Donald E. Born

HIF induces human embryonic stem cell markers in cancer cells.

Low oxygen levels have been shown to promote self-renewal in many stem cells. In tumors, hypoxia is associated with aggressive disease course and poor clinical outcomes. Furthermore, many aggressive tumors have been shown to display gene expression signatures characteristic of human embryonic stem cells (hESC). We now tested whether hypoxia might be responsible for the hESC signature observed in aggressive tumors. We show that hypoxia, through hypoxia-inducible factor (HIF), can induce an hESC-like transcriptional program, including the induced pluripotent stem cell (iPSC) inducers, OCT4, NANOG, SOX2, KLF4, cMYC, and microRNA-302 in 11 cancer cell lines (from prostate, brain, kidney, cervix, lung, colon, liver, and breast tumors). Furthermore, nondegradable forms of HIFα, combined with the traditional iPSC inducers, are highly efficient in generating A549 iPSC-like colonies that have high tumorigenic capacity. To test potential correlation between iPSC inducers and HIF expression in primary tumors, we analyzed primary prostate tumors and found a significant correlation between NANOG-, OCT4-, and HIF1α-positive regions. Furthermore, NANOG and OCT4 expressions positively correlated with increased prostate tumor Gleason score. In primary glioma-derived CD133 negative cells, hypoxia was able to induce neurospheres and hESC markers. Together, these findings suggest that HIF targets may act as key inducers of a dynamic state of stemness in pathologic conditions.

Neuronal nuclear antigen (NeuN): A marker of neuronal maturation in the early human fetal nervous system

Neuronal nuclear antigen (NeuN) immunocytochemistry was studied in 15 normal human fetal nervous systems of 8-24 weeks gestation and in four term neonates. Material was derived from products of conception or from autopsy. Antigen retrieval was enhanced for immunocytochemistry by microwave heating of formalin-fixed paraffin sections. NeuN appears highly specific as a marker of neuronal nuclei in human fetal brain. Only rare nuclei are recognized in the germinal matrix. Cerebellar external granule cells are more strongly immunoreactive than postmigratory internal granule cells until 24 weeks gestation; by term most internal and only a few external granule cells are recognized by NeuN antibody. In the cerebrum, some reactive nuclei are demonstrated along radial glial fibers, particularly near the cortical plate. Within the cortical plate, only deep neurons (future layers 4-6) are marked at 19-22 weeks, but by 24 weeks most neurons in the cortical plate exhibit immunoreactivity, though at term some in layer 2 are still non-reactive. Some neurons fail to be recognized by NeuN at all ages: Cajal-Retzius cells, Purkinje cells, inferior olivary and dentate nucleus neurons, and sympathetic ganglion cells are examples. Despite their common origin in the cerebellar tubercle, basal pontine neurons are strongly reactive even before midgestation, hence NeuN does not predict embryonic origin. Neurons of dorsal root and cranial nerve ganglia are reactive even at 8 weeks. This study of normal fetal central nervous system provides a basis for neuropathological evaluation and as a prelude to applications in cerebral dysgeneses.

Hippocampal GABA and glutamate transporter immunoreactivity in patients with temporal lobe epilepsy

Objective: Sodium-coupled transporters remove extracellular neurotransmitters and alterations in their function could enhance or suppress synaptic transmission and seizures. This study determined hippocampal gamma-aminobutyric acid (GABA) and glutamate transporter immunoreactivity (IR) in temporal lobe epilepsy (TLE) patients. Methods: Hippocampal sclerosis (HS) patients (n = 25) and non-HS cases (mass lesion and cryptogenic; n = 20) were compared with nonseizure autopsies (n = 8). Hippocampal sections were studied for neuron densities along with IR for glutamate decarboxylase (GAD; presynaptic GABA terminals), GABA transporter-1 (GAT-1; presynaptic GABA transporter), GAT-3 (astrocytic GABA transporter), excitatory amino acid transporter 3 (EAAT3; postsynaptic glutamate transporter), and EAAT2-1 (glial glutamate transporters). Results: Compared with autopsies, non-HS cases with similar neuron counts showed: 1) increased GAD IR gray values (GV) in the fascia dentata outer molecular layer (OML), hilus, and stratum radiatum; 2) increased GAT-1 OML GVs; 3) increased astrocytic GAT-3 GVs in the hilus and Ammon’s horn; and 4) no IR differences for EAAT3-1. HS patients with decreased neuron densities demonstrated: 1) increased OML and inner molecular layer GAD puncta; 2) decreased GAT-1 puncta relative to GAD in the stratum granulosum and pyramidale; 3) increased GAT-1 OML GVs; 4) decreased GAT-3 GVs; 5) increased EAAT3 IR on remaining granule cells and pyramids; 6) decreased glial EAAT2 GVs in the hilus and CA1 stratum radiatum associated with neuron loss; and 7) increased glial EAAT1 GVs in CA2/3 stratum radiatum. Conclusions: Hippocampal GABA and glutamate transporter IR differ in TLE patients compared with autopsies. These data support the hypothesis that excitatory and inhibitory neurotransmission and seizure susceptibility could be altered by neuronal and glial transporters in TLE patients.

Mycobacterium marinum Infection of Adult Zebrafish Causes Caseating Granulomatous Tuberculosis and Is Moderated by Adaptive Immunity

ABSTRACT The zebrafish, a genetically tractable model vertebrate, is naturally susceptible to tuberculosis caused by Mycobacterium marinum, a close genetic relative of the causative agent of human tuberculosis, Mycobacterium tuberculosis. We previously developed a zebrafish embryo-M. marinum infection model to study host-pathogen interactions in the context of innate immunity. Here, we have constructed a flowthrough fish facility for the large-scale longitudinal study of M. marinum-induced tuberculosis in adult zebrafish where both innate and adaptive immunity are operant. We find that zebrafish are exquisitely susceptible to M. marinum strain M. Intraperitoneal injection of five organisms produces persistent granulomatous tuberculosis, while the injection of ∼9,000 organisms leads to acute, fulminant disease. Bacterial burden, extent of disease, pathology, and host mortality progress in a time- and dose-dependent fashion. Zebrafish tuberculous granulomas undergo caseous necrosis, similar to human tuberculous granulomas. In contrast to mammalian tuberculous granulomas, zebrafish lesions contain few lymphocytes, calling into question the role of adaptive immunity in fish tuberculosis. However, like rag1 mutant mice infected with M. tuberculosis, we find that rag1 mutant zebrafish are hypersusceptible to M. marinum infection, demonstrating that the control of fish tuberculosis is dependent on adaptive immunity. We confirm the previous finding that M. marinum ΔRD1 mutants are attenuated in adult zebrafish and extend this finding to show that ΔRD1 predominantly produces nonnecrotizing, loose macrophage aggregates. This observation suggests that the macrophage aggregation defect associated with ΔRD1 attenuation in zebrafish embryos is ongoing during adult infection.

Multimodality treatment of brain arteriovenous malformations with microsurgery after embolization with Onyx: Single-center experience and technical nuances

OBJECTIVETo report our experience with the treatment of brain arteriovenous malformations (AVM) with microsurgical resection after embolization with Onyx liquid embolic agent (eV3, Irvine, CA). METHODSBetween August 2005 and December 2006, 28 patients were treated by the same surgical-endovascular team. Twenty-eight AVMs were embolized preoperatively in 55 sessions (71 pedicles) with Onyx. We analyzed the AVM size, volume, number of embolization sessions, degree of preoperative obliteration, time to embolization and resection after the bleed, intraprocedural complications, intraoperative blood loss, other complications, and postoperative outcome at 6 months. Technical nuances of the embolization and surgical resection of the embolized AVMs are illustrated in illustrative cases. RESULTSThe average size and volume of AVMs treated with Onyx were 3.56 cm (largest, 7.6 cm), and 13.03 ml, respectively. The average Spetzler-Martin grade was 2.75. The average preoperative volumetric obliteration was 74.1%. The average blood loss during resection of embolized AVMs was 348 ml. Complications related to embolization were stuck microcatheter (two patients), proximal vessel perforation (one patient), and anterior choroidal territory stroke (one patient). Surgical complications included wound infection (one patient), residual AVM nidus (one patient), normal pressure perfusion breakthrough with worsening of neurological deficit caused by embolization (one patient), and new-onset motor deficits in five patients. At the time of the 6-month follow-up examination, four patients with new-onset motor deficits had recovered completely or nearly completely, and one patient was disabled. One patient died, never recovering from the initial poor condition due to the bleed. Pathological examination of resected AVMs showed angionecrosis in 42.9%, foreign body giant cells in 39.3%, and evidence of recanalization of Onyx-embolized vessels in 14.3% of specimens. CONCLUSIONMultimodality treatment with microsurgery is safe and effective after embolization with Onyx. High occlusion rates and low complication rates were observed after Onyx embolization and were comparable to those in previous reports. Superselective intranidal or perinidal catheter positions and slow, controlled injections that protect the draining veins make the therapy safe even in complex AVMs and critical locations. We recommend resection of the AVM despite apparently complete embolization with Onyx. Team work and coordination between the surgeon and the interventional neuroradiologist are important to achieve a good outcome.

Afferent influences on brainstem auditory nuclei of the chick : nucleus magnocellularis neuronal activity following cochlea removal

Elimination of presynaptic elements often results in marked changes, such as atrophy and death, in postsynaptic neurons in the central nervous system. These transneuronal changes are particularly rapid and profound in young animals. In order to understand the cellular events underlying transneuronal regulation it is necessary to explore changes in the local environment of neurons following manipulations of their afferents. In previous investigations we have documented a variety of rapid and marked cellular changes in neurons of the cochlear nucleus of neonatal chicks (n. magnocellularis) following cochlea removal. In adult chickens, however, these transneuronal changes are either absent or minor. The goals of the studies presented here were to examine changes in the electrical activity of nucleus magnocellularis cells and their afferents following removal of the cochlea and to determine if these changes were similar in adult and neonatal animals. Two measures of electrical activity were used; multiunit recording with microelectrodes and incorporation of radiolabeled 2-deoxyglucose (2-DG). Microelectrode recordings revealed high levels of spontaneous activity in n. magnocellularis and n. laminaris, the binaural target of n. magnocellularis neurons. Neither puncturing of the tympanic membrane nor removal of the columella causes significant changes in spontaneous activity, although the latter results in a profound hearing loss (40-50 dB). Removal of the cochlea, on the other hand, results in immediate cessation of all extracellular electrical activity in the ipsilateral n. magnocellularis. Recordings from the same location for up to 6 h failed to reveal any return of spontaneous activity. When the electrode tip was placed in n. laminaris, unilateral cochlea removal had no discernible effect on extracellularly recorded spontaneous activity, probably due to the high levels of excitatory input from the intact ear. Bilateral cochlea removal, however, completely eliminated activity in n. laminaris. 2-DG studies conducted 1 h to 8 days following unilateral cochlea removal revealed marked decreases in 2-DG incorporation in the ipsilateral n. magnocellularis and bilaterally in the n. laminaris target of the ablated cochlea. No compensatory return of 2-DG incorporation was observed for up to 8 days. Comparisons of adult and neonatal chicks failed to reveal significant differences in the effects of cochlea removal on multiunit activity or 2-DG incorporation, suggesting that age differences in transneuronal regulation are due to intrinsic biochemical differences in young and adult neurons rather than differences in the proportion of synaptic input that has been abolished.

Outcome after surgery in patients with refractory temporal lobe epilepsy and normal MRI.

Our purpose is to determine predictors of outcome in patients with refractory temporal lobe epilepsy and normal high resolution magnetic resonance imaging (MRI) who undergo surgical therapy. We identified 23 patients who underwent temporal lobectomy and had normal pre-operative MRI, including surface coil phased array temporal lobe imaging. All were followed at least 2 years after surgery. We graded outcome as seizure-free, > 75% reduction in seizures, or < 75% reduction in seizures. We examined pre-operative interictal and ictal electroencephalographic (EEG) findings, age of onset, gender, duration of epilepsy, risk factors, family history, physical findings, age at operation, side of operation, and pathology of resected tissue in order to determine if any of these factors were associated with outcome. Overall, 48% (11/23) of patients were seizure-free, 39% (9/23) had > 75% reduction in seizures, while 13% (3/23) had < 75% reduction in seizures. Only the EEG findings were useful in predicting outcome. When ictal onsets arose from basal-temporal regions, 61% (11/18) of patients were seizure-free, while none (0/5) were seizure-free when seizures arose from mid-posterior temporal regions (P = 0.04). Interictally, if all epileptiform patterns were localized exclusively to one basal-temporal region, a finding that invariably correlated with ictal onsets, 78% (7/9) of patients were seizure-free, while only 29% (4/14) were seizure-free if discharges were bilateral or multifocal (P = 0.04). We conclude that surgery may be a reasonable treatment for some patients with intractable temporal lobe seizures and normal MRI. The best outcomes occur when seizure onsets and interictal epileptiform patterns are exclusive to one basal-temporal region. Unfavorable outcomes are most likely to occur when ictal origins are from mid-posterior temporal regions and when interictal discharges are bitemporal or multifocal in distribution.

Factors Predicting Outcome of Surgery for Intractable Epilepsy with Pathologically Verified Mesial Temporal Sclerosis

Summary:  Purpose: To examine the subgroup of patients with medically intractable epilepsy receiving temporal lobectomies who have pathologically verified mesial temporal sclerosis (MTS) and to determine the relation of demographic and clinical factors, results of diagnostic testing, and details of the surgical procedure with prognosis for achieving control of seizures.

Hippocampal N-methyl-D-aspartate receptor subunit mRNA levels in temporal lobe epilepsy patients.

Changes in the subunit stoichiometry of the N‐methyl‐D‐aspartate (NMDA) receptor (NMDAR) alters its channel properties, and may enhance or reduce neuronal excitability in temporal lobe epilepsy patients. This study determined whether hippocampal NMDA receptor subunit mRNA levels were increased or decreased in temporal lobe epilepsy patients compared with nonseizure autopsy cases. Hippocampal sclerosis (HS; n = 16), non‐HS (n = 10), and autopsy hippocampi (n = 9) were studied for NMDAR1 (NR1) and NR2A–D mRNA levels by using semiquantitative in situ hybridization techniques, along with neuron densities. Compared with autopsy hippocampi, non‐HS and HS patients showed increased NR2A and NR2B hybridization densities per dentate granule cell. Furthermore, non‐HS hippocampi showed increased NR1 and NR2B mRNA levels per CA2/3 pyramidal neuron compared with autopsy cases. HS patients, by contrast, showed decreased NR2A hybridization densities per CA2/3 pyramidal neuron compared with non‐HS and autopsy cases. These findings indicate that chronic temporal lobe seizures are associated with differential changes in hippocampal NR1 and NR2A–D hybridization densities that vary by subfield and clinical–pathological category. In temporal lobe epilepsy patients, these findings support the hypothesis that in dentate granule cells NMDA receptors are increased, and excitatory postsynaptic potentials should be strongly NMDA mediated compared with nonseizure autopsies. HS patients, by comparison, showed decreased pyramidal neuron NR2A mRNA levels, and this suggests that NMDA‐mediated pyramidal neuron responses should be reduced in HS patients compared with non‐HS cases.

Synaptophysin immunocytochemistry with thermal intensification: a marker of terminal axonal maturation in the human fetal nervous system.

Synaptophysin is a protein of synaptic vesicles and may be demonstrated in tissue sections of human brain and spinal cord by immunocytochemistry using a monoclonal antibody. Synaptophysin immunoreactivity was studied in paraffin-embedded sections of the central nervous system (CNS) in 14 normal human fetuses and neonates ranging in age from 8 to 41 weeks gestation, and in three brains with heterotopic neurons or malformations. A progressive expression of synaptophysin is seen in axonal terminals within grey matter in various parts of the CNS, beginning in the ventral horns of the spinal cord and brainstem tegmentum at 12-14 weeks. In the cerebellum, the molecular layer shows a band of reactivity from 18 weeks; by term two parallel bands of synaptophysin are seen in the molecular layer and reactivity also is demonstrated in the Purkinje and internal granular layers. In the cerebral neocortex, the molecular zone has weak synaptophysin reactivity as early as 10 weeks, though reactivity is not detected in the deep layers of the cortical plate until 19 weeks and in layers 2-4 until 25 weeks gestation. Synaptophysin reactivity is strong at the surface of neurons but not detected in their somatic cytoplasm; coarsely beaded reactivity within the neuropil probably corresponds to synaptic vesicles in terminal axons. Similar granular synaptophysin reactivity is seen around heterotopic neurons in the subcortical white matter, in dysgenesis of the cerebellar cortex and in the residual anencephalic forebrain. Thermal intensification by heating the incubating solution in a microwave oven often enhances immunoreactivity because of more complete antigen retrieval and is recommended for tissue stored in formalin or in paraffin for long periods. Synaptophysin provides a useful tissue marker of synaptogenesis during normal development and in cerebral dysgeneses, and may provide useful correlations with functional imaging of the brain in living patients. Used in conjunction with other neuronal markers, the expression of synaptophysin in terminal axons of distant neurons, in temporal relation to the maturation of the neurons they innervate, may provide clues to the pathogenesis of epilepsy in early infancy.