The influence of epileptic neuropathology and prior peripheral immunity on CNS transduction by rAAV2 and rAAV5.

Adeno-associated virus (AAV) provides a promising platform for clinical treatment of neurological disorders owing to its established efficacy and lack of apparent pathogenicity. To use viral vectors in treating neurological disease, however, transduction must occur under neuropathological conditions. Previous studies in rodents have shown that AAV5 more efficiently transduces cells in the hippocampus and piriform cortex than AAV2. Using the kainic acid (KA) model of temporal lobe epilepsy and AAV2 and 5 carrying a hybrid chicken ß-actin promoter driving green fluorescent protein (GFP), we found that limbic seizure activity caused substantial neuropathology and resulted in a significant reduction in subsequent AAV5 transduction. Nonetheless, this reduced transduction still was greater than AAV2 transduction in control rats. Although KA seizures compromise blood-brain barrier function, potentially increasing exposure of target tissue to circulating neutralizing antibodies, we observed no interaction between KA seizure-induced damage and immunization status on AAV transduction. Finally, while we confirmed the near total neuronal-specific transgene expression for both serotypes in control rats, AAV5-GFP expression was increasingly localized to astrocytes in seizure-damaged areas. Thus, the pathological milieu of the injured brain can reduce transduction efficacy and alter viral tropism- both relevant concerns when considering viral vector gene therapy for neurological disorders.

Restraint-induced fra-2 and c-fos expression in the rat forebrain: relationship to stress duration.

The protein product of the fra-2 gene (Fra-2), a fos-family member, can compete with Fos protein for participation in activating protein-1 (AP-1) transcription factor complexes and each protein can contribute different transactivational consequences to an AP-1 complex. To date, there is limited characterization of fra-2 mRNA expression in the rat forebrain. We examined basal and restraint-induced mRNA expression (in situ hybridization) of fra-2 in the rat forebrain and compared its temporal-spatial pattern to c-fos. In contrast to the very low basal expression of c-fos, fra-2 basal expression was moderately high throughout cortex and some subcortical structures, including prominent basal expression in the hypothalamic paraventricular nucleus (PVN). Restraint-induced fra-2 expression was quantified in the prefrontal cortex (PFC), lateral septum (LS) and PVN. Maximal fra-2 gene induction in the PFC and LS was delayed (60 min) after restraint onset with respect to c-fos (15 min), whereas in the PVN, fra-2 mRNA increased within 15 min of restraint. Additionally we compared c-fos and fra-2 gene expression in rats given shorter or longer restraint durations, but equal total time from stress onset to sample collection, to determine the extent to which the kinetics of gene induction matched that of a hypothalamic-pituitary-adrenal axis hormone response. Rats given 45 min recovery after 15 min restraint showed less c-fos expression in the PVN, less fra-2 expression in the prelimbic and infralimbic PFC, and no difference in the LS compared with rats restrained for 60 min. Thus, the expression of both genes was sensitive to stressor duration, but this sensitivity varied with brain region. Differential basal and stress-induced expression patterns of the fra-2 and c-fos genes are likely to have important functional consequences for AP-1 transcription factor dependent regulation of neural plasticity.

Activation of multiple molecular mechanisms for apoptosis in human malignant glioblastoma T98G and U87MG cells treated with sulforaphane.

Glioblastoma is the most malignant and prevalent brain tumor that still remains incurable. Recent studies reported anti-cancer effect of the broccoli-derived compound sulforaphane. We explored the mechanisms of sulforaphane-mediated apoptosis in human glioblastoma T98G and U87MG cells. Wright staining and ApopTag assay confirmed apoptosis in glioblastoma cells treated with sulforaphane. Increase in intracellular free Ca2+ was detected by fura-2 assay, suggesting activation of Ca2+-dependent pathways for apoptosis. Western blotting was used to detect changes in expression of Bax and Bcl-2 proteins resulting in increased Bax:Bcl-2 ratio that indicated a commitment of glioblastoma cells to apoptosis. Upregulation of calpain, a Ca2+-dependent cysteine protease, activated caspase-12 that in turn caused activation of caspase-9. With the increased Bax:Bcl-2 ratio, cytochrome c was released from mitochondria to cytosol for sequential activation of caspase-9 and caspase-3. Increased calpain and caspase-3 activities generated 145 kD spectrin breakdown product and 120 kD spectrin breakdown product, respectively. Activation of caspase-3 also cleaved the inhibitor-of-caspase-activated-DNase. Accumulation of apoptosis-inducing-factor in cytosol suggested caspase-independent pathway of apoptosis as well. Two of the inhibitor-of-apoptosis proteins were downregulated because of an increase in 'second mitochondrial activator of caspases/Direct inhibitor-of-apoptosis protein binding protein with low pI.' Decrease in nuclear factor kappa B and increase in inhibitor of nuclear factor kappa B alpha expression favored the process of apoptosis. Collectively, our results indicated activation of multiple molecular mechanisms for apoptosis in glioblastoma cells following treatment with sulforaphane.

Components of the kallikrein-kinin system in rat urine.

Using a direct radioimmunoassay and a kininogenase assay, we determined that 68% of rat urinary kallikrein was enzymatically active while 32% was in an inactive form which was activated by trypsin. Inorganic cations, at concentrations found in rat urine, were inhibitory in an amidase assay but appeared to potentiate kininogenase activity of pure rat urinary kallikrein. In random urines, kinin concentration was 4.2 +/- 0.7 ng/ml. Trypsinization of the urines generated 52.9 +/- 25.8 ng kinin/ml, indicating that kininogen was present. The rate of kinin formation in vivo may depend on the availability of kininogen and the concentration of inorganic cations in urine, as well as on other well-recognized factors, such as the kallikrein activity of the urine.

The effect of diuretics on components of the urinary kallikrein-kininogen-kinin system in man.

The effects of acute infusions of diuretics on components of the human urinary kallikrein-kininogen-kinin system were determined. Normal human subjects were given infusions of chlorothiazide and furosemide in doses calculated to produce a comparable natriuresis and diuresis. Alterations in urine electrolyte excretion, kinins, total and intact kininogen, and total active kallikrein were determined before and after the diuretic administration. Chlorothiazide caused a significant increase in total, but not active, kallikrein and had no effect on kinins and total or intact kininogen. Furosemide did not alter total or active kallikrein, or intact kininogen, but did decrease kinin and total kininogen excretion significantly. These differences in effects were not related to urinary sodium excretion or urinary flow because both diuretics produced comparable effects on these parameters. We conclude that acute infusions of diuretics do not activate the kallikrein-kininogen-kinin system and that some of the previously described effects of diuretics on this system may be related to their site of action.

Renal effects of angiotensin converting enzyme inhibitors in heart failure: a clinician's guide to minimizing azotemia and diuretic-induced electrolyte imbalances.

Angiotensin converting enzyme (ACE) inhibitors have proved to be valuable, life-saving medications in the management of heart failure. While reducing myocardial oxygen consumption, they increase cardiac output and thus renal plasma flow. Despite reports in the literature of adverse effects of these drugs on renal function, the risks of functional deterioration are predictable in patient populations and remediable. Patients at greatest risk of declining renal function during therapy with ACE inhibitors are those in whom maintenance of renal function is dependent on angiotensin II. Reducing the dose of the concomitant diuretic, liberalizing the dietary intake of sodium, and increasing the dose of the ACE inhibitor usually restores renal function to baseline. In patients with severe renal insufficiency, reducing the dose of the ACE inhibitor might be necessary to preserve the glomerular filtration rate.

Preventing transmission of hepatitis B virus from people with chronic infection.

BACKGROUND: People with chronic hepatitis B virus (HBV) infection are the major source of HBV transmission in the United States. The Public Health Service recommends prevention counseling for HBV-infected people and vaccination of their household contacts and sexual partners. OBJECTIVES: To describe the implementation of these recommendations by community physicians. METHODS: Telephone survey of 69 people with chronic HBV infection and their healthcare providers, October 1997 through November 1997, in San Diego, California. MAIN OUTCOME MEASURES: Counseling of people with chronic HBV infection and vaccination of their household contacts and sexual partners. RESULTS: Forty-three percent of providers reported providing prevention counseling to their HBV-infected patients to reduce transmission; 16% of patients reported receiving counseling. For the 32 pairs for which both the patient and provider could be reached and the patients were aware of their HBV infection, 20 (63%) providers reported counseling patients, and 10 (50%) of these providers' patients reported receiving counseling. Fifty-five percent of providers recommended vaccination of contacts; 13% of eligible adult household contacts and sexual partners and 20% of eligible child household contacts had begun hepatitis B vaccination. CONCLUSIONS: Prevention counseling of people with chronic HBV infection and vaccination of their contacts occur infrequently despite guidelines and an effective vaccine. Collaborative efforts between providers and people involved in public health are needed to improve delivery of these preventive health services.

Evaluation of enalapril/diltiazem ER in hypertensive patients with coexisting renal dysfunction. Enalapril/Diltiazem ER in Hypertensive Renal Disease Group.

Both enalapril and long-acting diltiazem have been shown to effectively lower blood pressure (BP) in hypertensive patients. Furthermore, in clinical studies, these two agents provided beneficial renal effects in these patients when administered on a long-term basis. A combination of enalapril/diltiazem ER was evaluated in 62 patients with Stage 1-3 hypertension and coexisting renal disease. This trial used a multicenter, randomized, double-blind, parallel group design. The study consisted of a 12-week double-blind phase followed by a 6-month open-label extension phase. The combination of enalapril/diltiazem ER was shown to reduce BP following both short-term and long-term treatment phases. Patients in Renal Group I (creatinine clearance CrCl): 30-59 ml/min/1.73 m2) had decreases of -18/-16 and -25/-20 mm Hg after 12 weeks and 9 months of therapy, respectively. Those in Renal Group II (CrCl: 10-29 ml/min/1.73 m2) had similar decreases of -23/-18 and -23/-19 mm Hg at these time points. The adverse events, in both phases, were those associated with the respective monotherapies. A reduction in CrCl with a coincident decrease in proteinuria was noted for both renal groups. The combination of enalapril/diltiazem ER lowered BP and was generally well tolerated by the patients. The combination of these two agents should improve the management of hypertensive patients.

Balance prosthesis based on micromechanical sensors using vibrotactile feedback of tilt.

A prototype balance prosthesis has been made using miniature, high-performance inertial sensors to measure lateral head tilt and vibrotactile elements mounted on the body to display head tilt to the user. The device has been used to study the feasibility of providing artificial feedback of head tilt to reduce postural sway during quiet standing using six healthy subjects. Two vibrotactile display schemes were used: one in which the individual vibrating elements, called tactors, were placed on the shoulders (shoulder tactors); another in which columns of tactors were placed on the right and left sides of the trunk (side tactors). Root-mean-square head-tilt angle (Tilt) and center of pressure displacement (Sway) were measured for normal subjects standing in a semi-tandem Romberg position with eyes closed, under four conditions: no balance aids; shoulder tactors; side tactors; and light touch. Compared with no balance aids, the side tactors significantly reduced Tilt (35%) and Sway (33%). Shoulder tactors also significantly reduced Tilt (44%) and Sway (17%). Compared with tactors, light touch resulted in less Sway, but more Tilt. The results suggest that healthy normal subjects can reduce their lateral postural sway using head tilt information as provided by a vibrotactile display. Thus, further testing with balance-impaired subjects is now warranted.

Thiazide induced hypotension: the role of plasma volume reduction and the urinary kallikrein system.

The hypotensive response the thiazide diuretics was studied in 15 males with moderate essential hypertension and correlated with serial changes in plasma volume, weight, plasma renin activity, urinary aldosterone, and urinary kallikrein, both total and activity. A greater than 10 mmHg fall in mean arterial pressure after four weeks of treatment defined the responders to therapy (n = 10) while all others were considered non-responders (n = 5). In responders, the fall in mean arterial pressure was accompanied by sustained reduction in plasma volume and weight. No sustained fall in plasma volume was noted in non-responders. Plasma renin activity and urinary aldosterone excretion increased in responders but not in non-responders. Urinary kallikrein, both total and active, increased in the responders but remained unchanged in the non-responders. The results are consistent with the hypothesis that a sustained reduction in plasma volume is necessary for the maintenance of a hypotensive response to thiazides. Stimulation of the renal kallikrein-kinin system may be necessary to balance the antinatriuretic and pressor effects of the renin-angiotensin-aldosterone system. If unopposed, this system would return plasma volume and blood pressure to pretreatment levels.

Urinary kininogen: a possible regulator of kinin formation in normal individuals and subjects with essential hypertension, end-stage renal and liver disease.

Most previous studies have not significantly correlated urinary kallikrein to urinary kinins. We investigated whether urinary kininogen might influence kinin formation within the urine. On an ad-lib diet the 24 hour excretion of total and intact kininogen, kinins and kallikrein was determined in 24 control subjects, 20 untreated essential hypertensives, 12 with end-stage renal disease and 8 subjects with liver disease. Kallikrein and kinins were measured by a direct radioimmunoassay. Total kininogen was determined from the sum of preformed kinins and kinins generated after trypsin (intact kininogen). Cross reactivity between purified human low molecular weight kininogen and bradykinin antiserum was 3%. Total and intact kininogen were significantly correlated with kinins in controls, essential hypertension and liver disease. In essential hypertension, end-stage renal and liver diseases kinins were significantly decreased. This was associated with a reduction in kininogen but not kallikrein in essential hypertension and liver disease, and a reduction in kallikrein but not kininogen in end-stage renal disease. Thus, renal kinin generation in various states may be affected by either or both kininogen and kallikrein.

Medial prefrontal cortex activity can disrupt the expression of stress response habituation.

Recent findings suggest that the expression of hypothalamic-pituitary-adrenal (HPA) axis stress response adaptation in rats depends on top-down neural control. We therefore examined whether the medial prefrontal cortex (mPFC) modulates expression of stress response habituation. We transiently suppressed (muscimol microinfusion) or stimulated (picrotoxin microinfusion) mPFC neural activity in rats and studied the consequence on the first time response to psychological stress (restraint) or separately on the development and expression of habituation to repeated restraint. We monitored both the hormonal (corticosterone) and neural (forebrain c-fos mRNA) response to stress. Inactivation of the mPFC had no effect on the HPA-axis response to first time restraint, however increased mPFC activity attenuated stress-induced HPA-axis activity. In a three day repeated restraint stress regimen, inactivation of the mPFC on days 1 and 2, but not day 3, prevented the expression of HPA-axis hormone response habituation. In these same rats, the mPFC activity on day 3 interfered with the expression of c-fos mRNA habituation selectively within the mPFC, lateral septum and hypothalamic paraventricular nucleus. In contrast, inactivation of the mPFC only on day 3, or on all 3 days did not interfere with the expression of habituation. We conclude that the mPFC can permit or disrupt expression of HPA-axis stress response habituation, and this control depends on alteration of neural activity within select brain regions. A possible implication of these findings is that the dysregulation of PFC activity associated with depression and post-traumatic stress disorder may contribute to impaired expression of stress-response adaptation and consequently exacerbation of those disorders.

Inescapable but not escapable stress leads to increased struggling behavior and basolateral amygdala c-fos gene expression in response to subsequent novel stress challenge.

Control over an aversive experience can greatly impact the organism's response to subsequent stressors. We compared the effects of escapable (ES) and yoked inescapable (IS) electric tail shocks on the hypothalamic-pituitary-adrenal (HPA) axis hormonal (corticosterone and adrenocorticotropic hormone (ACTH)), neural (c-fos mRNA) and behavioral (struggling) response to subsequent restraint. We found that although the HPA axis response during restraint of both previously stressed groups were higher than stress-naïve rats and not different from each other, lack of control over the tailshock experience led to an increase in restraint-induced struggling behavior of the IS rats compared to both stress-naïve and ES rats. Additionally, c-fos expression in the basolateral amygdala was increased selectively in the IS group, and relative c-fos mRNA expression in the basolateral amygdala positively correlated with struggling behavior. Restraint-induced c-fos expression in the medial prefrontal cortex, a brain area critical for mediating some of the differential neurochemical and behavioral effects of ES and IS, was surprisingly similar in both ES and IS groups, lower than that of stress-naïve rats, and did not correlate with struggling behavior. Our findings indicate that basolateral amygdala activity may be connected with the differential effects of ES and IS on subsequent behavioral responses to restraint, without contributing to the concurrent HPA axis hormone response.