammalian Hair Follicles.

Planar cell polarity (PCP) signaling controls the global orientation of surface structures, such as hairs and bristles, in both vertebrates and invertebrates. In Frizzled6(-/-) (Fz6(-/-)) mice, hair follicle orientations on the head and back are nearly random at birth, but reorient during early postnatal development to eventually generate a nearly parallel anterior-to-posterior array. We report the identification of a naturally occurring exon 5 deletion in Astrotactin2 (Astn2) that acts as a recessive genetic modifier of the Fz6(-/-) hair patterning phenotype. A genetically engineered Astn2 exon 5 deletion recapitulates the modifier phenotype. In Fz6(-/-);Astn2(ex5del/del) mice, hair orientation on the back is subtly biased from posterior-to-anterior, leading to a 180-degree orientation reversal in mature mice. These experiments suggest that Astn2, an endosomal membrane protein, modulates PCP signaling.

Functional assembly of accessory optic system circuitry critical for compensatory eye movements.

Accurate motion detection requires neural circuitry that compensates for global visual field motion. Select subtypes of retinal ganglion cells perceive image motion and connect to the accessory optic system (AOS) in the brain, which generates compensatory eye movements that stabilize images during slow visual field motion. Here, we show that the murine transmembrane semaphorin 6A (Sema6A) is expressed in a subset of On direction-selective ganglion cells (On DSGCs) and is required for retinorecipient axonal targeting to the medial terminal nucleus (MTN) of the AOS. Plexin A2 and A4, two Sema6A binding partners, are expressed in MTN cells, attract Sema6A(+) On DSGC axons, and mediate MTN targeting of Sema6A(+) RGC projections. Furthermore, Sema6A/Plexin-A2/A4 signaling is required for the functional output of the AOS. These data reveal molecular mechanisms underlying the assembly of AOS circuits critical for moving image perception.

Class 5 transmembrane semaphorins control selective Mammalian retinal lamination and function.

In the vertebrate retina, neurites from distinct neuronal cell types are constrained within the plexiform layers, allowing for establishment of retinal lamination. However, the mechanisms by which retinal neurites are segregated within the inner or outer plexiform layers are not known. We find that the transmembrane semaphorins Sema5A and Sema5B constrain neurites from multiple retinal neuron subtypes within the inner plexiform layer (IPL). In Sema5A⁻/⁻; Sema5B⁻/⁻ mice, retinal ganglion cells (RGCs) and amacrine and bipolar cells exhibit severe defects leading to neurite mistargeting into the outer portions of the retina. These targeting abnormalities are more prominent in the outer (OFF) layers of the IPL and result in functional defects in select RGC response properties. Sema5A and Sema5B inhibit retinal neurite outgrowth through PlexinA1 and PlexinA3 receptors both in vitro and in vivo. These findings define a set of ligands and receptors required for the establishment of inner retinal lamination and function.

Preclinical assessment of CNS drug action using eye movements in mice.

The drug development process for CNS indications is hampered by a paucity of preclinical tests that accurately predict drug efficacy in humans. Here, we show that a wide variety of CNS-active drugs induce characteristic alterations in visual stimulus-induced and/or spontaneous eye movements in mice. Active compounds included sedatives and antipsychotic, antidepressant, and antiseizure drugs as well as drugs of abuse, such as cocaine, morphine, and phencyclidine. The use of quantitative eye-movement analysis was demonstrated by comparing it with the commonly used rotarod test of motor coordination and by using eye movements to monitor pharmacokinetics, blood-brain barrier penetration, drug-receptor interactions, heavy metal toxicity, pharmacologic treatment in a model of schizophrenia, and degenerative CNS disease. We conclude that eye-movement analysis could complement existing animal tests to improve preclinical drug development.

Norrin, frizzled-4, and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization.

Disorders of vascular structure and function play a central role in a wide variety of CNS diseases. Mutations in the Frizzled-4 (Fz4) receptor, Lrp5 coreceptor, or Norrin ligand cause retinal hypovascularization, but the mechanisms by which Norrin/Fz4/Lrp signaling controls vascular development have not been defined. Using mouse genetic and cell culture models, we show that loss of Fz4 signaling in endothelial cells causes defective vascular growth, which leads to chronic but reversible silencing of retinal neurons. Loss of Fz4 in all endothelial cells disrupts the blood brain barrier in the cerebellum, whereas excessive Fz4 signaling disrupts embryonic angiogenesis. Sox17, a transcription factor that is upregulated by Norrin/Fz4/Lrp signaling, plays a central role in inducing the angiogenic program controlled by Norrin/Fz4/Lrp. These experiments establish a cellular basis for retinal hypovascularization diseases due to insufficient Frizzled signaling, and they suggest a broader role for Frizzled signaling in vascular growth, remodeling, maintenance, and disease.

Distinct roles of transcription factors brn3a and brn3b in controlling the development, morphology, and function of retinal ganglion cells.

Transcriptional regulatory networks that control the morphologic and functional diversity of mammalian neurons are still largely undefined. Here we dissect the roles of the highly homologous POU-domain transcription factors Brn3a and Brn3b in retinal ganglion cell (RGC) development and function using conditional Brn3a and Brn3b alleles that permit the visualization of individual wild-type or mutant cells. We show that Brn3a- and Brn3b-expressing RGCs exhibit overlapping but distinct dendritic stratifications and central projections. Deletion of Brn3a alters dendritic stratification and the ratio of monostratified:bistratified RGCs, with little or no change in central projections. In contrast, deletion of Brn3b leads to RGC transdifferentiation and loss, axon defects in the eye and brain, and defects in central projections that differentially compromise a variety of visually driven behaviors. These findings reveal distinct roles for Brn3a and Brn3b in programming RGC diversity, and they illustrate the broad utility of germline methods for genetically manipulating and visualizing individual identified mammalian neurons.

The optokinetic reflex as a tool for quantitative analyses of nervous system function in mice: application to genetic and drug-induced variation.

The optokinetic reflex (OKR), which serves to stabilize a moving image on the retina, is a behavioral response that has many favorable attributes as a test of CNS function. The OKR requires no training, assesses the function of diverse CNS circuits, can be induced repeatedly with minimal fatigue or adaptation, and produces an electronic record that is readily and objectively quantifiable. We describe a new type of OKR test apparatus in which computer-controlled visual stimuli and streamlined data analysis facilitate a relatively high throughput behavioral assay. We used this apparatus, in conjunction with infrared imaging, to quantify basic OKR stimulus-response characteristics for C57BL/6J and 129/SvEv mouse strains and for genetically engineered lines lacking one or more photoreceptor systems or with an alteration in cone spectral sensitivity. A second generation (F2) cross shows that the characteristic difference in OKR frequency between C57BL/6J and 129/SvEv is inherited as a polygenic trait. Finally, we demonstrate the sensitivity and high temporal resolution of the OKR for quantitative analysis of CNS drug action. These experiments show that the mouse OKR is well suited for neurologic testing in the context of drug discovery and large-scale phenotyping programs.

Melanopsin cells are the principal conduits for rod-cone input to non-image-forming vision.

Rod and cone photoreceptors detect light and relay this information through a multisynaptic pathway to the brain by means of retinal ganglion cells (RGCs). These retinal outputs support not only pattern vision but also non-image-forming (NIF) functions, which include circadian photoentrainment and pupillary light reflex (PLR). In mammals, NIF functions are mediated by rods, cones and the melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs). Rod-cone photoreceptors and ipRGCs are complementary in signalling light intensity for NIF functions. The ipRGCs, in addition to being directly photosensitive, also receive synaptic input from rod-cone networks. To determine how the ipRGCs relay rod-cone light information for both image-forming and non-image-forming functions, we genetically ablated ipRGCs in mice. Here we show that animals lacking ipRGCs retain pattern vision but have deficits in both PLR and circadian photoentrainment that are more extensive than those observed in melanopsin knockouts. The defects in PLR and photoentrainment resemble those observed in animals that lack phototransduction in all three photoreceptor classes. These results indicate that light signals for irradiance detection are dissociated from pattern vision at the retinal ganglion cell level, and animals that cannot detect light for NIF functions are still capable of image formation.

Emergence of novel color vision in mice engineered to express a human cone photopigment.

Changes in the genes encoding sensory receptor proteins are an essential step in the evolution of new sensory capacities. In primates, trichromatic color vision evolved after changes in X chromosome-linked photopigment genes. To model this process, we studied knock-in mice that expressed a human long-wavelength-sensitive (L) cone photopigment in the form of an X-linked polymorphism. Behavioral tests demonstrated that heterozygous females, whose retinas contained both native mouse pigments and human L pigment, showed enhanced long-wavelength sensitivity and acquired a new capacity for chromatic discrimination. An inherent plasticity in the mammalian visual system thus permits the emergence of a new dimension of sensory experience based solely on gene-driven changes in receptor organization.

Structure-function analysis of the bestrophin family of anion channels.

The bestrophins are a newly described family of anion channels unrelated in primary sequence to any previously characterized channel proteins. The human genome codes for four bestrophins, each of which confers a distinctive plasma membrane conductance on transfected 293 cells. Extracellular treatment with methanethiosulfonate ethyltrimethylammonium (MTSET) of a series of substitution mutants that eliminate one or more cysteines from human bestrophin1 demonstrates that cysteine 69 is the single endogenous cysteine responsible for MTSET inhibition of whole-cell current. Cysteines introduced between positions 78-99 and 223-226 are also accessible to external MTSET, with MTSET modification at positions 79, 80, 83, and 90 producing a 2-6-fold increase in whole-cell current. The latter set of four cysteine-substitution mutants define a region that appears to mediate allosteric control of channel activity. Mapping of transmembrane topography by insertion of N-linked glycosylation sites and tobacco etch virus protease cleavage sites provides evidence for cytosolic N and C termini and an unexpected transmembrane topography with at least three extracellular loops that include positions 60-63, 212-227, and 261-267. These experiments provide the first structural analysis of the bestrophin channel family.

Separate forms of pathology in the cochlea of congenitally deaf white cats.

Congenital deafness due to cochlear pathology can have an immediate or progressive onset. The timing of this onset could have a significant impact on the development of structures in the central auditory system, depending on the animal's hearing status during its critical period. In order to determine whether cats in our deaf white cat colony suffered from progressive hearing loss, they were tested repeatedly in 30-day intervals using standard auditory evoked brainstem response (ABR) methodology. ABR thresholds did not change over time, indicating that deafness in our colony was not progressive. Moreover, different forms of cochlear pathology were associated with deafness. One form (67% of the deaf ears) had a collapsed Reissner's membrane that obliterated the scala media, resembling what is called the Scheibe deformity in humans. A second form (18%) exhibited excessive epithelial growth within the bony labyrinth. A third form (15%) combined excessive epithelial growth in the apex and a collapsed Reissner's membrane in the base. Cochleae having an abnormally thin tectorial membrane and an outward bulging Reissner's membrane were associated with elevated thresholds (poor hearing).

Effects of congenital deafness in the cochlear nuclei of Shaker-2 mice: an ultrastructural analysis of synapse morphology in the endbulbs of Held.

It is well established that manipulation of the sensory environment can significantly alter central auditory system development. For example, congenitally deaf white cats exhibit synaptic alterations in the cochlear nucleus distinct from age-matched, normal hearing controls. The large, axosomatic endings of auditory nerve fibers, called endbulbs of Held, display reduced size and branching, loss of synaptic vesicles, and a hypertrophy of the associated postsynaptic densities on the target spherical bushy cells. Such alterations, however, could arise from the cat's genetic syndrome rather than from deafness. In order to examine further the role of hearing on synapse development, we have studied endbulbs of Held in the shaker-2 ( sh2 ) mouse. These mice carry a point mutation on chromosome 11, affecting myosin 15 and producing abnormally short stereocilia in hair cells of the inner ear. The homozygous mutant mice are born deaf and develop perpetual circling behavior, although receptor cells and primary neurons remain intact at least for the initial 100 days of postnatal life. Endbulbs of Held in 7-month old, deaf sh2 mice exhibited fewer synaptic vesicles in the presynaptic ending, the loss of intercellular cisternae, and a hypertrophy of associated postsynaptic densities. On average, postsynaptic density area for sh2 endbulbs was 0.23 +/- 0.19 microm(2) compared to 0.07 +/- 0.04 microm(2) ( p < 0.001) for age-matched, hearing littermates. These changes at the endbulb synapse in sh2 mice resemble those of the congenitally deaf white cat and are consistent with the idea that they represent a generalized response to deafness.

The effects of congenital deafness on auditory nerve synapses: Type I and Type II multipolar cells in the anteroventral cochlear nucleus of cats.

Sensory deprivation has been shown to exert detrimental effects on the structure and function of central sensory systems. Congenital deafness represents an extreme form of auditory deprivation, and in the adult white cat, synapses between auditory nerve endings and resident cells of the anteroventral cochlear nucleus exhibited abnormal structure. Endbulbs of Held were reduced in branching and displayed striking hypertrophy of their postsynaptic densities. So-called modified endbulbs showed no change in branching complexity but did exhibit hypertrophy of their postsynaptic densities. These differential pre- and postsynaptic effects prompted the question of how deafness might affect other primary endings and synapses. Thus, we studied type I and type II multipolar cells that receive bouton endings from auditory nerve fibers. Type I multipolar cells project to the contralateral inferior colliculus and have relatively few axosomatic endings; type II multipolar cells project to the contralateral cochlear nucleus and have many axosomatic endings. Compared with normal-hearing cats, bouton endings of congenitally deaf cats were smaller but there was no difference in synaptic vesicle density or size of postsynaptic densities. These data reveal that different classes of primary endings and second-order neurons exhibit different degrees of synaptic anomalies to deafness.

Hearing loss caused by progressive degeneration of cochlear hair cells in mice deficient for the Barhl1 homeobox gene.

The cochlea of the mammalian inner ear contains three rows of outer hair cells and a single row of inner hair cells. These hair cell receptors reside in the organ of Corti and function to transduce mechanical stimuli into electrical signals that mediate hearing. To date, the molecular mechanisms underlying the maintenance of these delicate sensory hair cells are unknown. We report that targeted disruption of Barhl1, a mouse homolog of the Drosophila BarH homeobox genes, results in severe to profound hearing loss, providing a unique model for the study of age-related human deafness disorders. Barhl1 is expressed in all sensory hair cells during inner ear development, 2 days after the onset of hair cell generation. Loss of Barhl1 function in mice results in age-related progressive degeneration of both outer and inner hair cells in the organ of Corti, following two reciprocal longitudinal gradients. Our data together indicate an essential role for Barhl1 in the long-term maintenance of cochlear hair cells, but not in the determination or differentiation of these cells.