Standardizing Randomized Controlled Trials in Celiac Disease: An International Multidisciplinary Appropriateness Study.

BACKGROUND & AIMS: There is a need to develop safe and effective pharmacologic options for the treatment of celiac disease (CeD); however, consensus on the appropriate design and configuration of randomized controlled trials (RCTs) in this population is lacking. METHODS: A 2-round modified Research and Development/University of California Los Angeles Appropriateness Method study was conducted. Eighteen gastroenterologists (adult and pediatric) and gastrointestinal pathologists voted on statements pertaining to the configuration of CeD RCTs, inclusion and exclusion criteria, gluten challenge, and trial outcomes. Two RCT designs were considered, representing the following distinct clinical scenarios for which pharmacotherapy may be used: trials incorporating a gluten challenge to simulate exposure; and trials evaluating reversal of histologic changes, despite attempted adherence to a gluten-free diet. Each statement was rated as appropriate, uncertain, or inappropriate, using a 9-point Likert scale. RESULTS: For trials evaluating prevention of relapse after gluten challenge, participants adherent to a gluten-free diet for 12 months or more with normal or near-normal-sized villi should be enrolled. Gluten challenge should be FODMAPS (fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) free, and efficacy evaluated using histology with a secondary patient-reported outcome measure. For trials evaluating reversal of villus atrophy, the panel voted it appropriate to enroll participants with a baseline villus height to crypt depth ratio ≤2 and measure efficacy using a primary histologic end point. Guidance for measuring histologic, endoscopic, and patient-reported outcomes in adult and pediatric patients with CeD are provided, along with recommendations regarding the merits and limitations of different end points. CONCLUSIONS: We developed standardized recommendations for clinical trial design, eligibility criteria, outcome measures, gluten challenge, and disease evaluations for RCTs in patients with CeD.

An Expert Consensus to Standardize Assessment of Bowel Cleansing for Clinical Trials of Bowel Preparations for Crohn's Disease.

BACKGROUND: Despite regular need for colonoscopy in patients with Crohn's disease (CD), the efficacy and tolerability of bowel preparation (BP) agents is rarely assessed in this population. Assessing BP quality with existing scales may be challenging in CD due to presence of inflammation, bowel resection, and strictures. AIMS: To provide recommendations for assessing BP quality in clinical trials for CD using a modified Research and Development/University of California, Los Angeles appropriateness process. METHODS: Based on systematic reviews and a literature search, 110 statements relating to BP quality assessment in CD were developed. A panel of 15 gastroenterologists rated the statements as appropriate, uncertain, or inappropriate using a 9-point Likert scale. RESULTS: Panelists considered it appropriate that central readers, either alone or with local assessment, score BP quality in clinical trials. Central readers should be trained on scoring BP quality and local endoscopists on performing high-quality video recording. Both endoscope insertion and withdrawal phases should be reviewed to score BP quality in each colonic segment and segments should align with endoscopic disease activity indices. The Harefield Cleansing Scale and the Boston Bowel Preparation Scale were considered appropriate. The final score should be calculated as the average of all visualized segments. Both total and worst segment scores should also be assessed. CONCLUSIONS: We developed a framework for assessing BP quality in patients with CD based on expert feedback. This framework could support the development or refinement of BP quality scales and the integration of BP quality assessment in future CD studies.

A systematic review of measurement of endoscopic disease activity and mucosal healing in Crohn's disease: recommendations for clinical trial design.

BACKGROUND: Crohn's disease (CD) is a chronic idiopathic inflammatory disorder of the gastrointestinal tract. Recently, mucosal healing has been proposed as a goal of therapy because clinical symptoms are subjective. Evaluative indices that measure endoscopic disease activity are required to define mucosal healing for clinical trials. The primary objective of this systematic review was to assess the existing evaluative indices that measure disease activity in CD and evaluate their role as outcome measures in clinical trials. METHODS: A systematic literature review was performed using MEDLINE (Ovid), EMBASE (Ovid), PubMed, the Cochrane Library (CENTRAL), and DDW abstracts to identify randomized controlled trials and controlled clinical trials that used a relevant evaluative index from inception to February 2013. The data obtained from these trials were reviewed and summarized. RESULTS: The initial literature searches identified 2300 citations. After duplicates were removed, 1454 studies remained. After application of the apriori inclusion and exclusion criteria, 109 articles were included and 3 were identified with handsearches. In total, 9 evaluative indices for CD were identified and reviewed. The Crohn's Disease Endoscopic Index of Severity (CDEIS) and the Simple Endoscopic Score in Crohn's Disease (SES-CD) are indices with the most extensively described operating properties. CONCLUSIONS: Both the endoscopic evaluative instrument selected and the definition chosen for mucosal healing affect the validity of assessing endoscopic disease activity during a clinical trial for CD. Currently, the CDEIS and SES-CD have the most data regarding operating properties; however, further validation is required.

Netrin 1 and Dcc regulate oligodendrocyte process branching and membrane extension via Fyn and RhoA.

The molecular mechanisms underlying the elaboration of branched processes during the later stages of oligodendrocyte maturation are not well understood. Here we describe a novel role for the chemotropic guidance cue netrin 1 and its receptor deleted in colorectal carcinoma (Dcc) in the remodeling of oligodendrocyte processes. Postmigratory, premyelinating oligodendrocytes express Dcc but not netrin 1, whereas mature myelinating oligodendrocytes express both. We demonstrate that netrin 1 promotes process extension by premyelinating oligodendrocytes in vitro and in vivo. Addition of netrin 1 to mature oligodendrocytes in vitro evoked a Dcc-dependent increase in process branching. Furthermore, expression of netrin 1 and Dcc by mature oligodendrocytes was required for the elaboration of myelin-like membrane sheets. Maturation of oligodendrocyte processes requires intracellular signaling mechanisms involving Fyn, focal adhesion kinase (FAK), neuronal Wiscott-Aldrich syndrome protein (N-WASP) and RhoA; however, the extracellular cues upstream of these proteins in oligodendrocytes are poorly defined. We identify a requirement for Src family kinase activity downstream of netrin-1-dependent process extension and branching. Using oligodendrocytes derived from Fyn knockout mice, we demonstrate that Fyn is essential for netrin-1-induced increases in process branching. Netrin 1 binding to Dcc on mature oligodendrocytes recruits Fyn to a complex with the Dcc intracellular domain that includes FAK and N-WASP, resulting in the inhibition of RhoA and inducing process remodeling. These findings support a novel role for netrin 1 in promoting oligodendrocyte process branching and myelin-like membrane sheet formation. These essential steps in oligodendroglial maturation facilitate the detection of target axons, a key step towards myelination.

Maintenance of axo-oligodendroglial paranodal junctions requires DCC and netrin-1.

Paranodal axoglial junctions are essential for the segregation of myelinated axons into distinct domains and efficient conduction of action potentials. Here, we show that netrin-1 and deleted in colorectal cancer (DCC) are enriched at the paranode in CNS myelin. We then address whether netrin-1 signaling influences paranodal adhesion between oligodendrocytes and axons. In the absence of netrin-1 or DCC function, oligodendroglial paranodes initially develop and mature normally but later become disorganized. Lack of DCC or netrin-1 resulted in detachment of paranodal loops from the axonal surface and the disappearance of transverse bands. Furthermore, the domain organization of myelin is compromised in the absence of netrin-1 signaling: K+ channels inappropriately invade the paranodal region, and the normally restricted paranodal distribution of Caspr expands longitudinally along the axon. Our findings identify an essential role for netrin-1 and DCC regulating the maintenance of axoglial junctions.

Dorsal column sensory axons lack TrkC and are not rescued by local neurotrophin-3 infusions following spinal cord contusion in adult rats.

By reducing the progressive degeneration and disconnection of axons following spinal cord injury the functional outcome should improve. After direct transection of dorsal column sensory axons, neurotrophin-3 (NT-3) treatment can reduce degeneration and promote regeneration of the proximal stumps. Here, we tested in adult rats whether NT-3 infusion at the site of a moderate T9 spinal cord contusion would rescue sensory connections to the gracile nucleus in the medulla. Sensory projections were anterogradely traced bilaterally with injections of cholera toxin B (CTB) into the sciatic nerve 3 days before analysis. Seven days after the contusion plus intrathecal (subarachnoid) vehicle infusion as a control, the CTB-positive innervation of the gracile nucleus was reduced to approximately 25% of sham-operated rats. Intrathecal infusion of 10 microg/day of NT-3 did not affect this reduced innervation. To ensure good tissue penetration and high concentrations of NT-3 early after the injury, other rats received intraparenchymal infusions of vehicle or NT-3 near the injury site starting 2 days before until 7 days after the injury. This NT-3 treatment also did not affect the reduced innervation. This suggests that local NT-3 treatments cannot protect sensory axons from secondary degeneration after a contusive spinal cord injury. These results are likely because TrkC is not present in axons of the dorsal columns or gracile nucleus, or in other dorsal column cell types, even after the contusion. Together with published results, our data suggest that NT-3 is a peripherally--but not centrally--derived neurotrophic factor for sensory neurons.

Developmental and injury-induced expression of alpha1beta1 and alpha6beta1 integrins in the rat spinal cord.

Loss and damage to blood vessels are thought to contribute to secondary tissue loss after spinal cord injury. Integrins might be therapeutic targets to protect the vasculature and/or promote angiogenesis, as their activation can promote tubule formation and survival of endothelial cells in vitro. Here, we show that immunostaining with an antibody against the alpha1beta1 integrin heterodimer is present only in blood vessels from postnatal day 1 (P1) through adulthood in Sprague-Dawley rats. After a spinal cord contusion at T9 in adults, the area of alpha1beta1 integrin positive blood vessels increases within 11 mm from the injury site at 3 days post-injury and remains prominent within the injured core only at 7 days. Staining for the alpha6beta1 integrin heterodimer increases in blood vessels between P10 and adulthood and is present in preganglionic neurons of the intermediolateral cell column (IML) at all ages. The alpha6beta1 integrin is also expressed by motor neurons postnatally, and oligodendrocyte precursors (OPCs), as previously reported. After the contusion, the area of alpha6beta1-stained blood vessels is increased at 3 days and most prominently, 1 mm from the injury site, followed by a significant reduction at 7 days, when alpha6beta1 integrin staining is most prominent around the injured core. Staining is also present in a subset of microglia and/or macrophages. These results raise the possibility that alpha1beta1 and alpha6beta1 integrins in blood vessels might be targeted to reduce blood vessel loss and promote angiogenesis, which may promote tissue sparing after spinal cord injury.

When a diffusible axon guidance cue stops diffusing: roles for netrins in adhesion and morphogenesis.

Netrins are a small family of secreted proteins that are best known for their role as secreted long-range chemotropic guidance cues. Extracellular gradients of netrin protein, established by diffusion, are thought to direct cell and axon migration during neural development. In addition to this long-range role, recent findings provide increasing support for short-range functions, in which secreted netrin protein remains closely associated with its cellular source. Emerging evidence for short-range actions of netrins suggests that they contribute to tissue morphogenesis by regulating cell-cell and cell-matrix adhesion.

The ADAMs family: coordinators of nervous system development, plasticity and repair.

A disintegrin and metalloprotease (ADAM) transmembrane proteins have metalloprotease, integrin-binding, intracellular signaling and cell adhesion activities. In contrast to other metalloproteases, ADAMs are particularly important for cleavage-dependent activation of proteins such as Notch, amyloid precursor protein (APP) and transforming growth factor alpha (TGFalpha), and can bind integrins. Not surprisingly, ADAMs have been shown or suggested to play important roles in the development of the nervous system, where they regulate proliferation, migration, differentiation and survival of various cells, as well as axonal growth and myelination. On the eleventh anniversary of the naming of this family of proteins, the relatively unknown ADAMs are emerging as potential therapeutic targets for neural repair. For example, over-expression of ADAM10, one of the alpha-secretases for APP, can prevent amyloid formation and hippocampal defects in an Alzheimer mouse model. Another example of this potential neural repair role is the finding that ADAM21 is uniquely associated with neurogenesis and growing axons of the adult brain. This comprehensive review will discuss the growing literature about the roles of ADAMs in the developing and adult nervous system, and their potential roles in neurological disorders. Most excitingly, the expanding understanding of their normal roles suggests that they can be manipulated to promote neural repair in the degenerating and injured adult nervous system.

A disintegrin and metalloprotease 21 (ADAM21) is associated with neurogenesis and axonal growth in developing and adult rodent CNS.

We have reported that alpha6beta1 integrin regulates the directed migration of neuroblasts from the adult rodent subventricular zone (SVZ) through the rostral migratory stream (RMS). ADAM (a disintegrin and metalloprotease) proteins bind integrins. Here, we show that ADAM21, but not ADAM2, -3, -9, -10, -12, -15, or -17, is expressed in adult rats and mice by ependyma and SVZ cells with long basal processes, and in radial glia at early postnatal times. ADAM21-positive processes projected into the RMS, contacted blood vessels, and were present within the RMS intermingled with neuroblasts up to where neuroblasts start their radial migration and differentiation in the olfactory bulb. Tissue inhibitors of metalloproteases (TIMPs) 1, 2, and 3 are present in the ependymal layer but not in the SVZ and RMS. Thus, ADAM21 could regulate neurogenesis and guide neuroblast migration through cleavage-dependent activation of proteins and integrin binding. ADAM21 is also present in growing axonal tracts during postnatal development and in growing primary olfactory axons in adults. In the olfactory nerve layer, ADAM21 often, but not always, colocalizes with OMP, a marker of mature olfactory neurons, but is not colocalized with the immature marker betaIII-tubulin. This suggests that ADAM21 is involved in the final axonal outgrowth phase and/or synapse formation. TIMP3 is present in periglomerular neurons, where it could restrict ADAM21-mediated axonal growth to the glomeruli. ADAM21's unique disintegrin and metalloprotease sequences and its restricted expression suggest that it might be a good target for influencing neurogenesis and neuronal plasticity.

Long distance selective fiber outgrowth of transplanted hNT neurons in white matter tracts of the adult rat brain.

Terminally differentiated neurons derived from a human teratocarcinoma cell line (NT2N or hNT neurons) are promising as a cell source for transplantation, as they have been shown to be safe for transplantation in humans. We have shown previously that hNT neurons can express a catecholaminergic phenotype in a rat Parkinson model. In this study, we investigated the long-term survival and ability of hNT neurons to express tyrosine hydroxylase and reconstruct the dopamine-denervated nigrostriatal pathway. Hemiparkinsonian rats received grafts of 400,000 viable hNT neurons into each of the denervated striatum and substantia nigra. Robust hNT grafts were detected up to 24 weeks posttransplantation, although few cells expressed tyrosine hydroxylase. Many hNT fibers were often associated with ipsilateral and contralateral white matter tracts--corpus callosum, rostral migratory stream, optic tract, and external capsule. Fewer fibers were associated with the superior cerebellar peduncle, medial lemniscus, and nigrostriatal pathway. Axons also projected into the frontal cortex and extended parallel to the surface of the brain in the superficial cortical layers. These pathways were seen in all grafted animals, suggesting that specific guidance cues exist in the adult brain governing hNT fiber outgrowth. Injured adult axons and transplanted embryonic neuronal axons rarely extend for such distances in the adult nervous system. We propose that elucidating the factors promoting and guiding hNT axonal outgrowth could provide important clues to enhancing regeneration and target reinnervation in the adult brain, two factors of critical importance for cell restoration strategies aimed at brain repair.

An adult rat spinal cord contusion model of sensory axon degeneration: the estrus cycle or a preconditioning lesion do not affect outcome.

A therapeutic strategy for acute spinal cord injury would be to reduce the progressive degeneration and disconnection of axons from their targets. Here, we describe a model to evaluate degeneration of the ascending sensory projections to the nuclei in the medulla following graded spinal cord contusions in adult female Sprague-Dawley rats. Cholera toxin B (CTB) labeling from the sciatic nerve of naive rats revealed effective labeling of the terminal fibers in the gracile nucleus at 3 days post-injection and a subpopulation of rapidly transporting fibers after 1 day. Seven days after contusions using the Infinite Horizon impactor the area of CTB-labeled terminal fibers had a negative correlation with increasing impact force. Moderate spinal contusions of around 150 kilodyne (kdyn or 0.15 x 10(-3) newton) caused a reduction to 40% in the fiber area which will enable the identification of protective as well as detrimental drugs and post-injury mechanisms. A preconditioning injury of the sciatic nerve reportedly can enhance growth of sensory axons but did not affect the terminal fiber area in the gracile nucleus. Estrogen and progesterone are protective in various systems and could therefore influence experimental outcomes when using females. However, the phase of the estrus cycle at the time of contusion or during the post-injury time did not affect the outcome of the contusion, indicating that female rats may be used without consideration of the estrus cycle. This model can readily be used to evaluate pharmacological agents for protection of sensory axons and pathophysiological mechanisms of their degeneration.

D3 dopamine receptors do not regulate neurogenesis in the subventricular zone of adult mice.

Testing the effects of drugs that stimulate endogenous neurogenesis in different species is important for the development of neural repair strategies in humans. We have previously shown in adult rats that a 14-day intracerebroventricular infusion of the D3 preferential agonist 7-hydroxydipropyl-amino-tetraline (7-OH-DPAT) increases BrdU labeling of neural precursors in the subventricular zone of the anterior lateral ventricle (SVZ). Here, we show that such a treatment failed to affect neurogenesis in C57Bl/6 and FVB mice, even at a high dose or when infused into the neostriatum. We confirmed that such a treatment was effective in adult rats. Moreover, D3 receptor inhibition or genetic knockout failed to affect the neurogenesis in mice. These results raise the possibilities that neurogenesis is not regulated by D3 receptors in all species and, therefore, that D3 agonists like pramipexole may not be useful to harness endogenous neurogenesis in cell replacement strategies for Parkinson's disease.

Dopaminergic nigrostriatal projections regulate neural precursor proliferation in the adult mouse subventricular zone.

An understanding of the regulators of neurogenesis in the normal and diseased brain is necessary in order to recruit endogenously produced neural precursors for cell replacement in neurodegenerative disorders such as Parkinson's disease. The location of dopaminergic projections from the midbrain to the neostriatum and nucleus accumbens overlaps with the most active region of neurogenesis in the adult brain, the subventricular zone of the anterior lateral ventricle. This suggests that dopamine may contribute to regulation of the subventricular niche of adult neurogenesis. Here, we show in adult mice that destruction of the dopaminergic neurons in the substantia nigra and ventral tegmental area in a 6-hydroxydopamine model of Parkinson's disease reduced the number of proliferating neural precursors in the subventricular zone of the anterior lateral ventricle by approximately 40%. The effect on neural precursor proliferation correlated with the extent of dopaminergic denervation in the neighboring neostriatum. This identifies dopamine as one of the few known endogenous regulators of adult neurogenesis with implications for the potential use of endogenous neural precursors in cell replacement strategies for Parkinson's disease.