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Aromatic l-amino acid decarboxylase (AADC) deficiency is a complex inherited neurological disorder of monoamine synthesis which results in dopamine and serotonin deficiency. The majority of affected individuals have variable, though often severe cognitive and motor delay, with a complex movement disorder and high risk of premature mortality. For most, standard pharmacological treatment provides only limited clinical benefit. Promising gene therapy approaches are emerging, though may not be either suitable or easily accessible for all patients. To characterize the underlying disease pathophysiology and guide precision therapies, we generated a patient-derived midbrain dopaminergic neuronal model of AADC deficiency from induced pluripotent stem cells. The neuronal model recapitulates key disease features, including absent AADC enzyme activity and dysregulated dopamine metabolism. We observed developmental defects affecting synaptic maturation and neuronal electrical properties, which were improved by lentiviral gene therapy. Bioinformatic and biochemical analyses on recombinant AADC predicted that the activity of one variant could be improved by l-3,4-dihydroxyphenylalanine (l-DOPA) administration; this hypothesis was corroborated in the patient-derived neuronal model, where l-DOPA treatment leads to amelioration of dopamine metabolites. Our study has shown that patient-derived disease modelling provides further insight into the neurodevelopmental sequelae of AADC deficiency, as well as a robust platform to investigate and develop personalized therapeutic approaches., Rossignoli et al. develop the first humanized neuronal model of AADC deficiency. They use this patient-derived neuronal system to elucidate disease mechanisms, and in particular to better define neurodevelopmental features, as well as to test precision therapy approaches.

Objective To investigate the molecular cause(s) underlying a severe form of infantile-onset parkinsonism and characterize functionally the identified variants. Methods A trio-based whole exome sequencing (WES) approach was used to identify the candidate variants underlying the disorder. In silico modeling, and in vitro and in vivo studies were performed to explore the impact of these variants on protein function and relevant cellular processes. Results WES analysis identified biallelic variants in WARS2, encoding the mitochondrial tryptophanyl tRNA synthetase (mtTrpRS), a gene whose mutations have recently been associated with multiple neurological phenotypes, including childhood-onset, levodopa-responsive or unresponsive parkinsonism in a few patients. A substantial reduction of mtTrpRS levels in mitochondria and reduced OXPHOS function was demonstrated, supporting their pathogenicity. Based on the infantile-onset and severity of the phenotype, additional variants were considered as possible genetic modifiers. Functional assessment of a selected panel of candidates pointed to a de novo missense mutation in CHRNA6, encoding the α6 subunit of neuronal nicotinic receptors, which are involved in the cholinergic modulation of dopamine release in the striatum, as a second event likely contributing to the phenotype. In silico, in vitro (Xenopus oocytes and GH4C1 cells) and in vivo (C. elegans) analyses demonstrated the disruptive effects of the mutation on acetylcholine receptor structure and function. Conclusion Our findings consolidate the association between biallelic WARS2 mutations and movement disorders, and suggest CHRNA6 as a genetic modifier of the phenotype.

We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner in a model of LPS-induced inflammation. This technology complements existing germline-transgenic models and may be applicable to other rodent disease models. ispartof: SCIENTIFIC REPORTS vol:10 issue:1 ispartof: location:England status: published

Recombinant adeno-associated virus (rAAV) has attracted a significant research focus for delivering genetic therapies to target cells. This non-enveloped virus has been trialed in many clinical-stage therapeutic strategies but important obstacle in clinical translation is the activation of both innate and adaptive immune response to the protein capsid, vector genome and transgene product. In addition, the normal population has pre-existing neutralizing antibodies against wild-type AAV, and cross-reactivity is observed between different rAAV serotypes. While extent of response can be influenced by dosing, administration route and target organ(s), these pose concerns over reduction or complete loss of efficacy, options for re-administration, and other unwanted immunological sequalae such as local tissue damage. To reduce said immunological risks, patients are excluded if they harbor anti-AAV antibodies or have received gene therapy previously. Studies have incorporated immunomodulating or suppressive regimens to block cellular and humoral immune responses such as systemic corticosteroids pre- and post-administration of Luxturna® and Zolgensma®, the two rAAV products with licensed regulatory approval in Europe and the United States. In this review, we will introduce the current pharmacological strategies to immunosuppress or immunomodulate the host immune response to rAAV gene therapy.

In the era of genomic medicine, diagnoses of rare paediatric neurological diseases are increasing. Many are untreatable and life-limiting, leading to an exceptional increase in gene therapy development. It is estimated that 20 gene therapy products will have received approval from the US Food and Drug Administration by 2025. With viral gene therapy considered a potential single-dose cure for patients with spinal muscular atrophy type 1 as one example, and contemporaneously tragically resulting in the deaths of three male children with X-linked myotubular myopathy receiving high-dose gene therapy in 2020, what is the current state of gene therapy? What is behind the decades of hype around viral gene therapy and is it high impact, but high risk? In this review, we outline principles of viral gene therapy development and summarize the most recent clinical evidence for the therapeutic effect of gene therapy in paediatric neurological diseases. We discuss adeno-associated virus and lentiviral vectors, antisense oligonucleotides, emerging genetic editing approaches, and current limitations that the field still faces. What this paper adds Viral gene therapy development and clinically used transgenes, regulatory elements, capsids, dosage, and delivery routes are summarized. Viral gene therapy for 18 childhood neurological disorders involving over 600 children in 40 clinical trials are reviewed.

BACKGROUND: Juvenile forms of parkinsonism are rare conditions with onset of bradykinesia, tremor and rigidity before the age of 21 years. These atypical presentations commonly have a genetic aetiology, highlighting important insights into underlying pathophysiology. Genetic defects may affect key proteins of the endocytic pathway and clathrin-mediated endocytosis (CME), as in DNAJC6-related juvenile parkinsonism. OBJECTIVE: To report on a new patient cohort with juvenile-onset DNAJC6 parkinsonism-dystonia and determine the functional consequences on auxilin and dopamine homeostasis. METHODS: Twenty-five children with juvenile parkinsonism were identified from a research cohort of patients with undiagnosed pediatric movement disorders. Molecular genetic investigations included autozygosity mapping studies and whole-exome sequencing. Patient fibroblasts and CSF were analyzed for auxilin, cyclin G-associated kinase and synaptic proteins. RESULTS: We identified 6 patients harboring previously unreported, homozygous nonsense DNAJC6 mutations. All presented with neurodevelopmental delay in infancy, progressive parkinsonism, and neurological regression in childhood. (123) I-FP-CIT SPECT (DaTScan) was performed in 3 patients and demonstrated reduced or absent tracer uptake in the basal ganglia. CSF neurotransmitter analysis revealed an isolated reduction of homovanillic acid. Auxilin levels were significantly reduced in both patient fibroblasts and CSF. Cyclin G-associated kinase levels in CSF were significantly increased, whereas a number of presynaptic dopaminergic proteins were reduced. CONCLUSIONS: DNAJC6 is an emerging cause of recessive juvenile parkinsonism-dystonia. DNAJC6 encodes the cochaperone protein auxilin, involved in CME of synaptic vesicles. The observed dopamine dyshomeostasis in patients is likely to be multifactorial, secondary to auxilin deficiency and/or neurodegeneration. Increased patient CSF cyclin G-associated kinase, in tandem with reduced auxilin levels, suggests a possible compensatory role of cyclin G-associated kinase, as observed in the auxilin knockout mouse. DNAJC6 parkinsonism-dystonia should be considered as a differential diagnosis for pediatric neurotransmitter disorders associated with low homovanillic acid levels. Future research in elucidating disease pathogenesis will aid the development of better treatments for this pharmacoresistant disorder. © 2020 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Approximately 40% of preterm births are preceded by microbial invasion of the intrauterine space: ascent from the vagina is the most common pathway. Within the cervical canal, antimicrobial peptides and proteins (AMPs) help to constitute a barrier which prevents ascending infection. We investigated whether expression of the AMP, human β-defensin-3 (HBD3), in the cervical mucosa prevented bacterial ascent from the vagina into the uterine cavity of pregnant mice. An adeno-associated virus vector containing both the HBD3 gene and GFP transgene (AAV8 HBD3.GFP) or control (AAV8 GFP), was administered intravaginally into E13.5 pregnant mice. Ascending infection was induced at E16.5 using bioluminescent E.coli (E.coli K1 A192PP-lux2). Bioluminescence imaging showed bacterial ascent into the uterine cavity, cellular events that led to premature delivery and a reduction in pups born alive, compared with uninfected controls. In addition, a significant reduction in uterine bioluminescence in the AAV8 HBD3.GFP-treated mice was observed 24 hours post-E.coli infection, compared to AAV8 GFP treated mice, signifying reduced bacterial ascent in AAV8 HBD3.GFP-treated mice. There was also an increase in the number of living pups in AAV HBD3.GFP-treated mice. We propose that HBD3 may be considered a possible candidate for augmenting cervical innate immunity to prevent ascending infection-related preterm birth.

Objectives To describe the spectrum of movement disorders and cerebrospinal fluid (CSF) neurotransmitter profiles in paediatric patients withPOLGdisease. Methods We identified children with genetically confirmedPOLGdisease, in whom CSF neurotransmitter analysis had been undertaken. Clinical data were collected retrospectively. CSF neurotransmitter levels were compared to both standardised age‐related reference ranges and to non‐POLGpatients presenting with status epilepticus. Results Forty‐one patients withPOLGdisease were identified. Almost 50% of the patients had documented evidence of a movement disorder, including non‐epileptic myoclonus, choreoathetosis and ataxia. CSF neurotransmitter analysis was undertaken in 15 cases and abnormalities were seen in the majority (87%) of cases tested. In many patients, distinctive patterns were evident, including raised neopterin, homovanillic acid and 5‐hydroxyindoleacetic acid levels. Conclusions Children withPOLGmutations can manifest with a wide spectrum of abnormal movements, which are often prominent features of the clinical syndrome. Underlying pathophysiology is probably multifactorial, and aberrant monoamine metabolism is likely to play a role.

Argininosuccinate lyase (ASL) belongs to the liver-based urea cycle detoxifying ammonia, and the citrulline-nitric oxide cycle synthesising nitric oxide (NO). ASL-deficient patients present argininosuccinic aciduria characterised by hyperammonaemia and a multi-organ disease with neurocognitive impairment. Current therapeutic guidelines aim to control ammonaemia without considering the systemic NO imbalance. Here, we observed a neuronal disease with oxidative/nitrosative stress in ASL-deficient mouse brains. A single systemic injection of gene therapy mediated by an adeno-associated viral vector serotype 8 (AAV8) in adult or neonatal mice demonstrated the long-term correction of the urea cycle and the citrulline-NO cycle in the brain, respectively. The neuronal disease persisted if ammonaemia only was normalised but was dramatically reduced after correction of both ammonaemia and neuronal ASL activity. This was correlated with behavioural improvement and a decrease of the cortical cell death rate. Thus, the cerebral disease in argininosuccinic aciduria involves neuronal oxidative/nitrosative stress not mediated by hyperammonaemia, which is reversed by AAV gene transfer targeting the brain and the liver, acting on two different metabolic pathways via a single vector delivered systemically. This approach provides new hope for hepatocerebral metabolic diseases.

Item does not contain fulltext Histone lysine methylation, mediated by mixed-lineage leukemia (MLL) proteins, is now known to be critical in the regulation of gene expression, genomic stability, cell cycle and nuclear architecture. Despite MLL proteins being postulated as essential for normal development, little is known about the specific functions of the different MLL lysine methyltransferases. Here we report heterozygous variants in the gene KMT2B (also known as MLL4) in 27 unrelated individuals with a complex progressive childhood-onset dystonia, often associated with a typical facial appearance and characteristic brain magnetic resonance imaging findings. Over time, the majority of affected individuals developed prominent cervical, cranial and laryngeal dystonia. Marked clinical benefit, including the restoration of independent ambulation in some cases, was observed following deep brain stimulation (DBS). These findings highlight a clinically recognizable and potentially treatable form of genetic dystonia, demonstrating the crucial role of KMT2B in the physiological control of voluntary movement.

Objective:To describe better the motor phenotype, molecular genetic features, and clinical course of GNAO1-related disease.Methods:We reviewed clinical information, video recordings, and neuroimaging of a newly identified cohort of 7 patients with de novo missense and splice site GNAO1 mutations, detected by next-generation sequencing techniques.Results:Patients first presented in early childhood (median age of presentation 10 months, range 0–48 months), with a wide range of clinical symptoms ranging from severe motor and cognitive impairment with marked choreoathetosis, self-injurious behavior, and epileptic encephalopathy to a milder phenotype, featuring moderate developmental delay associated with complex stereotypies, mainly facial dyskinesia and mild epilepsy. Hyperkinetic movements were often exacerbated by specific triggers, such as voluntary movement, intercurrent illnesses, emotion, and high ambient temperature, leading to hospital admissions. Most patients were resistant to drug intervention, although tetrabenazine was effective in partially controlling dyskinesia for 2/7 patients. Emergency deep brain stimulation (DBS) was life saving in 1 patient, resulting in immediate clinical benefit with complete cessation of violent hyperkinetic movements. Five patients had well-controlled epilepsy and 1 had drug-resistant seizures. Structural brain abnormalities, including mild cerebral atrophy and corpus callosum dysgenesis, were evident in 5 patients. One patient had a diffuse astrocytoma (WHO grade II), surgically removed at age 16.Conclusions:Our findings support the causative role of GNAO1 mutations in an expanded spectrum of early-onset epilepsy and movement disorders, frequently exacerbated by specific triggers and at times associated with self-injurious behavior. Tetrabenazine and DBS were the most useful treatments for dyskinesia.

Identifying immune escape mechanisms used by tumors may define strategies to sensitize them to immunotherapies to which they are otherwise resistant. In this study, we show that the antiapoptotic gene API5 acts as an immune escape gene in tumors by rendering them resistant to apoptosis triggered by tumor antigen-specific T cells. Its RNAi-mediated silencing in tumor cells expressing high levels of API5 restored antigen-specific immune sensitivity. Conversely, introducing API5 into API5low cells conferred immune resistance. Mechanistic investigations revealed that API5 mediated resistance by upregulating FGF2 signaling through a FGFR1/PKCδ/ERK effector pathway that triggered degradation of the proapoptotic molecule BIM. Blockade of FGF2, PKCδ, or ERK phenocopied the effect of API5 silencing in tumor cells expressing high levels of API5 to either murine or human antigen-specific T cells. Our results identify a novel mechanism of immune escape that can be inhibited to potentiate the efficacy of targeted active immunotherapies. Cancer Res; 74(13); 3556–66. ©2014 AACR.

The graft-healing potential of mesenchymal stromal cells (MSCs) derived from the remnants of ruptured anterior cruciate ligaments (ACLs) after ACL reconstruction may depend on the chronicity of the injury. The aim of this study was to assess the quantitative and phenotypic differences between MSCs isolated from ACL remnants in patients with (sub)acute and chronic tearing.Torn ACL remnants were harvested during ACL reconstruction from 41 patients, 24 with (sub)acute ACL (6 months from injury to surgery) and 17 with chronic ACL (time interval6 months) tears. MSCs isolated from these samples were assessed for quantitative and phenotypic differences, and the correlation between the proportion of MSCs and the chronicity of ACL tear was evaluated.At passage 0, the mean proportion of MSCs (CD34(-), CD44(+), CD90(+) and CD105(+)) was higher in (sub)acute than in chronic ACL tear samples (20.69% ± 7.82% versus 9.85% ± 8.01%, P 0.001). At passages 1 and 2, however, MSC proportions did not differ significantly in the two groups. Time interval showed a negative correlation with MSC proportion only at passage 0 (r = -0.505, P 0.001). The optimal cutoff value for time from injury to surgery yielding 10% freshly isolated ACL-MSCs, a percentage expected to have low tissue healing potential, was 23.5 months.The proportion of freshly isolated MSCs was higher in samples from patients with (sub)acute tearing than in chronic ACL tearing and negatively correlated with the time interval between trauma and surgery.

The monoamine neurotransmitter disorders are increasingly recognized as an expanding group of inherited neurometabolic syndromes caused by disturbances in the synthesis, transport and metabolism of the biogenic amines, including the catecholamines (dopamine, norepinephrine, and epinephrine) and serotonin. Disturbances in monoamine metabolism lead to neurological syndromes that frequently mimic other conditions, such as hypoxic ischemic encephalopathy, cerebral palsy, parkinsonism-dystonia syndromes, primary genetic dystonia and paroxysmal disorders. As a consequence, neurotransmitter disorders are frequently misdiagnosed. Early and accurate diagnosis of these neurotransmitter disorders is important, as many are highly amenable to, and some even cured by, therapeutic intervention. In this review, we highlight recent advances in the field, particularly the recent extensive characterization of known neurotransmitter disorders and identification of novel neurotransmitter disorders. We also provide an overview of current and future research in the field focused on developing novel treatment strategies.

Objective To assess the method and time to stand from supine and the time to run 10 m for normal young children. Study design Three hundred twenty-one normal children aged 2.8-7.8 years were recruited from primary schools. After standardization, each test was carried out twice, timed, and videoed. The influence of age, sex, height, weight, body mass index (BMI), and method of standing were analyzed. Charts for time to stand and running time were produced and assessment of reproducibility performed. Results For the time to stand from supine and the method used, there was a significant correlation with age. More than 50% of young children took >2 seconds. There was no significant association with BMI. Method of standing was associated with standing time in boys but not in girls. A Bland-Altman plot of standing times by 2 observers showed good reproducibility with no clinically significant difference. For the 10-m running test, there was a significant negative correlation with age, height, weight, and BMI. Conclusion There is considerable variability in the method used and time taken to stand from supine in young children. These change with age, permitting the creation of charts showing age-related normal values.

Aim To describe the frequency of renovascular abnormalities and hypertension in an opportunistic cohort of children with complex cerebrovascular disease from a single tertiary/quaternary referral centre. Method This was a retrospective case note and imaging review of children who had had cerebral and renal angiography, with a diagnosis of moyamoya or other occlusive cerebrovascular disease (OCVD). Hypertension was defined as at least three systolic blood pressure readings of the 95th centile or above. Results Of 34 children (12 males, 22 females; median age 5y 11mo, range 2mo-15y 3mo; 20 with moyamoya, 14 with OCVD), primary presentation was neurological in 29 (arterial ischaemic stroke, transient ischaemic attack, or headache) and with hypertension in five. Renovascular abnormalities were identified in 17, of whom 10 had main renal artery stenosis. Renovascular involvement was not predictable according to arteriopathy diagnosis. Blood pressure was rarely plotted on centile charts. Using the 50th height centile for blood pressure, and based on a median of five systolic blood pressure readings per patient, 20 out of 34 met the definition for hypertension (15/29 patients with primary neurological presentation). Interpretation Renovascular abnormalities were common in this group of children with complex cerebrovascular disease. Blood pressure was frequently abnormal but rarely measured and infrequently plotted on centile charts. Neurologists should be alert to potential systemic vascular involvement and its sequelae in children with complex cerebrovascular disease.

Deficits in the basal ganglia pathways modulating cortical motor activity underlie both Parkinson disease (PD) and Huntington disease (HD). Phosphodiesterase 10A (PDE10A) is enriched in the striatum, and animal data suggest that it is a key regulator of this circuitry. Here, we report on germline PDE10A mutations in eight individuals from two families affected by a hyperkinetic movement disorder due to homozygous mutations c.320A>G (p.Tyr107Cys) and c.346G>C (p.Ala116Pro). Both mutations lead to a reduction in PDE10A levels in recombinant cellular systems, and critically, positron-emission-tomography (PET) studies with a specific PDE10A ligand confirmed that the p.Tyr107Cys variant also reduced striatal PDE10A levels in one of the affected individuals. A knock-in mouse model carrying the homologous p.Tyr97Cys variant had decreased striatal PDE10A and also displayed motor abnormalities. Striatal preparations from this animal had an impaired capacity to degrade cyclic adenosine monophosphate (cAMP) and a blunted pharmacological response to PDE10A inhibitors. These observations highlight the critical role of PDE10A in motor control across species.

Vascular stroke syndromes in childhood (arterial ischemic stroke (AIS), cerebral sinovenous thrombosis and non-traumatic intracranial hemorrhage) are increasingly recognized as important causes of mortality and morbidity, with significant personal and societal economic costs. The disease mechanisms underlying childhood AIS differ significantly from adults; in particular, atheroma plays an extremely minor role in children. However, to date, treatment principles in children have been based on extrapolation from adult data, usually by consensus expert opinion.This review article summarizes the current pharmacological treatments of childhood AIS, the evidence supporting these and proposes future treatment targets directed at underlying disease mechanisms. This is categorized into acute management, secondary prevention, emerging treatments and future treatments.There is a paucity of robust data supporting current treatment approaches in childhood AIS. There are currently no recommended acute treatments; the role of thrombolysis in children remains to be determined. Aspirin, heparin and warfarin are used as secondary prevention therapies, but have not been shown to be superior to placebo. Non-atheromatous arteriopathy is now recognized as an important risk factor for recurrent AIS in children; further understanding of its etiology and pathophysiology could lead to identification of future therapeutic targets.

Hypoxic Ischemic Encephalopathy (HIE), occurs in 2-3 per 1000 live births. 25% of surviving affected newborns will suffer from severe life-long neurological disability. HIE is associated with reactive astrogliosis and inflammation. During this process the activity of STAT3 and NFkB transcription factors is increased, and glial fibrillary acidic protein (GFAP) expression is upregulated in astrocytes.Light producing transgenic mice, where luciferase expression is controlled by a surrogate promoter or by a minimal promoter downstream of tandem, synthetic, transcription factor binding elements, are used to provide an in vivo readout of disease processes. In this study, we aimed to deliver STAT3 and NFkB activated or GFAP promoter driven luciferase reporter constructs to brains of neonatal mice as a form of assessing the amount of astrogliosis and inflammation in a mouse model of HIE.A rodent GFAP promoter, and STAT3 and NFkB response elements were each cloned into a lentivirus vector upstream of the genes encoding a codon-optimised firefly luciferase and green fluorescent protein. Lentivirus vector pseudotyped with either gp64 or VSV-g viral envelope glycoproteins were injected intra-cranially into CD1 outbred neonatal (P0) mice, and luciferase expression was monitored continually by whole body bioluminescence imaging of conscious mice. Vector containing NFkB response element was also injected into P0 mice, which underwent HIE surgery at P7.The two pseudotypes gp64 and VSV-g exhibited different cellular tropisms within the central nervous system; VSV-g predominately targeting neuronal cells whereas gp64 transducing cells of fibrillary astrocytic morphology. Long-term expression of luciferase was observed. Following induction of HIE, the most severely affected mice lost weight. We observed a significant correlation between the luciferase expression 24 hours after surgery and weight 7 days after surgery (P = 0.0007) in mice which had received the gp64 pseudotyped NFkB biosensor; the most severely affected HIE mice had the lowest luciferase expression and weight. However, the HIE mice which had received the VSV-g pseudotyped NFkB biosensor failed to show this correlation between the luciferase expression and weight (P=0.479). This indicates that NFkB-controlled luciferase expression in astrocytes is predictive of brain injury. We are now investigating the mechanism of this predictive relationship.

The characteristics of anterior cruciate ligament (ACL)-derived mesenchymal stem cells (MSCs), such as proportion and multilineage potential, can be affected by donor age. However, the qualitative and quantitative features of ACL MSCs isolated from younger and older individuals have not yet been compared directly. This study assessed the phenotypic and functional differences in ACL-MSCs isolated from younger and older donors and evaluated the correlation between ACL-MSC proportion and donor age. Torn ACL remnants were harvested from 36 patients undergoing ACL reconstruction (young: 29.67 +/- 10.92 years) and 33 undergoing TKA (old: 67.96 +/- 5.22 years) and the proportion of their MSCs were measured. The mean proportion of MSCs was slightly higher in older ACL samples of the TKA group than of the younger ACL reconstruction group (19.69 +/- 8.57% vs. 15.33 +/- 7.49%, p = 0.024), but the proportions of MSCs at passages 1 and 2 were similar. MSCs from both groups possessed comparable multilineage potentiality, as they could be differentiated into adipocytes, osteocytes, and chondrocytes at similar level. No significant correlations were observed between patient age and MSC proportions at passages 0-2 or between age and MSC proportion in both the ACL reconstruction and TKA groups. Multiple linear regression analysis found no significant predictor of MSC proportion including donor age for each passage. Microarray analysis identified several genes that were differentially regulated in ACLMSCs from old TKA patients compared to young ACL reconstruction patients. Genes of interest encode components of the extracellular matrix (ECM) and may thus play a crucial role in modulating tissue homeostasis, remodeling, and repair in response to damage or disease. In conclusion, the proportion of freshly isolated ACL-MSC was higher in elderly TKA patients than in younger patients with ACL tears, but their phenotypic and multilineage potential were comparable.

Nat. Genet. 49, 223–237 (2017); published online 19 December 2016; corrected after print 20 April 2017 Following publication of this article, the authors were asked to remove a clinical image and some video footage of one of the affected individuals. Although consent was obtained, in keeping with their ethical consent framework, the authors allow for withdrawal of consent and are carrying out the wishes of the research subjects under their consent process.

Neurodegenerative monogenic diseases can also affect a broad range of tissues and organs throughout the body. An effective treatment would require a systemic approach. The intravenous administration of novel therapies is ideal but is hampered by the inability of such drugs to cross the blood-brain barrier and precludes efficacy in the central nervous system. A number of these early lethal intractable diseases also present devastating irreversible pathology at birth or soon after. Therefore, any therapy would ideally be administered during the perinatal period to prevent, stop or ameliorate disease progression. The concept of perinatal gene therapy has moved a step further towards being a feasible approach to treating such disorders. This has primarily been driven by the recent discoveries that particular serotypes of adeno-associated virus (AAV) gene delivery vectors have the ability to cross the blood-brain barrier following intravenous administration. Furthermore, this has been safely demonstrated in perinatal mice and non-human primates. This review focuses on the progress made in using AAV to achieve systemic transduction and what this means for developing perinatal gene therapy for early lethal neurodegenerative diseases.

Here we observe evidence that targeting mid-brain DA neurons is influenced by both delivery method and promoter following administration to the neonatal mouse brain.IV delivery using rTH and hSyn promoters showed the highest DA neuron transduction whilst IC delivery and GUSB promoter was least effective. Interestingly IC hSyn did not transduce TH positive neurons despite demonstrating the most extensive intracranial transduction. Targeting the neonatal mid-brain DA neurons remains a significant challenge and these data suggest that both promoter of choice and delivery method are important factors to consider.

Minor degrees of exomphalos have been known to be associated with vitellointestinal duct anomalies. The most frequent association is Meckel's diverticulum. It is unusual for a fistula to be communicating directly with the exomphalos sac. We report five neonates that presented with a fistula on the exomphalos sac over a 10-year period. The different variants of patent vitellointestinal duct in these cases have been discussed with a review of the literature.

Along with MHC class I (MHCI), 2B4 provides nonredundant NK-cell inhibition in mice. The immunoregulatory role of 2B4 has been increasingly appreciated in models of tumor and viral infection, however, the interactions among 2B4, MHCI, and other activating NK-cell receptors remain uncertain. Here, we dissect the influence of two distinct inhibitory pathways in modulating NK-cell-mediated control of tumors expressing strong activating ligands, including RAE-1γ. In vitro cytotoxicity and in vivo peritoneal clearance assays using MHCI(+) CD48(+) (RMA-neo), MHCI(+) CD48(+) RAE-1γ (RMA-RAE-1γ), MHCI(-) CD48(+) (RMA-S-neo), and MHCI(-) CD48(+) RAE-1γ (RMA-S-RAE-1γ) tumor lines demonstrated that NKG2D activation supersedes the inhibitory effect of both 2B4- and MHCI-mediated immune-tolerance systems. Furthermore, 2B4KO mice subcutaneously challenged with RMA-neo and RMA-S-neo exhibited reduced tumor growth and significantly prolonged survival compared with WT mice, implying that 2B4 is constitutively engaged in the NK-cell tolerance mechanism in vivo. Nevertheless, the inhibitory effect of 2B4 is significantly attenuated when NK cells encountered highly stressed tumor cells expressing RAE-1γ, resulting in an immune response shift toward NK-cell activation and tumor regression. Therefore, our data highlight the importance of the 2B4-mediated inhibitory system as an alternate self-tolerance mechanism, whose role can be modulated by the strength of activating receptor signaling within the tumor microenvironment.

Congenital myasthenic syndromes due to DOK7 mutations cause fatigable limb girdle weakness. Treatment with ephedrine improves muscle strength. Salbutamol, a β2-adrenergic receptor agonist with fewer side effects and more readily available, has been effective in adult and anecdotal childhood cases. This study reports the effects of salbutamol on motor function in childhood DOK7 congenital myasthenic syndrome. Nine children (age range 5.9–15.1years) were treated with oral salbutamol, 2–4mg TDS. The effect on timed tests of motor function, pre- and up to 28months post-treatment, was audited retrospectively. All 9 reported functional benefit within 1month, with progressive improvement to a plateau at 12–18months. Within the first month, all 3 non-ambulant children resumed walking with assistance. Although improvements were seen in some timed tests (timed 10m, arm raise time, 6min walk time) this did not fully reflect the observed functional benefits in daily living activities. No major side effects were reported. We conclude that oral salbutamol treatment significantly improves strength in children with DOK7 congenital myasthenic syndrome and is well tolerated. Outcome measures need to be refined further, both to accurately reflect functional abilities in children and to document progress and treatment response.

Germ line light producing transgenic mice, where luciferase expression is controlled by a surrogate promoter or by a minimal promoter downstream of tandem, synthetic, transcription factor binding elements, are used to provide an in vivo readout of disease processes. However, as every cell within the organism contains the luciferase reporter gene, it is therefore not specific to individual organs. We have developed a novel technology for the generation of light emitting somatic transgenic animals using lentiviral vectors. This allows signalling pathways in diseased organs to be monitored specifically, continually and in a non-invasive manner [1]. In this study, we aimed to deliver NFkB driving a luciferase reporter constructs to the nervous system of neonatal mice to generate somatic-transgenic mice using both lentivirus and adeno-associated viral (AAV) vectors. Lentivirus vector pseudotyped with VSV-G viral envelope glycoproteins or AAV8 serotyped vector carrying an NFkB response element was injected intracranially or intravenously to outbred CD1 neonatal (P1) mice and luciferase expression was monitored continually by whole body bioluminescence imaging of conscious mice. The ability to image conscious mice holds advantages when studying neuropathology. After weaning, pathological activation of NFKB was induced by intraperitoneal injection of lipopolysaccharide (LPS). Following intracranial injection, the VSV-G lentivirus NFkB biosensor showed transduction of the brain and spinal cord. Luciferase expression was upregulated 24 hours after administration of LPS. Immunohistochemistry revealed only modest spread throughout the brain. Conversely, intracranial injection of AAV8 NFkB biosensor showed a much wider spread and increased luciferase expression. Finally we administered AAV8 NFkB biosensor intravenously at P1. Whereas previous studies show AAV8 serotype transduces many systemic tissues [2] through this route; we observed luciferase expression predominantly in the brain and spine (see figurefigure). Using a standard Gateway® cloning system we have established a library of more than 25 lentivirus biosensors where some have been tested in vitro and in vivo. We plan on incorporating these response elements into the AAV backbone described above. Therefore, enabling the generation of somatic-transgenic mice which have a wider spread of AAV biosensor. This will complement existing germ line transgenic, light producing technology by maximising the use, and reducing the numbers, of animals used in biomedical research.View Large Image | Download PowerPoint Slide1. Buckley, SMK et al. 2015. In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters. Scientific Reports. 2. Inagaki, K et al. 2008. Frequency and spectrum of genomic integration of recombinant AAV serotype 8 vector in neonatal mouse liver. Journal of virology.

Hyperventilation-induced high-amplitude rhythmic slow activity with altered awareness (HIHARS) is increasingly being identified in children and is thought to be an age-related non-epileptic electrographic phenomenon. We retrospectively investigated the clinical outcome in 15 children (six males, nine females) with HIHARS (mean age 7y, SD 1y 11mo; range 4y 6mo-11y). The presenting feature in 11 cases was blank spells - two of these children also had generalized tonic-clonic seizures (GTCS) - and in one individual the main concern was deteriorating school performance. Three children had symptoms suggestive of focal motor seizures. Of the nine children presenting solely with blank spells, further follow-up (mean duration 18mo, SD 21mo) revealed full resolution of symptoms in six, but three had persistent symptoms. In our study, the symptoms of children with HIHARS presenting with blank spells in isolation appeared to resolve spontaneously and did not evolve into convulsive seizures or other paroxysmal events considered to be clearly epileptic. Children (with HIHARS) who presented with clinical features suggestive of GTCS or focal motor seizures (with or without blank spells) and/or had epileptiform discharges on interictal electroencephalography were subsequently diagnosed with epilepsy.

We describe the clinical course and treatment of three unrelated female patients ranging in age from 27 months to 14 years with anti-NMDA receptor encephalitis. The third case is reported as an addendum to the paper. None of the cases were paraneoplastic. All received initial immunotherapy consisting of steroids and IVIg, and two of them received 3 and 8 plasma exchanges respectively, without consistent or sustained clinical improvement. All three girls were then treated with monthly cycles of Cyclophosphamide. All had resolution of their movement disorder and a dramatic and sustained clinical improvement of their other symptoms in the domains of cognition, language and behaviour. The clinical improvement began after the first cycle in two and the second cycle in the third and continued with the subsequent cycles. None developed side-effects of treatment. In light of the recent review of the condition and our own clinical experience in the paediatric age group, we propose that second line immunotherapy should be considered early after failure of first line immunotherapy.

Objective: Partial or complete duplication of the renal tract may be an incidental finding or may be associated with significant pathology. Accurate assessment is not always easy. This retrospective review was undertaken to determine whether intravenous urography (IVU) in combination with a DMSA renal scan provides significant additional information. Patients and methods: Eighteen patients referred to our imaging department with a provisional diagnosis on ultrasound of renal tract duplication during a three year period were identified by searching the radiological computer files. The presenting features were urinary infection (13), abdominal pain (3) and abnormal antenatal sonography (2). Results: Four patients were found not to have renal duplication. There was concordance between the IVU and DMSA scan in seven. Additional clinically relevant information was obtained in three cases. A false negative result from the DMSA scan was found in four children and a false positive result in three. Conclusion: In a significant number of cases of possible renal tract duplication, additional relevant information can be obtained from an IVU. Patient distress is minimised by combining the IVU and DMSA in one single episode of venous access. Review of the radiological images during the investigation allows minimisation of radiation dosage.

Neurofibromatosis types 1 (NF1) and 2 (NF2) are genetically distinct conditions caused by mutations in tumour suppressor genes that share a number of phenotypic features. Childhood stroke and vasculopathy have been associated with NF1, but not with NF2. We describe a case of brainstem stroke in a child with NF2.

Purpose: To evaluate the diagnostic difficulties and pitfalls in establishing the diagnosis of congenital H-type tracheoesophageal fistula. Material and Methods: A retrospective review of all cases of H-type tracheoesophageal fistula that were diagnosed in a single unit over a 6-year period. The variables assessed were age at presentation, presenting symptoms, time to diagnosis, investigations, and time to surgical repair of H-type fistula. The investigations leading to a definitive diagnosis are assessed and discussed. Results: Between 1998 and 2004, five cases of H-type tracheoesophageal fistula presented to our unit. All cases had an upper gastrointestinal contrast study/tube esophagogram. In addition, four cases had a chest radiograph, three cases had a bronchoscopy, and one case an esophagoscopy. The median delay from the time of first presentation to diagnosis of H-type tracheoesophageal fistula was 14 days (7–58 days). Median age at surgery was 15 days (8–60 days). Conclusion: Although symptoms are usually present from birth, the diagnosis of H-type fistula is difficult and often delayed. The various diagnostic techniques are not entirely reliable and fistula identification can be elusive. The authors present recommendations for the diagnostic work-up, which may increase the diagnostic potential and avoid unnecessary delays in the diagnosis and management of H fistula.

Skull base osteomyelitis (SBO) arising from the sphenoidal paranasal air sinus infection without associated external otitis is rare. Initially SBO may have headache as the only symptom with cranial neuropathies occurring later. We report a 10-year-old immunocompetent girl with headache and chronic sinusitis, who developed a lateral medullary syndrome following streptococcal milleri sphenoidal osteomyelitis.

The antegrade continence enema is an effective method of treatment of fecal incontinence. We report our experience of a laparoscopic antegrade continence enema procedure and describe a simple approach to this procedure using a two-port technique.Over a 3-year period, 12 children with intractable constipation and fecal soiling underwent the antegrade continence enema procedure laparoscopically. All cases had full bowel preparation the day before surgery. This procedure was done through one 5-mm camera port and two 5-mm working ports in 8 cases, and using the camera port and only one additional 5-mm working port in 4 cases. The appendix was used in 5 cases and the cecum in 3 cases with the threeport technique while the appendix was used in all 4 cases with the two-port technique. The appendix or cecum was delivered extracorporeally through the 5-mm port site in the right lower quadrant. The mucocutaneous anastomosis was stented using a gastrostomy button.Between 2001 and 2004, 12 children (10 male, 2 female) underwent a laparoscopic antegrade continence enema procedure at a median age of 10.5 years (range, 7-14 years). This procedure was easy to perform and no case required conversion to an open procedure. The wash-outs via the MIC-KEY gastrostomy button (MIC-KEY, Kimberly-Clark) were commenced at a median of 3.5 days (range, 1-5 days). Median postoperative hospital stay was 2 days (range, 1-5 days). This procedure was effective in completely resolving fecal incontinence in 9 cases and partially resolving it in 3 cases. There were no episodes of stomal stenosis, leakage, or herniation. However, one case required a revision of antegrade continence enema due to wound breakdown and leakage of irrigation fluid around the stoma. The median follow-up period was 15.5 months (range, 5-32 months).The laparoscopic technique is a simple and effective approach in creating an antegrade continence enema. The use of a gastrostomy button can potentially reduce some of the complications commonly associated with an antegrade continence enema. We describe a procedure that incorporates the advantages of both laparoscopy and a button device, which is simple and easy to perform using just two ports.

H-type tracheoesophageal fistula is rare. Although symptoms are present from birth, its rarity and overlap of symptoms with other respiratory conditions makes diagnosis difficult and delayed. We report a case of H-type fistula in a 13-year-old girl that was detected incidentally during investigations for achalasia cardia.