Mistaken identity: Legionella micdadei appearing as acid-fast bacilli on lung biopsy of a hematopoietic stem cell transplant patient.

Legionella micdadei is a potential cause of invasive lung infections in immunocompromised hosts. On biopsy specimens, it can appear as an acid-fast bacillus (AFB) and can be mistaken for a member of genus Mycobacterium. As Legionella requires selective media to grow in culture, and the commonly used, commercially available urine antigen test for Legionella only detects Legionella pneumophila serogroup-1, but not L. micdadei, it is important to consider this organism in the differential diagnosis for AFB in immunocompromised hosts. We report a case of L. micdadei infection, which was initially treated empirically for non-tuberculous mycobacteria based on AFB staining of biopsy tissue before the final diagnosis was made.

Charles Bonnet syndrome in Asian patients in a tertiary ophthalmic centre.

AIMS: To describe the epidemiology of Charles Bonnet syndrome (CBS) among patients in an Asian tertiary ophthalmic centre and to describe the characteristics of the hallucinations experienced. METHODS: 1077 consecutive patients aged 50 years and above were asked a standardised question to determine if they had ever experienced formed visual hallucinations. All patients who experienced these symptoms were further interviewed using a detailed, standardised questionnaire to ascertain if they met the diagnostic criteria established for CBS. RESULTS: There were 491 men (45.6%) and 586 women (54.4%). The best corrected visual acuity ranged from 20/20 to light perception in the better seeing eye and from 20/20 to no light perception in the worse seeing eye. Four patients (0.4%) were diagnosed with CBS; two men and two women. There were two Chinese and two Indians. The average age of the CBS patients was 76.3 years (range 65-90 years). Two patients had cataracts, one had glaucoma, and one had both cataracts and glaucoma. A wide variety of visual hallucinations were reported. Three out of four patients experienced a negative reaction to their hallucinations. Only one patient had discussed his symptoms with a doctor. CONCLUSIONS: This is the first report on the epidemiology of CBS in Asian patients. The prevalence rate of CBS (0.4%) is slightly lower than in comparable studies in non-Asian populations. The nature of the hallucinations experienced were similar to those previously reported.

Neuroprotective effects of an adenoviral vector expressing the glucose transporter: a detailed description of the mediating cellular events.

Considerable knowledge exists concerning the events mediating neuron death following a necrotic insult; prompted by this, there have now been successful attempts to use gene therapy approaches to protect neurons from such necrotic injury. In many such studies, however, it is not clear what sequence of cellular events connects the overexpression of the transgene with the enhanced survival. We do so, exploring the effects of overexpressing the Glut-1 glucose transporter with an adenoviral vector in hippocampal cultures challenged with the excitotoxin kainic acid (KA). Such overexpression enhanced glucose transport, attenuated the decline in ATP concentrations, decreased the release of excitatory amino acid neurotransmitters, and decreased the total free cytosolic calcium load. Commensurate with these salutary effects, neuronal survival was enhanced with this gene therapy intervention. Thus, the neuroprotective effects of this particular gene therapy occurs within the known framework of the mechanisms of necrotic neuronal injury.

Calbindin d28k overexpression protects striatal neurons from transient focal cerebral ischemia.

BACKGROUND AND PURPOSE: Increased intracellular calcium accumulation is known to potentiate ischemic injury. Whether endogenous calcium-binding proteins can attenuate this injury has not been clearly established, and existing data are conflicting. Calbindin D28K (CaBP) is one such intracellular calcium buffer. We investigated whether CaBP overexpression is neuroprotective against transient focal cerebral ischemia. METHODS: Bipromoter, replication-incompetent herpes simplex virus vectors that encoded the genes for cabp and, as a reporter gene, lacZ were used. Sprague-Dawley rats received bilateral striatal injections of viral vector 12 to 15 hours before ischemia onset. With the use of an intraluminal occluding suture, animals were subjected to 1 hour of middle cerebral artery occlusion followed by 47 hours of reperfusion. Brains were harvested and stained with X-gal (to visualize beta-galactosidase, the gene product of lacZ). The number of remaining virally transfected, X-gal-stained neurons in both the ischemic and contralateral striata were counted and expressed as the percentage of surviving neurons in the ischemic striatum relative to the contralateral nonischemic striatum. RESULTS: Striatal neuron survivorship among cabp-injected animals was 53.5+/-4.1% (n=10) versus 26.8+/-5.4% among those receiving lacZ (n=9) (mean+/-SEM; P<0.001). CONCLUSIONS: We conclude that viral vector-mediated overexpression of CaBP leads to neuroprotection in this model of central nervous system injury. This is the first demonstration that CaBP overexpression protects neurons in a focal stroke model.

Indigenization and beyond: methodological relationalism in the study of personality across cultural traditions.

A brief intellectual history of the indigenization movement in Asia leads to the thesis that the generation of psychological knowledge is culture dependent. Indigenous psychologies go further and insist on viewing a target group from the natives' own standpoint. Psychological decentering underlies conceptions of human existence rooted in Asian intellectual traditions, in particular, relatedness between persons predominates in Confucianism. These conceptions demand new approaches to knowledge generation that signify a paradigmatic shift from methodological individualism to methodological relationalism. An implication is that relationships precede situations in the study of personality and social behavior. We define personality as the sum total of common attributes manifest in, and abstracted from, a person's behavior directly or indirectly observed across interpersonal relationships and situations over time. We rely on the notion that there are identifiable levels of cognition to develop a metatheoretical framework for reconstructing selfhood. Confronting the subject-object dichotomy opens the door to investigations of transcendent consciousness; confronting the self-other demarcation underlying Western theories leads to the construction of self-in-relations.

Gene therapies that enhance hippocampal neuron survival after an excitotoxic insult are not equivalent in their ability to maintain synaptic transmission.

Research shows that overexpression of cytoprotective genes can spare neurons from necrotic death, but few studies have addressed the functional status of surviving neurons. Overexpression of a brain glucose transporter, Glut-1, or the anti-apoptotic protein, Bcl-2, in rats decreases the size of hippocampal lesions produced by kainic acid (KA) treatment. In animals in which KA-induced lesions are reduced to similar extents by Glut-1 or Bcl-2 overexpression, spatial learning is spared by Glut-1, but not Bcl-2. We postulated that Glut-1 and Bcl-2 act differently to protect hippocampal function and investigated the effects of vector overexpression on synaptic physiology after KA treatment. Three days after KA and vector delivery to the dentate gyrus, mossy fiber-CA3 (MF-CA3) population excitatory postsynaptic potentials (EPSPs) were recorded in vitro. In addition to producing a lesion in area CA3, KA treatment reduced baseline MF-CA3 synaptic strength, posttetanic potentiation (PTP), and long-term potentiation (LTP). A similar reduction in the KA-induced lesion was produced by overexpression of Glut-1 or Bcl-2. Glut-1, but not Bcl-2, attenuated the impairments in synaptic strength and PTP. Overexpression of Glut-1 or Bcl-2 preserved LTP after KA treatment. Results indicate greater protection of MF-CA3 synaptic transmission with overexpression of Glut-1 compared to Bcl-2 and suggest that not all neuroprotective gene therapy techniques are equivalent in their ability to spare function.

Neuroprotective potential of a viral vector system induced by a neurological insult.

Gene transfer into neurons via viral vectors for protection against acute necrotic insults has generated considerable interest. Most studies have used constitutive vector systems, limiting the ability to control transgene expression in a dose-dependent, time-dependent, or reversible manner. We have constructed defective herpes simplex virus vectors designed to be induced by necrotic neurological insults themselves. Such vectors contain a synthetic glucocorticoid-responsive promoter, taking advantage of the almost uniquely high levels of glucocorticoids-adrenal stress steroids-secreted in response to such insults. We observed dose-responsive and steroid-specific induction by endogenous and synthetic glucocorticoids in hippocampal cultures. Induction was likely to be rapid enough to allow transgenic manipulation of relatively early steps in the cascade of necrotic neuron death. The protective potential of such a vector was tested by inclusion of a neuroprotective transgene (the Glut-1 glucose transporter). Induction of this vector by glucocorticoids decreased glutamatergic excitotoxicity in culture. Finally, both exogenous glucocorticoids and excitotoxic seizures induced reporter gene expression driven from a glucocorticoid-responsive herpes simplex virus vector in the hippocampus in vivo.

Limitations in the neuroprotective potential of gene therapy with Bcl-2.

Considerable attention has focused on the therapeutic transfer of genes with viral vectors into neurons for the purpose of protecting against neurological insults. A number of papers have reported that overexpression of the anti-apoptotic protein Bcl-2 can protect neurons both in vitro and in vivo against a variety of necrotic insults. An emerging literature suggests that the availability of energy tends to modulate a neuron towards dying apoptotically, rather than necrotically, in the aftermath of an insult. This suggests that an anti-apoptotic protein such as Bcl-2 should be minimally protective, at best, against purely energetic insults. In support of this idea, we report that overexpression of Bcl-2 with a herpes simplex viral vector fails to protect hippocampal neurons, either in vitro or in vivo, against the electron transport uncoupler 3-acetylpyridine (3AP). As a positive control, the same vector significantly protected against the excitotoxin kainic acid. This finding supports the view that neurotoxicity induced by 3AP is likely to have only minimal apoptotic facets. On a broader level, it suggests some limitations in the neuroprotective potential of gene therapy with Bcl-2.

An adenoviral vector expressing the glucose transporter protects cultured striatal neurons from 3-nitropropionic acid.

Considerable interest has focused on the possibility of using gene transfer techniques to introduce protective genes into neurons around the time of necrotic insults. We have previously used herpes simplex virus amplicon vectors to overexpress the rat brain glucose transporter, Glut-1 (GT), and have shown it to protect against a variety of necrotic insults both in vitro and in vivo, as well as to buffer neurons from the steps thought to mediate necrotic injury. It is critical to show the specificity of the effects of any such transgene overexpression, in order to show that protection arises from the transgene delivered, rather than from the vector delivery system itself. As such, we tested the protective potential of GT overexpression driven, in this case, by an adenoviral vector, against a novel insult, namely exposure of primary striatal cultures to the metabolic poison, 3-nitropropionic acid (3NP). We observed that GT overexpression buffered neurons from neurotoxicity induced by 3NP.

Calbindin D28K gene transfer via herpes simplex virus amplicon vector decreases hippocampal damage in vivo following neurotoxic insults.

Increases in cytoplasmic Ca2+ concentration ([Ca2+]i) can lead to neuron death. Preventing a rise in [Ca2+]i by removing Ca2+ from the extracellular space or by adding Ca2+ chelators to the cytosol of target cells ameliorates the neurotoxicity associated with [Ca2+]i increases. Another potential route of decreasing the neurotoxic impact of Ca2+ is to overexpress one of the large number of constitutive calcium-binding proteins. Previous studies in this laboratory demonstrated that overexpression of the gene for the calcium-binding protein calbindin D28K, via herpes simplex virus (HSV) amplicon vector, increases the survival of hippocampal neurons in vitro following energetic or excitotoxic insults but not following application of sodium cyanide. We now report that in vivo hippocampal infection with the calbindin D28K HSV vector increases neuronal survival in the dentate gyrus after application of the antimetabolite 3-acetylpyridine and increases transsynaptic neuronal survival in area CA3 following kainic acid neurotoxicity. The protective effects of infection with the calbindin D28K vector in an intact brain may prove to be beneficial during changes in Ca2+ homeostasis caused by neurological trauma associated with aging and certain neurological diseases.

Gene therapy with HSP72 is neuroprotective in rat models of stroke and epilepsy.

Brain areas damaged by stroke and seizures express high levels of the 72-kd heat shock protein (HSP72). Whether HSP72 represents merely a marker of stress or plays a role in improving neuron survival in these cases has been debated. Some induced tolerance experiments have provided correlative evidence for a neuroprotective effect, and others have documented neuroprotection in the absence of HSP72 synthesis. We report that gene transfer therapy with defective herpes simplex virus vectors overexpressing hsp72 improves neuron survival against focal cerebral ischemia and systemic kainic acid administration. HSP72 overexpression improved striatal neuron survival from 62.3 to 95.4% in rats subjected to 1 hour of middle cerebral artery occlusion, and improved survival of hippocampal dentate gyrus neurons after systemic kainic acid administration, from 21.9 to 64.4%. We conclude that HSP72 may participate in processes that enhance neuron survival during transient focal cerebral ischemia and excitotoxin-induced seizures.

Gene transfer of calbindin D28k cDNA via herpes simplex virus amplicon vector decreases cytoplasmic calcium ion response and enhances neuronal survival following glutamatergic challenge but not following cyanide.

Excitatory amino acid overstimulation of neurons can lead to a marked rise in cytoplasmic Ca2+ concentration ([Ca2+])i) and be followed by neuron death from hours to days later. If the rise in [Ca2+]i is prevented, either by removing Ca2+ from the extracellular environment or by placing Ca2+ chelators in the cytosol of the stimulated cells, the neurotoxicity associated with excitotoxins can be ameliorated. We have recently shown that neurons infected with a herpes simplex virus amplicon vector expressing cDNA for calbindin D28k responded to hypoglycemia with decreased [Ca2+]i and increased survival relative to controls. We now report that vector-infected neurons respond to glutamatergic insults with lower [Ca2+]i than controls and with increased survival. Infected neurons exposed to sodium cyanide did not respond with lower [Ca2+]i than controls, nor did they demonstrate increased survival postinsult. We examine these results in light of our earlier report and in the context of the potential of vectors like this for neuronal gene therapy.

Increased expression of calbindin D28k via herpes simplex virus amplicon vector decreases calcium ion mobilization and enhances neuronal survival after hypoglycemic challenge.

Disruption of Ca2+ homeostasis often leads to neuron death. Recently, the function of calcium-binding proteins as neuronal Ca2+ buffers has been debated. We tested whether calbindin D28k functions as an intracellular Ca2+ buffer by constructing bicistronic herpes simplex virus vectors to deliver rat calbindin cDNA to hippocampal neurons in vitro. Neurons were infected with vectors delivering calbindin or a negative control or were mock-infected. After 12 or 24 h of hypoglycemia, infected cells were made aglycemic during fura-2 calcium ratiometric imaging. In response to this challenge, neuronal overexpressing calbindin had less Ca2+ mobilized as compared with negative controls or mock-infected cells. Cells were assayed for survival after 12- or 24-h hypoglycemia or aglycemia. The calbindin vector decreased neuronal death due to hypoglycemia but not aglycemia. Here we demonstrate, in response to hypoglycemic challenge, both decreased Ca2+ mobilization and increased survival of cells infected with the calbindin vector.

Herpes simplex viral vectors expressing Bcl-2 are neuroprotective when delivered after a stroke.

Considerable interest has focused on the possibility of using viral vectors to deliver genes to the central nervous system for the purpose of decreasing necrotic neuronal injury. To that end, we have previously shown that a herpes simplex virus (HSV) vector expressing Bcl-2 could protect neurons from ischemia. In that study, vector was delivered before the ischemia. However, for such gene therapy to be of clinical use, vectors must be protective even if delivered after the onset of the insult. In the present study, we show that an HSV vector expressing Bcl-2 protects striatal neurons when delivered after focal ischemia. Rats were exposed to middle cerebral artery occlusion for 1 hour, followed by reperfusion, and damage was assessed 48 hours later. Delivery of the Bcl-2 vector 30 minutes after reperfusion (i.e., 1.5 hours after ischemia onset) prevented any significant loss of virally-targeted neurons in the striatum. In contrast, in rats microinfused with a vector only expressing a reporter gene, a highly significant loss of neurons occurred. By 4 hours into the reperfusion period (5 hours after ischemia onset), delivery of the Bcl-2 vector was no longer protective. These data show the efficacy of postinsult gene therapy strategies for the brain, underline the finite length of this temporal therapeutic window, and support the growing evidence attesting to the neuroprotective potential of Bcl-2.

Defective herpes simplex virus vectors expressing the rat brain stress-inducible heat shock protein 72 protect cultured neurons from severe heat shock.

Recently, preinduction of the heat shock response has been shown to protect CNS neurons undergoing various stressful insults, e.g., heat, ischemia, or exposure to excitotoxins. However, it is not known which of the proteins induced by the heat shock response mediate the protective effects. Previous correlative evidence points to a role for the highly stress-induced 72-kDa heat shock protein (hsp72). However, it is not known whether hsp72 expression alone can protect against a range of acute neuronal insults. We constructed a herpes simplex virus-1 vector carrying the rat brain stress-inducible hsp72 gene and the Escherichia coli lacZ (marker) gene. Infection with the vector caused hippocampal neurons to coexpress hsp72 and beta-galactosidase. Infection with a control vector led to marker gene expression only. Overexpression of hsp72 protected cultured hippocampal neurons against a heat shock but not against the metabolic toxin 3-nitropropionic acid or the excitotoxin glutamate. This is the first published report of protection following heat shock protein transfection in CNS neurons.

Inducible gene expression from defective herpes simplex virus vectors using the tetracycline-responsive promoter system.

Herpes simplex virus-based amplicon vectors have been used for gene transfer into cultured neurons and the adult CNS. Since constitutive expression of a foreign gene or overexpression of an endogenous gene may have deleterious effects, the ability to control temporal expression would be advantageous. To achieve inducible gene expression, we have incorporated the tetracycline-responsive promoter system into amplicon vectors and showed, both in vitro and in vivo, that expression can be modulated by tetracycline. Using the firefly luciferase as the reporter gene, maximal repression by tetracycline in hippocampal cultures was about 50-fold. Withdrawal of tetracycline derepressed gene expression, reaching maximal levels within 10-12 h. In contrast, addition of tetracycline to cultures without prior tetracycline exposure inhibited gene expression rapidly; luciferase activity was reduced to less than 8% within 24 h. In adult rat hippocampus, vectors expressing luciferase or the Escherichia coli lacZ were repressed by tetracycline 9- and 60-fold, respectively. Maximum gene expression from the vectors occurred 2-3 days post-infection and declined thereafter. Such decline impeded further induction of expression by withdrawing tetracycline. This study demonstrates the feasibility of incorporating a powerful inducible promoter system into HSV vectors. The development of such an inducible viral vector system for gene transfer into the adult CNS might prove to be of experimental and therapeutic value.

Energy and glutamate dependency of 3-Nitropropionic acid neurotoxicity in culture.

3-Nitropropionic acid (3-NP) irreversibly inhibits the activity of the mitochondrial enzyme succinate dehydrogenase, leading to selective striatal lesions when administered in vivo. We studied the effects of 3-NP on dissociated cultures of neurons and glia with the following findings: (a) 3-NP killed cultured striatal neurons with a median lethal dose of 2.5 mM after 20 h of incubation in 20.0 mM glucose medium. Despite its selective toxicity in vivo, cultured striatal, hippocampal, septal, and hypothalamic neurons were similarly sensitive to 3-NP incubation. (b) 3-NP's effects were remarkably energy substrate dependent, with the median lethal dose dropping over an order of magnitude when glucose concentrations were lowered to 3.0 mM, a condition that was itself nontoxic. Cultures exposed to 3-NP had a far greater sensitivity to energy availability than those exposed to glutamate. (c) Recent work suggests that 3-NP toxicity may be partially mediated by excitotoxins. Our experiments show that neither kynurenic acid, a nonspecific glutamate receptor antagonist, nor the NMDA-receptor antagonist, DL-2-amino-7-phosphonoheptanoic acid, either in combination or alone, reduced 3-NP toxicity in striatal cultures. However, the noncompetitive NMDA antagonist MK-801 did attenuate 3-NP toxicity.