Rapid detection of AAC(6')-Ib-cr production using a MALDI-TOF MS strategy.

Plasmid-mediated quinolone resistance mechanisms have become increasingly prevalent among Enterobacteriaceae strains since the 1990s. Among these mechanisms, AAC(6')-Ib-cr is the most difficult to detect. Different detection methods have been developed, but they require expensive procedures such as Sanger sequencing, pyrosequencing, polymerase chain reaction (PCR) restriction, or the time-consuming phenotypic method of Wachino. In this study, we describe a simple matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method which can be easily implemented in clinical laboratories that use the MALDI-TOF technique for bacterial identification. We tested 113 strains of Enterobacteriaceae, of which 64 harbored the aac(6')-Ib-cr gene. We compared two MALDI-TOF strategies, which differed by their norfloxacin concentration (0.03 vs. 0.5 g/L), and the method of Wachino with the PCR and sequencing strategy used as the reference. The MALDI-TOF strategy, performed with 0.03 g/L norfloxacin, and the method of Wachino yielded the same high performances (Se = 98 %, Sp = 100 %), but the turnaround time of the MALDI-TOF strategy was faster (<5 h), simpler, and inexpensive (<1 Euro). Our study shows that the MALDI-TOF strategy has the potential to become a major method for the detection of many different enzymatic resistance mechanisms.

Successful Treatment of Paraneoplastic Neuropathy and Pruritis With Scrambler Therapy: A Case Report.

Paraneoplastic neuropathy, including pruritis, remains a vexing problem as it often does not resolve even with successful treatment of cancer. Scrambler Therapy is a superficial form of neuromodulation that replaces the pain signal with "non-pain information" that is approved for chronic and neuropathic pain, with few side effects. We report here two cases of paraneoplastic neuropathy, one with additional pruritis, that both responded satisfactorily to Scrambler Therapy with no side effects.

An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria.

Mutations in Cu/Zn superoxide dismutase (SOD1) cause a subset of cases of familial amyotrophic lateral sclerosis. Four lines of mice accumulating one of these mutant proteins (G37R) develop severe, progressive motor neuron disease. At lower levels of mutant accumulation, pathology is restricted to lower motor neurons, whereas higher levels cause more severe abnormalities and affect a variety of other neuronal populations. The most obvious cellular abnormality is the presence in axons and dendrites of membrane-bounded vacuoles, which appear to be derived from degenerating mitochondria. Since multiple lines of mice expressing wild-type human SOD1 at similar and higher levels do not show disease, the disease in mice expressing the G37R mutant SOD1 must arise from the acquisition of an adverse property by the mutant enzyme, rather than elevation or loss of SOD1 activity.

Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.

Three glutamate transporters have been identified in rat, including astroglial transporters GLAST and GLT-1 and a neuronal transporter EAAC1. Here we demonstrate that inhibition of the synthesis of each glutamate transporter subtype using chronic antisense oligonucleotide administration, in vitro and in vivo, selectively and specifically reduced the protein expression and function of glutamate transporters. The loss of glial glutamate transporters GLAST or GLT-1 produced elevated extracellular glutamate levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis. The loss of the neuronal glutamate transporter EAAC1 did not elevate extracellular glutamate in the striatum but did produce mild neurotoxicity and resulted in epilepsy. These studies suggest that glial glutamate transporters provide the majority of functional glutamate transport and are essential for maintaining low extracellular glutamate and for preventing chronic glutamate neurotoxicity.

Vessel Wall MRI for Targeting Biopsies of Intracranial Vasculitis.

Central nervous system vasculitides are elusive diseases that are challenging to diagnose because brain biopsies have high false-negative rates. We sought to test the ability of contrast-enhanced, high-resolution 3D vessel wall MR imaging to identify vascular inflammation and direct open biopsies of intracranial target vessels and adjacent brain parenchyma. Eight of 9 specimens revealed vascular inflammation. We conclude that vessel wall MR imaging can identify inflamed intracranial vessels, enabling precise localization of biopsy targets.

TNF-alpha mediates SDF-1 alpha-induced NF-kappa B activation and cytotoxic effects in primary astrocytes.

Stromal-derived cell factor-1 alpha (SDF-1 alpha; CXCL12) and its receptor, CXCR4, are constitutively expressed on neuroepithelial cells and are believed to be involved in both development and pathological processes, such as AIDS-associated neurologic disorders. Here, we demonstrate that SDF-1 alpha activates NF-kappa B, stimulates production of chemokines and cytokines, and induces cell death in primary astrocytes, effects that depend on ongoing secretion of TNF-alpha. SDF-1 alpha upregulated TNF-alpha mRNA and protein secretion, as well as TNF receptor 2 expression. TNF-alpha treatment mimicked SDF-1 alpha induction of NF-kappa B, IL-1 alpha/beta, and RANTES, as well as cell death; neutralizing antibodies against TNF-alpha opposed these responses. We also found that SDF-1 alpha activated Erk1 and Erk2 (Erk1/2) MAPK in a biphasic fashion. Early Erk1/2 activation was stimulated directly by SDF-1 alpha and late activation was mediated by TNF-alpha. PD98059 suppression of early Erk1/2 activation correlated with reduction of SDF-1 alpha-induced TNF-alpha expression. Late Erk1/2 activation was involved in TNF-alpha-stimulated NF-kappa B activation and cytokine induction. SDF-1 alpha was induced in reactive CXCR4-positive astrocytes near axotomized spinal cord motor neurons, consistent with autocrine SDF-1/CXCR4 signaling in these cells. We propose that these novel effects of SDF-1 alpha are relevant to the pathogenic and developmental roles of SDF-1 alpha in the CNS.

Analysis of 30 Spinal Angiograms Falsely Reported as Normal in 18 Patients with Subsequently Documented Spinal Vascular Malformations.

BACKGROUND AND PURPOSE: The early diagnosis of spinal vascular malformations suffers from the nonspecificity of their clinical and radiologic presentations. Spinal angiography requires a methodical approach to offer a high diagnostic yield. The prospect of false-negative studies is particularly distressing when addressing conditions with a narrow therapeutic window. The purpose of this study was to identify factors leading to missed findings or inadequate studies in patients with spinal vascular malformations. MATERIALS AND METHODS: The clinical records, laboratory findings, and imaging features of 18 patients with spinal arteriovenous fistulas and at least 1 prior angiogram read as normal were reviewed. The clinical status was evaluated before and after treatment by using the Aminoff-Logue Disability Scale. RESULTS: Eighteen patients with 19 lesions underwent a total of 30 negative spinal angiograms. The lesions included 9 epidural arteriovenous fistulas, 8 dural arteriovenous fistulas, and 2 perimedullary arteriovenous fistulas. Seventeen patients underwent endovascular (11) or surgical (6) treatment, with a delay ranging between 1 week and 32 months; the Aminoff-Logue score improved in 13 (76.5%). The following factors were identified as the causes of the inadequate results: 1) lesion angiographically documented but not identified (55.6%); 2) region of interest not documented (29.6%); or 3) level investigated but injection technically inadequate (14.8%). CONCLUSIONS: All the angiograms falsely reported as normal were caused by correctible, operator-dependent factors. The nonrecognition of documented lesions was the most common cause of error. The potential for false-negative studies should be reduced by the adoption of rigorous technical and training standards and by second opinion reviews.

Ventral root avulsion: an experimental model of death of adult motor neurons.

The present study proposes a reproducible model of experimental degeneration of adult motor neurons in the rat. Avulsion of ventral roots in the adult lumbar cord transects motor axons at the root exit and leads to retrograde cell death of 80% of motor neurons 2 weeks later; this result follows a series of retrograde changes, including chromatolysis, loss of transmitter phenotype, and accumulation of phosphorylated neurofilaments in perikarya. Glial cells recruited at the site of retrograde injury express both microglia-specific epitopes (as exemplified by OX-42 immunoreactivity) and macrophage-specific markers (e.g., ED-1 immunoreactivity). Macrophage-specific markers become particularly intense 7 days postaxotomy and provide additional evidence of active phagocytosis of injured neurons. Ventral root avulsion is a very useful model for assessing mechanisms of motor neuron death and testing the ability of trophic factors and other agents to preserve the phenotype and promote the survival of adult motor neurons in vivo.

Human immunodeficiency virus and the peripheral nervous system workshop.

To provide a venue for a comprehensive multidisciplinary review of the current state of knowledge regarding the human immunodeficiency virus-associated peripheral neuropathies and to provide the institute with guidance in formulating future research initiatives, the National Institute of Neurological Disorders and Stroke (Bethesda, Md) convened a workshop on September 18 through 19, 2000. The participants were chosen from various disciplines and included clinicians, pathologists, neurobiologists, neurophysiologists, virologists, and neuroimmunologists. The present article summarizes the highlights of the meeting and includes the recommendations developed by the participants for future research. As might be expected in a rapidly evolving scientific field, the meeting was characterized by a lively and far-ranging discussion of data interpretation, experimental approaches, and priorities for future research. However, the recommendations presented at the end of this article constitute a consensus judgment reached by all of the participants of the most important areas for future research.

HIV in the brain: RNA levels and patterns of zidovudine resistance.

OBJECTIVE: To examine the association between HIV RNA levels, patterns of antiretroviral resistance, and neurologic status. METHODS: Autopsy samples from 13 HIV-infected subjects were examined for HIV-1 viral RNA (vRNA), and viral reverse transcriptase (RT) genotype was determined. All subjects had been clinically characterized using standard instruments before death. RESULTS: The median HIV-1 vRNA level in brain samples from subjects with moderate dementia was 7.79 log(10) copies/g (range 5.56 to 9.75 log(10) copies/g) compared with 5.44 log(10) copies/g (range 3.51 to 9.32 log(10) copies/g) for mildly demented subjects and 4.87 log(10) copies/g (3.51 to 6.86 log(10) copies/g) for those obtained from nondemented individuals. There were differences between subjects with moderate dementia and nondemented subjects (p = 0.0002) and between subjects with moderate and mild dementia (p = 0.0128). No significant differences among the groups were observed for vRNA levels in peripheral tissues. Some demented subjects had relatively low levels of HIV-1 vRNA, and paradoxically some nondemented subjects had high vRNA brain levels. Little subject effect in vRNA was noted in peripheral regions, but high regional variation in vRNA was noted within the brain. Patterns of the major zidovudine (ZDV) RT mutations in brain and peripheral tissues were concordant in most subjects. Subjects with longer duration of exposure to ZDV tended to have lower brain vRNA levels and a greater number of RT mutations than those with limited to no exposure. CONCLUSIONS: The presence and severity of HIV dementia correlates with the levels of productive HIV replication within the brain. Other pathophysiologic events (including macrophage activation) probably also contribute to neurologic dysfunction.

Detection of MHC class II-antigens on macrophages and microglia, but not on astrocytes and endothelia in active multiple sclerosis lesions.

Tissue sections of brains from patients with multiple sclerosis (MS) and from control individuals were immunostained with MHC class II and glial or vascular endothelial cell antibodies and analyzed by confocal microscopy. MHC class II was abundant in and around actively demyelinating MS lesions and was detected on microglia, phagocytic macrophages, and perivascular macrophages. Astrocytes and vascular endothelial cells were MHC class II-negative. Changes in the size and shape of MHC class II-positive cells associated with MS lesions suggest that microglia transform into phagocytic macrophages, and that they are actively involved in demyelination. Many MHC class II-positive perivascular macrophages within MS lesions contained abundant intracellular MHC class II immunoreactivity; these cells may be involved in antigen presentation and in T cell activation.

Histologic abnormalities associated with gadolinium enhancement on MR in the initial hours of experimental cerebral infarction.

PURPOSE: To determine the histologic changes associated with gadopentetate dimeglumine enhancement on MR images in acute focal cerebral ischemia. METHODS: In each of two baboons, a microcatheter was used to occlude partially the middle cerebral artery and reduce cerebral blood flow for approximately 3.5 hours. The catheter was then removed allowing reperfusion for approximately 3.5 hours. In two other baboons, cerebral blood flow was completely and irreversibly interrupted by injecting liquid adhesive into the middle cerebral artery. T2-weighted and serial enhanced T1-weighted MR images were obtained. Brain specimens were studied histopathologically. RESULTS: In the animals with incomplete and reversible reduction of cerebral blood flow, postcontrast T1-weighted images obtained during the initial 3 hours of ischemia showed focal areas of hypointensity. These areas were enhanced on later images. The areas of signal abnormality were subsequently found to be necrotic and were characterized by neuronal cytolysis and vascular "plugging." In the animals with complete and irreversible interruption of cerebral blood flow, no abnormal signal intensity or enhancement was observed. Histologic abnormalities were milder in these animals. CONCLUSIONS: Contrast enhancement on MR images in the initial hours of cerebral ischemia was associated with histologic evidence of tissue necrosis but was not associated with milder ischemic changes.

Irreversible regional cerebral ischemia: serial MR imaging and proton MR spectroscopy in a nonhuman primate model.

PURPOSE: To delineate the changes in proton MR spectroscopy and imaging that occur with acute, irreversible ischemia of the basal ganglia of a baboon. MATERIALS AND METHODS: The M1 segments of the middle cerebral arteries of six adult male baboons were occluded by endovascular means with microcatheters and N-butyl cyanoacrylate adhesive. Cerebral blood flow measurements were taken with positron emission tomography or radioactive microsphere techniques. Serial spatially localized proton MR spectroscopy of the basal ganglia and MR imaging of the brain were performed. The distribution of ischemic and infarcted tissue was demonstrated by histopathologic techniques or triphenyltetrazolium chloride staining. RESULTS: Radioactive microsphere or positron emission tomography measurements demonstrated no significant cerebral blood flow within the basal ganglia after occlusion of the middle cerebral artery. Proton MR spectroscopy of the basal ganglia demonstrated increasing cerebral lactate and decreasing N-acetyl aspartate within 30 minutes of middle cerebral artery occlusion. Changes in the MR imaging signal intensity of the basal ganglia were observed as early as 3.1 hours on T2-weighted, 3.3 hours on T1-weighted, and 6.1 hours on spin density-weighted images. The distribution of these changes correlated well with the histopathologic features of ischemia and infarction that were seen throughout the basal ganglia. CONCLUSION: Changes in MR imaging signal intensity corresponded to ischemia and infarction in our baboon model of acute irreversible ischemia of the basal ganglia. Increasing cerebral lactate and decreasing N-acetyl aspartate preceded changes in MR imaging signal intensity.

Biocompatibility of fixation materials in the brain.

Recent clinical reports documenting passive intracranial translocation of microplates and microscrews have prompted concerns regarding brain biocompatibility and neurotoxicity of fixation hardware used in craniofacial surgery. Although the effects of commercially pure titanium. Vitallium (cobalt-chromium-molybdenum), stainless steel, and various alloys have been well assessed in bone and soft tissues, there are no comprehensive studies of these materials in the brain. To investigate this, the biocompatibility of titanium, vitallium, and 316L stainless steel was evaluated in the rabbit brain and compared with silicone elastomer shunt tubing, a material that is used commonly as a neurosurgical implant material with well-established brain biocompatibility. Forty-eight juvenile New Zealand White rabbits were randomly assigned to one of three groups and underwent placement of either commercially pure titanium microscrews, vitallium microscrews, or 316L monofilament stainless steel wire into the parietal region. Silicone elastomer strips of similar size were implanted in the contralateral hemisphere of each rabbit. Animals were assessed daily for signs of neurotoxicity. Rabbits in each group were sacrificed at 2, 4, 8, and 26 weeks following implantation. Brains were sectioned at the implantation site and were examined by means of standard hematoxylin and eosin stains and with immunohistochemical markers sensitive to inflammatory changes in the brain. None of the animals showed any behavioral changes or neurologic deficits suggestive of either systemic or localized toxicity from the implant materials. Silicone clastomer was found to cause the least amount of inflammation relative to other materials tested at all sacrifice points, suggesting that as a standard neurosurgical implant material, it is an appropriate control for studies of brain biocompatibility. At 2 weeks, titanium was found to cause the largest inflammatory response in surrounding brain parenchyma based on analysis of markers for microglial proliferation, gliosis, and leukocyte infiltration. At the 26-week endpoint of our study, the biocompatibility of titanium was nearly equal to the biocompatibility of vitallium based on all studied markers of inflammation. A progressive increase in the inflammatory response surrounding stainless steel implants was noted at 8 and 26 weeks. Relative to all materials studied, at 26 weeks the greatest leukocyte response was found with stainless steel implants. Our results indicate that at the 26-week end-point of our study, titanium and vitallium incited a similar inflammatory response in the rabbit brain that was greater than the response found with silicone elastomer, a standard neurosurgical implant material, but less than that found with stainless steel wire, which is commonly recommended as an alternative fixation material.

Amyotrophic lateral sclerosis and Alzheimer disease. Lessons from model systems.

The human neurodegenerative diseases, including motor neuron disease and Alzheimer's disease (AD), are characterized by a selective involvement of certain regions of the brain/spinal cord and selected populations of neurons. Sporadic amyotrophic lateral sclerosis (ALS) is an age-associated disease with cytoskeletal abnormalities and death of motor neurons; familial ALS (FALS), an autosomal dominant disease linked to mutations in superoxide dismutase 1 (SOD1), is manifested by inclusions and degeneration of motor neurons. Autosomal dominant familial AD (FAD), linked to mutations in presenilin (PS1 and PS2) genes or the amyloid precursor protein (APP) gene, shows brain abnormalities (e.g., neurofibrillary tangles, deposits of .-amyloid A., and death of subsets of neurons) similar to those that occur in sporadic AD, the risk of which is enhanced by the presence of one or two copies of apolipoprotein E4 (apoE4) alleles. To examine the mechanisms of these diseases, investigators have used a variety of animal models, including experimentally produced, spontaneously occurring, or genetically engineered models of disease. Studies of models of degeneration of motor neurons (axotomy) and cytoskeletal abnormalities seen in motor neuron disease (i.e., axonopathy induced by .,.'-iminodipropionitrile (IDPN), hereditary canine spinal muscular atrophy (HCSMA), and neurofilament NF transgenic Tg mice) have demonstrated that NF-filled swellings of axons are related to alterations in the biology of NF transport. Tg mice with SOD1 mutations, which develop the clinical features of FALS, show selective degeneration of motor neurons, which is attributed to the acquisition of toxic properties by mutant SOD1. Models of AD include: aged monkeys that show both cognitive/memory deficits and cellular abnormalities (amyloid deposition/cytoskeletal abnormalities of neurons) in cortex and hippocampus; and Tg mice that express mutant human FAD-linked genes (i.e., APP and PS1) and show increased levels of A.42, amyloid deposits, dystrophic neurites, and local responses of astrocytes and microglia. This review discusses the behavioral/neuropathological features of AD, the results of investigations of mechanisms of disease in model systems, and potential utility of some of these models for testing new therapies.

Focal compression of the upper left thoracic intersegmental arteries as a potential cause of spinal cord ischemia.

BACKGROUND AND PURPOSE: This study was prompted by recurrent angiographic observations of focal stenoses involving the proximal segment of the left upper thoracic intersegmental arteries, a few centimeters distal to their origin. The purpose was to investigate the nature and prevalence of this anomaly. MATERIALS AND METHODS: The existence of non-ostial thoracic intersegmental artery stenoses was evaluated in 50 angiograms; the contribution of stenosed branches to the anterior spinal artery was recorded. Angiograms performed in 3 patients with right-sided aortas were also reviewed. The topographic relationships of the upper thoracic intersegmental artery with surrounding structures were investigated in 3 cadavers. RESULTS: Thirty-seven non-ostial stenoses were found in 26 patients (52%), predominantly on the left side (92%), between T3 and T8, most frequently at T4 and T5 (54%). Severe lesions were observed in 10% of cases. Patients with stenoses had fewer detectable anterior radiculomedullary arteries between T3 and T5 (35% versus 54%). Upper intersegmental artery stenoses, documented on the left side of all 3 specimens, appeared to be caused by the recurrent path of these intersegmental arteries related to the leftward position of the thoracic aorta, and by their course around reinforced paramedian longitudinal strands of the endothoracic fascia. CONCLUSIONS: Upper thoracic intersegmental artery stenoses are frequent. They result from the leftward deviation of the descending aorta and the existence of a fixed point along the course of the intersegmental arteries related to the endothoracic fascia. Because contributors to the spinal vascularization often originate at similar levels, these stenoses may play a role in the susceptibility of the upper and midthoracic spinal cord to ischemia.

Neuropathology of cervical dystonia.

The aim of this study was to search for neuropathological changes in postmortem brain tissue of individuals with cervical dystonia (CD). Multiple regions of formalin-preserved brains were collected from patients with CD and controls and examined with an extensive battery of histopathological stains in a two-stage study design. In stage one, 4 CD brains underwent a broad screening neuropathological examination. In stage two, these 4 CD brains were combined with 2 additional CD brains, and the subjective findings were quantified and compared to 16 age-matched controls. The initial subjective neuropathological assessment revealed only two regions with relatively consistent changes. The substantia nigra had frequent ubiquitin-positive intranuclear inclusions known as Marinesco bodies. Additionally, the cerebellum showed patchy loss of Purkinje cells, areas of focal gliosis and torpedo bodies. Other brain regions showed minor or inconsistent changes. In the second stage of the analysis, quantitative studies failed to reveal significant differences in the numbers of Marinesco bodies in CD versus controls, but confirmed a significantly lower Purkinje cell density in CD. Molecular investigations revealed 4 of the CD cases and 2 controls to harbor sequence variants in non-coding regions of THAP1, and these cases had lower Purkinje cell densities regardless of whether they had CD. The findings suggest that subtle neuropathological changes such as lower Purkinje cell density may be found in primary CD when relevant brain regions are investigated with appropriate methods.