Natural history of perihematomal edema after intracerebral hemorrhage measured by serial magnetic resonance imaging.

BACKGROUND AND PURPOSE: knowledge on the natural history and clinical impact of perihematomal edema (PHE) associated with intracerebral hemorrhage is limited. We aimed to define the time course, predictors, and clinical significance of PHE measured by serial magnetic resonance imaging. METHODS: patients with primary supratentorial intracerebral hemorrhage ≥ 5 cm(3) underwent serial MRIs at prespecified intervals during the first month. Hematoma (H(v)) and PHE (E(v)) volumes were measured on fluid-attenuated inversion recovery images. Relative PHE was defined as E(v)/H(v). Neurologic assessments were performed at admission and with each MRI. Barthel Index, modified Rankin scale, and extended Glasgow Outcome scale scores were assigned at 3 months. RESULTS: twenty-seven patients with 88 MRIs were prospectively included. Median H(v) and E(v) on the first MRI were 39 and 46 cm(3), respectively. Median peak absolute E(v) was 88 cm(3). Larger hematomas produced a larger absolute E(v) (r(2)=0.6) and a smaller relative PHE (r(2)=0.7). Edema volume growth was fastest in the first 2 days but continued until 12 ± 3 days. In multivariate analysis, a higher admission hematocrit was associated with a greater delay in peak PHE (P=0.06). Higher admission partial thromboplastin time was associated with higher peak rPHE (P=0.02). Edema volume growth was correlated with a decline in neurologic status at 48 hours (81 vs 43 cm(3), P=0.03) but not with 3-month functional outcome. CONCLUSIONS: PHE volume measured by MRI increases most rapidly in the first 2 days after symptom onset and peaks toward the end of the second week. The timing and magnitude of PHE volume are associated with hematologic factors. Its clinical significance deserves further study.

Irreversible thermodynamics of multicomponent fluids and its statistical mechanics basis.

The irreversible thermodynamics of a multicomponent fluid is reviewed. This includes a discussion of the role of individual component fluxes. It is argued that their differences vanish on the same time scale as that which establishes local thermodynamic equilibrium and thus do not play an independent role in fluid dynamics, but only arise in response to gradients in conserved thermodynamic variables. The contributions to the energy flux are examined and it is argued that there should be explicit contributions associated with the various component fluxes, which are not mentioned in standard kinetic theory presentations. Three different thermodynamic perspectives are discussed as to their form, with the respective equations for the entropy flux and production described and contrasted. The Onsager reciprocal relations are considered to be a consequence of the single-valuedness of the entropy production with the chemical potential gradients as the driving forces for diffusion. These are specialized to ideal gas mixtures using the component density gradients associated with Fick's laws and to using the mole fraction gradients that are standardly used in gas kinetic theory. The ideal gas Onsager relations are identical to those deduced from the Boltzmann equation. Irving and Kirkwood's statistical mechanics treatment of the evolution equations of a one-component fluid [J. Chem. Phys. 18, 817 (1950)JCPSA60021-960610.1063/1.1747782] is generalized to multicomponent fluids and agrees with the thermodynamic perspective that treats the energy transfers as reversible.

Thrombin stimulation of guanosine 3',5'-monophosphate formation in murine neuroblastoma cells (clone N1E-115).

Thrombin, the central regulatory enzyme in coagulation, when incubated in nanomolar concentrations with murine neuroblastoma cells produced a rapid and marked increase in tritiated guanosine 3',5'-monophosphate (cyclic GMP) formation that was blocked by hirudin and competitively antagonized by dansylarginine N-(3-ethyl-1,5-pentanediyl)amide. Diisopropylphosphofluoridate-inactivated thrombin as well as the serine protease trypsin were markedly less potent and less effective than alpha-thrombin in producing this effect. Thrombin-stimulated cyclic GMP formation was inhibited by mepacrine and nordihydroguaiaretic acid but unaffected by indomethacin, suggesting that lipoxygenase metabolites of arachidonic acid are involved in the response. These results suggest that a thrombin-like protease in the brain may be involved with the function of neurons or that thrombin interactions with nerve cells, such as those following cerebral hemorrhage or other trauma of the central nervous system, may be important in the subsequent neuropathology.

A potent nonpeptide antagonist of the substance P (NK1) receptor.

CP-96,345 [(2S, 3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)- methyl]-1-azabicyclo[2.2.2]octan-3-amine] is a potent nonpeptide antagonist of the substance P (NK1) receptor. CP-96,345 inhibited 3H-labeled substance P binding and was a classical competitive antagonist in the NK1 monoreceptor dog carotid artery preparation. CP-96,345 inhibited substance P-induced salivation in the rat, a classical in vivo bioassay, but did not inhibit NK2, NK3, or numerous other receptors; it is thus a selective NK1 antagonist. This compound may prove to be a powerful tool for investigation of the physiological properties of substance P and exploration of its role in diseases.

Changes in the phenotype of human small cell lung cancer cell lines after transfection and expression of the c-myc proto-oncogene.

Small cell lung cancer growing in cell culture possesses biologic properties that allow classification into two categories: classic and variant. Compared with classic small cell lung cancer cell lines, variant lines have altered large cell morphology, shorter doubling times, higher cloning efficiencies in soft agarose, and very low levels of L dopa decarboxylase production and bombesin-like immunoreactivity. C-myc is amplified and expressed in some small cell lung cancer cell lines and all c-myc amplified lines studied to date display the variant phenotype. To investigate if c-myc amplification and expression is responsible for the variant phenotype, a normal human c-myc gene was transfected into a cloned classic small cell lung cancer cell line not amplified for or expressing detectable c-myc messenger RNA (mRNA). Clones were isolated with one to six copies of c-myc stably integrated into DNA that expressed c-myc mRNA. In addition, one clone with an integrated neo gene but a deleted c-myc gene was isolated and in this case c-myc was not expressed. C-myc expression in transfected clones was associated with altered large cell morphology, a shorter doubling time, and increased cloning efficiency, but no difference in L dopa decarboxylase levels and bombesin-like immunoreactivity. We conclude increased c-myc expression observed here in transfected clones correlates with some of the phenotypic properties distinguishing c-myc amplified variants from unamplified classic small cell lung cancer lines.

Development of a homogeneous time-resolved fluorescence leukotriene B4 assay for determining the activity of leukotriene A4 hydrolase.

Leukotriene A4 (LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4 (LTB4), a potent lipid chemotactic agent and proinflammatory mediator. LTB4 has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA4 hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA4 hydrolase inhibitors, a specific and efficient assay to quantify LTB4 is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB4 (LTB4 HTRF assay) and its application to establish an HTRF-based LTA4 hydrolase assay for lead optimization. This LTB4 HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z' factor of 0.89 and a subnanomolar detection limit for LTB4. By coupling this LTB4 HTRF assay to the LTA4 hydrolase reaction, an HTRF-based LTA4 hydrolase assay was established and validated. Using a test set of 16 LTA4 hydrolase inhibitors, a good correlation was found between the IC50 values obtained using LTB4 HTRF with those determined using the LTB enzyme-linked immunoassay (R = 0.84). The HTRF-based LTA4 hydrolase assay was shown to be an efficient and suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4 hydrolase.

Lysine-derived cross-links in the egg shell membrane.

Egg shell membrane protein contains significant quantities of the lysine-derived aldehyde, allysine, and its aldol condensation product. NaB3H4 reduction followed by alkaline hydrolysis of purified protein revealed that there were six residues/1000 of both allysine and the reduced aldol while only traces of desmosine and isodesmosine were detected. The amino acid composition of the membrane protein did not resemble that of mammalian elastin.

Uptake by neuroblastoma cells of glucosylceramide, glucosylceramide glucosidase, its stimulator protein, and phosphatidylserine.

Serum-free cultured neuroblastoma cells (clone NlE-115) have been shown to absorb emulsified glucosylceramide, glucosylceramide glucosidase, an activator protein for the enzyme, and phosphatidylserine from a synthetic medium. Uptake of the enzyme was augmented by phosphatidylserine, and vice versa. Uptake of the enzyme-lipid complex was further augmented by the activator protein. It appears likely that the activator forms a complex only with the enzyme-lipid complex, not with the individual components. Two uptake mechanisms for the enzyme seem to be involved, one of which (the complex with activator proteins and acidic lipid) is sensitive to mannosyl phosphate groups. Hydrolysis of absorbed glucosylceramide was slow unless the medium was supplemented with the acidic phospholipid or glucosidase. The most rapid disappearance of stored glycolipid took place when the ternary mixture was added to the cell medium, enzyme + activator protein + phosphatidylserine. These findings may be relevant to enzyme replacement therapy for Gaucher disease.

Isolation of purified insoluble aortic collagen.

The chemistry of collagen from major blood vessels, such as the aorta, is poorly defined because of problems encountered in solubilization techniques. Normal extraction of calf aorta with acetic acid and/or pepsin does not yield significant quantities of collagen. However, treatment of the aorta with purified pancreatic elastase results in a residue containing a significant portion of the collagen. The amino acid analysis, the acrylamide gel electrophoretic patterns and the electron micrographs of this residue display characteristics consistent with relatively pure collagen. Using this purified collagen preparation approximately 90% of the collagenous material present can be solubilized by pepsin treatment.

Muscarinic receptor-stimulated Ca2+ signaling and inositol lipid metabolism in avian salt gland cells.

Activation of muscarinic cholinergic receptors was studied by measuring agonist-stimulated inositol lipid turnover and changes in [Ca2+]i in dissociated salt gland secretory cells. Carbachol stimulation of quin2-loaded cells results in a sustained 4-fold increase in [Ca2+]i, while incorporation of [32P]Pi into phosphatidylinositol (PI) and phosphatidate are similarly increased. [3H]Inositol phosphates, measured in the presence of Li+, increased 13-fold. The stimulated increment in [Ca2+]i required extracellular Ca2+, whereas [3H]inositol phosphate accumulation was independent of external Ca2+. Dose-response curves for carbachol-induced increments in [Ca2+]i, PI labeling, and labeled inositol phosphate release are similar, with EC50 values of 6, 4.5 and 8 microM, respectively. Dissociation constants for atropine vs. the quin2 and phospholipid responses are 0.59 +/- 0.3 nM and 0.48 +/- 0.28 nM, respectively. These cells thus provide a model system for the study of non-exocytotic secretion as a consequence of stimulated inositol lipid turnover.

Cerebrospinal fluid bombesin and calcitonin in patients with central nervous system metastases from small-cell lung cancer.

To determine whether levels of mammalian bombesin (MB) or calcitonin would be useful in detecting CNS metastases in patients with small-cell lung cancer (SCLC), we measured their concentrations in the CSF of 94 patients who underwent lumbar puncture for suspected CNS involvement. The MB concentration was significantly elevated in the 51 patients with definite CNS metastases as compared with the 30 patients without apparent CNS involvement (P less than .01). This significance was due to increased levels of MB in 18 patients with meningeal carcinomatosis. Whereas CSF MB was detectable (greater than 10 fmol/mL) in only 7% of patients without apparent CNS involvement, CSF MB was detectable in 21% with parenchymal CNS metastases and in 78% of those with meningeal carcinomatosis. Interestingly, 93% of patients having MB concentrations above 20 fmol/mL had meningeal metastases. The calcitonin concentration was significantly elevated in 42 patients with CNS metastases as compared with 27 patients without CNS involvement (P less than .01). Both the 15 patients with meningeal carcinomatosis and the 27 patients with only parenchymal metastases had significantly elevated levels of CSF calcitonin as compared with those without CNS metastases. Fifty-three percent of patients with meningeal carcinomatosis and 48% with parenchymal metastases had a CSF calcitonin level above 18 fmol/mL, whereas only 7% without apparent CNS metastases exceeded this level. Sixty-seven percent of all patients with CNS metastases had increased CSF levels of one of the two hormonal markers. Thus, in SCLC patients, an elevated CSF calcitonin strongly suggested CNS metastases and an elevated MB was very suggestive of the presence of meningeal carcinomatosis. However, only the latter observation seems of clinical importance due to the difficulties in establishing this diagnosis with current diagnostic measures.

A novel high-throughput screening format to identify inhibitors of secreted acid sphingomyelinase.

Secreted extracellular acid sphingomyelinase (sASM) activity has been suggested to promote atherosclerosis by enhancing subendothelial aggregation and retention of low-density lipoprotein (LDL) with resultant foam cell formation. Compounds that inhibit sASM activity, at neutral pH, may prevent lipid retention and thus would be expected to be anti-atherosclerotic. With the goal of identifying novel compounds that inhibit sASM at pH 7.4, a high-throughput screen was performed. Initial screening was run using a modification of a proven system that measures the hydrolysis of radiolabeled sphingomyelin presented in detergent micelles in a 96-well format. Separation of the radiolabeled aqueous phosphorylcholine reaction product from uncleaved sphingomyelin lipid substrate was achieved by chloroform/methanol extraction. During the screening campaign, a novel extraction procedure was developed to eliminate the use of the hazardous organic reagents. This new procedure exploited the ability of uncleaved, radiolabeled lipid substrate to interact with hydrophobic phenyl-sepharose beads. A comparison of the organic-based and the bead-based extraction sASM screening assays revealed Z' factor values ranging from 0.7 to 0.95 for both formats. In addition, both assay formats led to the identification of sub- to low micromolar inhibitors of sASM at pH 7.4 with similar IC(50) values. Subsequent studies demonstrated that both methods were also adaptable to run in a 384-well format. In contrast to the results observed at neutral pH, however, only the organic extraction assay was capable of accurately measuring sASM activity at its pH optimum of 5.0. The advantages and disadvantages of both sASM assay formats are discussed.

Characterization of a small molecule PAI-1 inhibitor, ZK4044.

Plasminogen activator inhibitor-1 (PAI-1) is a key negative regulator of the fibrinolytic system. In animal studies, inhibition of PAI-1 activity prevents arterial and venous thrombosis, indicating that PAI-1 inhibitors may be used as a new class of antithrombotics. In this study, we characterize a small molecule PAI-1 inhibitor, ZK4044, which was identified by high throughput screening and chemically optimized. In a chromogenic substrate-based urokinse (uPA)/PAI-1 assay and a tissue-type plasminogen activator (tPA)-mediated clot lysis assay, ZK4044 inhibited human PAI-1 activity with IC50 values of 644+/-255 and 100+/-90 nM, respectively. ZK4044 had no detectable inhibitory activity toward other serpins such as antithrombin III, alpha1-antitrypsin and alpha2-antiplasmin, indicating that ZK4044 is a specific PAI-1 inhibitor. ZK4044 was shown to bind directly to PAI-1 and prevent the binding of PAI-1 to tPA in a dose-dependent manner in surface plasmon resonance Biacore-based experiments. ZK4044 also prevented PAI-1/tPA complex formation, as analyzed by SDS/PAGE. ZK4044 had little effect on elastase-mediated cleavage of active PAI-1, indicating that the primary mode of action of ZK4044 is most likely to directly block the PAI-1/tPA interaction rather than to convert active PAI-1 to latent PAI-1. In the chromogenic substrate-based uPA/PAI-1 assay, ZK4044 was approximately 2-fold less potent against a mutant PAI-1 (14B-1), which contains four mutations at N150H, K154T, Q319L and M354I, compared with wild-type PAI-1, suggesting that the ZK4044 binding site on the surface of PAI-1 is close to these mutant residues. Together, our data show that ZK4044 represents a new class of small molecule PAI-1 inhibitors with anti-thrombotic potential.

Ubiquitous expression of the calcitonin-i gene in multiple tissues in response to sepsis.

Calcitonin precursors (CTpr), including procalcitonin, are important markers and also potentially harmful mediators in response to microbial infections. The source and function of CTpr production in sepsis, however, remains an enigma. In the classical view, the transcription of the CT-I gene is restricted to neuroendocrine cells, in particular the C cells of the thyroid. To better understand the pathophysiology of CTpr induction in sepsis, we used an animal model analog to human sepsis, in which bacterial infection is induced in hamsters by implanting Escherichia coli pellets ip. Compared with control hamsters, levels of CTpr were elevated several fold in septic plasma and in nearly all septic hamster tissues analyzed. Unexpectedly, CT-messenger RNA was ubiquitously and uniformly expressed in multiple tissues throughout the body in response to sepsis. Notably, the transcriptional expression of CT-messenger RNA seemed more widely up-regulated in sepsis than were classical cytokines (e.g. tumor necrosis factor-alpha and interleukin-6). Our findings, which describe a potentially new mechanism of host response to a microbial infection mediated by CTpr, introduce a new pathophysiological role for the CT-I gene.

Serum calcitonin precursors in sepsis and systemic inflammation.

High serum levels of the calcitonin (CT) prohormone, procalcitonin (pro-CT), and its component peptides occur in systemic inflammation and sepsis. Using two different assays, we undertook a prospective study to determine the utility of serum precalcitonin peptides (pre-CT) as markers in this condition. Twenty-nine patients meeting criteria for the systemic inflammatory response syndrome were studied daily in two intensive care units. Sera were collected, and APACHE II scores were determined until recovery or death. All patients had markedly elevated serum pre-CT. Prognostically, peak values were the most important. The highest values portended mortality, and a lower level could be ascertained below which all patients survived. Peak pre-CT levels were significantly higher in patients with infection documented by blood cultures than in those patients with no documented infection from any source (P < 0.05). Mature CT remained normal or only moderately elevated. Compared with the serum pre-CT levels, receiver operating characteristic curve analysis revealed that the APACHE II scores, although more cumbersome, were better overall predictors of mortality. Thus, pre-CT is an important serum marker for systemic inflammatory response syndrome and is predictive of outcome. It also provides data concerning the presence of severe infection and may prove to be clinically useful for proactive patient care.

Immunoneutralization of procalcitonin as therapy of sepsis.

Prior studies have demonstrated that the prohormone, procalcitonin (ProCT), and its component calcitonin precursors (CTpr) are increased in the serum of septic patients, correlate with the severity of the illness, and persist for relatively long periods of time. Animal studies in septic hamsters have revealed that the administration of ProCT is toxic and that immunoneutralization with IgG that is reactive to this molecule significantly improves survival. A large animal model of a very rapidly lethal polymicrobial sepsis has been developed in the pig in order to measure continuous physiological and metabolic parameters and also to compare the effects in this animal of an immunoneutralization, which is performed late in the course of the disease, to an identical, but early, therapy. Based upon the physiological and metabolic parameters, the late therapy, which was initiated during the fourth hour at a time when pigs were nearly moribund, was found to be as beneficial as early therapy. In both late and early therapy, the only animals to survive at the predetermined time of euthanasia were those which had received immunoneutralization therapy.

Amitriptyline supersensitizes a central cholinergic mechanism.

The withdrawal of tricyclic antidepressants produces symptoms characteristic of cholinergic overdrive states. The authors previously proposed that these states are the consequence of the pharmacological induction of cholinergic system supersensitivity by chronic treatment with antidepressants, combined with a reduction in the plasma level of a competitive muscarinic receptor antagonist when the dose of a tricyclic is decreased. This is consistent with the facts that all tricyclic antidepressants are antimuscarinic agents and that classical antimuscarinic compounds, such as scopolamine, up-regulate and supersensitize muscarinic cholinergic systems. The authors present evidence that chronic treatment with amitriptyline supersensitizes a central cholinergic mechanism. Core body temperature is subject to influence by a central (hypothalamic) muscarinic mechanism, which is rendered supersensitive to cholinomimetic challenge by treatment with scopolamine. The authors telemetrically measured the hypothermic responses of adult male rats to various doses of the muscarinic agonist oxotremorine before and in the course of chronic treatment with amitriptyline. Treatment with amitriptyline resulted in marked enhancement of the cholinomimetic-induced hypothermia. Methylscopolamine nitrate, a peripherally active antimuscarinic agent, did not block the hypothermic response to oxotremorine, whereas scopolamine, a centrally active antimuscarinic compound, did. This study indicates that the chronic administration of amitriptyline can produce supersensitivity of a central muscarinic cholinergic mechanism. Clinical and theoretical implications of this finding are discussed.

Phenytoin and cerebellar lesions. Similar effects on cerebral catecholamine metabolism.

Phenytoin has been shown to inhibit catecholamine (CA) metabolism in vitro. The present investigation examined its longer-term in vivo effects in rats. Phenytoin 100 mg/kg/day for two weeks, caused an increase in hindbrain norepinephrine (NE) concentration, a slight decrease in forebrain NE concentration, and little change in dopamine (DA) levels. The turnover rates of forebrain DA and NE estimated by synthesis inhibition, were increased by 70% and 100%, respectively. Surgical lesions of the anterior cerebellar vermis produced similar (but not additive) increases in turnover. It is concluded that long-term phenytoin use stimulates CA metabolism in the forebrain and that this effect may be mediated indirectly by the cerebellar vermis.

Effects of methylphenidate on rat endurance performance and neuromuscular transmission in vitro.

The studies were designed to evaluate the effects of methylphenidate on endurance performance in vivo and on neuromuscular transmission in the isolated rat phrenic nerve-diaphragm preparation. Methylphenidate produced a biphasic effect on treadmill endurance performance, increasing running times by 41-61% at 2.5-5 mg/kg, while reducing running times by 35% at 20 mg/kg. A biphasic effect on nerve-stimulated muscle concentrations was also observed, with twitch tension increased by up to 49-106% at low concentrations (0.1-0.3 mM) and blocked at high concentrations (0.6-1.0 mM). Tissues obtained from rats pretreated with alpha-methyl-p-tyrosine or reserpine exhibited no change in twitch height. Methylphenidate failed to protect against irreversible blocking of the twitch by alpha-bungarotoxin and did not modify the resting membrane potential, miniature endplate potential (MEPP) frequency or nerve-stimulated acetylcholine release. High concentrations reduced the amplitudes of the MEPP and endplate potential. Whereas methylphenidate and amphetamine both produced biphasic effects on skeletal muscle contractions in vitro, they act by different neuropharmacological mechanisms. Unlike amphetamine, the biphasic effects of methylphenidate are produced by mechanisms that are independent of cholinergic or adrenergic interactions and may involve direct effects on the muscle.

Cholinergic-independent effects of amphetamine on mammalian skeletal muscle contractions.

Studies were designed to investigated the cholinergic-independent mechanism(s) by which (+)-amphetamine produces a biphasic modification of directly stimulated contractions of skeletal muscle in the rat phrenic nerve-diaphragm preparation. In tissues pretreated with alpha-bungarotoxin, low concentrations of (+)-amphetamine (2.7-1.08 x 10(-4) M) enhanced muscle blockade. In other studies (Gerald, Meldrum and Skau, Res. Commun, chem. Path. Pharmac., 1982), high K+ concentrations or low Na+ concentrations antagonized amphetamine-enhancement of the twitch, while potentiating the blockade; in contrast K+-free media augmented amphetamine-induced enhancement of contractions. Low concentrations of amantadine, tetracaine, and tetrodotoxin increased the facilitatory response to (+)-amphetamine while the inhibitory effects of (+)-amphetamine were potentiated by higher concentrations of these antagonists; similar biphasic effects were observed with (-)-amphetamine and tetracaine. (+)-Amphetamine reserved the marked enhancement of the twitch produced by veratridine while, conversely, this neurotoxin failed to alter muscle contractions after (+)-amphetamine pretreatment. These findings suggest that amphetamine-induced enhancement and blockade of directly-stimulated skeletal muscle resulted from alterations in Na+ fluxes, possibly through interactions with membrane ionic channels.