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1.  Lis1 Reduction Causes Tangential Migratory Errors in Mouse Spinal Cord 
The Journal of comparative neurology  2012;520(6):1198-1211.
Mutations in human LIS1 cause abnormal neuronal migration and a smooth brain phenotype known as lissencephaly. Lis1+/− (Pafah1b1) mice show defective lamination in the cerebral cortex and hippocampal formation, whereas homozygous mutations result in embryonic lethality. Given that Lis1 is highly expressed in embryonic neurons, we hypothesized that sympathetic and parasympathetic preganglionic neurons (SPNs and PPNs) would exhibit migratory defects in Lis1+/− mice. The initial radial migration of SPNs and PPNs that occurs together with somatic motor neurons appeared unaffected in Lis1+/− mice. The subsequent dorsally directed tangential migration, however, was aberrant in a subset of these neurons. At all embryonic ages analyzed, the distribution of SPNs and PPNs in Lis1+/− mice was elongated dorsoventrally compared with Lis1+/+ mice. Individual cell bodies of ectopic preganglionic neurons were found in the ventral spinal cord with their leading processes oriented along their dorsal migratory trajectory. By birth, Lis1+/− SPNs and PPNs were separated into distinct groups, those that were correctly, and those incorrectly positioned in the intermediate horn. As mispositioned SPNs and PPNs still were detected in P30 Lis1+/− mice, we conclude that these neurons ceased migration prematurely. Additionally, we found that a dorsally located group of somatic motor neurons in the lumbar spinal cord, the retrodorsolateral nucleus, showed delayed migration in Lis1+/− mice. These results suggest that Lis1 is required for the dorsally directed tangential migration of many sympathetic and parasympathetic preganglionic neurons and a subset of somatic motor neurons.
doi:10.1002/cne.22768
PMCID: PMC4079006  PMID: 21935943
lissencephaly; pafah1b1; reeler; sympathetic preganglionic neurons; parasympathetic preganglionic neurons; somatic motor neurons
2.  Outcomes in Systemic Sclerosis-related Lung Disease following Lung Transplantation 
Transplantation  2013;95(7):975-980.
Background
Lung disease (LD) is the leading cause of death in systemic sclerosis (SSc). The diagnosis of SSc-related LD (SSc-LD) is often a contraindication to lung transplantation (LT) due to concerns that extra-pulmonary involvement will yield worse outcomes. We sought to evaluate post-transplant outcomes in persons with SSc-LD with esophageal involvement compared to persons with non-connective tissue disease related interstitial lung disease (nCTD-ILD).
Methods
From 1998-2012, persons undergoing LT for SSc-LD were age and gender matched in a 2:1 fashion to controls undergoing LT for nCTD-ILD. Esophageal function was assessed by pH testing and manometry. We defined esophageal dysfunction as the presence of a DeMeester score >14 or dysmotility more severe than “mild non-specific disorder”. The primary outcome was post-transplant survival. Secondary outcomes included freedom from bronchiolitis obliterans syndrome (fBOS) and rates of acute rejection. Survival and fBOS were estimated with Kaplan-Meier methods. Acute rejection was compared with Students t-test.
Results
Survival was similar in 23 persons with SSc-LD and 46 controls who underwent LT (p=0.47). For the SSc-LD group, 1- and 5-year survival was 83% and 76% compared to 91% and 64% in the nCTD-ILD group. There were no differences in fBOS (p=0.83). Rates of acute rejection were less in SSc-ILD (p=0.05). Esophageal dysfunction was not associated with worse outcomes (p>0.55).
Conclusions
Persons with SSc-LD appear to have similar survival and fBOS as persons transplanted for nCTD-ILD. The risk of acute rejection after transplant may be reduced in persons with SSc-LD. Esophageal involvement does not appear to impact outcomes.
doi:10.1097/TP.0b013e3182845f23
PMCID: PMC3616420  PMID: 23545509
Lung Transplantation; Systemic Sclerosis; Interstitial Lung Disease; survival; esophageal dysmotility; bronchiolitis obliterans syndrome
3.  Association of Large-Airway Lymphocytic Bronchitis with Bronchiolitis Obliterans Syndrome 
Rationale: Lung transplantation offers great promise for otherwise terminal lung diseases, but the development of bronchiolitis obliterans syndrome (BOS) continues to limit survival. Although acute rejection and lymphocytic bronchiolitis have been identified as risk factors for the development of BOS, it is unclear whether large-airway lymphocytic inflammation conveys the same risk.
Objectives: We evaluated lymphocytic bronchitis on endobronchial biopsies as a risk factor for BOS and mortality.
Methods: Endobronchial biopsies were collected and graded during surveillance after lung transplantation. We assessed samples with negative cultures collected in the first 90 days from 298 subjects and compared large-airway lymphocytic bronchitis assessed by a 0–2 “E-score” and with standard A and BR pathology scores for acute rejection and small-airway lymphocytic bronchiolitis, respectively.
Measurements and Main Results: We found surprisingly little association between large- and small-airway lymphocytic inflammation scores from a given bronchoscopy. Endobronchial lymphocytic bronchitis was more prevalent in subjects in BOS stage 0p and BOS stages 1–3 at the time of biopsy. Within 90 days after transplantation, increasing maximum E-score was associated with greater risk of BOS (adjusted hazard ratio, 1.76; 95% confidence interval, 1.11–2.78; P = 0.02) and in this analysis 90-day maximum E-scores were the only score type predictive of BOS (P < 0.01).
Conclusions: These results support a multicenter study to evaluate endoscopic biopsies for the identification of patients at increased risk for BOS. The association of endobronchial lymphocytic inflammation and BOS may have mechanistic implications.
doi:10.1164/rccm.201206-1025OC
PMCID: PMC3603592  PMID: 23239157
bronchiolitis obliterans; graft rejection; bronchoscopy; lung transplantation
4.  Rhinovirus and Other Respiratory Viruses Exert Different Effects on Lung Allograft Function That Are Not Mediated Through Acute Rejection 
Clinical transplantation  2012;27(1):E64-E71.
Background
Community acquired respiratory virus (CARV) infections in lung transplant recipients (LTR) have been associated with adverse outcomes, including acute rejection (AR) and decline in allograft function, in some but not all studies.
Methods
Spirometry and transbronchial biopsy results of LTR diagnosed with CARV infection over a 2-year period were extracted from clinical records. Primary outcomes, studied at 1-2.5 months post-infection, were: (1) incidence of biopsy-proven AR (grade>A0) and (2) allograft function, defined by forced expiratory volume in 1 second (FEV1). A reference group of biopsies (n=526) collected during the study period established the baseline incidence of AR. Rhinovirus (RV) and non-rhinovirus (non-RV) infections were analyzed as subgroups.
Results
87 cases of CARV infection were identified in 59 subjects. Incidences of AR were similar in the post-CARV and reference groups, and did not differ significantly after RV vs non-RV infection. Allograft function declined significantly after non-RV infection, but not after RV infection.
Conclusions
In LTR, CARV infections other than RV are associated with allograft dysfunction at 1-2.5 months after infection. However, CARVs do not appear associated with AR at this time point. The impact of specific CARVs on lung allografts, including the development of chronic allograft rejection, merits further study.
doi:10.1111/ctr.12054
PMCID: PMC3558613  PMID: 23278569
Lung transplantation; graft rejection; adenoviridae infections; influenza; paramyxoviridae infections; picornaviridae infections
5.  Neuronal loss in Pelizaeus–Merzbacher disease differs in various mutations of the proteolipid protein 1 
Acta neuropathologica  2009;118(4):10.1007/s00401-009-0562-8.
Mutations affecting proteolipid protein 1 (PLP1), the major protein in central nervous system myelin, cause the X-linked leukodystrophy Pelizaeus–Merzbacher disease (PMD). We describe the neuropathologic findings in a series of eight male PMD subjects with confirmed PLP1 mutations, including duplications, complete gene deletion, missense and exon-skipping. While PLP1 mutations have effects on oligodendrocytes that result in mutation-specific degrees of dysmyelination, our findings indicate that there are also unexpected effects in the central nervous system resulting in neuronal loss. Although length-dependent axonal degeneration has been described in PLP1 null mutations, there have been no reports on neuronal degeneration in PMD patients. We now demonstrate widespread neuronal loss in PMD. The patterns of neuronal loss appear to be dependent on the mutation type, suggesting selective vulnerability of neuronal populations that depends on the nature of the PLP1 disturbance. Nigral neurons, which were not affected in patients with either null or severe misfolding mutations, and thalamic neurons appear particularly vulnerable in PLP1 duplication and deletion patients, while hippocampal neuronal loss was prominent in a patient with complete PLP1 gene deletion. All subjects showed cerebellar neuronal loss. The patterns of neuronal involvement may explain some clinical findings, such as ataxia, being more prominent in PMD than in other leukodystrophies. While the precise pathogenetic mechanisms are not known, these observations suggest that defective glial functions contribute to neuronal pathology.
doi:10.1007/s00401-009-0562-8
PMCID: PMC3876935  PMID: 19562355
6.  The 7th Edition AJCC Staging System for Cutaneous Squamous Cell Carcinoma Accurately Predicts Risk of Recurrence for Heart and Lung Transplant Recipients 
Background
Cutaneous squamous cell carcinoma (cSCC) is the most common malignancy after solid organ transplantation, with an increased risk of recurrence and metastasis over the general population. The newly updated 7th edition American Joint Committee on Cancer (AJCC) staging system for cSCC is based on consensus expert opinion and requires validation in large cohort studies and in specific patient subpopulations.
Objective
Our objective was to evaluate the risk of cSCC recurrence in a high-risk population of heart and lung transplant recipients, based on the 7th edition AJCC staging system.
Methods
We performed a 10-year retrospective cohort study of all primary cSCC diagnosed in heart and lung transplant recipients at a tertiary care academic dermatology center.
Results
The cumulative incidence of local recurrence was 4% for cSCC in situ and 19% for Stage I cSCC at 5 years, and 54% for Stage II cSCC at 3 years. Stage II tumors had a 10-fold greater risk of recurrence than stage I, and a 43-fold greater risk of recurrence than in situ tumors.
Limitations
This study is limited to a specific patient subgroup at a tertiary care center, and may not be generalizable to all populations.
Conclusions
Heart and lung transplant recipients are at high risk for local recurrence of cSCC. These data substantiate the prognostic accuracy of the newly updated 7th edition AJCC staging system for stage 0, I and II cSCC in this population and demonstrate the aggressive behavior of this cancer in immunosuppressed patients.
doi:10.1016/j.jaad.2012.01.010
PMCID: PMC3345300  PMID: 22285618
Squamous Cell Carcinoma; Lung Transplantation; Heart Transplantation; Staging
7.  High Cumulative Dose Exposure to Voriconazole is Associated with Cutaneous Squamous Cell Carcinoma in Lung Transplant Recipients 
BACKGROUND
Lung transplant recipients (LTR) have an increased risk of cutaneous squamous cell carcinoma (SCC) due to immunosuppressive therapy. Voriconazole, which is associated with phototoxic side effects in some patients, may be an additional risk factor for SCC in this population.
METHODS
To test whether voriconazole is a risk factor for developing SCC in LTR, we evaluated cumulative exposure to voriconazole in 327 adults who underwent lung transplantation at a single center between 1991 and 2010. Voriconazole exposure was assessed as a time-varying covariate. We analyzed risk of developing SCC over time using survival analysis methods.
RESULTS
Exposure to voriconazole was associated with a 2.6-fold increased risk for SCC. This phenomenon was dose-dependent; the risk for SCC increased by 5.6% with each 60-day exposure at a standard dose of 200mg twice daily. At five years posttransplant, voriconazole conferred an absolute risk increase for SCC of 28%.
CONCLUSIONS
These results suggest that caution should be taken when using voriconazole in LTR, as this drug increases the already high risk for SCC in this population.
doi:10.1016/j.healun.2012.02.033
PMCID: PMC3371090  PMID: 22484291
Voriconazole; Squamous Cell Carcinoma; Lung Transplantation; Skin Cancer; Risk Factors
8.  Lung mast cell density defines a subpopulation of patients with idiopathic pulmonary fibrosis 
Histopathology  2012;61(1):98-106.
Aims
The relationship of mast cells to the pathogenesis of lung fibrosis remains undefined despite recognition of their presence in the lungs of patients with pulmonary fibrosis. This study was performed to characterize the relationship of mast cells to fibrotic lung diseases.
Methods and results
Lung tissues from patients with idiopathic pulmonary fibrosis (IPF), chronic hypersensitivity pneumonitis (HP), systemic sclerosis (SSc)-related interstitial lung disease (ILD) and normal individuals were subjected to chymase immunostaining and the mast cell density quantified. Eosinophils were quantified by immunostaining for eosinophil peroxidase. Changes in lung function were correlated with mast cell density. Lung tissue obtained from IPF patients had a higher density of chymase-immunoreactive mast cells than that from patients with HP, SSc-related ILD or normal lungs. IPF lung tissue had a higher density of eosinophils than normal lung. There was no correlation between mast cell density and eosinophil density in IPF lung. IPF patients with high mast cell density had a slower rate of decline in forced vital capacity (FVC) than IPF patients with low mast cell density.
Conclusions
Mast cell density in IPF lungs is higher than in other fibrotic lung diseases and normal lungs. Increased mast cell density in IPF may predict slower disease progression.
doi:10.1111/j.1365-2559.2012.04197.x
PMCID: PMC3371307  PMID: 22394225
chloroacetate esterase; eosinophil; hypersensitivity pneumonitis; idiopathic interstitial pneumonia; systemic sclerosis
9.  Delayed Myelination in an Intrauterine Growth Retardation Model Is Mediated by Oxidative Stress Upregulating Bone Morphogenetic Protein 4 
Intrauterine growth retardation (IUGR) is associated with neurological deficits including cerebral palsy and cognitive and behavioral disabilities. The pathogenesis involves oxidative stress that leads to periventricular white matter injury with a paucity of mature oligodendrocytes and hypomyelination. The molecular mechanisms underlying this damage remain poorly understood. We employed a rat model of IUGR created by bilateral ligation of the uterine artery at embryonic day 19 that results in fetal growth retardation and oxidative stress in the developing brain. The IUGR rat pups showed significant delays in oligodendrocyte differentiation and myelination that resolved by 8 weeks. Bone morphogenetic protein 4 (BMP4), which inhibits oligodendrocyte maturation, was elevated in IUGR brains at postnatal time points and returned to near normal by adulthood. Despite the apparent recovery, behavioral deficiencies were found in 8-week-old female animals, suggesting that the early transient myelination defects have permanent effects. In support of these in vivo data, oligodendrocyte precursor cells cultured from postnatal IUGR rats retained increased BMP4 expression and impaired differentiation that was reversed with the BMP inhibitor noggin. Oxidants in oligodendrocyte cultures increased BMP expression, which decreased differentiation; however, abrogating BMP signaling with noggin in vitro and in BMP-deficient mice prevented these effects. Together, these findings suggest that IUGR results in delayed myelination through the generation of oxidative stress that leads to BMP4 upregulation.
doi:10.1097/NEN.0b013e31825cfa81
PMCID: PMC3390978  PMID: 22710965
Bone morphogenetic protein (BMP); Intrauterine growth retardation; Myelin; Oligodendrocytes; Oxidative stress; Periventricular white matter injury
10.  Microcephaly gene links Trithorax and REST/NRSF to control neural stem cell proliferation and differentiation 
Cell  2012;151(5):1097-1112.
SUMMARY
Microcephaly is a neurodevelopmental disorder causing significantly reduced cerebral cortex size. Many known microcephaly gene products localize to centrosomes, regulating cell fate and proliferation. Here, we identify and characterize a nuclear zinc finger protein, ZNF335/NIF-1, as a causative gene for severe microcephaly, small somatic size, and neonatal death. Znf335-null mice are embryonically lethal and conditional knockout leads to severely reduced cortical size. RNA-interference and postmortem human studies show that Znf335 is essential for neural progenitor self-renewal, neurogenesis, and neuronal differentiation. ZNF335 is a component of a vertebrate-specific, trithorax H3K4-methylation complex, directly regulating REST/NRSF, a master regulator of neural gene expression and cell fate, as well as other essential neural-specific genes. Our results reveal ZNF335 as an essential link between H3K4 complexes and REST/NRSF, and provide the first direct genetic evidence that this pathway regulates human neurogenesis and neuronal differentiation.
doi:10.1016/j.cell.2012.10.043
PMCID: PMC3567437  PMID: 23178126
11.  Mitochondrial tRNA-serine (AGY) m.C12264T mutation causes severe multisystem disease with cataracts 
Discovery medicine  2012;13(69):143-150.
Progressive multisystem disease should invoke consideration of potential mitochondrial etiologies. Mitochondrial disease can affect any organ system at any time, particularly involving neurologic, cardiac, muscular, gastroenterologic, and/or ophthalmologic manifestations. We report here a 19-year-old Caucasian man who was followed since birth in multiple pediatric subspecialty clinics for myelomeningocele complications. However, he progressively developed a host of additional problems that were not readily attributable to his neural tube defect but involved developmental, ophthalmologic, cardiac, muscular, endocrine, and intermediary metabolic manifestations. Clinical diagnostic testing limited to analysis for common point mutations and deletions in his blood mitochondrial DNA (mtDNA) was not revealing. Skeletal muscle biopsy revealed abnormal mitochondrial morphology and immunostaining, mitochondrial proliferation, and mildly reduced respiratory chain complex I–III activity. Whole mitochondrial genome sequencing analysis in muscle identified an apparently homoplasmic, novel, 12264C>T transition in the tRNA serine (AGY) gene. The pathogenicity of this mutation was supported by identification of it being present at low heteroplasmy load in his blood (34 percent) as well as in blood from his maternal grandmother (1 percent). Interestingly, the proband developed severe nuclear cataracts that proved to be homoplasmic for the pathogenic mtDNA 12264C>T mutation. This case highlights the value of pursuing whole mitochondrial genome sequencing in symptomatic tissues in the diagnostic evaluation of suspected mitochondrial disease. Furthermore, it is the first report to directly implicate an mtDNA mutation in the pathogenesis of ocular cataracts and clearly illustrates the important contribution of normal metabolic activity to the function of the ocular lens.
PMCID: PMC3618896  PMID: 22369973
12.  Differential effects of a polyalanine tract expansion in Arx on neural development and gene expression 
Human Molecular Genetics  2011;21(5):1090-1098.
Polyalanine (poly-A) tracts exist in 494 annotated proteins; to date, expansions in these tracts have been associated with nine human diseases. The pathogenetic mechanism by which a poly-A tract results in these various human disorders remains uncertain. To understand the role of this mutation type, we investigated the change in functional properties of the transcription factor Arx when it has an expanded poly-A tract (ArxE), a mutation associated with infantile spasms and intellectual disabilities in humans. We found that although ArxE functions normally in the dorsal brain, its function in subpallial-derived populations of neurons is compromised. These contrasting functions are associated with the misregulation of Arx targets through the loss of the ability of ArxE to interact with the Arx cofactor Tle1. Our data demonstrate a novel mechanism for poly-A expansion diseases: the misregulation of a subset of target genes normally regulated by a transcription factor.
doi:10.1093/hmg/ddr538
PMCID: PMC3277309  PMID: 22108177
13.  Distinct DNA binding and transcriptional repression characteristics related to different ARX mutations 
Neurogenetics  2012;13(1):23-29.
Mutations in the Aristaless-related homeobox gene (ARX) are associated with a wide variety of neurologic disorders including lissencephaly, hydrocephaly, West syndrome, Partington syndrome, and X-linked intellectual disability with or without epilepsy. A genotype-phenotype correlation exists for ARX mutations, however the molecular basis for this association has not been investigated. To begin understanding the molecular basis for ARX mutations, we tested the DNA binding sequence preference and transcriptional repression activity for Arx, deletion mutants and mutants associated with various neurologic disorders. We found DNA binding preferences of Arx are influenced by the amino acid sequences adjacent to the homeodomain. Mutations in the homeodomain show a loss DNA binding activity, while the T333N and P353R homeodomain mutants still possess DNA binding activities, although less than wild type. Transcription repression activity, the primary function of ARX, is reduced in all mutants except the L343Q, which has no DNA binding activity and does not functionally repress Arx targets. These data indicate that mutations in the homeodomain result in not only a loss of DNA binding activity but also loss of transcriptional repression activity. Our results provide novel insights into the pathogenesis of ARX related disorders and possible directions to pursue potential therapeutic interventions.
doi:10.1007/s10048-011-0304-7
PMCID: PMC3279587  PMID: 22252899
ARX; lissencephaly; X-linked intellectual disability and Homeodomain
14.  Polymicrogyria Includes Fusion of the Molecular Layer and Decreased Neuronal Populations, But Normal Cortical Laminar Organization 
Malformations of cortical development are frequently identified in surgical resections for intractable epilepsy. Among the more frequently identified are cortical dysplasia, pachygyria and polymicrogyria. The pathogenesis of these common developmental anomalies remains uncertain. Polymicrogyria is particularly vexing because there are multiple described forms (2, 4 and 6 layer) that have been attributed to multiple etiologies (e.g. ischemic, genetic, infectious, and toxic). We reviewed the pathology in 19 cases and performed cortical laminar analysis in 10 of these cases. Our data indicate that a defining feature of polymicrogyria is fusion of the molecular layer and that most often there is a well-defined grey matter-white matter junction. Unexpectedly, the cortical lamina were normally positioned but there were reduced neuronal populations within these lamina, particularly in the subgranular layers. Based on these data, we propose that the categorization of polymicrogyria according to the number of lamina is artificial and should be abandoned and polymicrogyria should be defined according to the presence or absence of coexisting neuropathological features. Furthermore, our data indicate that polymicrogyria is not a cell migration disorder and rather that it should be considered a post-migration malformation of cortical development.
doi:10.1097/NEN.0b013e31821ccf1c
PMCID: PMC3113653  PMID: 21572338
Cell migration; Cerebral cortex; Cortical lamina; Malformation of cortical development; Polymicrogyria; Seizures
15.  XLMR candidate mouse gene, Zcchc12 (Sizn1) is a novel marker of Cajal-Retzius cells 
Gene expression patterns : GEP  2010;11(3-4):216-220.
Sizn1 (Zcchc12) is a transcriptional co-activator that positively modulates BMP (Bone Morphogenic Protein) signaling through its interaction with Smad family members and CBP. We have demonstrated a role for Sizn1 in basal forebrain cholinergic neuron specific gene expression. Furthermore, mutations in SIZN1 have been associated with X-linked mental retardation. Given the defined role of SIZN1 in mental retardation, knowing its complete forebrain expression pattern is essential to further elucidating its role in cognition. To better define the dynamic expression pattern of Sizn1 during forebrain development, we investigated its expression in mouse brain development from embryonic day 8.0 (E8.0) to adult. We found that Sizn1 is primarily restricted to the ventral forebrain including the medial ganglionic eminence, the septum, amygdala, and striatum. In addition, Sizn1 expression is detected in the cortical hem and Pallial-subpallial boundary (PSB; anti-hem); both sources of Cajal-Retzius cells. Sizn1 expression in the dorsal forebrain is restricted to a subset of cells in the marginal zone that also express Reln, indicative of Cajal-Retzius cells. These data provide novel information on brain regions and cell types that express Sizn1, facilitating further investigations into the function of Sizn1 in both development and the pathogenesis of mental retardation.
doi:10.1016/j.gep.2010.12.005
PMCID: PMC3065530  PMID: 21172456
16.  SDF1 regulates leading process branching and speed of migrating interneurons 
Cell migration is required for normal embryonic development, yet how cells navigate complex paths while integrating multiple guidance cues remains poorly understood. During brain development, interneurons migrate from the ventral ganglionic eminence to the cerebral cortex within several migratory streams. They must exit these streams to invade the cortical plate. While SDF1-signaling is necessary for normal interneuron stream migration, how they switch from tangential stream migration to invade the cortical plate is unknown. Here we demonstrate that SDF1-signaling reduces interneuron branching frequency by reducing cAMP levels via a Gi-signaling pathway using an in vitro mouse explant system, resulting in the maintenance of stream migration. Blocking SDF1-signaling, or increasing branching frequency, results in stream exit and cortical plate invasion in mouse brain slices. These data support a novel model to understand how migrating interneurons switch from tangential migration to invade the cortical plate in which reducing SDF1-signaling increases leading process branching and slows the migration rate, permitting migrating interneurons to sense cortically directed guidance cues.
doi:10.1523/JNEUROSCI.3118-10.2011
PMCID: PMC3038431  PMID: 21289183
Interneuron; migration; branching; SDF1; guidance
17.  Leading Process Branch Instability in Lis1+/− Nonradially Migrating Interneurons 
Cerebral Cortex (New York, NY)  2009;20(6):1497-1505.
Mammalian forebrain development requires extensive migration, yet the mechanisms through which migrating neurons sense and respond to guidance cues are not well understood. Similar to the axon growth cone, the leading process and branches of neurons may guide migration, but the cytoskeletal events that regulate branching are unknown. We have previously shown that loss of microtubule-associated protein Lis1 reduces branching during migration compared with wild-type neurons. Using time-lapse imaging of Lis1+/− and Lis1+/+ cells migrating from medial ganglionic eminence explant cultures, we show that the branching defect is not due to a failure to initiate branches but a defect in the stabilization of new branches. The leading processes of Lis1+/− neurons have reduced expression of stabilized, acetylated microtubules compared with Lis1+/+ neurons. To determine whether Lis1 modulates branch stability through its role as the noncatalytic β regulatory subunit of platelet-activating factor (PAF) acetylhydrolase 1b, exogenous PAF was applied to wild-type cells. Excess PAF added to wild-type neurons phenocopies the branch instability observed in Lis1+/− neurons, and a PAF antagonist rescues leading process branching in Lis1+/− neurons. These data highlight a role for Lis1, acting through the PAF pathway, in leading process branching and microtubule stabilization.
doi:10.1093/cercor/bhp211
PMCID: PMC2871376  PMID: 19861636
branching; leading process; microtubules; migration; PAFAH1b
18.  Does Chronic Microaspiration Cause Idiopathic Pulmonary Fibrosis? 
The American journal of medicine  2010;123(4):304-311.
Idiopathic pulmonary fibrosis is a diffuse fibrotic lung disease of unknown etiology with no effective treatment. Emerging data support a role for chronic microaspiration (i.e. subclinical aspiration of small droplets) in the pathogenesis and natural history of idiopathic pulmonary fibrosis. However, the precise relationship between chronic microaspiration and idiopathic pulmonary fibrosis remains unknown. Gastroesophageal reflux, a presumed risk factor for microaspiration, has been strongly associated with idiopathic pulmonary fibrosis with an estimated prevalence of 90%. This review aims to describe the relationship between chronic microaspiration and idiopathic pulmonary fibrosis by laying out the clinical and biologic rationale for this relationship and exploring the scientific evidence available. The gaps in our current understanding of the diagnosis of chronic microaspiration and idiopathic pulmonary fibrosis and the ongoing uncertainties in management and treatment will be highlighted. Defining the role of chronic microaspiration in idiopathic pulmonary fibrosis is essential as it has potential clinical, pathobiological and treatment implications for this deadly disease.
doi:10.1016/j.amjmed.2009.07.033
PMCID: PMC2851633  PMID: 20362747
pulmonary fibrosis; respiratory aspiration; gastroesophageal reflux; etiology; diagnostic techniques and procedures
19.  The Combined Functions of Proapoptotic Bcl-2 Family Members Bak and Bax Are Essential for Normal Development of Multiple Tissues 
Molecular cell  2000;6(6):1389-1399.
Summary
Proapoptotic Bcl-2 family members have been proposed to play a central role in regulating apoptosis. However, mice lacking bax display limited phenotypic abnormalities. As presented here, bak–/– mice were found to be developmentally normal and reproductively fit and failed to develop any age-related disorders. However, when Bak-deficient mice were mated to Bax-deficient mice to create mice lacking both genes, the majority of bax–/–bak–/– animals died perinatally with fewer than 10% surviving into adulthood. bax–/–bak–/– mice displayed multiple developmental defects, including persistence of interdigital webs, an imperforate vaginal canal, and accumulation of excess cells within both the central nervous and hematopoietic systems. Thus, Bax and Bak have overlapping roles in the regulation of apoptosis during mammalian development and tissue homeostasis.
PMCID: PMC3057227  PMID: 11163212
20.  Steady-State Intrapulmonary Pharmacokinetics and Pharmacodynamics of Posaconazole in Lung Transplant Recipients▿  
This prospective study evaluated the plasma and intrapulmonary pharmacokinetics and pharmacodynamics (PKPD) of posaconazole (POS) in lung transplant recipients. Twenty adult lung transplant patients were instructed to take a 400-mg POS oral suspension twice daily (BID) with a high-fat meal for a total of 14 doses. Pulmonary epithelial lining fluid (ELF) and alveolar cell (AC) samples were obtained via bronchoalveolar lavage, and blood samples were collected at the approximate time of bronchoscopy. POS concentrations were assayed using liquid chromatography with tandem mass spectrometry. The maximum concentrations (Cmax) (mean ± standard deviation [SD]) in plasma, ELF, and AC were 1.3 ± 0.4, 1.3 ± 1.7, and 55.4 ± 44.0 μg/ml. POS concentrations in plasma, ELF, and AC did not decrease significantly, indicating slow elimination after multiple dosing. Mean concentrations of POS in plasma, ELF, and AC were above the MIC90 (0.5 μg/ml) for Aspergillus species over the 12-h dosing interval and for 24 h following the last dose. Area under the concentration-time curve from 0 to 12 h (AUC0-12)/MIC90 ratios in plasma, ELF, and AC were 21.98, 22.42, and 1,060. We concluded that a dose of 400 mg BID resulted in sustained plasma, ELF, and AC concentrations above the MIC90 for Aspergillus spp. during the dosing interval. Confirmation of the therapeutic value of these observations requires further investigation. The intrapulmonary PKPD of POS may be favorable for treatment or prevention of aspergillosis, although further research on the relevant PKPD parameters and the effect of POS protein binding is required.
doi:10.1128/AAC.01396-09
PMCID: PMC2934990  PMID: 20516276
21.  Intrapulmonary Pharmacokinetics and Pharmacodynamics of Micafungin in Adult Lung Transplant Patients▿  
Invasive pulmonary aspergillosis is a life-threatening infection in lung transplant recipients; however, no studies of the pharmacokinetics and pharmacodynamics (PKPD) of echinocandins in transplanted lungs have been reported. We conducted a single-dose prospective study of the intrapulmonary and plasma PKPD of 150 mg of micafungin administered intravenously in 20 adult lung transplant recipients. Epithelial lining fluid (ELF) and alveolar cell (AC) samples were obtained via bronchoalveolar lavage performed 3, 5, 8, 18, or 24 h after initiation of infusion. Micafungin concentrations in plasma, ELF, and ACs were determined using high-pressure liquid chromatography. Noncompartmental methods, population analysis, and multiple-dose simulations were used to calculate PKPD parameters. Cmax in plasma, ELF, and ACs was 4.93, 1.38, and 17.41 μg/ml, respectively. The elimination half-life in plasma was 12.1 h. Elevated concentrations in ELF and ACs were sustained during the 24-h sampling period, indicating prolonged compartmental half-lives. The mean micafungin concentration exceeded the MIC90 of Aspergillus fumigatus (0.0156 μg/ml) in plasma (total and free), ELF, and ACs throughout the dosing interval. The area under the time-concentration curve from 0 to 24 h (AUC0-24)/MIC90 ratios in plasma, ELF, and ACs were 5,077, 923.1, and 13,340, respectively. Multiple-dose simulations demonstrated that ELF and AC concentrations of micafungin would continue to increase during 14 days of administration. We conclude that a single 150-mg intravenous dose of micafungin resulted in plasma, ELF, and AC concentrations that exceeded the MIC90 of A. fumigatus for 24 h and that these concentrations would continue to increase during 14 days of administration, supporting its potential activity for prevention and early treatment of pulmonary aspergillosis.
doi:10.1128/AAC.01647-09
PMCID: PMC2916355  PMID: 20439610
22.  Impact of the Lung Allocation Score on Lung Transplantation for Pulmonary Arterial Hypertension 
Rationale: In 2005, lung allocation for transplantation in the United States changed from a system based on waiting time to a system based on the Lung Allocation Score (LAS).
Objectives: To study the effect of the LAS on lung transplantation for idiopathic pulmonary arterial hypertension (IPAH) compared with other major diagnoses.
Methods: We studied 7,952 adults listed for lung transplantation between 2002 and 2008. Analyses were restricted to patients with IPAH, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF). Transplantation, waiting list mortality, and post-transplant mortality were compared between diagnoses for patients listed before and after implementation of the LAS.
Measurements and Main Results: The likelihood of transplantation from the waiting list increased for all diagnoses after implementation of the LAS. Waiting list mortality decreased for every diagnosis, except for IPAH, which remained unchanged. Implementation of the LAS was not associated with changes in post-transplant mortality for any diagnosis. Under the LAS system, patients with IPAH were less likely to be transplanted than patients with IPF (hazard ratio [HR], 0.53; P < 0.001) or CF (HR, 0.49; P < 0.001) and at greater risk of death on the waiting list than patients with COPD (HR, 3.09; P < 0.001) or CF (HR, 1.83; P = 0.025) after adjustment for demographics and transplant type. Post-transplant mortality for IPAH was not statistically different from that of other diagnoses.
Conclusions: Implementation of the LAS has improved the likelihood of lung transplantation for listed patients with IPAH, but mortality on the waiting list remains high compared with other major diagnoses.
doi:10.1164/rccm.200810-1603OC
PMCID: PMC2742763  PMID: 19520906
lung transplantation; pulmonary arterial hypertension; lung allocation score
23.  Targeted loss of Arx results in a developmental epilepsy mouse model and recapitulates the human phenotype in heterozygous females 
Brain  2009;132(6):1563-1576.
Mutations in the X-linked aristaless-related homeobox gene (ARX) have been linked to structural brain anomalies as well as multiple neurocognitive deficits. The generation of Arx-deficient mice revealed several morphological anomalies, resembling those observed in patients and an interneuron migration defect but perinatal lethality precluded analyses of later phenotypes. Interestingly, many of the neurological phenotypes observed in patients with various ARX mutations can be attributed, in part, to interneuron dysfunction. To directly test this possibility, mice carrying a floxed Arx allele were generated and crossed to Dlx5/6CRE-IRES-GFP(Dlx5/6CIG) mice, conditionally deleting Arx from ganglionic eminence derived neurons including cortical interneurons. We now report that Arx−/y;Dlx5/6CIG (male) mice exhibit a variety of seizure types beginning in early-life, including seizures that behaviourally and electroencephalographically resembles infantile spasms, and show evolution through development. Thus, this represents a new genetic model of a malignant form of paediatric epilepsy, with some characteristics resembling infantile spasms, caused by mutations in a known infantile spasms gene. Unexpectedly, approximately half of the female mice carrying a single mutant Arx allele (Arx−/+;Dlx5/6CIG) also developed seizures. We also found that a subset of human female carriers have seizures and neurocognitive deficits. In summary, we have identified a previously unrecognized patient population with neurological deficits attributed to ARX mutations that are recapitulated in our mouse model. Furthermore, we show that perturbation of interneuron subpopulations is an important mechanism underling the pathogenesis of developmental epilepsy in both hemizygous males and carrier females. Given the frequency of ARX mutations in patients with infantile spasms and related disorders, our data unveil a new model for further understanding the pathogenesis of these disorders.
doi:10.1093/brain/awp107
PMCID: PMC2685924  PMID: 19439424
Epilepsy; development; conditional knockout; genetic model; interneurons
24.  Undifferentiated Connective Tissue Disease-Associated Interstitial Lung Disease: Changes in Lung Function 
Lung  2010;188(2):143-149.
Undifferentiated connective tissue disease (UCTD) is a distinct clinical entity that may be accompanied by interstitial lung disease (ILD). The natural history of UCTD-ILD is unknown. We hypothesized that patients with UCTD-ILD would be more likely to have improvement in lung function than those with idiopathic pulmonary fibrosis (IPF) during longitudinal follow-up. We identified subjects enrolled in the UCSF ILD cohort study with a diagnosis of IPF or UCTD. The primary outcome compared the presence or absence of a ≥5% increase in percent predicted forced vital capacity (FVC) in IPF and UCTD. Regression models were used to account for potential confounding variables. Ninety subjects were identified; 59 subjects (30 IPF, 29 UCTD) had longitudinal pulmonary function data for inclusion in the analysis. After accounting for baseline pulmonary function tests, treatment, and duration between studies, UCTD was associated with substantial improvement in FVC (odds ratio = 8.23, 95% confidence interval, 1.27–53.2; p = 0.03) during follow-up (median, 8 months) compared with IPF. Patients with UCTD-ILD are more likely to have improved pulmonary function during follow-up than those with IPF. These findings demonstrate the clinical importance of identifying UCTD in patients presenting with an “idiopathic” interstitial pneumonia.
doi:10.1007/s00408-009-9226-7
PMCID: PMC2837880  PMID: 20069430
Idiopathic interstitial pneumonia; Systemic rheumatic disease; Undifferentiated connective tissue; Collagen vascular disease; Autoimmune disease
25.  Evidence that SIZN1 is a Candidate X-Linked Mental Retardation Gene 
An estimated 1-3% of individuals within the United States are diagnosed with mental retardation (MR), yet the cause is unknown in nearly 50% of the patients. While several environmental, genetic and combined teratogenetic etiologies have been identified, many causative genes remain to be identified. Furthermore, the pathogenetic mechanisms underlying MR are known for very few of these genes. Males have a much higher incidence of MR implicating genes on the X-chromosome. We have recently identified a novel gene, SIZN1, on the X-chromosome and showed that it functions in modulating the BMP signaling pathway. Furthermore, we have shown this gene is necessary for basal forebrain cholinergic neuron (BFCN) specific gene expression. Given that cognitive function is impaired when BFCNs are lost or functionally disrupted, we undertook a screen of cognitively impaired males for SIZN1 mutations. We report on four different sequence variants in SIZN1 in 11 individuals with nonsyndromic X-linked mental retardation. Our data implicate SIZN1 as a candidate gene for X-linked mental retardation and/or as a neurocognitive functional modifier.
doi:10.1002/ajmg.a.32472
PMCID: PMC2575800  PMID: 18798319
BMP; SIZN1(ZCCHC12); forebrain cholinergic neuron; X-linked mental retardation

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