A latitudinal cline in disease resistance of a host tree.

The possible drivers and implications of an observed latitudinal cline in disease resistance of a host tree were examined. Mycosphaerella leaf disease (MLD) damage, caused by Teratosphaeria species, was assessed in five Eucalyptus globulus (Tasmanian blue gum) common garden trials containing open-pollinated progeny from 13 native-forest populations. Significant population and family within population variation in MLD resistance was detected, which was relatively stable across different combinations of trial sites, ages, seasons and epidemics. A distinct genetic-based latitudinal cline in MLD damage among host populations was evident. Two lines of evidence argue that the observed genetic-based latitudinal trend was the result of direct pathogen-imposed selection for MLD resistance. First, MLD damage was positively associated with temperature and negatively associated with a prediction of disease risk in the native environment of these populations; and, second, the quantitative inbreeding coefficient (QST) significantly exceeded neutral marker FST at the trial that exhibited the greatest MLD damage, suggesting that diversifying selection contributed to differentiation in MLD resistance among populations. This study highlights the potential for spatial variation in pathogen risk to drive adaptive differentiation across the geographic range of a foundation host tree species.

In vivo skin absorption and distribution of the nerve agent VX (O-ethyl-S-[2(diisopropylamino)ethyl] methylphosphonothioate) in the domestic white pig.

The purpose of this study was to characterize the skin absorption and distribution of VX (O-ethyl-S-[2 (diisopropylamino)ethyl] methylphosphonothioate) in the domestic pig in order to evaluate the animal as a potential model for assessing pretreatments against toxic anti-cholinesterase compounds. A liquid droplet (equivalent to a 2 x LD50 dose) of radiolabelled VX was applied to the inner ear-skin of each anaesthetized animal. Blood and tissue samples (liver, lung, kidney, heart and skin exposure sites) were obtained post-mortem. The amount of radioactivity in each sample was measured by liquid scintillation counting, from which the skin absorption rate and dose distribution of VX were calculated. A substantial proportion (22 +/- 3%) of the applied dose remained within the skin at the site of application. It is conceivable that strategies to minimize or remove this reservoir may be of benefit in the early treatment of VX-exposed casualties. Image analysis of autoradiographs of exposed skin sites indicated that each milligram of radioactive VX covered an area of 1.2 +/- 0.5 cm2. The average skin absorption rate of 14C-VX was 661 +/- 126 microg/cm2 per hour. Comparison of these data with previous studies suggests that human skin is less permeable to VX than pig skin, but VX spreads over a greater surface area when applied to human skin. Thus, paradoxically, while pig-ear skin is more permeable than human skin, the difference in skin surface spreading may lead to the absorption of an equivalent systemic dose.

Clinical manifestations of VX poisoning following percutaneous exposure in the domestic white pig.

Nerve agents are a class of organophosphorus chemicals that inhibit certain cholinesterase enzymes (ChE). If untreated, percutaneous exposure to nerve agents, such as VX (O-ethyl-S-[2(diisopropylamino)ethyl] methylphosphonothioate) can cause paralysis, apnoea and death. Much of the information concerning the percutaneous absorption and subsequent toxicity of nerve agents has been obtained using various rodent models. However, the most relevant 'skin model' is arguably the pig. Therefore, the purpose of this study was to examine the clinical manifestations of VX intoxication in the domestic white pig following a 2 LD50 (120 microg/kg) percutaneous challenge. There was a consistent onset of signs (where present) in each animal: mastication was followed by miosis, salivation, fasciculations and apnoea. Whilst ChE activity did not correlate with the onset of signs, there was a qualitative relationship in that mastication preceded substantial ChE inhibition, miosis lagged behind the linear decrease in acetylcholinesterase (AChE) activity and fasciculations and apnoea occurred after maximum ChE inhibition had been attained (5-10% of normal). These observations may be of use for the triage of patients exposed to VX. In comparison with similar studies with GD, VX did not affect glucose utilization. However, VX was similar to GD in that it caused a mild hyperkalaemia and hyperphosphataemia, although the significance of this observation was not clear. There was substantial lateral diffusion of the initial droplet of VX over the application site, indicating that, when decontaminating exposed skin, attention should also be directed to areas peripheral to the original site of exposure.

Magnetic resonance imaging predicts neuropathology from soman-mediated seizures in the rodent.

Intoxication by the organophosphate compound soman causes prolonged seizures that lead to neuropathology in the brain. This MRI-based study describes the temporal and spatial evolution of brain pathology that follows soman-induced convulsions. We observed significant decreases in apparent diffusion coefficients (ADC; 23% below control) of the hippocampus and thalamus by 12 h after soman treatment. The ADC then returned to near normal values in all regions at 24 h but declined again during the next 7 days. These data suggest that the initial cellular degradation may be resolved but is ultimately followed by regional cellular remodeling. T2 relaxation values declined significantly at 12 h (37% decrease) returning to near normal values by 24 h. These data lend detail to the model suggesting that injured tissues experience an edematous influx that is resolved by 24 h. The imaging data was fully supported by histopathological comparisons where moderate cell loss and swelling within the hippocampus and piriform cortex was observed. This is the first report providing excellenttemporal and spatial resolution of emerging soman-mediated, seizure-induced neuropathology using MRI with histological correlation.

A comparison of the effects of sarin and succinylcholine on respiratory parameters in anesthetized domestic swine.

Differences in the "respiratory paralysis" caused by sarin (GB) and succinylcholine (SDC) were observed in a domestic swine model using a bedside pulmonary dynamics monitor. GB was administered intravenously (9 micrograms/kg/30 min) and compared with SDC administered intravenously (20 mg/30 min). All animals developed respiratory insufficiency indicated by decreased respiratory frequency. Minute ventilation was relatively maintained in animals that received GB by increasing tidal volume, whereas both of these parameters decreased in animals that received SDC. GB animals showed an increase in airway resistance and work of breathing. The former was unchanged and the latter was decreased in animals that received SDC. Mouth occlusion pressure at 100 milliseconds and tidal volume were relatively maintained in GB animals but decreased in SDC animals, suggesting a central mechanism for respiratory paralysis with GB and a peripheral mechanism for respiratory paralysis with SDC.

Variation in utilization of computed tomography scanning for the investigation of minor head trauma in children: a Canadian experience.

OBJECTIVES: To compare the utilization rates of CT scans in investigating minor head trauma in children in Canada, to identify the injuries determined by these scans, and to identify clinical findings that are highly associated with its diagnosis and the injury itself. METHODS: A retrospective cohort study involving nine pediatric hospitals in Canada was conducted. A structured data collection method was used. Inclusion criteria included age 16 years or less, history of blunt head trauma, and a Glasgow Coma Scale score (GCS) greater than or equal to 13. Data collected included demographic information, type of injury, relevant clinical information, computed tomography (CT) scan data, and clinical outcome. Clinical findings associated with CT scan and positive CT scan were identified using logistic regression. RESULTS: One thousand one hundred sixty-four children were included in the study. One hundred seventy-one (15%) had a CT scan, of which 60 (35%) were abnormal. There was a significant difference in the rate of ordering of CT scans among the participating hospitals, but no significant difference in the rate of abnormal CT scans. Mechanism of injury, GCS, and loss of consciousness were significantly related to the presence of an abnormal CT scan. CONCLUSIONS: Although there is a significant difference in the utilization of CT scans to investigate minor head trauma in children across Canada, there is no significant difference in the frequency of head injuries in these patients. This suggests that it may be possible to determine clinical criteria that are predictive of a head injury in these patients.

Effect of intracellular calcium modulation on sulfur mustard cytotoxicity in cultured human neonatal keratinocytes.

Previous studies in human skin keratinocyte cultures have shown that sulfur mustard (HD) induces an immediate and irreversible increase in internal free calcium levels that was independent of external calcium concentrations. These findings suggested a role for calcium in the aetiology of HD-induced cell death and that modulation of intracellular calcium concentrations may assist in providing protection against this agent. In the current work, actively proliferating and confluent cultures of first passage neonatal human skin keratinocytes were used to assess the effect of altered intra- and extracellular calcium levels on HD toxicity. Treatment of cultures with the endoplasmic reticulum calcium ATPase inhibitor thapsigargin, or the calcium chelator BAPTA-AM, which reduce HD-induced elevation of intracellular free calcium, did not modulate the toxicity of HD. Furthermore, alteration of external calcium concentrations during these same experiments failed to elicit any change in the viability of HD-exposed cells. Treatment of confluent cultures with ionomycin at either low (100 microM) or high (1.2 mM) external calcium concentrations also failed to modulate the toxicity of HD in any way. It appears that in neonatal human skin keratinocytes in culture, HD-induced intracellular calcium perturbation does not play a major role in HD-induced cytotoxicity.

Modification of cytosolic free calcium concentrations in human keratinocytes after sulfur mustard exposure.

Exposure of confluent cultures of human skin keratinocytes to sulfur mustard (SM) induced an immediate and irreversible rise in internal free Ca(2+) levels that was independent of external Ca(2+) concentrations. The response was rapid, beginning within 1min after addition of SM to the cells, and sensitive, with significant effects observed at 100 mum. The rise in [Ca(2+)](INT) was unaffected by zero external Ca(2+) but was blocked by prior incubation with thapsigargin. The sensitivity to and irreversibility of the effects of SM on Ca(2+) levels was paralleled by cellular toxicity as assessed using three different cell viability assays. In addition, the time course of the onset of irreversible toxicity in our cultures coincides with the time course of effects on [Ca(2+)](INT). SM was also found to displace specifically bound ATP from purinergic (P(2)) receptors. These results suggest that therapies aimed at protecting internal stores of Ca(2+) from disruption by SM, perhaps at P(2) receptors, may provide substantial benefit in protecting human skin cells from the toxic effects of this vesicant.

Concurrent aneurysm rupture and thrombosis of high grade internal carotid artery stenosis: report of two cases.

BACKGROUND: The simultaneous presentation of aneurysmal subarachnoid hemorrhage and thrombosis of a high-grade internal carotid artery stenosis is rare, and their management raises several treatment dilemmas. METHODS: Two such patients with ruptured aneurysms are presented: one with high-grade internal carotid artery stenosis that progressed to occlusion and one with acute internal carotid artery occlusion. RESULTS: Both patients were treated with craniotomy for clipping of the ruptured aneurysm followed by carotid thromboendarterectomy. CONCLUSIONS: We advocate urgent surgical treatment of both lesions, dealing with the most symptomatic lesion first. These two cases demonstrate the importance of reestablishing blood flow in patients with an acutely thrombosed carotid artery.

Focal midbrain glioma: long term survival in a cohort of 16 patients and the implications for management.

BACKGROUND: Focal gliomas involving the midbrain tectum and tegmentum have been identified as having a better prognosis than diffuse tumors affecting the brain stem. However, only limited information is available concerning treatment effectiveness and long term outcome for these patients. METHODS: A retrospective, population-based cancer registry survey was performed to assess the clinical features and treatment courses of patients with focal midbrain tumors. RESULTS: Sixteen patients with midbrain gliomas were identified; eight had tectal gliomas and eight tegmental gliomas. Thirteen patients presented with symptoms related to hydrocephalus, and 12 required a ventriculoperitoneal shunt. Seven patients underwent surgery directed at the tumor. Eight patients underwent initial radiation therapy and none had initial chemotherapy. One patient diagnosed at age 18 months had a rapidly growing tumor after 14 months of follow up which has responded to chemotherapy. The mean survival of this patient population was 84 months (range 3-280 months) after diagnosis, with only one tumor related death occurring (280 months after diagnosis). Survival was not affected by tumor location within the midbrain (tegmental or tectal) or by whether radiation therapy was or was not administered. CONCLUSIONS: Patients with focal midbrain gliomas require symptom control aimed at treatment of hydrocephalus, or mass effect from the tumor. However the extended survival of this population suggests that routine aggressive surgical debulking is often not required. Furthermore, the routine use of radiation therapy or chemotherapy for all such patients is questioned.

Revascularization and aneurysm surgery: current techniques, indications, and outcome.

Revascularization is an important component of treatment for complex aneurysms that cannot be directly clipped and instead require parent vessel occlusion. A consecutive series of 61 patients with 63 aneurysms requiring cerebral revascularization is presented. Aneurysms were located along the petrous internal carotid artery (ICA) (n = 5), the cavernous ICA (n = 16), the supraclinoid ICA (n = 12), the middle cerebral artery (n = 17), the anterior cerebral artery (n = 4), the vertebral artery/posterior inferior cerebellar artery (n = 5), and the midbasilar artery (n = 4). Aneurysms were treated by direct clipping (n = 8), trapping (n = 28), proximal vessel occlusion (n = 9), distal vessel occlusion (n = 1), excision (n = 15), and thrombotic occlusion (n = 2). Revascularization was performed with petrous to supraclinoid ICA bypass (n = 12), superficial temporal artery to middle cerebral artery bypass (n = 15), superficial temporal artery to middle cerebral artery bypass with saphenous graft (n = 5), superficial temporal artery to superior cerebellar artery bypass (n = 4) long saphenous bypass (n = 11), in situ bypass (n = 3), and primary reanastomosis (n = 13). Fifty-seven patients (93%) had good outcomes, and one patient died (surgical mortality, 2%). This experience demonstrates that revascularization can be performed with low morbidity and mortality. We think that the cumulative risks of not performing revascularization in patients who tolerate ICA balloon occlusion exceed the surgical risk of revascularization. We therefore favor revascularization in patients with complex aneurysms treated by surgical arterial occlusion.

Posterior skull base transfacial approaches.

Transfacial approaches to the midline skull base provide direct access for resection of complex lesions. The transfrontal nasal-orbital and transmaxillary (Levels III and V) approaches have become the "work horses" of our skull base team, in that either alone or in combination, they provide access to most parts of the anterior skull base and clivus. The flexibility offered by the ability to select a Level I or Level II approach in place of a Level III approach, or a Level VI approach in place of a Level V approach, however, greatly enhances the individualization of treatment strategy. The overlapping exposure shared by these techniques provides flexibility in the choice of surgical exposure and allows multiple factors to be considered, including tumor location and size.

Multimodality treatment of deep arteriovenous malformations: thalamus, basal ganglia, and brain stem.

THE THERAPEUTIC APPROACH toward arteriovenous malformations (AVMs) located in the basal ganglia, thalamus, and brain stem has evolved from microsurgical resection as the predominant therapy to a combination of microsurgery, embolization, and radiosurgery. This multimodality treatment was used in the management of 32 patients with deep AVMs of all sizes. Twenty-two patients with surgically accessible AVMs (i.e., typically located in the brain stem and thalamus) underwent microsurgical resection. The AVMs of half of these patients were devascularized preoperatively with transfemoral embolization. Five patients with residual AVMs were then treated with radiosurgery. Ten patients had AVMs, typically located in the basal ganglia, that were surgically inaccessible. These patients underwent embolization to reduce the AVM size, and the postembolization nidus was then treated with radiosurgery. For patients treated early in the series with a predominantly surgical approach, the complete resection rate was 43%. For patients treated later in the series after radiosurgery was incorporated into the management scheme, the complete elimination rate was 72%. Overall, there were no deaths in this series, and the permanent treatment-associated morbidity rate was 9%. These results indicate that an individualized, multimodality approach can be used to eliminate both large and small deep AVMs with an acceptably low morbidity and mortality rate.

Effect of ruthenium red on voltage-sensitive Ca++ channels.

The organometallic dye, ruthenium red (RR) inhibited Ca++ influx, omega-conotoxin GVIA-sensitive Ca++ binding and Ca(++)-dependent neurotransmitter release in a qualitatively similar manner in rat and chicken synaptosomes and in mammalian neuromuscular preparations, but had no effect on Ca(++)-dependent processes mediated by the dihydropyridine-sensitive Ca++ channel. These effects are specific for the RR complex, as RuCl3 affected neither Ca++ influx, omega-conotoxin GVIA binding nor neurotransmitter release, but did, however, in contrast to RR, displace [3H]nitrendipine from synaptosomes. RR, in a manner similar to omega-conotoxin MVIIC and omega-agatoxin IVA (AgaIVA), also effectively inhibited the response of the rat diaphragm to nerve stimulation and blocked AgaIVA-sensitive Ca++ influx in the rat brain, suggesting a significant interaction at the P-type voltage-sensitive Ca++ channel. These effects of RR suggest that this amino complex affects both the N and the P domain of the Ca++ channel in the chicken brain and both the N- and P-type Ca++ channel which is intimately coupled to the Ca++ influx and neurotransmitter release in rat synaptosomes. Its ability to block all of the Ca++ influx in mammalian brain preparations and to inhibit completely the nerve-stimulated rat neuromuscular junction certainly indicates a significant action at the P-type voltage-sensitive Ca++ channel, similar to omega-conotoxin MVIIC or AgaIVA. RR should prove to be a valuable synthetic, inexpensive tool with which to probe the neuropharmacology of the mammalian neurotransmitter-linked voltage-sensitive Ca++ channels.

The hemocyanin of the Californian black sea hare, Aplysia vaccaria Winkler.

The hemocyanin of the Californian black sea hare. Aplysia vaccaria exists in solution largely as a di-decameric protein with a molecular weight of close to 8.0 x 10(6) and a sedimentation coefficient of about 92 S. Light-scattering measurements at pH 8.0, 0.1 M Tris, 0.05 M Mg2+, 0.01 M Ca2+ gave a molecular weight of 8.0 +/- 0.6 x 10(6), and scanning transmission electron microscopic determinations (STEM) gave a slightly higher particle mass of 8.49 +/- 0.41 x 10(6) daltons. Measurements using the STEM method gave a particle mass of 4.27 +/- 0.26 x 10(6) daltons for the dissociated half-molecules or decamers. Light-scattering measurements on the dissociated monomers at pH 11.1 and in 8.0 M urea gave molecular weights of 4.74 x 10(5). Sedimentation measurements in the presence of 0.01 M Mg2+ indicate that the hemocyanin of A. vaccaria is largely in the di-decameric form in the pH region from about 5.0 to 8.0. Above pH 8.0 the hemocyanin di-decamers are found to dissociate to half-molecules or decamers, followed by dissociation to dimers and monomers as the pH is increased above pH 9.0.

Insulin reduction of cerebral infarction due to transient focal ischemia.

Insulin has recently been shown to ameliorate damage in models of global brain ischemia. To determine whether insulin is also neuroprotective in focal ischemia, 20 rats were given 2 to 3 IU/kg insulin and 10 did not receive treatment prior to normothermic transient middle cerebral artery occlusion for 2 hours at a blood pressure of 60 mm Hg. To further elucidate whether infarction volume is influenced by variations in blood glucose levels within the physiological range, blood glucose was raised in 10 of the insulin-treated animals to levels comparable with the untreated controls. At 1-week survival, damage was assessed using quantitative neuropathological examination of 25 coronal planes. It was found that preischemic insulin lowered the mean intraischemic blood glucose level from 8.4 +/- 0.2 mM (mu +/- standard error of the mean) in the control group to 3.4 +/- 0.2 mM and reduced total damage (atrophy plus cortical and striatal necrosis), expressed as the percentage of the normal hemisphere, from a control of 28.5% +/- 2.9% to 14.5% +/- 1.6% (p < 0.005). Coadministration of glucose and insulin resulted in a mean intraischemic blood glucose level of 10.1 +/- 0.5 mM, with 27.0% +/- 2.4% total damage (p = 0.96, compared with control). Total ischemic damage showed an independent correlation with blood glucose levels (r = 0.67, p = 0.0018). The findings indicate that insulin benefits transient focal ischemia and that reducing the blood glucose from 8 to 9 mM to the low-normal range of 3 to 4 mM with insulin dramatically reduces subsequent infarction. The data suggest that the neuroprotective mechanism of insulin action in focal middle cerebral artery occlusion is mediated predominantly via alterations in blood glucose levels. In comparison to global ischemia, focal ischemia appears to show only a minor direct central nervous system effect of insulin. In clinical situations in which transient focal ischemia to the hemisphere can be anticipated, insulin-induced hypoglycemia of a mild degree may be beneficial.

Radical resection of anterior skull base tumors.

The transfacial approaches give the surgeon wide exposure for resecting skull base lesions. The classification system, using six levels, helps plan the best surgical strategy. Our experience with the transfacial approaches has been associated with acceptably low rates of morbidity and mortality. Our small experience with carotid sacrifice reflects our practice of preserving the ICA whenever possible. We recommend preserving the ICA with benign tumors because they do not invade the artery, or they invade it to a limited extent. In contrast, we recommend radical tumor resection and sacrifice of the ICA with malignant tumors, because they directly threaten the integrity of the ICA and a patient's survival. The ICA should not be considered a limitation to radical tumor resection, because the ICA can be safely reconstructed with an appropriate bypass procedure.

Identification of noradrenaline in venom from the funnel-web spider Hololena curta.

Venom from the funnel-web spider Hololena curta was added to the Krebs-Henseleit solution bathing isolated ring preparations of rat thoracic aorta, suspended in water-jacketed organ baths, for the purpose of tension recordings. Hololena curta venom at dilutions of 1:100,000 to 1:1000 caused a marked vasoconstriction, which was completely inhibited by the alpha 1 adrenoceptor antagonist prazosin (1 microm). The vasoconstriction appears to be due to the direct effects on alpha 1 adrenoceptors of a venom constituent, which we have identified using HPLC/ECD as the catecholamine, noradrenaline.

Pharmacological identification of a novel Ca2+ channel in chicken brain synaptosomes.

Ca2+ influx was measured in rat and chicken brain synaptosomes in the presence of a number of pharmacological tools which have recently been used to define voltage-sensitive Ca(2+)-channel (VSCC) types. In chicken brain synaptosomes. VSCCs which, because of their sensitivity to inhibition by omega-conotoxin (omega-CgTx), are thought to be exclusively N-type, the P-type VSCC polyamine inhibitor FTX (from Agelenopsis aperta venom; 1 microliters/ml), its synthetic analogue, sFTX (1-5 mM) and the polypeptides AgaIVA (IC50 0.29 microM) and omega-CgTx MVIIC (IC50 0.0022 microM) inhibited 70-100% of the measurable K+ stimulated Ca2+ influx. The prototypical N-channel VSCC inhibitor omega-CgTx GVIA (IC50 0.014 microM), Cd2+ (50 microM) and diluted venom from Hololena curta (1:2,500) also caused complete or almost complete, inhibition of Ca2+ influx. In comparable studies using rat brain synaptosomes, sFTX (1-10 mM) caused a dose-dependent reduction of Ca2+ influx, while FTX (1 microliters/ml) and AgaIVA (IC50 0.02 microM) completely inhibited Ca2+ influx. Similar to the findings in chicken synaptosomes, Cd2+ (50 microM) and H. curta (1:2,500 dilution) both inhibited K+ stimulated influx by > 80% whereas omega-CgTx (1 microM) only caused a maximum 25% inhibition. Both sFTX and its congener spermine, inhibited [125I]omega-CgTx binding to rat and chicken synaptosomal membranes. These results strongly implicate P-type channels as the major VSCC in rat brain. The results also clearly demonstrate a heretofore unrecognized, novel, FTX/AgaIVA/omega-CgTx GVIA/omega-CgTx MVIIC-sensitive VSCC in chicken brain.

A comparison of intra-arterial and intravenous tissue-type plasminogen activator on autologous arterial emboli in the cerebral circulation of rabbits.

BACKGROUND AND PURPOSE: The efficacy of thrombolytic therapy for treatment of embolic stroke has been a subject of both experimental and clinical examination. The aim of this study was to compare the efficacy, in regard to reduction of volume of ischemic brain, of two different modes of administration (ie, intra-arterial and intravenous) of tissue-type plasminogen activator (TPA) given 30 minutes after experimental embolic stroke in rabbits. METHODS: A randomized, blinded, controlled experimental trial was undertaken. Embolic stroke was simulated in rabbits by injecting fragments of autologous arterial thrombus into one internal carotid artery. Thirty minutes after embolization, the rabbits were blindly treated with 2 mg/kg intra-arterial TPA, 2 mg/kg intravenous TPA, or saline (all n = 10). Six hours after embolization the rabbits were killed. The brains were perfused with triphenyltetrazolium chloride and cut into 0.5-cm-thick coronal sections, and the areas of ischemia were measured. RESULTS: Administration of TPA resulted in a significant reduction in the volume of ischemic cerebral injury (P < .0001): control animals sustained ischemic injury to 20.1 +/- 4.6% (mean +/- SD) of total brain compared with 4.6 +/- 4.1% for animals treated with intra-arterial TPA and 3.4 +/- 2.6% for those treated with intravenous TPA. The difference between intra-arterial and intravenous TPA treatment was not significant (P = .786). CONCLUSIONS: In this rabbit model of embolic stroke, administration of TPA within 30 minutes resulted in a dramatic reduction in the amount of ischemic injury, with equal efficacy for the two modes of administration. These results favor the treatment of acute embolic stroke with intravenous TPA, given the rapidity with which intravenous therapy can be established in the clinical setting.