Dora Ho

Abstract

Malaria results in over 650 000 deaths each year; thus, there is an urgent need for an effective vaccine. Pre-clinical studies and recently reported human trials suggest that pre-erythrocytic stage vaccines can provide protection against infection. A Phase 1, randomized, placebo-controlled, dose-escalation study was conducted with a vaccine composed of a replication-deficient adenovirus-35 backbone with P. falciparum circumsporozoite (CS) surface antigen (Ad35.CS.01). Healthy adult subjects received three doses of 10 (8), 10 (9), 10 (10), or 10 (11) vp/mL Ad35.CS.01 vaccine or saline placebo intramuscularly at 0, 1, and 6-mo intervals. Adverse events were assessed and anti-CS antibody responses were determined by ELISA. Seventy-two individuals were enrolled, with age, gender, and ethnicity similar across each study arm. While the vaccine was generally well tolerated, adverse events were more frequent in the highest dose groups (10 (10) and 10 (11) vp/mL). More robust humoral responses were also noted at the highest doses, with 73% developing a positive ELISA response after the three dose series of 10 (11) vp/mL. The Ad35.CS.01 vaccine was most immunogenic at the highest dosages (10 (10) and 10 (11) vp/mL). Reactogenicity findings were more common after the 10 (11) vp/mL dose, although most were mild or moderate in nature and resolved without therapy.

View details for DOI 10.4161/hv.26038

View details for PubMedID 23955431

View details for DOI 10.4161/hv.26038

View details for Web of Science ID 000330383200015

Abstract

Objectives: To describe and discuss the utility and potential pitfalls of ribosomal RNA locus sequencing for direct identification of invasive fungi from fresh and formalin-fixed, paraffin-embedded specimens. Methods: DNA was extracted from fresh and formalin-fixed, paraffin-embedded tissue and subjected to real-time polymerase chain reaction (PCR) targeting ITS2 and D2 regions of fungal ribosomal RNA locus. Cycle sequencing was performed on PCR products, and the identity of sequences was determined using a public database. Results: Four clinical cases of invasive fungal infection are presented to illustrate the utility of DNA sequencing for determining etiology when microbiological culture is negative, for shortening the time to identification of slow-growing fungi, for guiding antifungal therapy, and for shedding light on the pathogenesis of disseminated fungal infection. Conclusions: Fungal ribosomal RNA locus sequencing from fresh or formalin-fixed, paraffin-embedded specimens is a powerful tool for rapid and accurate diagnosis of patients with culture-negative or uncultured invasive mycosis.

View details for DOI 10.1309/AJCPNSU2SDZD9WPW

View details for Web of Science ID 000322149600010

View details for PubMedID 23897255

Abstract

The clinical significance of the detection of low copy numbers of cytomegalovirus (CMV) DNA in immune-suppressed patients remains unclear. In this study, we compared the artus CMV Rotor-Gene PCR, utilizing an automated nucleic acid extraction and assay setup (the artus CMV protocol), with the COBAS Amplicor CMV Monitor test (our reference protocol). We then analyzed the results of all CMV PCR tests ordered following the implementation of the artus CMV protocol at our institution and followed 91 adult patients with positive test results. The artus CMV protocol had a linear range extending from 2.0 to 7.0 log(10) copies/ml and had a lower limit of 95% detection of 57 copies/ml. With archived plasma samples, this protocol demonstrated 100% sensitivity and 94% specificity for the detection of CMV DNA. Following implementation of the artus CMV protocol, 320 of 1,403 (22.8%) plasma samples tested positive (compared with 323/3,579 [9.0%] samples in the preceding 6 months), and 227 (16.2%) samples had copy numbers of <400/ml. Ninety-one adult patients had at least one positive test. The data were analyzed using a threshold of 200 copies/ml, and in 22 episodes, the viral load increased from <200 copies/ml to ? 200 copies/ml on sequential tests. In 21 of these 22 episodes, either the viral load continued to increase or antiviral treatment was initiated in response to the repeat value. In summary, we evaluate the performance characteristics of a protocol utilizing the artus CMV PCR and identify clinically meaningful changes in CMV DNA copy numbers even when they are initially detected at a low level.

View details for DOI 10.1128/JCM.06800-11

View details for Web of Science ID 000307360800033

View details for PubMedID 22518866

Abstract

The reconstitution of immune function after hematopoietic cell transplant (HCT) plays an important role in the control of viral infections. Both donor and recipient cytomegalovirus (CMV) serostatus has been shown to contribute to effective immune function; however, the influence of a nonmyeloablative preparative (NMA) regimen using total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) on antiviral immune reconstitution has not yet been described. In 117 recipients of NMA HCT patients following ATG and TLI, not unexpectedly, CMV viremia was seen in approximately 60% of the seropositive patients regardless of donor serostatus, and recipient seropositivity significantly increased the odds of CMV viremia after transplant in a multivariate analysis. The administration of ATG and TLI resulted in a strikingly earlier viremia in the posttransplant period when compared to the previously reported timing of viremia following myeloablative preparative regimens, especially for transplant recipients who were seropositive for CMV with seronegative donors. Furthermore, early viremia in the setting of a CMV naïve donor was associated with a delay in functional antiviral control. These observations demonstrate the dynamic nature of immunity in relation to CMV antigen exposure in the complex environment resulting from NMA conditions where both donor and residual recipient immune response affect viral control.

View details for DOI 10.1016/j.bbmt.2010.08.010

View details for Web of Science ID 000290061500012

View details for PubMedID 20736077

Abstract

Phaeoacremonium parasiticum is an environmental fungus usually associated with subcutaneous infections. We report the first documented case of central nervous system involvement with brain abscess formation in a patient with chronic granulomatous disease and review the literature on Phaeoacremonium parasiticum infections.

View details for DOI 10.1128/JCM.00830-10

View details for Web of Science ID 000287967100072

View details for PubMedID 21191052

Abstract

Infection because of herpes simplex virus (HSV) that is resistant to acyclovir (ACV) poses treatment challenges in hematopoietic cell transplant (HCT) patients. We present a series of patients with ACV-resistant HSV following HCT who were successfully treated with continuous infusion high-dose ACV after failing standard treatment regimens for ACV-resistant HSV.

View details for DOI 10.1016/j.bbmt.2010.06.020

View details for Web of Science ID 000287350400010

View details for PubMedID 20615475

Abstract

Haematological patients with neutropenic fever are frequently evaluated with chest computed tomography (CT) to rule out invasive fungal infections (IFI). We retrospectively analysed data from 100 consecutive patients with neutropenic fever and abnormal chest CT from 1998 to 2005 to evaluate their chest CT findings and the yield of diagnostic approaches employed. For their initial CTs, 79% had nodular opacities, with 24.1% associated with the halo sign. Other common CT abnormalities included pleural effusions (48%), ground glass opacities (37%) and consolidation (31%). The CT findings led to a change in antifungal therapy in 54% of the patients. Fifty-six patients received diagnostic procedures, including 46 bronchoscopies, 25 lung biopsies and seven sinus biopsies, with a diagnostic yield for IFI of 12.8%, 35.0% and 83.3%, respectively. In conclusion, chest CT plays an important role in the evaluation of haematological patients with febrile neutropenia and often leads to a change in antimicrobial therapy. Pulmonary nodules are the most common radiological abnormality. Sinus or lung biopsies have a high-diagnostic yield for IFI as compared to bronchoscopy. Patients with IFI may not have sinus/chest symptoms, and thus, clinicians should have a low threshold for performing sinus/chest imaging, and if indicated and safe, a biopsy of the abnormal areas.

View details for DOI 10.1111/j.1439-0507.2009.01760.x

View details for Web of Science ID 000284900600009

View details for PubMedID 19793207

Abstract

Dosage-sparing strategies, adjuvants and alternative substrates for vaccine production are being explored for influenza vaccine development. We assessed the safety and immunogenicity of a Vero cell culture-grown inactivated whole virus influenza A/H5N1 vaccine with or without aluminum hydroxide adjuvant [Al(OH)(3)] in healthy young adults. Vaccines were well tolerated, but injection site discomfort was more frequent in groups receiving Al(OH)(3). Dose-related increases in serum antibody levels were observed. Neutralizing antibody titers varied significantly when tested by two different laboratories. Al(OH)(3) did not enhance HAI or neutralizing antibody responses, and contributed to increased injection site pain. Because influenza antibody titers vary significantly between different laboratories, international standardization of assays is warranted.

View details for DOI 10.1016/j.vaccine.2009.03.015

View details for Web of Science ID 000272056300022

View details for PubMedID 19773098

Abstract

To augment the available influenza vaccine supply, a phase III study was conducted to evaluate the immunogenicity, safety, and consistency of a new trivalent inactivated influenza vaccine manufactured by CSL Limited. Healthy adults (ages 18-64) were randomized to receive either a single dose of TIV from multi-dose vials with thimerosal, TIV from pre-filled syringes without thimerosal, or placebo. Of the TIV recipients, 97.8% achieved a post-vaccination titer > or =40 against H1N1, 99.9% against H3N2 component, and 94.2% against influenza B. Few local or systemic adverse events were noted after vaccination with either TIV presentation. TIV was well tolerated and immunogenic.

View details for DOI 10.1016/j.vaccine.2008.05.024

View details for Web of Science ID 000258610900014

View details for PubMedID 18602726

Abstract

Disseminated Fusarium infection can cause significant morbidity and mortality in immunocompromised patients. We present a case of disseminated fusariosis in a patient with neutropenic fever successfully treated using both liposomal amphotericin B and voriconazole. Combination anti-fungal therapy may be considered for such patients, particularly for those failing single-drug therapy.

View details for DOI 10.1111/j.0933-7407.2006.2006.01346.x

View details for Web of Science ID 000246151400013

View details for PubMedID 17472622

Abstract

Necrotic insults such as seizure are excitotoxic. Logically, membrane hyperpolarization by increasing outwardly conducting potassium channel currents should attenuate hyperexcitation and enhance neuron survival. Therefore, we overexpressed a small-conductance calcium-activated (SK2) or voltage-gated (Kv1.1) channel via viral vectors in cultured hippocampal neurons. We found that SK2 or Kv1.1 protected not only against kainate or glutamate excitotoxicity but also increased survival after sodium cyanide or staurosporine. In vivo overexpression of either channel in dentate gyrus reduced kainate-induced CA3 lesions. In hippocampal slices, the kainate-induced increase in granule cell excitability was reduced by overexpression of either channel, suggesting that these channels exert their protective effects during hyperexcitation. It is also important to understand any functional disturbances created by transgene overexpression alone. In the absence of insult, overexpression of Kv1.1, but not SK2, reduced baseline excitability in dentate gyrus granule cells. Furthermore, while no behavioral disturbances during spatial acquisition in the Morris water maze were observed with overexpression of either channel, animals overexpressing SK2, but not Kv1.1, exhibited a memory deficit post-training. This difference raises the possibility that the means by which these channel subtypes protect may differ. With further development, potassium channel vectors may be an effective pre-emptive strategy against necrotic insults.

View details for DOI 10.1046/j.1471-4159.2003.01880.x

View details for Web of Science ID 000184723000003

View details for PubMedID 12911616

Abstract

To report a case of anaphylactoid reaction in an HIV-negative patient associated with the administration of intravenous ciprofloxacin.A 79-year-old Armenian man developed an anaphylactoid reaction following a first-time exposure to intravenous ciprofloxacin. This reaction was characterized by severe hypotension, wheezing, tachypnea, tachycardia, and pruritus. The patient had complete recovery once ciprofloxacin treatment was terminated and supportive care was provided.Fluoroquinolones are important therapeutic agents in the management of infectious diseases and are generally safe and well tolerated. Anaphylactoid and anaphylactic reactions have been documented as adverse effects of ciprofloxacin, ofloxacin, norfloxacin, levofloxacin, and moxifloxacin. To date, >33 cases have been reported with ciprofloxacin, of which at least 10 occurred in HIV-positive patients. In Europe, 15 cases of anaphylactoid reactions to ofloxacin have been reported and, more recently, with moxifloxacin. Since anaphylactoid reactions are potentially life threatening, the administration of fluoroquinolones to patients who have experienced a prior reaction to any of these agents should be avoided, unless tolerance has been confirmed by oral challenge tests.The anaphylactoid reaction in our patient was probably induced by ciprofloxacin as validated by the Naranjo probability scale. Although anaphylactoid/anaphylactic reactions are rare adverse effects of ciprofloxacin and other fluoroquinolones, clinicians should be aware of this potentially fatal event.

View details for DOI 10.1345/aph.1C498

View details for Web of Science ID 000184075800014

View details for PubMedID 12841811

Abstract

There is an increasing recognition of the damaging role played by oxygen radicals in mediating necrotic neuronal injury. As such, it becomes important to understand the transport mechanisms that help maintain appropriate levels of small molecule antioxidants such as ascorbate in the brain. It has long been known that the transport of dehydroascorbate (DHA) into a variety of cell types is accomplished through the Glut-1 glucose transporter. In this paper, we characterize interactions among the transports of ascorbate, DHA and glucose in hippocampal cultures. We find: (a) sodium-dependent transport of ascorbate in mixed neuronal/glial, pure glial, and neuron-enriched hippocampal cultures; in contrast, we observed no such transport of DHA; (b) such ascorbate transport appeared to be independent of the glucose transporter, in that glucose did not compete for such transport, and overexpression of the Glut-1 glucose transporter did not alter ascorbate uptake; (c) in contrast, ascorbate, at concentrations ranging from 1 to 20 mM inhibited 2-dexogyglucose transport in mixed, glial and enriched neuronal hippocampal cultures; (d) potentially, ascorbate, by acting as an electron donor, could impair the function of molecules involve in the transport or metabolism of glucose. We observed mild inhibition of glucose transport by one unrelated electron donor (glutathione). Moreover, transport was also inhibited by an ascorbate analog which is not an electron donor. Thus, we conclude that ascorbate transport in hippocampal neurons and glia occurs independent of the glucose transporter but that, nevertheless, ascorbate, at concentrations generally thought to be supraphysiological, has the potential for disrupting glucose transport.

View details for Web of Science ID 000171812300015

View details for PubMedID 11597599

Abstract

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.

View details for Web of Science ID 000170095100005

View details for PubMedID 11457430

Abstract

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.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.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).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.

View details for Web of Science ID 000167951300038

View details for PubMedID 11283407

Abstract

Numerous studies have demonstrated that gene therapy interventions can protect neurons from death after neurological insults. In nearly all such studies, however, "protection" consists of reduced neurotoxicity, with no demonstrated preservation of neuronal function. We used a herpes simplex virus-1 system to overexpress either the Glut-1 glucose transporter (GT) (to buffer energetics), or the apoptosis inhibitor Bcl-2. Both decreased hippocampal neuron loss to similar extents during excitotoxic insults in vitro and in vivo. However, the mediating mechanisms and consequences of the two interventions differed. GT overexpression attenuated early, energy-dependent facets of cell death, blocking oxygen radical accumulation. Bcl-2 expression, in contrast, blocked components of death downstream from the energetic and oxidative facets. Most importantly, GT- but not Bcl-2-mediated protection preserved hippocampal function as assessed spatial maze performance. Thus, gene therapeutic sparing of neurons from insult-induced death does not necessarily translate into sparing of function.

View details for Web of Science ID 000165225800082

View details for PubMedID 11058147

Abstract

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.

View details for Web of Science ID 000165119800016

View details for PubMedID 11031094

Abstract

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.

View details for Web of Science ID 000088608000091

View details for PubMedID 10908682

Abstract

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.

View details for Web of Science ID 000086080600003

View details for PubMedID 10719065

Abstract

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.

View details for Web of Science ID 000085998400003

View details for PubMedID 10720611

Abstract

Herpes simplex virus type-1 (HSV) amplicon vectors containing neuroprotective genes can alter cell physiology and enhance survival following various insults. However, to date, little is known about effects of viral infection itself (independent of the gene delivered) on neuronal physiology. Electrically-evoked synaptic responses are routinely recorded to measure functional alterations in the nervous system and were used here to assess the potential capability of HSV vectors to disrupt physiology of the hippocampus (a forebrain structure involved in learning that is highly susceptible to necrotic insult, making it a frequent target in gene therapy research). Population excitatory post-synaptic potentials (EPSPs) were recorded in the dentate gyrus (DG) and in area CA3 in vivo 72 h after infusion of an HSV vector expressing a reporter gene (lacZ) or vehicle into the DG. Evoked perforant path (PP-DG) or mossy fiber (MF-CA3) EPSPs slope values measured across input/output (I/O) curves were not altered by infection. Paired-pulse facilitation at either recording site was also unaffected. X-gal-positive granule cells surrounded the recording electrode (PP-DG recording) and stimulating electrode tracts (MF-CA3 recording) in animals that received vector, suggesting that we had measured function, at least in part, in infected neurons. Because of the negative electrophysiological result, we sought to deliver a gene with an HSV amplicon which would affect the measured endpoints, as a positive control. Delivery of calbindin D28kpotentiated PP-DG synaptic strength, indicating that our recording system could detect alterations due to vector expression. Thus, the data indicate that HSV vectors are benign, in regard to effects on synaptic function, and support the use of these vectors as a safe method to deliver selected genes to the central nervous system.

View details for Web of Science ID 000082957100006

View details for PubMedID 10516716

Abstract

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.

View details for Web of Science ID 000082037000035

View details for PubMedID 10461912

View details for Web of Science ID 000082191900048

Abstract

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.

View details for Web of Science ID 000076316300002

View details for PubMedID 9778256

Abstract

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.

View details for Web of Science ID 000075478400013

View details for PubMedID 9721726

View details for Web of Science ID 000073930600021

View details for PubMedID 9624676

Abstract

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.

View details for Web of Science ID A1997XR46700017

View details for PubMedID 9282926

Abstract

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.

View details for Web of Science ID A1997XR34500003

View details for PubMedID 9270490

View details for Web of Science ID A1997WZ79800033

View details for PubMedID 9163944

Abstract

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.

View details for Web of Science ID A1997WJ84200009

View details for PubMedID 9048741

Abstract

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.

View details for Web of Science ID A1996VF39200026

View details for PubMedID 8883953

Abstract

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.

View details for Web of Science ID A1996UF07800013

View details for PubMedID 8620928

Abstract

Herpes simplex virus vectors bearing a glucose transporter (GT) gene and a marker gene were found to protect neurons against a 1-h focal ischemic insult. Rats receiving the GT vector v alpha22beta gal alpha4GT exhibited a 67.4 +/- 35.3% survival of virally targeted neurons in the ischemic hemisphere compared with the contralateral control (n = 7), whereas rats receiving a control vector exhibited only 32.8 +/- 17.9% survival (n = 9). This significant improvement in survival (105%, p=0.022) suggests that energy failure is an important contributor to the neuropathology of ischemic damage in the striatum, and that it can be alleviated by gene transfer. This is the first demonstration of protection against ischemic cerebral injury by the direct transfer of GT genes to neurons.

View details for Web of Science ID A1996TW39300001

View details for PubMedID 8594048

Abstract

Previous studies have demonstrated that overexpression of the proto-oncogene bcl-2 can protect neuron and neuron-like cell lines from growth factor deprivation, calcium ionophores, glutamate excitotoxicity, hypoglycemia, free radicals, and lipid peroxidation. To determine whether Bcl-2 exhibits a similar protective effect in CNS neurons, we generated defective herpes simplex virus (HSV) vectors capable of overexpressing Bcl-2 in primary cultures and in the intact brain. Infection of hippocampal cultures with Bcl-2 vectors enhanced neuron survivorship after exposure to adriamycin, a potent oxygen radical generator. Furthermore, dichlorofluorescein measurements indicated that there was a significant reduction in the accumulation of oxygen radicals associated with this insult. Bcl-2 vectors also enhanced survival in cultured neurons after exposure to glutamate and hypoglycemia. Most significantly, the in vivo delivery of the vector protected neurons against adriamycin toxicity in the dorsal horn of the dentate gyrus and focal ischemia in the striatum.

View details for Web of Science ID A1996TP51900007

View details for PubMedID 8551333

Abstract

The use of herpes simplex virus vectors offers an attractive means for the in vitro and in vivo transfer of novel genes into postmitotic neurons. Such an approach allows for the introduction of genes with the potential to protect neurons from necrotic insults. Toward that end, we have previously constructed a bicistronic herpes viral vector expressing the gene for the Glut-1 rat brain glucose transporter (GT), along with the Escherichia coli lacZ reporter gene. We observed that this vector enhances glucose uptake both in primary hippocampal cultures and in the hippocampus itself. Moreover, we have found that this vector will protect a variety of types of cultured neurons from necrotic insults and protect hippocampal neurons in vivo from seizure-induced damage. In the present report, we further demonstrate the neuroprotective potential of this GT-expressing vector. 3-Acetylpyridine, an electron transport uncoupler which is preferentially toxic to the dentate gyrus, was microinfused into the dorsal hippocampus of rats. Infection of dentate neurons with GT vectors at the time of exposure to the toxin significantly decreased damage, whereas infection with a physiologically neutral control vector did not. Moreover, there was a window of opportunity for this intervention, as microinfusion of the GT-expressing vector up to 1 h, but not 4 h, after the insult was still neuroprotective.

View details for Web of Science ID A1996TQ62700005

View details for PubMedID 8566211

Abstract

We have generated herpes simplex virus (HSV) vectors vIE1GT and v alpha 4GT bearing the GLUT-1 isoform of the rat brain glucose transporter (GT) under the control of the human cytomegalovirus ie1 and HSV alpha 4 promoters, respectively. We previously reported that such vectors enhance glucose uptake in hippocampal cultures and the hippocampus. In this study we demonstrate that such vectors can maintain neuronal metabolism and reduce the extent of neuron loss in cultures after a period of hypoglycemia. Microinfusion of GT vectors into the rat hippocampus also reduces kainic acid-induced seizure damage in the CA3 cell field. Furthermore, delivery of the vector even after onset of the seizure is protective, suggesting that HSV-mediated gene transfer for neuroprotection need not be carried out in anticipation of neurologic crises. Using the bicistronic vector v alpha 22 beta gal alpha 4GT, which coexpresses both GT and the Escherichia coli lacZ marker gene, we further demonstrate an inverse correlation between the extent of vector expression in the dentate and the amount of CA3 damage resulting from the simultaneous delivery of kainic acid.

View details for Web of Science ID A1995RM72200024

View details for PubMedID 7638175

Abstract

Because neurons are postmitotic, they are irreplaceable once they succumb to necrotic insults such as hypoglycemia, ischemia, and seizure. A paucity of energy can exacerbate the toxicities of these insults; thus, a plausible route to protect neurons from necrotic injury would be to enhance their glucose uptake capability. We have demonstrated previously that defective herpes simplex virus (HSV) vectors overexpressing the rat brain glucose transporter (GT) gene (gt) can enhance glucose uptake in adult rat hippocampus and in hippocampal cultures. Furthermore, we have observed that such vectors can maintain neuronal metabolism during hypoglycemia and reduce kainic acid-induced seizure damage. In this study, we have developed bicistronic vectors that coexpressed gt and Escherichia coli lacZ as a reporter gene, which allows us to identify directly neurons that are infected with the vectors. Overexpression of GT from these vectors protected cultured hippocampal, spinal cord, and septal neurons against various necrotic insults, including hypoglycemia, glutamate, and 3-nitropropionic acid. Our observations demonstrate the feasibility of using HSV vectors to protect neurons from necrotic insults. Although this study has concentrated on the delivery of gt, other genes with therapeutic or protective capability might also be used.

View details for Web of Science ID A1995RJ65700048

View details for PubMedID 7616244

Abstract

With its natural propensity to infect and establish life-long latency in neurons, herpes simplex virus type 1 (HSV-1) has been successfully employed by various laboratories as vectors for gene transfer into neurons. However, analysis of its cytopathic effects in vivo and in vitro has been limited. In this study, we examined the cytopathic effects of 2 HSV-1 alpha 4 mutants (ts756 and d120) on adult rat hippocampus and striatum and of d120 on hippocampal neurons in culture. We assessed damage by stringent counting of surviving neurons after infection and demonstrated that while neither ts756 nor d120 infection resulted in any gross anatomical or behavioral changes of the animals, ts756, but not d120, produced a significant amount of damage in the CA4 cell field and dentate gyrus of the hippocampus. Thus, since crude examination is insufficient to detect subtle but significant degrees of neuron loss, the cytopathic effects of HSV or any vector system must be carefully analyzed. Furthermore, we also observed that uninfected cell lysates damaged neurons, both in vivo and in vitro. This cytotoxicity occurred within the first 24 h post-inoculation and probably arose through the activation of glutamate receptors. For the preparation of HSV vectors, purification of the virus from soluble cellular components by a simple pelleting step can significantly decrease such acute toxicity.

View details for Web of Science ID A1995QU14500010

View details for PubMedID 7609584

Abstract

The application of gene transfer techniques to organ transplantation offers the potential for modulation of immunity directly within an allograft without systemic side effects. Expression vectors and promoter elements are important determinants of gene transfer and expression. In this study, various vectors (naked plasmid DNA, retroviral vector, herpes simplex viral vector, and adenoviral vector) with various promoters (RSV-LTR, SV40, MuLV-LTR, HCMVie1) were directly compared to demonstrate the successful gene transfer and expression of beta-galactosidase in murine myoblasts in vitro and within murine heterotopic, nonvascularized cardiac isografts or allografts in vivo. Expression of transferred genes was not toxic to cells and strength of expression varied according to the type of vector. Plasmid DNA was expressed in myocytes, retroviral vector was expressed in the graft infiltrating cells, and herpes simplex and adenoviral vectors were expressed in both myocytes and graft-infiltrating cells. Preliminary studies evaluated the ability of these vectors to deliver immunologically important signals. Allografts injected with pSVTGF-beta 1, a plasmid-encoding transforming growth factor beta 1 (TGF-beta 1) under the control of the SV40 promoter, showed significant prolongation of graft survival of 26.3 +/- 2.5 days compared with 12.6 +/- 1.1 days for untreated allografts, and 12.5 +/- 1.5 days for the allografts injected with control plasmid (P < 0.05). Allografts injected with MFG-vIL-10, a retroviral vector encoding viral interleukin-10 under the control of the MuLV-LTR, showed prolongation of graft survival of 36.7 +/- 1.3 days versus 12.6 +/- 1.1 days for the untreated allograft, and 13.5 +/- 2.0 days for the allografts injected with control retroviral vector (P < 0.001). Both vectors were transcriptionally active in vivo and did not appear to have toxic effects. Gene therapy for transplantation can induce transient expression of immunologically relevant molecules within allografts that impede immune activation while avoiding the systemic toxicity of conventional immunosuppression.

View details for Web of Science ID A1995QQ42500002

View details for PubMedID 7701573

View details for Web of Science ID A1994BB61Z00009

View details for PubMedID 7823862

Abstract

Because of their postmitotic nature, neurons are difficult subjects for gene transfer. To circumvent this, we have used a defective herpes simplex virus vector to overexpress the rat brain glucose transporter (GT) gene under the control of the human cytomegalovirus ie1 promoter. This vector, designated vIE1GT, was propagated using a herpes simplex virus type 1 temperature-sensitive mutant, ts756. GT expressed from vIE1GT was readily immunoprecipitated from membrane fractions of vIE1GT-infected Vero cells. By using indirect double immunofluorescence techniques, vIE1GT was shown to be capable of enhancing GT expression in cultured hippocampal neurons and glia. Glucose transport in such vIE1GT-infected cultures was increased approximately 2-fold relative to controls. The efficacy of this system in vivo was then tested by microinjection of vIE1GT into adult rat hippocampus. When examined 2 days later, GT expression from vIE1GT was demonstrated in hippocampal neurons by in situ hybridization; a small but significant increase in glucose transport was detected in tissue immediately surrounding the injection site by 2-deoxy[14C]glucose uptake and autoradiography. Such injections did not cause marked cytopathology. Thus, this approach can be used to alter central nervous system physiology in vitro and in vivo.

View details for Web of Science ID A1993KX81600112

View details for PubMedID 8386379

Abstract

We constructed a recombinant thymidine kinase-negative herpes simplex virus type 1 (HSV-1) that expressed the rotavirus major outer capsid glycoprotein, VP7. In the recombinant HSV-1, a promoter from the 5' noncoding region of the HSV-1 glycoprotein B locus regulated the expression of VP7 as a HSV-1 gamma 1 gene product. HSV-1-expressed VP7 resembled rotavirus-expressed VP7 in its SDS-PAGE mobility, high mannose-type glycosylation, disulfide bonding, perinuclear to cytoplasmic localization, intracellular retention, and reactivity with polyclonal antisera and nonneutralizing antibodies. Unlike rotavirus-expressed VP7, HSV-1-expressed VP7 lacked several neutralizing epitopes by immuno-histochemical staining and by ELISA. One neutralizing epitope identified on HSV-1-expressed VP7 by ELISA was masked by paraformaldehyde fixation of recombinant HSV-1- but not rotavirus-infected cells. Neutralizing epitopes were restored to HSV-1-expressed VP7 by coinfection of cells with the HSV-1 recombinant and a heterologous rotavirus that lack the neutralizing epitopes. The recovered neutralizing epitopes were detected on double-shelled rotavirus particles produced in the coinfected cells. This study indicates that the formation of several neutralizing epitopes on rotavirus VP7 requires interaction of VP7 with other rotavirus proteins. In addition, HSV-1 was a useful vector for studying the localization, processing, and antigenicity of an RNA virus glycoprotein.

View details for Web of Science ID A1992HD62800003

View details for PubMedID 1371025

View details for Web of Science ID A1992HP43100003

View details for PubMedID 1317601

Abstract

During latent infection by herpes simplex virus (HSV), an abundant latency-associated transcript (LAT) that is antisense to a predominant viral alpha gene (ICP0) is found localized in the nucleus of sensory neurons. We disrupted both copies of the LAT gene in the HSV-1 genome by insertion of the Escherichia coli lacZ gene under LAT promoter control. The resulting recombinant virus, RH142, does not express any detectable LAT in either latently or productively infected cells, although beta-galactosidase expression is readily detectable in sensory neurons of latently infected mice. Expression was first detectable 3 days postinoculation and continued at approximately the same level for the entire experimental period (56 days). beta-Galactosidase expression was not detectable at any time during RH142 replication in Vero cells. Thus, the kinetics of expression and cell-type specificity of the LAT gene are distinct from other HSV-1 genes that are expressed during productive growth. When latently infected trigeminal ganglia were explanted, RH142 reactivated from latency with the kinetics and an efficiency indistinguishable from the parental wild-type virus. These studies argue against any possible antisense regulatory mechanism of LAT in the regulation of viral gene expression or any role of LAT-encoded protein during the establishment or maintenance of latency in the mouse.

View details for Web of Science ID A1989AT78200066

View details for PubMedID 2552449

Abstract

We have inserted a modified Escherichia coli lacZ gene, placed under the control of herpes simplex virus alpha 4 or beta 8 regulatory signals, into the HSV-1 genome disrupting the viral thymidine kinase gene. Using beta-galactosidase as an in situ indicator of viral gene expression, we detected expression from these recombinant HSV in dermal and neural tissues of the BALB/c mouse. Our detection of beta-galactosidase expression in neuronal cells indicates that TK-deficient viruses are capable of invading mouse neuronal cells and expressing up to the beta class of gene product.

View details for Web of Science ID A1988Q895500033

View details for PubMedID 2847416