[Molecular mechanisms of antioxidant action of dalargin on the liver in experimental cholestasis].

Changes of antioxidant activity of dalargin in the liver after naloxone (100 micrograms/kg) administration were examined in experiment on 144 rats with cholestasis. It was found that dalargin inhibited the activity of xanthine oxidase by 32-37% in different time periods after the injection. Dalargin and naloxone, when used in combination, had no effect on the enzyme activity. Glutathione-S-transferase activity rose by 38.0% and 21.8% on hour 1 and 3 after the injection, respectively, while simultaneous injection of dalargin and naloxone induced no changes in the enzyme activity after 1 hour, though decreased it by 36.8% and 26.4% on hour 3 and 5, respectively. Dalargin inhibited lipid peroxidation by 29-35%, simultaneous injection of dalargin and naloxone raised lipid peroxidation by 109.2%, 80.7% and 25.7% after 1, 3 and 5 hours, respectively. Dalargin injection elucidated a marked tendency to lowering of blood release of the liver-specific enzymes histidase and urokaninase in line with enhancement of their activity in the liver. A combined injection of dalargin and naloxone promoted high release of histidase and urokaninase in blood and did not change histidase activity in the liver in all cases. Urokanidase activity elevated in 5 hours. It was noticed that dalargin raised leu-enkephalin levels in the liver 3.5-fold 1 h after the injection. The reduced dalargin antioxidant effect coupled with naloxone pretreatment demonstrated indirect action of the neuropeptide on the liver via neuron receptors of the liver.


[A comparative study on the effects of amiridin on learning and memory of old rats during passive avoidance test].

18-month rats showed much less learning ability in comparison with that of 3-month rats. 20-days treatment of old rats with amiridin, tacrine, and piracetam improved latency in passive avoidance test to the level of 3-month rats. The activity of acetylcholine esterase (AChE) from homogenates of old rat cortex was reduced as compared with that of young rats. After treatment with amiridin the activity of the enzyme remained unchanged, tacrine stimulate the decrease of activity of AChE while piracetam increased activity of AChE to the level of 3-month rats. The aging of rats is followed by the reduction of unsaturated fatty acids, the increase of cholesterol content and the increase of microviscosity of membranes of brain synaptosomes. Multiple treatment with amiridin, piracetam and tacrine normalized these indices.


[Effects of combined action of heavy metal salts and phenol on energetics of isolated rat liver mitochondria].

Experiments were carried out to investigate effects of heavy metal salts and phenol both alone and in different combinations on respiration rate and coupling of respiration and oxidative phosphorylation by rat liver mitochondria. Oxygen consumption measurements were obtained using a Clark electrode and lactate plus glutamate as substrates. It is shown that a 50% decrease of the respiratory rate was achieved in less concentrations of substances in the mixture compared with the effects of the individual substances. For respiratory control recovery by EDTA titration more amounts of complexion for model mixtures was necessary. The results evidence synergism of the substances responsible for a considerable increase in toxicity. Mitochondrial test-systems are suggested for rapid prognosis of toxicity of multicomponent mixtures of substances.


[Effects of propranolol on the gastroduodenal myoelectrical activity].

The effects of propranolol on the myoelectrical activity of the stomach, pylorus and duodenum were investigated in chronic experiments on rabbits. During the first phase of propranolol action a positive gastro-chronotropic effect was accompanied by a negative cardio-chronotropic effect. The gastro-chronotropic effect was 3 times higher than cardio-chronotropic one. The second phase of the propranolol effect manifested with periodical fluctuation of the gastroduodenal myoelectrical activity during stable bradycardia. Marked reactivity of the gastro-duodenum and periodical fluctuation of its activity are evidence of vital importance of the autoregulation mechanisms for gastroduodenal myoelectric activity.


[Role of hematopoietic cells-precursors in the regeneration of hematopoiesis after cytostatic action].

Cyclophosphamide was injected at a dose of 150 mg/kg once to CBA mice and caused expressed hypoplasia of bone marrow hemopoiesis (decreased myelokaryocyte count, CFU-S and CFU-D) from day 1 of the experiment. The rise of CFU-S (8) and CFU-S (12) to a baseline level was noted on day 4 (the time of intensive regeneration processes), whereas CFU-D levels did not change. It is suggested that cytostatic action may trigger "shunt" hemopoiesis mechanisms which enhance differentiation of primitive hemopoietic cells and proliferative activity of mature hemopoietic elements.


[Zinc contents of pancreatic islets in animals of various species after administration of diabetogenic agent, dithizone].

A decrease of zinc content in pancreatic islet was shown in animals which received diabetogenic agent dithizone. The changes of islet zinc content occurred mainly on account of insulin-producing cells and in coincidence with glycemia changes.


[Specificity of the effect of polyene phosphatidylcholine depending on the mode of administration and animal species].

The effects of polyene phosphatidylcholine (PPC) treatment at oral and intravenous administration to rats and rabbits in hypercholesterolemic diet were studied. No aorta damage was observed in either of rat groups. But fatty liver appeared, and it was the greatest in rats, who received cholesterol and PPC. The result may be attributed to adaptive protection of peripheral tissues due to high experiment duration (18 months) in the state of active reverse cholesterol transport (RChT). No antiatherogenic effect was noted in rabbits at PPC administration (170 mg/kg), while its intravenous injection (50 mg/kg) resulted in marked reduction of plasma cholesterol level, elevation of HDL cholesterol and decrease of the extent of aorta damage. The conclusion is drawn on the ppc high antiatherogenic effect predominantly at intravenous administration, and on advisability of its use in cases of RChT deficiencies, as its activator.


[Effects of morphine and naloxone administered to pregnant rats on the adrenal glands and testes of the offspring].

Relative testis and adrenal weights, testosterone and corticosterone levels in the blood were determined in 9-, 16-day and 2-month-old rats born to females injected with morphine (10 mg/kg/day), naloxone (10 mg/kg/day) or saline throughout the 15-18 days of gestation. Opioid receptors agonist morphine caused a long-lasting inhibition of the testes and activation of the adrenals. Saline injections to the females, that are known to be a stressor for them, also inhibited the testis of the neonatal offsprings. The block of the opioid receptors by the naloxone prevented the effect of prenatal stress on the testis, but inverted the negative correlation between the testes and the adrenals, that can be observed in the normal development.


[Effects of phenothiazine and dibenzazepine derivatives on the muscarinic cholinergic system].

Interactions of some dialkylaminoalkyl (DAL) and dialkylaminoacyl (DAC) derivatives of phenothiazine and dibenzazepine with muscarinic cholinergic receptors (MR) of rabbit striatum and heart and rat brain were investigated. DAC derivatives were more active at brain and heart MR in some cases. The most active preparation was G-512, DA-analogue of chlorpromazine. Some cardiotropic properties of antianginal preparation nonachlazine may be connected with its central antimuscarinic activity.


[Immunocorrecting properties of antibiotics in secondary immunodeficiency].

The study investigated immunocorrecting properties of penicillin G, streptomycin, gentamycin in cyclophosphamide--induced immunodeficiency in mice. It was determined, that antibiotics in sub-bactericidal doses possess pronounced immunocorrecting properties. This effect was observed in both humoral and cell-mediated immune response.


[Antiviral factor production by infected chick embryo fibroblasts].

The secretion of antiviral factor (AF) by infected cell cultures was examined. Activity of AF depended on the cell culture used. AF produced by infected chick embryo fibroblasts had maximal activity. No activity was registered in BHK-21 cells, whereas human embryo fibroblasts and cell line Vero produced a low level of activity. Actinomycin D and cycloheximide prevented the production of AF. The results indicate that VEE virus-infected chick embryo fibroblasts produce AF which may be attributed to nonspecific factors of cell defense.


[Effects of IL-1 and TNF-alpha on the in vitro growth of fibroblasts].

Purified mouse IL-1 at doses higher than 5 u/ml strongly inhibits the growth of passaged rabbit bone marrow fibroblasts. Rec, human TNF-alpha at doses 0.05-3 u/ml slightly stimulates and at higher doses inhibits the in vitro growth of passaged fibroblasts from rabbit and human bone marrow. At the same time the in vitro growth of human skin fibroblasts is stimulated by rec. human TNF-alpha.


[Expression of human LSB 32/67 KD gene in E. coli and analysis of its interactions with laminin].

Earlier we have cloned cDNA coding for a polypeptide that reacts with monoclonal antibodies specific for some cytoskeleton structures. This gene is homologous to the laminin receptor 67 KD. However, cDNA suffices only for a polypeptide of 32 Da, far smaller than the 67 rDa laminin receptor. We have constructed a vector that produces the fusion protein LBP-TrpE in the bacterial strain CAG-456. Our studies show that hybrid protein LBP-TrpE is able to interact with laminin. This result was confirmed by using following methods: immunoblotting, "ELISA" and affinity chromatography on laminin.


[Athymic mice of the 101/HY strain with a new allele at the nude locus].

The spontaneous autosomal recessive mutation at locus nude (nuY) was discovered in 101/HY mice imperfect in respect to repair of induced chromosome damage and this is maintained in conventional conditions at RLEBM. Homozygous mutants have no hair and thymic differentiation. The new mutant form can be a valuable model for medical and biological experiments requiring T-cell immune deficiency.


[Erythrophagocytosis and pigment cells of the amphibian liver].

The ultrastructure of Kupffer liver cells of adult frogs collected in winter and Kupffer cells during amphibian metamorphosis when larval red cells are replaced by adult red cells were investigated. It was revealed that Kupffer cells of the animals investigated had very large size and consisted of either the whole senescent erythrocyte or many phagosomes with small electron dense granules resembling ferritin. Phagosomes are oriented among numerous vesicular and vermiform profiles of agranular endoplasmic reticulum and big Golgi complex. Comparing our morphologic evidence with data of the literature that granules of melanin are synthesized and localized in frog liver Kupffer cells, we came to conclusion that pigment cells were formed as a result of erythrophagocytosis and therefore they were the depo of catabolism products. The comparison of functions of melano-macrophage centers of fish liver and of pigment cells of amphibia liver were discussed.


[Dependence of epidermal growth factor activation on its affinity and oligomerization].

Epidermal growth factor receptor (EGF-R) oligomerization has been followed on A-431 cells using covalent labeling by 125I-EGF and EGF-dependent autophosphorylation of receptor-kinase. High molecular weight complexes corresponding to monomeric, dimeric, and trimeric forms of EGF-R are detected. The process of oligomerization occurs effectively at 37 degrees C while at 4 degrees C no oligomer formation is detected. PMA or ATP treatment reduces the number of high-affinity EGF-binding sites but has no influence on dimer formation. Dimerisation of the EGF-R in the absence of the ligand has been established on formalin-fixed A-431 cells.


[Effects of phytoadaptogens on biorhythms of indicators of the hemostasis system in dogs].

Circadian rhythms in dog hemostasis were studied in winter. The animals were fed with eleutherococcus and liquorice extracts with bread in different time of the day. Hemostatic investigations revealed chronosensitivity of hemostasis to the phytoadaptogens which is important for assessment of their chronopharmacological properties and prospects of their use.


[Development of a pseudointima in a synthetic prosthesis in experimental hypercholesterolemia].

Under study was the dynamics of formation of pseudointima in a synthetic lavsan prosthesis of the aorta of 32 rabbits, 22 of them were on the atherogenic diet. Against the background of experimental hypercholesterolemia the proliferation of cells and the obliteration of the vessel lumen were found to proceed more rapidly than in normal animals.


[Effects of hypothermia on the development of ischemic lesions of skeletal muscles in rats].

The influence of hypothermia on the development of the ischemic disorders was studied using allotransplantation of the rat skeletal muscle (m. lumbricalis) to the anterior chamber of the eye after different period of ischemia. The morphological and immunohistochemical (monoclonal antibodies to heavy chain of the fast myosin, PAP-method) data were found confirming that hypothermia (2-4 degrees C) prolongs the period of the ischemic disorders first appearance by 5 h (from 6 to 11 h) if compared with development of ischemia in the muscle at 21-23 degrees C.


[Light optical and electron microscopic changes in striated muscle tissue under the effects of sea water and thymogen in experimental animals].

Effect of sea-water with t degrees +4 degrees to +8 degrees C with time of exposure from 30 minutes to 24 hours on pelvic extremities was studied in 200 rabbits, optical and ultrastructural studies were carried out. It was revealed that effect of sea-water causes damage of striated tissues' structure and death of animals. Prophylactic injections of thymogen stabilized ultrastructure and raised survival, if time of exposure did not exceed 12 hours.


[Changes in gas composition of expired air during electric stimulation of the ventral medulla oblongata].

The experiments on urethane-anesthetized cats with the electrically stimulated ventral brain stem revealed that caudal ventral medulla at the depth of 1500 microns possesses structures whose electrical activation increases the level of carbon dioxide in arterial blood and in the end portion of expirate, on the one hand, and decreases the oxygen content in expirate and arterial blood, on the other hand.


[Formation of alcohol motivation under blockade of protein synthesis by cycloheximide].

The influence of protein blocker cycloheximide on ethanol intake in rats under condition of development of alcohol motivation was studied. It was found that intracerebroventricular injection of cycloheximide caused inhibition of alcohol intake in rats which had free choice between water and 20% ethanol solution. The blockade action of cycloheximide on the development of alcohol motivation was dependent on initial preference of ethanol in rats and was more strong in rats with originally low preference for ethanol.


[Changes in brain monoamine oxidase activity in conditioned passive avoidance reaction in rats].

Activity of the enzyme monoamine oxidase (MAO) and kinetic parameters (Km, Vmax) for the 5-hydroxytryptamine (5-HT) and dopamine deamination were examined in the brain of rats with conditioned passive avoidance recall. Changes of the 5-HT and dopamine deamination were found in amygdala, striatum and frontal cortex. MAO activity was not changed in hippocampus. In amygdala the rate of 5-HT deamination was significantly increased and kinetic studies revealed increased affinity of the enzyme for 5-HT. The metabolism of dopamine in amygdala was unchanged. In frontal cortex the deamination of 5-HT was not changed, but the dopamine deamination significantly decreased. This decrease was due to lowering of MAO affinity for dopamine. In striatum the metabolism of both 5-HT and dopamine was reduced, and kinetic studies showed the lowering of Vmax for 5-HT and dopamine deamination.


[Effects of neuropeptide Y on rat body temperature in normal conditions and after ethanol administration].

It was shown that intracerebroventricular (icv) administration of 2 micrograms neuropeptide Y (NPY) increased the rectal temperature in rats 2.5 hours postinjection. During 5 days we analysed dynamics of the effect of NPY on alcohol-induced hypothermia in this particular interval. 2 micrograms of NPY were given daily 30 min prior to 25% solution of ethanol (3 g/kg weight rat) intraperitoneal injection. It was found that NPY can prevent the attenuation of alcohol hypothermia on the 3-d and 4-th injection day. It was supposed that the inhibitory effect of NPY on the development of alcohol tolerance may be due to the capacity of NPY to increase food behavior. So it's known that activation of other competitor motivation may inhibit the development of alcoholism.


[Effects of magnesium and nickel ions on penicillin-induced focal epileptic activity in the cerebral cortex of rats].

In experiments on freely moving male Wistar rats on the model of penicillin-induced focal epileptic activity (EA) (the application onto the sensorimotor cortex of a filter paper soaked with benzylpenicillin sodium salt solution) it was shown that addition of MgSO4 (series 1) and NiCl2 (series 2) into the solution of penicillin significantly weakened EA. The combination of Mg2+ and Ni2+ with penicillin (series 3) produced a more significant suppression of EA as compared with separate application of the above-mentioned ions: the latency period of appearance of interictal discharges (IID) increased, the frequency and amplitude of IID decreased much more, no ictal discharges appeared in any animal, the duration of epileptic foci reduced to a much greater extent. This effect can be explained by the blockade of Ca current by the above-mentioned ions. One can suppose that the amplification of antiepileptic effects of combined action of Mg2+ and Ni2+ was due to an increase in the number of blocked voltage-dependent and NMDA-operated calcium channels.


[Study of blood and lymph in experimental myocardial ischemia and arterial hypertension].

The peripheral blood and central lymph of rats under experimental myocardial infarction was studied by means of light microscopy and electric conductivity measurement. Both hypertensive rats and animals 3 days after myocardial infarction had similar quantity of neutrophils in peripheral blood. Lymph cells count of hypertensive rats by middle lymphocytes is similar to the animals 1 day after myocardial infarction. The correlation between lymph and blood electric conductivity and its cell composition was noted.


[Use of 2,4-dinitrophenol for immunosuppressive action on the recipient in implantation of fetal organs in mice].

In experiments performed on 118 white mice and 10 laboratory rats, it was shown that cutaneous application of 2,4-dinitrophenol microdoses once a week around the sites of fetal organ implantation leads to a significant prolongation of the implant life with minimal trouble for the recipient's health. The maximal effect was reached with 10(-3) M concentration of DNP, the applications beginning 2-3 weeks before fetal pancreas implantations.


[Effects of carnosine on systemic hemodynamics and myocardial metabolism in rats in the early postresuscitation period].

It was demonstrated in experiments on male rats that acute lethal blood loss and subsequent resuscitation after 4- and 6-min clinical death induce lipid peroxidation processes, decreased antioxidant enzyme activity, cause activation of anaerobic glycolysis in the myocardium. This metabolic heart impairment causes hemodynamic instability in postresuscitation period. 25 mg/kg of carnosine injected during resuscitation decreased functional-metabolic heart impairments and hemodynamic disarrangement as well as early postresuscitation lethality. The authors attribute positive carnosine effect to its significant antioxidant activity.


[Possibilities of restoration of the regional lymphatic channel by the use of sorbents in the treatment of experimental generalized purulent peritonitis].

Regional lymphatic bed in local application of mineral sorbent SUMS-2p in 80 white noninbred rats with experimental generalized purulent peritonitis is studied with the use of indirect intravital lympho-roentgenography. The correlation between lympho-dynamics and generalized purulent peritonitis stage was observed. It is shown that the peritoneo-sorption with the mineral sorbent SUMS-2p led to early restoration of lympho-drainage.


[Effects of the respiratory epithelium on the tracheal smooth muscle in guinea pigs].

We studied the influence of respiratory epithelium on tracheal smooth muscle tone of guinea pigs. Mechanical removal of the epithelium induced an increase of the contractile response to histamine. In preparation of smooth muscle previously contracted by histamine, isoproterenol induced dose--dependent relaxation, the level of which was significantly greater in tracheal smooth muscle with epithelium than without it. These results suggest an important role of respiratory epithelium on the contractile activity of smooth muscle.


[Effects of synthetic antioxidant ionol on bioelectric activity of cardiomyocytes and arrhythmia in global ischemia and reperfusion of the isolated rat heart].

Isolated rat hearts were subjected to global ischemia (15 min) and reperfusion (20 min). Transmembrane potentials were recorded on the epicardial surface and contractile force was measured. Ischemia reduced the resting potential, the action potential (AP) amplitude and the AP duration (APD) in control animals. Pretreatment with synthetic antioxidant ionol (BHT, 50 mg/kg, per os) didn't influence the time course of changes in the resting potential and AP amplitude during ischemia, but significantly increased APD. In the pretreated group, 5 min after the aorta clamping, the APD at 50% and 90% levels of repolarization was 36% (p less than 0.05) and 13% (p less than 0.1) higher in comparison to the preischemic level and 10 min after clamping by 27% (p less than 0.1) and 29% (p less than 0.05), respectively. By the end of ischemia in the pretreated group, APD re-decreased almost to basal level, but in control group, it remained decreased. During reperfusion BHT improved the recovery of bioelectrical activity and the contractile function. The BHT 10-fold reduced the malignant arrhythmias duration and 2.5-fold the incidence of ventricular tachycardia and fibrillation during reperfusion. These results indicate that the induced by BHT increase in APD can contribute to the mechanism of BHT antiarrhythmic action.


[Beta-mannosidase of trophoblast and humana skin fibroblasts].

Conditions for assay of beta-mannosidase activity in human chorionic villi were studied using the fluorogenic substrate 4-methylumbelliferyl-beta-D-mannopyranoside. Comparison of the biochemical properties of the chorionic villi beta-mannosidase with those of the enzyme from human cultured fibroblasts showed their similarity. Like the enzyme from skin fibroblasts, the chorionic villi beta-mannosidase had rather high activity. Both enzymes had virtually the same pH optimum (4.2-4.7) and Km value. The data presented suggest that chorion biopsy specimens can be used for prenatal determination of beta-mannosidase activity at the early stage of development.


[Study of pantothenic acid derivatives as cardiac protectors in a model of experimental ischemia and reperfusion of the isolated heart].

An isolated heart model with experimental ischemia and reperfusion was used to show effective decrease in lactate, increase in ATP content and prevention of conjugated dienes accumulation in the myocardium by derivatives of pantothenic acid: panthenol (9.0 mg/kg), calcium pantothenate (15.6 mg/kg) and by these ones applied simultaneously as ingredients of perfusate (25 microM) in postischemic period. In that way derivatives of pantothenic acid should be regarded as cardiac protectors.


[Hemispheric asymmetry of the nigrostriatal system of the brain in rats genetically predisposed to catalepsy].

Hemispheric asymmetry of nigro-striate system in a strain of rats GC bred from Wistar for a predisposition to cataleptic reaction was studied by means of biochemical and morphological methods. Hemispheric asymmetry was found in GC and Wistar rats with respect to aminopeptidase activity in neurons of caudate nucleus, with a more pronounced left-side increase in GC rats, the asymmetry index being 13.7%. Acetylcholine esterase activity in subcellular particles of caudate nucleus showed an inversion of asymmetry with higher activity in the left hemisphere of Wistar and right hemisphere of GC rats, and asymmetry index of 15.5%. With respect to the number of astroglia cells in S. nigra, and astroglia and oligodendroglia in N. accumbens there was also an inversion of asymmetry in GC rats who had more cells in the structures of the left hemisphere, whereas Wistar rats had more in the right hemisphere. The asymmetry index was high and equal to 29.8% for astroglia in S. nigra, and 17% for astroglia and 21.4% for oligodendroglia in N. accumbens. However, in S. nigra the number of neurons and oligodendroglia cells was equally increased in the right hemisphere in GC and Wistar rats. The data suggest that the mechanism of hereditary pathology of brain nigro-striate system involves both enhancement and inversion of the hemispheric asymmetry.


[Pharmacokinetics of antipyrine, nifedipine and diazepam in experimental myocardial infarct].

It has been shown, that antipyrine, nifedipine, diazepam pharmacokinetics changes in different ways after myocardial infarction. On day 7, 14, and 21 after myocardial ischemia antipyrine T1/2 increased considerably, and antipyrine Cl and Kel decreased. Nifedipine T1/2 increased on day 7 only. There were no diazepam pharmacokinetic changes during restoration period. However, microsomal diazepam metabolism changed significantly. Diazepam hydroxylation increased on day 7 of myocardial infarction, and on day 14 and 21 did not differ from the control. Diazepam metabolites content changed considerably during restoration period. Under myocardial infarction cytochrome P-450 isoenzymes, oxidizing present substances seem to be altered to different extent.


[Reduction of voluntary alcohol consumption under the effects of prolonged-action zinc].

It was revealed that an involuntary rats' intoxication with 10% ethanol solution during 8 months caused a reduction of zinc content in the brain. A subcutaneous depot administration of highly dispersed zinc powder in a dose of 5 mg/kg reduced ethanol consumption under conditions of free choice for two weeks. This effect of the preparation was associated with normalization of zinc content and increase of a malondialdehyde level in the brain.


[Action of GABA-positive preparations on uterus-stimulating effects of activating neuromediators, prostaglandin F2 alpha and oxytocin].

Experiments on isolated strips of the non-pregnant rabbit and rat uterus showed the ability of dopamine, noradrenaline, serotonin, acetylcholine, prostaglandin F2 alpha, oxytocin to increase the uterine strips contractile activity. On the other hand, GABA, GABAB receptors agonist phenibut and diazepam inhibit the stimulating effects of the above mentioned substances, thus showing the properties of physiological antagonists of these neuromediators, prostaglandin and oxytocin.


[Thymectomy and splenectomy of adult mice have no influence on the level of suppressor activity of T-lymphocytes specific to antigens of H-2 complex].

I.v. immunization of mice with irradiated (1500 rad) allogeneic spleen cells induces T cells specific to histocompatibility antigens and capable to suppress proliferation in mixed lymphocyte cultures. The lymph node cells appeared to be almost as capable as spleen cells of producing suppression. Splenectomy experiments showed that the spleen is not essential for induction of suppressive activity of T cells. The precursors of T cells with suppressive activity do not belong to the pool of short-living cells as they are insensitive to adult thymectomy, produced 6 weeks before immunization.


[Serum antibodies to brain protein antigens in cerebral palsy in children].

Using special brain antigen test-system ELITEST-24, ELISA assays of the serum of children with cerebral palsy were conducted. The data obtained were compared to relevant characteristics of the sera from neurologically and somatically healthy persons and patients with diagnoses of multiple sclerosis, schizophrenia, epilepsy and hepato-cerebral dystrophy according to special PC program VIZUAL and DIAGNOST. High specificity of the cerebral palsy anti-brain antigens reactivity was revealed by ELITEST technique. Moreover, the comparison of the mother-child pair immunoreactivity has been conducted. Evidence for hypothesis of epigenetically performing of children antibodies repertoires by mother-during-pregnancy immune status were obtained. Possible immunopathologic mechanisms of the disease are discussed.


[Effect of T-activin on the formation of conditioned reflex and manifestations of unconditioned reflex of avoidance in August strain rats].

It has been revealed that intraperitoneal injection of T-activin (humoral factor of the thymus) to August rats leads to more rapid and stable conditioned reflex formation to a sound and to a decrease of avoidance time when electric current is given to a shuttle chamber. Furthermore, less amount of uneffective series in testing unconditioned avoidance is registered in the test animals. A positive T-activin effect on conditioned reflex formation and unconditioned reflex manifestation is probably connected with its ability to alter hippocampus functional parameters and (or) with anti-stressor properties of the preparation.


[Induction of stromal cell transformation in xenografts of human colonic cancer].

Two transformed stromal cell lines FM-7 and FR-7 were obtained from human xenografts of colon tumor (HCT-7), propagated in nude mice and nude rats, respectively. These two cell lines were confirmed to be murine and rat's by a cytogenetic study and were tumourigenic in BALB/c nude mice with the morphology of fibrosarcoma. Human DNA sequences were not detected by hybridization with pBlur8 probe in DNA of FR-7 cell line grown in vitro. These results indicate that human cancer cells from xenograft HCT-7 can induce malignancy in adjacent normal cells of nude mice and nude rats in the absence of DNA transfer.


[Stimulation of skin wound contraction and epithelialization by soluble collage].

It is found that local applications of the unguent with soluble collagen, but not solution of the collagen, stimulate healing of erosions and full-thickness excision wounds in the rat skin. Not all the stages of healing were stimulated, but only two of them--contraction and epithelialization.


[Effects of helium-neon laser on regeneration capacity of skeletal muscles of adult guinea pigs].

The muscle regeneration was studied in across-cut gastrocnemius muscle of adult guinea pigs. Both hind guinea pig legs were exposed to laser therapy (3-9 procedures for 5 min each) after operation. It was shown that laser therapy accelerated fibrin resorption and wound healing, decreased the muscle fiber degeneration, increased DNA and RNA synthesis in regenerating tissues, stimulated regenerative muscle fiber capacity. However, the regenerating muscle tissue did not connect both muscle stumps, and the narrow connective tissue scar was formed. Before it was shown that the same laser therapy conditions were more effective for rats. Probably, the stimulating effect of laser rays on regenerative tissue ability depends on species peculiarities of animals.


[Ultrastructural and morphometric analysis of the Paneth cell reaction to administration of cholera toxin].

Experiments were made on 40 immature guinea--pigs which were divided into two groups. 20 animals received intraduodenal injections of cholera toxin and 20 others were injected normal saline (groups I and II, respectively). Group I animals exhibited a rapid fall in one--cell secretion of XS recorded on hour 6-12 after the injection of cholera toxin. Inhibition of functional activity was associated with defected synthesis of granules in the cells of Paneth.


[A new method of a morphometric study of the kidney using a computer-assisted morphometric complex "Diamorph"].

The use of a computer--based morphometric complex "DIAMORPH" for automated morphometric analysis of renal glomeruli is described. By combining the principles of morphological and functional approaches to diagnosis with the capabilities of present--day research equipment, a new informative morphometric parameter--the extent of capillary--mesangium contact--has been identified. With this parameter and its derivatives, it has been possible to differentiate, in a statistically significant way (p less than 0.01), healthy subjects (controls) from patients with manifest diabetes mellitus of up to I year in duration who could not be distinguished through light microscopic or conventional morphometric studies.


[Effects of different doses of hyperbaric oxygenation on morphology and transcription of cortical neurons of rats with experimental cerebral ischemia].

Its effect of hyperbaric oxygenation (the doses 1.2 and 2 ata) on cortical pyramids of the rats with one-or both-side ligation of common arteria carotis 2.5 or 24 hours after operation was studied. The neuron survival and transcription activity were estimated. In all experimental situations except the most serious, e.g. 24 hours after the both-side artery ligation, the dose 2 ata was more effective. On the contrary, in most serious cases the dose 1.2 ata or applying in the baro-camera the air instead of oxygen provided the better result.


[Obtaining and morphological characteristics of primary monolayer cell cultures of human somatotropinomas].

This paper describes the development of method of preparing cell suspension obtained from surgical material of patients with pituitary adenomas and acromegaly as well as the procedure of subsequent long-term cultivation in monolayers of the cells isolated. As judged by visual inspection and measurement of growth hormone and prolactin secretion, tumor pituitary cells kept viability and functional activity for at least 6 days of growing in vitro. Immunocytochemical visualization of somato- and lactotrophs of the same histological preparations permitted us to show that vast majority of cultured cells is represented by somatotrophs; however, a small portion of cell population is represented by lactotrophs and lactosomatotrophs. The peculiarities of cytoarchitectonics in two types of cell cultures of human somatotropinomas were studied.


[Effects of low intensity infra-red laser irradiation on ultrastructure and proliferation of liver cells in experimental hepatitis and cirrhosis].

With the aid of light, electron transmission and scanning electron microscopy and radioautography and stereometry, the influence of low-intensive laser irradiation (LILI) (infrared) was studied in normal rat liver and in experimental cirrhosis and hepatitis. It was revealed that arsenide-gallium laser irradiation causes the change of intracellular structure. These changes show the intensification on their specific function manifest in an increase of relative volume of intracellular structures. The changes of microvessels show the activation of microcirculation. The elevation of the index of the labelled nuclei testify to increased proliferation. The similar influences of LILI on the liver ultrastructure and proliferation in hepatitis and cirrhosis are accompanied by the reduction of the pathological changes of the liver--the hepatocytes oedema, granular, vacuolar and fatty dystrophy.


[Morpho-biochemical study of catecholamine metabolism in the myocardium of dogs with fibrillation and autolysis].

The balance of catecholamines (CA) and their metabolites in the canine heart during myocardial fibrillation and 1, 2, 3, 4, 5, 6 hours after death was studied. The fibrillation was caused by low myocardial electrostimulation. The comparative neuro-morphometric analysis of the CA-containing structures in 1 and 5 min of the fibrillation and the determination of the norepinephrine, its precursors and metabolites by HPLC-ED method was done. There was an increase of the myocardial norepinephrine (20%), 3-4-dihydroxyphenylethyleneglycol (25%) and decrease of the dopamine (50%) contents without any qualitative changes of CA metabolism. Autolysis leads to the successive decrease of the norepinephrine and dopamine contents without any qualitative changes of the CA metabolism too. These data suggest that autopsies could be used for the determination of the myocardial CA content and the qualitative characteristics of the CA metabolism before death in those who died suddenly.


[Histophotometric characteristics of structural-metabolic heterogeneity of hepatocytes in acute hemorrhage and pulmonary artery thromboembolism].

Basing on the data obtained at early autopsies, we compared the structure and metabolism of liver acini in acute blood loss and pulmonary artery thromboembolism. Morpho- and pathogenetic impairment of hepatocytes of various acinic zones was correlated and studied histo-enzymatically.


[Structural-metabolic characteristics of acute hepatic failure].

Basing on early autopsy data, the study was made of structural and metabolic characteristics of the liver in marked intoxication and blood loss. Histologic, histoenzymatic and biochemical methods were used to evaluate morpho- and pathogenesis of acute liver failure arising in the above conditions.


[Neurohistological characteristics of regeneration of the ends of the injured nerve under measured traction].

A resection of sciatic nerve in 25 dogs was performed out and the ends of cut nerve were connected by sutures with special apparatus for quantified traction of nerve stumps. 5 days after resection traction was stented at the rate of 0.25 mm twice a day. A histological analysis performed in 5, 12, 19, 26, 33 days after resection an 2-12 months after nerve suture has shown that stress strain effects on intercalation of nerve structures (Ilizarov's effect). Newly formed Schwann chains free of products of axonal and myelin disruption have got a perfect longitudinal orientation and that's why can be considered as an optimum substratum for axonal regeneration.


An advance in implant-supported overdentures utilizing rigid attachments.

Two cases are described in which a bar overdenture design is utilized to meet the aesthetic, phonetic, and functional requirements of patients with one or more edentulous arches. The implant-supported overdenture can be a creative solution for anatomic and skeletal discrepancies.


Treatment of a discolored, endodontically treated tooth with home bleaching and composite resin.

Internal bleaching is frequently used to treat discolorations of endodontically treated teeth, and home bleaching of stained vital teeth has also become popular. This article describes a case in which carbamide peroxide home bleaching was used to enhance the appearance of an internally bleached, endodontically treated tooth.


In-office fabrication of indirect composite-resin restorations.

Indirectly fabricated composite-resin inlay and onlay posterior restorations are becoming more popular. By utilizing a flexible die material, these restorations can be expeditiously completed by the dentist or by a well-trained dental auxiliary, often in one appointment.


Placement of implants into fresh extraction sites using a resorbable collagen membrane: case reports.

The ability to place endosseous implants has recently expanded to include placement in fresh extraction sites. In this technique, the use of some form of occlusive membrane is often beneficial. In the future, resorbable membranes may provide a predictable clinical result in these types of cases.


Provisionalizing periodontally involved dentition with implants.

Many patients who are partially or fully edentulous have practiced poor oral hygiene. However, implants can benefit even those patients who are not ideal candidates. In cases of severe periodontal compromise, implants may enhance the survival of the natural teeth, minimize the difficulties of transition if additional tooth loss occurs, and also offer significant psychological benefit to the patient.


Visible light-activated glass ionomer cement: use as liners/bases and restoratives.

Traditional glass ionomer cements were sensitive to moisture during the initial state and were subject to dehydration as the material began to harden. Preliminary findings from in vitro studies and clinical trials indicate that a new light activated glass ionomer cement exhibits high shear bond values, reduced microleakage and improved aesthetics.


The interdental papilla pedicle graft: a simple and aesthetic approach to mucogingival correction.

The interdental papilla represents a potential source of donor tissue for effecting augmentation of the zone of gingiva. Root surfaces that have previously been exposed due to gingival recession also can be covered.


Restoration of Class V abrasions with a new light-cured glass ionomer restorative.

The clinical performance of conventional glass ionomer materials have demonstrated clinical success in Class V restorations, but the physical properties and limited aesthetics of these materials have limited their widespread utilization. This article describes the clinical application of a new Type II light-cured glass ionomer restorative material, demonstrating improved physical properties and aesthetics.


Direct proximal restoration of a permanent first molar: a window of opportunity.

Glass ionomer and composite resin direct application bonding has become an important part of modern restorative dentistry. This article describes the technique of glass ionomer/composite resin stratification restoration of the mesial surface of a permanent molar.


Achieving anterior aesthetics with an anti-rotational abutment.

Single-tooth implants of various minerals and metals have been used, with some success, since ancient times. However, single-tooth implant survival was unpredictable until Branemark and his co-workers developed the theory and technique for obtaining a predictable direct bone to implant interface, which has been termed "osseointegration." Retrospective studies suggest excellent survival of single-tooth implants that predictably achieve this direct bone to implant interface. Case reports of single-tooth implants in the anterior and posterior areas of the jaws suggest the efficacy of this treatment, although scientific prospective studies evaluating the safety and efficacy of this treatment have only recently begun.


The composite resin-amalgam window preparation.

Amalgam restorations with a very visible mesiofacial aspect can be aesthetically modified. Clinical experience has proven the amalgam window preparation to be an economical, expedient, and effective treatment approach when patients request tooth-colored fillings in posterior maxillary teeth.


The bonded porcelain margin in porcelain-fused-to-metal prosthetics.

Progress in dentistry is often made when existing techniques and products are combined with new developments. For example, although porcelain has been used for decades and Buonocore reported on its use in 1955, only in early 1980 did bonded porcelain become popular. Today, we assume that porcelain facings and inlays are an integral part of dentistry.


The plunging ball technique: Class II direct composite resins.

New polymer resin materials appear to be superior in wear resistance and may have less polymerization shrinkage than many other posterior restorative materials. With the use of the plunging ball technique, the ability to obtain tight contact and adequate contour results in fewer postoperative problems.


Direct oven-tempered hybrid composite-resin laminate veneers.

The difficulty in achieving optimal aesthetics of the single-tooth indirect laminate veneer has prompted the author to develop a direct technique for fabricating oven-tempered hybrid composite resin veneers. These restorations exhibit excellent physical properties, marginal integrity, and aesthetics.


Conservative soft tissue management with the low-powered pulsed Nd:YAG dental laser.

Lasers can be operated in a continuous or pulsed mode, either in contact or out of contact with the target tissue. This article discusses laser systems and their resultant effects, and presents histologic findings and clinical examples.


Perception aesthetics and light-cured composites.

Perception aesthetics is the aspect of aesthetic dentistry concerned with patients' perception of their dental appearance. This article discusses how perception aesthetics can be affected by various factors, including the size, color, and shape of the teeth.


Progress in understanding the pathogenesis of the anemia of chronic disease.

Improved understanding of the inflammatory response and the identification and characterization of the specific cytokines involved, as well as improved understanding of erythropoiesis, and the availability of recombinant human growth factors such as EPO, have greatly enhanced our appreciation of the pathogenesis of ACD by allowing development of a number of informative models for studying this syndrome. It appears that a variety of cytokines are involved in all aspects of the pathogenesis of ACD, from the inhibition of erythroid progenitors and EPO production to impairment of iron release. A schematic of the contributions of some of these cytokines to the development of ACD is shown in Fig 6. The exact biochemical mechanisms by which these effects occur is still to be determined. The progress outlined in this report has allowed us to develop a more precise understanding of the pathogenesis of this common and important clinical syndrome. In 1983, Hansen subtitled a review of ACD "A Bag of Unsolved Questions." Although this description is still accurate, our understanding of ACD has now developed to the point where we can offer a more defined subtitle: "A Bag of Cytokines."


Chromosome 11q23 translocations in both infant and adult acute leukemias are detected by in situ hybridization with a yeast artificial chromosome.

The yeast artificial chromosome (YAC-13HH4), which spans a 440-kb region of DNA just distal to the CD3 locus on chromosome 11 at band q23, has been used to characterize a range of chromosomal translocations in acute leukemias from both adults and infants. In situ hybridization was performed on metaphase cells from bone marrow of 17 leukemias and two cell lines with a variety of chromosome 11q23 abnormalities. It was established that in infant leukemias the translocations t(11;19), t(4;11), and t(5;11) had occurred in the region defined by YAC 13HH4. Additionally, the translocations t(4;11), t(6;11), t(9;11), t(X;11), and t(10;11) in other leukemias were found to disrupt the same region of chromosome 11q23, although an exception was found in one t(6;11) translocation for which the breakpoint was distal to the YAC. One patient had a t(9;11) translocation in a therapy-related leukemia, suggesting that this class of etoposide-related malignancy has similar breakpoints to those occurring in de novo leukemias. An example of a lymphoma-derived translocation t(4;11) was shown to involve a deletion of the region defined by YAC 13HH4. A leukemia with a deletion on chromosome 11 (q23-q25) was also studied and it was shown that the YAC sequence was unaffected. It was concluded that, with a few exceptions, the translocations at 11q23 in a wide range of acute infant and adult leukemias occur in a common region and may result from a common underlying mechanism.


Low-risk intensive therapy for multiple myeloma with combined autologous bone marrow and blood stem cell support.

To improve the safety of autotransplantation for myeloma, peripheral blood stem cell (PBSC) collection was attempted in 75 previously treated patients after the administration of high-dose cyclophosphamide (HD-CTX; 6 g/m2) with or without granulocyte-macrophage colony-stimulating factor (GM-CSF). Sixty patients subsequently received melphalan 200 mg/m2 (57 patients) or melphalan 140 mg/m2 and total body irradiation (850 cGy) (3 patients) supported by both autologous bone marrow and PBSC; 38 patients received GM-CSF posttransplantation. Among 72 patients undergoing PBSC apheresis, "good" mobilization (greater than 50 colony-forming units granulocyte-macrophage [CFU-GM] per 10(5) mononuclear cells) was achieved when prior chemotherapy did not exceed 1 year and when GM-CSF was used post-HD-CTX; similarly, rapid platelet recovery to 50,000/microL within 2 weeks was associated with "good" PBSC mobilization. These same variables also predicted for rapid engraftment after autotransplantation, so that hematologic recovery (granulocytes greater than 500/microL and platelets greater than 50,000/microL) proceeded within 2 weeks among the 37 patients with "good" PBSC collection. As a result of rapid neutrophil recovery (greater than 500/microL) within a median of 2 weeks, infectious complications both post-HD-CTX and posttransplant were readily manageable, resulting in only one treatment-related death post-HD-CTX. The cumulative response rate (greater than or equal to 75% cytoreduction) for all 75 patients was 68%, with 12-month event-free and overall survival projections of about 85%. Using both bone marrow and PBSC together with GM-CSF, autotransplants are safe and appear effective in myeloma, especially when prior therapy had been limited to less than 1 year. More than 80% of transplanted patients achieved complete hematologic recovery within a median of 1 month posttransplant (granulocytes greater than 1,500/microL; platelets greater than 100,000/microL; hemoglobin greater than 10 g%), thus providing sufficient hematopoietic reserve for further chemotherapy in the event of posttransplant relapse.


Enhanced marrow recovery by short preincubation of marrow allografts with human recombinant interleukin-3 and granulocyte-macrophage colony-stimulating factor.

We studied an alternative method of using hematopoietic growth factors (HGFs) to enhance hematopoietic recovery in patients undergoing bone marrow transplantation (BMT), by short in vitro preincubation. Twenty consecutive patients with leukemia received T-cell-depleted allografts using Campath-1G. Two thirds of the marrow was infused on the scheduled day of transplant and one third of the marrow following preincubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) on day 4. Engraftment parameters and duration of hospitalization were compared by actuarial analysis to those of 40 historical controls. Patients receiving the incubated boost had significantly faster platelet recovery (P = .017) and shorter hospitalization period (P = .001) when compared with the control subjects. Platelet count reached greater than 25 x 10(9)/L on day 17 (median) in the study group and on day 23 in the controls. The median duration of hospitalization was 20 and 36 days, respectively. In the early posttransplantation follow-up, two of four patients in the study group died as a result of graft rejection, while all 13 deaths in the control group resulted from complications associated with marrow suppression. We suggest that pretransplant in vitro activation of bone marrow cells with IL-3 and GM-CSF may prove to be an efficient method for enhancing marrow recovery after BMT.


The role of macrophages in the regulation of erythroid colony growth in vitro.

Depletion of macrophages from murine marrow by the use of a monoclonal anti-macrophage antibody resulted in a significant increase in the number of erythroid burst forming units (BFU-E). This increase could be neutralized by the addition back to culture of macrophages or macrophage conditioned medium indicating that the suppression was mediated by soluble factors. To further characterize this effect, the addition to culture, either alone or in combination, of interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the growth of BFU-E and the colony-forming unit granulocyte-macrophage (CFU-GM) was examined in macrophage-containing and macrophage-depleted cultures. The addition of IL-1 alpha to culture stimulated the release of both TNF alpha and GM-CSF and acted synergistically with both cytokines, resulting in a dose-dependent suppression of BFU-E and stimulation of CFU-GM growth. The increase in CFU-GM caused by the addition of IL-1 alpha was mediated by GM-CSF but not by TNF alpha as the increase was prevented by the addition of a monoclonal anti-GM-CSF antibody but not by anti-TNF alpha. When either TNF alpha or GM-CSF was neutralized by monoclonal antibodies the addition of IL-1 alpha resulted in a significant increase in BFU-E growth. The addition of GM-CSF to culture caused a dose-dependent suppression of BFU-E that was mediated by TNF alpha, as colony number was not reduced when GM-CSF and a monoclonal anti-TNF alpha antibody were simultaneously added to culture. TNF alpha-induced suppression of BFU-E only occurred in the presence of macrophages. In macrophage-depleted cultures, a dose-dependent suppression of BFU-E could be induced if subinhibitory concentrations of IL-1 alpha or GM-CSF were simultaneously added with increasing concentrations of TNF alpha. The effects of IL-1 alpha or GM-CSF and TNF alpha were markedly synergistic so that the doses required to induce suppression when added simultaneously was only 10% of that required when either were added to culture alone. Suppression of BFU-E by GM-CSF or the combined addition of GM-CSF and TNF alpha did not require IL-1 alpha because inhibition was not neutralized by the addition of anti-IL-1 alpha antibody.(ABSTRACT TRUNCATED AT 400 WORDS)


Identification and characterization of osteoclast progenitors by clonal analysis of hematopoietic cells.

We have identified a distinct population of colony-forming cells that give rise to mononuclear cells expressing an enzyme marker and other features of the osteoclast in bone marrow cultures stimulated by conditioned medium of a murine tumor cell line. These colony-forming cells were defined as osteoclast colony-forming units (CFU-O). The tumor cell-derived activity was recently isolated and was named osteoclast colony-stimulating factor (O-CSF). To understand the development of osteoclast progenitors and to clarify the relationship of osteoclast progenitors to other hematopoietic progenitors, we examined CFU-O in hematopoietic tissues obtained from normal adult mice, mouse fetuses, and mice with 5-fluorouracil (5FU) treatment. CFU-O were present in the adult mouse bone marrow, adherent cell-depleted marrow, in the spleen, and in the day 14 fetal liver, with an incidence similar to other hematopoietic progenitors. The culture period required for the development of CFU-O-derived colonies in vitro and the manner in which CFU-O responded to 5FU suggested that CFU-O belonged to a relatively primitive progenitor population; they are clearly more immature than macrophage progenitors that respond to macrophage-CSF, but more mature than multilineage progenitors that respond to stem cell factor. Our studies have defined and characterized an osteoclast progenitor and distinguished it from other hematopoietic progenitors for the first time.


CD4 is expressed on murine pluripotent hematopoietic stem cells.

We show here for the first time that pluripotent hematopoietic stem cells express the CD4 antigen. CD4+ cells isolated from mouse marrow repopulated all hematopoietic lineages in both the long-term repopulation assay and the competitive repopulation assay. This finding indicates that the CD4+ population contains primitive stem cells with extensive repopulation capacity. Interestingly, the CD4- population had significant life-sparing activity, even though this population was depleted of long-term repopulating stem cells when compared with CD4+ cells. The majority of the cells that respond to the stroma in Whitlock-Witte cultures with B-cell differentiation were recovered in the CD4- population. Thus, this bone marrow (BM)-derived B-cell precursor lacks CD4, which is in contrast to myeloid precursors and thymus-derived lymphoid precursors that reportedly express CD4. We show further that the CD4 molecule expressed on BM cells is similar in molecular weight and epitope makeup to the CD4 antigen found on thymocytes. Detection of CD4 on BM cells is dependent on using high concentrations of antibodies. Thus, it is not surprising that expression of CD4 on pluripotent stem cells has been missed previously. Taken together, our data suggest that the CD4 molecule may play an important role in lineage definition in early hematopoietic differentiation.


A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells.

Members of the CCAAT/enhancer binding protein (C/EBP) family have been shown to regulate the terminal differentiation of adipocytes and hepatocytes. In these cell lineages, high levels of C/EBP alpha are found only in mature, nondividing cells. Using Western blotting and immunohistochemical staining, we have determined the temporal order of expression for C/EBP alpha, C/EBP beta, and C/EBP delta in differentiating myelomonocytic marrow cells. These studies show a unique temporal pattern of C/EBP isoform expression in the myeloid lineage. In particular, C/EBP alpha expression is very high in proliferative myelomonocytic cells, and diminishes during phenotypic maturation. While we have detected C/EBP alpha, C/EBP beta, and C/EBP delta in multiple myeloid leukemia cell lines, and C/EBP alpha in normal myeloid cells and in de novo human myeloid leukemias, we have not detected these C/EBP isoforms in either erythroid or lymphoid cells. Finally, we show that C/EBP alpha, C/EBP beta, and C/EBP delta protein and messenger RNA levels correlate in maturing granulocytic cells. The formation of tissue-specific combinations of C/EBP homodimers and heterodimers may allow this family of transcription factors to regulate different sets of genes in adipocytes, hepatocytes, and myelomonocytes.


Evidence that postoperative fibrinolytic shutdown is mediated by plasma factors that stimulate endothelial cell type I plasminogen activator inhibitor biosynthesis.

Postoperative fibrinolytic shutdown has been attributed to an increase in plasma levels of type I plasminogen activator inhibitor (PAI-1) activity and may contribute to postoperative venous thrombosis. The purpose of this study was to determine whether the postoperative increase in PAI-1 is contributed to by a plasma mediator(s) that stimulates PAI-1 synthesis and secretion by vascular endothelium. Plasma samples collected from patients (N = 11) before and after surgery for total hip replacement were (1) assayed for endogenous plasma PAI-1 antigen and activity, and (2) incubated with cultured human umbilical vein endothelial cells (HUVECs) and PAI-1 antigen and activity measured in the conditioned medium (CM). Eighteen hours after surgery, endogenous plasma levels of PAI-1 antigen and activity were increased by 225% (P = .003) and 190% (P = .04), respectively over the preoperative values. In addition, compared with preoperative plasma, postoperative plasma increased HUVEC secretion of PAI-1 antigen and activity by 99% (P = .001) and 66% (P = .002), respectively. This increase in HUVEC PAI-1 secretion reflects an increase in PAI-1 mRNA expression and protein biosynthesis as confirmed by metabolic radiolabeling, immunoprecipitation, and Northern blot analysis. Ultra-filtration experiments indicate that the postoperative plasma mediator(s) that stimulates HUVEC PAI-1 biosynthesis is in a molecular weight (MW) range of approximately 30 to 100 Kd. Heat treatment (56 degrees C; 30 minutes) of postoperative plasma abolished the induction of HUVEC PAI-1 production. Enzyme-linked immunosorbent assay and immunoneutralization experiments indicate that tumor necrosis factor-alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) do not contribute to the postoperative plasma effect on HUVEC PAI-1 synthesis. These observations demonstrate that postoperative patient plasma contains a factor(s) that may stimulate endothelial cell PAI-1 biosynthesis in vivo and thus mediate postoperative fibrinolytic shut-down.


Heterogeneous immunophenotype of granular lymphocyte expansions: differential expression of the CD8 alpha and CD8 beta chains.

In this study, we have evaluated 14 large granular lymphocyte (LGL) expansions, 11 of which were CD8+. Analysis of the membrane expression of the alpha and beta chains of the CD8 antigen, using specific monoclonal antibodies (MoAbs), has shown that LGL expansions with the CD3+, CD4+, CD8+, CD57+ T-cell receptor (TcR) alpha beta phenotype bear the CD8 alpha/alpha isoform, while the CD3+, CD4-, CD8+, CD57+ TcR alpha beta samples were positive for both the CD8 alpha and CD8 beta chains. These data were confirmed also by messenger RNA analysis. One additional case, with a peculiar phenotype (CD3-, CD2-, CD4-, CD8+, CD57-) and a germline configuration of the TcR beta and gamma chain genes, expressed only the CD8 alpha chain. After additional phenotypic analysis with a wider panel of MoAbs, it was found that the beta chain of the interleukin-2 receptor was constitutively expressed on the majority of the samples tested, and that most of the monoclonal samples coexpressed CD45RA/R0 antigens. Using MoAbs directed against the variable regions of the TcR beta chain, we could show a preferential V beta region restriction in the CD8+ monoclonal cases. This more extensive characterization of CD8+ LGL expansions has further documented the marked heterogeneity within this rare condition and allowed a better phenotypic dissection between the monoclonal and polyclonal cases.


Detection and quantitation of malignant cells in the peripheral blood of multiple myeloma patients.

One of the distinguishing features of multiple myeloma (MM) is the proliferation of plasma cells that home to the bone marrow (BM). However, there still remains some uncertainty concerning the presence of related malignant cells in the peripheral blood of myeloma patients. Using consensus oligonucleotide primers, we amplified the third complementary determining region (CDR3) of rearranged immunoglobulin heavy chain alleles from MM marrow samples by polymerase chain reaction (PCR). From the sequences of the products, we derived allele-specific oligonucleotides (ASO), and these were used in subsequent amplification reactions to detect malignant clones in the peripheral blood of myeloma patients. This method is highly specific and sensitive to 1 malignant cell in the background of 10(5) normal cells. Using this method we detected circulating malignant cells in 13 of 14 previously untreated MM patients. Furthermore, by applying ASO-PCR to artificial titrations of initial BM DNA sample into normal peripheral blood lymphocyte (PBL) DNA we were able to generate standard curves and quantitate the amount of tumor in the patient PBL. We observed a wide variation in the amount of circulating tumor between patients. In addition, we found that the incidence of circulating tumor cells was independent of BM tumor burden and stage of disease. The detection and quantitation of circulating tumor cells in the PBL of MM patients may offer an alternative assessment of the disease and may be an important consideration in the use of peripheral stem cells in bone marrow transplantation.


Identification of breakpoints in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ETO, with similarity to Drosophila segmentation gene, runt.

We have developed a restriction map of the chromosome 21 breakpoint region involved in t(8;21)(q22;q22.3) acute myelogenous leukemia (AML) and have isolated a genomic junction clone containing chromosome 8 and 21 material. Using probes from these regions, rearrangements have been identified in each of nine cases of t(8;21) AML examined. In addition, we have isolated cDNA clones from a t(8;21) AML cDNA library that contain fused sequences from chromosome 8 and 21. The chromosome 8 component, referred to as ETO (for eight twenty-one), is encoded over a large genomic region, as suggested by the analysis of corresponding yeast artificial chromosomes (YACs). The DNA sequence of the chromosome 21 portion of the fusion transcript is derived from the normal AML1 gene. A striking similarity (67% identity over 387 bp, with a corresponding 69% amino acid identity) was detected between AML1 and the Drosophila segmentation gene, runt. The critical consequence of the translocation is the juxtaposition of 5' sequences of AML1 to 3' sequences of ETO, oriented telomere to centromere on the der(8) chromosome.


Acute graft-versus-host disease: analysis of risk factors after allogeneic marrow transplantation and prophylaxis with cyclosporine and methotrexate.

Previous studies of risk factors for acute graft-versus-host disease (GVHD) involved patients receiving predominantly single-agent prophylaxis. Therefore, a retrospective analysis was performed on 446 patients, from a single institution, who received transplants of marrow from HLA-identical siblings and the combination of cyclosporine (CSP) and methotrexate (MTX) to determine risk factors for acute GVHD associated with this more effective form of GVHD prophylaxis. The incidences of Grades II-IV and Grades III-IV (severe) acute GVHD were 35% and 16%, respectively. Increased clinical grades of acute GVHD in patients without advanced malignant disease were associated with a decreased survival. In a multivariate Cox regression analysis, risk factors associated with the onset of Grades II-IV acute GVHD were sex mismatch and donor parity (P = .001), increased dose of total body irradiation (TBI) (P = .001), and reduction to less than 80% of the scheduled dose of MTX (P = .02) or CSP (P = .02). The multivariate analysis indicated a relative risk of 1.37 for acute GVHD in a group defined as having advanced malignant disease at transplant; however, this difference failed to reach conventional levels of statistical significance (P = .07). Reduction of MTX and CSP occurred in up to 36% and 44% of patients, respectively, primarily because of renal or hepatic dysfunction. The periods of increased risk for the onset of acute GVHD were up to 1 week after a reduction of MTX and 2 weeks after a reduction in CSP. When only patients who developed Grades II-IV acute GVHD were considered, the more severe acute GVHD of Grades III-IV was associated with increased patient age of 40 years or greater (P = .05) and dose reductions of CSP (P = .008). Serologic status of patient and donor for cytomegalovirus (CMV), HLA antigens in the A and B loci, and isolation in a laminar air flow room during marrow transplantation, all previously identified as risk factors for acute GVHD, were not confirmed as risk factors in this study population. The toxicity of MTX and CSP and the development of acute GVHD from inadequate immunosuppression because of dose reduction warrants further trials with potentially less toxic immunosuppressive agents. Risk factors for acute GVHD should be considered in clinical management and in the design of clinical trials.


Anti-CD3 + interleukin-2 stimulation of marrow and blood: comparison of proliferation and cytotoxicity.

The proliferation and in vitro cytolytic activity of interleukin-2 (IL-2)-activated and anti-CD3 + IL-2-stimulated marrow mononuclear cells (MMC) and peripheral blood mononuclear cells (PBMC) were studied. Samples from 8 normal donors, 15 patients with acute lymphoblastic leukemia (ALL), and 7 patients with non-Hodgkin's lymphoma (NHL) in remission were cultured in IL-2 (100 U/mL) or IL-2 (100 U/mL) plus anti-CD3 (10 ng/mL). MMC as well as PBMC samples demonstrated significant synergy between IL-2 and anti-CD3 in the promotion of proliferation as measured by 3H thymidine incorporation on day 5 (P less than .001) or fold increase in cell number on day 14. Cryopreserved marrow specimens had equally rapid proliferation as fresh MMC when cultured in the presence of anti-CD3 + IL-2. Anti-CD3 concentrations of 3, 11, 33, and 100 ng/mL augmented proliferation similarly in the presence of IL-2 (0.1 to 100 U/mL). Mean fold increases in cell number of both marrow- and blood-derived cultures after 14 days were significantly higher for anti-CD3 + IL-2-stimulated cultures compared with cultures stimulated with IL-2 only (50- to 200-fold increase in cell number; P = .01). Comparison of remission MMC and PBMC from ALL and NHL patients with normal controls showed equivalent growth rates of activated cultures at 7, 14, and 21 days. Marrow purging with immunotoxin anti-CD19 pokeweed antiviral protein plus 4HC had no significant effect on proliferation of anti-CD3 + IL-2-stimulated MMC cultures in patients with ALL. Cytolytic activity of IL-2- and IL-2 + anti-CD3-activated PBMC and MMC cultures was assessed in 51Cr release assays using K562 (natural killer ([NK]-sensitive), Daudi (Burkitt's lymphoma-, NK-resistant), and Nalm-6 (ALL-, lymphokine-activated killer [LAK]-resistant) cell lines and cryopreserved ALL blasts. Cytolytic activity on a per-cell basis (percent cytotoxicity at an effector:target ratio of 30:1) was similar in IL-2-activated PBMC- and MMC-derived cultures from ALL patients. MMC activated with anti-CD3 plus IL-2 killed Daudi significantly less well than IL-2-activated cultures on days 12 and 19 (P = .03); no significant differences were observed in lysis of LAK-resistant Nalm-6 or cryopreserved ALL blast targets. Dose response of anti-CD3 augmentation of Daudi and Nalm-6 killing was different in IL-2- and IL-2 + anti-CD3-stimulated cultures.(ABSTRACT TRUNCATED AT 400 WORDS)


Thrombotic microangiopathies in the 1980s: clinical features, response to treatment, and the impact of the human immunodeficiency virus epidemic.

We reviewed the medical records of 44 adults with 50 consecutive episodes of thrombotic thrombocytopenia purpura (TTP) or hemolytic uremic syndrome (HUS) seen at the University of California, San Francisco affiliated hospitals during the past decade. Patients were treated according to a uniform plan in which initial therapy included daily large volume plasmapheresis using fresh frozen plasma. Patients not responding completely to initial therapy were treated with a salvage regimen including splenectomy, dextran, and corticosteroids. At the time of diagnosis, the lactate dehydrogenase (LDH) was elevated in 98% of cases, with a median value of 1,208 U/L. Other clinical features were present inconsistently, and only 34% of "TTP" episodes involved the classic pentad of hemolytic anemia, thrombocytopenia, neurologic disorders, noninfectious fever, and renal impairment. Primary treatment with plasma exchange produced complete remission in 56% (27 of 48) of the episodes. Previously splenectomized patients uniformly responded to plasma therapy (12 of 12). In patients not responding completely to primary therapy, salvage splenectomy produced complete responses in 81% (13 of 16) of the cases. The pattern of clinical response to therapy was consistent, with initial resolution of neurologic dysfunction (median, 3 days) followed by normalization of LDH levels (5 days) and platelet count (7 days). Normalization of renal function occurred significantly later (15 days). Although short-term responses to plasma therapy in human immunodeficiency virus (HIV)-seropositive patients did not differ from other patients, no HIV-positive patient survived more than 2 years from diagnosis of thrombotic microangiopathy (TMA). We conclude that the diagnosis of TMA requires a high degree of clinical suspicion and that the diagnostic criteria should consist of microangiopathic hemolytic anemia, thrombocytopenia, and an elevated LDH. Initial therapy with plasma exchange leads to disease control in the majority of cases, but an optimal treatment strategy requires the use of alternative methods if initial remission is transient or not achieved. Salvage therapy with splenectomy, steroids, and dextran is highly effective in this setting.


Differentiation and erythropoietin receptor gene expression in human erythroid progenitor cells.

Partially purified human burst-forming unit-erythroid (BFU-E) cells from peripheral blood were cultured for 6 to 8 days to obtain colony-forming unit-erythroid (CFU-E) cells. When these BFU-E-derived CFU-E were further purified and recultured in liquid suspension cultures with erythropoietin (EPO), they matured and differentiated into reticulocytes in vitro. A maximum rate of hemoglobin synthesis was observed at day 10 of cumulative culture time by measuring 59Fe incorporation into heme. Withdrawal of EPO from erythroblast cultures at various times during development showed that between day 10 and day 11 (when the majority of the cells are in the polychromatic erythroblast stage), these cells became independent of EPO. The timing of the disappearance of the EPO requirement in these cells coincided with the marked decline in proliferation. Measurement of EPO receptor messenger RNA (mRNA) levels by Northern analysis showed that there is a slight decline during the day 8 to day 10 time period, followed by a rapid decline between days 10 and 14. Binding of 125I-EPO to erythroblasts also showed a steady decline of the cell surface binding during maturation and terminal differentiation. The half-life of the human EPO receptor was 90 minutes in the presence of the transcriptional inhibitor actinomycin D and the half-life measured at two different times during the 8- to 14-day culture period remained constant. These results indicate that human EPO receptor mRNA must be transcribed continuously to maintain the levels seen by Northern analysis. The human cell system described here is well suited for the study of a wide variety of biochemical events during late erythroid differentiation.


Fibrinogen Marburg: a homozygous case of dysfibrinogenemia, lacking amino acids A alpha 461-610 (Lys 461 AAA-->stop TAA).

In the A alpha-chain gene coding for an abnormal fibrinogen (fibrinogen Marburg) we identified a single base substitution (A-->T) that changes the codon A alpha 461 AAA (Lys) to TAA (Stop). The propositus was found to be homozygous for the mutation, whereas the father and five siblings were heterozygous, and three other siblings contained only the normal sequence. The stop codon at position 461 results in the deletion of the carboxyl-terminal segment A alpha 461-610. Purified fibrinogen Marburg contained an A alpha-chain with a relative molecular weight of approximately 47,000. The FpA release by thrombin was not affected by this deletion, whereas the fibrin polymerization was strongly decreased. The binding of endothelial cells to immobilized fibrinogen Marburg was almost completely abolished compared with normal fibrinogen. Fibrinogen Marburg contained a substantial amount of albumin linked to the fibrinogen molecule by disulfide bonds, and these fibrinogen-albumin complexes were also present in plasma. The plasma fibrinogen concentration of the propositus was measured by three different methods: a functional method (< 0.25 mg/mL), an immunologic method using polyclonal antibodies (0.6 mg/mL), and an immunologic method based on two monoclonal antibodies specific for the amino-terminus and carboxyl-terminus of the A alpha-chain (< 0.05 mg/mL). Using the two immunologic methods, it appeared that only 10% to 15% of the plasma fibrinogen of the heterozygous siblings was abnormal.


Generation of vasoactive peptide bradykinin from human umbilical vein endothelium-bound high molecular weight kininogen by plasma kallikrein.

High molecular weight kininogen (HK) is a multifunctional plasma glycoprotein that occupies a critical position in pathways that link inflammation and coagulation. It is an inhibitor of sulfhydryl proteases and has procoagulant properties. It is also a source of the vasoactive peptide bradykinin (BK). It has been previously shown that HK binds to human umbilical vein endothelial cells (HUVEC) in culture. We have further characterized that interaction herein. Immunohistochemical experiments have indicated that when freshly obtained umbilical vein segments were treated with HK, washed, and probed with anti-HK antibodies, HK was localized on the endothelium. We next determined whether HUVEC-bound HK can be cleaved by plasma kallikrein to release BK. Cultured HUVEC were incubated with unlabeled HK for varying times, washed, and the kinetics of BK release by plasma kallikrein were assayed by radioimmunoassay. Results indicated that kallikrein released BK from HUVEC in proportion to the initial amount of bound HK. No release of BK occurred in the absence of kallikrein. Also, there was no BK release upon kallikrein treatment of the HUVEC not treated with exogenous HK. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of HUVEC-bound 125I-HK indicated that addition of kallikrein resulted in cleavage of HK, thus corroborating the BK release experiments. Comparison of cleavage patterns has also indicated that cell-bound HK is slightly less susceptible to digestion by kallikrein than free HK. Therefore, our data suggest that human HK can bind to vascular endothelium in situ and that plasma kallikrein can recognize endothelial-bound HK as a substrate and liberate the vasoactive peptide BK.


Factor Xa interacts with two sites on monocytes with different functional activities.

Studies were performed to elucidate the functional significance of factor Xa interactions at the monocyte membrane in the presence and absence of factor Va, with respect to prothrombin and factor IX cleavage. Factor Xa-catalyzed prothrombin activation at the monocyte surface was absolutely dependent on the addition of factor Va, indicating that thrombin was generated solely by a membrane-bound complex of factors Va and Xa. In contrast, in the absence of added factor Va, factor Xa bound to monocytes catalyzed the cleavage of factor IX to the nonenzymatic intermediate factor IX alpha through a reaction that was dependent on both monocyte and factor Xa concentration. At limiting factor Xa concentration, added factor Va inhibited the factor Xa-catalyzed cleavage of factor IX, suggesting that a monocyte-bound complex of factors Va and Xa did not recognize factor IX as a substrate. These combined data suggest that factor Xa interacts with the monocyte through two sites which can be distinguished by their requirement for added factor Va and their expression of different functional activities. Both functional sites could be distinguished also by their differential susceptibility to inhibition by a monoclonal antibody directed against the light chain of factor Va (alpha-HFV1). At the monocyte surface, the factor Va/Xa-catalyzed activation of prothrombin was maximally inhibited with 0.25 mumol/L alpha-HFV1, whereas 1.0 mumol/L alpha-HFV1 was required to effect 50% inhibition of the factor Xa-catalyzed cleavage of factor IX. The ability of factor Va to modulate factor Xa substrate specificity was investigated further. Factor Xa bound to thrombin-activated platelets either through platelet-released factor Va or added factor Va did not cleave factor IX. Consistent with this result, a plasma concentration of factor IX had no effect on thrombin generation catalyzed by a platelet-bound complex of factors Va and Xa. In marked contrast, factor Xa bound to phospholipid vesicles either independently or in complex with factor Va catalyzed factor IX cleavage with equal efficiency. These combined data indicate that factor Va bound to cell surfaces modulates factor Xa substrate specificity, whereas no discriminatory effect is conferred by factor Va bound to phospholipid vesicles. Thus, by providing two distinct sites at its membrane surface, the monocyte modulates factor Xa binding and the functional activity expressed by the bound enzyme, depending on the availability of factor Va.


Maturation of the hemostatic system during childhood.

The hemostatic system is assumed to be similar in children and adults and reference ranges established for adults are commonly used to evaluate children suspected of having congenital or acquired hemostatic problems. However, we know that the hemostatic system is not fully mature by 6 months of age and comprehensive studies of healthy older children have not been published. Therefore, we conducted a prospective cohort study of the hemostatic system in healthy children having minor, elective day surgery. After obtaining informed consent, a 3-mL blood sample was obtained at the time routine preoperative blood work was drawn. The plasma was fractioned and stored at -70 degrees C for batch assaying. We measured the concentration of 33 components of the hemostatic system (functional and immunologic assays) and the bleeding time (automated pediatric device) in 246 children aged 1 to 16 inclusive (a minimum of four subjects at each age). Eleven components of hemostasis (fibrinogen, prekallikrein, high-molecular weight kininogen, factors VIII and XIII, antithrombin III [ATIII], heparin cofactor II [HCII], alpha 1-antitrypsin [alpha 1AT], protein S, plasminogen, alpha 2-antiplasmin [alpha 2AP]) had mean values and ranges of normal that were similar to adults. Mean values of seven coagulants (II, V, VII, IX, X, XI, XII) were significantly lower than adult values and varied with age. Values for three inhibitors, alpha 2-macroglobulin (alpha 2M), protein C, and protein C1-inhibitor (C1-Inh) also differed from adults. Alpha 2M and C1-Inh inhibitor levels were elevated throughout childhood, whereas protein C levels were low, with a lower limit of normal of 0.40 U/mL until the age of 11. Finally, the upper limit of normal for the bleeding time was longer in children during the first 10 years of life, but decreased to adult values in the teenage years. In summary, there are important physiologic differences in the hemostatic system in children compared with adults. The decreased levels of several critical coagulants and increased levels of alpha 2M may contribute in part to the lower risk of thrombotic events in childhood. Age-matched controls should be used for evaluation of the hemostatic system in children with suspected congenital or acquired defects.


Antiplatelet drugs and generation of thrombin in clotting blood.

Platelets participate in formation of thrombin through secretion of coagulation factors and by providing a catalytic surface on which prothrombinase complex is assembled. We studied the effects of four antiplatelet drugs on thrombin formation in healthy volunteers. Thrombin generation was monitored both in vitro--in recalcified plasma--and ex vivo--in blood emerging from a standardized skin microvasculature injury, which also served to determine bleeding time. A mathematical model has been developed to describe the latter reaction. It is based on estimation of the rate of increase in fibrinopeptide A (FPA), a specific marker of thrombin activity, in blood emerging from skin incisions. Two hours after the ingestion of 500 mg of aspirin, thrombin formation became significantly impaired both in vitro and ex vivo. In contrast, 2 hours after the oral administration of placebo, indomethacin 50 mg, or OKY-046 (a thromboxane synthase inhibitor) 400 mg, thrombinogenesis remained unaltered. Ticlopidine, studied either 3 hours after 500 mg oral administration, or after 5 days of intake at a daily dose of 500 mg, had no effect on thrombin generation. Thus, aspirin, contrary to other antiplatelet drugs, depresses thrombin formation in clotting blood, a phenomenon that might be of clinical relevance. It is suggested that aspirin exerts this effect by acetylating prothrombin and/or macromolecules of platelet membrane.


Detection of bcl-2 protein and bcl-2 messenger RNA in normal and neoplastic lymphoid tissues by immunohistochemistry and in situ hybridization.

bcl-2 protein has been detected in surgical specimens and cultured permanent cell lines of non-Hodgkin's lymphomas and leukemias using enzyme immunohistochemistry and immunofluorescence with anti-bcl-2 monoclonal antibodies. Of 40 surgical specimens, bcl-2 protein was expressed in 50% of B-cell and 41% of T-cell lymphomas, both with and without the bcl-2 gene rearrangement. In investigations of 38 hematopoietic cell lines, bcl-2 protein was detected not only in lymphoid cell lines but also in myeloid cell lines. In situ hybridization and immunohistochemical analysis of reactive lymph nodes showed that lymphocytes in mantle zones and paracortical areas expressed bcl-2 protein consistent with the messenger RNA distribution and that germinal center cells showed abundant bcl-2 transcript, despite the absence of detectable bcl-2 protein. These results suggest that bcl-2 protein is broadly expressed in various hematopoietic neoplasms not restricted in t(14; 18) lymphomas and that germinal center cells may be involved in some arrest of bcl-2 protein expression at the posttranscriptional level.


Identification and expression of a common missense mutation (L302P) in the acid sphingomyelinase gene of Ashkenazi Jewish type A Niemann-Pick disease patients.

Types A and B Niemann-Pick disease (NPD) result from the deficient activity of acid sphingomyelinase (ASM; E.C. 3.1.4.12) and the resultant lysosomal accumulation of sphingomyelin. Type A disease is a fatal, neurodegenerative disorder of infancy, whereas type B disease has no neurologic manifestations and is characterized primarily by reticuloendothelial involvement and survival into adulthood. Both disorders occur more frequently among individuals of Ashkenazi Jewish ancestry than in the general population. Recently, a missense mutation in the ASM gene (designated R496L) was detected in more than 30% of the ASM alleles from Ashkenazi Jewish type A NPD patients. We report a second, common mutation that resulted from a T to C transition at nucleotide 905 and predicted a leucine to proline substitution at ASM codon 302 (designated L302P). Notably, the L302P mutation occurred in 23.5% (8 of 34) of the Ashkenazi Jewish type A NPD alleles studied. In contrast, it was not found in any of the ASM alleles from non-Jewish type A patients, in 36 alleles from type B patients, or in 100 ASM alleles from normal Ashkenazi Jewish individuals. To confirm the authenticities of the L302P and R496L mutations, each nucleotide change was separately introduced into the full-length ASM cDNA by site-directed mutagenesis and transiently expressed in COS-1 cells. Neither mutation expressed ASM catalytic activity, consistent with the type A phenotype of homoallelic patients. The identification of the L302P mutation should further facilitate molecular carrier detection for NPD in the Ashkenazi Jewish population, particularly because the L302P mutation can be easily detected using the restriction enzyme, AlwNl.


Rapid diagnosis of beta-thalassemia mutations in Chinese by naturally and amplified created restriction sites.

We developed a rapid and simple method to diagnose the molecular defects of beta-thalassemia in Chinese patients. This method involves the selective amplification of a DNA fragment from human beta globin gene with specific oligonucleotide primers, followed by digestion with restriction enzymes that recognize artificially created or naturally occurring restriction sites. To detect the 4-nucleotide deletion of codon 41-42, we introduced a single mismatch nucleotide into the 3' end of the upstream primer to create an artificial Taq I restriction site. With a similar approach, an artificial Rsa I site was generated to detect the nucleotide 654 mutation (C-->T) of IVS-2, and Alu I restriction site was created to detect the codon 17 mutation (A-->T), and EcoRI restriction site was created for the -28 mutation (A-->G), a Rsa I restriction site was created for the nucleotide 5 mutation (G-->C) of IVS-1, and a Spe I restriction site was created to distinguish the codon 71 (+T) and codon 71/72 (+A) mutations from a normal sequence. The other eight rare mutations that occur in the genes of the Chinese people naturally create or abolish restriction sites. Using this kind of approach, we are able to provide a simple, rapid, accurate, and nonradioactive method to detect the genetic defects of beta-thalassemia in the Chinese population. It should be used not only for routine screening but also for prenatal diagnosis.


A deletional frameshift mutation of the beta-spectrin gene associated with elliptocytosis in spectrin Tokyo (beta 220/216).

A novel spectrin variant carrying a truncated beta-chain and designated Spectrin Tokyo (beta 220/216) is presented. It was associated with elliptocytosis and moderate uncompensated hemolysis. The dimer self-association was reduced. An increase of the alpha I 74-Kd fragment was detected upon partial trypsin digestion. Analysis of cDNA and genomic DNA showed a 1-base deletion in codon 2059 (GCC AGC-->GCA GCT; Ala-Ser-->Ala-Ala) that belongs to exon X of spectrin beta-gene. A missense sequence extended down to (new) codon 2075. Serine 2060, a potential phosphorylation site, was replaced by alanine. The shortened beta-chain failed to undergo phosphorylation in vitro. Spectrin Tokyo shared the same stop codon, overlapping normal codons 2076 and 2077 (CTG AAA), as Spectrin Nice (beta 220/216), which is caused by a dinucleotide insertion in codon 2046 and contains 2076 amino acids. However, for some reason, Spectrin Tokyo had a lower incorporation level into the membrane than Spectrin Nice.


Fetal compensation of the hemolytic anemia in mice homozygous for the normoblastosis (nb) mutation.

The mouse autosomal recessive mutation nb causes a deficiency of erythroid ankyrin and generates a life-threatening hemolytic anemia in adult mice; however, at birth, nb/nb mice appear to be robust and show no pallor. In our study, the time of disease onset was sought by comparison of nb/nb and +/? mice both in utero and postnatally. Erythroid ankyrin messenger RNA (mRNA) is expressed in fetal erythroid progenitors from normal mice, but is reduced to 10% of normal levels in mutant fetuses. Despite the deficiency of erythroid ankyrin mRNA, 16 and 18 day nb/nb fetuses have normal levels of red blood cells (RBCs) and the RBCs are morphologically normal by scanning electron microscopy. The earliest signs of any clinical anomaly are an increase in the number of circulating reticulocytes and the deposition of minor amounts of iron just before birth in the 18 day fetal nb/nb liver, suggesting that RBCs are being destroyed. Within 24 hours after birth, nb/nb neonates have a slight but significant decrease of their RBC counts. During the next 5 days, the nb/nb RBC counts decrease markedly, the reticulocyte counts assume the mutant adult levels of 60%, the erythrocytes become microcytic and fragmented, and iron deposits accumulate in the liver. The rapid onset of clinical disease postnatally, coupled with our findings that the erythroid ankyrin gene is transcribed in fetal erythroid cell precursors from normal mice, suggest that mechanisms exist in the nb/nb fetus to compensate for the erythroid ankyrin deficiency.


Allogeneic leukocytes but not therapeutic blood elements induce reactivation and dissemination of latent human immunodeficiency virus type 1 infection: implications for transfusion support of infected patients.

Various immunologic stimuli and heterologous viral regulatory elements have been shown to increase susceptibility to, and replication of, human immunodeficiency virus type 1 (HIV-1) in lymphocytes and monocytes in vitro. Transfusion of allogeneic blood components from heterologous donors constitutes a profound immunologic stimulus to the recipient, in addition to being a potential route of transmission of lymphotropic viral infections. To investigate the hypothesis that transfusions, and particularly those containing leukocytes, activate HIV-1 replication in infected recipient cells, we cocultured peripheral blood mononuclear cells (PBMC) from three anti-HIV-1-positive individuals with allogeneic donor PBMC, as well as partially purified populations of donor lymphocytes, monocytes, granulocytes, platelets, and red blood cells (RBC) and allogeneic cell-free plasma. Allogeneic PBMC induced a dose-related activation of HIV-1 expression in in vivo infected cells, followed by dissemination of HIV-1 to previously uninfected patient cells. Activation of HIV-1 replication was observed with donor lymphocytes, monocytes, and granulocytes, whereas no effect was seen with leukocyte-depleted RBC, platelets, or plasma (ie, therapeutic blood constituents). Allogeneic donor PBMC were also shown to upregulate HIV-1 expression in a "latently" infected cell line, and to increase susceptibility of heterologous donor PBMC to acute HIV-1 infection. Studies should be performed to evaluate whether transfusions of leukocyte-containing blood components accelerate HIV-1 dissemination and disease progression in vivo. If so, HIV-1-infected patients should be transfused as infrequently as possible and leukocyte-depleted (filtered) blood components should be used to avoid this complication.


Evidence that the antigens of the Yt blood group system are located on human erythrocyte acetylcholinesterase.

The Yt blood group system comprises two antigens, Yta and Ytb. Human anti-Yta and human anti-Ytb immune precipitate a component of the same apparent molecular weight as acetylcholinesterase from radioiodinated erythrocytes of appropriate Yt phenotype. Immune precipitates obtained with anti-Yta and anti-Ytb contained acetylcholinesterase activity. In contrast, immune precipitates obtained with human anti-Gya and murine monoclonal anti-CD55, which identify other glycosylphosphatidylinositol-linked erythrocyte surface proteins, did not have acetylcholinesterase activity. Quantitative binding assays using murine monoclonal antiacetylcholinesterase antibodies (AE-1 and AE-2) gave 3,000 to 5,000 binding sites/cell for IgG and 7,000 to 10,000 sites/cell for Fab fragments. Endo F digestion of immune precipitates obtained with AE-1 and anti-Yta indicated that approximately 10% of the enzyme comprises N-glycans. These results indicate that the Yt antigens define an inherited polymorphism on erythrocyte acetylcholinesterase and that the recent assignment of the Yt blood group locus to the long arm of chromosome 7 (Zelinski et al, Genomics 11:165, 1991) provisionally identifies the position of the acetylcholinesterase gene.


Point mutations of rhodopsin gene found in Japanese families with autosomal dominant retinitis pigmentosa (ADRP).

The mutations of codon 17, 23, 58, and 347 of rhodopsin gene were investigated in 24 unrelated Japanese families including 33 patients with autosomal dominant retinitis pigmentosa (ADRP). A patient with codon 17 mutation (Thr-17-Met, ACG-->ATG) and a family including 4 patients with codon 347 mutation (Pro-347-Leu, CCG-->CTG) were detected among them. Their clinical findings were extremely different between the two mutations. The former showed type 2 and the latter showed type 1 ADRP. No mutation of codon 23 and 58 was detected in any families so far analyzed in the present study. Clinical findings associated with the mutation in codon 17 and 347 of the rhodopsin gene show an existence of allelic heterogeneity.


Assignment of the human cytochrome P-450 nifedipine oxidase gene (CYP3A4) to chromosome 7 at band q22.1 by fluorescence in situ hybridization.

We have used a full length cDNA clone (2.2 kb) for the human cytochrome P-450 nifedipine oxidase (CYP3A4) enzyme as a probe to determine its chromosome localization by fluorescence in situ hybridization. CYP3A4 was mapped on R-banded human prometaphase chromosomes, and the precise localization of CYP3A4 on chromosome 7 was further confirmed by a delineation of G-banded pattern on the same prometaphase chromosomes through a combination of UV-filter. We assigned CYP3A4 to chromosome 7 at q22.1.


Detection of variation in the ribosomal RNA gene clusters by a modified fluorescence in situ hybridization method.

Physical mapping of genes by fluorescence in situ hybridization (FISH) has become routine using fluorescein isothiocyanate (FITC) for probe detection and propidium iodide (PI) for chromosome staining. We have modified this conventional FISH method in a way that utilizes Texas red (TR) for signal detection and quinacrine mustard (QM) for chromosome banding. Using this Texas red and quinacrine (TRQ) method, we were able to identify individual acrocentric chromosomes with varying degrees of ribosomal RNA gene clusters. Two acrocentric chromosomes were found to carry extremely small number of rRNA gene copies as compared to the other eight counterparts in human diploid lymphoblastoid cell line GM00130B. Thus, the TRQ method allows one to probe for a specific sequence while identifying individual chromosomes and will be powerful for the chromosomal localization of various genes.


Genetic polymorphism of human factor H (HF, beta 1H globulin) in Chinese Han population in northeast China.

The distribution of human factor H of serum phenotypes were studied using ultrathin polyacrylamide gel isoelectric focusing (PAGIEF) and subsequent immunoblotting techniques in 203 Chinese of Han population in Liaoning Province of northeast China. The gene frequencies of HF*A and HF*B were 0.4828 and 0.5172, respectively. All the observed numbers of the phenotypes were in agreement with the expected numbers under the Hardy-Weinberg equilibrium. The gene frequencies among Chinese, Japanese, and Caucasian populations were compared.


DNA analysis of two patients with a non-fluorescent Y chromosome.

Results of DNA study on two patients of gonadal dysgenesis with a 45,X/46,X,Ynf (non-fluorescent Y chromosome) karyotype are described. In one patient who developed gonadoblastoma, all 12 loci on the non-fluorescent part of Yq were detected. Another patient did not have gonadoblastoma at 20 years, and only the proximal 6 loci out of 12 were detected.


Terminal deletion of the short arm of chromosome 3.

A boy with growth and mental retardation, flat occiput, high and broad forehead, blepharoptosis, narrow palpebral fissures, low set, malformed ears, short neck, anal atresia, deep sacral dimple is reported. High-resolution banding analysis showed terminal deletion of the short arm of chromosome 3 (46,XY,del(3)(p25.3)). Deletions of the short arm of chromosome 3 are relatively rare. The clinical features of the patient are compared with those of 19 previously reported cases.