Analysis of CA/GT microsatellite polymorphism in IVS8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene: a study of Italian CF families.

Forty-six CF Italian patients and their parents were screened for a highly polymorphic microsatellite consisting of a variable number of CA/GT repeats in intron 8 of the CFTR gene. A strong degree of association was found between alleles 2 and 6 and the CF mutation delta F508. Moreover, considering the haplotypes at the closely linked locus D7S23 and the microsatellite's alleles, a strong linkage disequilibrium was again found for delta F508 and also for non-delta F508 CF chromosomes and the eight commonest haplotypes (B2, B6, C7, A6, A7, B7, D2 and D7). These data, compared with those described in the Spanish population, further support the common origin of the delta F508 mutation in Southern European populations.


Quantification of the in vitro activity of some compounds with spermicidal activity.

The in vitro spermicidal activity of the commonly used surfactant spermicides and the antiseptic chlorhexidine, were quantified in a statistically reproducible manner, using donor semen and image capture analysis. The spermicidal activity was expressed as the ED50 under defined assay conditions. Using these parameters, the order of spermicidal activity was: Menfegol > nonoxynol-9 approximately benzalkonium chloride > sodium docusate > chlorhexidine. These differences were statistically significant.


A comparative study of allergenic and potentially allergenic enzymes from Dermatophagoides pteronyssinus, D. farinae and Euroglyphus maynei.

The presence of the enzymatically active allergens equivalent to Der p I (cysteine protease), Der p III (serine protease) and amylase in extracts of Dermatophagoides pteronyssinus, D. farinae and Euroglyphus maynei was determined using appropriate enzymatic techniques. Biochemical equivalents of all three allergens were present in each extract studied. Studies also showed that the mite extracts contained a variety of other biochemically active enzymes including trypsin, chymotrypsin, carboxypeptidase A and B, glucoamylase and lysozyme. Marked differences in the relative concentrations of some of these enzymes in different mite extracts were observed, particularly trypsin and carboxypeptidase A. The enzymes were physicochemically similar to equivalent enzymes from vertebrate and invertebrate sources. Chromatofocusing studies of faecal extracts derived from D. pteronyssinus and D. farinae showed that several isoforms of each enzyme were present. The data indicated that there were more trypsin isoforms, with pI over a wider range, in extracts prepared from D. pteronyssinus. Proteases and carbohydrases were also found in extracts prepared from faecally enriched material suggesting that they were endoperitrophic and associated with mite digestion. The data suggest that not only are the group I, III and amylase allergens a consistent feature of most pyroglyphid dust mites but also that other proteases and carbohydrases present in mite faeces are allergenic.


Comparative efficacy, safety and pharmacokinetics of verapamil SR vs verapamil IR in hypertensive patients.

The antihypertensive effects of the regular immediate release formulation of verapamil (verapamil IR) and the newer sustained release formulation of verapamil (verapamil SR) were compared in Hispanic patients with untreated essential hypertension. Verapamil IR was given in 3 divided doses (80 or 160mg 3 times daily) and verapamil SR was given either as a single daily dose of 240mg or as 240mg every 12 hours. With both formulations there was a significant reduction in systolic (SBP) and diastolic blood pressure (DBP); a greater lowering of BP was observed with verapamil 480 mg/day than with 240 mg/day. With verapamil SR 480 mg/day, 91% of patients had reductions in SBP and DBP greater than 20 and 15mm Hg, respectively. In addition, 83% of patients reached normotension. With the lower dose (240mg once daily), 83% of patients had decreases in DBP greater than 10mm Hg and 73% of patients achieved normotension. Comparable effects were achieved with verapamil IR. With verapamil IR there was a more rapid fall in BP which peaked 3 to 4 hours postdose, whereas with verapamil SR a more gradual and sustained BP reduction was observed. Only small changes in heart rate (HR) were observed with verapamil IR and verapamil SR. For verapamil SR, the mean increase in HR was 5 beats/min (to 80 beats/min) and the mean decrease in HR was 13 beats/min (to 62 beats/min). Both verapamil SR and verapamil IR prolonged the PR interval of the ECG. An equal degree of PR prolongation was observed with 240 and 480 mg/day. The incidence of side effects (headache, palpitations, dizziness and flushing) was dose dependent, decreased with continuous treatment and was much higher with verapamil IR than with verapamil SR. Steady-state plasma verapamil concentrations were monitored. Compared with verapamil IR, verapamil SR produced a more gradual rise and a more sustained elevation of plasma verapamil and norverapamil concentrations. Comparable trough verapamil concentrations (Cmin) were observed with verapamil IR (98 micrograms/L) and SR (81 micrograms/L); morning Cmin verapamil concentrations were higher than daytime Cmin values. The normalised area under the plasma concentration-time curve (AUC) and maximum concentration (Cmax) were 10 to 20% greater for verapamil IR than SR. The 2-fold increase in oral dose produced a 2.2- and 2.4-fold increase in AUC for verapamil IR and SR, respectively, associated with a 20% reduction in metabolism to norverapamil. Fasting increased the rate and extent of absorption of verapamil.(ABSTRACT TRUNCATED AT 400 WORDS)


Use of cell culture for optimisation of direct antiatherogenic therapy with verapamil.

The potential of verapamil to prevent atherogenesis induced by atherogenic serum in cell culture was investigated. Smooth muscle cells were cultured from human aortic intima and incubated with blood serum from patients with coronary artery disease. Only serum causing a significant rise in total cholesterol content in cultured cells during a 24-hour incubation period was used. The addition of verapamil to cells decreased serum-induced cholesterol accumulation. The maximum antiatherogenic effect of verapamil in vitro was recorded at 10(-5) to 10(-4) mol/L. Blood serum obtained from patients before and after oral verapamil administration was added to cultured cells. The atherogenic potential of serum obtained after verapamil administration was significantly lower than the predose value. To optimise direct antiatherogenic therapy with verapamil, the minimum dose that produced the maximum effect was established as 30 to 40mg. Using a 40mg dose, the maximum effect of oral verapamil was observed 3 hours after administration. A second 40mg dose was given 5 hours after the first dose, when the blood serum atherogenic potential was about 40% of the predose value and rising. This second dose maintained the atherogenic potential of serum at about 40%. Thus, to decrease atherogenic potential of serum and to maintain it at a low level, verapamil should be administered at a dose of 40mg 5 times daily with a 4- to 5-hour interval between doses.


Vascular calcium overload. Physiological and pharmacological consequences.

Pronounced vascular calcium overload, especially of compliance arteries, is an important aspect of aging, and of various age-related vascular diseases such as hypertension and atherosclerosis. The development of new drugs specifically aimed at attenuating the evolution and/or pathological consequences of vascular calcium overload is hindered by the paucity of animal models available. Although several laboratory animals exhibit vascular calcium overload, this evolves slowly and is far less marked than in man. Nevertheless, Fleckenstein and associates (1989) suggested a suitable animal model involving the treatment of young rats with vitamin D3 and nicotine; such treatment produces pronounced vascular calcium overload within a few weeks. This paper describes the cardiovascular effects of such treatment, and our preliminary attempts to pharmacologically modify vascular calcium overload and its cardiovascular consequences.


Arterial calcinosis, chronic renal failure and calcium antagonism.

A progressive rise in arterial calcium content is the most characteristic age-associated alteration in the arterial wall and the decisive factor in arteriosclerotic degeneration. Experimental studies have demonstrated that calcium antagonists can prevent or retard the development of arterial calcinosis associated with vitamin D overload, hypertension or alloxan-induced diabetes. Although similar effects are more difficult to observe in humans, they have been demonstrated in patients with coronary artery disease and in patients with end-stage renal disease, which is characterised by an acceleration of the normal arterial aging process.


Metabolism of a new HIV-1 reverse transcriptase inhibitor, 3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-methylpyridin-2(1H)-one (L-696,229), in rat and liver slices.

L-696,229 is a potent and specific inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and is currently undergoing clinical evaluation. In vivo metabolism in rats was investigated using an intravenous bolus dose of 5 mg/kg [3H]L-696,229. The amount of radioactivity eliminated in bile and urine over a period of 6 hr was 60 and 22%, respectively. Radiochromatographic analysis of the bile and urine showed that L-696,229 was metabolized rapidly and completely to several common metabolites. Sequential oxidation at the alpha-position of the 5-ethyl group to an acetyl moiety, aromatic hydroxylation of the benzoxazole group (position C4', C6', or C7'), and subsequent sulfate conjugation were the major metabolic pathways as determined by the application of enzymatic hydrolysis, FAB-MS, and 1H- and 13C-NMR spectroscopies. The in vitro metabolism of this 2-pyridinone derivative with rat liver slices resulted primarily in hydroxylation at the 6-methyl and 5-ethyl groups. The 6-hydroxymethyl- and 5-alpha-hydroxyethyl analogs were also inhibitors of HIV-1 reverse transcriptase.


Use of ambulatory blood pressure monitoring in the management of antihypertensive therapy.

The recent development of ambulatory blood pressure (ABP) monitoring techniques has improved recording of blood pressure in therapeutic trials and in the clinical setting. The application of ABP differs according to which of these 2 applications is being considered. In therapeutic trials, a placebo control is required. The large quantity of precise data acquired with ABP monitoring allows the study of a limited number of patients; it also allows individual study of patients with a 'white coat' response (i.e. elevated blood pressure in response to examination by the clinician). Analysis of data from ABP monitoring may include the following: comparison of mean blood pressure values over 24 hours, daytime or night-time, or over any other selected time period; 24-hour blood pressure profiles, or analysis hour-by-hour, giving true chronotherapy, and providing data regarding the wearing-off of a drug effect or loss of therapeutic control; analysis of blood pressure at particular times, such as on waking; or specific examination of nonresponders. In individual patients, ABP monitoring should be reserved for specific indications. It can be used before initiation of treatment to confirm the necessity for treatment, especially in the context of hypertension at rest or the 'white coat' effect. With established treatment, ABP monitoring can be used in patients with resistant hypertension, in severe hypertension to examine loss of blood pressure control over time or inversion of the day/night cycle, and in patients with a specific illness, e.g. diabetes, in order to obtain the lowest blood pressure readings possible. Examination of these factors assists clinicians to accurately decide upon the timing and frequency of antihypertensive therapy.


Can the coronary atherosclerotic process be influenced by calcium antagonists?

Cell culture experiments and various animal models have shown that calcium antagonists can inhibit atheroma development. A number of antiatherosclerotic mechanisms have been proposed and the promising results of these in vitro and animal experiments prompted clinical trials. Retrospective analyses indicated that verapamil might retard disease progression and the development of new stenoses. Prospective clinical studies, however, have revealed no effect by calcium antagonists on pre-existing coronary stenoses (> or = 20%) and it is suggested that the study duration (2 to 3 years) may have been too short and that advanced stages of atheromatous disease are not influenced by calcium antagonists. Current data suggest that only early stages of coronary atheromatosis may be affected by treatment with calcium antagonists; however, optimum drug, dosage and suitable patients are yet to be defined.


Preliminary clinical experience with calcium antagonists in atherosclerosis. Verapamil in Hypertension Atherosclerosis Study Investigators.

Animal experiments have shown that the administration of calcium antagonists can prevent or slow the progression of atherosclerosis by inhibiting calcium overload and interfering with lipid metabolism and deposition. These encouraging results have prompted clinical trials to evaluate the effects of calcium antagonists (dihydropyridines and diphenylalkylamines) on atherosclerotic plaque formation. In patients with coronary heart disease, several studies have already shown that calcium antagonists can have a positive effect on plaque evolution, while in hypertensive patients no such study has been published to date. The Verapamil in Hypertension Atherosclerosis Study is an ongoing multicentre randomised double-blind parallel group trial comparing the antihypertensive efficacy of verapamil SR 240 mg/day with that of chlorthalidone 25 mg/day in 1464 patients with essential hypertension aged 40 to 65 years. In a randomised subgroup of patients (n = 550), who will be followed up for 3 years, B-mode ultrasonography is being employed to evaluate the effects of the 2 drugs on carotid wall thickness and carotid plaque development. Ultrasonographic evaluations are performed at baseline, after 3 months, and 1, 2 and 3 years after a standardised protocol to determine intimal-medial thickness in 4 segments of the extracranial carotid tree. The most interesting result to date is the high incidence of carotid alterations, with plaques present in 35% and arterial wall thickening in 31.8% of the 311 asymptomatic hypertensive patients processed so far. A preliminary evaluation of the antihypertensive efficacy of the trial medications after 6 months of double-blind treatment indicates a 63.5% response rate to monotherapy and a 7.8% drop-out rate because of drug inefficacy or intolerance.


Quality of life in the treatment of hypertension. The effect of calcium antagonists.

The effects of calcium antagonists on psychological well-being, cognitive function, activity and physical symptoms in hypertensive patients are reviewed. Effects on these aspects of quality of life appear to differ according to whether a dihydropyridine calcium antagonist such as nifedipine is employed or verapamil, which is a phenylalkylamine derivative. Nifedipine has been associated with a self-assessment of impaired cognitive function in 2 clinical trials. Nifedipine was also associated with more symptomatic complaints than both atenolol and verapamil in different studies. The problems with nifedipine centred on oedema, flushing and palpitations. Verapamil was associated with constipation. Compared with other classes of antihypertensive drugs, the position of calcium antagonists with respect to the maintenance of patients' quality of life is presently unclear. Verapamil has been associated with improved quality of life compared with propranolol (a beta-blocker) and nifedipine. Verapamil appears to have similar effects on quality of life as atenolol and the angiotensin converting enzyme (ACE) inhibitor, captopril. The position of nifedipine remains unclear.


Does a reduction in left ventricular hypertrophy reduce cardiovascular morbidity and mortality?

Left ventricular hypertrophy is an important risk factor for sudden death and other cardiovascular morbidity and mortality irrespective of the level of arterial blood pressure. Left ventricular hypertrophy, i.e. an increase in wall thickness at the expense of left ventricular volume, is an adaptive mechanism observed in patients with long standing arterial hypertension. Severe left ventricular hypertrophy is associated with a reduction in left ventricular compliance, impaired coronary reserve, ventricular ectopy, and impaired contractile function. Left ventricular hypertrophy can be reduced by antihypertensive therapy; however, not all antihypertensive agents have the same effect on left ventricular hypertrophy despite their similar effects on arterial blood pressure. Angiotensin converting enzyme (ACE) inhibitors appear to be the most powerful agents for reducing left ventricular hypertrophy, followed by the nondihydropyridine calcium antagonists. In addition to reducing left ventricular mass and arterial blood pressure, certain calcium antagonists also improve left ventricular filling, suppress ventricular ectopy, and maintain or enhance contractile function. However, despite these beneficial effects, it is not known whether the risk of cardiovascular morbidity and mortality can be prevented or reduced by specific antihypertensive agents.


Choosing the correct drug for the individual hypertensive patient.

With the availability of a wide selection of antihypertensive drugs acting by different mechanisms, it should be possible to match the requirement of individual patients with the pharmacological and clinical properties of an appropriate agent. Although the concept of stepped-care therapy is now largely outdated, therapy must be initiated with one agent. Diuretics remain a first-choice option in the elderly and in Black patients, as do calcium antagonists. In patients with ischaemic heart disease or enhanced adrenergic drive, beta-blockers are preferred. Calcium antagonists or ACE inhibitors are finding increasing use as initial therapy when quality of life is important and metabolic neutrality is required. The choice of antihypertensive agent may be limited by adverse effects, e.g. pedal oedema with nifedipine, constipation with verapamil, and cough with ACE inhibitors. Certain advantages are evident for both calcium antagonists and ACE inhibitors. Calcium antagonists are more likely to be effective first-line therapy than ACE inhibitors in Black patients, in those with a high salt intake, in patients with Raynaud's disease, and when angina pectoris is present. ACE inhibitors are preferred for use in combination with diuretic agents, and in the presence of congestive heart failure or low salt intake. Combination therapy between these 2 drug classes is finding increasing acceptance because of its many theoretical advantages, and may provide a means of maximising benefit.


Ambulatory blood pressure monitoring.

This paper reviews the evidence that, in patients with hypertension, end-organ damage correlates more closely with blood pressure values obtained by ambulatory blood pressure monitoring than with those obtained by conventional sphygmomanometry. However, ambulatory blood pressure monitoring is not suitable for routine use in the clinical setting because of a lack of standard reference values and data regarding the prognostic significance of this method. Ambulatory blood pressure values are reproducible and this method avoids the so-called placebo effect; thus, this method is useful in clinical studies investigating the efficacy and duration of action of antihypertensive drugs. Data from 1 study in which hypertensive patients were treated with slow release verapamil 240 mg, enalapril 20 mg, nitrendipine 20 mg and placebo, given once daily for 8 weeks according to a double-blind parallel group design, showed that mean 24-hour ambulatory blood pressure was reduced by all 3 drugs compared with placebo. Verapamil and enalapril showed similar antihypertensive efficacy and both drugs reduced night-time blood pressure more effectively than nitrendipine.


Calcium overload--an important cellular mechanism in hypertension and arteriosclerosis.

Arterial hypertension and arteriosclerosis are dramatic consequences of vascular calcium overload. Acute intracellular calcium overload of vascular smooth muscle cells produces hypercontractility. Hypertension develops if a general increase in systemic arteriolar tone leads to a rise in peripheral flow resistance. Moreover, progressive elevation of calcium destroys the structural integrity of the arterial and arteriolar walls. Thus, in various animals models, calcium overload initiates lesions of an arteriosclerotic character. Interestingly, conventional human coronary plaques also represent a calcium-dominated type of arteriosclerosis. With the advent of specific calcium antagonists, the pathogenic effects of calcium overload and its deleterious consequences have become, for the first time, accessible to therapeutic intervention. Accordingly, adequate treatment with calcium antagonists prevents calcium overload and can thereby protect arteries and arterioles from functional disturbances and structural damage. In spontaneously hypertensive rats, specific calcium antagonists of the verapamil, nifedipine and diltiazem type normalise blood pressure (BP) by reducing transmembrane calcium influx into vascular smooth muscle cells. However, in addition to controlling BP, these drugs also act as tissue protective agents. The long term effects of calcium antagonists such as verapamil in experimental hypertension include the prevention of severe arteriosclerosis, myocardial hypertrophy, and malignant nephrosclerosis. In humans, the antihypertensive efficacy of verapamil is well documented. Further clinical studies have yet to evaluate the antiarteriosclerotic and tissue protective potential of verapamil in humans.


Secondary prevention with calcium antagonists after acute myocardial infarction.

Experimental studies have demonstrated that the 3 calcium antagonists nifedipine, diltiazem, and verapamil have a comparable effect in the prevention of myocardial damage during ischaemia. Secondary prevention trials after acute myocardial infarction, which aimed at improving survival and preventing reinfarction, nevertheless demonstrated pronounced differences between the 3 drugs. Nifedipine had no effect on reinfarction or death. Diltiazem had no overall effect but prevented first reinfarction or cardiac death (cardiac events) in patients without heart failure, and increased cardiac events in patients with heart failure before randomisation. Verapamil prevented first reinfarction or death (major events); the most pronounced effect was found in patients without heart failure before randomisation. Verapamil did not have detrimental effects in patients treated for heart failure before randomisation. Differences between trials and between drugs explaining the different clinical findings are evaluated.


Cardiac hypertrophy and aortic compliance after calcium channel antagonism in hypertension.

Calcium antagonists are potent vasodilators. They improve arterial compliance by their action at the level of the large arteries. Several experimental and clinical studies have shown decreases of varying magnitudes in hypertension-induced cardiac hypertrophy after long term treatment. It appears that the mechanisms of this reduction in cardiac mass are complex. As arterial compliance is one of the determinants of left ventricular hypertrophy in hypertension, it may be considered that an improvement in cardiac mass can result not only from the blood pressure reduction but also from the increase in arterial compliance associated with calcium antagonist treatment in essential sustained hypertension.


Regional myocardial blood flow and coronary reserve in hypertensive patients. The effect of therapy.

Patients with essential arterial hypertension demonstrate abnormal vasodilator capacity either during increased cardiac metabolic demand or during pharmacological vasodilation. Structural and functional damage to the coronary microcirculation has been proposed as one of the major causes of impaired coronary reserve in this disease. To assess the role of microvascular impairment in regional myocardial blood flow (MBF), 27 patients with essential hypertension were evaluated by dynamic positron emission tomography (PET) at rest, during atrial pacing and after dipyridamole infusion and compared with 13 healthy subjects. All patients had normal coronary arteries, 17 had moderate to severe hypertension and 10 had mild hypertension. Baseline mean MBF of 0.97 +/- 0.25 ml/min/g was significantly increased to 1.60 +/- 0.38 during atrial pacing and 2.35 +/- 0.95 after dipyridamole infusion (p < 0.01); however, mean flow during atrial pacing and after dipyridamole infusion was significantly lower than in healthy subjects (2.15 +/- 0.73 and 3.71 +/- 0.86 ml/min/g, p < 0.05 and p < 0.01, respectively). The MBF response to atrial pacing and dipyridamole infusion was similarly depressed in patients with mild and severe hypertension. The study was repeated after 6 months of antihypertensive treatment with the calcium antagonist verapamil or the angiotensin converting enzyme (ACE) inhibitor enalapril in a subgroup of 20 patients as part of a randomised, single-blind clinical trial. This study is still in progress; the initial 16 patients treated with verapamil or enalapril showed an obvious improvement in MBF values during atrial pacing and after dipyridamole infusion after 6 months of therapy (mean MBF: 2.10 +/- 0.64 and 2.99 +/- 1.63 ml/min/g, respectively, p < 0.05 vs pretreatment values). In conclusion, obvious impairment of MBF during atrial pacing and after dipyridamole infusion was observed in hypertensive patients with normal coronary arteries and this appeared unrelated to the severity of hypertension. Therapy with verapamil or enalapril improved coronary reserve and MBF response to an increase in myocardial oxygen demand.


The J-curve. The importance of gradual reduction of blood pressure.

Even if the treatment goal in hypertension is 'the lower the better', it is obvious that blood pressure (BP) cannot be reduced without reaching a point at which organ perfusion may become compromised and where mortality and morbidity will increase rather than decrease. In 1979, a 5-fold increase in myocardial infarction among patients whose diastolic BP was reduced to below 95mm Hg was reported. In 1987, these results were confirmed, and a J-shaped relationship between diastolic BP and death from myocardial infarction in those patients with evidence of ischaemic heart disease at entry into the trial was shown. In recent years, several studies have demonstrated this J-shaped curve with a J-point between 85 and 91mm Hg. However, all the data were obtained in retrospective analyses of a small number of patients and prospective properly designed studies are necessary before changing our treatment criteria. The Behandla Blodtryck Battre Study fulfils these criteria and preliminary results demonstrate that it is possible to achieve a further reduction in BP in well controlled hypertensive patients without increasing the incidence of side effects.


Is salt restriction necessary in hypertensive patients treated with calcium antagonists?

A moderate restriction of salt intake is recommended as a nonpharmacological approach to reducing high blood pressure, and can be used as an adjuvant to antihypertensive drug treatment. Noncompliance with a salt-restricted diet is the main limitation to this therapeutic approach. Evidence to date has shown that hypertensive patients treated with a calcium antagonist may not require a salt-restricted diet to achieve target blood pressure reductions. Further long term studies are required to confirm this.


Risk-benefit aspects of antihypertensive drugs.

Several long term trials using traditional antihypertensive therapy with diuretics and beta-blockers have shown that antihypertensive therapy reduces the overall risk of cardiovascular complications. However, even after several years of therapy the cardiovascular risk in hypertensive patients cannot be lowered to that in the normotensive population. Antihypertensive therapy can reduce the incidence of cerebrovascular complications in patients with hypertension by about 65%. However, the effect of such therapy in preventing coronary events has been disappointing, as these events are 3 to 4 times more common than cerebrovascular complications in hypertensive patients. It is now apparent that adverse pharmacological effects of diuretics and beta-blockers on lipid metabolism persist for many years. Thus, treatment with these agents constitutes a new risk factor for coronary heart disease and may, at least in part, explain the failure of traditional antihypertensive therapy to reduce the incidence of myocardial infarction and sudden death as effectively as that of cerebrovascular accidents. On the other hand, titration of these antihypertensive agents to the lowest possible dose in order to avoid metabolic alterations and subjective adverse effects has frequently resulted in the administration of subtherapeutic doses, particularly for hydrochlorothiazide. Until comparative long term clinical trials with older and newer antihypertensive agents and morbidity and mortality as end-points are completed, the debate on first-line drugs for antihypertensive treatment will not be satisfactorily resolved.


Swiss hypertension treatment programme with verapamil and/or enalapril in diabetic patients.

The purpose of the present study was to assess the efficacy and tolerability of diuretic-free antihypertensive therapy with a calcium antagonist and/or an angiotensin converting enzyme (ACE) inhibitor in patients with diabetes mellitus. 54 hypertensive [blood pressure (BP) above 140/90mm Hg] patients with diabetes mellitus type 1 (n = 7) or 2 (n = 47) and normal serum creatinine levels (mean 82 +/- 6 mumol/L) received either verapamil or enalapril after a 2-week washout and a 4-week placebo phase. If BP remained elevated, both agents were combined. Verapamil or enalapril alone normalised diastolic BP (to less than 90mm Hg) in 36 patients; verapamil decreased BP from 159/98 to 147/87mm Hg (n = 19, p < 0.001) and enalapril decreased BP from 166/99 to 146/88mm Hg (n = 17, p < 0.001). In 18 patients who remained hypertensive after 10 weeks of monotherapy, a combination of both drugs decreased BP from 169/104 to 151/90mm Hg (p < 0.001). Overall, 87% of patients achieved a target BP response at 30 weeks. Urinary albumin as related to creatinine excretion (UAE; micrograms albumin:mg creatinine) was on average not significantly changed after verapamil or enalapril treatment, alone or combined. Nevertheless, in patients with initial microalbuminuria, UAE decreased (p < 0.05) during enalapril treatment. Serum potassium, total lipids, high density lipoprotein cholesterol, low density lipoprotein cholesterol, glycosylated haemoglobin, serum C peptide and fructosamine levels were not significantly modified by treatment. Subjective tolerability of the drugs was also generally good. Thus, in hypertensive patients with diabetes, a diuretic-free therapy based on the calcium antagonist verapamil or the ACE inhibitor enalapril, alone or combined, can effectively decrease BP without adversely affecting carbohydrate and lipid metabolism.


Effects of treatment with verapamil SR and captopril on the lipid profile of hypertensive patients.

The potential beneficial effects of antihypertensive drugs on cardiovascular morbidity and mortality may be compromised by their adverse effects on serum lipid levels. In our study we compared verapamil and captopril and evaluated their effects on blood pressure and on serum lipid and lipoprotein levels, with particular attention to lipoprotein(a) [Lp(a)]. 20 hypertensive patients were treated with sustained release verapamil 240mg once daily or captopril 25mg twice daily for 3 months in a double-blind randomised study. Diastolic blood pressure was reduced from 100 +/- 3mm Hg to 87 +/- 6mm Hg (p < 0.01) and from 100 +/- 5mm Hg to 92 +/- 7mm Hg (p < 0.05) in the verapamil and captopril groups, respectively. Small but significant changes in serum lipid levels were noted: total cholesterol was reduced from 6 to 5.8 mmol/L (verapamil) and from 6.1 to 5.9 mmol/L (captopril); low density lipoprotein (LDL) cholesterol was reduced from 4 to 3.8 mmol/L (verapamil) and from 4.2 to 3.9 mmol/L (captopril); apolipoprotein C-III was reduced from 0.3 +/- 0.07 to 0.2 +/- 0.06 mmol/L (9.7 +/- 2.5 to 9.2 +/- 2.3 mg/dl) [verapamil] and from 0.2 +/- 0.1 to 0.2 +/- 0.09 mmol/L (9.1 +/- 3.7 to 8.3 +/- 3.4 mg/dl) [captopril]; apolipoprotein A-II increased only with verapamil (p < 0.02). Lp(a) levels showed only minor changes in individual patients. In conclusion, in our study verapamil and captopril were effective antihypertensive agents and did not adversely effect the lipid profile.


Sodium intake does not influence the effect of verapamil in hypertensive patients with mild renal insufficiency.

Information is limited regarding the efficacy of antihypertensive drugs in patients with hypertension associated with renal insufficiency. To address this question, a group of 14 outpatients with essential hypertension and mild renal insufficiency received slow release verapamil 240 mg/day for 14 days after a 4-week washout period. Patients were randomly assigned to a low (4 g/day) or high (11 g/day) salt diet, and crossed over to the alternative diet after 7 days. 24-Hour blood pressure monitoring was performed at the end of the washout period and after 7 and 14 days during verapamil treatment. Verapamil induced a significant fall in mean 24-hour blood pressure that was similar for patients on both diets (p < 0.01). As expected, natriuresis increased significantly during high sodium intake (p < 0.01), and bodyweight fell significantly when sodium intake was reduced (p < 0.05). Meanwhile, serum creatinine and creatinine clearance remained stable. These results indicate that the antihypertensive effect of verapamil is independent of sodium intake even in the presence of mild renal insufficiency.


Is renal protection with calcium antagonists possible?

Organ protection is the main goal in the treatment of high blood pressure. Consequently, this protective capacity should be one of the main characteristics of any drug used in the treatment of hypertension. A renal protective agent should protect the kidney from intrinsic renal vasoconstrictors and exogenous agents, and should also protect, or at least delay, the decline in renal function in the presence of renal insufficiency, by mechanisms other than increasing glomerular filtration pressure. Verapamil protects mesangial cells from the reduction in surface area induced by endothelin in vitro. In human subjects, it minimises the renal impairment provoked by the administration of cisplatin, and in mice it protects superficial cortical blood flow from the vasoconstriction elicited by cyclosporin. Finally, verapamil may protect from glomerulosclerosis as a result of its capacity to inhibit mesangial cell replication.


Patterns of cytodistribution of prosomal antigens on the vimentin and cytokeratin networks of monkey kidney cells.

Prosomes were found as mRNA-associated ribonucleoprotein particles (RNP) and cofactors of untranslated (ribosome-) free mRNP. Previous data have shown the presence of prosomal networks in the cytoplasm of PtK1 and HeLa cells and their superposition onto the intermediate filaments (IF) of cytokeratin type but little if any of vimentin type. Here it is shown that in LLC-MK2 cells various prosomal antigens are present on both, vimentin and cytokeratin networks, individual prosomal antigens superposing to variable degrees onto the IF subnetworks. Some prosomal antigens in variable relative concentrations were also observed in the nuclei of these cells. We suggest the existence of prosomal subnetworks specific for each prosomal antigen superposing to a variable extent onto the IF of both types.


Characterization of heterotypic adherence between transformed human lymphoblastic cells and marrow stromal cells: VCAM-1 is a ligand for one of the leukemia cell adhesion proteins.

Marrow stromal cells are important in normal myelopoiesis and support growth of leukemia/lymphoma (LL) cells in vitro. We have previously described the heterotypic adherence of a human B-lymphoblastic cell line (UTMB-460) to marrow stromal cells (MSC). We have extended these observations to a human T-lymphoblastic cell line (CEM) and characterized the heterotypic adherence of B- and T-lymphoblastic cell lines to human MSC. Electron microscopy demonstrated UTMB-460 cells were in very close apposition to the MSC, but no specific intercellular junctions were noted. Under the conditions employed, these MSC express extracellular fibronectin, collagen types I and IV, intracellular laminin, and vimentin, but no factor VIII-R antigen. In addition, the MSC had receptors for the lectin Ulex europaeus agglutinin I. UTMB-460 and CEM cells do not adhere to extracellular matrix (ECM) proteins secreted by the MSC, i.e., fibronectin, collagen types I, III, or IV, or laminin. Monoclonal antibodies (MoAbs) against CD11a, CD11b, CD18, and CD54 and a polyclonal anti-human fibronectin antibody do not inhibit attachment of either B- or T-lymphoblastic cells to MSC. Peptides GRGES and GRGDS did not inhibit adherence of UTMB-460 and CEM cells to MSC. In contrast, the anti-vascular cell adhesion molecule (VCAM)-1 MoAb (4b9) caused significant inhibition (p < 0.01) of the adherence of both UTMB-460 and CEM cells to normal human MSC monolayers. These data suggest: (1) that MSC to which lymphoblastic cells adhere are specialized mesenchymal cells; (2) that the membrane interactions between T- and B-lymphoblastic cells and MSC involve close apposition of cell membranes of MSC and the lymphoblastic cells; (3) that the heterotypic adherence between B- and T-lymphoblastic cell lines (UTMB-460 and CEM) and MSC does not involve the RGD recognition sequence of the integrin family, the B2 leukocyte integrins, CD44, LAM-1, or the ECM proteins examined; and (4) that VCAM-1 may at least be partially responsible for heterotypic adherence between human MSC and B- and T-lymphoblastic cells.


Extracellular matrix stimulation of guinea pig megakaryocyte proplatelet formation in vitro is mediated through the vitronectin receptor.

We have used an in vitro culture system to study the role of extracellular matrix (ECM) in the fragmentation of guinea pig bone marrow megakaryocytes (MK) and the formation of proplatelet fragments from these cells. Proplatelet formation is stimulated by culturing the cells on a hydrated type I collagen gel in the presence of serum. MK express integrin proteins alpha 5, alpha 6, beta 1, and the alpha v beta 3 complex as demonstrated by immunofluorescence. A monoclonal antibody, LM 609, to the alpha v beta 3 vitronectin receptor blocked proplatelet formation, whereas the monoclonal antibodies to the beta 1, alpha 5, and alpha 6 integrin proteins did not inhibit proplatelet formation. The tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits proplatelet formation; however, there was no inhibition by the fibronectin receptor-specific peptide GRGDdSP. The fibrinogen gamma chain peptide HHLGGAKQAGDV, which binds to the platelet membrane glycoprotein complex IIb-IIIa but not to the vitronectin receptor (VnR), did not inhibit proplatelet formation, nor did two different laminin peptides. In the absence of serum, 5.7% of MK spontaneously formed processes or fragmented. The addition of 50 micrograms/ml of vitronectin to serum-free cultures increased proplatelet formation to 21.5% of the MK, equal to cultures with 10% serum. Stimulation of proplatelet formation by vitronectin in serum-free cultures was inhibited by LM 609. Antibody staining with anti-bovine vitronectin antibody showed that MK contain intracellular vitronectin. These data show that guinea pig MK express alpha 5, alpha 6, beta 1, and alpha v beta 3 integrins. However, only the MK vitronectin receptor and its interaction with vitronectin plays an essential role in proplatelet formation in vitro.


Characterization of a muscle-associated antigen from Wuchereria bancrofti.

A recombinant clone, WbN1, isolated from a genomic expression library of Wuchereria bancrofti and showing restricted specificity at the DNA level (Southern and PCR analyses) for Wuchereria bancrofti and Brugia malayi has been previously described. Sequence analysis of WbN1 indicated that it had notable similarity to myosin. Further characterization using in situ hybridization has localized the mRNA in the muscle of the adult parasite and in the microfilariae. Rabbit polyclonal antiserum, raised against the recombinant WbN1 fused to the maltose-binding protein, recognized a 200-kDa polypeptide in immunoblots containing B. malayi antigen extracts. The same antibody also recognized myosin extracted from Brugia pahangi, Onchocerca volvulus, and Caenorhabditis elegans. Localization using the rabbit antiserum revealed the presence of the antigen in the adult muscle tissue and in the microfilariae; the same antibody inhibited the binding of a monoclonal antibody 28.2 (directed toward MHC B of C. elegans myosin) to the recombinant WbN1 antigen and also to purified C. elegans myosin. Based on homology data, structural location, competitive ELISA, and immunoblot we conclude that WbN1 is related to myosin or a similar myofibrillar protein.


E-selectin and intercellular adhesion molecule-1 are released by activated human endothelial cells in vitro.

Endothelial cells respond to several cytokines by a rapid increase in expression of the adhesion molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1), followed by a gradual decline. The fate of these molecules, which was so far unknown, was studied. Specific sandwich ELISA for the detection of soluble (s)E-selectin and sICAM-1 were developed. In supernatant, centrifuged 3 hr at 100,000 g to remove microparticles, from human umbilical vein endothelial cells (HUVEC) activated with tumour necrosis factor (TNF), interleukin-1 (IL-1) or lipopolysaccharide (LPS), E-selectin and ICAM-1 molecules could be detected. Biochemical analysis revealed that sE-selectin migrated as a band of approximately 94,000 MW. The amount of soluble adhesion molecules released was directly correlated with cell surface expression. Maximal release of E-selectin was observed 6-12 hr after activation of HUVEC and decreased to below detection limit 24 hr after activation. After activation, release of ICAM-1 gradually increased with ICAM-1 cell surface expression, and reached a plateau after 24 hr, which was constant for 3 days. Since E-selectin and ICAM-1 are highly expressed at inflammatory sites, the resulting high concentrations of released E-selectin and ICAM-1 may affect interactions of leucocytes with endothelial cells. The physiological role, however, of the release of E-selectin and ICAM-1 remains to be elucidated.


B-T lymphocyte interactions in experimental autoimmune myasthenia gravis: autoantibody mediated up-regulation of the response of acetylcholine receptor-specific T lymphocytes.

Twenty-six monoclonal antibodies of five different isotypes and reactive against distinct parts (alpha, beta, gamma, delta-subunits) of the nicotinic acetylcholine receptor (AChR) from Torpedo californica were screened for their capacity to enhance activation of AChR-specific CD4+ autoreactive T cells. The T-cell line (LR) used in this study recognized an epitope (98-116) on the alpha-subunit of Torpedo AChR. Four monoclonal antibodies bearing a gamma 2b isotype and recognizing an epitope on the Torpedo AChR alpha-subunit, especially the main immunogenic region (MIR), were able to enhance T-cell activation in a dose-response manner. Four further gamma 2b isotype monoclonal antibodies, recognizing epitopes other than the AChR alpha-subunit, had no effect. Monoclonal antibodies of other isotypes (IgM, IgG1, IgG2a, IgG2c), irrespective of their subunit specificity, were unable to influence the T-cell response. Thus, the enhancement requires a IgG2b isotype, and both the antibody and the T-cell recognize an epitope on the same subunit. We have previously shown that AChR-specific B cells are directly able to present antigen to AChR-specific T-cell lines in a privileged way. The present data demonstrate that B cells are also capable of enhancing indirectly the immunogenicity of autoantigens via their humoral antibodies.


Systemic lupus erythematosus murine monoclonal DNA-binding antibodies recognize cytoplasmic and nuclear phosphorylated antigens that display cell cycle redistribution in HEp-2 cells.

The immunological basis for the production of autoantibodies characteristic of systemic lupus erythematosus (SLE) against a wide range of antigens remains obscure. The specificity of (NZB x NZW)F1 (BWF1) or MRL/Mp-lpr/lpr (MRL/lpr) mouse monoclonal antibodies (mAb) was examined by immunofluorescence, immunoblotting and immunoprecipitation techniques. Using non-synchronized HEp-2 cells as substrate, the murine mAb were classified by indirect immunofluorescence into five groups on the basis of their staining patterns of subcellular components in interphase and mitotic stages of the cell cycle. The nature of the antigens recognized by the murine lupus was assessed by immunoblotting experiments in total, cytoplasmic and nuclear cell extracts from HEp-2 cells. The six antibodies used recognized in total cell extracts a range of polypeptides with apparent molecular weights from 25,000 to 210,000. Three polypeptides of 130,000, 110,000 and 45,000 MW were recognized by all six antibodies in both nuclear and cytoplasmic extracts. Immunoprecipitation of total cellular extracts labelled with [35S]methionine showed almost the same pattern as obtained in the immunoblotting assay. The labelling in vivo of HEp-2 cells with [32P], followed by the immunoprecipitation of the [32P]cell lysate showed that these mAb recognized phosphorylated proteins. The progressive decrease in reactivity of these mAb following treatment with higher concentrations of alkaline phosphatase in both [32P]cell lysate or nitrocellulose membranes indicates that these mAb recognize phosphorylated epitopes.


The distribution of IgE plasma cells in lymphoid and non-lymphoid tissues of high-IgE responder rats: differential localization of antigen-specific and 'bystander' components of the IgE response to inhaled antigen.

Repeated exposure of high-IgE responder rats to antigen-containing aerosols stimulates IgE responses which last for several weeks, and are eventually terminated with the onset of immunological tolerance. Studies on the distribution of total and antigen-specific IgE plasma cells and IgE mRNA during antibody production, identified the lymph nodes draining the lower respiratory tract as the primary site for initiation of the IgE response to inhaled antigen; subsequently the response seeded to mucosa-associated lymph nodes but not to central lymphoid organs. A vigorous 'bystander' IgE response (approx. x 10 the specific response) was also observed, but this was restricted to areas directly draining sites of deposition of inhaled antigen, including non-lymphoid respiratory mucosal tissues. Despite the rapid termination of the specific IgE response after the fourth week of exposure, the bystander component persisted. These results are discussed in relation to the role of cognate/non-cognate T-B interactions in the IgE response to inhaled antigens, and the relative susceptibility of each component to T-cell regulation in vivo.


Diversity of anti-HLA-DR antibodies elicited by distinct anti-idiotypic monoclonal antibodies recognizing idiotopes co-expressed by the immunizing monoclonal antibody.

Analysis at the clonal level of the idiotypic network has identified differences in fine specificity between antigen-binding anti-anti-idiotypic (anti-anti-id) monoclonal antibody (mAb) and the original mAb as well as among antigen-binding anti-anti-idiotypic (anti-id) mAb. However, the diversity of humoral immune responses elicited by anti-id mAb recognizing idiotopes co-expressed on the immunizing mAb has not been analysed. Since this information may contribute to our understanding of the role of anti-id antibodies in the generation of diversity in the course of an immune response, we have compared the fine specificity and idiotype profile of two subsets of anti-HLA-DR mAb generated with the anti-id mAb F5-444 and F5-830. The latter mAb recognize idiotopes co-expressed in the antigen-combining site of the immunizing anti-HLA-DR1,4,w14,w8,9 mAb AC1.59. These investigations showed that: (1) the two subsets of anti-HLA-DR mAb overlap only partially in their reactivity patterns with HLA-DR+ cells; (2) both subsets of anti-HLA-DR mAb recognize spatially close epitopes; (3) each subset of anti-HLA-DR mAb has unique reactivity patterns with soluble HLA-DRw16 and DRw17 antigens; and (4) each subset of anti-anti-id mAb displays a distinct idiotype profile. The subtle differences in the fine specificity and idiotype profile of the two subsets of anti-HLA-DR mAb suggest that anti-id antibodies may play a role in the generation of diversity in the course of a humoral immune response.


Identification of an immunodominant B-cell epitope in bovine type II collagen and the production of antibodies to type II collagen by immunization with a synthetic peptide representing this epitope.

Using epitope scanning of 272 short, synthetic peptides representing the amino acid sequence of the CB-11 peptide of type II collagen, we have shown that five strains of rat, immunized with type II collagen, produce antibodies to a region 37-45 amino acids from the amino end of CB-11 peptide. Antibodies to this region always gave the highest binding values suggesting that it is an immunodominant region. Wistar rats immunized with a synthetic peptide representing this region, coupled to keyhole limpet haemocyanin, produced antibodies to this peptide which could still be detected at 1:4000 to 1:8000 dilution but none developed clinical arthritis. All sera also showed binding of antibodies to denatured bovine type II collagen but not to native type II collagen, keyhole limpet haemocyanin or to bovine serum albumin by ELISA. Sera from peptide-immunized rats were examined for antibody binding to the 272 short peptides of the CB-11 peptide and to the synthetic peptides representing shortened forms of the immunodominant region and forms of it with substituted amino acids. These results showed that the antibodies in the peptide-immunized rats were not identical to those produced to that peptide by rats immunized with type II collagen but may represent subpopulations of them. These findings suggest caution in interpreting the role of antibodies to individual peptides in arthritis induction without knowledge of their fine specificity.


The use of GM-CSF in AIDS.

Hematopoietic growth factors may mitigate the cytopenias that frequently complicate HIV disease or its treatment. Clinical and in vitro studies have indicated the ability of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) or erythropoietin (EPO) to overcome the myelosuppression of HIV or many of the drug therapies used in the care of HIV-infected individuals. In addition, neutrophil or monocyte functional abnormalities observed in AIDS patients may be improved by the use of GM-CSF. Issues which may distinguish the use of hematopoietic growth factors in AIDS as compared with in other clinical settings include: 1) interaction of the growth factor with other cytokines which are aberrantly expressed, 2) direct effects of the growth factor on the replicative activity of HIV, and 3) potential interactions of the growth factor with other concurrently administered medications. This review focuses on the potential roles and limitations of growth factor use in AIDS and reviews the clinical studies using GM-CSF in HIV-infected individuals.


Induction of neointima formation by platelet derived angiogenesis fraction in a small diameter, wide pore, PTFE graft.

Enhancement of endothelialization and patency of a small diameter (2 mm), wide pore, PTFE graft was attempted by coating the luminal surface with a platelet derived angiogenesis fraction (PDAF) and implanting it in a rat model. PDAF was delivered to the grafts by combining it with a carrier polymer. PDAF-treated grafts were initially implanted in the retroperitoneum for 21 days followed by removal of one for histology and in situ end to side bypass to the infrarenal aorta for the other. Vascularized grafts were examined at 14 days for patency and 100 days for patency and histology. Significant differences were noted in transmural ingrowth of capillaries and tissue at 21 days post implantation in PDAF-treated versus untreated grafts. Similarly, near significance was noted in capillary ingrowth and significance was noted in tissue ingrowth at 100 days in PDAF-treated grafts. Despite favorable trends particularly early in the time course, no significant differences in graft patency, endothelialization, or hydroxyproline content was demonstrated between PDAF-treated and untreated grafts. Results of this preliminary study are encouraging for further study of PDAF-treated PTFE grafts and the potential that rapid vascularized neointima formation results improved in graft patency rates.


Post-antibiotic effect of ceftazidime, ciprofloxacin, imipenem, piperacillin and tobramycin for Pseudomonas cepacia.

The post-antibiotic effects (PAE) of ceftazidime, ciprofloxacin, imipenem, piperacillin and tobramycin were studied for ten strains of Pseudomonas cepacia isolated from patients with cystic fibrosis. Antibiotic concentrations used for exposure were either the MIC of each agent for the sensitive isolates or the recommended sensitivity breakpoint concentrations for the resistant isolates. After 2 h of exposure, cultures were rapidly diluted 1000-fold to eliminate the antibiotic. Out of the ten isolates, there were eight sensitive to ceftazidime, six to ciprofloxacin, six to imipenem, nine to piperacillin and five to tobramycin. All antibiotics tested demonstrated PAE for some isolates of P. cepacia, however, each antibiotic failed to produce a PAE for at least one isolate. The mean PAE was 1.35 h for ceftazidime, 2.38 h for ciprofloxacin, 2.39 h for imipenem, 2.16 h for piperacillin and 1.77 h for tobramycin. Imipenem demonstrated PAE of > or = 0.5 h for all sensitive isolates tested; ceftazidime, piperacillin, ciprofloxacin and tobramycin demonstrated PAE of > or = 0.5 h for 6/8, 8/9, 5/6 and 2/5 sensitive isolates, respectively. These data indicate that several antibiotics have significant (> or = 0.5 h) PAE for isolates of P. cepacia.


Investigation of the role of Von Korff fibers during murine dentinogenesis.

The existence and significance of Von Korff fibers during early dentinogenesis are still very controversial. The purpose of the present study was to re-examine the questions of the existence, nature and significance of Von Korff's fibers using light microscopy and immunohistochemistry. Specimens were obtained from 3 days-old CD-I mice and mandibles were carefully dissected under constant irrigation and immediately fixed in 10% neutral buffered formalin for light microscopy. Sections were treated or not with collagenase prior to silver staining. For immunohistochemistry, specimens were fixed in 95% ethanol and embedded in paraffin. Sections were reacted with goat anti-human-bovine type I or type III collagen and a rhodamine (RITC) labelled rabbit anti-goat IgG was then reacted as a secondary antibody. Slides were then examined under a Zeiss II photomicroscope equipped with epifluorescence. Our results have confirmed the presence of argyrophilic material concentrated at the periphery of the dental papilla and stretching from the subodontoblastic layer to the future dentino enamel junction. The distribution of type III collagen was very similar to the distribution of the silver staining at the cervical loop area. Type I collagen distribution was different and concentrated in areas where odontoblasts were fully differentiated. Our study showed that Von Korff fibers are not artefactual. We have established the presence of an apical compartment containing type I collagen fibers and a basal compartment containing type III collagen to explain the image of continuous silver staining crossing the entire thickness of the odontoblast layer.


Langerhans cell dynamics in human gingiva during experimentally induced inflammation.

The changes in the number of Langerhans cells within the gingiva during a 21 day experimental gingivitis episode were investigated immunohistochemically. Monoclonal antibodies to CD1a (specific for Langerhans cells and thymocytes) and HLA-DR (class II major histocompatibility antigens - (MHC)) were used to identify Langerhans cells within gingival biopsies taken on days 0, 7, 14 and 21. HLA-DR antibody stained dendritic cells within the oral epithelium which were morphologically identical to the CD1a+ Langerhans cells. Class II MHC LC numbers rose and plateaued between day 7 and 14 then decreased to baseline by day 21. As plaque accumulated and initial inflammation developed there was an increase in the number of CD1a+ Langerhans cells which peaked at day 7 and stayed high (day 14). As inflammation developed there was a statistically significant decrease in the number of CD1a+ Langerhans cells by day 21 (p = 0.028). The initial increase, followed by a decrease in CD1a+ Langerhans cells as inflammation developed, suggests that migration of Langerhans cells occurs within the gingival epithelium and this may represent an important early event in the gingival immune response to plaque.


Effects of culture passages on collagen immunostaining in human dental pulp and gingival fibroblasts.

In this study, a scanning microscopic computer-assisted image analysis system was used for the immunocytochemical characterisation of collagen types I, III and V in normal human fibroblasts from pulp and gingival explants, using specific purified antibodies and peroxidase labeling. The culture conditions were standardized in order to evaluate simultaneously the expression of the three antigens in four different culture passages of the two fibroblast types. The optical density values of immunostaining intensities were quantified, the integrated optical density per cell was calculated, and the results were analyzed by a variance test. It was found that all three collagen types were present in the tissues, and in both gingival and pulp fibroblasts after three to nine culture passages. A non-parametric statistical analysis of the staining intensity variances revealed significant differences between antigenic levels depending on the tissue origin of the fibroblasts and an effect of culture passages. The results seemed to justify application of this technique at the light microscope level for the evaluation of collagen production, the principal function of fibroblasts, but the tissue origin and number of culture passages should be taken into consideration for in vitro biocompatibility testing of dental materials.


Projections to the inferior colliculus from the dorsal column nuclei. An experimental electron microscopic study in the cat.

The projections from the dorsal column nuclei (DCN) to the inferior colliculus (IC) were investigated in cats by means of electron microscopy. The DCN were destroyed unilaterally by electrocoagulation and the animals survived 3 or 4 days. A variable number of degenerating synaptic boutons were observed bilaterally in the IC-external nucleus and in the intercollicular area. The greatest number of degenerating terminals was encountered in the rostral portions of the IC-external nucleus and in the intercollicular area contralateral to the DCN lesion. In these regions some series of ultrathin sections the degenerating boutons comprised 6-13% of the entire synaptic bouton population of the examined area. In the caudal portion of the contralateral IC-external nucleus the number of degenerating boutons greatly diminished and the latter showed a patchy distribution. Ipsilateral to the DCN lesion the number of degenerating terminals was low, and a more substantial number was found only in the rostroventral portion of the IC-external nucleus and in the intercollicular area. The degenerating synaptic boutons displayed markedly diverse patterns of alterations. Most often was the dark (electron-dense) degeneration pattern, followed by the filamentous degeneration. More rare were the light (electron-lucent) degenerating boutons, and a very limited number of terminals displayed pinocytotic degeneration changes. The DCN boutons contained round and oval synaptic vesicles and terminated mainly on proximal dendritic trunks, followed by the perikarya of the efferent neurons, and smaller, distal dendrites. Rarely the degenerating terminals contacted dendritic spines, and no contacts were found with the small (interneuronal) perikarya and with other vesicle containing profiles. The axodendritic contacts were asymmetrical and the axosomatic--from the intermediate type. In agreement with previous light microscopic data, the present findings demonstrate the existence of substantial excitatory projection from the DCN to the polysensory nuclei of the IC that integrate converging auditory and tactile information, and are involved in acoustico-motor behavior.


Symbolic play development in toddlers with expressive specific language impairment (SLI-E).

Twenty toddlers with expressive specific language impairment (SLI-E) and 20 toddlers with normal language development were compared in their symbolic play development. The groups did not differ in amount of engagement with the toys or in functional conventional play behaviors. However, the children with SLI-E displayed less decentered play (use of play schemes with a doll or another person), less well-developed sequential play, and fewer occurrences of symbolic play transformations (use of a neutral object or an absent object to carry out pretending). The provision of structure in the form of thematically related toy sets, instructions, and modeling did not reduce the discrepancy between demonstrated play behaviors of toddlers with SLI-E and their normally developing peers. Three possible explanations for this discrepancy are considered: a "stylistic" difference in play, a developmental lag in symbol use, or a deficit in retrieval of stored symbolic representation.


Spoken and written language relationships in language/learning-impaired and normally achieving school-age children.

Students with language/learning impairment (LLI) and three groups of normally achieving children matched for chronological age, spoken language, and reading abilities wrote and told stories that were analyzed according to a three-dimensional language analysis system. Spoken narratives were linguistically superior to written narratives in many respects. The content of written narratives, however, was organized differently than the content of spoken narratives. Spoken narratives contained more local interconnections than global interconnections; the opposite was true for written narratives. LLI and reading-matched children evidenced speaking-writing relationships that differed from those of the age- and language-matched children in the way language form was organized. Further, LLI children produced more grammatically unacceptable complex T-units in their spoken and written stories than students from any of the three matched groups. The discussion focuses on mechanisms underlying the development of speaking-writing differences and ramifications of spoken-language impairment for spoken and written-language relationships.


Contextual and linguistic correlates of intelligibility in children with developmental phonological disorders.

Listeners' glosses of children's intended words provided data for two studies of the potential influence of selected contextual and linguistic variables on word intelligibility. Several regularities associated with the occurrence of unintelligible words were identified. In Study I, intelligibility outcomes were associated with utterance length and fluency, word position, intelligibility of adjacent words, phonological complexity, and grammatical form. In Study II, intelligibility outcomes were associated with phonological complexity, syllabic structure, and grammatical form. Discussion considers the implications of these and other regularities associated with the occurrence of unintelligible words for a comprehensive perspective on the utterance-to-utterance intelligibility deficits of children with phonological disorders of unknown origin.


Symbol vocabulary and the focus of conversations: augmenting language development for youth with mental retardation.

Communication devices designed to augment the language development of individuals with severe cognitive disabilities and little or no functional speech typically contain primarily nouns because they seem easiest to acquire and evaluate. In this study, the effect of a more diverse vocabulary was assessed. Systematic observations of the use of computerized speech-output devices by 12 youth with moderate or severe mental retardation and severe spoken language disability and by their partners were made over a 2-year period. Social-regulative symbols (e.g., "please," "I'm finished") were used as soon as they were introduced, and their availability expanded the focus of conversations both at home and at school. Implications for conceptualizing variation in early language use and for the design of language intervention programs are discussed.


Effects of configuration on the paired-associate learning of blissymbols by preschool children with normal cognitive abilities.

Translucency appears to be a potent variable in the learning of Blissymbols by preschool children with normal cognitive abilities. Complexity does not appear to influence learning for these individuals when there is a concurrent condition of high translucency. However, a condition of high complexity seems to enhance the learnability of Blissmbols having low translucency. For the present experiment, an attempt was made to determine if symbol configuration affects the learning of highly complex Blissymbols that bear little relationship to their referents. A paired-associate learning paradigm was used to present 30 Blissymbols to 20 cognitively normal preschool children. These symbols were evenly divided into superimposed and nonsuperimposed groups. Results indicated that the children learned more superimposed symbols than their nonsuperimposed counterparts. The implications of this finding for the teaching of Blissymbols are discussed.


Interactions of lithium with the adrenergic system in electrically-stimulated guinea pig myenteric plexus isolated preparations. Actions of a phosphodiesterase inhibitor.

The possible involvement of lithium in the mechanism of action of norepinephrine has been studied in electrically-stimulated preparations isolated from guinea pig myenteric plexus. Results show that concentrations of lithium above 0.5 x 10(-2) M significantly inhibit the norepinephrine effect. The results obtained when preparations were preincubated with alpha-adrenergic blocking agents (phenoxybenzamine and phentolamine) suggest a beta-adrenergic action of lithium since these substances induced 74% and 37% inhibition of the lithium effects, respectively. When preparations were preincubated with beta-adrenergic blocking agents (propranolol, toliprolol, atenolol and sotalol) the action of lithium was unchanged. A phosphodiesterase inhibitor also led to 50% inhibition of the lithium effects. These results, together with the fact that the adenylate cyclase cAMP system is linked directly to the beta-adrenoceptors, suggest that the inhibitory action of lithium on norepinephrine, in this preparation, is related to its beta-adrenergic action, which agrees with the results obtained in brain by other authors.


The effect of hypoxia on brain edema--the promoting effect of superimposed hypercapnia or hypertension.

The effects of hypoxia and superimposed hypercapnia or hypertension during hypoxia on brain tissue water content, pH, and electric activity were studied in Sprague-Dawley and stroke-prone spontaneously hypertensive rats. Auditory brainstem responses and sensory evoked potentials were recorded during the experiment as the indices for cerebral oxygen metabolism. The brains were removed immediately, 1 day, and 2 days after hypoxic insult for gravimetric study. The brain water content increased in all groups on the 1st and 2nd days after hypoxia. The percentage change from the control water content increased only on the 1st day in hypoxic rats. In contrast, it increased on both the 1st and 2nd days after hypoxia in hypercapnic or hypertensive rats. The evoked potentials of hypoxic and hypercapnic-hypoxic rats showed that peak latencies were prolonged significantly during hypoxia and recovered 1 and 2 days after hypoxia. The brain tissue pH decreased during hypoxia and recovered after hypoxia. This study suggests that brain edema develops within 2 days of hypoxic insult and that superimposed hypercapnia or hypertension promotes the brain edema.


Effectiveness of interferon-beta, ACNU, and radiation therapy in pediatric patients with brainstem glioma.

Sixteen pediatric patients with brainstem glioma were treated with a combination of interferon-beta, 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride (ACNU), and radiation therapy (IAR therapy). All patients received 1-1.5 million IU/day of interferon-beta intravenously for 1 week of each 6-week cycle. In addition, ACNU (2-3 mg/kg) was given on the 2nd day of each cycle. Conventional focal irradiation (1.5-2 Gy/day for 5 days to a total dosage of 40-60 Gy) was administered beginning on day 3. Patients underwent at least two 6-week cycles. Adverse effects included nausea, vomiting, and myelosuppression, but were mild and transient. Response to treatment was evaluated by the reduction in tumor size measured on postcontrast computed tomographic scans and magnetic resonance images. Responses occurred in 10 of 11 patients with the intrinsic type of brainstem glioma, including three complete and seven partial responses. Two of five patients with exophytic type gliomas partially responded. The median survival was 15.7 months, a remarkable improvement over the natural course of this disease. These results indicate that IAR therapy is a useful primary treatment for pediatric patients with brainstem gliomas.


Prognostic factors in patients surviving for more than 1 or 5 years after removal of metastatic brain tumors.

Favorable and unfavorable prognostic factors were identified in 28 patients surviving for more than 1 year after surgery for metastatic brain tumor and 18 patients surviving for more than 5 years. A high incidence of favorable factors, including neurological grade and location of the brain lesion, indicated significance in the prognosis. A low incidence of favorable factors, including tumor-free interval and presence or absence of extracranial tumor, indicated little significance to the prognosis.


Suprachiasmal carotid-ophthalmic artery aneurysm--report of two cases.

Two rare suprachiasmal carotid-ophthalmic artery aneurysms, one large and one giant, were discovered incidentally. The patients had no visual disturbances. Angiography showed superomedial projection of the sac. The aneurysms were clipped via an ipsilateral pterional approach. A suprachiasmal carotid-ophthalmic artery aneurysm is indicated when preoperative angiography reveals a superomedial carotid-ophthalmic artery aneurysm without visual disturbances. Direct surgery to clipp a suprachiasmal aneurysm should be carried out to prevent rupture of these frequently large aneurysms.


Subfrontal schwannoma--case report.

A rare case of subfrontal schwannoma occurred in a 33-year-old male with the chief complaint of headache. Computed tomography demonstrated a low-density mass in the subfrontal region. Magnetic resonance imaging indicated the mass extension into the ethmoidal sinus. The tumor was totally removed via a subfrontal approach. The histological diagnosis was schwannoma. The most likely origin of the tumor is the meningeal branches or anterior ethmoidal nerve.


Abnormal continuous flow in giant intracranial aneurysm detected by transcranial Doppler sonography--case report.

Transcranial Doppler sonography demonstrated a huge, non-thrombosed intracranial aneurysm in a 62-year-old male preoperatively as abnormal continuous flow, which disappeared after aneurysmal neck clipping. Transcranial Doppler sonography is very useful to assess hemodynamics in the parent artery and the dome of the aneurysm.


Aneurysm of the cervical internal carotid artery associated with Marfan's syndrome--case report.

An aneurysm of the internal carotid artery associated with Marfan's syndrome occurred in a 23-year-old female with a 2-year history of a pulsating lesion in the left neck, which progressively increased in size. Left carotid angiography demonstrated a giant saccular aneurysm at the origin of the internal carotid artery. The aneurysm was excised and end-to-end anastomosis performed without postoperative morbidity.


Contralateral development of acute subdural hematoma following surgery for chronic subdural hematoma--case report.

An unusual case of acute subdural hematoma developed after drainage of chronic subdural hematoma in a 71-year-old male. The acute subdural hematoma was located over a membranous layer in the subdural space similar to the outer membrane of the chronic subdural hematoma. Intraoperatively, bleeding from the bridging vein and oozing from the superior sagittal sinus were observed. The membranous layer probably separated from the dura mater following decompression after drainage of the contralateral hematoma, and this separation then damaged the bridging vein and superior sagittal sinus, resulting in the acute subdural hematoma.


Papillary craniopharyngioma of the third ventricle--case report.

A papillary craniopharyngioma localized in the third ventricle occurred in a 45-year-old male. The clinical presentation was unusual and the neuroradiological appearance resembled a choroid plexus papilloma of the third ventricle. The tumor originated from the right anterolateral wall of the third ventricle, forming a discrete mass with prominent papillae formation. The tumor was totally removed by a transcallosal approach without neurological or endocrinological sequelae.


Malignant fibrous histiocytoma in the skull--case report.

A 45-year-old male presented with a large calvarial mass caused by a malignant fibrous histiocytoma arising primarily from the frontal bone and extending into the intracranial space. Magnetic resonance imaging revealed a well-demarcated, mottled enhanced bulky tumor with several low-signal separations. He underwent extensive tumor and bone removal, followed by radiation therapy. Histological examination showed pleomorphic spindle cells in a storiform pattern. The tissue stained positive for alpha-1-antitrypsin and alpha-1-antichymotrypsin by immunohistochemical techniques.


Variant forms of galanin isolated from porcine brain.

In a peptide concentrate, prepared from acid extracts of porcine brain, several galanin-like immunoreactive peptides were detected and two of these were purified. Characterization of the peptides by sequence analysis, mass spectrometry, and capillary zone electrophoresis identified them as a N-terminally nine residue elongated form of galanin, preprogalanin(24-61) amide, and as an N-terminally four residue truncated form of galanin corresponding to preprogalanin(37-61) amide. The former finding suggests that the removal of the signal peptide in preprogalanin occurs by enzymatic cleavage between glycine-23 and leucine-24. The presence of truncated galanin might refer to a mechanism, where galanin is inactivated by removal of functionally important amino acid residues from the N-terminus.


Orofacial pain increases mRNA level for galanin in the trigeminal nucleus caudalis of the rat.

The changes of preprogalanin mRNA levels in the superficial dorsal horn neurons (laminae I and II) of the trigeminal nucleus caudalis in response to orofacial pain induced by the injection of 5% formalin into the lips of rats was investigated and compared to those of preproenkephalin A mRNA and preprodynorphin mRNA in the same region by means of in situ hybridization histochemistry. Rapid and marked increases of preprogalanin and preprodynorphin mRNA were observed on the side of the injection, but the increase of preproenkephalin A mRNA level was less pronounced than that of the other two mRNAs, indicating that these peptides have different roles in the dorsal horn analgesic mechanism and that galanin, in addition to opioid peptides, may have a highly specific role in this mechanism.


Circulating neuropeptide Y in dog plasma consists of multiple peptide fragments.

Neuropeptide Y-like immunoreactivity (NPY-LI) in dog plasma was characterized and quantified using three extraction methods (Sep-Pak:acetonitrile, HCl:ethanol, and ethanol). Sep-Pak extraction yields the best recovery and preserves the integrity of the peptide. Oxidized NPY is not generated during blood collection. Using two antisera of different specificities, at least three peptide forms in normal dog arterial and venous plasma were detected. A peptide with retention times similar to oxidized NPY or peptide YY is the major component of plasma NPY-LI under basal conditions, but NPY(1-36) predominates during sympathetic stimulation. The mature peptide in dog plasma is similar to human NPY. The antiserum ABII provides a more accurate measure of circulating NPY(1-36) and its oxidized form. The antiserum ABI is useful for detecting NPY-like fragments.


The role of rigid oesophagoscopy in oesophageal carcinoma.

The efficacy and safety of rigid oesophagoscopy in diagnostic and therapeutic settings in a consecutive series of 404 patients with oesophageal carcinoma were studied and compared to that for flexible oesophagoscopy in the same group. In addition, we examined the same parameters in a smaller group who had undergone radiotherapy with subsequent malignant stricturing. We performed 328 rigid procedures and 118 flexible procedures in a single regional surgical referral unit over a 7 year period. The combined perforation rate was 1.3%, with an overall mortality of 1% from 446 procedures. We conclude that rigid oesophagoscopy in the presence of carcinoma retains an important diagnostic and therapeutic role which can be achieved with low incidence of perforation in high-risk patients.


Prenatal diagnosis of spina bifida aperta after first-trimester valproate exposure.

In the context of a prospective study on the adverse effects of anti-epileptic drugs on fetal outcome, we evaluated our experience with prenatal diagnosis by ultrasonography and alpha-fetoprotein (AFP) determination in amniotic fluid. We compared these results with AFP values in maternal serum obtained prior to amniocentesis. From November 1985 to July 1990, amniocentesis at 16-18 weeks of gestation was performed in 267 pregnancies of 237 different women using anti-epileptic drugs. Among 92 pregnancies with maternal valproic acid use, five (including one concordantly affected monozygotic twin-pair) were terminated because of a spina bifida aperta, all prenatally diagnosed by AFP determination and acetylcholinesterase electrophoresis in amniotic fluid. The maternal serum AFP level was raised (> or = 2.5 multiples of the median (MOM) for singleton pregnancies and > or = 4.5 MOM for twin pregnancies) in only two of these five affected pregnancies. We emphasize that maternal serum AFP levels may be unreliable for prenatal screening for fetal neural tube defects in women taking valproate and recommend that amniocentesis and fetal ultrasound examination should be offered directly.


Prospective intervention trial of a screening protocol to identify fetal trisomy 18 using maternal serum alpha-fetoprotein, unconjugated oestriol, and human chorionic gonadotropin.

Two prenatal centres in New England, routinely using a screening protocol for fetal Down syndrome that included maternal serum alpha-fetoprotein (AFP), unconjugated oestriol (uE3), and human chorionic gonadotropin (hCG) measurements in combination with maternal age, adopted a separate screening protocol for trisomy 18. That protocol identified a pregnancy as being at high risk when AFP, uE3, and hCG measurements all fell at or below specified cut-offs (0.75, 0.60, and 0.55 multiples of the median, respectively), regardless of maternal age. Among the first 19,491 women screened, 98 (0.5 per cent) were found to have values which placed them in the high-risk category. Four of these women were subsequently found not to be pregnant. In two others, samples from non-pregnant individuals were found to have been incorrectly submitted for analysis in place of the samples from the pregnant women. All of the remaining 92 women were counselled and offered amniocentesis and fetal karyotyping. Eighty-eight (96 per cent) accepted. Karyotypes or birth outcomes were available on all 92 pregnancies. Six cases of trisomy 18 and one case of Turner syndrome were identified by karyotype. One case of trisomy 18 was identified for every 14 unaffected pregnancies offered amniocentesis. In the present prospective study, an estimated 85 per cent of the cases of trisomy 18 were identified. However, given the small number of cases (six), the 95 per cent confidence interval for the detection rate is broad (40-95 per cent).


Regulation of galanin secretion from pituitary cells in vitro by estradiol and GHRH.

The effects of estradiol and growth hormone-releasing hormone (GHRH) on galanin release from anterior pituitary cells were examined in vitro. 17-beta-Estradiol (0.001-10 nM) increased galanin secretion from anterior pituitary cells in a concentration-dependent manner. Estradiol (10 nM) increased galanin release 300 and 600% from pituitary cells of ovariectomized and male rats, respectively. Immunocytochemical studies demonstrated that estradiol (10 nM) increased the number of galanin-containing cells twofold after 4 days in culture. Growth hormone-releasing hormone (1 and 10 nM) increased and SRIF (1 and 10 nM) decreased galanin release from pituitary cells of ovariectomized and male rats. We conclude that estradiol increases galanin release by a direct effect on pituitary cells, in part by increasing the number of pituitary cells synthesizing galanin. In addition, GHRH stimulates galanin release when estradiol levels are low.


Selective labeling of alpha- or epsilon-amino groups in peptides by the Bolton-Hunter reagent.

Incorporation of N-succinimidyl-3(4-hydroxyphenyl) propionate (Bolton-Hunter reagent) or its 125I-labeled derivative into peptides can be selectively directed towards either alpha- or epsilon-amine functions by modifying the pH of the reaction. Acylation of alpha-amino groups is favored at pH 6.5 whereas epsilon-amino groups react more readily at pH 8.5. We have taken advantage of this result to prepare two new 125I-labeled analogues of substance P and neurotensin that bind selectively and reversibly to their respective receptors. The method described here is of general interest and can be used to incorporate various reporter groups into peptide structures.


Synaptic connections of physiologically identified geniculocortical axons in kitten cortical area 17.

Single geniculocortical axons were recorded in the cortical white matter of kittens and adult cats by using micropipettes filled with horseradish peroxidase (HRP). Of 41 axons recovered in 4-5 week old kittens, three well-filled axons arborized in area 17; the remainder were incomplete or arborized in area 18. One axon had Y-like physiological properties, two were X-like. They were recovered from two 34-day-old kittens. All three axons formed clustered arborizations, mainly in layer 4A. Electron microscopic (EM) analysis of 50 boutons from kitten and 38 boutons from adult controls revealed that the boutons from kitten made synapses more frequently on spines (91% of targets) than did the boutons from the adult (71%). One X-like axon in kitten also had a collateral projection that made synapses in layer 1; this has not been seen in adult cats. In overall extent, the axons from kitten fell within the adult range.


Reciprocal role of the inward currents ib, Na and i(f) in controlling and stabilizing pacemaker frequency of rabbit sino-atrial node cells.

Experiments and computations were done to clarify the role of the various inward currents in generating and modulating pacemaker frequency. Ionic currents in rabbit single isolated sino-atrial (SA) node cells were measured using the nystatin-permeabilized patch-clamp technique. The results were used to refine the Noble-DiFrancesco-Denyer model of spontaneous pacemaker activity of the SA node. This model was then used to show that the pacemaker frequency is relatively insensitive to the magnitude of the sodium-dependent inward background current ib, Na. This is because reducing ib, Na hyperpolarizes the cell and so activates more hyperpolarizing-activated current, i(f), whereas the converse occurs when ib, Na is increased. The result is that i(f) and ib, Na replace one another and so stabilize nodal pacemaker frequency.


Different channel-gating properties of two classes of cyclic GMP-activated channel in vertebrate photoreceptors.

We report that two types of cGMP-activated channel coexist in the photoreceptor plasma membrane, with the most commonly encountered class appearing broadly similar to the channel reported in previous patch-pipette experiments. However, we find that flickering of this channel between the open and closed states is so rapid that a discrete single-channel conductance cannot unequivocally be resolved; the occurrence of flickering is largely independent of membrane voltage and of the presence of cytoplasmic Ca2+ or Mg2+. In recordings from the inner segment we occasionally find a second class of cGMP-gated channel, with activity resembling that reported for cloned channels. This channel does not flicker, but instead exhibits distinct open-close transitions. Our results suggest that the predominant form of channel in vivo differs significantly from cloned channels, and that its gating properties are not as simple as reported previously.


Poisson sampling-based inference for single ion channel data with time interval omission.

Patch-clamp recording allows investigations of the gating kinetics of single ion channels. Statistical analysis of kinetic data can enhance our understanding of channel gating at a molecular level. Experimental channel records suffer from time interval omission, i.e. failure to detect brief channel openings and closings. It is important to incorporate this phenomenon into statistical analyses of ion channel data. When time interval omission is ignored, the method of maximum likelihood can usually be used to estimate gating parameters from a single channel record. However, it is far more difficult to apply this method when time interval omission is incorporated. We present an alternative approach to parameter estimation based on Poisson sampling. A simulated homogeneous Poisson process is superimposed onto the channel record and inference is based on the numbers of points in successive open and closed sojourns, rather than on the sojourn times themselves. We describe the method for the two-state Markov model C<-->O, although it is applicable to more general models. Computer-simulated data are used to demonstrate the efficacy of the method. Modifications of the method are discussed briefly.


Single-channel conductances of NMDA receptors expressed from cloned cDNAs: comparison with native receptors.

To cast light on the subunit composition of native NMDA-type glutamate receptors, four cloned subunits of the NMDA receptor have been expressed, in pairs, in Xenopus oocytes, and their single-channel properties have been measured. The conductances of the channels, and their characteristic patterns of sublevel transitions, turn out to be useful diagnostic criteria for subunit composition. The NR1-NR2A and NR1-NR2B combinations (which have identical TM2 sequences) are very similar to each other. Both have 50 pS openings and brief 40 pS sublevels (in 1 mM external Ca2+), with similar mean lifetimes and frequencies. They also show close quantitative resemblance to the channels of hippocampal CA1 and dentate gyrus cells and of cerebellar granule cells, except that the NR1-NR2A combination has a lower glycine sensitivity than the native channels. In contrast, the NR1-NR2C combination produces a channel with 36 pS and 19 pS conductances of similar (brief) duration; these closely resemble the 38-18 pS channels that have previously been observed in large cerebellar neurons in culture (together with 50 pS channels).


Rapid decay of averaged single-channel NMDA receptor activations recorded at low agonist concentration.

The NMDA class of glutamate receptors have the unique property of binding some agonists, including glutamate, for a very long period of time. One manifestation of this is that brief (1 ms) application of glutamate (1 mM) produces a slowly decaying current, the major component of which has a time constant of approximately 200 ms. Application of glutamate at low concentrations allows identification of groups ('superclusters') of openings in the data record that probably correspond to a single period during which one or more molecules of glutamate are bound to the receptor, i.e. a single activation of the channel. The length of such superclusters is long on average (74 ms); the longest component of the distribution has a duration of approximately 300 ms, and comprises about 25% of the area. However, aligning many superclusters to obtain an average current reveals that the decay is mainly fast; the major component has a time constant of around only 5 ms. It is shown that incorporation of a distribution of first latencies (from the time of the jump to the first opening) can explain at least part of this discrepancy.


A combination of human alpha 1 and beta 1 subunits is required for formation of detectable GABA-activated chloride channels in Sf9 cells.

The baculovirus expression system was used to produce alpha 1 and beta 1 subunits of the human GABAA receptor in Sf9 cells. In cells infected with both alpha 1 and beta 1 recombinant viruses, GABA elicited an outwardly rectifying chloride current that was blocked by bicuculline and potentiated by pentobarbitone. GABA did not produce detectable currents in cells infected with either alpha 1 or beta 1 recombinant viruses alone. In these cells, and in control (non-infected) Sf9 cells, pentobarbitone depressed the leakage current (Ki = 55 microM). Fluorescently labelled monoclonal antibodies to the alpha 1 subunit showed greater amounts of the alpha 1 subunit in cells infected with only the alpha 1 recombinant virus than in cells co-infected with the alpha 1 and beta 1 recombinant viruses. Fluorescence of the plasma membrane was seen in cells co-infected with the alpha 1 and beta 1 recombinant viruses, but was absent in cells infected with only the alpha 1 recombinant virus. It was concluded that the alpha 1 subunit normally interacts with the beta 1 subunit to be transported to the plasma membrane in Sf9 cells.


[The autistic child, his thoughts, his body and his images. Or the footprint of a step not yet taken].

The author studies the autistic mind through the concept of image. This concept is differentiated from the processes of representing (and mainly self-representation), figuration and communication. The author then discusses different aspects of the image: mental image, mirror image and physical image. Finally is discussed the narcissistic or objectal polarity of image production in the autistic child and the author argues that in these cases the transformation from the original to the primary level is essentially disturbed.


Biochemical and morphological alterations in the lungs and livers of mice following exposure to polluted air in a traffic tunnel.

The air in the Lin-Shen tunnel in Taipei is heavily polluted due to heavy traffic. To elucidate the biologic effects of air pollution, we studied the biochemical alterations of Polycyclic aromatic hydrocarbons metabolic enzymes and histological changes in the livers and lungs in experimental mice under long-term and short-term exposure to the tunnel air. The concentrations of 16 Polycyclic aromatic hydrocarbons in the air were measured by HPLC, and the mutagenicity of the extract of air particulates was assayed by the Ames test. We found that the benzo(a)pyrene hydroxylase and cytochrome P-450 in the livers and nitroreductase in the lungs of the 30-day exposed animals were significantly increased as compared to the unexposed control (p < 0.005). The nitroreductase in the liver also increased but not significantly. The lungs of long-term (6 mo.) exposed mice showed anthracosis, chronic inflammation, and emphysema (10/10). The livers of the experimental animals did not show significant alteration. A whitish circular nodule appeared on the liver surface of one mouse (1/10). Microscopically, the nodule showed necrosis and hyperchromatic cells around the central vein. It is concluded that the polluted air in the traffic tunnel has evident biohazardous effects on the lungs and livers of mice under the experimental conditions.


[Benign prostatic hypertrophy: the therapeutic options].

BPH is a very diffuse condition among males increasing in incidence with the age. This explains the large interest in the evaluation of the different strategies concerning BPH treatment. The gold standard has still to be considered the transurethral resection of the prostate which all the other treatments must be compared to. More recently as alternatives to the invasive therapy, so called minimally invasive treatments have been proposed (thermotherapy, prostate dilatation, intraprostatic stents) whose real significance is not completely evaluable yet. As far as medical treatment is concerned, phyto-therapeutic agents have been partially replaced by different substances (alfa- blockers; 5-alfa reductase blockers) that are expected to have a promising future.


Endothelin, vasopressin, and substance P like immunoreactivity in cultured and intact epithelium from rabbit trachea.

The control of airways reactivity is essential to our understanding of disease processes such as asthma. Many studies have examined the neural control of the airways, but more recently there has been evidence to show that the epithelium lining the airways may influence airways reactivity. Rabbit tracheal epithelial cells were dispersed with enzymes, grown in primary culture and fixed. Tissue from intact tracheas was also sampled and fixed. Localisation of the vasoactive substances endothelin-1, arginine-vasopressin, and substance P was investigated by immunolabelling techniques. Scattered immunolabelling to endothelin-1, arginine-vasopressin, and substance P was found throughout the cultures (with < 20% of cells staining positively to each antibody). At the ultrastructural level this immunoreactivity was found in the cytoplasmic matrix. In addition, immunoreactivity of intact tissue to endothelin-1, arginine-vasopressin, and substance P was examined and positively staining cells were found to be scattered through the epithelium. The presence of these vasoactive substances within the epithelium lining the airways supports the view that epithelial cells may provide an additional mechanism in the control of airways reactivity.


Nodular lesions of the liver in multiple myeloma: a role for cytoadhesion molecules?

Two patients with multiple myeloma and in vivo macroscopic nodular lesions of the liver are presented. The clinical aspects of this very unusual condition are briefly reviewed. In particular, the expression on neoplastic plasma cells of the cytoadhesion molecules CD56 and CD11a, which are involved in the cellular process of recirculation and homing, suggests a possible role for such markers in this atypical localization of the disease.


Epirubicin, methotrexate and bleomycin (EMB) in the treatment of recurrent epidermoid cancer of the head and neck.

During the period May 1989 to November 1990, at the "O. Alberti" Radium Institute of Brescia's General Hospital, 35 patients affected by epidermoid head and neck carcinoma were treated every 28 days with the salvage chemotherapy regimen EMB (epirubicin, 50 mg/m2 i.v. day 1; methotrexate, 40 mg/m2. i.v. days 1, 18; bleomycin, 10 mg/m2 i.v. days 4, 11, 18). Sixteen patients had been previously treated with surgery, 15 with radiotherapy and 4 with chemotherapy. Six patients (Group A) received only 1 cycle of chemotherapy because of disease progression and subsequent death. In another 15 patients (Group B) it was possible to administer 2 cycles of EMB, and 9 of them showed local disease progression and died. Among the remaining 6 patients, evaluated as PR, 1 refused further therapy and 5 were amenable to a previously impossible radiotherapy (4 of them are still alive). Fourteen patients received 3 or more cycles of EMB (Group C): 8 subjects showed progression and died; 1 reached CR and is alive without any evidence of tumor; 5 are in PR (3 of them underwent subsequent radiotherapy and 1 chemotherapy with CDDP). Out of 35 patients, 12 (34%) reached a favorable response (CR or PR) and 8 (22%) are still alive. As regards toxicity, the following adverse events were recorded (< or = 2 Miller's scale): leukopenia (8.5%), thrombocytopenia (5.7%), anemia (14.2%), stomatitis (5.7%), vomiting (5.7%), alopecia (8.5%), and fever (11.4%). It can be concluded that the EMB regimen is very well tolerated and shows good effects in the treatment of patients with relapsed head and neck carcinoma.