[An experimental model of apneusis and periodic respiration].

In experiments on sodium pentobarbital (40 mg/kg, i. p.) anesthetized mongrel cats of either sex weighting from 2.0 to 4.0 kg, it was stated, that sodium or lithium hydroxybutyrate (HOB) (200 mg/kg, i. v.) effectively slowed down breathing with inspiratory holdings. Thus 3-5 minutes after HOB administration, eupneic pattern of respiration was changed firstly to inspiratory apneustic one (100% of cats), and then to periodic one (80% of cats). This pattern persisted for 60-90 minutes, and after that the respiratory pattern usually changed its direction to the opposite one. In these conditions alterations of arterial blood composition (pH, pO2, pCO2, SO2) and hemodynamic variables (heart rate, arterial pressure) can not be considered as the cause of apneustic pattern of respiration. It is suggested, that HOB can be used for simulating such terminal respiratory patterns as apneustic and periodic ones.


[Aldosterone and electrolyte content in blood and myocardium of rats after single physical load in acute alcoholic intoxication].

It is shown that acute alcoholic intoxication causes essential changes of aldosterone content and balance of electrolytes in blood plasma of Wistar rat males. The SPL test revealed the inadequate reaction in the mineral-corticoid function of the adrenal glands in the rats with acute alcoholic intoxication. The method of the free choice of the load by the experimental rats is the most optimal way to assess. The rats with acute alcoholic intoxication displayed smaller tolerance to physical load.


[Organization of reflex sympathetic influence on rate and force of cardiac contraction].

In acute experiments on 21 cats it was proved that the change of afferent impulse on vagus nerves by means of either freeze-block or electrostimulation of their central ends results in differential reflex influences on rhythm and force of the cardiac contractions caused by sympathetic nervous system. The cut of the lower cardiac nerves may cause 'break-up' of the observed reflex, removing or inverting its ino- or chronotropy component. The given phenomenon was revealed in the experiments with high arterial pressure and with absence of tonic chronotropy influences of the left lower cardiac nerve.


[Effects of inflammation on adhesive interactions of keratinocytes].

The reversible prolonged increase of intercellular adhesion in the epidermis along the margins of the wound as well as in the scar epidermis during the full-thickness skin wound healing was found. This effect must have adaptive significance.


[Quantitative characteristics of interactions of blood lipoproteins and steroid hormones].

Human and serum lipoproteins interaction with steroid hormones (corticosterone and hydrocortisone) were studied. Methods of fluorescence quenching titration and equilibrium dialysis were used for quantitative evaluation of VLDL, LDL and HDL glucocorticoids binding ability. Association constants were found to be 0.6-2.0 x 10 M for corticosterone and 4.0-8.0 x 10 M for hydrocortisone. The number of binding sites ranged from 3 to 300 for different classes of lipoproteins. Our data suggest high specificity of serum lipoproteins binding with corticosterone and hydrocortisone.


[Antioxidant activity of high density lipoproteins in vivo].

The paper deals with antioxidant effect of high density lipoproteins (HDL) in vivo. The effect of intravenous injection of large-dose HDL3 (200 mg protein), isolated from human plasma to rabbits with experimental hypercholesterolemia, on the content of primary products of lipid peroxidation in rabbit blood, the correlation between HDL cholesterol level and the content of lipid peroxide products in blood plasma of healthy persons and patients with IHD were studied. Intravenous injection of HDL3 to rabbits with experimental hypercholesterolemia has been shown to lead in 6 hr to a 24-hr significant (p < 0.01) decrease of conjugated dienes and trienes. The existence of negative correlation between HDL-cholesterol level and the content of conjugated dienes in blood plasma of healthy persons (N = 47) and patients with IHD (n = 64) is revealed. Basing on the data obtained conclusion of universal character of protective antiatherogenic effect of HDL is drawn.


[Activation of free radicals reaction and changes in the state of antioxidant protection in blood in toxic experimental influenza infection].

The paper presents an experimental model of toxic influenza infection. The toxic form of influenza was shown to result in the activation of free radical processes accompanied by the accumulation of both lipid peroxidation products and methemoglobin and the decrease of alpha-tocopherol in erythrocytes, by the accumulation of NO. and nitrizyl complexes of heme iron in the blood. The activation of free radical processes was followed by the stimulation of antioxidant system in the blood. Thus, there was an increase of both superoxide dismutase activity in erythrocytes and ceruloplasmin content in the blood. The data obtained support the important role of the active products of free radical processes in the development of toxicosis under acute virus infections.


[Microsomal hydroxylating system of the mouse liver in toxic forms of influenza infection].

The paper presents the experimental model of toxic influenza infection induced by A/Victory/35/72 (H3N2) strain adapted to CBA mice. The virus toxicosis was shown by means of ESR technique to be accompanied by a decrease of both the content of the active form of cytochrome P-450 and the activity of p-nitroanisole o-demethylase. In microsomes there was activation of lipid peroxidation (LP) and an increase of microviscosity of lipid matrix. LP activation in microsomes was not accompanied by the change of alpha-tocopherol content.


[Effects of cerebrocrast on local cerebral circulation and EEG in cats with cerebral hemorrhage].

In experiments on awake cats before and after intracerebral hemorrhage influence of cerebrocrast and nimodipine intravenous infusion (1 microgram/kg-1/min-1 during 60 min) on cerebral blood flow (CBF) in the cortex, thalamus and reticular formation and power of (alpha + beta)-, theta-, and delta-waves was investigated. During cerebrocrast infusion increasing CBF and improving bioelectrical activity were demonstrated.


[Dynamics of calmodulin in cerebral structures under the action of modulated UHF electromagnetic fields].

The influence of modulated UHF-electromagnetic fields (low intensity) on calmodulin levels in several brain structures was studied. It was shown that UHF-electromagnetic fields influence calmodulin levels in the hypothalamus and sensorimotor cortex. Its effect depends on modulation regimes.


[Activation of ATP-sensitive K+-channels of cardiomyocytes by endogenous cardiopeptides].

The "patch-clamp" method was applied to rat ventricular cardiomyocytes to study the effect of endogenous cardiopeptides (a component of a cardiologic medicine "cardialin") on single K(+)-channel currents. It was noted that ATP-sensitive K(+)-channel activity increased only in the presence of 10(-8)-10(-6) mg.ml-1 peptide solution in the patch-clamp micropipette. To check up the suggestion that G-protein takes part in a direct reaction of K(+)-channel by external ligand we studied the effect of GTP-gamma-S (0.1 mM) in the presence of Mg2+ (1 mM) on the K(+)-channel in "run-down" state in the "inside-out" configuration. It was demonstrated experimentally that reactivation of the ATP-sensitive K(+)-channel develops in the presence of GTP-analogue.


[Prolonged action of tuftsin in penicillin epilepsy].

The free behaviour experiments on rats showed during the first day the tuftsin-induced (0.3 mg/kg i. p.) increment of epileptic discharge in all the structures simultaneously. Open field behaviour depended on the sequence of penicillin and tuftsin injections. The rats with tuftsin used after the epileptiformic activity has been developed showed the behaviour analogical to the penicillin-injected animals. The preventive injections of tuftsin resulted in restoration of all behavioural indications by the third day. The optimal corrective effect has been obtained with tuftsin used preventively.


[Mechanisms of hepatoprotective action of new nicotinic acid derivatives in experimental CCL4-induced liver injury].

New biologically active compounds (BAC) created on the basis of nicotinic acid possess hepatoprotective action. The preparations were introduced preventively in doses of 10 mg/kg during 14 days. Litonit and nicogamol increased survival of experimental animals by 36.8% and nicotinic acid by 26.8%. ALT, AST, GGT activity in the blood serum was reduced. The activity of the main antioxidant enzymes (SOD and catalase) grew in the rat liver tissue in parallel with inhibition of DK and MDA activity. Morphological picture of the rat liver, most evident after application of litonit improved. Hepatoprotective action of these BAC are attributed to their membrano stabilizing effects.


[Non-quantal secretion of a mediator as factor determining consequences of acetylcholinesterase inhibition].

The mechanism of shortening MEPC decay phase after initial prolongation due to acetylcholinesterase inhibition by armine and neostigmine was studied by use of two-electrode voltage-clamp at the mice diaphragm Factors which switch off non-quantal secretion of acetylcholine from the nerve (acute denervation in vitro, ouabain, high concentration of magnesium ions) only slightly reduced the prolongation of MEPC caused by AChE inhibition. So, postsynaptic potentiation of MEPC by nonquantal ACh is not significant immediately after AChE inhibition. At the same time these factors abolished the process of shortening MEPC decay phase. It is concluded, that desensitization of the postsynaptic membrane induced by nonquantal ACh is the main mechanism of the MEPC shortening and that this mechanism can compensate insufficient AChE activity.


[Development and use of a method of determination of specific immunoglobulin G based on immunoenzyme analysis in the diagnosis of pulmonary aspergillosis].

Enzyme-linked immunoassay (ELISA) was employed for determination of specific immunoglobulin G to Aspergillus fumigatus (A.f.). Culture filtrate of A.f. was used as antigen. Optimal conditions of ELISA were found. Serum of 138 asthmatic patients with total IgE level more than 500 PRU/ml were analysed for determining the limits of IgG to A.f. concentrations.


[Effects of alpha-interferon on the level of adenosine metabolism enzymes and bactericidal activity of macrophages in staphylococcal infection].

In staphylococcal infection the changes in functional ability of macrophages occur: their oxygen-depending bactericidity and adenosine-desaminase activity are depressed 5-nucleotidase ability increases. Introduction of homologous alpha-IFN in the dose of 1 x 10(3) u/mouse leads to enhancing macrophage bactericidity of the animals infected, inhibits their 5-nucleotidase activity and enhances adenosine desaminase one. Influence of alpha-IFN on the activity of adenosine metabolism enzymes in macrophages can be considered one of the most important mechanisms of its modulating effect in bacterial infections.


[Effects of modulators and cytostatics on catabolism of I-125 desoxyuridine in tumors (express-method of antineoplastic modulator screening)].

El-4 and P-815 murine tumor cells labelled by 125I-deoxyuridine or 51Cr were administered in 7-day subcutaneous syngeneic tumors or subcutaneously. At the same time different groups of mice were treated by LPS plus MDP, beta-C7H15-MDP, dexal-MDP, polyacrylamide-MDP-phosphatidylethanolamine, adriblastin or cyclophosphamide. It was shown that cytostatics and immunomodulators significantly delayed catabolism and withdrawing of 125I-deoxyuridine (that has not been incorporated in DNA) from tumor cells. This delay was correlated with the inhibition of tumor nodes growth rate. It is concluded that influence of cytostatics and immunomodulators on catabolism and withdrawing rate of 125I-deoxyuridine from tumor cells relates to their cytostatic effect and may be used at the earliest screening step of immunomodulator analysis.


[Radiation protective properties of a synthetic antagonist of glucocorticoids RU 38486].

Radioprotective properties of synthetic antiglucocorticoid RU 38,486 were investigated. It was demonstrated that this antagonist of glucocorticoids possesses radioprotective effect in vitro and in vivo systems. Radioprotective properties at molecular level exhibited in inhibition of postirradiation endonuclease activation and in prevention of internucleosome chromatin degradation. Involvement of cytosol glucocorticoid receptors in initiation of radiation-induced programmed cell death is discussed.


[Effects of atrial natriuretic peptide AP II on the morphofunctional state of the adrenal cortex of white rats].

The effect of atrial natriuretic peptides synthetic analog AP II on adrenal zona glomerulosa and zona fasciculata physiological regeneration have been studied on male rats. The 3H-thymidine incorporation into DNA in adrenal cortical cells was evaluated in 4 and 24 h after intraperitoneal injection of 10 or 100 mcg/kg AP II. Besides, we have investigated the influence of AP II on adrenal cortical cells karyometric parameter in 4 and 24 h and aldosterone plasma concentration in 1 h after injection. 10 mcg/kg AP II increased the fraction of labelled nuclei in zona glomerulosa and decreased the aldosterone plasma level. No significant changes were seen in zona fasciculata cells proliferation. 100 mcg/kg AP II inhibited the DNA synthesis process in adrenal zona fasciculata, but had no significant influence on zona glomerulosa physiological regeneration and aldosterone plasma concentration. No nuclear morphometric parameter changes were observed in adrenal glomerulosa and fasciculata cells of AP II--treated animals.


[Seasonal and circadian fluctuations in blood biochemical indicators in mice in natural conditions and exposed to constant light].

The fluctuations of activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and those of the levels of protein, glucose, cholesterol, bilirubin, creatinine, blood urea nitrogen, K+, Cl-, Na+ in blood plasma of mice in natural conditions (NC) and exposed to constant light (CL) were studied in different seasons of the year (in January, April, July, October) on days 18, 24, 6 (at 12 o'clock). Most indices both in NC and CL animals had seasonal rhythm similar for each of them. This proves a primary effect of environmental geoclimatic factors of formation of circadian periodicals as compared to desynchronization in constant light revealed by Kosinor analysis in winter (acrophase from 14.16 till 16.32 o'clock) and autumn (acrophase from 23.03 til 4.40 o'clock). During the same seasons one can observe the maximum desynchronization influences of constant light, which leads to abrupt falling (to the 10-fold and more) of the fluctuations amplitude and in some cases to stabilization of circadian rhythm.


[Neural regulatory effects on the contractility of heart ventricles].

As a result of acute experiments on cats we revealed the approaches to choosing the best indices of the contractility of the left and the right cardiac ventricles in the concrete conditions of the experiment on the basis of the original criterion of optimality. For revealing neural inotropy influences we suggest the index based on the combined changes of optimum indices. A new approach to revealing reflex influences on contractility in the conditions of intact blood circulation is worked out, which is based on the comparison of these influences with the control changes of haemodynamic parameters.


[Morphological aspects of broad-plasma lymphocyte migration through ependymal cell of mammalian vascular plexus].

A morphological study of the cerebral vascular plexus was performed to find out the ways of blood cell migration from the vascular lumen into the liquor and in the opposite direction, as well as through ependymocytes. The material of the study consisted of cattle (cows and goats) with the body weight of 500 kg and 30-40 kg, respectively. From each animal species 16 animals were used in the study. Investigational methods included light and electron microscopy. Sequential stages of broad-plasma lymphocyte migration from the vascular lumen through endotheliocytes, capillary basal layer, connective tissue stroma, basal membrane of ependymocytes, as well as through proper ependymocytes have been revealed. The authors refer the phenomenon of broad-plasma lymphocyte migration to the phenomenon of emperipolesis.


[Morphofunctional aspects of genetic analysis of osteogenesis imperfecta in cultured skin fibroblasts].

Osteogenesis imperfecta (OI) is a disease with biochemical evidence of abnormality in collagen biosynthesis. We report here that expression of the OI phenotype extends to the level of dermal fibroblast morphology in vitro. Growth characteristics and morphology of control (n = 3) and 01 cell strains (n = 10) were compared. Our results show that (1) OI fibroblasts take longer time (16 days) than that with control cells (13 days) to reach stationary phase; (2) OI fibroblasts achieve a lower cell density (1.0 +/- 0.09) at stationary phase compared to control cells (1.47 +/- 0.1); p < 0.01; (3) cell shape (expressed as the width/length ratio) was also abnormal in OI cells: they have significantly increased ratios (p < 0.05) compared to control. These changes were associated with an increased level of fibronectin concentration and engorged cytoplasmic vesicles in dermal fibroblasts in vitro. We have reason to suspect that dysmorphology of fibroblasts may be related to aberrant collagen metabolism that leads to inadequacy of extracellular substratum, that in its turn hinders normal adhesion of cells as well as their spreading, morphology and fibronectin concentration.


[Effects of different regimens of hyperbaric oxygenation on transcriptional activity and morphology of frontal cortex neurons in occlusion of the common carotid artery in rats].

The effect of different regimens of hyperbaric oxygenation (HBO) on cortical pyramids morphology and transcription were studied under occlusion of common arteria carotis. The effect of early (3 hours after occlusion) and late (14 hours after occlusion) use of 1.2 and 2.0 ata of HBO was estimated. The rats were killed 15 minutes or 24 hours after HBO session. The effect of early use of HBO was normalizing, stabilizing and independent on the dose. Protecting effect of the late HBO on morphology and transcription activity of nuclear chromatin of pyramids was higher in the 1.2 ata regimen. In case of late HBO both pressures produced depressing effect on the nucleoplasmic chromatin, transcription.


[A method of multivariate analysis of data in the study of the effects of space flight factors on the rat brain neuron structure].

The usage of some methods of multivariate analysis for neuro-morphological studies is discussed. The alterations of dendritic trees of pyramidal neurons of layer III of visual cortex of the rat exposed to the influence of space flight aboard biosputnik "Cosmos-1887" were studied and the results are described to illustrate the methods power. o-technique of factor analysis was shown to be suitable for processing of the data obtained from an experiment with complex environmental conditions.


[Is transferrin a neurotrophic factor controlling myosin composition of the skeletal muscle?].

After transferrin injection to the slow m. soleus of guinea pig no immunohistochemical changes in myosin composition with monoclonal antibodies (AB) against fast myosin heavy chain (HCf) have been found. All muscles fibers in intact and experimental animals were identified as slow ones (type I of muscle fibers). After colchicine treatment of nerve trunk innervating slow muscle, some muscle fibers reacting with monoclonal AB against myosin HCf have been found. However, after colchicine treatment of nerve trunk and transferrin injection also some muscle fibers in slow muscle reacting with monoclonal AB against myosin HCf have been found. In appears that transferrin probably can not be the factor of neurotrophic control for myosin composition in skeletal muscle.


[Effects of minced muscle tissue implantation and subsequent laser therapy on regeneration of skeletal muscles in guinea pigs].

It is known that regenerative capacity of skeletal muscles in guinea pigs is less than in rats. The guinea pig muscle regenerates are characterized by smaller muscle fibers and greater amounts of interstitial connective tissue. The present experiments were designed to stimulate the regenerative capacity of muscle tissue in adult guinea pigs, using tissue and laser therapy. It was shown that the minced muscle tissue implantation to the site of injury in m. gastrocnemius increased the quantity of regenerating muscle tissue. When the same operation was combined with following laser therapy the increase of muscle tissue was observed as well but not by far. At the same time laser therapy promoted noticeable acceleration of fibrin resorption, wound healing and regenerative modifications in implanted muscle fragments. Thus, the present methods stimulated relatively law regenerative capacity of guinea pig muscle tissue and promoted more qualitative recovery of injured area.


Health promotion in the British workplace: a suitable case for treatment?

Over the years, the British workplace has provided a fertile bed for the growth of health-related surveillance, research and intervention. A brief review of the literature on this topic, highlights the plethora of different groups involved, and their differing agenda in relation to the health of the workforce. Key participants, including the workforce, have different perceptions of, and commitment to, the promotion of health. Health promotion should be concerned with reducing inequalities in health experience and opportunity but, because the large majority of British workplaces have no access to appropriate occupational health services, most health promotion activity is concentrated in large profit-making concerns with suitable existing facilities. In this article, the background to health-related initiatives in the British workplace is given, and the various contributions to workplace health promoting activities are noted. Their potential for mis-use is discussed and the need for a more comprehensive and integrated approach to primary care, including health promotion, is suggested. In the present economic climate in Britain there is a need to create a comprehensive database of all health promotion activities so that, with collaboration, integration and coordination of all initiatives, regardless of venue, we can put our present meagre resources to best use.


Tuberculosis screening in health service employees: who needs chest X-rays?

There is uncertainty in the NHS about which individuals should be offered pre-employment screening by chest X-ray and whether this procedure is of value in the detection of tuberculosis. To provide evidence for practice, pre-employment chest X-ray and tuberculin skin test status were examined retrospectively for employees of a health district. Cases were those with an abnormal chest X-ray; referents were a sample of the remainder with a normal chest X-ray. The majority of the population had positive tuberculin skin tests and there was no difference between cases (58 positive out of 68) and referents (170 positive out of 212). Most of the X-ray abnormalities were trivial; four findings were thought significant, but would have caused no problems if undetected and none of these findings was related to tuberculosis. We conclude that in new employees in Hampstead health district, X-ray abnormalities are rare and not predicted by testing tuberculin skin reactivity; neither procedure is justified routinely as a means of screening for tuberculosis. The situation in districts with a high incidence of tuberculosis needs to be investigated.


Investigation into the environmental factors associated with the incidence of skin disease following an outbreak of Miliaria rubra at a coal mine.

A retrospective study was undertaken to examine by interactive linear modeling a possible association between environmental conditions and the incidence of skin conditions (excluding athletes foot) in a coal mine following an increase in skin rashes, mainly 'prickly heat' (Miliaria rubra). An increase in the incidence was found with increasing surface temperature and underground air velocity; a decreased incidence with increased underground air quantity (air volume per unit time); whilst the relationship with underground temperature was found to be quadratic with incidence decreasing until 27 degrees C wet bulb (although this is not necessarily the optimum temperature for other purposes) and then increasing. The association between skin incidence and environmental factors was strongest (in terms of variance explained) when the incidence was associated with the environmental conditions of 4 to 8 weeks previously. The constant nature of relative humidity over the period prevented an examination of its relationship with rash incidence. Possible strategies for reducing the incidence of 'prickly heat' are discussed.


Relationship between occupational health care and absenteeism.

A connection is generally assumed between occupational health care (in Dutch 'bedrijfsgezondheidszorg', or BGZ) and a reduction in absenteeism. In the Dutch literature various authors and researchers make great claims for the effect of occupational health care in reducing absenteeism. As yet, however, they have not concerned themselves very much with providing proof for the validity of their position. They speculate as to the positive influence of occupational health care on the health and welfare of employees and assume a direct connection between it and the reduction of absenteeism, but they do not have at their disposal empirical research results to support their speculations. So far as we know solid research of an experimental or quasi-experimental design into the relationship between occupational health care and absenteeism has never been carried out. Recently, however, an experiment with occupational health care for teaching personnel (in Dutch: Experiment met BGZ voor Onderwijspersoneel, or EBO) did take place, providing the opportunity to determine the influence of occupational health care on absenteeism in a quasi-experimental setting. Unfortunately, the results of the experiment indicate that the relationship between occupational health care and absenteeism is more complex than is generally assumed. In the course of the experiment, and in particular during the final year, the level of absence in the experimental group rose considerably, while in the control group it fell slightly. That rise was mainly due to the increase in the number of persons who were sick for a long period of time (at least 6 months).


Effects of a change in shift work on health.

Aspects of health were studied in a sample of factory workers who changed their pattern of working from 'fortnight about' to three advancing shifts. Within the same factory and doing similar work there was a comparison group who were working permanent days only. The change in shifts was associated with more difficulty with sleep, more indigestion, higher alcohol consumption, reduced alertness and a worse social life. There was more minor psychiatric disturbance in those who worked three shifts than in those who worked days only.


Impact of the new European legislation regarding vocational driving on London Transport Medical Service: the doctor's role.

The outcome of the first 1000 medical examinations undertaken by London Transport Medical Service (LTMS) to comply with the regulations for non-passenger carrying public carriage vehicle licence holders was analysed to assess where the primary role of the examining doctor affected the final outcome. It was found that the primary involvement of the doctor possibly altered the outcome in only two cases. In 49 cases, the medical practitioner had an interpretative role in the final assessment, or in the pursuit of further information, such as clarification of past history from other sources. In these cases, actual contact with the applicant did not affect the final outcome.


Acoustic trauma in a telephone operator.

This case report describes an incident where a telephone operator suffered acoustic trauma from a subscriber blowing a whistle down a telephone mouthpiece, the sound being transmitted into the ear of the operator. Evidence of hearing loss persisted for several months before complete recovery was achieved. Comparison is made with hearing loss associated with the use of cordless telephones.


Skin testing with recombinant Mycobacterium intracellulare antigens.

The immunoreactivity of four recombinant Mycobacterium intracellulare beta-galactosidase fusion proteins, which correspond to 22, 40, 43 and 85 kDa M. intracellulare antigens, was assessed. Lymphoproliferative assays demonstrated that Escherichia coli lysates containing each of the fusion proteins stimulated T cells in vitro. Purified preparations of three of these recombinant M. intracellulare antigens (22, 43 and 85 kDa) also induced delayed-type hypersensitivity (DTH) reactions in sensitized guinea pigs. However, the skin test responses evoked by each of these antigens was not species-specific. Given these results, the potential utility as skin test reagents of the purified antigens or peptides derived from these proteins is discussed.


In vitro activity of meropenem and imipenem against mycobacteria: development of a daily antibiotic dosing schedule.

The activity of two carbapenem antibiotics, meropenem and imipenem, against Mycobacterium species was assessed by means of the Bactec 460 radiometric apparatus. This system allowed a result to be read in 4-10 days. The instability of imipenem, particularly, in the test system necessitated the development of a regimen of daily addition of antibiotic to permit a more accurate assessment of antibiotic activity to be made. When antibiotic-containing media are incubated for long periods, as is the case when determining antimycobacterial activity, the stability of test antibiotics is an important factor that must be considered.


The humoral immune response after BCG vaccination: an immunoblotting study using two purified antigens.

The specific IgG response induced by BCG vaccination was investigated in 27 adults using two purified BCG antigens in a dot immunoblotting assay. The reflectance values increased significantly after vaccination when P64 but not when P32 was used as the antigen. The values measured after vaccination were compared with those obtained when testing sera from patients with tuberculosis. We observed that the level of anti-P64 IgG reached after BCG immunization was high enough to preclude a serological differential diagnosis with tuberculosis.


Occurrence of mycobacteria in biofilm samples.

The occurrence of mycobacteria was studied in 50 biofilm samples from water treatment plants, domestic water supply systems and aquaria. Mycobacteria were found in 90% of the samples and their densities usually ranged between 10(3) and 10(4) cfu/cm2 (maximum density 5.6 x 10(6) cfu/cm2). Organic substances such as plastics and rubber were usually colonized by larger numbers of mycobacteria than inorganic substances such as copper and glass. The highest mycobacterial densities were found on plastic surfaces which were continuously perfused with water at temperatures between 22 and 30 degrees C. The species identified include Mycobacterium chelonae, M. flavescens, M. fortuitum, M. gordonae, M. kansasii, and M. terrae/nonchromogenicum. The occurrence in microcolonies indicate that biofilms may be an important replication site of aquatic mycobacteria.


Proposal of an improved score method for the diagnosis of pulmonary tuberculosis in childhood in developing countries.

210 children aged less than 5 years, referred to the Arua Regional TB Centre (Uganda) for suspected pulmonary tuberculosis (PTB), were examined by anamnesis, clinical examination, Mantoux test, gastric washing, chest X-ray. The response to treatment criterion was applied to the patients treated. According to the score method suggested by Ghidey and Habte, 31 children were diagnosed as PTB patients. 30 of the 31 children with PTB tested positive for alcohol acid-fast bacilli (AAFB) in the aspirated juice. The Mantoux test and X-rays gave a minor contribution to diagnosis. The clinical results are commented. A statistical analysis was carried out to evaluate the role of gastric washing in the diagnosis of PTB in children under 5 years of age (sensitivity, 96.8%; specificity, 92.2%; positive predictive value, 68.2%; negative predictive value, 99.4%). The response to treatment was also evaluated. A modified enlarged score method (based on gastric washing and including response to treatment) is proposed to be applied in developing countries where chest X-ray and other facilities are often lacking.


The impact of changing BCG coverage on tuberculosis incidence in Swedish-born children between 1969 and 1989.

In April 1975, the mass vaccination of newborns against tuberculosis was replaced by selective vaccination of groups at risk. BCG coverage fell from more than 95% before 1974 to 1.8% between 1975 and 1982 and thereafter reached an average of 13.7% up to 1989. The cumulative incidence of tuberculosis before 5 years of age was estimated among children born in Sweden during periods of high, low and moderate increasing BCG coverage. The incidence figures per 100,000 children was 0.8, 3.9 and 2.9, respectively, for children born to Swedish parents and 2.6, 39.4 and 13.2, respectively, for those born to foreign parents. The observed incidence of tuberculosis among non-BCG vaccinated children born to Swedish parents was within the expected limits given by a prognostic model based on the natural change of the risk of infection. The effectiveness of the selective BCG vaccination programme, which was intensified after 1981 for the second generation of immigrants, was estimated to 0.82 (95% confidence interval 0.38, 0.95) assuming that there was no change of the risk of infection for children born to foreign parents over the period studied. From April 1975 to December 1989, tuberculosis was notified in 85 children born in Sweden during the same period, 7 of them were BCG vaccinated and 78 non-vaccinated, 45 were symptomatic, 3 of them with disseminated tuberculosis.


Tuberculosis control programme in Beijing.

Since 1978 a new tuberculosis control programme based on modern concepts has been started in Beijing. The main goals of the programme were: (1) To prioritize the control of sources of infection; (2) to instigate fully supervised treatment (FST) for new smear-positive patients; (3) to extend services to the rural areas. A four-level network of tuberculosis control services has been organized both in urban and rural areas. Tuberculosis control has been integrated into rural primary health care. Coverage of FST among new smear-positive cases increased to 93% in 1990. A standard regimen, 1HS/11H2S2 has been used for initial treatment. A reserve regimen of RFP and EMB was added to those who failed to convert in 6 months. Since 1988 a 6-month intermittent short-course regimen has been introduced as standard regimen. Case-finding has been intensified since 1980 through symptomatics. Control of chronic excreters was achieved through FST of new smear-positive cases and fully supervised retreatment of old chronic cases. The new tuberculosis control programme has proved highly successful in controlling sources of infection. The prevalence of smear-positive pulmonary tuberculosis in Beijing during the national surveys in 1979, 1984-1985 and 1990 was 127, 56 and 16 per 100,000 respectively. The average annual reduction rate was 17%. This successful experiment has been recognized as a model for the whole country.


Early diagnosis of tuberculosis by fibreoptic bronchoscopy.

We carried out a retrospective study of the methods used to achieve an early diagnosis of 67 patients with pulmonary tuberculosis treated at our institute between 1984 and 1989. Sputum bacteriology was positive in 56 of the 67 patients, 22 were positive on microscopical examination of smears and on culture and 34 on culture alone. The 11 patients with negative sputum bacteriology were all diagnosed by fibreoptic bronchoscopy. In addition, 21 of the 34 smear-negative/culture-positive patients were examined by fibreoptic bronchoscopy and the initial diagnosis was made in 7 of these. Thus the initial diagnosis was made by sputum bacteriology in 49 cases and by fibreoptic bronchoscopy in 18 cases. The median number of days between obtaining a specimen and starting therapy was 7 days for sputum microscopy, 41 days for sputum culture, 7 days for microscopic examination of bronchoscopy specimens, 51 days for culture of the same and 19 days for biopsy. Fibreoptic bronchoscopy is therefore useful for the diagnosis of cases of tuberculosis in which tubercle bacilli are not detected in sputum and, in some instances, for an earlier diagnosis of smear-negative/culture-positive patients.


Defaults, defaulter action and retrieval of patients during studies on tuberculous meningitis in children.

Chemotherapy studies were undertaken on 180 children, aged between 1 and 12 years, with tuberculous meningitis. They received therapy for 12 months. As approximately one-half of the patients came from outside Madras city, retrieval action was not possible in all cases of default. Despite this, a high rate of compliance and punctuality was achieved as a result of initial and periodic motivation. This report highlights the role and importance of motivation in the prevention of default and the retrieval of defaulters during the treatment of children with tuberculous meningitis.


An unusual presentation of oesophageal tuberculosis.

Oesophageal tuberculosis secondary to tuberculous mediastinal lymphadenopathy is a very unusual presentation of adult tuberculosis. We report a young patient who presented with anorexia and weight loss. The chest radiograph and CT scan revealed mediastinal lymphadenopathy causing extrinsic oesophageal compression on the barium swallow. This was confirmed by upper gastrointestinal endoscopy. Four weeks later, because of spontaneous partial relief in dysphagia, upper gastrointestinal endoscopy was repeated and revealed an ulcerated lesion with nodular margins at the mid-oesophagus. Biopsy from the ulcer margin revealed non-caseating granulomas. The patient had complete relief of dysphagia and other symptoms within 3 weeks of start of antituberculosis therapy.


The medical treatment of a tuberculous tracheo-oesophageal fistula.

Tracheo-oesophageal (TE) fistulas caused by Mycobacterium tuberculosis are rare and usually require both surgical treatment and medical treatment with antituberculosis drugs. A patient with a tuberculous TE fistula and mediastinal lymphadenitis was successfully treated by medical management alone.


Clinical evaluation of 99mTc-MIBI myocardial tomography for detecting coronary artery disease.

To evaluate the clinical efficacy of 99mTc-MIBI myocardial tomography for detecting coronary artery disease, 34 consecutive patients who underwent both 99mTc-MIBI study and coronary arteriography were included in the present study. Ten without and 24 with significant coronary artery disease were documented by coronary arteriography. The overall sensitivity and specificity of 99mTc-MIBI study for detecting coronary artery disease were 91.8% (22/24) and 80.0% (8/10), respectively. In subset studies, 16 of 17 (94.1%) patients with previous myocardial infarction and 6 of 7 (85.7%) patients with significant CAD but without MI were identified as abnormal. Reversible perfusion abnormalities were shown in 4 of 5 patients undergoing both exercise and rest studies. Rest perfusion abnormalities were shown in 2 patients with unstable angina pectoris who underwent only rest study. In conclusion, 99mTc-MIBI myocardial tomography provides a reliable method for detecting coronary artery disease. Rest study has high sensitivity for identifying patients with MI, and exercise/rest study can identify patients with significant CAD but without MI.


Growth inhibition and differentiation of promyelocytic cells (HL-60) induced by BC-4, an active principle from Boswellia carterii Birdw.

The induction of cell differentiation and growth inhibition of HL-60 cells by Bc-4, an active principle from Boswellia carterii, was studied in vitro and in vivo. The proliferation of HL-60 cells was found to be inhibited by Bc-4 at a concentration of 5-10 micrograms/ml. Under the action of Bc-4, the acid phosphatase and NBT reduction activities in HL-60 cells were increased, and phagocytosis of cells was also induced. All these activities were concentration dependent. The HL-60 cells were induced by Bc-4 to differentiate into more mature cells morphologically. The in vivo growth of HL-60 cells in mouse subrenal capsules (SRC) and in diffusion chambers inoculated into mice was inhibited by Bc-4 at a dose of 50 mg/kg. The morphology of HL-60 cells treated by Bc-4 in diffusion chambers exhibited characteristics of mature granulocytic cells.


The use of an external tibial fixator in the treatment of genu varum.

This paper reports 72 cases of genu varum treated by a U-shaped osteotomy of the tibia and an oblique osteotomy of the fibula in combination with immobilization effected by a specially designed external tibial fixator. Owing to this modified approach, the patients were able to stand up and move about much earlier than usual. Our trial yielded 80.7% excellent, 16.6% good and 2.7% fair results. The treatment course was shortened to two thirds of the time required when using previously described techniques.


The application of Dick instrumentation in spine surgery.

From June, 1989 to March, 1991, 24 cases with various spinal disorders were treated in our department using the Dick technique. The results were as follows: In the fresh fracture group (7 cases), all the spine fractures were reduced anatomically: the 2 cases without neurological defects returned to work 3 months after operation; the 3 cases with incomplete paraplegia had rapid neurological recovery; and the 2 cases with complete paraplegia showed no recovery after operation. In the late fracture group (10 cases), traumatic kyphotic curves were partially reduced and back pain was decreased markedly in all: Muscle power was increased significantly in 3 cases; spasticity was remarkably improved in 2 cases; 3 cases obtained complete cure of incontinence; and 4 cases had no significant improvement. In 3 cases with ankylosing spondylitis, the initial average kyphotic curve was 73.3 degrees, while the postoperative average curve was 28.3 degrees. The result of treatment of spinal stenosis due to degenerative spondylolisthesis (1 case) was good; slipping vertebrae were stabilized and fused with the Dick system after thorough decompression. In 1 tumor and 2 Tb-spine cases, the patients recovered and were ambulatory soon after operation.


The quantitative alteration of 5s rRNA during the development of mammalian erythroid cells and its effect on DNA synthesis in SP2/0 mouse myeloma cells.

Mouse myeloma cells (SP2/0) were incubated with 125I-5s rRNA from rabbit reticulocytes and processed for autoradiography. The results indicated that 5s rRNA could pass into the nuclei of mouse myeloma cells. In a separate experiment, SP2/0 were incubated with cold 5s rRNA, then with 3H-TdR and processed for autoradiography. It was found that in the mouse myeloma cells, DNA synthesis and cell division were obviously suppressed. In another series of experiments, rRNA was extracted from rabbit bone marrow, reticulocytes and erythroid cells and from rat embryonic liver and erythroid cells. The rRNA was analyzed by agarose electrophoresis. It was found that the amount of 5s rRNA in various stages of erythroid development changed along with the denucleating process. Thus it seems likely that 5s rRNA from mammalian erythroid cells could play a role in reversing the malignant phenotype of tumor cells and denucleation of mammalian erythroid cells through inhibiting DNA synthesis.


Molecular cytogenetic study of short arm aberrations in human D, G group chromosomes.

Six cases of Dp+/Gp+, 10 cases of D/G translocation, 1 case of supernumerary marker chromosome, and 1 case of Yqs were studied using molecular and cytogenetic techniques. The Ag-NOR frequencies of the Dp+ and Gp+ groups were found to be higher than those of normal controls, while their satellite association frequencies were lower. Autoradiographic silver grains were not significantly distributed along the p+ part of the marker chromosome as revealed by chromosomal in situ hybridization using an rRNA probe. This result differs from our previous report. It is suggested that there might be different mechanisms for the formation of p+ on acrocentric chromosomes. D/G translocation cases were found to have lost their NOR. A study of supernumerary marker chromosomes and Yqs cases suggested that the marker chromosome and Yqs exerted no phenotypic effect. The mechanism of their formation is discussed.


The relationship between the changes in plasma atrial natriuretic peptide and renin angiotensin contents and hemodynamics in pulmonary hypertensive rats.

Pulmonary artery pressure (PAP) increased significantly in rats after hypobaric hypoxic exposure for 15 days (CH15) (P less than 0.001) and was maintained at the same level after prolonged hypoxic exposure for 40 days (CH40). There were no significant changes in carotid systolic and diastolic blood pressure in CH15 and CH40 rats. The contractility of the right and left ventricles of CH15 and CH40 rats showed no difference as compared with the control group. Plasma atrial natriuretic peptide (ANP) content in CH40 rats was obviously increased (P less than 0.001), while angiotensin II (AII) dropped markedly (P less than 0.05). Plasma renin activity was increased in both hypoxic groups (P less than 0.05 respectively). These results indicate that during chronic hypoxia, plasma ANP and the renin-angiotensin system (RAS) play an important role in the modulation of pulmonary as well as systemic hemodynamics. They may work cooperatively to limit the development of pulmonary hypertension and to maintain systemic blood pressure within the normal range.


STD epidemiologic analysis at national surveillance spots in the period 1987-1990.

This article summarizes surveillance data of STDs at 16 urban monitoring centers in 1987-1990. During the four year period, 125,493 STD cases were reported. The average annual incidence was 77.80/100,000. Because the rate of increase in females (65.85% per year) was greater than that in males (36.81% per year), the male-to-female ratio fell from 2.39:1 (1987) to 1.34:1 (1990). The number of STD cases reported from all centers tended to increase, and the overall rate of increase was 46.61% per year. The major disease was gonorrhea, with a constituent ratio of 59.22% (1990). However, condyloma acuminatum and nongonococcal urethritis had greater rates of increase (105.03% and 85.14% per year, respectively). The 20-39 year age group accounted for 82.10% of total cases, for STD patients were mostly in the sexually active population. The STD incidence among self-employed businessmen (1206.06/100,000, in 1990) was highest among professional populations. According to analysis of different regions, STD incidence was highest in the southern cities (203.00/100,000, 1990). The greatest rate of increase of STDs was in Yangtze River Valley cities, where the average annual rate of increase was 71.41%.


Breast volume measurement in 125 young Chinese women.

The authors carried out research on breast volume and body surface anatomy of 125 unmarried young women. As a result, an average breast volume for young Chinese women was obtained (325.36 +/- 12.66 ml), and a table together with several linear equations for calculating breast volume were derived. These will provide plastic surgeons with useful tools for designing mammaplasty procedures.


A comparative study of the outcome of renal transplantation in peritoneal dialysis and hemodialysis patients.

Fifty-four cases of renal transplantation performed in peritoneal dialysis (PD) patients were compared with 48 cases in hemodialysis (HD) patients. Three immunosuppressive treatments were used: prednisolone, azathioprine and cyclosporine. Methylprednisolone was used to treat rejection, and polyclonal Atgem or monoclonal OKT3 antibodies were used if there was no response. There was no difference in sex, age, donor source, immunosuppressive regime, duration of dialysis before transplantation, or duration of follow-up between the two groups. Following transplantation, there was no significant difference in patient mortality and survival or graft survival between the groups. The incidences of infections were also similar in the two groups. PD is commonly used in developing countries as an alternative to hemodialysis for the treatment of chronic renal failure. This study has shown that renal transplantation can be successfully performed in patients treated by this method.


Comparative study of 99mTc-HM-PAO SPECT brain imaging, EEG and CT scanning in epileptic patients during the interictal period.

Forty patients with epilepsy in the interictal period were studied with 99mTc (HM-PAO) SPECT brain imaging, EEG and CT scanning. Four cases of generalized epilepsy had normal findings on EEG, SPECT imaging and CT scanning. Two cases of childhood benign partial seizures had abnormal EEG, but the SPECT brain imagings were normal. Regional hypoperfusions of various extents were found in 20 of the 34 patients with complex partial seizures. Among the 34 patients, 17 had abnormal EEG and 8 had abnormal CT scans. A good topographic concordance between regional HM-PAO hypofixation and EEG foci was found (39.1%). Localization of the lesion by SPECT imaging and CT scan was coincident in 5 cases (27.8%). The correlation between these three examination methods is discussed.


Studies on intrahepatic cholelithiasis in west China.

Intrahepatic cholelithiasis is widespread in Asia and brings much suffering to the Chinese people. This paper presents a brief sketch of our clinical observations, research and some related views.


Skin test inhibition by astemizole.

For the purpose of evaluating the therapeutic effect of antihistamines, we have set up an assay method called the "Skin Test Inhibition Index" (STII). Twenty subjects with hay fever were given astemizole (10 mg/d) for 7 days. Skin titration tests were carried out before and after treatment. Significant inhibition of the skin test reaction by astemizole was demonstrated (STII = 91). Another group of 6 hay fever patients was given astemizole (10 mg/d) for 10 days, and STII was determined on days 5 and 10 and on days 7, 14 and 21 after treatment. STII were calculated as 12, 108, 90, 10 and 7, respectively. These results demonstrate that astemizole is a long-acting antihistamine.


Hepatocyte growth factor is a synergistic factor for the growth of hematopoietic progenitor cells.

Bone marrow (BM) stromal cells, which include macrophages, fibroblasts, endothelial cells, and adipocytes, have been shown to produce several factors that modulate the growth of BM progenitors. Hepatocyte growth factor (HGF) is a fibroblast-derived factor and has recently been shown to be a ligand for the c-met proto-oncogene, a member of the receptor class of tyrosine kinases. c-met messenger RNA (mRNA) is predominantly expressed in epithelial cells, but has been detected in several murine hematopoietic progenitor cell lines, suggesting that HGF and met might function during hematopoiesis. Here, BM cells were found to express both met mRNA and protein. Moreover, HGF was shown to synergize with interleukin-3 and granulocyte-macrophage colony-stimulating factor to stimulate colony formation of hematopoietic progenitor cells in vitro. These results show that, in addition to its activity on epithelial cells, HGF is a new member of the functionally related group of factors that modulate hematopoiesis.


Recombinant human tissue plasminogen activator for the treatment of established severe venocclusive disease of the liver after bone marrow transplantation.

Seven patients were treated with recombinant human tissue plasminogen activator (tPA) for severe hepatic venocclusive disease (VOD) that developed after bone marrow transplantation for hematologic malignancy. Recombinant human tPA (10 mg/d x 2 days) and heparin (1,000 U bolus followed by continuous intravenous infusion of 150 U/kg/d x 10 days) were begun a median of 9 days (range, 4 to 18 days) posttransplant. The median total serum bilirubin and percent weight gain from baseline were 19.4 mg/dL (range, 14.6 to 34.9 mg/dL) and 9.1% (range, 1% to 18.5%), respectively, at the start of tPA administration. Five patients responded to therapy with prompt reduction in total serum bilirubin within 96 hours of starting tPA. Three patients are alive 178 to 379 days posttransplant without evidence of VOD. No patient had significant hemorrhagic complications with tPA. We conclude that recombinant human tPA can be administered to patients with severe VOD at the dosage described. Whereas preliminary data suggests that recombinant human tPA can alter the natural history of severe VOD, further study is necessary to determine its efficacy.


Clinical and cytogenetic responses to granulocyte-macrophage colony-stimulating factor in therapy-related myelodysplasia.

We treated 10 patients with a therapy-related myelodysplastic syndrome with escalating doses of granulocyte-macrophage colony-stimulating factor (GM-CSF; sargramostim) in a phase II trial and used sequential cytogenetic analyses to determine whether there was stimulation of nonclonal hematopoiesis. The GM-CSF was administered by continuous intravenous infusion over 2 hours daily for 14 days, followed by a 14-day rest period. The initial starting dose was 60 micrograms/m2/d. The GM-CSF dose was escalated within individual patients to 125 micrograms/m2, 250 micrograms/m2, and then 500 micrograms/m2/d until the peripheral blood neutrophil count at least doubled and exceeded 1,000/microL. GM-CSF treatment then continued in monthly maintenance cycles. During 57 treatment courses, the neutrophil count increased in 52 but only doubled and exceeded 1,000/microL in 21. Mild eosinophilia was stimulated in five patients, but only two had greater than 1,000 eosinophils/microL. In only three patients was any stimulation of platelet or red blood cell production observed, and thus, little change in transfusion requirements occurred. The bone marrow karyotypes from individual patients either remained completely abnormal or became increasingly abnormal over the course of treatment. We found no evidence that GM-CSF preferentially stimulated normal marrow stem cells to proliferate or had the ability to eradicate the cytogenetically abnormal clone by inducing terminal differentiation. Although the effect on granulopoiesis was transient and dependent on continued GM-CSF treatment, the increase in the neutrophil count was clinically important in some patients, allowing more effective control of ongoing infections.


Favorable outcome of B-cell acute lymphoblastic leukemia in childhood: a report of three consecutive studies of the BFM group.

In 1981 the BFM group introduced a new treatment strategy for B-cell acute lymphoblastic leukemia (B-ALL). A cytoreductive prephase (prednisone/cyclophosphamide) was followed by eight 5-day courses of chemotherapy. Fractionated cyclophosphamide, methotrexate (MTX) 0.5 g/m2 (24-hour infusion), and MTX intrathecally were administered at each course and cytosine arabinoside (ARA-C)/teniposide (VM-26) was given alternately with doxorubicin. In study ALL-BFM-83, central nervous system (CNS) chemotherapy was intensified by adding dexamethasone, while MTX/ARA-C was administered intraventricularly. Therapy duration was reduced to six courses. In study ALL-BFM-86, MTX 0.5 g/m2 was replaced by high-dose (HD) MTX, 5 g/m2 (24-hour infusion), and MTX/ARA-C/prednisolone intrathecal therapy was introduced. Doses of ARA-C and VM-26 were increased and fractionated, cyclophosphamide was partially replaced by ifosfamide, and vincristine was added. CNS irradiation was 24 Gy for prevention and 30 Gy for overt disease in studies ALL-BFM-81 and -83, but was omitted in ALL-BFM-86. In all, 87 patients were enrolled, 22 (8 CNS-positive) in study All-BFM-81, 24 (7 CNS-positive) in study ALL-BFM-83, and 41 (0 CNS-positive) in study ALL-BFM-86. The estimated 5-year duration of event-free survival (EFS) was 43% in study ALL-BFM 81, 50% in study ALL-BFM-83, and 78% in study ALL-BFM-86 (minimal follow-up, 25 months). Nineteen of 24 relapses occurred while on therapy or shortly thereafter. In study ALL-BFM 81, the CNS was the most frequent site of failure. In ALL-BFM-83, there were no isolated CNS relapses, but more bone marrow (BM) relapses occurred. In ALL-BFM-86, localized manifestations were the predominant site of failure, no isolated BM relapses occurred, and only one CNS relapse was diagnosed. No single parameter exerted a consistent influence on outcome with one exception. The presence of residual disease after the first two courses was correlated with an increased risk of therapy failure. We conclude that an intensive, short-pulse therapy delivered within a 4-month period is highly effective in the treatment of B-ALL. In addition to fractionated cyclophosphamide/ifosfamide, a 24-hour infusion of HD MTX 5 g/m2 in conjunction with an i.th. therapy is an important component for prevention of both systemic and CNS relapses. CNS irradiation is not needed for CNS-negative patients.


Differential effects of sequential, simultaneous, and single agent interleukin-3 and granulocyte-macrophage colony-stimulating factor on megakaryocyte maturation and platelet response in primates.

Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) following interleukin-3 (IL-3) priming has been shown to increase thrombopoiesis. To elucidate the comparative abilities of IL-3 and GM-CSF in influencing megakaryocyte development in vivo, serial bone marrow analyses were performed on rhesus monkeys treated with 5 micrograms/kg/d of IL-3 and 5 micrograms/kg/d of GM-CSF sequentially for 4 days each, simultaneously for 8 days, and as single agents for 8 days. Platelet counts maximally increased to a mean of 7.5 x 10(5)/microL (n = 3) on days 11 through 12 in monkeys treated with sequential IL-3/GM-CSF. In contrast, neither IL-3 alone nor simultaneously administered IL-3/GM-CSF elicited increases in thrombopoiesis between days 3 and 15. GM-CSF elicited a variable platelet response. Megakaryocyte ploidy distributions were significantly (P < .001) shifted between days 7 and 10 in monkeys treated sequentially and between days 3 and 15 in monkeys treated with combined IL-3/GM-CSF and with GM-CSF alone but not in monkeys treated with IL-3 alone. The changes in mean DNA content and megakaryocyte size, as determined by digital image analysis, were larger in monkeys treated with sequential IL-3/GM-CSF and with GM-CSF alone than in simultaneously treated monkeys. In addition, sequentially but not simultaneously treated monkeys showed increased numbers of megakaryocytes on bone marrow biopsy. We conclude that administration of IL-3 followed by GM-CSF treatment increases thrombopoiesis by sequentially increasing megakaryocyte numbers and maturation and that these effects are diminished by simultaneous administration of the two cytokines.


Bryostatin 1 modulates the proliferation and lineage commitment of human myeloid progenitor cells exposed to recombinant interleukin-3 and recombinant granulocyte-macrophage colony-stimulating factor.

The activity of protein kinase C (PK-C) has been implicated in the regulation of the growth and differentiation of both normal and neoplastic hematopoietic cells. We have examined the effects of the PK-C-activating agents phorbol 12,13-dibutyrate (PDBu), mezerein, and bryostatin 1 on the proliferation and lineage commitment of CD34+ human myeloid progenitor cells stimulated by recombinant interleukin-3 (rIL-3) and/or recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Although each of the PK-C activators administered alone induced no colony formation, coadministration of these agents with plateau concentrations of each cytokine (eg, 50 ng/mL) increased the number of day 14 granulocyte-macrophage colony-forming units by 100% to 150%. The number of pure and mixed neutrophil and macrophage colonies was substantially enhanced in the presence of PK-C activators, whereas the percentage and, in most cases, the absolute number of eosinophilic colonies was significantly reduced. The inhibition of eosinophilic colony formation was not overcome by the addition of rIL-5. Although addition of bryostatin 1 24 hours before rIL-3 abrogated the increase in total colony formation observed with simultaneous administration of factors, the inhibition of eosinophilic colonies and the increase in neutrophil/macrophage colonies persisted under these conditions. The addition of bryostatin 1 for up to 144 hours after rIL-3 continued to potentiate total colony formation, whereas the inhibition of eosinophilic commitment was lost after 120 hours. Together, these results suggest that pharmacologic interventions at the level of PK-C may regulate both the proliferation as well as the lineage commitment of human hematopoietic progenitors exposed to rGM-CSF and rIL-3.


Roles of erythropoietin, insulin-like growth factor 1, and unidentified serum factors in promoting maturation of purified murine erythroid colony-forming units.

We have used 75% to 90% pure murine erythroid colony-forming units (CFU-E) to delineate the processes and factors underlying their maturation. These CFU-E form 32 cell colonies and are drawn from what we term generation I of a six-generation long maturation sequence (Landschulz et al, Blood 79:2749, 1992). Applying assays of 59Fe-heme biosynthesis and colony numbers as measures of maturation and analyses of DNA degradation as an index of programmed cell death, we find that (1) erythropoietin (Epo) enhances maturation throughout most of its course; (2) Epo first seems able to forestall DNA degradation when CFU-E reach generation II; (3) the processes that Epo elicits thereafter start to persist when Epo is withdrawn; (4) insulin-like growth factor I (IGF-I) also forestalls DNA breakdown, but later loses effectiveness; (5) IGF-I adds little to maturation when Epo levels are high, but when Epo levels are low, enhances it substantially; and (6) for maturation to be entirely optimal, an unidentified serum factor(s) is probably required when Epo levels are high and is certainly needed when Epo levels are like those in normal animals. Quantitatively, about 40% of optimal in vitro erythropoiesis at normal Epo levels depends on Epo alone, another 30% or less on the addition of IGF-I, and the remaining 30% or more on the addition of unidentified serum factor(s). Applied together, these three or more factors lead to two-thirds of the maximum maturation realized with saturating Epo levels. Because we also find that heme accumulated in CFU-E culture can closely approach levels in red blood cells, we suppose that our conclusions apply as well to CFU-E maturation in vivo.


Release of interleukin-1 and interleukin-6 from human monocytes by antithymocyte globulin: requirement for de novo synthesis.

Antithymocyte globulin (ATG) is an effective treatment in patients with severe aplastic anemia (SAA). Its mechanism of action remains unclear, although it has been assumed to be immunosuppressive. However, ATG has also been shown by several laboratories to be immunostimulatory. Recently, interleukin-1 (IL-1) production has been found to be decreased in lipopolysaccharide-stimulated peripheral blood monocytes obtained from SAA patients. We have investigated the ability of ATG to function as an immunostimulatory agent via the production of IL-1 and IL-6 by normal human monocytes in vitro. Supernatants from ATG-stimulated monocytes were assayed for biologically active and immunoreactive IL-1 and IL-6. We have found that ATG, via its F(ab')2 fragment is a powerful inducer of IL-1 and IL-6 production. Furthermore, ATG induction of both cytokines from normal monocytes required de novo synthesis, as determined by 35S-methionine incorporation. Because these two cytokines synergize with other cytokines at both the stem cell and progenitor levels, these stimulatory properties of ATG may be relevant to the treatment of SAA. This would favor the hypothesis of a bimodal mechanism for ATG as an inducer of hematopoietic growth factors and as an immunosuppressive agent.


Involvement of nuclear factor-kappa B in induction of the interleukin-6 gene by leukemia inhibitory factor.

Recent studies have indicated that the leukemia inhibitory factor (LIF) induces secretion of interleukin-6 (IL-6) in myeloid cells. We here show that synthesis of IL-6 by human mononuclear phagocytes exposed to recombinant human (rh) LIF is preceded by an increase of IL-6 transcript levels as a result of transcriptional activation of the IL-6 gene. Analysis of deleted fragments of the IL-6 promoter indicated that transcriptional activation of the IL-6 promoter was associated with enhanced binding activity of the transcription factor nuclear factor (NF)-kappa B. Binding of activation protein (AP)-1 and NF-IL-6, also known to transcriptionally activate the IL-6 promoter, was not inducible by LIF. Furthermore, introduction of the NF-kappa B sequence into a heterologous promoter construct, but not of AP-1- and NF-IL-6-binding sequences, conferred inducibility by LIF to this promoter. Deletion of the NF-kappa B binding site in the IL-6 promoter was associated with loss of inducibility by LIF, lending further support for the notion that the NF-kappa B binding site is crucial for LIF-mediated induction of the IL-6 promoter. Taken together, our results show that rhLIF induces IL-6 gene expression in mononuclear phagocytes through transcriptional gene activation involving NF-kappa B.


Isolation of a cDNA clone encoding a zinc finger protein highly expressed in T-leukemia lines.

A cDNA clone encoding a novel zinc finger protein expressed in lymphoid cells has been isolated. This protein contains 5 repeats of the C2H2 motif previously described in the Drosophila gap gene, Krüppel, which is involved in embryo segmentation. Northern blot analysis showed that the messenger RNA (mRNA) encoding this protein is expressed at high levels in a variety of T-leukemia cell lines, at lower levels in some B cells, but is not observed in nonlymphoid cells. Within the T lineage, the mRNA is found at high levels in both alpha beta and gamma delta T cells. These data suggest that this cDNA, designated Hkr-T1, represents a gene that may contribute to the determination of the differentiation and the specificity within lymphoid cells.


Alteration of the proliferative rate of acute myelogenous leukemia cells in vivo in patients.

Ten patients with active acute myelogenous leukemia (AML) received either 13 cis retinoic acid (RA) + alpha interferon (IFN) or recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for 3 days. Cell cycle measurements were performed before and at the conclusion of administration of the bioactive agent(s). The proliferative rate of the leukemia cells in vivo decreased in four of five patients receiving RA+IFN whereas in one patient proliferation accelerated. The proliferative rate of AML cells accelerated in three of the five patients who received rhGM-CSF and slowed in two patients. These data show that while the proliferative rate of AML cells can be altered in vivo, the effect produced by bioactive agents may be the opposite of the desired effect. Furthermore, the studies described here demonstrate the usefulness of marrow biopsies for measuring the percent S-phase cells and the importance of measuring the duration of S phase so that the effects of bioactive agents on the cell cycle time of the leukemia cells can be determined.


Hexamethylene bisacetamide in myelodysplastic syndrome and acute myelogenous leukemia: a phase II clinical trial with a differentiation-inducing agent.

Hexamethylene bisacetamide (HMBA) is a potent inducer of differentiation of a number of transformed cell lines in vitro. We report results of a phase II clinical trial in 41 patients with myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML) to whom HMBA was administered by continuous infusion for 10 days and repeated after an interval of 18 to 75 days. HMBA induced a complete remission (CR) in three patients and a partial remission (PR) in six patients. The median duration of CR was 6.8 months (range 1.3 to 16 months) and 3.7 months for PR (range 1 to 7 months). No significant difference was observed between responders and nonresponders with respect to the mean HMBA plasma levels, which were 0.86 +/- 0.04 mmol/L and 0.87 +/- 0.12 mmol/L, respectively. In certain patients morphologic and chromosome analyses provided evidence that HMBA induced differentiation of transformed hematopoietic precursors. The most prominent toxicity was thrombocytopenia, generally reversible on cessation of administration of HMBA.


Compound heterozygosity in a complete erythrocyte bisphosphoglycerate mutase deficiency.

Erythrocyte bisphosphoglycerate mutase (BPGM) deficiency is a rare disease associated with a decrease in 2,3-diphosphoglycerate concentration. A complete BPGM deficiency was described in 1978 by Rosa et al (J Clin Invest 62:907, 1978) and was shown to be associated with 30% to 50% of an inactive enzyme detectable by specific antibodies and resulting from an 89 Arg-->Cys substitution. The propositus' three sisters exhibited the same phenotype, while his two children had an intermediate phenotype. Samples from the family were examined using polymerase chain reaction and allele-specific oligonucleotide hybridization and sequencing techniques. Amplification of erythrocyte total RNA from the propositus' sister around the 89 mutation indicated the presence of two forms of messenger RNAs, a major form with the 89 Arg-->Cys mutation and a minor form with a normal sequence. Sequence studies of the propositus' DNA samples indicated heterozygosity at locus 89 and another heterozygosity with the deletion of nucleotide C 205 or C 206. Therefore, the total BPGM deficiency results from a genetic compound with one allele coding for an inactive enzyme (mutation BPGM Créteil I) and the other bearing a frameshift mutation (mutation BPGM Créteil II). Examination of the propositus' two children indicated that they both inherited the BPGM Créteil I mutation.


Evidence that sustained growth suppression of intestinal anaerobic bacteria reduces the risk of acute graft-versus-host disease after sibling marrow transplantation.

The influence of intestinal bacterial decontamination on the occurrence of grades II to IV acute graft-versus-host disease (GVHD) was retrospectively analyzed in 194 predominantly adult patients treated by genotypically identical sibling marrow transplantation under conditions of strict protective isolation and intestinal antimicrobial decontamination. Forty-five patients (23%) developed acute GVHD and univariate analysis identified four features that significantly increased the risk for this reaction: chronic myeloid leukemia as the underlying disease, as compared with all other disease categories (P < .0001); female marrow donors for male recipients, as compared with other gender combinations (P < .005); ineffective, as compared with sustained growth suppression of intestinal anaerobic bacteria (P < .006); and methotrexate as the sole immunoprophylactic compound, as compared with cyclosporine containing regimens (P < .05). Using the duration of anaerobic growth suppression as a time-dependent explanatory variable, proportional hazards regression analysis confirmed these features as independent predictors for acute GVHD with relative risk estimates of 1.9 (95% confidence interval [CI], 1.3 to 2.7) for the immunoprophylactic regimen (P < .0004), of 1.8 (95% CI, 1.3 to 2.5) for the underlying disease (P < .0005), of 1.7 (95% CI, 1.2 to 2.5) for anaerobic decontamination (P < .002), and of 1.3 (95% CI, 1.1 to 1.6) for the donor/recipient gender combination (P < .008), respectively. Best subset selection modeling also identified the quality of anaerobic decontamination as the third most important predictor for acute GVHD, when all four significant features were included. Estimates of acute GVHD stratified by the quality of anaerobic bacterial growth suppression showed a strong influence of anaerobic decontamination in patients burdened by at least one of the other unfavorable factors (P < .009). In conclusion, this study provides strong evidence that sustained growth suppression of intestinal anaerobic bacteria after clinical sibling marrow transplantation can independently modulate the occurrence of grades II to IV acute GVHD, which is in concordance with previous results from animal transplantation models. Antimicrobial chemotherapy specifically targeted to the intestinal anaerobic bacterial microflora may be complementarily useful in preventing acute GVHD and should be investigated in a prospective trial.


Impaired erythropoietin response to anemia after bone marrow transplantation.

Delayed erythroid recovery is common after bone marrow transplantation (BMT), with some patients continuing to require red blood cell (RBC) transfusion support for as long as 1 year. While the etiology is multifactorial, inadequate stimulation of erythroid progenitors by the erythroid growth factor, erythropoietin, may play a role. In this study, the erythropoietin response to anemia of 70 consecutive patients undergoing BMT at the Johns Hopkins Oncology Center was compared with the erythropoietin response in uncomplicated iron deficiency anemia. Erythropoietin levels were elevated for the degree of anemia early after BMT; however, at the time of marrow recovery, erythropoietin levels were significantly suppressed in both allogeneic and autologous BMT patients compared with the iron-deficient patients. Patients with acute graft-versus-host disease (GVHD) had a more marked suppression of the erythropoietin response to anemia. In the patients who remained anemic for extended periods of time (up to 12 months after BMT), an inadequate erythropoietin response to anemia persisted. Delayed erythroid recovery after BMT is associated with inadequate erythropoietin levels. Therefore, recombinant human erythropoietin may be useful in the treatment of the anemia associated with both autologous and allogeneic BMT.


Molecular basis of inherited human antithrombin deficiency.

Figures 1 and 4 summarize the various AT mutations that have been described. The molecular elucidation, over the past decade, of the various AT deficiency types has provided important new insights into functional-structural relationships of AT. This knowledge, together with data provided by monoclonal antibodies and x-ray crystallographic studies of related molecules, has provided important new insights as to how the AT molecule functions in vivo. Finally, such knowledge might, in the foreseeable future, lead to the production of AT molecules that are specifically genetically engineered to be of use in a variety of clinical situations.


Breakpoints at 11q23 in infant leukemias with the t(11;19)(q23;p13) are clustered.

We have analyzed a series of nine infant leukemias that carry a t(11;19)(q23;p13). They had the morphologic features of acute lymphoblastic leukemia (ALL) and expressed markers typical of B-cell progenitor ALL or pre-B ALL; one coexpressed myeloid markers in addition to lymphoid markers (biphenotypic). Two probes (P/S4 and 98.40) subcloned from a yeast artificial chromosome (YAC) known to span the breakpoint in the t(4;11) were used to investigate DNA isolated from the leukemic cells of these patients. A total of approximately 15 kb of genomic DNA in the vicinity of the probes was examined by conventional Southern blot analysis using a series of restriction enzymes. In eight of the nine cases, the breakpoint could be mapped to an approximately 10-kb BamHI fragment disclosed by hybridization to the P/S4 probe.


All-transretinoic acid followed by intensive chemotherapy gives a high complete remission rate and may prolong remissions in newly diagnosed acute promyelocytic leukemia: a pilot study on 26 cases.

We entered 26 patients with newly diagnosed acute promyelocytic leukemia (APL) in a pilot study of all-transretinoic acid (ATRA) followed by intensive chemotherapy. Median age was 46 (range 25 to 63). No patient presented with leukocytes > 10 x 10(9)/L or had the microgranular APL variant. Cytogenetic analysis (25 patients) found a t(15;17) in 24 cases. Patients were scheduled to receive ATRA (45 mg/m2/d) until complete remission, followed by an intensive daunorubicin (DNR) + Ara C course ("4 + 7" course), then three "2 + 5" DNR + Ara C courses and maintenance chemotheapy. However, the "4 + 7" course was administered in emergency if hyperleukocytosis rapidly developed to prevent leukostasis. Twenty-five patients (96%) achieved CR, 14 with ATRA alone and 11 after the addition of the "4 + 7" course on day 2 to 30 of treatment, because leukocytes rapidly increased (9 cases), because of resistance to ATRA (1 case), and development of organomegaly (1 case). The remaining patient died on day 6, from CNS bleeding. Apart from hyperleukocytosis, side effects were usually moderate. In the 11 patients who could be studied in vitro, a very good correlation was found between in vivo and vitro differentiation and proliferation of APL blasts with ATRA. Three patients were allografted after the "4 + 7" course. Four patients did not receive this course but received the subsequent "2 + 5" courses and maintenance. The remaining patients followed the scheduled protocol. Three patients relapsed after 8, 11, and 15 months (including one allografted patient). Two patients died in CR, after 6 and 17 months. The other 20 patients remained in CR after 18+ to 34+ months (median 21). Actuarial disease free interval (DFI) and event free survival (EFS) were 87% and 77%, respectively, after 18 months. These results were compared to those obtained in our previous APL 84 trial with chemotherapy alone in newly diagnosed APL (after excluding patients included in this trial who presented with hyperleukocytosis). In APL 84 trial, the CR rate was 76%, the actuarial DFI and EFS were 59% and 48% after 18 months, respectively. Differences with the pilot study of ATRA followed by chemotherapy were significant for DFI (P = .02), EFS (P = .006), but not for CR rate (P = .08). Although this is a historical comparison, these results suggest that ATRA followed by chemotherapy may prove superior to chemotherapy alone in newly diagnosed APL, by slightly increasing the CR rate, but perhaps more importantly by reducing the relapse rate.(ABSTRACT TRUNCATED AT 400 WORDS)


A controlled trial of recombinant human granulocyte-macrophage colony-stimulating factor after total body irradiation, high-dose chemotherapy, and autologous bone marrow transplantation for acute lymphoblastic leukemia or malignant lymphoma.

Infections during granulocytopenia are major complications of autologous bone marrow transplantation (ABMT). Since recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF) has proved to accelerate bone marrow recovery after cytostatic chemotherapy, we studied its effects on hematopoietic regeneration and on infectious complications after total body irradiation (TBI) and high-dose chemotherapy followed by ABMT. Eighty-one patients with acute lymphoblastic leukemia (ALL) in complete remission (CR) or with non-Hodgkin's lymphoma (NHL) in CR or partial remission were randomized in a double-blind, placebo-controlled trial. They received either rhuGM-CSF 250 micrograms/m2 (Escherichia coli-derived) daily by continuous infusion after ABMT, or placebo. Treatment was continued until the neutrophil counts reached greater than 500/microL for 1 week. The maximum treatment duration was 30 days. Thirty-nine patients in the rhuGM-CSF group and 40 patients in the placebo group were evaluable. The median time needed to reach a neutrophil count of 500/microL was 15 days with rhuGM-CSF and 28 days with placebo (P = .0001). Bacterial infections occurred in 14 (35.9%) of the patients with rhuGM-CSF and in 25 (62.5%) of the patients given the placebo (P = .024). Nine of the 14 bacterial infections in the rhuGM-CSF group and 20 of the 25 infections in the placebo group were diagnosed within the first 10 days after ABMT. Capillary leakage and a reversible fluid retention were seen in five of the rhuGM-CSF-treated patients. Patients treated with rhuGM-CSF had lower serum protein and albumin levels than patients in the placebo group. There was no statistically relevant difference in overall survival between the two groups (P = .47). Relapse occurred in 14 (34%) patients with rhuGM-CSF and in 18 (45%) patients with placebo. We conclude that continuous infusion of rhuGM-CSF after ABMT accelerates the regeneration of granulocytes and reduces the number of bacterial infections.


Changes in T-helper cell function in human immunodeficiency virus-infected children during didanosine therapy as a measure of antiretroviral activity.

Didanosine has shown activity against the human immunodeficiency virus (HIV) in both children and adults. We prospectively assessed T-helper cell (Th) function as determined by in vitro interleukin-2 (IL-2) production in response to a panel of T-cell stimuli in 22 HIV-infected children before and during didanosine therapy and we correlated the incidence of opportunistic and recurrent bacterial infections with changes in p24 antigen and CD4 counts. Didanosine (270, 360, or 540 mg/m2/d) was administered orally for periods ranging from 8 to 40 weeks (mean, 24 weeks). Five of six asymptomatic patients (Centers for Disease Control P-1) compared with 6 of 16 symptomatic (P-2) patients exhibited improved Th function (greater than threefold increase in IL-2 production to at least 2 of the 4 stimuli) during therapy. Of 12 patients without infections during therapy, 9 (75%) showed improvement in Th function, compared with only 2 of 10 patients with infections (P = .03). Notably, the incidence of infections was not correlated with improvements in CD4 count or decreases in p24 antigen. Improvement in Th function during didanosine therapy is correlated with decreased incidence of infections. Assessment of Th function may provide an additional measurement of immunologic response to antiretroviral therapy.


Monoclonal antibody against the common beta subunit (beta c) of the human interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor receptors shows upregulation of beta c by IL-1 and tumor necrosis factor-alpha.

High-affinity receptors for human granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are composed of two distinct subunits, alpha and beta. Each receptor has its own ligand-specific alpha subunit, and the three receptors share the common beta subunit, beta c. Using a transfectant of NIH3T3 cells expressing the high-affinity human GM-CSF receptor, monoclonal antibodies (MoAbs) against beta c were generated. These MoAbs specifically bound to cells bearing beta c and immunoprecipitated the beta c protein of 120 Kd. Using these MoAbs, expression of beta c was examined. It is known that IL-1 augments the proliferative response of a human factor-dependent hematopoietic cell line TF-1 to either GM-CSF, IL-3, or IL-5, and that it upregulates the high-affinity receptors for GM-CSF, IL-3, and IL-5. Antibody binding and immunoprecipitation demonstrated that IL-1 increased cell surface expression of beta c. This enhancement by IL-1 was accompanied by an increased level of beta c mRNA. In addition, we found that tumor necrosis factor-alpha (TNF-alpha) also increased the expression of beta c, although it did not augment the proliferative response of TF-1 to GM-CSF, IL-3, and IL-5.


Role of monocytes in the expansion of human activated natural killer cells.

We have studied the mechanisms underlying expansion of recombinant interleukin-2 (rIL-2)-stimulated natural killer (NK) cells in vitro. A population of NK cells expressing the CD56+/CD3-phenotype (98.9% +/- 0.42%) was obtained from normal human peripheral blood mononuclear cells (PBMNC) by fluorescence-activated cell sorting (FACS). Culture of NK cells in media containing rIL-2 (1,000 U/mL) for 18 days resulted in a population of activated NK cells (ANK) with significantly enhanced cytotoxicity, but only 2.6 +/- 0.56-fold expansion of cell number compared with the starting NK population. Culture of starting NK populations and autologous PBMNC in a Transwell system (Costar, Cambridge, MA), providing separation of the two cell populations by a 0.4-microns pore membrane, resulted in a dose-dependent increase in fold expansion of ANK (expansion = 19.9 +/- 4.0-fold; P < .001; n = 22) significantly greater than that observed when NK were cultured alone. Further experiments using the Transwell system showed that the stimulatory effect of autologous PBMNC on ANK progenitor proliferation resides in the CD14+ monocyte fraction (maximal expansion = 14.5 +/- 1.5-fold; n = 17) and not in the CD5+ T-lymphocyte or CD19+ B-lymphocyte fractions. Direct coculture of purified NK and autologous monocytes in the same compartment, thus permitting cell-cell contact, resulted in significantly greater expansion of the ANK population (30.6 +/- 4.7-fold expansion, P < .001; n = 10) than that observed when NK and monocytes were separated by the Transwell membrane. Finally, depletion of PBMNC of cells bearing CD5 and CD8 by panning on antibody-coated plastic flasks resulted in a starting cell population enriched for NK progenitors and for monocytes. Cultures of this resultant population for 18 days in the presence of rIL-2 yielded an ANK population similar to that obtained when CD56+/CD3- cells obtained by FACS were cocultured with autologous monocytes. These results suggest that proliferation of ANK requires autologous monocytes and is in part mediated by humoral factors, but is enhanced when NK and monocytes are in direct cell-cell contact. Depletion of cells bearing CD5 and CD8 from PBMNC is a single efficient method for obtaining a starting population capable of producing large numbers of ANK in culture that may lead to new therapeutic uses for the ANK population.


Calpain activity in patients with thrombotic thrombocytopenic purpura is associated with platelet microparticles.

Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia and disseminated platelet thrombi throughout the microvasculature. Studies by our group have demonstrated calcium-dependent proteolytic activity (calpain) that is no longer detectable in the serum of patients with acute TTP after their recovery. The purpose of this study was to investigate if the protease activity of TTP was detectable in plasma and, therefore, not an in vitro phenomenon secondary to the formation of serum. Additionally, we looked for evidence of membrane association of the active protease in the patients' samples, which would explain the persistence of its activity in the presence of plasma inhibitors. Acute TTP samples, both serum and plasma, were collected from 10 patients with TTP. Calpain was measured using bioassays for enzyme activity and also by detection of the protein using immunoblotting with an anticalpain monoclonal antibody (MoAb). In all instances, calpain could be detected both functionally and antigenically in the acute TTP sera and plasma. No calpain activity could be detected in any of the controls, although antigenic calpain was detectable in one sample from a patient who had undergone cardiopulmonary bypass surgery. To investigate whether the calpain was associated with microparticles in the plasma, the TTP plasma samples were ultrafiltered and ultracentrifuged. Activity was not lost by passage across a 0.2-micron filter but was detectable only in the pellet following ultracentrifugation. Membrane association of the calpain in the microparticles also was demonstrated using solubilization with Triton X-100. Immunoprecipitation studies demonstrated that the calpain activity could be removed by MoAbs against platelet membrane glycoproteins (IX and IIb/IIa) but not by a MoAb against red blood cell membrane glycophorin. These studies indicate that active calpain is associated with platelet microparticles in plasma from patients with TTP.


Characterization of platelet-releasable forms of beta-amyloid precursor proteins: the effect of thrombin.

Activated platelets release a potent inhibitor of factor XIa previously identified as a Kunitz proteinase inhibitor domain-containing form of the beta-amyloid precursor proteins (beta APP). Two carboxy-terminal truncated forms of the beta APP, beta APP-751 and beta APP-770, are shown to be the predominant isoforms secreted by platelets. The release of beta APP from platelets is responsible for the higher concentration of beta APP in serum compared with plasma, and thrombin dose-response data show that release of beta APP is most consistent with alpha granule localization within the platelet. Thrombin induces a limited and specific proteolysis of platelet-secreted beta APP, resulting in loss of a carboxy-terminal fragment. This phenomena is dependent on both thrombin concentration and duration of incubation and is inhibited by the thrombin-specific inhibitor hirudin, characteristics that can be duplicated in a mixture of purified recombinant beta APP-751 and thrombin. A similar effect of thrombin on full-length transmembrane forms of beta APP would result in a membrane-bound remnant containing the intact beta-amyloid protein.


Response of human platelets to activating monoclonal antibodies: importance of Fc gamma RII (CD32) phenotype and level of expression.

Certain monoclonal antibodies (MoAbs) specific for platelet membrane glycoproteins are known to be capable of activating platelets, and it is generally thought that platelets from normal subjects are equally susceptible to stimulation by such MoAbs. We found that platelets from 20 normal donors varied significantly in their sensitivity to three IgG1 murine MoAbs specific for membrane glycoproteins CD9, GPIV (CD36), and the GPIIb/IIIa complex (CD41), respectively. The response of platelets to these MoAbs was blocked by prior addition of MoAb IV.3 specific for the Fc gamma RII receptor, indicating that activation was Fc receptor mediated. Platelets that responded poorly to these MoAbs failed to bind the MoAb 41H.16, specific for the "responder" form of Fc gamma RII, but platelets that responded well reacted with this MoAb. The average number of Fc gamma RII receptors on platelets from "responders" and "non-responders" was approximately the same. However, the number of Fc gamma RII receptors expressed influenced sensitivity of a subgroup of "responder" platelets to the anti-CD41 MoAb. These platelets were judged on the basis of MoAb binding studies to be heterozygous for the two alleles of Fc gamma RIIA. In contrast to their varying sensitivity to IgG1 MoAbs, members of the platelet panel responded equally well to 50H.19, an IgG2a MoAb specific for CD9, and these responses could not be blocked by MoAb IV.3 in the presence of plasma. This appears to be because of dual actions of 50H.19 on platelets: one FcR-dependent and the other complement-dependent. Our findings confirm previous reports that certain IgG1 MoAbs activate platelets through binding of their Fc domains to Fc gamma RII receptors and demonstrate that this response is influenced both by Fc gamma RII phenotype and (in the case of the anti-CD41 MoAb) by the number of Fc gamma RII receptors expressed. The failure of nonresponding platelets to bind detectable amounts of MoAb 41H.16, which is thought to recognize all Fc gamma RII receptors except for one allele of the Fc gamma RIIA gene, is consistent with the possibility that Fc gamma RIIA gene products, but not Fc gamma RIIB or Fc gamma RIIC gene products, are expressed on platelets.


Presence of active and latent type 1 plasminogen activator inhibitor associated with porcine platelets.

Data from a number of laboratories indicate that human platelets contain type I plasminogen activator inhibitor (PAI-1) primarily in a latent form; however, one report (Biochemistry 28:5773, 1989) indicated that it is predominantly the active form of PAI-1 that is present in and can be purified from an ammonium sulfate precipitate of porcine platelets. To clarify this situation, we investigated and compared the status of PAI-1 in porcine and human platelets. Immunologic analysis of the ability of PAI-1 to form complexes with immobilized t-PA indicated that porcine and human platelets contained 3.7 +/- 0.4 and 1.7 +/- 0.3 U of PAI activity per 10(8) platelets (n = 6; +/- SD), respectively; sodium dodecyl sulfate (SDS)-activation of the lysates increased PAI-1 activity to 10.8 +/- 3.0 and 3.8 +/- 0.5 U per 10(8) platelets. Platelet lysates were also treated with an excess of soluble t-PA, which formed complexes with active PAI-1, whereas the latent form was detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography. Furthermore, immobilized t-PA was able to deplete active PAI-1 from the platelet extracts, and the latent form remaining in the absorbed extract could be quantitated by activation with 4 mol/L guanidine. To investigate the differences between our observations and the published data, porcine platelets were extracted, and PAI-1 was partially purified as described in the literature. For quantitative analysis, porcine platelet PAI-1 was also purified to homogeneity using standard chromatographic procedures optimized in our laboratory for endothelial PAI-1, and the purified protein was used to develop an enzyme-linked immunoabsorbent assay for porcine PAI-1 antigen. Our results indicate that: (1) latent PAI-1 in concentrated ammonium sulfate precipitates of porcine platelet lysates cannot be detected unless the precipitates are diluted before treatment with denaturants; and (2) active and latent porcine platelet PAI-1 can be separated by gel filtration over molecular sieving columns. In summary, this report documents that PAI-1 in porcine platelets is present in both an active and a latent form.


Prothrombin Himi: a compound heterozygote for two dysfunctional prothrombin molecules (Met-337-->Thr and Arg-388-->His).

A congenitally dysfunctional form of prothrombin, Prothrombin Himi, shows reduced fibrinogen clotting activity, although it retains full hydrolytic activity toward synthetic substrates. To elucidate the structural abnormality of the variant prothrombin, we first performed genetic analysis of dysprothrombin. Polymerase chain reaction amplification of the exons 8 through 14 of the proband and her family members' prothrombin genes, which code the thrombin moiety, followed by single-strand conformation polymorphism analysis, identified two variant conformers in exon 10 specific to this family. One variant allele detected in the father was inherited by the proband and one of her sisters, and the other detected in the mother was also inherited by them. This result indicates that the proband has two different base pair changes in the gene. Sequencing showed two novel point mutations in the proband's gene. One is a T to C transition at position 8751, resulting in the substitution of threonine for methionine at codon 337 (Thrombin Himi I). The other is a G to A transition at 8904, resulting in the substitution of histidine for arginine at codon 388 (Thrombin Himi II). By sequencing analysis of her parents, it was determined that Thrombin Himi I was inherited from the father and Thrombin Himi II from the mother. These results confirm that Prothrombin Himi is compound heterozygous for two dysfunctional prothrombin molecules.


Importance of platelets in experimental venous thrombosis in the rat.

Venous thrombosis was induced by ligature of the inferior vena cava in rats whose blood was made hypercoagulable by intravenous (IV) administration of tissue thromboplastin. From a dose-response showing that the administration of increasing doses of tissue thromboplastin resulted in a subsequent progressive increase of thrombus weight, two concentrations of tissue thromboplastin were chosen: a high dose (550 microL/kg, IV) where thrombus formation was optimal and a concentration (7 microL/kg, IV) where tissue thromboplastin-hypercoagulability was intermediate. In both experimental conditions, leukopenia provoked by a myelotoxic drug did not influence the development of venous thrombosis. However, after thrombocytopenia induced by an antiplatelet antiserum, a dramatic decrease in thrombus formation was observed in animals that had been pre-challenged with the lower dose of tissue thromboplastin, whereas decrease in platelet count did not affect venous thrombosis under high thrombogenic challenge. When administered orally 2 hours before thrombosis induction, the ticlopidine analogue clopidogrel showed dose-dependent inhibition of thrombus formation in animals that were pre-challenged with a low dose of tissue thromboplastin (ED50 = 7.9 +/- 1.5 mg/kg, orally) but remained ineffective against high tissue thromboplastin-induced venous thrombosis. We further determined the effect of heparin and hirudin, and showed that both of these drugs exhibited a more potent antithrombotic activity after injection of the lower dose of tissue thromboplastin than after injection of a high dose of tissue thromboplastin. Therefore, using our model of stasis and hypercoagulability, platelet activation played a major role in the development of venous thrombosis when the thrombogenitic stimulus was mild.