Transport of digoxin by human P-glycoprotein expressed in a porcine kidney epithelial cell line (LLC-PK1). This article represents the first evidence that the renal secretion of the commonly used drug, digoxin, is mediated by P-glycoprotein. In this study, it was demonstrated that digoxin is a substrate of P-glycoprotein, and the mechanism of a clinically important drug interaction, such as digoxin-quinidine, was elucidated. Human P-glycoprotein was expressed on the apical membrane of the porcine kidney epithelial cell line, LLC-PK1 by transfecting with human MDR1 cDNA. The expression and function of P-glycoprotein were confirmed by Southern and Western blotting, RNase protection assay, immunostaining and transporting activity for vinblastine. The transepithelial transport of [3H]digoxin was measured across the cell monolayers grown on microporous polycarbonate membrane filters. The transfectant cells exhibited markedly greater basal-to-apical transport and less apical-to-basal transport than the host cells, and the former was 8-fold greater than the latter. The augmented transepithelial transport resulted from the increased efflux from cells to apical side. This oriented transport was inhibited by the presence of 20 microM vinblastine, quinidine or verapamil. The rate of efflux to the apical side was 2-fold greater than that to the basal side. Quinidine inhibited the efflux to the apical side but did not affect transport into the basal side. These findings demonstrate that digoxin is transported by human P-glycoprotein, which is a previously undiscovered drug transport system in the kidney other than organic cation and anion transport systems, and suggest a molecular mechanism for the renal tubular secretion of digoxin as well as clinically important digoxin-quinidine interaction via P-glycoprotein.


Cholinergic excitation of GABAergic interneurons in the rat hippocampal slice. 1. Intracellular recordings were made from CA1 pyramidal cells in the rat hippocampal slice to study the cholinergic modulation of GABAergic inhibition. The cholinergic receptor agonist, carbamylcholine (carbachol), depressed evoked excitatory postsynaptic potentials (EPSPs) and evoked inhibitory postsynaptic potentials (IPSPs), but enhanced small spontaneously occurring membrane potential fluctuations that resembled IPSPs. Both atropine (1 microM) and picrotoxin (25-60 microM) abolished the small fluctuations. 2. Recording from cells with potassium or caesium chloride (KCl or CsCl)-filled microelectrodes enhanced and inverted spontaneous Cl(-)-dependent GABAA-mediated IPSPs. These events appeared to result from the spontaneous firing of GABAergic interneurons since they could be inhibited by picrotoxin or bicuculline and nearly eliminated by tetrodotoxin. 3. Muscarinic acetylcholine (ACh) receptor activation significantly increased the frequency of spontaneous-activity-dependent IPSPs from 1.7 +/- 0.4 s (mean +/- S.E.M.) in control saline to 7.0 +/- 1.1 s in carbachol (10-50 microM)-containing saline, although evoked IPSPs were inhibited. All effects of carbachol were completely reversed by atropine. 4. The increase in frequency of spontaneous IPSPs observed in carbachol was not secondary to changes in the postsynaptic cell and was not blocked by high doses of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5-10 microM) and 2-amino-5-phosphonovaleric acid (APV, 10-20 microM), which abolished evoked excitatory transmission. Amplitude histograms showed an increase in mean size as well as of frequency of spontaneous IPSCs in carbachol. 5. Stimulation of cholinergic afferents in stratum oriens in the presence of the acetylcholinesterase inhibitor eserine (1 microM) also increased spontaneous IPSP frequency, and the time course of this response was similar to that of the muscarinic slow EPSP. Postsynaptic factors or the activation of glutamatergic excitatory pathways could not account for this effect. 6. Evoked monosynaptic IPSCs in CNQX and APV were diminished by carbachol. 7. We conclude that GABAergic inhibitory interneurons possess muscarinic receptors, that activation of these receptors increases the excitability of the interneurons and that synaptically released ACh increases interneuronal activity. Cholinergic reduction of the monosynaptic IPSC may point to additional complexity in cholinergic regulation of the GABA system.


Somatosensory stimulation causes autonomic vasodilatation in cat lip. 1. Electrical stimulation of the infra-orbital nerve and the maxillary buccal gingiva caused an increase in ipsilateral lip blood flow in a stimulus intensity-dependent manner in anaesthetized cats. 2. The reflex vasodilator response was resistant to blockade by antimuscarinic (atropine), antiadrenergic (phentolamine and propranolol) and antihistaminergic (tripelennamine) but sensitive to ganglionic blocking agent (hexamethonium). 3. The reflex vasodilator response was unaffected by section of the ipsilateral cervical sympathetic trunk and facial nerve root, but was completely abolished by ipsilateral section of the glossopharyngeal nerve root. 4. The vasodilator response elicited by stimulation of the facial and glosso-pharyngeal nerves was never affected by lesion of the ipsilateral pterygopalatine ganglion. 5. Local anaesthesia or section of the inferior alveolar nerve abolished the vasodilator effects of stimulation of the facial and the glossopharyngeal nerves. 6. These results suggest that there is a somato-autonomic reflex vasodilator system mediated via final neurons that are not cholinergic, and that the parasympathetic vasodilator fibres emerge from the brain stem with the glossopharyngeal nerve and reach the blood vessels via the otic ganglion and the inferior alveolar nerve in the cat mandibular lip.


Pharmacological and kinetic properties of alpha 4 beta 2 neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes. 1. Co-injection of RNA synthesized from cloned neuronal acetylcholine receptor (nAChR) subunits (alpha 4 and beta 2) in Xenopus oocytes produced functional receptors. In macroscopic voltage-clamp experiments, the agonist-induced current exhibited a strong inward rectification. 2. Voltage jumps from +50 mV to more negative potentials produced relaxations of the agonist-induced current with a single exponential time course. The relaxation rate constant was only weakly voltage dependent. 3. At the single-channel level, three conductances were recorded of 12, 22 and 34 pS. Their burst durations were similar and varied only weakly with voltage (e-fold for 120 to 370 mV), consistent with the poorly voltage-dependent relaxation rate constants. However, the burst durations were less than 10 ms, or less than 1/5 the value expected from voltage-jump relaxations. 4. Hexamethonium (Hex, 0.5 to 8 microM) inhibited the agonist-induced current and produced voltage-jump relaxations characterized by a rapid conductance increase and a slower conductance decrease. Analysis of these relaxations suggested that the Hex-receptor interaction is open-channel blockade characterized by a forward binding rate of 1 x 10(7) M-1 s-1 and a dissociation rate constant of about 25 s-1. 5. For the relaxations produced by QX222, the slowest phase was a conductance increase, suggesting that the dissociation rate constant for QX222 is 10-30-fold greater than for Hex. 6. Hex but not QX222 produced an additional use-dependent blockade that was manifest during repetitive hyperpolarizing pulses. 7. With mouse muscle ACh receptors expressed in oocytes, the blockade by Hex did not depend strongly on voltage. Neither Hex nor QX222 produced appreciable use-dependent block on muscle ACh receptors. 8. Of the four conditions studied (neuronal and muscle receptors, Hex and QX222), only the blockade of the neuronal AChR by Hex is characterized by a residence time longer than the normal open time. 9. It is concluded that the modest differences in primary amino acid sequence between muscle and neuronal receptors lead to profound changes in their interactions with channels blockers.


Dopamine D2 receptor stimulation differentially affects voltage-activated calcium channels in rat pituitary melanotropic cells. 1. Whole-cell voltage clamp recordings were made from 141 rat pituitary melanotropic cells in short-term, serum-free, primary culture. The effects of the dopamine D2 receptor agonist, LY 171555, on sodium, potassium and barium currents were investigated. 2. Application of 1 microM-LY 171555 did not affect the inward sodium and outward potassium currents. 3. Application of LY 171555 reversibly inhibited barium currents, with the strongest inhibition on the early inward current. The effect was dose dependent (IC50 = 4 x 10(-8) M), maximal inhibition of the total current was 30% and the LY 171555-induced block (1 microM) was reversibly antagonized by (+/-)sulpiride (4 microM). 4. Using barium-selective saline solutions, different types of barium current (T, N, and two L components) were identified on the basis of their voltage-dependent kinetics. Their relative amplitudes differed between cells. 5. The T-type current activated at potentials positive to -60 mV, reaching peak amplitude between -20 and -10 mV. At -30 mV, this current was inhibited up to 30% by 1 microM-LY 171555. The time constants of activation (10-3 ms) and inactivation (50-20 ms) as well as the voltage dependence of inactivation (potential of half-maximal inactivation (H), -61 mV; slope factor (S), 4.9 mV) were not affected by LY 171555 application. 6. A rapidly inactivating (time constants 100-50 ms), high threshold current component was identified as an N-type current. This current activated at command potentials positive to -30 mV and reached a maximal amplitude at +10 mV. The steady-state inactivation was described by a single Boltzmann equation with H = -65 mV and S = 11.7 mV. Application of 1 microM-LY 171555 completely suppressed this current. 7. The slowly inactivating (time constants > 1500 ms), high-threshold, L-type current displayed the same voltage dependence of activation as the N current. The voltage dependence of inactivation was modelled by the sum of two Boltzmann equations (L1: H1 = -45 mV, S1 = 13.0 mV; L2:H2 = -11 mV, S2 = 6.0 mV), indicating the existence of two L channel populations. Neither time course, nor voltage dependence of inactivation were influenced by LY 171555. However, LY 171555 induced a slow-down in the time course of activation, which necessitated the use of two time constants to model the activation kinetics. One of these (approximately 2 ms) was also observed under control conditions.(ABSTRACT TRUNCATED AT 400 WORDS)


Quantal transmitter release mediated by strontium at the mouse motor nerve terminal. 1. In isolated mouse diaphragm, nerve stimulation in the presence of Sr2+ evokes phasic quantal transmitter release (endplate potentials, EPPs) with the same time course as in the presence of Ca2+. 2. Brief tetanic trains of nerve stimuli in the presence of Sr2+ cause an increase in quantal content of EPPs accompanied by an increase in the frequency of miniature EPPs (MEPPs); the latter persists as a 'tail' that subsides within about a second. Pseudo-random stimulation sequences were used to characterize these changes. 3. The fourth root of MEPP frequency during or after stimulation rose and fell in accordance with first order kinetics with the same time constants for rising and falling phases, in agreement with a 'residual ion' model in which (a) each nerve impulse causes the same entry of Sr2+ into the nerve terminal, (b) transmitter release (MEPP frequency) is proportional to the fourth power of [Sr2+] at release sites, and (c) Sr2+ removal is a first order process with a time constant of about 250 ms. 4. After exposure to bis (O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, acetoxymethyl ester form (BAPTA AM), 'Sr2+ tails' of MEPP frequency were reduced in magnitude and prolonged. 5. During stimulation trains, growth of phasic transmitter release rates (and quantal content of EPPs) were related to growth of MEPP frequency in almost exact agreement with a residual ion model, in which 'phasic' release (EPPs) and MEPP frequency are governed by the same equation, with the same parameters, and without any effect of depolarization per se to affect phasic release. 6. Prolonged (1 s) nerve terminal depolarizations in the presence of Sr2+ produce increased MEPP frequency with a time course corresponding to a model in which depolarization per se has little or no effect to increase transmitter release. 7. It was concluded that in the presence of Sr2+ the intense 'phasic' acceleration of quantal release induced by nerve impulse manifest in an EPP can be attributed to a transient rise of intracellular [Sr2+] in the vicinity of release sites, while the modulation of 'phasic' release by antecedent nerve impulses can be attributed to residual Sr2+ which is also manifest in a rise in MEPP frequency.


Activation and desensitization of N-methyl-D-aspartate receptors in nucleated outside-out patches from mouse neurones. 1. Activation and desensitization of N-methyl-D-aspartate (NMDA) receptors were studied in large outside-out patches excised from cultured embryonic neurones dissociated from mouse forebrain. The patches were exposed to rapid changes of NMDA or L-glutamate concentrations in the presence of glycine at concentrations (10-20 microM) saturating the modulatory site of the NMDA receptor. 2. Immediately after formation of the patch the responses to NMDA and L-glutamate showed a slow and small desensitization, even with high concentrations of agonist. During the following hour, the peak response either decreased or remained relatively stable, but in all cases the desensitization increased and accelerated until it stabilized. In this 'stabilized' state, the desensitization produced by high concentrations of NMDA (1 mM) or L-glutamate (300 microM) had an exponential time course, with a time constant of about 30 ms. The ratio of the peak over the steady-state current was in the order of 40 for NMDA and about 30 for L-glutamate. 3. Concentration-response curves were built for the peak and the plateau responses, for NMDA and for L-glutamate. The comparison of these curves indicated that (i) the EC50 of the peak (K(app) was always higher than the EC50 of the plateau (Kss); (ii) the two EC50 values for NMDA (K(app) and Kss) were higher than those for L-glutamate; (iii) the Hill coefficient was close to 1.4 for each of the four curves. 4. The application of NMDA or L-glutamate at a low concentration for 3 s periods reduced the response to a subsequent application of the same agonist at a saturating concentration. The IC50 of this 'predesensitization', termed Kpre, was lower than the EC50 of the steady-state response, Kss. 5. The onset rates of desensitization increased with the concentration of agonist. The EC50 of this relation was close to the value of K(app). 6. The decay of the currents at the end of a 3 s application of agonist was usually well described by the sum of two exponentials both of which were faster for NMDA than for L-glutamate. 7. The recovery from desensitization after a long (3 s) pulse of agonist was approximately exponential, with a time constant of about 0.5 s for NMDA and about 3.5 s for L-glutamate.(ABSTRACT TRUNCATED AT 400 WORDS)


Glutamatergic motoneurons in the stomatogastric ganglion of the mantis shrimp Squilla oratoria. 1. Transmitters of motoneurons in the stomatogastric ganglion (STG) of Squilla were identified by analyzing the excitatory neuromuscular properties of muscles in the posterior cardiac plate (pcp) and pyloric regions. 2. Bath and iontophoretic applications of glutamate produce depolarizations in these muscles. The pharmacological experiments and desensitization of the junctional receptors elucidate the glutamatergic nature of the excitatory junctional potentials (EJPs) evoked in the constrictor and dilator muscles. The reversal potentials for the excitatory junctional current (EJC) and for the glutamate-induced current are almost the same. 3. Some types of dilator muscle show sensitivity to both glutamate and acetylcholine (ACh) exogenously applied. The pharmacological evidence and desensitization of the junctional receptors indicate the glutamatergic nature of neuromuscular junctions in these dually sensitive muscles. The reversal potentials for the EJC and for the ACh-induced current are not identical. 4. Glutamate is a candidate as an excitatory neuro-transmitter at the neuromuscular junctions which the STG motoneurons named PCP, PY, PD, LA and VC make with the identified muscles. Kainic and quisqualic acids which act on glutamate receptors are potent excitants of these muscles. Extrajunctional receptors to ACh are present in two types of the muscle innervated by LA and VC. 5. Neurotransmitters used by the STG motoneurons of stomatopods are compared to those of decapods.


The morphology, innervation and neural control of the anterior arterial system of Aplysia californica. The morphology, innervation, and neural control of the anterior arterial system of Aplysia californica were investigated. Immunocytochemical and histochemical techniques generated positive reactions in the anterior arterial system for several neuroactive substances, including SCPB, FMRFamide, R15 alpha 1 peptide, dopamine and serotonin. Three neurons were found to innervate the rostral portions of the anterior arterial tree. One is the identified peptidergic neuron R15 in the abdominal ganglion, and the other two are a pair of previously unidentified neurons, one in each pedal ganglion, named pedal arterial shorteners (PAS). The endogeneously bursting neuron R15 was found to innervate the proximal anterior aorta. It also innervates a branch of the distal anterior aorta, the left pedal-parapodial artery. Activity in R15 causes constriction of the left pedal-parapodial artery. This effect is presumed to direct hemolymph towards the genital groove and penis on the right side in vivo. This vasoconstrictor action of R15 is mimicked by the R15 alpha 1 peptide. The PAS neuron pair causes longitudinal contraction of the rostral anterior aorta and the pedal-parapodial arteries. In vivo, the pair is active during behaviors involving head withdrawal and turning. By adjusting the length of the arteries during postural changes, the PAS neurons may prevent disturbances in blood flow due to bending or kinking of the arterial walls.


Local research ethics committees. Report of the 2nd National Conference. The major theme of this conference was the present variable performance of local research ethics committees (LRECs). Opening the meeting, Dame Margaret Turner-Warwick pointed to the tremendous advances that were taking place in medical practice and science and that this was therefore an opportune moment to 'see where we are, and share solutions as well as problems in the discussion and debate of this meeting'.


Who will care for our elderly people?

Who will care for our elderly people? A conference on the topic 'Who will care for our elderly people?' was held at the Royal College of Physicians on 16 March 1992. It began with a consideration of the challenge in terms of numbers and problems.


General medicine and ophthalmology: common interests. A conference entitled 'General Medicine and Ophthalmology' was held at the Royal College of Physicians on 1 June, 1992. Eye diseases are frequently a manifestation of systemic conditions; it is therefore in the patient's best interest for ophthalmologists and physicians to co-operate in their management. Without such co-operation there is the risk that patients fall between stools and neither condition is adequately treated. Medical specialties in which eye conditions are particularly prominent include dermatology, endocrinology, neurology, rheumatology, and cardiovascular diseases. The advantages of joint clinics in medicine and ophthalmology were demonstrated by Professors Alex Crombie and Pat Kendall-Taylor for Graves' disease, by Mr Philip Murray and Dr David Young for uveitis, and by Professor Eva Kohner for diabetes. These included more expert assessments of patients leading to quicker and more complete diagnoses, earlier recognition of complications, and access to a wider range of investigations and treatments, opportunities for collaborative research, improved education for patients and doctors, increased patient convenience, and a stimulus for better control of factors which can worsen the disease.


Immunohistochemical localization of 3 beta-hydroxysteroid/delta 5-delta 4-isomerase, tyrosine hydroxylase and phenylethanolamine N-methyl transferase in adrenal glands of sheep fetuses throughout gestation and in neonates. Immunoreactive 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) was localized in adrenal glands of sheep fetuses in cortical-type cells, but not in medullary-type cells, from day 43 of gestation to term and in 2-4-day-old neonates. From day 54 of gestation, the formation of distinct zones within the adrenal cortex was apparent and immunoreactive 3 beta-HSD was found in cortical cells in the zona fasciculata and in groups and cords of cortical cells within the developing medulla, with weak positive staining in the zona glomerulosa. At this stage, most medullary cells were positive for immunoreactive tyrosine hydroxylase, and some of these cells with a juxtacortical distribution also stained positively for immunoreactive phenylethanolamine N-methyl transferase (PNMT). Between days 65 and 130, the adrenal medulla increased in size with little change in the width of the cortex. Organization and zonation of immunoreactive 3 beta-HSD staining cells were evident in the zona fasciculata and in groups of cells in the medulla. Between day 130 and term, uniform immunoreactive 3 beta-HSD staining was found throughout the zona fasciculata, and there was also staining in single cells and small clusters of cells throughout the medulla. At this stage, immunoreactive tyrosine hydroxylase was distributed in most cells throughout the medulla, but in two distinct patterns: cells staining intensely for immunoreactive tyrosine hydroxylase in the central region of the medulla, and cells exhibiting weaker staining for immunoreactive tyrosine hydroxylase localized in a juxta-cortical position. These juxta-cortical cells were also positive for immunoreactive PNMT.(ABSTRACT TRUNCATED AT 250 WORDS)


Neuroleptic-associated hyperprolactinemia. Can it be treated with bromocriptine? Six stable psychiatric outpatients with hyperprolactinemia and amenorrhea/oligomenorrhea associated with their neuroleptic medications were treated with bromocriptine. Daily dosages of 5-10 mg corrected the hyperprolactinemia and restored menstruation in four of the six patients. One woman, however, developed worsened psychiatric symptoms while taking bromocriptine, and it was discontinued. Thus, three of six patients had their menstrual irregularity successfully corrected with bromocriptine. This suggests that bromocriptine should be further evaluated as potential therapy for neuroleptic-associated hyperprolactinemia and amenorrhea/galactorrhea.


Human T cell leukemia virus-I carriers and gonarthrosis. A preliminary report. Gonarthrosis in 11 human T cell leukemia virus-1 (HTLV-I) carriers were studied for sera, synovial fluids (SF) and cerebrospinal fluids (CSF). Atypical lymphocytes similar to adult T cell leukemia cells were noticed in the SF of all the HTLV-I carriers and ranged from 19 to 1%, while those of peripheral white blood cells ranged from 3 to 0%. Anti-HTLV-I antibodies in the SF were equal to or lower than those in the sera. The CSF from 7 of them demonstrated positive titers of anti-HTLV-I antibodies, while no evidence of neurological disorders manifested. The histopathological findings of synovial membranes on hematoxylin and eosin staining method showed no statistical difference between those of gonarthrosis of HTLV-I carriers and noncarriers.


Long-term effects on insulin sensitivity and sodium transport in glucose-intolerant hypertensive subjects when beta-blockade is replaced by captopril treatment. Long-term effects on insulin sensitivity and leucocyte sodium transport were studied in 42 glucose-intolerant hypertensives on beta-blockers, randomly assigned to continuous beta-blockade (beta group) or a switch to captopril treatment (mean daily dose 69.1 mg) (ACE group). In the ACE group, despite a tendency towards improvement, glucose uptake during the euglycaemic insulin clamp procedure did not change significantly from the baseline value of 4.0(0.5-12.9) mg/kg/min, values at 6 and 12 months being 4.8(1.3-14.7) and 4.4(1.5-9.8) mg/kg/min, respectively: the corresponding values for the beta group were 4.2(1.1-15.3), 3.6(1.2-8.9) and 4.0(1.5-12.0) mg/kg/min. The 22Na efflux rate constant, both baseline and follow-up values, was similar in both groups, and unrelated to insulin sensitivity. Owing to the surprisingly great variation in peripheral glucose uptake, the subgroup with values below the median for the population as a whole (4.9 mg/kg/min) was evaluated separately: those switched to captopril treatment manifested a 60% improvement in glucose disposal at 6 months and persisting at 12 months, the respective values being 2.1(0.5-4.8) (baseline), 3.5(1.3-6.3) and 3.4(1.5-6.1) mg/kg/min, (P = 0.012). The body mass index (BMI) was not significantly affected. Values for BMI, peripheral insulin and triglycerides were higher in the subgroup with glucose disposal below the median than in the subgroup with values above the median. Correlation between BMI and glucose uptake was highly significant (r = -0.75, P = 0.0001). The present findings suggest that captopril may be a better alternative than beta-blockers for treating the highly insulin-resistant, glucose-intolerant patients, predominantly to be found among the overweight.


Effect of a histamine H2 type receptor antagonist (WY 45, 727) on the healing of gastric ulcers in ponies. Using video gastroscopy, the efficacy of a Histamine-H2 type receptor antagonist (WY 45, 727) was investigated in young ponies with spontaneous and experimentally induced gastric ulcers. Oral administration of WY 45, 727 at 2 mg/kg and 10 mg/kg of body weight every 12 hours for 14 days resulted in complete healing of spontaneous gastric ulcers in the non-glandular portion of the stomach in 2/5 (40%) and 3/4 (75%) of the ponies, respectively, compared (P < 0.05) to 0/5 (0%) placebo-treated ponies. After intramuscular administration of flunixin meglumine at 1.5 mg/kg body weight every 8 hours for 6 days, 9/18 ponies had ulcers in the non-glandular portion of the stomach. Oral administration of WY 45, 727 at 10 mg/kg body weight every 12 hours for 14 days resulted in complete healing of the non-glandular gastric ulcers in 3/4 (75%) compared with (P < 0.05) 1/5 (20%) placebo-treated ponies. This study indicates that 1) the occurrence of subclinical ulcers may be common in young ponies; 2) flunixin meglumine at 1.5 mg/kg intramuscularly every 8 hours for 6 days may result in ulcers of the non-glandular stomach in ponies; and 3) WY 45, 727, a histaminergic H2 type receptor antagonist could be of value in the therapeutic management of ulcers in the non-glandular stomach of foals and adult horses.


Influence of mild to moderate treated hypertension on 9-11 year mortality in patients with pre-existing coronary heart disease. Hypertension is a known risk factor in the genesis of coronary artery disease. However, the effect of pre-existing hypertension on the long-term mortality in patients with established coronary heart disease is not clear. The present cohort study analysed the influence of baseline mild to moderate treated hypertension in cases of known coronary heart disease with cardiac mortality as end point. Data from a cohort of 511 patients including 266 normotensives and 245 controlled hypertensives was analysed over a follow-up period of 9 to 11 years. The baseline data were identical regarding other major risk factors like age, gender, smoking, diabetes, cholesterol levels and congestive heart failure on univariate analysis. There were more cases of myocardial infarction in the normotensive group. The number of patients receiving beta-blockers or aspirin were similar in both groups. However, more patients in the hypertensive group received nifedipine. Actuarial analysis of survival showed that mortality was the same in both groups with an overall cardiac mortality of 65 (26.5%) in the hypertensive group and 86 (32.3%) in the normotensive group (P greater than 0.1). The survival curves also showed no significant difference in mortality at any point in time (logrank test = 2.37, P greater than 0.1). Analysis of mortality after adjusting for myocardial infarction at first presentation also showed no significant difference. These data indicate that in patients with coronary heart disease the presence of mild to moderate hypertension does not add to the risk of cardiac mortality.


Synthesis and hypoglycemic activity of substituted 8-(1-piperazinyl)imidazo[1,2-a]pyrazines. A series of alkyl- and halo-substituted 8-(1-piperazinyl)imidazo[1,2-a]pyrazines were prepared using two approaches, the condensation of alpha-halocarbonyl derivatives with an aminopyrazine or the oxidation-dehydration of a [(beta-hydroxyalkyl)amino]pyrazine. These imidazo[1,2-a]pyrazines were evaluated for their binding affinity to the alpha 1, alpha 2, beta 1, and beta 2 adrenergic receptors as well as their ability to lower blood glucose in insulin resistant hyperglycemic ob/ob mice. Modifications on 8-(1-piperazinyl)imidazo[1,2-a]pyrazine (4) reduced alpha 2 binding, lowered hypoglycemic potency, and showed variations in binding to the alpha 1, beta 1, and beta 2 adrenergic receptors. In addition to 4, the 2-methyl, 3-methyl, and 5-methyl 8-(1-piperazinyl)imidazo[1,2-a]pyrazines (16k, 25m, and 16f, respectively) displayed high affinity for the alpha 2 receptor and were potent hypoglycemic agents when compared to 2-amino-7,8-dihydro-4-(1-piperazinyl)-6H-thiopyrano[3,2- d]pyrimidine (MTP-1403, 2). Receptor binding was modified by use of a 4-methylpiperazine moiety which reduced alpha 1 and beta 1 binding while retaining some hypoglycemic activity. The structure-activity relationship for heterocyclic alkyl and halo substitution on biological activity is discussed.


Synthesis and dopaminergic activity of some 3-(1,2,3,6-tetrahydro-1-pyridylalkyl)indoles. A novel conformational model to explain structure-activity relationships. The synthesis and dopaminergic properties of a novel type of dopamine agonist is described. The number and kind of essential structural elements differ significantly from that of the rigid apomorphine-type dopamine agonists. Using standard molecular modeling techniques, a conformational model is developed proposing a U-shaped conformation which might be energetically preferred through aromatic pi-pi-interactions between both of the electron rich aromatic structural elements of this class of compounds. Superimposition of conformations of the lead compound 28 with apomorphine yields a novel model explaining the atypical structure-activity relationships found in this class of indolealkylamines.


Synthesis of biologically active taxol analogues with modified phenylisoserine side chains. Taxol (1) is a highly potent antitumor agent, exerting its mechanism of action by promoting the assembly of stable microtubules in cells. We are reporting on the first synthesis and biological evaluation of taxol derivatives with substituted phenyl rings at the C-13 N-benzoyl-(2'R,3'S)-3'-phenylisoserine side chain of taxol (1). Two taxol derivatives were synthesized, one possessing a N-(p-chlorobenzoyl)-(2'R,3'S)-3'-phenylisoserine side chain (2) and the other one a N-benzoyl-(2'R,3'S)-3'-(p-chlorophenyl)isoserine side chain (3). The synthesis of the novel phenylisoserine side chains was achieved through the asymmetric synthesis of 3-hydroxy-4-aryl-2-azetidinone derivatives via the ester enolate-imine cyclocondensation reaction. The 2-azetidinones 14 and 15 were acylated with p-chlorobenzoyl chloride and benzoyl chloride, respectively, to form the N-acyl beta-lactams 16 and 17. Subsequent coupling of 16 and 17 to 7-(triethylsilyl)baccatin III (6) in the presence of pyridine and DMAP afforded, after removal of the protecting groups, the desired taxol analogues 2 and 3 in excellent yields. The newly synthesized derivatives 2 and 3 were tested in the tubulin assembly assay and also evaluated for their cytotoxicity against B16 melanoma cells. It was found that the taxol derivatives 2 and 3 had activity comparable to taxol (1).


Large de novo DNA deletion in a patient with sporadic neurofibromatosis 1, mental retardation, and dysmorphism. A mildly dysmorphic, mentally retarded male with neurofibromatosis 1 (NF1) was found to have a de novo deletion of chromosome 17. The deletion occurred on the paternally derived chromosome 17 as shown by the absence of a D17S73 paternal allele. Densitometric analysis indicated that, in addition to the D17S73 locus, the patient has only one copy of four other adjacent loci. The deletion involved the loci D17S120, NF1, D17S57, D17S115, and D17S73 and was estimated to encompass more than 380 kb of DNA. The deletion of the entire paternal NF1 allele argues strongly that this disorder is not caused by the action of an abnormal NF1 protein. The extent of the deletion suggests that the mental retardation and dysmorphism of this patient may result from a deletion involving both the NF1 gene and contiguous genetic material.


Difficult diagnosis of the fragile X syndrome made possible by direct detection of DNA mutations. Genetic recombination near the fragile X locus (Xq27.3) has frequently been a problem in linkage studies of families in which the fragile X is segregating. This case report illustrates the resolution of a difficult situation in a fragile X family for whom cytogenetic studies were inconclusive and where recombination had twice confounded attempts at prenatal DNA diagnosis by RFLP analysis. Using a newly developed DNA probe, StB12.3, for direct detection of DNA instability in the fragile X locus, the presence of the fragile X was ascertained definitively in a prenatal DNA sample.


The adherence of verocytotoxin-producing Escherichia coli to rabbit intestinal cells. Verocytotoxin-producing Escherichia coli (VTEC) have been recognised recently as an important cause of human disease. The adherence of VTEC to rabbit intestinal tract and the relationship between adherence and other virulence traits were studied. Twenty clinical isolates of VTEC (O157:H7 and other serotypes) and a control, commensal E. coli strain, were examined. Bacteria were evaluated for the presence of surface fimbriae, plasmid profile and hybridisation with a 3.4 kb DNA probe derived from the 60-MDa plasmid of such strains. Adherence was determined by electronmicroscopy and quantitatively with radio-labelled bacteria. Of the VTEC strains, 12 (60%) had surface fimbriae; all O157:H7 and 10 (70%) of 14 of the non-O157:H7 strains hybridised with the probe. No isolate was negative for both of these virulence traits and there was no correlation between their presence. The plasmid profiles varied among the strains, with no correlation to virulence traits. The adherence of VTEC strains differed significantly, ranging from 0.3 to 34.0 bacteria/intestinal cell. The mean adherence of fimbriate strains was greater than that of non-fimbriate strains (3.9 versus 2.7 bacteria/cell), although marked variability was noted in both groups. This study showed that VTEC strains differed markedly in their adherence capability and that neither the presence of fimbriae nor hybridisation with the 3.4-kb probe was essential for adherence. Several distinct mechanisms probably play a role in VTEC adherence.


Variation in the CD4+ and CD8+ populations in lymph nodes does not reflect that in the blood during SIVMNE/E11S infection of macaques. The decline in the CD4% and CD4/CD8 ratios have been compared in lymph nodes and blood from SIVMNE/E11S infected rhesus macaques. The results indicate that loss from the LN CD4+ cell pool does not occur until CD4/CD8 ratios of less than 0.5 is reached in blood. These changes also correlate with the ability to isolate virus from the blood and the transition of CD45RAhi to highly activated CD45RAlo CD8+ cells both of which may play a role in eliminating CD4+ cells. In end-stage disease, CD8+ cells also decline in LN and mitogen responsiveness no longer exists in any nodes. Interestingly at this stage, the circulating CD8% increases significantly and represents the only source of functional T cells remaining in the body.


Clinical correlates of MDR1 (P-glycoprotein) gene expression in ovarian and small-cell lung carcinomas. Expression of the MDR1 (P-glycoprotein) gene causes resistance to several classes of lipophilic anti-cancer drugs, but MDR1 expression in untreated ovarian and lung carcinomas is rarely detectable by standard assays. This study was designed to measure the MDR1 messenger RNA (mRNA) content of ovarian and lung carcinomas and to analyze clinical correlations of MDR1 expression in these tumors. A sensitive assay based on the polymerase chain reaction (PCR) was used in a retrospective study to measure MDR1 mRNA in biopsy samples of 100 solid tumors, including 60 ovarian and 32 lung carcinomas. The levels of MDR1 mRNA were correlated with history of chemotherapeutic treatment for all tumors; for ovarian and small-cell lung carcinomas (SCLCs), these levels were also correlated with subsequent tumor response to chemotherapy. Among previously untreated patients, MDR1 mRNA was expressed in 68% (50 of 74) of all tumors. Among patients pretreated with chemotherapy regimens that included at least one P-glycoprotein-transported drug (MDR regimens), 95% (20 of 21) of all tumors expressed MDR1 mRNA though the incidence of high-level MDR1 expression was decreased among the treated tumors. MDR1 mRNA was expressed in only one of five tumors treated with regimens that included no P-glycoprotein substrates (non-MDR regimens). Subsequent tumor response to chemotherapy was evaluated in 35 patients with ovarian carcinoma and seven patients with SCLC. The presence of even very low levels of MDR1 mRNA correlated with the lack of response to MDR regimens in these tumor types (P < .035 for ovarian carcinomas, P < .029 for SCLCs, and P < .0005 for both tumor types; Fisher's Exact Test). Low-level expression of MDR1 mRNA correlates with clinical resistance to combination chemotherapy in ovarian cancer and SCLC. We hypothesize that MDR1 is expressed in a subpopulation of more malignant tumor cells possessing multiple mechanisms of drug resistance. The presence of MDR1-expressing tumor cells may be useful as a predictive marker for clinical resistance to combination chemotherapy in ovarian cancer and SCLC. Prospective studies are needed to confirm this hypothesis.


Effect of P-glycoprotein expression on sensitivity to hormones in MCF-7 human breast cancer cells. Data obtained from studies of primary human breast cancers and established cell lines indicate that overexpression of the MDR1 gene (also known as PGY1) is associated with decreased expression of steroid hormone receptors and increased expression of epidermal growth factor (EGF) receptors. Other study results indicate that both progestins and triphenylethylene antiestrogens may be substrates for P-glycoprotein, the product of the MDR1 gene. These findings together suggest an association between overexpression of the MDR1 gene and cross-resistance to progestin and antiestrogen therapies. This study was designed to determine (a) the ability of MDR1 expression to alter tumor sensitivity to hormone therapy and (b) the role of MDR1 expression in expression of functional hormone receptors in human breast cancer. We transduced MCF-7 cells with MDR1 complementary DNA, using a retroviral vector directing the constitutive expression of the MDR1 gene. Transduced cells (MCF-7MDR1) were examined for ability to produce P-glycoprotein, expression of steroid hormone receptors, and responsivity to antiestrogens. For comparison, we used MCF-7ADR human breast cancer cells, which overexpress MDR1 and have also lost the requirement for 17 beta-estradiol supplementation to form tumors in nude mice. We also investigated the level of EGF-R mRNA expression by using a sensitive RNase protection analysis. MCF-7MDR1 cells retained both estrogen receptor and progesterone receptor expression as well as sensitivity to 4-hydroxytamoxifen. Expression of the estrogen-inducible pS2 and EGF receptor genes was similar in parental MCF-7 and transduced MCF-7MDR1 cells. EGF receptor expression was increased, and pS2 expression was lost (undetectable) in MCF-7ADR cells. The data indicate that overexpression of the MDR1 gene alone confers a multidrug-resistant phenotype, but it does not directly result in either cross-resistance to antiestrogens or a loss of steroid hormone receptor expression. MCF-7MDR1 cells provide an important model for study of the interactions of cytotoxic drugs, hormones, and the MDR1 glycoprotein in human hormone-responsive breast cancer cells.


Correlates of SIV encephalitis in rhesus monkeys. Necropsy records from 204 SIV-infected rhesus monkeys that had been inoculated with various strains of SIV and had died of SIV-related disease were reviewed. The relationship of SIV encephalitis with other parameters was evaluated. Encephalitis was associated with the presence of syncytial cells in other tissues, with persistent or early recurrent antigenemia, with a selective decrease in CD4+CD29+ blood lymphocytes, and with a shortened time of survival. Monkeys whose lymphocytes produced high levels of virus in culture also had a higher incidence of encephalitis. SIV was more frequently isolated from the brains of animals with encephalitis. No other clear associations were detected.


Phase I trial of Taxotere: five-day schedule. Taxotere, a semisynthetic compound structurally related to taxol, has a broad spectrum of activity in murine transplantable tumors; in the B16 melanoma model, it caused a total log cell kill 2.5 times greater than that caused by taxol at equitoxic doses. We conducted a phase I study of Taxotere (a) to determine its qualitative and quantitative toxic effects and a starting dose for phase II trials, (b) to investigate its clinical pharmacology, and (c) to document its antitumor activity. Taxotere was given as a 1-hour infusion at a starting dose of 1 mg/m2 per day for 5 consecutive days. The 5-day course of therapy was repeated every 21 days. Thirty-nine cancer patients with advanced disease were entered in the study; at least three patients were entered at each dose level. Initial dose escalations were planned at 100% increments until biologic activity was observed; subsequent escalations were planned at 50% increments until grade 2 toxicity (the National Cancer Institute's Common Toxicity Criteria) occurred and then at 25% increments until the maximum tolerated dose was established. Thirty-nine patients were entered in the study. Successive dose levels used were 1, 4, 8, 16, 12, and 14 mg/m2 per day. The dose-limiting toxic effects were granulocytopenia and concurrent mucositis. Grade 4 granulocytopenia associated with grade 3 mucositis developed in six of 12 patients treated at a dose of 16 mg/m2 per day, two of 10 treated at 12 mg/m2 per day, and two of eight treated at 14 mg/m2 per day. Because these toxic effects occurred concurrently, all patients so affected developed neutropenic fevers and required hospitalization. Neither cardiac nor neurologic toxic effects were noted. Anti-tumor activity was observed in six patients with ovarian cancer and in one with breast carcinoma. Although pharmacokinetic parameters were consistent between day 1 and day 5 for individual patients, considerable variation existed among those treated at the same dose level. A relationship was observed between the area under the curve for plasma concentration of drug x time (AUC) on day 1 and the percentage decrease in absolute granulocyte counts. Granulocytopenia associated with oral mucositis is the dose-limiting toxicity of this schedule. We recommended a starting dose of 14 mg/m2 per day for phase II studies of this 5-day schedule. Dose modifications on days 2-5 based on the day-1 AUC may allow individualized dosing.


High-dose oral tamoxifen, a potential multidrug-resistance-reversal agent: phase I trial in combination with vinblastine. P-glycoprotein mediates resistance to natural-product anti-neoplastic agents like vinblastine through an active transport process resulting in reduced intracellular concentration of these agents. The triphenylethylene antiestrogen tamoxifen and its major metabolite N-desmethyltamoxifen at concentrations of 4-6 microM enhance the intracellular concentration of natural-product antineoplastics and augment the cytotoxicity of such drugs three-fold to 10-fold in a variety of human and murine cell lines. On the basis of these preclinical findings, we conducted a phase I clinical trial of high-dose, oral tamoxifen administered in conjunction with a 5-day continuous infusion of vinblastine. We studied 53 patients with advanced epithelial tumors. Tamoxifen was given orally as a loading dose on day 1, followed by two doses a day on days 2-13. Vinblastine was given as a 120-hour continuous infusion (1.5 mg/m2 per day) on days 9-13 of each tamoxifen course. The starting dose of tamoxifen was 40 mg/m2 administered twice a day following a loading dose of 150 mg/m2. The maximum dose was 260 mg/m2 twice a day following a loading dose of 680 mg/m2. Treatment cycles were repeated every 28 days. The dose-limiting toxic effects of tamoxifen were neurologic and began within 3-5 days after the start of treatment. They consisted of tremor, hyperreflexia, dysmetria, unsteady gait, and dizziness. One patient experienced a grand mal seizure 24 hours after the last tamoxifen dose. Toxic effects were rapidly reversible. Asymptomatic prolongation of the QT interval on electrocardiogram occurred at doses of tamoxifen of 80 mg/m2 or higher given twice a day. No coagulation or ophthalmologic abnormalities occurred. Tamoxifen did not enhance the toxicity of vinblastine. Mean plasma concentrations of tamoxifen or N-desmethyltamoxifen at 260 mg/m2 tamoxifen given twice a day for 13 days were 6.04 and 6.56 microM, respectively. There was no relationship between plasma antiestrogen content and the development of neurotoxic effects. Tamoxifen at 150 mg/m2 given twice a day following a loading dose of 400 mg/m2 results in plasma levels of tamoxifen and N-desmethyltamoxifen of 4 and 6 microM, respectively, without dose-limiting toxicity. We recommend this dose for phase II trials of tamoxifen to modulate P-glycoprotein-mediated drug resistance. Our study demonstrates that high-dose tamoxifen can be safely administered and that plasma concentrations that may inhibit P-glycoprotein function can be achieved.


In vitro and in vivo inhibition of lysyl oxidase by aminopropionitriles. Inhibition of lysyl oxidase (protein-lysine 6-oxidase, EC 1.4.3.13) decreases the rate of collagen and elastin cross-link formation and produces osteolathyrism in animals. Organic nitriles, including beta-aminopropionitrile (BAPN), have been shown to irreversibly inhibit lysyl oxidase in vitro. Both BAPN and 3,3'-iminodipropionitrile (IDPN) have been shown to produce osteolathyric changes when administered to animals. To date compounds that have been reported to inhibit this enzyme possess a primary amine functional group. In this study a series of primary and substituted aminopropionitriles was studied for their ability to inhibit lysyl oxidase activity both in vitro and in vivo. Our results show that of the compounds tested, BAPN was the most potent inhibitor of the enzyme. Reversible inhibition of lysyl oxidase in vitro was found with two secondary aminonitriles, IDPN and monomethylaminopropionitrile (MMAPN). There was no inhibition of enzyme activity associated with the tertiary compound 3,3'-dimethylaminopropionitrile (DMAPN) or propionitrile, a compound lacking an amine functional group. IDPN was found to produce a slight irreversible inhibition of the enzyme both in vitro and in vivo. Pretreatment of rats with pargyline, an inhibitor of monoamine oxidase, was found to increase the inhibitory potential of BAPN (p < or = .1). Pargyline pretreatment did not alter the inhibitory potential for any of the other aminonitriles tested. These results suggest that the presence of a primary amino functional group is not a strict requirement for inhibition of lysyl oxidase. In addition, reversible and irreversible mechanisms of inhibition may be involved in the production of osteolathyric changes associated with IDPN exposure.


Phenotypic plasticity of avian embryonic sympathetic neurons grown in a chemically defined medium: direct evidence for noradrenergic and cholinergic properties in the same neurons. Avian embryonic sympathetic ganglia possess both catecholaminergic and cholinergic features and can synthesize noradrenaline (NAd) and acetylcholine (ACh) simultaneously. In the present study we sought to determine (1) whether or not this coproduction of NAd and ACh corresponds to the existence of two non-overlapping populations, and (2) to what extent the levels of synthesis are influenced by non-neuronal ganglion cells. We have focused on the correlation between the immunocytochemically demonstrable presence of the noradrenergic and cholinergic enzymes tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT), respectively, and the synthesis of the corresponding neurotransmitters in embryonic quail sympathetic neuronal and non-neuronal cells purified by fluorescence-activated cell sorting. We show that (1) freshly sorted neurons synthesize both NAd and ACh, whereas non-neuronal cells produce neither; (2) the overwhelming majority of the sympathetic neurons display TH immunoreactivity; (3) about half of the TH-positive neurons are recognized by an anti-ChAT antibody in an artificial medium that selectively enhances synthesis and/or accumulation of ACh; (4) the non-neuronal cells are important for survival of the neurons and potentiate their synthesis of ACh in this medium, and (5) finally, we present evidence that expression of TH in noradrenergic neurons and in small intensely fluorescent cells of sympathetic ganglia is differentially regulated.


Clinical evaluation of pediatric ethylene glycol monobutyl ether poisonings. Ethylene glycol butyl ether, CAS 111-76-2, an ingredient in many popular commercial window/glass cleaners, is known to produce equal if not greater toxicity than ethylene glycol when administered to animals. Treatment recommendations for human poisonings are based upon animal data and include the use of ethanol therapy to inhibit the production of toxic metabolites. No human experiential data exist to accurately assess human toxicity or to verify treatment modalities. A 5 month retrospective review of all glass cleaner ingestions reported to a regional poison information center disclosed 24 pediatric patients, ages 7 mo to 9 y, who ingested 5-300 mL of a liquid glass cleaning product containing ethylene glycol butyl ether. All ingestions were reported within 5 min of ingestion, and all 24 children were asymptomatic at that time and subsequently. The product concentrations of ethylene glycol butyl ether ranged from 0.5% to 9.9%. Two of the 24 children ingested > 15 mL and were treated by gastric emptying and 24 h hospital observation. Neither hospitalized child suffered symptoms consistent with hemolysis, nervous system depression, acidosis, or renal compromise. Dilution with oral fluids at home is considered appropriate treatment of pediatric ingestions of < 10 mL of a commercial liquid glass/window cleaners containing < 10% ethylene glycol butyl ether.


A comparison of the sedative effects of three alpha 2-adrenoceptor agonists (romifidine, detomidine and xylazine) in the horse. The sedative effects of a new alpha 2-adrenoceptor agonist, romifidine, were compared with those of xylazine and detomidine. Five horses were treated with two doses of romifidine (40 micrograms/kg body weight and 80 micrograms/kg body weight), two doses of detomidine (10 micrograms/kg body weight and 20 micrograms/kg body weight) and one dose of xylazine (1 mg/kg body weight) given by intravenous injection using a Latin-square design. The dose of 80 micrograms/kg romifidine appeared equipotent to 1 mg/kg xylazine and 20 micrograms/kg detomidine, although at these doses both xylazine and detomidine had a shorter action. Detomidine 20 micrograms/kg and xylazine both produced greater lowering of the head and a greater degree of ataxia than romifidine at either dose. Romifidine produced sedation similar to that of the other drug regimes. The effect upon imposed stimuli was similar.


Immunodeficiency and the risk of death in HIV infection. To describe the rate of development of immunodeficiency in human immunodeficiency virus (HIV) infection and to relate this to the risk of death. Inception cohort followed up for up to 12 years from HIV seroconversion until January 1, 1992. A regional hemophilia center based in a major teaching hospital. All 111 patients with hemophilia who seroconverted to HIV-1 between 1979 and 1985 were registered at the center. Patients have been closely followed up clinically and immunologically. Development of immunodeficiency, defined by a CD4 lymphocyte count falling beneath 0.20 and 0.05 x 10(9)/L, and death. Kaplan-Meier estimates suggest that almost half (46%; 95% confidence interval [CI], 26% to 66%) of patients alive 12 years after seroconversion will have a CD4 lymphocyte count that has remained above 0.05 x 10(9)/L. Thirty-five percent (95% CI, 22% to 48%) remain above 0.20 x 10(9)/L. Thirty-seven patients died of HIV-related causes, and there was a 52% probability (95% CI, 35% to 69%) of HIV-related mortality by 12 years from seroconversion. Mortality risk was closely associated with severe immunodeficiency. There was only a 15% chance (95% CI, 6% to 25%) of HIV-related death occurring before a CD4 count of below 0.05 x 10(9)/L had been reached. There was an average of one HIV-related death per 96.7 patient-years of observation before the CD4 count had fallen below 0.05 x 10(9)/L, as compared with one death per 2.5 patient-years of observation after the CD4 count had fallen below this level (P < .0001). In patients with HIV infection who are closely followed up, the risk of death is low before the CD4 lymphocyte count has fallen to 0.05 x 10(9)/L, a count many patients remain above up to 12 years after seroconversion.


Immunologic aspects of cardiovascular disease. The spectrum of vasculitis is diverse, and numerous entities do not fit the aforementioned broad categories. Examples of these include Buerger's disease; vaso-occlusive vasculitis of the lower extremities associated with cigarette smoking; Behçet's disease, which is prevalent in the Orient and Middle East and is characterized by recurrent aphthous stomatitis, genital ulcerations, uveitis, meningoencephalitis, and phlebitis; and isolated central nervous system vasculitis, a rare disease with a poor prognosis that primarily affects intracranial arteries without a systemic acute-phase response. Improvement in the classification and definitive therapy of vasculitis awaits identification of etiologic agents and definition of host factors and the immune response responsible for the pathology.


[Evaluation of the timing principle with small priming doses of vecuronium]. The intubating conditions using the timing principle combined with small priming doses of vecuronium were evaluated in forty patients who underwent elective surgery. They were randomly assigned to one of two groups: 1) timing, 2) timing with priming. In timing group, vecuronium 0.15 mg.kg-1 was administered, and at the onset of clinical muscle weakness, thiopental 4-5 mg.kg-1 was given promptly. Sixty seconds after thiopental, patients were intubated. In the timing with priming group, vecuronium 0.005 mg.kg-1 was administered as priming doses. Four minutes later vecuronium 0.15 mg.kg-1 was given. The administration of thiopental and the intubation were done in the same way as in timing group. The time to onset of clinical weakness after the administration of vecuronium 0.15 mg.kg-1 was significantly shorter in the timing with priming group than that in the timing group (46.1 +/- 4.8 vs. 57.6 +/- 7.8, P < 0.01). There were no significant differences in intubating score, T1, TR, onset time, and duration between the two groups. We conclude that the timing principle combined with small priming doses of vecuronium might be safe and useful for rapid tracheal intubation.


Genetic analysis of dilated cardiomyopathy--HLA and immunoglobulin genes may confer susceptibility. To identify genetic factors in the immune system which control the susceptibility to dilated cardiomyopathy (DCM), HLA class II DNA typing was performed in 61 Japanese patients, using PCR/SSO probe analyses. The frequencies of HLA-DQB1*0503 (15% vs 5%; RR = 3.06, chi 2 = 7.19) and DQB1*0604 (21% vs 10%; RR = 2.41, chi 2 = 6.20) were significantly increased and that of HLA-DQB1*0502 (RR = 1.74) was slightly increased in the DCM patients. The frequency of DQB1*0303 (16% vs 31%; RR = 0.44, chi 2 = 5.16) was significantly decreased in the patients. The increased HLA-DQB1 alleles have a histidine residue in common at the 30th codon for the HLA-DQ beta chain. Among the genetic markers studied by Southern blot analyses, IGLV (immunoglobulin lambda light chain, pV3.3) showed a strong association with DCM, i.e. A2/A2 genotype was found in 37.7% of patients whereas it was observed in only 18.9% of the control subjects (RR = 2.6, chi 2 = 7.77). The frequency of this genotype was higher in patients under age 45 years at the time of diagnosis (45.5%, RR = 3.6, chi 2 = 10.02). These results suggest that HLA and immunoglobulin genes are closely linked to susceptibility to DCM.


[Effects of epithelium removal and cooling on tracheal contraction in guinea pigs]. We investigated the effects of epithelium removal and cooling (32 degrees C, 27 degrees C, 22 degrees C) on isolated tracheal smooth muscle contraction induced by bethanechol (BCh), acetylcholine (ACh), histamine (Hist), and KCl in guinea pigs. The pD2 values (-logED50) increased in the epithelium damaged group compared with the intact group when ACh was administered at 37 degrees C, 32 degrees C, 27 degrees C and 22 degrees C, with histamine at all four temperature conditions, and with KCl at 37 degrees C, 32 degrees C and 27 degrees C. In both groups, the pD2 values for BCh, ACh, and KCl tended to decrease as temperature was lowered, and significant decrease in pD2 was observed at 27 degrees C. In contrast, the pD2 value for histamine tended to increase as temperature was lowered in every group. Concerning these agents, we conclude that epithelium removal and cooling exert neither significant nor consistent effect on tracheal epithelial function.


[Rapid induction with vecuronium/fentanyl combinations for coronary artery surgery: influence on cardiovascular responses and plasma fentanyl concentrations]. Cardiovascular effects of fentanyl (60 micrograms.kg-1.min-1)/vecuronium combination for rapid induction were studied in fifty five patients for coronary artery bypass grafts. Cardiovascular and cerebral profiles of patients who had rapid fentanyl administration (39 patients) or slow fentanyl administration (15 patients) were compared with base line values and between each group. Plasma samples were assayed for epinephrine, norepinephrine, growth hormone, anti diuretic hormone and fentanyl. There were no differences in all hemodynamic parameters between each group. However, in the rapid administration group, statistically significant changes were detected in the heart rate, cardiac index, stroke volume index and left ventricular stroke work index. The changes in the heart rate were small and returned to the baseline value after sternotomy. A decrease in cardiac index after induction depends on a decrease in stroke volume index rather than in the heart rate. In rapid administration group, the EEG showed low frequency activity within a minute. The hormonal stress responses were significantly attenuated in both groups. High plasma concentrations of fentanyl could be achieved at the intubation. The data demonstrate that rapid induction by fentanyl is safe and convenient.


[Varicella-zoster virus infection after peripheral blood stem cell autografts in children with leukemia or non-Hodgkin's lymphoma]. Thirty-two patients with leukemia or non-Hodgkin's lymphoma (NHL) who underwent high-dose chemotherapy without total body irradiation (TBI) and peripheral blood stem cell autografts (PBSCT) were evaluated for the occurrence of clinically manifested varicella-zoster virus infection (VZV). In a minimum follow-up of 3 mo, there were 14 cases with VZV; 12 patients were treated with intravenous acyclovir and 2 patients received additional intravenous VZV hyperimmune immunoglobulin preparations. History of previous varicella-zoster at some time before PBSCT was the only significant risk factor for the development of VZV. Furthermore, a significant association between VZV and higher disease-free survival rate after PBSCT was proved. Development of minor and reversible VZV is rather common event after high-dose chemotherapy without TBI and PBSCT. VZV appears to be one of the factors which reflects basic physiologic mechanism preventing relapse of leukemias or NHL after PBSCT.


[Inversion of chromosome 16 observed in acute myeloblastic leukemia (M2) with biphenotypic surface markers lacking monocytosis and eosinophilia]. Inversion of chromosome 16 was found in a 73-year-old female with acute myeloblastic leukemia (FAB:M2). Complete remission was achieved by combined chemotherapy (DNR, Ara-C, 6-MP, Prednisolone), but she relapsed 6 months later without CNS involvement and died of respiratory failure presumably due to cerebrovascular accident during remission reinduction chemotherapy. Biphenotypic surface markers (CD2+ and CD13+) were observed on relapse. Eosinophilia was not observed throughout. Our patient and the other reported case suggest that biphenotypism and the lack of eosinophilia and monocytosis in inv (16) leukemia may be correlated with a poor prognosis.


[Function and molecular structure of IL-12]. Natural killer cell stimulatory factor (NKSF), also called cytotoxic lymphocyte maturation factor (CLMF) or interleukin-12, is a novel cytokine of a 70 kDa heterodimer, composed of two covalently linked glycosylated chains of 40 and 35 kDa. NKSF was originally identified in the supernatant fluid of human EB virus-transformed B lymphoblastoid cell lines. NKSF has been shown to: (a) induce interferon-gamma production from both T and NK cells and synergize in this effect with interleukin-2 (IL-2), mitogens, phorbol diesters, anti-CD3 antibodies, and allogeneic antigens; (b) exert a comitogenic effect on fresh resting T cells together with lectins or phorbol diesters; (c) mediate a direct mitogenic effect on activated T or NK cell blasts; (d) enhance the cytotoxic activity of resting peripheral blood NK cells; and (e) synergize with IL-2 in the generation of lymphokine-activated killer cells.


Production of immunoreactive corticotropin-releasing hormone in various neuroendocrine tumors. The concentrations of immunoreactive (IR) corticotropin-releasing hormone (CRH) in 218 neuroendocrine tumors were determined by CRH radioimmunoassay. The tumors examined were 86 pancreatic endocrine tumors (PET), 22 neuroblastic tumors (NBT), 26 carcinoid tumors (CA), 24 pheochromocytomas (PHEO), 40 small cell lung carcinomas (SCLC) and 20 medullary thyroid carcinomas (MTC). IR-CRH was detectable in 21 neuroendocrine tumors (10 PET, four NBT, three CA, two PHEO and two SCLC) at levels of 10-2,700 ng/g wet weight (9.6%). The 21 patients with these CRH-producing tumors showed no clinical symptoms suggestive of Cushing's syndrome. The levels of plasma IR-CRH extracted by immunoaffinity chromatography were < 7.5 pg/ml in five normal subjects and a patient with a neuroblastic tumor containing 55 ng/g wet weight IR-CRH, but in a patient with a thymic carcinoid tumor containing 1,000 ng/g wet weight IR-CRH, the plasma level was elevated to 180 pg/ml. This patient did not have Cushing's syndrome nor an elevated plasma adrenocorticotropic hormone (ACTH) level. The concentrations of nine peptides (growth hormone-releasing hormone, somatostatin, ACTH, calcitonin, gastrin-releasing peptide, glucagon, vasoactive intestinal peptide, neuropeptide tyrosine and pancreatic polypeptide) were determined in extracts of the 21 IR-CRH-producing tumors. Some of these peptides were frequently found to be produced concomitantly with CRH. The results indicate IR-CRH to be produced by various neuroendocrine tumors, but Cushing's syndrome, due to the CRH, to be very rare. The results also show that CRH-producing tumors produce multiple hormones.


Marked increase of CD57+CD16- cells in long-term survivors of graft-versus-host disease after allogeneic bone marrow transplantation. We have evaluated long-term serial changes in the immunological state from soon after allogeneic bone marrow transplantation (BMT) In 44, mainly leukemia patients with respect to changes in lymphocyte surface markers. Absolute numbers of cluster designation (CD)2+, CD20+ and human lymphocyte antigen-DR+ (HLA-DR+) cells recovered to within their normal ranges three months, one year and two years, respectively, after BMT. The reversal of the CD4+: CD8+ ratio persisted for five years or more but returned to normal after six years. CD57+CD16- cells were markedly increased from three mo up to a maximum of five years after transplantation; they were increased between three and six months after transplantation irrespective of graft-versus-host disease (GVHD), but changes after one year or more differed among patients without GVHD, with acute GVHD, with acute and chronic GVHD or with chronic GVHD. Absolute numbers of CD57+CD16- cells tended gradually to return to normal after one year or more in the group without GVHD but only after six years in patients in the other three GVHD groups.


Prognostic factors for recurrent breast cancer: univariate and multivariate analyses including histologic grade and amplification of the c-erbB-2 proto-oncogene. Post-recurrence survival was examined in 62 breast cancer patients who had undergone curative radical mastectomies between 1974 and 1976 and suffered recurrences within 127 months of surgery. The prognostic value of 11 clinical, histological and genetic factors, including histologic grade of malignancy and amplification of oncogenes was analyzed using univariate and multivariate analyses. Not only the site of first recurrence, clinical stage and size of primary tumor at initial surgery, and disease-free period, but also histologic grade and amplification of the c-erbB-2 proto-oncogene were significant prognostic indicators of recurrent breast cancer. Multivariate analysis using Cox's regression model, histologic grade and amplification of c-erbB-2 in the primary tumor, as well as clinical stage at surgery and site of first recurrence, were shown to be major independent prognostic factors of recurrent breast cancer. Because post-recurrence prognosis was strongly influenced by the clinical, histological and genetic status of the primary breast cancer, appropriate evaluation of the primary tumor for the grade of aggressiveness of the cancer cells, as well as the extent of cancer spread, seem to be important.


Sympathetic vasoconstrictor tone induced by pentobarbital anesthesia in hindquarters of rats. Hindquarter (terminal aortic) blood flow (HQF) and arterial pressure (AP) were observed in rats with an electromagnetic flow probe implanted around the terminal aorta and an arterial indwelling cannula. Hindquarter peripheral resistance (HQR) was calculated by dividing mean AP by HQF. Under pentobarbital anesthesia, HQR was decreased significantly (p less than 0.001) by ganglionic blockade with hexamethonium bromide (C6). Since C6 does not change HQR significantly without anesthesia, we interpret that pentobarbital anesthesia generated a sympathetic vasoconstrictor tone in hindquarter resistance vessels. This was further substantiated by the observation that the increase in HQR on infusion of vasopressin was obscure under pentobarbital anesthesia: Presumably, the increase was offset by reflex inhibition of the hindquarter tone induced by anesthesia. The generation of hindquarter vasoconstrictor tone by pentobarbital was for the most part ascribable to the baroreceptor reflex to compensate for the depressor effect of this anesthetic, because it was greatly diminished after severance of the buffer nerves.


Conditioning stimulation of the central amygdaloid nucleus inhibits the jaw-opening reflex in the cat. To elucidate a function of the central amygdaloid nucleus (ACE) in the trigeminal system, the ACE conditioning effect on the jaw-opening reflex (JOR) regarded as a nociceptive reflex was investigated in the cat anesthetized with pentobarbital sodium. The JOR to molar tooth pulp stimulation with an intensity 1.2-1.5 times the threshold was recorded in the ipsilateral digastric muscle. As conditioning stimulation, a train of 33 rectangular pulses (0.5 ms in duration) at 330 Hz with an intensity of 300 microA was applied to the ipsilateral ACE. The conditioning stimulation inhibited a JOR that had a latency of 7.90 +/- 0.88 ms (n = 36). The inhibition was maximum (83.1 +/- 11.2%) at a conditioning-test (C-T) interval of 110 ms and continued for C-T intervals of up to 1,000 ms. Likewise, microinjection of 0.5 M monosodium glutamate (10 microliters) into the ACE-inhibited the JOR for approximately 10 min. Additionally, the ACE conditioning stimulation inhibited the JOR induced by the stimulation of the sensory trigeminal nuclear complex in a similar manner, but not the jaw-opening response induced by the stimulation of the trigeminal motor nucleus (Mo V). Also, the conditioning stimulation influenced neither the evoked potentials induced by the tooth pulp stimulation at the main sensory and rostral nuclei nor the jaw-closing reflex induced by the stimulation of the mesencephalic trigeminal nucleus (Mes V). These results suggest that the excitation of the cell bodies in the ACE exerts an inhibitory modulation on the JOR with no effect on the non-nociceptive reflex such as the jaw-closing reflex at the level of Mo V.


Effects of a new histamine H2-receptor antagonist, Z-300, on gastric secretion and gastro-duodenal lesions in rats: comparison with roxatidine. We examined the effects of a new compound, N-[3-[3-(piperidinomethyl)phenoxy]-propyl]-2-(2-hydroxyethyl-1- thio)acetamido.2-(4-hydroxy benzoyl)benzoate (Z-300), on the histamine H2-receptor, gastric secretion in rats and dogs, and acute gastro-duodenal lesions or chronic gastric ulcers in rats. Roxatidine acetate hydrochloride (roxatidine), a known histamine H2-receptor antagonist, was used as a reference compound. The pA2 values for Z-300 and roxatidine for the isolated guinea pig atrium were 6.8 and 7.0, respectively. These agents at less than 10(-5) M did not affect the contraction of guinea pig ileum in response to carbachol. Z-300, administered either orally or parenterally, significantly inhibited the basal and histamine-stimulated gastric acid secretion in rats. Gastric acid secretion stimulated by histamine, pentagastrin or carbachol in Heidenhain pouch dogs was also significantly inhibited by the compound. The effect persisted for greater than 7 hr in the case of histamine-stimulation. Oral Z-300 significantly protected the gastric mucosa from water-immersion stress-, indomethacin-, aspirin- and HCl.ethanol-induced lesions and protected the duodenal mucosa against mepirizole- and cysteamine-induced ulcers. These effects on gastric secretion and lesion formation were, as a whole, stronger than those observed with roxatidine. Z-300, but not roxatidine, significantly accelerated the spontaneous healing of acetic acid ulcers induced in rats and prevented the delay in ulcer healing caused by indomethacin. The mechanism of action of Z-300 on acute lesions and chronic ulcers appears to be mostly related to its potent antisecretory and mucosal-protective activities.


In vitro pharmacological profile of the novel alpha 1-adrenoceptor antagonist HSR-175. The pharmacological profile of HSR-175, a new alpha 1-adrenoceptor antagonist, was studied in vitro and compared with those of other alpha 1-antagonists. HSR-175, prazosin, bunazosin and yohimbine competitively antagonized the contractile responses induced by noradrenaline in the dog mesenteric arteries and the rabbit thoracic aorta. The pA2 values for HSR-175 in the dog mesenteric arteries and the rabbit aorta were 10.38 and 9.63, respectively, which were significantly higher than those for prazosin (8.39 and 8.80), bunazosin (8.44 and 8.75) and yohimbine (7.34 and 6.10). HSR-175 also inhibited the sympathetic adrenergic contraction induced by electrical transmural stimulation in the dog mesenteric arteries, and the inhibitory effect of HSR-175 was more potent than those of prazosin and bunazosin. Although HSR-175 also possessed competitive antagonist properties at pre- and postsynaptic alpha 2-adrenoceptors in the rat vas deferens and the dog saphenous veins, those affinities (pA2 = 6.41 and 7.05) were much lower than those at postsynaptic alpha 1-adrenoceptors. Furthermore, HSR-175 at concentration of 10(-6) M showed no inhibition on the contractile responses to 5-HT, histamine, KCl and angiotensin II in the rabbit thoracic aorta. These results indicate that HSR-175 is a very potent and selective alpha 1-adrenoceptor antagonist.


Characteristics of the association of brotizolam, a thieno-triazolo diazepine derivative, with the benzodiazepine receptor: a selective and high affinity ligand of the central type I benzodiazepine receptor. Characteristics of the association of brotizolam, a thieno-triazolo diazepine derivative, to central benzodiazepine receptors were examined. Brotizolam significantly displaced the [3H]flunitrazepam and [3H]beta-carboline carboxylate ethylester bindings to crude synaptic membrane from the rat brain. This agent had the highest affinity for benzodiazepine receptors in the cerebellum, and it was found to be 2.1 times that in the spinal cord. Furthermore, a low concentration of brotizolam potentiated the GABA-stimulated 36Cl- influx into membrane vesicles. In contrast, the bindings of [3H]8-hydroxy-2-(di-n-propylamino)tetralin to 5-hydroxytryptamine1A receptors and [3H]ketanserin to 5-hydroxytryptamine2 receptors were not affected by brotizolam. The present results suggest that brotizolam may be a selective and high affinity ligand for the type I central benzodiazepine receptor. The anxiolytic and hypnotic actions of brotizolam seem to be not due to the association with 5-hydroxytryptamine receptor, but due to the activation of the GABAA receptor complex. Furthermore, the present results suggest that the lower affinity of brotizolam to benzodiazepine receptors in the spinal cord than those in the cerebellum may be related to the low muscle relaxation action of this drug.


Tryptophan inhibits the [3H]glutamate uptake into Xenopus oocytes injected with rat brain mRNA. We characterized the glutamate (Glu) uptake in Xenopus oocytes injected with rat brain mRNA. The Glu uptake into oocytes was higher in mRNA-injected oocytes than in vehicle-injected ones. Na+ omission or addition of tryptophan inhibited the uptake in mRNA-injected oocytes, although it did not affect that in vehicle-injected oocytes. These results suggest that Glu transporters with a tryptophan sensitivity different from that of Glu transporters in native oocytes are expressed after injection of rat brain mRNA.


[Bronchial hyperresponsiveness to acetylcholine during acute pulmonary congestion in guinea pigs]. To understand the precise mechanism of bronchial hyperresponsiveness in patients with congestive heart failure, we studied the effect of mild pulmonary congestion on bronchial responsiveness to inhaled acetylcholine (ACh) in guinea pigs. We induced mild pulmonary congestion by inflation of a balloon placed in the left atrium, and maintained the left atrial pressure (Pla) at 10 mmHg for 30 minutes with continuous monitoring of lung resistance (RL) and dynamic compliance (Cdyn). Furthermore, we determined the provocative concentration of ACh producing 100% increase in RL (PC100-ACh), before and during balloon inflation. In animals with propranolol pretreatment, but not in animals without propranolol pretreatment, mild pulmonary congestion caused slight increase in RL (N.S.) and significant decrease in Cdyn (p less than 0.01) and PC 100-ACh (p less than 0.01). Cutting of bilateral vagal nerves partially inhibited the decrease of PC100-ACh, but pretreatment with either phenoxybenzamine, indomethacin, AA-861 or OKY-046 had not effect. These results suggest that blockade of beta-adrenergic receptors and the vagal reflex, but not of alpha-adrenergic receptors or arachidonates, contributes to bronchial hyperresponsiveness during acute pulmonary congestion.


Characterization of primary human fetal dissociated central nervous system cultures with an emphasis on microglia. We undertook a systematic examination of human fetal central nervous system dissociated cell cultures, particularly with respect to microglia and their dynamic interactions with other central nervous system cell components. The growth and differentiation of astrocytes, microglia, and small immature neuronal cells in mixed and single cell type-enriched cultures were followed by phase contrast microscopy, immunocytochemistry, flow cytometry, electron microscopy and immunoelectron microscopy, in regard to their phenotype change, proliferation and class II major histocompatibility complex antigen expression. Both astrocytes and microglia expressed marked phenotype heterogeneity, but they were consistently positive for glial fibrillary acidic protein and CD68, respectively. Highly enriched astrocyte and microglial cultures suitable for biochemical or molecular biologic studies were obtained. Using double immunocytochemical labeling techniques with the proliferating cell nuclear antigen and cell-type specific markers, astrocytes grown in serum-containing media showed a high labeling index, whereas microglia seldom exhibited a similar degree of proliferative activity. Microglia, however, proliferated in response to certain growth factors, such as granulocyte macrophage colony-stimulating factor. Both microglia and astrocytes expressed high basal levels of class II MHC antigens, which further increased with addition of interferon-gamma. The microglia in dissociated cultures existed in two forms, ameboid and ramified. Microglial ramification was induced when ameboid microglia were co-cultured with a monolayer of astrocytes, suggesting a role for astrocytes in microglial differentiation. In addition, lipopolysaccharide in nanogram amounts consistently enhanced microglial survival and morphologic differentiation. The small bipolar cells, the most frequent cell type in mixed culture, were glial fibrillary acidic protein (-) and CD68 (-), and their size and shape remained unchanged under our culture conditions. A subpopulation of small bipolar cells was stained positive with an antibody to surface ganglioside, A2B5. Electron microscopic examination showed that their processes contained parallel microtubules bearing side arms consistent with immature neurons. Highly purified astrocyte and microglial cultures are obtained using the currently described technique. The current culture system is a valuable tool in studying human central nervous system biology and disease.


Mechanisms of somatostatin action in RINm5F cells in culture: preliminary evidence for possible altered G protein function. Octreotide (SMS), a somatostatin analogue, is an established antigrowth peptide, but it does not effectively inhibit the growth of insulinoma cells. In order to study the mechanisms that underlie this apparent lack of an antiproliferative effect on insulinoma tumor cells we established the rat insulinoma cell line, RINm5F, in culture. Cells in culture were tested by incubation in media with and without SMS. To study tritiated [3H]-thymidine incorporation into extracted DNA (TTID), 2 muCi/well of 3H was added for 24 hr, and cells were harvested and assayed for TTID (cpm/microgram DNA). Insulin (IRI) and intracellular cAMP (cAMPi) were measured by RIA. To study the effects of SMS on insulin secretion, conditioned media were sampled after 24 hr. To study the effects of cAMPi, conditioned medium was used to extract cAMPi following incubation with SMS for 15 min. Increasing concentrations of SMS had no significant effect on TTID in the presence of 1% FBS. Trypan blue exclusion tests showed > 90% viable cells throughout all stages of these experiments. There were no significant differences in cell numbers and protein content in the presence of SMS. There was a significant decrease in the secretion of insulin and intracellular cAMP levels in response to 50 nM SMS. However, SMS significantly inhibited TTID in RINm5F cells following a 4-hr pretreatment with pertussis toxin (PT) (23553 +/- 1747 vs 20635 [cpm/microgram DNA] +/- 1983 [SEM], P < 0.01). We conclude that the inhibition of insulin secretion by SMS is associated with an attenuation of cAMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)


Proliferative activity in gastric cancer determined with cell cycle-related monoclonal antibodies Ki-67 and p105: analysis by flow cytometry. The proliferative activity of gastric cancer cells was determined by DNA flow cytometric (FCM) analysis and labeling rates of Ki-67 and monoclonal antibodies and proliferation-associated nuclear antigen (p105) autoantibodies in 28 patients with fresh human gastric cancer cells. By setting the cutoff line at the level as used in a negative control study without primary antibody in the same sample, the Ki-67 and p105 labeling rates were calculated by the dual fluorescence analysis. A total of 43 experiments was performed on FCM analysis for each antigen: 28 with Ki-67 and 15 with p105. The mean Ki-67 labeling rate of gastric cancer cells was 45.1% (13.9-76.3%). The Ki-67 labeling rates were significantly higher for larger size tumor, peritoneal metastasis, and advanced clinical stage. A significant correlation was found between Ki-67 labeling rate and p105 labeling rate (P < 0.05). Bivariate FCM may be an easy method for obtaining useful information of cell kinetics.


Structural alterations of the p53 gene in human leukemias. We have investigated alterations of the p53 gene in human leukemias by polymerase chain reaction-mediated restriction fragment length polymorphism analysis. This method detects the codon 72 polymorphism of the p53 gene, allowing identification of loss of heterozygosity (LOH) of the p53 gene. In this study, at least two specimens were obtained from each patient to compare the allele status at different points of clinical course. Of 21 cases examined 7 were heterozygous at this polymorphic site and were evaluable for LOH study. Only one patient of Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL) lost the heterozygosity in the specimen of her last relapse. Leukemic cells from her repeated relapses including the last one revealed to have the clonally rearranged immunoglobulin H chain gene of the same size, indicating that alteration of the p53 gene in this patient might account for the lethal relapse as a clonal evolution event. Northern-blot analysis of the p53 gene showed that one case of CD7(+) ALL had altered p53 mRNA. Overall, it is demonstrated that alterations of the p53 gene might be involved in the pathogenesis or progression of at least some human leukemias, although the alterations in leukemias seemed to be not as frequent as in solid tumors.


In vitro electroreceptor organs for pharmacological studies. Electroreception is a well-established sensory faculty in aquatic vertebrates. The general physiology of the receptor organs is comprehensively documented. The transduction mechanism of the receptor cells and the synaptic transmission mechanism are less well understood. Research has been hampered by the inaccessibility of the synaptic site. This paper describes how to prepare an in vitro preparation of ampullary electroreceptor organs which allows exposure of both the mucosal and the serosal sides of the receptor cells to superfusion of test solutions. The preparation is quite robust and has been shown to function reliably for more than 8 h. Furthermore, the use of in vitro electroreceptors organs as a model for pharmacological studies is evaluated.


In situ hybridization of whole-mounts of Aplysia ganglia using non-radioactive probes. A protocol for carrying out in situ hybridization with non-radioactive, digoxigenin-labelled probes has been developed for whole-mounts of Aplysia ganglia. Whole-mount preparations preserve the anatomical relationships of neurons within intact ganglia and facilitate the precise identification of a particular neuron in live preparations so that functional studies can be correlated with biochemical attributes of an identified neuron. The protocol was developed with the use of probes to messenger RNAs that are abundant in Aplysia neurons. In situ hybridization with a cDNA probe to a neuronal form of beta-actin stained all neurons, including their processes, whereas use of a cDNA probe for the neuropeptide FMRFamide resulted in staining of a select group of Aplysia neurons.


Intensive conventional chemotherapy can lead to a precocious overshoot of cytogenetically normal blood stem cells (BSC) in chronic myeloid leukemia and acute lymphoblastic leukemia. Forty patients with Ph-positive blastic phase (BP) (28 patients) or chronic phase (CP)-CML (3 patients) and relapsed adult acute lymphoblastic leukemia (ALL) (9 patients) with cytogenetical translocations [t(8;14):2 patients; t(4;8):2 patients; t(4;11):3 patients; t(9;22):2 patients], received an intensive conventional chemotherapy. During early recovery from marrow aplasia, when WBC reached 0.3-1.5 x 10--9/L, peripheral blood stem cells (BSC) were collected by 4-8 leukapheresis consecutively. BSC collected from the 2/3 patients with CP-CML resulted Ph-negative and PCR negative. In 8 out of 26 BP-CML patients, BSC resulted Ph-negative and in two cases PCR negative. Of the nine ALL patients, 6 patients lost the cytogenetic translocations, one patient died during aplasia, two patients did not have cytogenetic modifications and died in few weeks of leukemia and one patient out of six responding patients relapsed before transplant. After complete recovery, 15 patients (BP-CML:8 patients; CP-CML:2 patients; ALL:5 patients) were subsequently given high-dose therapy (VP-16 +/- Cy+TBI in single dose) followed by reinfusion of "normal" BSC. Both the patients in CP-CML and 5/5 patients with ALL maintain clinical and cytogenetic remission; all the patients transplanted in BP-CML relapsed 5-18 months post-transplant. It is concluded that intensive conventional chemotherapy employed in CML and ALL can lead to a precocious overshoot of cytogenetically normal BSC.


Biological markers and minimal residual disease in hairy cell leukemia. Soluble Interleukin-2 Receptor (sIl-2R) and Tumor Necrosis Factor-alpha (TNF-alpha) have been found significantly increased in serum samples of patients with HCL at diagnosis and a strict correlation with leukemic burden has been reported. Furthermore, following therapy, serological monitoring of these cytokines may be considered a useful tool for controlling therapeutic efficacy and for detection of minimal residual disease. Eighteen HCL patients, treated with 2-Chlorodeoxyadenosine (2-CdA) at a dose of 0.1 mg/kg daily for 7 days, entered the study all of them showing increased levels of sIL-2R and TNF-alpha prior to therapy. After therapy, serum levels were reassessed and a remarkable decrease was recorded in all cases. In particular, after 1 month by the end of treatment sIL-2R and TNF-alpha decreased from 3,377 +/- 2,303 to 149 +/- 96 pM/ml (p = 0.00003) and from 38 +/- 41 to 18 +/- 18 pg/ml (p = 0.015) respectively. The only 3 patients who did not normalize sIL-2R and TNF-alpha levels showed also an evident persistence of the disease in the marrow. In conclusion, 2-CdA leads to a rapid normalization of the increased levels of sIL-2R and TNF-alpha in the majority of HCL patients. Furthermore, monitoring of these cytokines represents a useful tool for detecting minimal residual disease.


Myeloablative regimens in peripheral stem cell transplantation. There is considerable opportunity for research in identifying the relative merits of various preparatory regimens utilized for high dose therapy in autologous peripheral stem cell transplantation. These include studies of the dose level of a particular regimen (e.g., CBV). There is some suggestion in the literature that a dose response effect exists even at high doses. Studies of the relative merits of different drugs such as cyclophosphamide, melphalan, and hydroxyurea that have all been added to the CBV regimen need to be carried out. The contribution of TBI also remains uncertain. However, these studies require prospective, randomized trials that will probably have to be performed on a multi-institutional basis. It is also possible that future uses of hematopoietic growth factors will significantly modify concerns about safety with any regimen.


Medical therapy for ocular allergy.

Medical therapy for ocular allergy. The ocular manifestations of allergy have traditionally been classified into four categories--namely, hay fever conjunctivitis, vernal keratoconjunctivitis, atopic keratoconjunctivitis, and contact lens-associated giant papillary conjunctivitis. Typically, hay fever conjunctivitis is characterized by mild conjunctival inflammation, whereas the other disorders may have severe inflammation and clinical manifestations. Potentially blinding corneal complications may result from vernal keratoconjunctivitis and atopic keratoconjunctivitis. Although hay fever conjunctivitis is clearly an immediate hypersensitivity reaction, the immunologic mechanisms that cause vernal keratoconjunctivitis, atopic keratoconjunctivitis, and giant papillary conjunctivitis are primarily unknown and speculative. Treatment of patients with ocular allergies is often challenging and may necessitate collaborative efforts of an ophthalmologist and an allergist. Herein we discuss conventional therapy and new, promising antiallergy drugs.


Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Endothelial dysfunction is an early event in experimental studies of atherogenesis, preceding formation of plaques. We have devised a non-invasive method for testing endothelial function, to find out whether abnormalities are present in symptom-free children and young adults at high risk of atherosclerosis. With high-resolution ultrasound, we measured the diameter of the superficial femoral and brachial arteries at rest, during reactive hyperaemia (with increased flow causing endothelium-dependent dilatation), and after sublingual glyceryl trinitrate (GTN; causing endothelium-independent dilatation) in 100 subjects--50 controls without vascular risk factors (aged 8-57 years), 20 cigarette smokers (aged 17-62 years), 10 children with familial hypercholesterolaemia (FH; aged 8-16 years), and 20 patients with established coronary artery disease (CAD). Adequate scans were obtained in all but 6 cases. Flow-mediated dilatation was observed in arteries from all control subjects. Dilatation was inversely related to baseline vessel diameter (r = -0.81, p < 0.0001); in arteries of 6.0 mm or less, mean dilatation was 10 (SE 2)%. In smokers, FH children, and adults with CAD, flow-mediated dilatation was much reduced or absent (p < 0.001 for comparison with each relevant control group). Dilatation in response to GTN was present in all groups. Endothelial dysfunction is present in children and adults with risk factors for atherosclerosis, such as smoking and hypercholesterolaemia, before anatomical evidence of plaque formation in the arteries studied. This may be an important early event in atherogenesis.


Randomised controlled trial of laparoscopic versus mini cholecystectomy. The McGill Gallstone Treatment Group. Laparoscopic cholecystectomy (LC) has gained wide acceptance for treatment of cholelithiasis in preference to open cholecystectomy, though it has not been formally compared with mini cholecystectomy (MC). We have compared these two techniques in a randomised trial. 70 patients with ultrasound-proven cholelithiasis were randomly allocated LC (38) or MC (32); 37 and 25, respectively, underwent the assigned procedure. The mean hospital stay (including 1 preoperative day) was significantly shorter in the LC than the MC group (median 3 [interquartile range 2-3] vs 4 [3-5], p = 0.001) as was duration of convalescence (mean 11.9 [SD 9.1] vs 20.2 [16.5] days, p = 0.04). The rate of return to normal activities was 1.77 times greater in the LC group than in the MC group (95% confidence interval 1.01-3.11, p = 0.03). In regression analysis, the type of cholecystectomy done was the only variable significantly associated with the duration of convalescence. Although there was significant postoperative improvement in all of three quality of life scores in both groups, LC patients improved more quickly than did MC patients. This randomised trial shows the superior effectiveness of LC over MC in treating cholelithiasis.


Effect of vitamin and trace-element supplementation on immune responses and infection in elderly subjects. Ageing is associated with impaired immune responses and increased infection-related morbidity. This study assessed the effect of physiological amounts of vitamins and trace elements on immunocompetence and occurrence of infection-related illness. 96 independently living, healthy elderly individuals were randomly assigned to receive nutrient supplementation or placebo. Nutrient status and immunological variables were assessed at baseline and at 12 months, and the frequency of illness due to infection was ascertained. Subjects in the supplement group had higher numbers of certain T-cell subsets and natural killer cells, enhanced proliferation response to mitogen, increased interleukin-2 production, and higher antibody response and natural killer cell activity. These subjects were less likely than those in the placebo group to have illness due to infections (mean [SD] 23 [5] vs 48 [7] days per year, p = 0.002). Supplementation with a modest physiological amount of micronutrients improves immunity and decreases the risk of infection in old age.


Adult coronary artery disease probably due to childhood Kawasaki disease. We have surveyed adult survivors of childhood Kawasaki disease (KD) who had coronary artery disease that could be ascribed to KD. In response to questionnaires sent to cardiologists throughout Japan, 21 patients (17 men, 4 women, aged 20-63 years) with coronary lesions and a definite (2) or suspected (19) history of KD were reported. 5 patients had presented with acute myocardial infarction, 6 previous myocardial infarction, 9 angina pectoris, and 1 dilated cardiomyopathy. 16 patients had obstructions in two or more coronary arteries. 3 had died and 18 were alive with serious sequelae (mitral regurgitation, arrhythmias, congestive heart failure). Childhood KD should be included in the differential diagnosis of coronary artery disease in young adults.


Electrically stimulated gracilis sphincter for treatment of bladder sphincter incontinence. Correction of total urinary incontinence due to sphincter damage is done with an artificial sphincter prosthesis or urinary diversion. In this pilot study we used graciloplasty around the bladder neck followed by electrical stimulation of this muscle with an implanted stimulator, which could be switched off and on by a magnet. Stimulus variables could be changed externally. With the stimulator on, urethral pressures of about 50 cm H2O were obtained. Of three patients who underwent the procedure, two became continent and one improved but remained incontinent. Dynamic graciloplasty can restore urinary incontinence.


Usefulness of clinical case-definitions in guiding therapy for African children with malaria or pneumonia. The World Health Organisation has developed disease-specific clinical case-definitions to guide management of children with fever or cough, the cardinal signs of malaria and pneumonia. To assess the usefulness of the case-definitions and to investigate their interaction, we studied children with fever or cough brought to a hospital in Lilongwe, Malawi. For all children, a thick blood smear was examined for Plasmodium falciparum parasites. Chest radiography was done only for children with parasitaemia and those who satisfied the clinical case-definition for pneumonia; others were assumed to have normal chest radiographs. Of 1599 enrolled children, 566 (35%) had parasitaemia and 116 had radiographic evidence of pneumonia; 43 had both pneumonia and parasitaemia. Of the 471 children who met the clinical definition for pneumonia, 449 (95%) also met the malaria clinical definition. Among children with radiographic evidence of pneumonia, the clinical definition for malaria was not predictive of parasitaemia (sensitivity 93%, specificity 5%). Whether malaria parasitaemia was present or absent, the pneumonia clinical definition distinguished children with and without radiographic evidence of pneumonia (sensitivity and specificity > 60%). Children who satisfied the pneumonia clinical definition were more likely to have radiographic evidence of pneumonia (odds ratio 10.4, 95% confidence interval 5.2-20.7), parasitaemia (1.6, 1.2-2.2), or both at the same time (4.2, 2.1-8.4) than were children who did not meet the definition. Children who satisfy the malaria and pneumonia clinical definitions need treatment for both disorders. Scarce diagnostic methods, especially microscopy, are needed for more specific treatment of children with fever and cough.