A novel patient-derived tumorgraft model with TRAF1-ALK anaplastic large-cell lymphoma translocation.

Although anaplastic large-cell lymphomas (ALCL) carrying anaplastic lymphoma kinase (ALK) have a relatively good prognosis, aggressive forms exist. We have identified a novel translocation, causing the fusion of the TRAF1 and ALK genes, in one patient who presented with a leukemic ALK+ ALCL (ALCL-11). To uncover the mechanisms leading to high-grade ALCL, we developed a human patient-derived tumorgraft (hPDT) line. Molecular characterization of primary and PDT cells demonstrated the activation of ALK and nuclear factor kB (NFkB) pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells, lacking PRDM1/Blimp1 and carrying c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to the downregulation of p50/p52 and lymphoma growth inhibition. Moreover, a NFkB gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Although a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, nevertheless the disease could not be eradicated. These data indicate that the activation of NFkB signaling contributes to the neoplastic phenotype of TRAF1-ALK ALCL. ALCL hPDTs are invaluable tools to validate the role of druggable molecules, predict therapeutic responses and implement patient specific therapies.

Demonstration of neuronal and extraneuronal uptake of circulating norepinephrine in the forearm.

Disturbances in peripheral norepinephrine release or removal by neuronal and extraneuronal uptake may have pathogenetic significance in cardiovascular disease states. We investigated the mechanisms of removal of norepinephrine in the forearm of healthy subjects under basal conditions, using measurements of arterial and venous plasma norepinephrine concentrations, blood pressure, heart rate, and forearm blood flow. The specific inhibitor of neuronal uptake, desipramine, was infused intra-arterially into the brachial artery of five subjects. Net norepinephrine overflow from the forearm increased markedly, revealing considerable local release of norepinephrine. Six other subjects received four intra-arterial infusions of norepinephrine, 1.18 pmol/kg/min, with various doses of desipramine and the extraneuronal uptake-inhibiting drug hydrocortisone. The forearm extraction rate for circulating norepinephrine decreased with increasing doses of desipramine (from 69.4 +/- 3.0 [SEM] to 35.3 +/- 8.4%; p less than 0.001). Increasing doses of hydrocortisone during continued inhibition of neuronal uptake resulted in decreased forearm extraction of norepinephrine (from 63.3 +/- 4.9 to 40.6 +/- 4.4%; p less than 0.01). In six other subjects who received the highest dose of hydrocortisone without concomitant inhibition of neuronal uptake, forearm extraction of norepinephrine decreased from 57.1 +/- 4.9 to 51.5 +/- 4.7% (p less than 0.05). These results suggest that neuronal uptake contributes markedly to the removal of circulating and endogenously released norepinephrine in the forearm. For circulating norepinephrine, a corticosteroid-sensitive mechanism of extraneuronal uptake was also demonstrated. These results indicate that neuronal and extraneuronal uptake can be estimated separately in this vascular bed. Similar organ-specific studies in patients may reveal disturbances in mechanisms of norepinephrine removal.

Norepinephrine removal and release in the forearm of healthy subjects.

The relevance of local removal and release of norepinephrine (NE) for antecubital venous plasma NE concentration was studied in 22 healthy subjects. Arterial and venous plasma NE and forearm blood flow were measured during intra-arterial infusion of two doses of NE, intra-arterial NE infusion with two doses of sodium nitroprusside, intravenous infusion of NE with intra-arterial infusion of four doses of sodium nitroprusside, and lower body negative pressure of -20 mm Hg for 15 minutes. The venous plasma NE concentration-time curves during the infusions of the two doses of NE indicated first-order kinetics for forearm extraction: forearm NE extraction rate during the low dose infusion was 67 +/- 4.1% (SEM) and correlated with basal forearm blood flow (r = -0.64, p less than 0.03, n = 12). Local sodium nitroprusside-induced vasodilatation during the intra-arterial and intravenous NE infusions was accompanied by dose-dependent decreases in forearm extraction rates for NE and epinephrine. During lower body negative pressure, taking into account the high basal forearm extraction rate for NE, local and systemic release of NE was indicated by increases in arterial and venous plasma and the venous-arterial plasma NE concentration difference (p less than 0.05 for all). These data show that removal of NE from forearm circulation is a process with a high extraction ratio obeying first-order kinetics and that this extraction process inversely relates to forearm blood flow. Thus, antecubital venous plasma NE is likely to be derived mainly from local release and not from the arterial plasma NE input.

Does regional norepinephrine spillover represent local sympathetic activity?

Regional spillover of norepinephrine (NE), based on isotope dilution and single-compartment steady-state kinetics, is considered one of the best parameters for estimating organ sympathetic activity. However, the effects of local changes in clearance of NE on the spillover have not yet been investigated. We studied local NE kinetics and clearance in the forearm of 10 healthy subjects using intra-arterial infusions of NE, tritiated NE, the neuronal uptake inhibitor desipramine, and tyramine, which competes with NE for the neuronal uptake carrier. Before and during complete blockade of neuronal uptake by desipramine the venous concentration-time curves for tritiated NE and for NE released by tyramine were biexponential, consistent with the presence of (at least) two compartments for circulating tritiated NE and for locally released NE. The time constants for tyramine-induced release of NE and, in the same subjects during desipramine infusion, for tritiated NE were almost equal at the same level of forearm blood flow. This argues against possible diffusion or transport differences for NE to and from the circulation and the synapse. The regional intrinsic clearance capacity (a measure of the maximal ability of an organ to irreversibly remove drug by all pathways in the absence of any flow limitations) for NE decreased in the forearm by 65% (p less than 0.01) during neuronal uptake blockade by desipramine; the forearm clearance decreased by 59% (p less than 0.001), whereas the spillover rate of NE increased from 33 +/- 5 to 63 +/- 11 pmol.min-1 (p less than 0.05). Nitroprusside-induced increments in blood flow increased the spillover of NE from 18 +/- 4 to 35 +/- 6 pmol.min-1 (p less than 0.01); the clearance of circulating NE also increased (by 58%, p less than 0.05), and the intrinsic clearance capacity remained unchanged. This demonstrates that regional spillover of NE is markedly influenced by local changes in clearance and flow. The new parameter plasma appearance rate of NE is proposed. Although also derived from isotope dilution, this parameter may better approximate the regional entry of NE into the blood pool than spillover. This is corroborated by the nonsignificant changes of plasma appearance rate of NE during our desipramine and nitroprusside infusions.

The effect of a converting enzyme inhibitor upon renal damage in spontaneously hypertensive Fawn Hooded rats.

OBJECTIVE: To determine the effect of angiotensin converting enzyme inhibition (CEI) upon renal function and the incidence of glomerulosclerosis in spontaneously hypertensive Fawn Hooded rats (FHR). DESIGN: Male FHR were treated with captopril from the age of 5 months when mild hypertension, proteinuria and glomerulosclerosis are present, and sacrificed at 12 months of age. Renal function was determined in separate groups of FHR at 6 months of age. METHODS: Proteinuria, body weight and systolic blood pressure were determined at regular intervals. Blood pressure was measured by the tail-cuff method. Kidneys were prepared for histological examination by standard methods. Renal function was determined by inulin clearance and urinary kallikrein by an amydolitic assay. RESULTS: In untreated FHR blood pressure, proteinuria and glomerulosclerosis increased with time. Captopril normalized blood pressure and stabilized proteinuria at pretreatment levels. At the end of the study, the incidence of glomerulosclerosis was significantly lower and comparable with the incidence at 5 months. Glomerular volume did not show a correlation with the incidence of glomerulosclerosis. Hemodynamic studies showed a significant increase of glomerular filtration rate in captopril-treated rats. No statistically significant effect was seen on renal plasma flow or filtration fraction. Urinary excretion of kallikrein was increased in captopril-treated rats. CONCLUSIONS: CEI is effective in protecting the kidney from structural damage in hypertensive FHR even when treatment is started under conditions of established glomerular injury. The protection given by captopril is probably related to intrarenal effects.

Binding of [3H]flunitrazepam and [3H]spiperone to melanoma cell membrane preparations.

Binding of [3H]flunitrazepam and [3H]spiperone to membrane preparations isolated by high speed centrifugation of hamster, rabbit and human melanoma cell homogenates was analyzed. All melanoma cell types expressed a high density of specific binding sites for [3H]flunitrazepam (3-4 pmol/mg protein) with a high affinity (Kd about 30 nM). This binding was independent of melanin content of cells and could be classified, based on competition experiments, as a Ro 5-4864-like binding type. Specific [3H]spiperone binding to these cell lines clearly revealed at least two types of binding sites: a low affinity, high capacity type of binding site (Kd greater than 100 nM, Bmax about 50 pmol/mg protein) and a high affinity, low capacity binding site (Kd less than 1 nm, Bmax 30 fmol/mg protein). Binding of spiperone to the low affinity, high capacity site appeared displaceable by NM 113 and dependent on melanin content of the cells and probably represents binding to melanin. Analysis of drug binding to melanoma membrane cell preparations and correlation with drug effects should include the possible involvement of binding to melanin.

Monitoring the specific activities of dopamine and its metabolites in striatum and olfactory tubercle after intravenous administration of l-[(3)H]tyrosine: Complex relations, indicating more than two compartments.

It is still a matter of debate whether in dopaminergic nerve endings dopamine (DA) is present in different functional and/or metabolic compartments. To investigate this, DA metabolism was studied in vivo by measuring the specific activity of DA and its metabolites after intravenous administration of l-[3,5-(3)H]tyrosine (200 ?Ci/rat) to freely moving animals. The incorporation of (3)H into DA and metabolites was determined in striatum and olfactory tubercle at 5, 10, 20, 40, 60 and 80 min after [(3)H]tyrosine administration. In both structures the level of [(3)H]tyrosine initially declined monoexponentially, but deviated from that pattern later on. The curves representing the formation in time of [(3)H]DA and [(3)H]metabolites were very similar in both structures, although as a whole, the levels in the olfactory tubercle were higher. The relative patterns of the specific activities of DA and those of its metabolites, a possible clue to DA compartmentation, neither indicated a clearcut metabolic one-compartment, nor a two-compartment system. The flow of radioactivity through DA metabolism could in fact only be explained by assuming more complex metabolic relations.

Repeated administration of HA-966 and haloperidol to rats: similar tolerance to striatal dopamine accumulation after HA-966 challenge, but dissimilar effects on striatal [3H]spiperone binding.

Repeated administration of 1-hydroxy-3-amino-pyrrolidone-2 (HA-966) to rats induces tolerance, as shown by a decreased, drug-stimulated accumulation of dopamine (DA) in the striatum. In the present study we compared the adaptive response of the striatal dopaminergic system to repeated administration of HA-966 with the adaptive response observed after repeated haloperidol. These treatments deprive dopamine (DA) receptors from their agonist and cause a blockade of DA receptors, respectively. Tolerance to HA-966 was not accompanied by a change in the specific binding of [3H]spiperone to striatal membranes. This is in contrast to the well-documented up-regulation of DA receptors that occurs with tolerance to haloperidol. Repeated haloperidol pretreatment also diminished DA accumulation following a challenge dose of HA-966, to a similar extent as that caused by repeated pretreatment with HA-966. These similar effects of pretreatment with HA-966 or haloperidol on the response to the HA-966 challenge are in line with, and strengthen, the idea that an increased sensitivity of presynaptic DA receptors is responsible for the decreasing effect of HA-966 after its repeated administration. Haloperidol and HA-966 clearly have different effects on postsynaptic DA receptors, as is shown by their differential effects on striatal [3H]spiperone binding.

Localization of rat striatal monoamine oxidase activities towards dopamine, serotonin and kynuramine by gradient centrifugation and nigro-striatal lesions.

Subfractionation of the crude synaptosomal-mitochondrial fraction of rat striatum in a continuous sucrose gradient in a zonal rotor led to the following results. The distribution pattern of monoamine oxidase (MAO) activity towards dopamine (DA) was very similar to the pattern of MAO activity towards serotonin (5HT), but differed from the pattern of MAO activity towards kynuramine (KYN). As 5HT is specifically deaminated by MAO-A while KYN is a common MAO substrate, this supports earlier suggestions that in rat striatal preparations DA is deaminated preferentially by MAO-A. The patterns of the MAO activities towards DA and 5HT were clearly dissimilar, despite considerable overlap, to the patterns of tyrosine hydroxylase (TH) and DOPA decarboxylase (DD) activity, both marking the presence of striatal dopaminergic synaptosomes. The peak activities were separated and all patterns were symmetrical without showing a shoulder. This indicates that rat striatal MAO activity towards DA and 5HT is not specifically or for the greater part localized in dopaminergic terminals. We also investigated the effects of electrolytic and 6-hydroxydopamine lesions of the substantia nigra, both causing extensive degeneration of striatal dopaminergic terminals as appeared from the large decrease of striatal TH and DD activity. However, neither type of lesion induced a reduction of the MAO activity towards any of the substrates used. It is concluded that the amount of MAO activity towards DA and 5HT (probably MAO-A activity) present in dopaminergic terminals is very low compared with the total activity of this enzyme in rat striatal tissue.

Subcellular fractionation of striatum: sedimentation properties of dopaminergic synaptosomes.

Linear sucrose density gradient centrifugation of a crude synaptosomal-mitochondrial preparation of rat striatum was performed at 82,500g for 7.5, 15 and 30 min and 1, 4 and 20 h. After centrifugation various marker enzyme activities were measured throughout the gradients, viz. tyrosine hydroxylase (TH) and DOPA decarboxylase (DD) as markers of dopaminergic synaptosomes, lactate dehydrogenase (LDH) as a general synaptosomal marker and monoamine oxidase (MAO) as a mitochondrial marker. At all centrifugation times the distribution patterns of TH and DD activity coincided almost perfectly. Notable differences were found between the sedimentation properties of these TH/DD-containing particles and LDH-containing particles: TH and DD were symmetrically distributed in the gradient much sooner than LDH, at all centrifugation times the top of the TH and DD curves was lying deeper in the gradient than the highest LDH activity, and TH and DD became enriched in the gradients to a much greater extent than LDH. It is concluded that rat striatal dopaminergic synaptosomes form a relatively homogeneous population of particles sedimenting faster into the gradients than the bulk of striatal synaptosomes does. This distinct sedimentation behaviour of the dopaminergic synaptosomes can be usefully applied for analytical purposes.

Neuronal and extraneuronal uptake of norepinephrine in the human forearm.

The effect of the neuronal uptake (U1) inhibiting drug desmethylimipramine (DMI) and the extraneuronal uptake (U2) inhibiting drug hydrocortisone (HC) upon the forearm extraction (FE) of norepinephrine (NE) was investigated in six healthy male subjects. Four infusions of NE 0.2 ng/kg per min were given intra-arterially (i.a.) concomitantly with three doses of DMI and subsequently with three doses of HC combined with one dose of DMI. All doses of DMI and the highest two doses of HC decreased the FE of NE significantly. As a result of local U1 and possibly also U2 inhibition, the venous-arterial plasma NE difference over the forearm increased between the start of the first infusion and the start of the last infusion, indicating an important degree of local neuronal release of NE in basal conditions. It is concluded that the extensive removal of NE by U1 and U2, and the local release of NE limit the use of peripheral venous NE concentration as a measure of overall sympathetic activity.

Tritium isotope effect in reversed-phase high-performance liquid chromatographic analysis of dopamine and dihydroxyphenylacetic acid.

A tritium isotope effect has been demonstrated in the high-performance liquid chromatographic analysis of dopamine and its acidic metabolite dihydroxyphenylacetic acid. The chromatographic system consisted of tributyl-n-phosphate, bound to a ChromSpher C8 column, as stationary phase, and a citrate buffer, containing the ion-pairing agent perchlorate, as the mobile phase. For detection we used continuous electrochemical monitoring (for the total amount of solutes) and discontinuous liquid scintillation counting (for radiolabelled molecules) of the column effluent. [3H]Dopamine and [3H]dihydroxyphenylacetic acid were biosynthesized by incubation of homogenates of striatal tissue from rat brains with 3H-labelled L-tyrosine. The tritium-labelled compounds were eluted before the corresponding unlabelled analogues. The capacity factor reduction increased with the number of tritium atoms incorporated in the molecules: for single, double and triple tritium-labelled dopamine the separation factors amounted to 1.015, 1.028 and 1.033, respectively. No isotope separation was observed for 7-14C-labelled dopamine and dihydroxyphenylacetic acid. The isotope effect observed is ascribed to a decrease in lipophilicity following tritium substitution for hydrogen.

Beta 1-adrenoceptor selectivity of single oral doses of bisoprolol and atenolol.

The hemodynamic and biochemical properties of bisoprolol, a new highly beta 1-adrenoceptor selective blocker, were compared with those of atenolol. The studies were performed in eight healthy volunteers and consisted of two separate parts. In the first part the effects of oral doses of 20 mg bisoprolol, 100 mg atenolol, and placebo, given in a double-blind randomized order, on the hemodynamic and biochemical changes induced by multiple doses of intravenous isoproterenol and bicycle exercise were investigated. Blood pressure and heart rate and the plasma concentrations of magnesium, potassium (K+), glucose, lactate, renin activity, norepinephrine, and epinephrine were determined. In the second part the effects of the same oral doses of bisoprolol and atenolol on changes in forearm blood flow (FBF) induced by intra-arterial (i.a.) infusions of isoproterenol and epinephrine were investigated. After both beta blockers approximately 10-times higher doses of isoproterenol i.v. were needed to obtain similar hemodynamic responses as after placebo. The hemodynamic and biochemical changes during isoproterenol i.v. and bicycle exercise were almost identically influenced by bisoprolol and atenolol. However, after bisoprolol, isoproterenol lowered plasma K+ more (p less than 0.02) and the exercise-induced increase in renin activity was less (p less than 0.02) than after atenolol. The effect of isoproterenol i.a. on FBF was attenuated to the same extent by bisoprolol and atenolol. The vasodilatory component of epinephrine i.a. was reduced only by atenolol (p less than 0.05). It is concluded that bisoprolol and atenolol are selective beta 1 antagonists. In the doses used, bisoprolol showed only slightly more potency and selectivity for beta 1 adrenoceptors than atenolol.

Haemodynamic effects of physiological concentrations of circulating noradrenaline in man.

1. To define the role of circulating noradrenaline in cardiovascular regulation, threshold concentrations for haemodynamic effects were determined in arterial and venous plasma of eight healthy volunteers. 2. Five doses of noradrenaline, 0-54 ng min-1 kg-1, were infused intravenously in random order and single-blind for 15 min per dose. Changes in intra-arterial blood pressure, heart rate, forearm blood flow and forearm vascular resistance were determined, and plasma noradrenaline was measured in arterial and venous blood samples. 3. Significant increases in systolic and diastolic blood pressure were found at arterial and venous plasma noradrenaline concentrations (means +/- SEM) of 3.00 +/- 0.23 and 1.35 +/- 0.12 nmol/l, respectively. A significant decrease in heart rate was found at arterial and venous plasma noradrenaline concentrations of 8.99 +/- 0.69 and 3.09 +/- 0.60 nmol/l, respectively. The lower doses of noradrenaline tended to increase forearm blood flow and to decrease forearm vascular resistance, whereas the higher doses had no consistent effect on forearm haemodynamics. 4. During the noradrenaline infusions 73 +/- 5% of the increase in arterial plasma noradrenaline concentration was extracted in the forearm. 5. The venous plasma noradrenaline threshold concentration was found to be much lower than previously reported. It is concluded that arterial and venous plasma noradrenaline concentrations which are readily encountered in physiological circumstances elicit haemodynamic effects.

Stimulation of norepinephrine release by peripheral presynaptic beta-adrenoceptors.

Increases in plasma norepinephrine (NE) concentration induced by beta-agonist infusion have been taken as evidence for the existence of peripheral presynaptic beta-adrenoceptors, facilitating NE release. Concomitant hemodynamic changes, however, could invoke reflex mechanisms and thus hamper the interpretation of the results. We studied the presence of peripheral presynaptic beta-adrenoceptors by giving intra-arterial infusions of EPI in the forearm before and during uptake inhibition, and the effects of systemic infusions of very low-dose epinephrine (EPI) and isoproterenol (ISO) on hemodynamics and arterial and venous plasma NE concentrations. An i.a. infusion of EPI, 0.1 ng.kg-1.min-1, increased the net overflow of NE from 0.3 +/- 1.0 to 5.4 +/- 1.8 pmol.min-1 (M +/- SEM, p less than 0.05) and raised the calculated arterial plasma EPI concentration to 0.92 +/- 0.22 nmol.l-1. The uptake inhibition increased the net overflow of NE to 15.4 +/- 2.4 pmol.min-1 and during addition of EPI, 0.1 ng.kg-1.min-1, it further increased to 23.0 +/- 4.1 pmol.min-1 (p less than 0.05), while it decreased during addition of an effective beta-blocking dose of propranolol, 50 ng.kg-1.min-1 i.a., to 18.2 +/- 4.1 pmol.min-1 (n.s.). Systemic infusions of EPI and ISO increased blood pressure, heart rate, and forearm blood flow before significant changes in arterial or venous plasma NE concentrations were found. It is concluded that the increases in net NE overflow from the forearm during local EPI infusions indicate the presence of peripheral presynaptic beta-adrenoceptors which are responsive to physiologic concentrations of EPI. Systemic infusions of EPI or ISO elicit hemodynamic effects, and thus may invoke reflex mechanisms, before an influence on arterial or venous plasma NE concentration is found.

Functional and structural determinants of glomerulosclerosis in the fawn-hooded rat.

The effect of uninephrectomy (UN) at 4 months of age was studied on several parameters involved in the development of glomerulosclerosis (GS) in male spontaneously hypertensive Fawn-Hooded rats. Protein excretion per animal was significantly more increased in UN rats at 2 months after operation compared to sham operated controls (202 +/- 104 vs. 88 +/- 37 mg 24 h-1, P = 0.005) and remained significantly higher throughout the rest of the observation period. At 11 months of age UN rats had a marked increase in the incidence of GS, 37 +/- 16% compared to 5 +/- 3% (P less than 0.001) in controls. No differences were observed in mean arterial blood pressure. Functional studies in separate groups of rats at 5 months of age showed an increase in single kidney glomerular filtration rate in UN rats (0.40 +/- 0.07 vs. 0.28 +/- 0.09 ml min-1 100 g, P = 0.006). Single kidney renal plasma flow and filtration fraction were not altered. Mean glomerular volume was increased 1 month after UN (1.86 +/- 0.25 vs. 1.39 +/- 0.25 x 10(6) microns 3, P = 0.003). Urinary noradrenaline excretion per animal (24-h) showed a high sympathic nervous tone in both sham and UN rats. Total urinary dopamine and kallikrein excretion per animal were not influenced by UN. These data indicate that after UN the development of GS in this rat strain is accelerated in association with compensatory hyperfiltration and glomerular volume expansion, which may play a role in the pathogenesis of GS.

4-[123I]iodospiperone as a ligand for dopamine DA receptors: in vitro and in vivo experiments in a rat model.

Radioiodinated spiperone is of interest for dopamine (DA) receptor studies in the living human brain by single photon emission computed tomography (SPECT). Stimulated by data obtained with [11C]-N-methyl-spiperone we synthesized 4-[123I]iodospiperone and investigated the in vitro binding characteristics of this ligand to the striatal membrane of the rat and the in vivo distribution over various rat brain regions. The in vitro binding experiments showed that this radioligand displays about 10 times less affinity for the DA receptor than spiperone and specific binding, as shown with [3H]spiperone, was not observed. Displacement by butaclamol was not observed. The in vivo studies demonstrated that both 4-[123I]iodospiperone and [3H]spiperone concentrate in striatal tissue, respectively, 1.9 and 3.5 times as high as in cerebellar tissue. Haloperidol pretreatment largely prevented this accumulation. In view of the obtained target-to-non-target ratios we believe, however, that this accumulation in brain areas rich in DA-receptors does not offer prospects for clinical receptor imaging with SPECT.

Diurnal variation of essential and physiological tremor.

Evaluations were made of the diurnal variations of tremor power at rest, after fatigue and after mass loading, and plasma norepinephrine in patients with familial essential tremor and normal subjects. Diurnal tremor power rhythms for both essential and physiological tremor pursued identical temporal profiles. Plasma norepinephrine levels followed a congruent diurnal pattern with later peak values than the peak values of tremor power. Sympathetic nervous system activity is unlikely to be the cause of diurnal tremor power variation. The consistent diurnal rhythm of tremor power may affect dosage schemes of tremorolytic drugs.