Selective overexpression of excitatory amino acid transporter 2 (EAAT2) in astrocytes enhances neuroprotection from moderate but not severe hypoxia-ischemia.

Attempts have been made to elevate excitatory amino acid transporter 2 (EAAT2) expression in an effort to compensate for loss of function and expression associated with disease or pathology. Increased EAAT2 expression has been noted following treatment with beta-lactam antibiotics, and during ischemic preconditioning (IPC). However, both of these conditions induce multiple changes in addition to alterations in EAAT2 expression that could potentially contribute to neuroprotection. Therefore, the aim of this study was to selectively overexpress EAAT2 in astrocytes and characterize the cell type specific contribution of this transporter to neuroprotection. To accomplish this we used a recombinant adeno-associated virus vector, AAV1-glial fibrillary acidic protein (GFAP)-EAAT2, designed to selectively drive the overexpression of EAAT2 within astrocytes. Both viral-mediated gene delivery and beta-lactam antibiotic (penicillin-G) treatment of rat hippocampal slice cultures resulted in a significant increase in both the expression of EAAT2, and dihydrokainate (DHK) sensitive glutamate uptake. Penicillin-G provided significant neuroprotection in rat hippocampal slice cultures under conditions of both moderate and severe oxygen glucose deprivation (OGD). In contrast, viral-mediated overexpression of EAAT2 in astrocytes provided enhanced neuroprotection only following a moderate OGD insult. These results indicate that functional EAAT2 can be selectively overexpressed in astrocytes, leading to enhanced neuroprotection. However, this cell type specific increase in EAAT2 expression offers only limited protection compared to treatment with penicillin-G.

Overexpression of hippocampal Ca2+/calmodulin-dependent protein kinase II improves spatial memory.

Hippocampal alpha-calcium/calmodulin-dependent protein kinase II (alphaCaMKII) has been implicated in neuronal plasticity and spatial learning. In the present experiment, an adeno-associated virus (AAV) vector was designed to express alphaCaMKII driven by the U6 promotor. Microinfusion of this vector into the rat hippocampus increased alphaCaMKII immunoreactivity by approximately 73% (Western analysis) and improved performance in a water maze task. Locomotor activity and exploratory behavior in an open field task were not altered by the overexpression of alphaCaMKII. These data support a role for alphaCaMKII in spatial or explicit memory storage. The advantages of viral vectors for manipulating target proteins expression compared with genetically modified mouse models are discussed.

In vivo inhibition of hippocampal Ca2+/calmodulin-dependent protein kinase II by RNA interference.

Hippocampal alpha-Ca2+/calmodulin-dependent protein kinase II (alpha-CaMKII) has been implicated in spatial learning, neuronal plasticity, epilepsy, and cerebral ischemia. In the present study, an adeno-associated virus (AAV) vector was designed to express green fluorescent protein (GFP) from the CBA promoter and a small hairpin RNA targeting alpha-CaMKII (AAV-shCAM) driven from the U6 promoter. The AAV-shCAM or control vector was microinfused into the rat hippocampus and behavioral testing conducted 19-26 days following surgery. Expression of the marker gene and alpha-CaMKII was evaluated 31 days following AAV infusion. GFP expression was localized to the hippocampus and extended +/-2 mm rostral and caudal from the injection site. Hippocampal alpha-CaMKII was significantly reduced following AAV-shCAM treatment as demonstrated using immunohistochemical and Western analysis. This suppression of alpha-CaMKII was associated with changes in exploratory behavior (open field task) and impaired place learning (water maze task). These results demonstrate the efficacy of a viral-based delivered shRNA to produce gene suppression in a specific circuit of the brain.

Relapse prevention by citalopram in SAD patients responding to 1 week of light therapy. A placebo-controlled study.

OBJECTIVE: We have tested the relapse-preventive effect of citalopram when compared with placebo in 282 patients with Seasonal Affective Disorder (SAD) responding to 1 week of light therapy. METHOD: The response rate to 1-week light therapy and relapse during the continuation phase of 15 weeks were assessed by use of the Hamilton Depression Rating Scale (HAM-D17), the six-item subscale (HAM-D6), the Melancholia Scale (MES), and the combined HAM-D/SIGH-SAD. RESULTS: The response rate to light therapy was 62.5% on the HAM-D17 and the HAM-D6, 56.1% on the HAM-D/SIGH-SAD, 52.8% on the MES. In the continuation phase, citalopram was found superior to placebo on all scales, but the difference was only of statistical significance on the HAM-D6 and the MES. Mean citalopram dose was 26.3 mg. CONCLUSION: Light therapy was found to have and early onset of action. On the HAM-D6 and the MES citalopram significantly reduced the relapse rate in the continuation phase.

Outcomes associated with supportive periodontal therapy in smokers and nonsmokers.

PURPOSE: This study evaluated the effects of smoking status on retrospective clinical and radiographic measures of periodontal disease and compared these to prospective changes in digital radiographic bone height. METHODS: Clinical data on moderate (4 to 6 mm) and severe (> 6 mm) periodontal pocket depths, and bleeding on probing, were obtained from 95 subjects on suggested three-month supportive periodontal therapy (SPT) for AAP Class III/IV periodontitis. Standardized radiographic data were obtained concerning posterior interproximal alveolar bone height from 36 of the 95 subjects using computer-assisted digital technology at baseline and one year later. The subjects were divided into groups by smoking status: current, former, and never. Data were evaluated using a general linear statistical model. RESULTS: Evaluation of clinical data showed that current smokers exhibited a significantly higher percentage of moderate (18%) and severe (1%) periodontal pockets than nonsmokers (10% and 0%, respectively; p < 0.002). Baseline radiographic interproximal bone height loss also was greater in current smokers (5.75 +/- 1.07 v. 4.64 +/- 1.16 mm). Bone loss over one year occurred in 5% of the sites, but was not significantly different among groups. CONCLUSION: Clinical periodontal pockets and bone loss accumulated more rapidly in smokers, even though they submitted to regular supportive periodontal therapy. Although this population was clinically compliant over a one year period, digital radiography showed a high incidence of detectable bone loss. The impact of smoking, however, may require longer than one year to show longitudinal changes. It is recommended that a periodic radiographic analysis on bone height be considered during SPT, and longer term studies be conducted in order to accurately identify the outcome of smoking status on this variable.

An oral vaccine against NMDAR1 with efficacy in experimental stroke and epilepsy.

The brain is generally considered immunoprivileged, although increasing examples of immunological responses to brain antigens, neuronal expression of major histocompatibility class I genes, and neurological autoimmunity have been recognized. An adeno-associated virus (AAV) vaccine generated autoantibodies that targeted a specific brain protein, the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor. After peroral administration of the AAV vaccine, transgene expression persisted for at least 5 months and was associated with a robust humoral response in the absence of a significant cell-mediated response. This single-dose vaccine was associated with strong anti-epileptic and neuroprotective activity in rats for both a kainate-induced seizure model and also a middle cerebral artery occlusion stroke model at 1 to 5 months following vaccination. Thus, a vaccination strategy targeting brain proteins is feasible and may have therapeutic potential for neurological disorders.

Increased neurovirulence of polytropic mouse retroviruses delivered by inoculation of brain with infected neural stem cells.

Following intraperitoneal (IP) inoculation of neonatal mice, the polytropic recombinant murine leukemia virus (MuLV), Fr98, induces a severe brain disease characterized by ataxia, seizures and death. In contrast, no apparent clinical neurological disease is seen after IP infection with Fr54, a polytropic MuLV differing from Fr98 in its envelope gene sequences. In the brain both Fr98 and Fr54 infect primarily capillary endothelial cells and microglia. However, the level of microglial infection by Fr98 is twofold higher than by Fr54, which might account for the difference in neurovirulence. In the present study, in order to test directly whether an increase in the number of microglia infected by Fr54 would be sufficient to induce clinical disease, we attempted to increase the level of Fr54 in the brain by changing the route of infection. After intraventricular inoculation with Fr54-infected neural stem cells (clone C17.2), a well-established vehicle for delivery of viruses and genes to the brain, mice became ataxic and died 4 weeks postinfection. In these mice induction of brain disease was correlated with a higher level of viral antigen in the cerebrum and an increase in the number of infected microglial cells in all brain regions examined compared with mice inoculated IP. In contrast, mice inoculated with neural stem cells infected with an ecotropic nonneurovirulent murine leukemia virus, FB29, developed no clinical disease in spite of evidence for widespread infection of microglia in brain. Since the main differences between Fr54 and FB29 are in the SU (gp70) region of the envelope gene, this region is most likely to account for the differences in induction of CNS disease seen in the current experiments.

Mapping of a neurovirulence determinant within the envelope protein of a polytropic murine retrovirus: induction of central nervous system disease by low levels of virus.

Murine leukemia virus (MuLV) clone Fr98 is a recombinant polytropic virus that causes neurological disease characterized by ataxia in susceptible mouse strains. The envelope gene of Fr98 has been previously shown to encode at least two separate neurovirulence determinants. In the present study, the determinant encoded within the EcoRI/AvrII fragment of the envelope gene was further defined. In these experiments, neurovirulence was associated with a change from a serine to an arginine at position 195 and a glycine to an alanine at position 198 within the envelope protein. Neurovirulent and nonvirulent virus clones, which differed only at these two amino acid residues, showed no difference in the type or location of cells infected. Furthermore, equivalent levels of viral p30 capsid protein were detected in the brains of mice infected with either the neurovirulent or nonvirulent virus clones. These results were consistent with the interpretation that the envelope protein of the neurovirulent virus differed from that of the nonvirulent virus by having a greater toxic effect on central nervous system function.

Research notes: Comparison of disease susceptibility and resistance in three lines of chickens experimentally infected with infectious laryngotracheitis virus.

The susceptibility of three F1 hybrid lines of chickens to graded doses of infectious laryngotracheitis virus (ILTV) was investigated. The three F1 hybrid lines, each produced from mating two inbred lines, included the SC (B2B2) and TK (B15B21) lines and the 15I5 x 7(1) (B2B15) line. Although at 1 d of age all three lines were susceptible to ILTV, SC birds were significantly less susceptible (10%) than TK (80%) or 15I5 x 7(1) (50%) birds when exposed to 5,000 pfu of virus at 4 wk of age. The ability of each inbred F1 hybrid line to establish a protective immune response to ILTV was also determined. The SC birds required a smaller immunizing dose of virus (500 pfu) to mount a protective immune response to ILTV than the 15I5 x 7(1) line (5,000 pfu). A 5,000 pfu immunizing dose did not elicit a protective immune response in the TK line to a 10(6) pfu challenge dose of ILTV. These results also correlated with the ability to produce ILTV-specific antibodies. This study confirms and expands on observations that lines of chickens differ with respect to their susceptibility and resistance to ILTV.

Characterization of the assembly and processing of infectious laryngotracheitis virus glycoprotein B.

Infectious laryngotracheitis virus (ILTV) is an alpha-herpesvirus that causes severe upper respiratory infections in chickens. Although ten putative ILTV glycoprotein genes have been identified by sequence analysis, no ILTV glycoprotein has been extensively characterized. In order to delineate the synthesis and processing pathway of ILTV glycoprotein B (gB), rabbit polyclonal antibodies were raised against a Cro-gB-beta-galactosidase fusion protein. Through immunoprecipitation analysis of ILTV-infected chicken embryo liver cells it was determined that ILTV gB is initially synthesized as a 110 kDa monomeric precursor protein which rapidly assembles into homodimers composed of 100 kDa subunits. The dimer form of ILTV gB is rapidly cleaved to form two disulphide-linked species of 58 kDa. The apparent reduction in mass (from 110 to 100 kDa) of the mature form of gB during processing in the Golgi apparatus appears to be a common feature of avian herpesvirus gB proteins.

The use of bulk segregant analysis to identify a RAPD marker linked to leaf rust resistance in barley.

An F2 population from a cross between barley accession Q21861 and the Australian barley variety 'Galleon' was used to develop RAPD markers for resistance to barley leaf rust (Puccinia hordei). Resistant and susceptible DNA bulks were constructed following the classification of F2 plants by leaf rust infection type. Bulked segregant analysis was then used to identify a 2.7-kb marker, designated OU022700 and located approximately 12cM from RphQ, a leaf rust resistance gene in Q21861. The marker was generated by PCR with the oligonucleotide primer OPU-02 (Operon). Infection types of F3 progeny were used to confirm assignment of F2 genotypes. OU022700 was shown, retrospectively, to be useful in the identification of individual F2 plants that had been originally misclassified as having susceptible infection types. Both the RAPD marker and RphQ will be potentially useful in the development of new barley cultivars.

Identification of the infectious laryngotracheitis virus glycoprotein gB gene by the polymerase chain reaction.

The infectious laryngotracheitis virus (ILTV) homologue of the herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) gene was identified by PCR amplification of genomic ILTV DNA. A 488-bp amplified DNA fragment was used to identify and clone two adjacent PstI fragments from genomic ILTV DNA. Sequence analysis of the region surrounding the amplified fragment identified a 2619-bp open reading frame that has 39% homology with both the nucleotide and amino-acid sequences of the HSV-1 gB gene. Northern blot analysis using a portion of the open reading frame as a probe identified a 2.7-kb RNA transcript in ILTV-infected chicken embryo liver cells. Analysis of the predicted amino acid sequence of the ILTV protein indicated that it shares structural features with the gB glycoproteins of other herpesviruses.

Quality of life, side effects and efficacy of lisinopril compared with metoprolol in patients with mild to moderate essential hypertension.

In a double-blind, parallel-group multicentre study in general practice, lisinopril (10-20 mg once daily) was compared with metoprolol (100-200 mg once daily) in 360 patients whose diastolic blood pressure (DBP) was in the range 91-115 mmHg despite diuretic treatment. Following a three week run-in period during which the diuretic was withdrawn, monotherapy with either lisinopril or metoprolol was given for two months with dose doubled after one month if DBP remained greater than 90 mmHg. Quality of life was assessed using established and validated questionnaires at the time of cessation of diuretic treatment and again after two months's active treatment. Both metoprolol and lisinopril achieved statistically significant BP reduction relative to baseline (P less than 0.001). Significantly fewer adverse events were experienced on lisinopril and metoprolol than on diuretic treatment. Frequency of withdrawals due to adverse events were statistically significantly lower on lisinopril than metoprolol P = 0.01. Before treatment approximately 35% of the patients had quality of life problems measured by General Health Questionnaire (GHQ), which was reduced to 17% on lisinopril and 23% on metoprolol. Thus both metoprolol and lisinopril were effective and safe in the treatment of mild to moderate essential hypertension with lisinopril being better tolerated. From patients' self-assessments of quality of life, lisinopril was found to be superior to metoprolol in some aspects of emotional, cognitive and social functioning.

[Treatment of hypertension in general practice, consequences of therapeutic changes. A multipractice study of the therapeutic patterns, blood pressure control, drug administration schedule and incidence of side effects]. / Behandlingen af hypertension i almen praksis, konsekvenser af behandlingsskift. En multipraksisundersøgelse over behandlingsmønstre, blodtrykskontrol, doseringshyppighed og bivirkningsforekomst.

The antihypertensive treatment schedules were investigated in 1,153 consecutive patients in 65 general practices. Treatment consisted mainly of diuretics or betablockers singly or combined. In a sample of 303 of these patients, treatment was altered in a randomised, single-blind, cross-over design to consist of a single daily dose of metoprolol, or a single daily dose of metoprolol plus a single dose of a placebo. The effect of multiple dosages on the quality of life was investigated simultaneously with investigation of the efficacy of single drug treatment with a beta-blocker on blood-pressure control as compared with the previous treatment. A slight increase in side-effects was observed which might be ascribed to increased awareness by the patient and also by the doctor. The more simplified regimen with a single daily dose of the betablocker showed the same blood-pressure levels as in the previous more complicated regimen.

[Measurement of the quality of life as a element in assessing therapeutic effects in essential arterial hypertension. Methodologic aspects]. / Livskvalitetsmaling som effektstørrelse ved behandling af essentiel arterial hypertension. Metodologiske betragtninger.

The quality of life has become an important element in assessment of medicinal treatment. As an independent expression, the quality of life covers the human disappointments which a medical disease may involve ("discomfort") whereas the clinical dysfunction caused by the disease is covered by the expression "disability". In the treatment of essential arterial hypertension, the clinical symptoms of the disease are few or none ("asymptomatic hypertension"). In an investigation of 303 patients in general practice for arterial hypertension, the significance of the frequency of dosage of metoprolol (Seloken) was assessed by means of measurement of the quality of life by the General Health Questionnaire (GHQ), a questionnaire which contains components such as fatigue, sleep, anxiety and depression. No advantages in the quality of life were found in administering antihypertensive drugs once or twice daily. From the methological point of view, the following conditions were found which should be taken into consideration in future investigations in this field: The GHQ was found to be easy for the patients to employ. This is important as the quality of life is a personally experienced dimension. The questionnaire correlated adequately with the visual analogue scales. Approximately 1/3 of the patients had reduced quality of life at the commencement of the investigation as assessed by GHQ. A multiple regression analysis revealed that the reduced well-being was due to social, life events and side effects. It is therefore recommended that these aspects should be described when patients commence a course of medicinal treatment i.e. the social life events and side effects of the medicine in connection with the actual subjective dimension of quality of life.

Integration of DSM-III and ICD-8 to be used in a consultation-liaison psychiatric service. Preliminary experiences.

In 1983 a total of 405 patients received psychiatric supervision in somatic departments in the general hospital. At this supervision, these patients were registered by means of a five-axial diagnostic coding according to the DMS III principle, and this was combined with a quantitative global assessment of the severity of the condition. Reliability testing was undertaken by five supervising physicians with a total of 15 patients. The total number of supervisions constituted one supervision per somatic bed per annum. Women were overrepresented, and medical departments made the greatest use of psychiatric supervision. Reactive conditions dominated parallel with a high relative incidence of alcohol-related conditions. In patients with diagnoses of psychoses, only slight to moderate psychiatric symptoms were encountered. This held true also for personality deviations. 50% of the patients had experienced significant psychosocial stress, but 10% of these were diagnosed as having non-reactive psychoses, 52% of the patients had moderate to pronounced disturbances of social function. Half of the patients supervised in this manner could be investigated or treated in the referring departments. Approximately half of the patients in whom referral to private psychiatric specialists was made did not keep these appointments. Reliability testing in the material shows the employability of the diagnostic armamentarium. All in all, the investigation suggests that extension of the liaison psychiatric service in somatic departments would result in a relative increase in the number of patients who could be treated in the referring department and an increase in the number of psychiatric conditions diagnosed. Establishment of a psychiatric outpatient clinic in the somatic environment appears to be indicated.

Discomfort or disability in patients with chronic pain syndrome.

In the present study of 253 patients with chronic pain syndrome we have made a multidimensional approach. All patients have been included in the study independent of coexisting states of anxiety or depression. We included criteria for diagnosis, duration, generability and intensity of pain, anxiety and depression, psychosocial stressors and social functioning. Using this system we have evaluated the antipain effectiveness of clomipramine and mianserin in a double-blind, placebo-controlled trial. By use of the Melancholia Scale 16 patients (6%) had a major depression, and by use of the Hamilton Anxiety Scale, 72 patients (28%) had a generalized anxiety disorder. The results showed no statistically significant difference between the three treatments, when using a visual analogue scale (VAS 10 cm with cut-off score 2 cm) for severity of pains as outcome criteria or the results of VAS and Global Clinical Impression Scale using the criteria of reduction of 50% or more between the pretreatment and posttreatment scores. By use of all the assessments it is possible to make an improvement curve for each patient expressed by the area under the curve, and not even there we found a difference between the three treatments. Clomipramine and mianserin were significantly superior to placebo in the topographical pain subgroup with headache using area under the improvement curves as criteria (p less than 0.05). When the 60-item General Health Questionnaire was used to identify minor psychiatric morbidity 44% was found. We can use this as a measure of quality of life. Our results have indicated that placebo-controlled studies are still needed in this field of research.