Pattern of irregular dorsal root discharge in the spinal cat. Pattern of irregular type of dorsal root discharge (DRD) in non-anaesthetized spinal cats was inferred from the distribution of its interspike intervals. Interspike interval histograms were compiled from antidromic spike potentials of irregular DRD which were recorded in the central ends of 33 single dorsal root fibres of L7 dorsal root. The majority of histograms was unimodal. The mean preferred interval of irregular DRD which indicated the most frequently occurring interspike interval was 6.1 +/- 0.5 ms. The shortest and longest intervals were 3.7 +/- 0.3 ms and 45.9 +/- 4.4 ms, respectively. The coefficient of symmetry of the histogram was 0.06, indicating highly unsymmetrical distribution of intervals of irregular DRD. Conduction velocity of dorsal root fibres carrying irregular antidromic discharge amounted to 52.3 +/- 5.3 m/s (n = 23). There were no significant correlations between three analysed interspike intervals and conduction velocity. The results are discussed in view of the hypothesis of the modulating effect of antidromic discharge on the afferent inflow. Comparison of preferred interspike interval of irregular DRD with the existing data on the rate of orthodromic activity in afferent nerve fibres suggests that irregular antidromic discharge on many occasions is able to block othodromic impulses by collision.


The 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine causes a differential incorporation of hexadecanol into neutral ether ester glycerolipids of 2 variant cell lines of rat colon carcinoma. The 1-O-octadecyl 2-O-methyl-sn-glycerophosphocholine (ET-18-O-CH3), when incubated for 24-48h with cells in culture, exerts a highly selective cytotonic activity against a variety of tumor cells that is not seen in normal ones. In this study, we present data which indicate that this exogenous molecule altered the endogenous synthesis of the neutral ether, ester-sn-glycerols, in 2 variant cell lines of a rat colon carcinoma. ET-18-O-CH3, at 20 microM in the medium and for an incubation of 48h, inhibited the growth rates of the PRO cells which have the ability to metastasize and of the REG cells (the regressive cell line), by, respectively, 54 and 67%, as measured after [3H] thymidine uptakes. The synthesis of the ether, ester-glycerolipids was followed after an incorporation of [3H] hexadecanol into the cell lipids. The radiospecific activity of the alcohol in the ether, ester-glycerolipids was higher for the PRO cells than for the REG cells. ET-18-O-CH3 activated the incorporation of [3H] hexadecanol in the neutral ether, ester-sn-glycerols: 1.55 fold in the PRO cells, but 2.15 fold in the REG cells. No change was observed in the alkyl (alkenyl) acyl-sn-glycerophospholipids. Most of the transformed cells have a low etherase activity and are known to accumulate the ether, ester-glycerolipids, (neutral and ionic structures).(ABSTRACT TRUNCATED AT 250 WORDS)


Simultaneous effect of initial weight, initial crowding, temperature and O2 concentration on the nutritional use of food by rainbow trout (Oncorhynchus mykiss). The simultaneous effects of initial weight, initial crowding, temperature and O2 concentration on the following ratios: relative growth rate percent (RGRP), feed efficiency (FE), protein efficiency ratio (PER) and protein productive value (PPV) were studied in the rainbow trout. Multivariant equations were obtained for each of the mentioned indices. The joint effects of these factors were evidenced by means of a multiple correlation analysis. The influence of temperature and, to a lesser extent, of crowding, and O2 concentration on the nutritional use of food by the trout was demonstrated, their fundamental dependence on factors extrinsic to the animal being underlined. The non proportional changes in PER and PPV as temperature rises revealed that an increasing part of the ingested aminoacids were used for synthesis of fat, non for proteins edification.


Parathyroid hormone-related peptide inhibits oxytocin-induced rat uterine contractions in vitro. Synthetic human parathyroid hormone-related peptide (hPTHrP)-(1-34) fragment was compared with parathyroid hormone (bovine sequence, 1-34; bPTH-(1-34) for inhibiting oxytocin or prostaglandin F2 alpha (PGF2 alpha)-induced contractions on rat uterus in vitro. bPTH exhibited a potent (ED50 = 7 x 10(-9) M) inhibition on oxytocin-induced contractions. Both bPTH-(1-34) and hPTHrP-(1-34) were devoided of any significant effect upon PGF2 alpha-induced uterine contractions. Human PTHrP also inhibited oxytocin-induced uterine contractions (ED50 = 77 x 10(-9) M) and this effect, like that of bPTH, was dose dependent. Human PTHrP-(140-173) fragment had no significant effect on oxytocin-induced uterine contractions. The inhibitory effect of hPTHrP-(1-34) disappeared after pretreatment with [Tyr]34-bPTH-(7-34)-NH2, a competitive reversible antagonist of bPTH-(1-34). Thus PTHrP might be involved in the control of myometrial activity.


In the rat, intestinal lymph carries a significant amount of ingested glucose into the bloodstream. Intestinal lymph flow in rats receiving an oral load of 3 mmoles of glucose was determined by measuring the amount of tritiated gavage water entering via the portal route [computed differential arterio-venous tritium water concentrations multiplied by portal blood flow) and measuring the total amount of tritium transferred from the intestinal lumen to the rest of the rat in a given time [from the blood tritium specific activity and the water space (795 ml.kg-1) of the rat]. The approximate figure obtained (79.8 ml.kg-1.h) was used for the evaluation of the amount of glucose (from 14C-labelled glucose in the gavage) transfer via intestinal lymph from the label present in the lymph and its flow. Lymph glucose concentration (c. 11 mM) and specific radioactivity were higher than those of blood. The amount of glucose actually taken up from the intestinal lumen was determined measuring the remaining label in the alimentary canal. The portal contribution to label distribution was measured as for tritium water transfer. Most glucose label passed through in the form of glucose, with much smaller proportions as CO2 and even smaller as lactate and other labelled compounds. The results suggest that the contribution of lymph circulation to dietary glucose incorporation into the bloodstream may be significant, amounting to values higher than 10% of the glucose actually incorporated.


Cholinergic mechanisms and bronchoconstriction in guinea-pigs. Seventy-four guinea-pigs have been actively sensitized against ovalbumin. The effects of bilateral cervical vagotomy and of cholinergic efferent blockade on the bronchoconstriction induced by inhaled acetylcholine, histamine or antigen have been quantified. Animals were curarized then tested during controlled ventilation to ensure constant conditions throughout the whole experiment by suppressing the post-vagotomy apneusis. Bronchial sensitivity was estimated by calculating the bronchoconstrictor dose-threshold provoking respiratory asynchronism. Corresponding variation of total lung resistance and dynamic compliance were also measured as indices of bronchial reactivity. The data obtained in 27 guinea-pigs indicated that the vagi section produced no significant effect on the direct bronchial response to histamine (P greater than 0.5), to acetylcholine (P greater than 0.1) or to antigen (P greater than 0.5). In 47 guinea-pigs, atropine at doses 5 and 10 times higher than the acetylcholine blocking one did not alter the direct bronchial response to histamine (P greater than 0.5). Higher doses induced a progressive decrease in bronchial sensitivity to antigen (.05 greater than P greater than .01) which could be related to the repetitive administration of the antigen. According to the present results, peripheral cholinergic mechanisms play no significant role in guinea-pigs neither in histamine nor in anaphylactic bronchoconstriction. Curarization has no influence on resting bronchial tone.


[Tolerance to exertion after sleep reduction and after taking a hypnotic: zolpidem]. The aim of the present study was to investigate the effects of a delayed bedtime (3 a.m.), an advanced rising (3 a.m.), a sleep under placebo and under a hypnotic compound, i.e. 10 mg of zolpidem (Stilnox) on sleep structure and on the adaptations to a subsequent exercise in 8 male athletes. The chronology of these nights was randomized and each treatment administered in a double blind fashion. During each experimental night, subjects were monitored with conventional EEG/EOG/EMG polygraphic recordings. The next day, athletic performance was tested using a bicycle ergometer. A codified exercise was performed and consisted to a 10 min warm up followed by a 30 min steady state cycling corresponding to 75% of predetermined VO2 max. Then the work load increased progressively by steps of 10 W every minute until exhaustion. The recovery lasted 30 min. Heart rate, ventilation, VO2, ERO2 were monitored during all exercise and recovery. Plasma lactates and catecholamines were also measured at the same time. The data concerning sleep recordings showed that both nights with partial sleep deprivation resulted in a drop of time spent in slow wave sleep II (decrease of 55%) and in rapid eye movement sleep (decrease of 45%) while the amount of slow wave sleep III and IV were identical to that observed after the reference night. The sleep onset latency and the amount of sleep in stage I was reduced only after the delayed bedtime. Sleep data under zolpidem did not show any significantly difference in the amount of different sleep stages.(ABSTRACT TRUNCATED AT 250 WORDS)


Thiamin uptake by rat isolated enterocytes: relationship between transport and phosphorylation. The membrane and intracellular steps of thiamin (T) uptake were studied in rat isolated enterocytes. The membrane step was investigated by using enterocytes deenergized with 25 microM rotenone and a short (30 s) incubation time, while the intracellular metabolic step was assessed by using normoenergized (normal) cells incubated for a longer (30 min) time. Evaluation of Km, Jmax and KD (apparent passive permeability coefficient) values, at 30 s incubation, indicated that deenergization slightly reduced both the affinity and the capacity of the saturable component of T uptake, as well as the capacity of the non-saturable component. At 30 min incubation, deenergization suppressed completely the saturable component, without affecting the non-saturable component. Comparison of the Km and Jmax values of the saturable components in normoenergized enterocytes at 30 s and 30 min indicated that intracellular metabolic phosphorylation is probably the event responsible for the active process involved in T uptake.


Measurement of rat lung blood flow with labelled microspheres. A modification of the usual microsphere injection method is presented which is appropriate for rat lung blood flow measurement. The injection of microspheres into the abdominal cava vein, together with the sampling of a reference flow from the right ventricle, allowed to calculate pulmonary blood flows in the same range than the cardiac output. The differences between the two figures (about 30%) are attributed to arterio-venous shunts. The tissue distribution of the microspheres bypassing the lung capillary beds agree with this interpretation.


Regional amines levels in the rat brain following intra-accumbens cholecystokinin and intraperitoneal amphetamine pre-treatment. Cholecystokinin octapeptide (CCK8) coexists with dopamine (DA) in a subpopulation of mesolimbic DA neurons including the projections to the nucleus accumbens. In this structure, CCK8 has been reported to exert agonist-like or antagonist-like effects on DA-mediated behaviours and on amphetamine's locomotor-activating effects in rodents. These findings raise the possibility that CCK8 plays a role in modulating the neurochemical mechanisms underlying the effects of DA and amphetamine in the mesolimbic DA system. The purpose of this study was to determine regional tissue monoamine levels in the rat brain and their modulation following injection of CCK8 in the nucleus accumbens. The same paradigm was used to determine the effects of this octapeptide on changes in amine levels induced by amphetamine administered intraperitoneally. DA, norepinephrine (NE), serotonin (5-HT) and their primary metabolites, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were measured using a reversed-phase ion pair high-pressure liquid chromatography system with electrochemical detection (HPLC-ED). Frontal cortex displayed the highest DOPAC/DA ratio and the lowest DOPAC/HVA ratio in contrast to the olfactory tubercle. The intraperitoneal injection of amphetamine (1 mg/Kg) followed by the intra-accumbens administration of 0.15M saline decreased the levels of DOPAC and increased DA, 5-HT and 5-HIAA both in nucleus caudatus and nucleus accumbens. The DA agonist induced a decrease in the level of NE in olfactory tubercle and frontal cortex. The direct administration of CCK8 (300 pmol) into the nucleus accumbens decreased the level of DA, DOPAC and 5-HT mainly in olfactory tubercle and nucleus accumbens itself.(ABSTRACT TRUNCATED AT 250 WORDS)


[Structure and function of blood platelets]. Blood platelets are discoid cellular fragments without nucleus originating from megakaryocytes. Platelets are able to respond to a great variety of agonists which bind to specific receptors localized on the plasma membrane. This process takes place when blood vessels are cut. Platelets then change their shape, adhere to newly exposed subendothelial tissues, release the content of numerous secretory granules and aggregate together. During this process, a great numbers of biochemical reactions are triggered such as phospholipases activation, synthesis of mediators and protein phosphorylation. These events result from increased cytoplasmic free calcium originating through calcium channels from the extracellular medium and from internal stores. Involvement of blood platelets in cardiovascular diseases may result from an exaggeration of these mechanisms by risk factors and are also discussed.


Changes in carbohydrate metabolism of Pila globosa in response to crustacean hyperglycemic hormone. Hyperglycemia was caused in the snail Pila globosa by the injection of the hyperglycemic hormones obtained from fresh water crabs (Oziotelphusa senex senex) and marine tiger prawns (Penaeus monodon). Tissue glycogen and total carbohydrates presented a significant decrease which indicated that the source of hyperglycemia was the tissue carbohydrates. The hyperglycemic principles also increased the tissue phosphorylase activity and provided evidence for a possible action of the crustacean hyperglycemic hormones in non-arthropod species.


Alkaline phosphatase and ATPases in brush-border membranes of rat jejunum: distinct effects of divalent cations and of some inhibitors. The effects of divalent cations and of some inhibitors on the activities of alkaline phosphatase and ATPase were examined in rat jejunal brush-border membranes (BBM) isolated by tha Ca(2+)-(BBMCa) or the Mg(2+)-precipitation method (BBMMg). Similar results were found in BBMCa and BBMMg though generally higher in BBMCa. Alkaline phosphatase activity was stimulated by 5 mM MgCl2 (30% to 44%), but not by 5 mM CaCl2 or 0.1 mM ZnCl2, at pH 9.5 or 7.4. ATPase activity was equally stimulated by 5 mM MgCl2 and by 5 mM CaCI2 (about 150%). Alkaline phosphatase activity was significantly inhibited by 1 mM vanadate, 5 mM diamox, 5.0 mM L-leucine and 1 mM theophylline. In contrast, Ca(2+)-ATPase and Mg(2+)-ATPase activities were not depressed by those alkaline phosphatase inhibitors, but were inhibited by 0.1 mM trifluoperazine (more than 70%). 0.1 mM ZnCl2 also appeared to be inhibitory to Ca(2+)-ATPase and Mg(2+)-ATPase, but not to alkaline phosphatase activity even in the presence of Ca2+ and Mg2+. These results suggest that Ca(2+)-ATPase and Mg(2+)-ATPase activities of the rat jejunal BBM are not merely manifestations of alkaline phosphatase, but rather belong to (a) distinct enzyme(s).


[RNA synthesis and transport in the rat liver under the effects of pesticide cotoran (fluometuron)]. The experiments on the investigation of pesticide cotoran-effect on RNA synthesis and transport were carried out. Cotoran was shown to destroy considerably the processes of RNA biosynthesis in rat liver, that results in the decrease of RNA transport from nuclei into cytoplasm. By special experiments it was established that functional activity and the integrity of nuclear membrane (according to the alteration in the activity of nuclear membrane enzyme Mg2-dependent ATP-ase) was not destroyed.


Absorbability of oleic and palmitic acid in young chicks. Effect of yolk sac ablation. The aims of the present study were: 1) to describe the changes in the absorption of free oleic and palmitic acids in young chicks during the first two weeks after hatching; 2) to find out if yolk sac ablation induces any change in the absorbability of such fatty acids. The study was carried out in normal and yolk-sac-ablated broiler chicks aged 2, 8 and 13 days. 14C-labelled oleic and palmitic acids were given p.o. in suspension. Stools were collected for 48h and the recovery of radioactivity was determined and considered as an indicator of the extent of absorption. Our results indicate that the absorption of palmitic acid decreased with age (p less than 0.05) whereas the absorption of oleic acid increased significantly (p less than 0.005). The changes in the absorptive capacity for each fatty acid occurred during the first week. The absorption of both fatty acids was greater in yolk-sac-ablated chicks (p less than 0.05). The relative contents of different fatty acids in yolk lipids was investigated. The results clearly indicate that the ratio OA/PA linearly increased from hatching until the 4th day. Thus the possibility that yolk sac lipids passing to the intestine through the yolk stalk are able to modify the absorptive processes cannot be excluded.


[Effects of intranasally administered substance P in parkinsonian syndrome]. The effect of intranasal substance P injection on parkinsonian syndrome and the generator of pathologically enhanced excitation (GPEE) in caudate nuclei (CN) was investigated. MPTP or reserpine administration in old rats induced oligokinesia, rigidity and tremor followed by the high amplitude slow and rapid waves in both CN. The bilateral intranasal injection of substance P (25 micrograms/kg) resulted in an increase in motor activity and almost completely abolished the rigidity and tremor. The reduction of extrapyramidal symptoms was considered as a result of the inhibition of GPEE in CN. The possibility of substance P entry from nasal cavity into the brain was discussed. The changes of the substance P balance in nigrostriatal system was suggested to be on of the pathogenetic links of parkinsonian syndrome.


[Release of acetylcholine and its regulation]. The mechanism of acetylcholine (ACh) release and its regulation is a widely studied subject still underdebated. Although the vesicular hypothesis for ACh release is at present largely accepted, alternative theories have been proposed. ACh release is triggered by calcium influx through specific presynaptic Ca2+ channels. The modulation of this calcium influx appears as the main mechanism through which ACh release is regulated. This can be achieved by direct modification of the presynaptic Ca2+ channel opening or indirectly by a change in the polarization level of the presynaptic membrane due to the opening or closing of other presynaptic channels (usually K+ channels). The increase in the intracellular Ca2+ concentration that triggers ACh release is also under the control of Ca2+ membrane exchanges and intracellular Ca2+ buffers. ACh synthesis that takes place in the cytoplasm of the terminal, can itself be modulated leading to changes in the quantity of ACh available for release. All these regulatory mechanisms can be initiated by the activation of presynaptic receptors to either ACh itself (autoreceptors) or to other transmitters (heteroreceptors). Most often, these presynaptic receptors seem to require the transducing role of G proteins and the involvement of various second messengers. Some illnesses concerning the cholinergic system can be related to a disfunction of one of these presynaptic regulatory mechanisms.


Identification of three-element windkessel model: comparison of time and frequency domain techniques. The problem of the parameter identification of the three-element windkessel model is studied. Minimization by least-square technique--LSQ--in time domain and frequential techniques--FFT--are compared. Continuous pressure and flow curves were recorded in the proximal aorta of an open chest dog. Comparison shows very high correlations between the parameter estimations obtained by LSQ and FFT methods. However, systematic differences appear between the calculated values, but do not seem to endanger physiological interpretation of the results.


[Cellular localization of human secretory beta globulin in normal and tumor tissues]. Distribution of secretory beta-globulin (S beta G) which possesses affinity for steroids was investigated immunohistochemically. Tissue specificity of S beta G, produced in adult secretory epithelial cells of the seminal vesicles, salivary glands, prostate, bronchi and mammary gland was discovered. The protein was not detected in fetal and embryonal tissues. S beta G synthesis is abnormal in neoplasms: its expression partly preserves in breast cancer cells and increases in epithelium of mammary ducts near the focus of malignancy. In lung cancer and bronchial glands cells near the focus of neoplastic transformation S beta G positive reaction was not observed.


[Studies of kinin-like properties of a new peptide isolated from bovine brain]. Kinin-like properties of two new peptides NRP-11 and P7 which have structural similarity with neurotensin (NT) and kallidin (K) were investigated. It was found that, unlike NT and K, the new peptides possess reduced myotropic and hypotensive activity. On the other hand, similarly to NT and K, the new peptides exhibited a high histamine-releasing activity in rat peritoneal mast cells. Possible central effects are implied for peptide NRP-11 isolated from bovine brain and its fragment P7.


[Study of the possibility to abolish the action of immunosuppressive factors of tumor cells]. We investigated the efficacy of IL-2, LPS, MDP, TRA, ionomycin and contrykal on proliferation of lymphocytes treated by tumor cell immunosuppressive factors (ISF). IL-2, LPS and/or MDP did not abolish the influence of P815 and B16 ISF on Con A or alloantigen-induced lymphocyte proliferation. TPA and in less extent ionomycin and combination of the above preparations totally abrogated the suppression of Con A-induced lymphocyte proliferation. In inverted experiments Con A abrogated ISF-mediated suppression of lymphocyte proliferation induced by TPA plus ionomycin.


[Morphogenesis and modifications of the respiratory epithelium]. The tracheobronchial tree begins to form during the fourth week of development through a series of dichotomic divisions of an entoblastic evagination. The morphogenesis and maturation of the respiratory tract depend both on the nature of the extracellular matrix which facilitates cell migration and on epithelial-mesenchymal interactions which induce the proliferation and differentiation of epithelial cells. The study of the plasticity and the phenotypic modifications of secretory cells during both development and inflammatory remodeling of the tracheobronchial mucosa suggests an important role for secretory cells during ciliogenesis and repair.


[Cultures of islet cells obtained from the fetal bovine pancreas]. The pancreas from bovine fetuses of 27-35 cm crown-rump length were used as a source of islet cell cultures. Pancreatic tissue was treated by collagenase, filtered through the metal sieve and incubated in MEM for 24 h. Using this method, cultures similar to so-called pseudo-islets were obtained. Aldehyde-fuchsin staining and electron microscopy revealed a significant number of beta-cells within the pseudo-islets, the insulin-producing activity of which was confirmed by RIA.


[Effects of mechanical stimulation on vascularization of the wall of major arteries]. In vivo mechanical stretching induced angiogenesis in the t. adventitia of large artery. Vasa vasorum penetrate from t. adventitia into t. media with formation of vessel sprouting and migration of the endothelial cells.


[Cellular mechanism of muscle contraction of bronchial smooth muscle]. Airway smooth muscle is one of the main effector of bronchial reactivity. The understanding of the cellular mechanisms involved in the contraction of this muscle has advanced in the recent past since isolated cells in culture can now be studied. Extracellular messengers (neurotransmitters and mediators) as well as their specific membrane receptors have been analyzed in some details. The membrane transduction of extracellular messengers brings about the formation (or the increase in the concentration) of the intracellular second messenger which, in airway smooth muscle, is the cytosolic calcium (Ca2+i) via activation of calcium channels which depend on surface membrane potential changes (electromechanical coupling) on the one hand and mainly via mechanisms independent of surface membrane potential changes-so-called the pharmacomechanical coupling--which involves membrane phosphoinositides metabolism. Changes in Ca2+i activate contractile proteins leading the muscle to shorten and to develop force via several controlled steps such as phosphorylation of myosin or changes in the sensitivity to Ca2+ of the contractile elements. Experimental techniques that enable to simultaneously study different aspects of the cellular response are being developed in airway smooth muscle and are likely to provide complementary information about the cellular physiology and pathophysiology of this muscle.


[Structure and secretory functions of the respiratory epithelium]. Airway secretions actively participate in respiratory epithelium protection. Apart from its main participation in transport of inhaled microorganisms and particles by mucociliary clearance, respiratory mucus also contributes to other protective purposes such as the control of airway humidification. Biochemical components found in secretions, such as mucins, lipids, antibacterial agents (secretory IgA, lysozyme, lactoferrin), antioxidant and antiprotease components, contribute significantly to the airway epithelium defense.


Interaction of human bone marrow fibroblasts with megakaryocytes: role of the c-kit ligand. Human kit ligand (KL), also known as stem cell factor (SCF), steel factor, or mast cell growth factor, is a recently identified hematopoietic growth factor whose receptor is the product of the c-kit proto-oncogene. Alternative splicing of the pre-mRNA of KL/SCF results in secreted and membrane-bound forms of the protein. We and others have recently shown that the c-kit gene product is expressed on human megakaryocytes and that soluble KL/SCF in combination with granulocyte-macrophage colony-stimulating factor, interleukin-3 (IL-3), or IL-6 increased megakaryocyte progenitor colony formation (CFU-MEG) and stimulated mature megakaryocytes. Here we show that adhesion of human megakaryocytes to bone marrow stromal fibroblasts, which express the membrane-bound form of KL/SCF (mKL/SCF), is mediated in part by the interaction between mKL/SCF and the c-kit protein. This interaction also results in marrow fibroblast-stimulated proliferation but not an increase in ploidy of megakaryocytes; when the two cell types were separated by a transoluble membrane, proliferation did not occur. Adhesion and proliferation of human megakaryocytes to an immortalized murine stromal cell line SI/SI lacking the KL/SCF gene was impaired, whereas transfection of SI/SI cells with human mKL/SCF significantly increased both adhesion and proliferation. Marrow stromal fibroblast mKL/SCF may serve both as an adhesion structure and as a growth-potentiating factor for megakaryocytes in the bone marrow.


Recombinant human interleukin-9 induces protein tyrosine phosphorylation and synergizes with steel factor to stimulate proliferation of the human factor-dependent cell line, M07e. Human interleukin-9 (IL-9) was originally identified and cloned based on its stimulatory effect on proliferation of human myeloid cell line, M07e. IL-9 synergized with Steel factor, the ligand for the c-kit product, to stimulate M07e cell proliferation. To investigate potential mechanisms for this, IL-9 was assessed for effects on protein tyrosine kinase activities in M07e cells by immunoblotting with anti-phosphotyrosine monoclonal antibody; results were compared with those of Steel factor alone and in combination with IL-9, and those of 12-0-tetradecanoyl phorbol-13-acetate (TPA). Recombinant human IL-9 (10 ng/mL) rapidly and transiently induced or enhanced at least four tyrosine phosphorylated protein bands with molecular weights of 105, 97, 85, and 81 Kd. This tyrosine phosphorylation pattern was different from that generated by recombinant murine Steel factor or TPA stimulation and the combination of IL-9 and Steel factor did not change the IL-9-induced pattern. IL-9-induced tyrosine phosphorylated bands were completely blocked by treatment of IL-9 with anti-IL-9 antibody under conditions that also neutralized the synergistic effect of IL-9 with Steel factor on M07e cell proliferation. Genistein, a tyrosine kinase inhibitor, blocked phosphorylation of IL-9 and Steel factor-induced bands. Unlike Steel factor or TPA, IL-9 did not appear to stimulate phosphorylation of 42-Kd mitogen-activated protein (MAP) kinase or Raf-1, or enhance MAP kinase activity. MAP kinase and Raf-1 are serine/threonine kinases that are phosphorylated and activated by many growth factors and by agonists for protein kinase C. While the combination of IL-9 plus SLF did not appear to induce phosphorylation of new bands not already seen with either IL-9 or SLF alone, or enhance the phosphorylation of those bands seen with either cytokine alone, the results suggest that IL-9 activates specific and unique signal transduction pathways.


CD34+ marrow cells, devoid of T and B lymphocytes, reconstitute stable lymphopoiesis and myelopoiesis in lethally irradiated allogeneic baboons. CD34+ cells devoid of detectable mature and immature T and B lymphocytes, expressing the CD2, CD10, and CD20 antigens, were isolated from marrows of three pairs of sex-mismatched, mixed lymphocyte culture (MLC) nonreactive, sibling baboons. Reciprocal transplants were performed between members of each pair, using the sex chromosomes, identified by standard cytogenetic techniques, as markers of the transplanted cells. Five animals from these three pairs were transplanted with 0.6 to 2.1 x 10(6)/kg of isolated cryopreserved and/or fresh isolated cells that were greater than 95% to 97% CD34+. Before transplantation, animals were treated with either single (920 or 1,020 cGy) or split (700 cGy x 2) dose total body irradiation. All animals engrafted with donor cells, as demonstrated by cytogenetic analysis of bone marrow metaphase cells 4 weeks after transplantation, with days to white blood cell count (WBC) greater than 500 being 19 +/- 2, to WBC greater than 1,000 23 +/- 2, to absolute neutrophil count greater than 500 24 +/- 3, and to platelets greater than 20,000 30 +/- 7. Three animals died of infectious-related complications at 34, 42, and 109 days after transplantation with evidence of host and donor cells (mixed chimerism) in marrow. Two animals remain alive and healthy more than 545 and 455 days after transplantation with stable mixed chimerism in marrow and blood. For these two animals, cytogenetic analysis of granulocyte/macrophage and erythroid colonies derived from marrow precursors between weeks 25 and 42 posttransplant showed evidence of mixed chimerism. Cytogenetic studies of CD2+ T cells and CD20+ B cells isolated from blood of these two animals between weeks 21 and 51 posttransplant showed the presence of mixed chimerism in both lymphocyte populations. Thus, isolated allogeneic CD34+ marrow cells devoid of detectable mature and immature T and B lymphocytes can engraft and reconstitute stable long-term myelopoiesis and lymphopoiesis in lethally irradiated baboons. These results are consistent with the hypothesis that CD34+ marrow cells contain pluripotent hematopoietic stem cells capable of fully reconstituting lymphohematopoiesis in the transplanted host.


Interferon-gamma increases the expression of the gene encoding the beta subunit of the granulocyte-macrophage colony-stimulating factor receptor. Granulocyte-macrophage colony-stimulating factor (GM-CSF) activates a broad range of myeloid cells through binding to high-affinity receptors (GM-CSF-R) consisting of at least two distinct subunits, GM-CSF-R alpha and GM-CSF-R beta. The genes of these GM-CSF-R subunits have been identified recently, but little is known about the regulation of their expression. In this study, we investigated the expression of the GM-CSF-R subunit genes in normal human monocytes. Out of a panel of various cytokines and factors tested, only interferon-gamma (IFN-gamma) affected the expression of one of the GM-CSF-R subunit genes by increasing the GM-CSF-R beta mRNA expression threefold to sixfold with no effect on GM-CSF-R alpha. Maximal effects occurred 2 to 4 hours after stimulation with 500 to 5,000 U/mL IFN-gamma. Nuclear run-on assays and mRNA half-life studies showed that IFN-gamma modestly enhanced the transcription of the GM-CSF-R beta gene and stabilized the GM-CSF-R beta mRNA, with the latter mechanism predominant. Pretreatment of the monocytes with cycloheximide did not abrogate the increase of GM-CSF-R beta mRNA expression induced by IFN-gamma, indicating that de novo protein synthesis was not required for this activity. When monocytes were exposed to IFN-gamma for 6 to 24 hours, the number of GM-CSF-R per cell was increased 79% as compared with controls, whereas the receptor affinity remained unchanged. These data indicate that the GM-CSF-R expression in monocytes may be upregulated by IFN-gamma via an increased expression of the beta subunit gene, involving both transcriptional and post-transcriptional mechanisms.


Stem cell factor enhances proliferation, but not maturation, of murine megakaryocytic progenitors in serum-free culture. The effects of recombinant rat stem cell factor (SCF/c-kit ligand) on murine megakaryocytopoiesis were studied using partially purified bone marrow cells derived from normal and 5-fluorouracil (5-FU)-treated mice in a serum-free culture system. SCF alone did not support the formation of megakaryocyte (M) and granulocyte-macrophage-megakaryocyte (GMM) colonies. However, the addition of SCF to cultures containing interleukin-3 (IL-3) resulted in a significant increase in the number of M and GMM colonies formed by bone marrow cells from normal mice, whereas IL-6 augmented only M colony growth. The stimulatory effect of SCF was approximately three to four times as high as that of IL-6 on the primitive progenitors capable of megakaryocytic-lineage expression derived from 5-FU-treated mice. In addition, SCF, but not IL-6, significantly increased the number of constituent cells in the individual M colonies supported by IL-3. On the other hand, SCF did not exert any effect on the size and DNA content of megakaryocytes in IL-3-dependent M and GMM colonies, whereas IL-6 enhanced the maturation of megakaryocytes. These results suggest that SCF stimulates the proliferative process in megakaryocytic progenitors and that the main activity of IL-6 is the promotion of megakaryocyte maturation.


Hematopoietic cytokines inhibit apoptosis induced by transforming growth factor beta 1 and cancer chemotherapy compounds in myeloid leukemic cells. Transforming growth factor-beta 1 (TGF-beta 1) induces cell death in myeloid leukemia by apoptosis. In the M1 myeloid leukemia, this induction of apoptosis was inhibited by granulocyte colony-stimulating factor (G-CSF) or interleukin-6 (IL-6) and to a lesser extent by IL-1 alpha. IL-3 and stem cell factor/mast cell growth factor (SCF) showed only a marginal effect, and granulocyte-macrophage and macrophage CSFs (GM-CSF and M-CSF, respectively) were inactive. The induction of apoptosis by TGF-beta 1 in a different myeloid leukemia (7-M12) was inhibited by GM-CSF and IL-3 but not by the other cytokines. In the absence of TGF-beta 1, both M1 and 7-M12 leukemic cells were independent of hematopoietic cytokines for cell viability and growth. The cytotoxic compounds vincristine, vinblastine, adriamycin, cytosine arabinoside, cycloheximide, and sodium azide, some of which are used in cancer chemotherapy, induced cell death by apoptosis in both leukemias. As with TGF-beta 1, apoptosis induced by these cytotoxic compounds was inhibited by GM-CSF (7-M12 leukemia) and by G-CSF or IL-6 (M1 leukemia). Cyclosporine A decreased cell multiplication in M1 cells without inducing apoptosis, and G-CSF and IL-6 inhibited the cytostatic effect of cyclosporine A. It is suggested that the clinical use of cytokines to correct therapy-associated myelosuppression should be carefully timed to avoid protection of malignant cells from the cytotoxic action of the therapeutic compounds.


Involvement of tumor necrosis factor (TNF) receptors p55 and p75 in TNF responses of acute myeloid leukemia blasts in vitro. Tumor necrosis factor (TNF)-alpha and TNF-beta have multiple effects on human acute myeloid leukemia (AML) cells in vitro, including (1) synergistic stimulation of proliferation with interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) and upregulation of interleukin-3 (IL-3) and GM-CSF receptors; (2) inhibition of granulocyte-CSF (G-CSF)-induced growth and rapid downmodulation of G-CSF receptors; and (3) induction of autocrine growth. Recently, two distinct TNF receptors (TNF-Rs), TNF-R(p55) and TNF-R(p75), have been identified. In this study, we show that both receptor types may be expressed by AML blasts. It has been investigated whether the different effects of TNF on AML blasts can be explained by differential activation of the distinct TNF-R structures. For this purpose, we used the monoclonal antibodies HTR-1 and HTR-9, specifically recognizing TNF-R(p55), and UTR-1, specific for TNF-R(p75). TNF-(alpha and -beta) mediated synergistic activation with IL-3/GM-CSF, upregulation of IL-3/GM-CSF receptors, inhibition of G-CSF-induced growth, and rapid downmodulation of G-CSF receptors exclusively result from activation of TNF-R(p55). In certain cases in which TNF-alpha, rather than TNF-beta, induces AML growth through an autocrine mechanism, both TNF-R(p55) and (p75) are involved. These data indicate that the variety of TNF responses observed in AML can only be partially explained by differential activation of the TNF-R(p55) and (p75) structures, and that TNF-R(p55) on AML blasts can transduce both positive (synergism with IL-3/GM-CSF) and negative regulatory signals (inhibition of G-CSF-induced proliferation) following TNF activation.


Acute lymphoblastic leukemia in patients aged 60 years and over: a population-based study of incidence and outcome. Data on all patients aged 60 or older in whom a diagnosis of acute lymphoblastic leukemia (ALL) was made in the Northern Health Region of the United Kingdom, over a period of 8 1/2 years, were studied to determine the incidence and range of biological subtypes, and the outcome of a variety of therapeutic approaches. Forty-nine cases were diagnosed (31% of all adult ALL), giving an incidence of 0.9 per 100,000 age-specific population (ASP). Immunophenotyping was performed in 41 patients. Eighteen had common, two T-ALL, 10 null, and 7 B-ALL. Four were biphenotypic with both lymphoid and myeloid markers. One patient refused treatment and four were considered too frail for chemotherapy. Twenty-two were treated palliatively. Aggressive "curative" treatment was planned for 22, but three died before it started. The actuarial survival for the whole group was 4%. The median survival of the palliatively treated group was 1 month, compared with 3 months for those given aggressive treatment. Only two patients (aged 74 and 67) survived more than 2 years in complete remission (CR), both having been treated aggressively. We conclude that the prognosis for those over 60 years of age is extremely poor, but that for a small number of patients a useful prolongation of good quality life may be achieved. Age should not, by itself, be a bar to treatment intended to induce remission. Patients and their relatives should be made aware of the overall poor prognosis and advised of the therapeutic goals when decisions regarding management are being made.


Regulation of human embryonic globin genes zeta 2 and epsilon in stably transformed mouse erythroleukemia cells. Previous work has suggested that the promoter regions of the human embryonic zeta 2 and epsilon globin genes contain negative regulatory regions that could play a role in the repression of these genes in postembryonic erythroblasts. We have examined this possibility by studying the expression of these genes in mouse erythroleukemia cells, an adult erythroid cell line that might be expected to contain repressor molecules that would bind to the putative negative regulatory regions. When attached to appropriate upstream regulatory elements (alpha HS-40 and beta HS1,2) both the zeta and epsilon genes were expressed in these cells at a low level, but no increase in expression was observed when similar constructs lacking the proposed negative regulatory sequences were introduced into these cells. These results cast doubt on the possibility that these sequences play a major role in the developmental repression of the embryonic globin genes, unless they function only in a normal chromosomal organization.


Nitric oxide modulation of human leukemia cell differentiation and gene expression. Nitric oxide (NO) functions as an intercellular messenger molecule in such varied contexts as neurotransmission, immune regulation, and the control of vascular tone. We report that NO, delivered as purified gas or released from the pharmacologic NO donors sodium nitroprusside or 6-morpholino-sydnonimine, caused monocytic differentiation of cells of the human myeloid leukemia cell line HL-60 and altered gene expression. The treated cells stopped proliferating, became spread and vacuolated, had increased expression of nonspecific esterase and the monocyte marker CD14, and displayed increased capacity to produce hydrogen peroxide. Furthermore, these treated cells had increased steady-state expression of messenger RNA (mRNA) for tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), but decreased expression of mRNA for the proto-oncogenes c-myc and c-myb. The increase in TNF-alpha and IL-1 beta mRNA levels was due (at least in part) to a new transcription of these specific mRNAs. NO elaborated in the bone marrow microenvironment may have a role in normal and malignant hematopoietic cell growth and differentiation.


Dissociation of human cytokine receptor expression and signal transduction. We have examined the relationship between granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) receptor expression and signal transduction in populations of HL-60 cells differing in proliferative capacity to these cytokines. GM-CSF or IL-3 stimulation of HL-60 cells pretreated with either dimethyl sulfoxide (DMSO) or retinoic acid results in increases in proliferative response as well as both tyrosine and serine phosphorylation. In contrast, neither GM-CSF or IL-3 stimulation of parental HL-60 cells (those not treated with DMSO or retinoic acid) produced any changes in either proliferation or protein phosphorylation. Thus, although parental HL-60 cells expressed both GM-CSF and IL-3 receptors, treatment with either DMSO or retinoic acid was necessary to confer the capacity for signal transduction as assessed by both a biologic and biochemical response. Pretreatment of cells with genistein, a protein tyrosine kinase inhibitor, resulted in inhibition of GM-CSF-induced protein tyrosine phosphorylation as well as proliferation. These data show a strong correlation between cytokine-induced increases in protein phosphorylation and subsequent biologic responses. Further, this work demonstrates that cytokine receptor expression and signal transduction can be disassociated and suggests the potential for independent regulation of these two components of signal transduction.


Interaction of fibroblast growth factor (FGF) with megakaryocytopoiesis and demonstration of FGF receptor expression in megakaryocytes and megakaryocytic-like cells. We have investigated the interaction of fibroblast growth factor (FGF) with megakaryocytopoiesis. Acidic FGF (aFGF) stimulated the proliferation of murine megakaryocytes and human erythroleukemia (HEL) cells in a concentration-dependent manner. The concentrations of aFGF required to elicit half-maximum and maximum effects were similar for HEL and megakaryocytic colony formation. The effect of aFGF was comparable to that of basic FGF (bFGF) in both cell types. The effect of both FGFs was found to be synergistic with interleukin-3 (IL-3), and was abrogated by a monoclonal anti-IL-6 antibody. A specific cell surface receptor complex of approximately 120 Kd was detected for FGF by crosslinking experiments on HEL cells and total bone marrow (BM) cells. Single-cell autoradiography of megakaryocytes in BM smears and BM cultures showed binding sites for 125I-aFGF. Northern blot analysis of messenger RNA (mRNA) from total BM and HEL cells showed a 4.4-kb mRNA specific for FGF receptors type 1 (flg) and type 2 (bek). This was confirmed by polymerase chain reaction, which also showed the presence of FGF receptor mRNA in megakaryocytic-like cells, normal megakaryocytes, and platelets. Together, these results indicate that FGF is involved in megakaryocytopoiesis and suggest that this interaction may be mediated via FGF receptor type 1 and type 2 located on the megakaryocytic lineage or on accessory cells responsible for the release of megakaryocytic growth-promoting activities.


Enhanced hematopoiesis in vivo and in vitro by splenic stromal cells derived from the mouse with recombinant granulocyte colony-stimulating factor. Splenic stromal cells (CF-1 cells) were established from a mouse administered recombinant human granulocyte colony-stimulating factor (rG-CSF) to clarify the mechanism of splenic extramedullary hematopoiesis induced by the factor. The cells were negative for alkaline phosphatase, factor VIII-related antigen, mac I, and phagocytosis. They were positive for acid phosphatase, collagen type I, collagen type III, and fibronectin. CF-1 cells were not converted to adipocytes in a confluent culture with 10(-6) mol/L hydrocortisone. [35S]rG-CSF bound to CF-1 cells specifically in the growth phase but not in the resting phase. The CF-1 cells had greater colony-stimulating activities than the normal splenic stromal cells. When CF-1 cells were added to bone marrow cells in the spleen colony-forming cells (CFU-S) assay, the number of colonies in the spleen increased between 1.4 and 1.8 times the control without these stromal cells. On the other hand, the normal splenic stromal cells had no effect on increasing the number of CFU-S colonies. Therefore, these data suggest that a factor-dependent hematopoietic microenvironment is generated in the spleen by rG-CSF, and the stromal cells that have the hematopoietic potency become dominant in splenic extramedullary hematopoiesis induced by rG-CSF.


Erythropoietin-induced tyrosine phosphorylations in a high erythropoietin receptor-expressing lymphoid cell line. Retroviral gene transfer of the murine erythropoietin receptor (EpR) cDNA into the pro-B-cell line, Ba/F3, was used to generate cells expressing high EpR levels. One of the resulting clones, Ba/F3 clone C5, contained 5 integrated copies of the gene and expressed, at the cell surface, a single affinity class of EpRs at 10 to 15 times the level present on spleen cells from phenylhydrazine-treated mice. Cross-linking studies with clone C5, using 125I-Ep, yielded the same two 105- and 88-Kd major species as that seen with typical erythroid cells. This was distinct from that obtained with EpR-transfected COS cells or L cells, which gave species of 88 and 65 Kd. This suggests that the biologically active EpR complex generated in this Ba/F3 cell line may closely resemble that present in native Ep-responsive erythroid progenitor cells. Tyrosine phosphorylation experiments showed that several proteins in clone C5 cells were rapidly phosphorylated on tyrosine residues in response to Ep, one being the EpR itself. The proportion of cell surface EpRs tyrosine phosphorylated in response to Ep was substantial, reaching a maximum of approximately 10% within 30 minutes of incubation at 37 degrees C. A comparison of Ep- and murine interleukin-3 (mIL-3)-induced tyrosine phosphorylation patterns in clone C5 cells showed that both growth factors stimulated the tyrosine phosphorylation of proteins with molecular weights of 135, 93, 70, and 55 Kd. This could suggest that the Ep and mIL-3 receptors are capable of using the same tyrosine kinase in these cells.


BALB/3T3 fibroblast-conditioned medium attracts cultured mast cells derived from W/W but not from mi/mi mutant mice, both of which are deficient in mast cells. Proliferation of murine mast cells is induced by both T-cell-derived and fibroblast-derived growth factors. Because the most potent T-cell-derived mast cell growth factor, interleukin-3, promotes the migration of mast cells, we investigated whether fibroblast-derived growth factors had the chemoattractive activity as well. Conditioned medium (CM) of BALB/3T3 fibroblasts induced the migration of cultured mast cells (CMC) derived from normal (+/+) mice. BALB/3T3-CM contained the mast cell growth factor (MGF)/stem cell factor (SCF)/kit ligand (KL), which is the ligand for the receptor encoded by the W (c-kit) gene. CMC derived from the spleen of W/W mice lack the extracellular domain of the W (c-kit) receptor, and W/W CMC did not proliferate in response to BALB/3T3-CM. However, W/W CMC did migrate normally toward BALB/3T3-CM and, moreover, the antibody to the extracellular domain of the W (c-kit) receptor did not inhibit the chemoattractive activity of +/+ CMC toward BALB/3T3-CM. These results indicated that MGF/SCF/KL itself did not represent the major chemoattractive activity. On the other hand, BALB/3T3-CM induced neither proliferation nor migration of CMC derived from mi/mi mice. Both W/W and mi/mi mice are deficient in mast cells, but the present results suggest that the mechanism of the abnormality is different between W/W and mi/mi mice.


Specific growth inhibition of primitive hematopoietic progenitor cells mediated through monoclonal antibody binding to major histocompatibility class II molecules. In a previous study using a canine model, we reported that a certain anti-class II monoclonal antibody (MoAb H81.9), which recognizes an epitope formed by the alpha and beta subunits of HLA-DR, prevented long-term engraftment of autologous marrow cells if administered intravenously during the first 4 days after 9.2 Gy of total body irradiation. Another MoAb (B1F6), reactive with only the beta subunit of HLA-DR and -DP, had no adverse effect on engraftment, although both MoAbs detect antigens on hematopoietic long-term repopulating cells as determined from complement-mediated lysis experiments. In the present study, continuous exposure of unfractionated human marrow to MoAb H81.9 specifically inhibited the growth of primitive progenitor cells that require multiple hematopoietic growth factors for proliferation (high proliferative potential colony forming cells [HPP-CFC] and burst-forming units-erythroid [BFU-e]), but had no effect on more mature, single factor responsive (CFU-GM), progenitor cells. In contrast, MoAb B1F6 did not impair primitive progenitor cell growth cultured as unfractionated marrow. However, when cell dose-response experiments were performed using CD34-positive cells plated at low cell densities, the marked inhibitory effects of MoAb H81.9 on HPP-CFC and BFU-e colony formation were not seen. These findings suggest that MoAb H81.9 may not inhibit primitive hematopoietic cells directly, but rather indirectly through the action of potent mediators derived from other HLA-DR-positive marrow cells.


Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products. Mature circulating polymorphonuclear cells (PMN) have the shortest half-life among leukocytes and undergo rapid programmed cell death in vitro. In this study, we have examined the possibility that inflammatory signals (cytokines and bacterial products) can regulate PMN survival. PMN in culture were found to rapidly die, with percentages of survival at 24, 48, 72, and 96 hours of 97.3% +/- 1.9%, 36.8% +/- 5.3%, 14.5% +/- 3.1%, and 4.2% +/- 2.9%, respectively (mean +/- SE of 20 different donors). PMN incubated with interleukin-1 beta (IL-1 beta), tumor necrosis factor, granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, and interferon-gamma (IFN-gamma), but not with prototypic chemoattractants (fMLP, recombinant C5a, and IL-8), showed a marked increase in survival, with values ranging at 72 hours of incubation from 89.5% +/- 5.8% for IL-1 beta to 47.6% +/- 6.4% for IFN-gamma. The calculated half-life was 35 hours for untreated and 115 hours for IL-1-treated PMN. PMN activated with lipopolysaccharide (LPS) or inactivated streptococci also showed a longer survival compared with untreated cells (94.4% +/- 3.2% and 95.5% +/- 2.4%, respectively, at 72 hours). PMN surviving in response to LPS or IL-1 beta retained the capacity to produce superoxide anion when treated with phorbol esters or fMLP. All inducers of PMN survival protect these cells from programmed cell death because they reduced cells with morphologic features of apoptosis and the fragmentation of DNA in multiples of 180 bp. Thus, certain cytokines and bacterial products can prolong PMN survival by interfering with the physiologic process of apoptosis. Prolongation of survival may be important for the regulation of host resistance and inflammation, and may represent a crucial permissive step for certain cytokines and microbial products that activate gene expression and function in PMN.


CD10/NEP is expressed on Thy-1low B220+ murine B-cell progenitors and functions to regulate stromal cell-dependent lymphopoiesis. To further characterize the function of the common acute lymphoblastic leukemia antigen (CALLA; CD10, neutral endopeptidase 24.11, NEP) in early lymphoid development, we have identified murine lymphoid progenitors expressing CD10/NEP and analyzed the effects of inhibiting the enzyme in in vitro assays of murine lymphoid differentiation. CD10/NEP transcripts and enzymatic activity were primarily restricted to the subpopulation of murine lymphoid progenitors, termed pro-B cells, which were isolated from bone marrow (BM) and modified Whitlock-Witte cultures and defined by coexpression of B220 and low levels of Thy-1. CD10/NEP transcripts and cell surface enzymatic activity were also detected in BM stromal cells known to support the development of B-lymphoid progenitors. In contrast, Abelson and H-ras transformed pre-B-cell lines were CD10/NEP- as were Thy-1-B220+ pre-B cells from BM and modified Whitlock-Witte cultures and Thy-1lowLin- (B220-Mac-1-GR-1-Ly-2/3-) uncommitted hematopoietic progenitors from BM. The expression of CD10/NEP on murine pro-B cells and BM stromal cells suggests a role for the enzyme in early B-cell ontogeny. In modified Whitlock-Witte cultures in which Thy-1lowLin- progenitors plated on BM stromal cells differentiate into Thy-1lowB220+ pro-B and Thy-1-B220+ pre-B cells, the addition of specific CD10/NEP inhibitors increased the number of lymphoid colonies at days 5 through 7 by 34% (P < .001). The results suggest that CD10/NEP participates in the regulation of the earliest stages of stromal cell-dependent B lymphopoiesis.


Thrombocytosis in patients with tumors producing colony-stimulating factor. We investigated the cause of thrombocytosis in 14 patients with tumors producing colony-stimulating factor (CSF). Of the 14 patients, 10 had tumors producing granulocyte-CSF (G-CSF) and 4 had tumors producing granulocyte-macrophage--CSF (GM-CSF). Thrombocytosis of greater than 400 x 10(9)/L was noted in 8 of 10 patients with G-CSF-producing tumors and all 4 patients with GM-CSF-producing tumors. Median peak platelet counts were, respectively, 511 x 10(9)/L (range, 384 to 694 x 10(9)/L) and 579 x 10(9)/L (range, 526 to 910 x 10(9)/L) in patients with tumors producing G-CSF and GM-CSF. In most patients, thrombocytosis declined towards the terminal stage. High interleukin-1 (IL-1) and IL-6 levels were found in addition to CSFs in the plasma or culture supernatants of tumor cells obtained from most patients. In patients with GM-CSF-producing tumors, these specimens had megakaryocyte-CSF (Meg-CSF) activity, which was abolished by anti-GM-CSF antibody. These specimens also had megakaryocyte potentiating (Meg-Pot) activity attributable to both GM-CSF and IL-6. In patients with G-CSF-producing tumors, only Meg-Pot activity due to IL-6 was detected. These results indicate that the thrombocytosis in GM-CSF-producing tumors was caused by both the Meg-CSF activity of GM-CSF and the Meg-Pot activity of IL-6 plus GM-CSF, while that in G-CSF-producing tumors was due to the Meg-Pot activity of IL-6.


Interferon-alpha downregulates the abnormal intracytoplasmic free calcium concentration of tumor cells in hairy cell leukemia. Hairy cell leukemia (HCL) is a B-cell tumor affecting the preplasma stage of B-cell differentiation. One important feature of the disease is its exquisite sensitivity to interferon-alpha (IFN-alpha) therapy. Because we showed earlier that the CD20 molecule is consistently hyperphosphorylated in hairy cells and because previous studies showed that CD20 is involved in regulating intracytoplasmic free calcium concentrations ([Ca2+]i) in normal B lymphocytes, we measured [Ca2+]i in tumor cell samples from patients with HCL and studied the effect of IFN-alpha on this parameter. Using the Ca(2+)-sensitive fluorophore fura-2, we observed that hairy cells display a slightly but consistently higher [Ca2+]i than normal 48-hour-activated B cells or other leukemic cells. Furthermore, both in vitro preincubation of cell samples with IFN-alpha and in vivo administration of this cytokine reduced the [Ca2+]i in hairy cells. This effect was observed together with a decrease in transmembrane Ca2+ influx. However, preincubation with IFN-gamma had no effect. The in vivo correlation between the diminution of CD20 phosphorylation and [Ca2+]i in tumor cell samples from patients at the beginning of IFN-alpha therapy suggests that these two parameters are connected.


Genomic hypomethylation in human chronic lymphocytic leukemia. DNA samples from patients with chronic lymphocytic (CLL), chronic myelocytic (CML), acute myelocytic (AML), and acute lymphocytic leukemia (ALL), as well as samples from patients with multiple myeloma (MM) and healthy volunteers, were analyzed for their genomic methylation status using Hpa II and Msp I digestions followed by a simple gel electrophoresis and ethidium bromide staining. A densitometric method was developed to measure more accurately the extent of methylation in genomic DNA samples and the results were confirmed by high-performance liquid chromatography (HPLC) analysis of hydrolyzed DNA. Southern analysis with ornithine decarboxylase (ODC) gene probe was also employed, and the levels of ODC mRNA were determined with the aid of polymerase chain reaction (PCR). The results indicated that a general genomic hypomethylation was present in almost all of the samples obtained from patients with B-cell CLL. This hypomethylation was most striking among the patients who were freshly diagnosed and among untreated chronic patients. CML appeared to be a heterogenous group, comprising patients with normal methylation status in addition to patients with slight hypomethylation. Patients with ALL, AML, or MM did not show any signs of DNA methylation changes in comparison to healthy volunteers. Although all analyzed samples from patients with B-CLL showed hypomethylation of ODC sequences, little correlation existed between the mRNA levels and the extent of hypomethylation of ODC gene.


Involvement of CD36 on erythrocytes as a rosetting receptor for Plasmodium falciparum-infected erythrocytes. Plasmodium falciparum-infected erythrocytes (parasitized red blood cells [PRBCs]) can adhere to uninfected erythrocytes (RBCs) to form rosettes, and adhere to the endothelial cell (EC) surface antigen CD36. These adherence phenomena have previously been considered quite different. We show that anti-CD36 monoclonal antibodies (MoAbs) reverse rosetting of PRBCs from both a culture-adapted line (Malayan Camp [MC] strain) and a natural isolate, GAM425. Three MoAbs that block adherence of PRBCs to ECs or C32 melanoma cells also reversed rosetting by greater than 50% at levels of less than 1 microgram/mL (OKM5, OKM8, and 8A6). Two other MoAbs that react with purified CD36 (1D3 and 1B1), but do not react with the surface of C32 cells, failed to reverse rosetting. When rosettes were disrupted and the RBCs and PRBCs were pretreated separately with antibodies before mixing to allow rosette reformation, only pretreatment of RBCs had an effect. MoAb 8A6 pretreatment of RBCs blocked rosette reformation, while MoAb 1B1 pretreatment did not. Rosetting was also reversed by purified human platelet CD36. In conjunction with evidence that CD36 is expressed on normal human erythrocytes (van Schravendijk et al, Blood 80:2105, 1992), we conclude that this CD36 is able to act as a host receptor for rosetting in the MC strain and some natural isolates of P falciparum.


Normal human erythrocytes express CD36, an adhesion molecule of monocytes, platelets, and endothelial cells. We have recently shown that rosetting of Plasmodium falciparum (MC R+ line)-infected erythrocytes (parasitized red blood cells [PRBCs]) with uninfected erythrocytes (RBCs) is blocked by coating of the RBCs with anti-CD36 monoclonal antibodies (MoAbs; Handunnetti et al, Blood 80:2097, 1992). Adult RBCs have previously been considered negative for CD36. However, using fluorescence-activated cell sorter analysis with the anti-CD36 MoAbs 8A6, OKM5, and OKM8, which reverse rosetting, we consistently detect CD36 on the majority of normal adult RBCs. Absorption of the MoAb solutions with CD36-transfected Chinese hamster ovary (CHO-CD36) cells removed the reactivity against both CHO-CD36 cells and RBCs, whereas absorption with CHO cells had no effect. By comparison with staining for glycophorin A, LFA-3, and CR1, the level of expression of CD36 appeared to be low. Nevertheless, normal RBCs were capable of adhering to plastic coated with anti-CD36 MoAbs. RBCs from one African malaria patient were identified as deficient in CD36 and these RBCs did not rosette with the patient's own P falciparum PRBCs, even though these PRBCs were capable of rosetting with RBCs from a normal donor in a CD36-dependent manner. Therefore, the level of expression of CD36 on normal RBCs is sufficient to be important in cell adherence, and may have a biologic role in normal individuals as well as in the pathology of P falciparum malaria.


Double NOR is not a good indicator of risk for Down syndrome. The silver staining technique was applied to study the nucleolar organizer regions (NORs) of 38 parents of children with Down syndrome and 40 parents of healthy offspring. Double NOR (dNOR) variants were found in both groups of parents. We compared the incidence of dNOR between these two groups. Our results indicate that different conclusions can be drawn from the same data depending on how the analyses are carried out. It seems to us that dNOR is not a useful marker for predicting whether or not its carrier is at a higher risk of having children with Down syndrome.


Endocrine therapy for advanced breast cancer: a review. More than 45,000 women will die of metastatic breast cancer in the United States in 1991. Endocrine therapy remains a major option for treatment of such patients, and results in complete plus partial response rates of 30% with a median duration of approximately one year. Postmenopausal status, increased age, a prolonged disease-free interval, bone and soft tissue metastases, and positive estrogen and progesterone receptors are all associated with an increased response to endocrine therapy. The use of additive hormonal therapy, specifically antiestrogens, progestins, and aromatase inhibitors, have replaced surgical ablative procedures in the majority of patients; response rates to antiestrogen therapy, progestin therapy, and aromatase inhibitors are similar, but antiestrogens have generally been associated with the most favorable therapeutic index. At present, there is no convincing evidence that either combinations of endocrine therapies or endocrine therapy combined with chemotherapy are associated with an improvement in survival for patients with metastatic disease. Future research efforts directed at defining the molecular mechanisms of endocrine activity should facilitate clinical trials of newer and potentially more effective agents. All patients with metastatic breast cancer should be considered for at least one trial of endocrine therapy provided their metastatic disease is not rapidly progressive or life-threatening.


Synthesis and macromolecular organization of gastrointestinal mucin. Mucus glycoproteins (mucins), the principal determinants of mucus protective qualities and mucosal defense, are studied extensively to define pathological aberrations in the relation to gastrointestinal disease and to develop the mucous barrier strengthening agents. Recent work from our laboratory provided evidence as to the initial stages of the gastrointestinal mucin synthesis, molecular size of the apomucin, its macromolecular organization and interaction with other elements of gastrointestinal mucus. Using monoclonal antibodies against apomucin (clone 1H7), O-glycosylated with N-acetylgalactosamine apomucin (clone 2B4), and that against carboxyl terminal of the apomucin (clone 3G12), the mucin synthesizing polysomes were isolated and glycosylated peptides ranging in size from 6-60 kDa identified. The in vitro synthesis in the cell-free system also afforded 60-64 kDa products recognized by 1H7 and 3G12 antimucin MAbs. The obtained results provided evidence that the mucin core consists of 60 kDa peptide which at cotranslational stage is O-glycosylated with N-acetylgalactosamine. Studies on mucin polymer assembly revealed that mucin preparations prepared by equilibrium density gradient centrifugation and Sepharose 2B chromatography (Mantle, M., Mantle, D., and Allen, A. (1981) Biochem. J. 195, 277-285) are not completely purified and contain DNA and extraneous proteins. The evidence was obtained that so called mucin "link protein", 118 kDa glycopeptide, is a N-glycosylated fragment of fibronectin, whereas the supposedly native undegraded mucin isolated by Carlstedt et al. (Biochem. J. (1983) 211, 13-22) was found to contain mucin-fibronectin-DNA complexes. The general picture that emerged from the studies is that the pure mucin consists of 60 kDa glycosylated peptides only. The carboxyl terminal (8-12 kDa fragment) of these peptides is not glycosylated (naked) and is responsible for mucin interaction with fibronectin and other fibronectin-like extracellular matrix proteins. While the formation of the mucosal coat depends on many other factors and extracellular components, our findings on mucin structure and interaction with the extracellular matrix proteins provide explanation as to the possible mechanism of mucin adherence to the epithelial surfaces.


Fibronectin and fibrinogen degradation products stimulate PMN-leukocyte and mast cell degranulation. The ability of various peptides cleaved by plasmin from human fibrinogen and fibronectin or fibrinogen- and fibronectin- related synthetic peptides to induce histamine release from mast cells and collagenase and elastase from PMN-leukocytes was examined. Low molecular weight fibrinogen degradation products showed dose dependent secretion of collagenase. These peptides (mol. wt. 1.4 kD) at the concentration of 10(-5) M released about 47% of collagenase and 13% of elastase. Synthetic fibrinopeptides A and B had a similar strong collagenase releasing potency and also released histamine from mast cells. Peptides from plasmin digestion of fibronectin containing cell attachment site with sequence Arg-Gly-Asp-Ser and also synthetic peptide reproducing this amino-acid sequence at the concentration of 1000 micrograms/ml released about 50% of collagenase and 55% of elastase from PMN-leukocytes. Moreover peptides containing cell attachment and gelatin binding site induced histamine release from mast cells. The association of fibrinogen and fibronectin degradation with activation of mast cells may motivate the treatment with antihistaminic drugs of all pathological conditions where the intensive protein degradation takes place.


The continent ileal reservoir--an experimental study. This thesis describes new concepts pertaining to the continent ileostomy. The aim of the study was twofold: to counter valve desinvagination and to simplify pouch construction. In Chapter 1 a survey is given of the history, the present situation of the technique and the complications of the continent ileostomy. It appears that on the one hand the operation improves the quality of the patient's life, but on the other hand the operation gives rise to many complications. The number of complications quoted in the literature varies and has been reported to be as high as 43%. Most of the time this led to repeat surgery, with equally uncertain results. This is the reason why the operation is not very frequently performed. Most of the time the complications concern the valve system and to a lesser degree the reservoir. In order to obtain a better insight into the origin of and possible gain better control over these complications an investigation was carried out on laboratory animals. This investigation involved: the complications of the valve system, the effect of the suturing method on the function of the reservoir and the simplification of the construction of the reservoir. In Chapter 2 the aim of the investigation was formulated in three questions. 1. Is it possible to diminish the chance of complications of the valve system of the continent ileostomy? 2. Does the method of suturing influence the function of the reservoir? 3. Is it possible to simplify the construction of the reservoir, so that the duration of the operation can be shortened? Chapter 3 is the general materials and methods section. Chapter 4 is about the research on the valve system. Up to now, no method of suturing the valve has consistently produced results good enough to make subsequent re-operations unnecessary. In this study two types of valve experiments have been carried out. First the feasibility of circumventing the problems of the nonpermanent form of the valve was investigated combining a short papilla with a 10 centimeter long, anti-peristaltic sutured section of the ileum. The assumption was that permanence of the form would be more easily obtained in a shorter valve (2 centimeter instead of 5 centimeter). After an initial period of continence, incontinence occurred on the average after 10 months. Autopsy showed that the short papilla had disinvaginated.(ABSTRACT TRUNCATED AT 400 WORDS)


Acute phase proteins in alcoholics with or without liver injury. Acute phase proteins behaviour has been examined in chronic alcoholics to verify the hypothesis that chronic alcohol consumption stimulates the hepatic synthesis of acute phase proteins. Certain acute phase proteins were studied in two groups of alcoholics, one with and one without liver damage, and in a third group of healthy volunteers. The results show that the acute phase proteins were similar in the two groups of alcoholics, but differed when compared to the control group. The authors have concluded from these results, that chronic alcohol consumption causes a serum increase of: mucoproteins (p less than 0.001), alpha 1 acid glycoprotein (p less than 0.05), haptoglobin (p less than 0.05) and fibrinogen (p less than 0.02). Such increases are independent from the existence of liver damage.


Metallic stents in biliary disease.

Metallic stents in biliary disease. The development of self-expanding metallic endoprostheses which can be implanted easily, with minimal trauma, has revolutionized the non-surgical treatment of both benign and malignant biliary strictures. The Wallstent (Medinvent SA, Lausanne, Switzerland), a pliable, tubular stainless steel mesh, is the metallic stent of choice for treatment of malignant strictures and can be implanted in a single session resulting in a shortened hospital stay for patients undergoing palliation of irresectable biliary tumours. Although follow-up is currently rather limited, it appears that the occlusion rate of Wallstents will be lower than that of plastic endoprostheses and no cases of stent migration have been reported. The Gianturco zigzag stent (Cook Inc., Bloomington, Ind, USA) should not be used in malignant strictures because of rapid occlusion due to tumour ingrowth through the struts. However, it exerts a strong, continuous, outward radial force and is ideally suited for use in the small, but difficult to manage, group of patients with benign biliary strictures which recur despite surgery and repeated balloon dilations.


Accurate histochemical definition of argon-laser-induced tissue necrosis. We present a histochemical method using the redox dye nitro blue tetrazolium chloride (NBTC) for the evaluation of argon-laser-induced tissue damage. By this method viable cells are stained blue, devitalized cells not. Our findings suggest that the NBTC staining method is more appropriate for the accurate definition of the necrosis and cell viability after laser treatment than routine histology.


[Demonstration of thymidine phosphorylase activity in human healthy, adenomatous and cancerous prostate]. The presence of thymidine phosphorylase in human healthy, adenomatous and cancerous prostate was demonstrated. The enzyme was responsible for the cleavage and synthesis of thymidine and for the transfer of deoxyribose from one deoxyribonucleoside to a pyrimidic base. The enzyme from normal and adenomatous prostate was retained on DEAE-Sephadex gel. In PC-3 cells, two enzymes with thymidine phosphorylase activity were present, one was retained on the gel, the second was excluded from it. Thymidine phosphorylase activity was higher in adenomatous and cancerous tissues that in healthy ones. In all tissues, the reactions of thymidine synthesis and of deoxyribose transfer were more important than that of thymidine cleavage.


Evaluation of complement in patients with eosinophilic pneumonia. Complement evaluation was performed in two patients with active eosinophilic pneumonia and in one in remission, to determine the role of complement activation in the pathogenesis of this disorder. All three had cough, dyspnea, malaise, and blood eosinophilia; two patients also had pyrexia. In all 3 cases the pulmonary eosinophilic infiltrates (radiographic findings) and symptoms responded rapidly to steroid administration. The two patients with active eosinophilic pneumonia showed elevated CR3 but reduced FcrR on the PMN before and during steroid administration. In contrast PMN from four patients with bronchial asthma exhibited slightly elevated expression of both CR3 and FcrR during their asthma attack. It is suggested that clinical symptoms disappear soon after the beginning of steroid but changes of complement receptors on PMN may last for longer periods. On the basis of the combined results, this study indicates that estimation of complement activation may provide a useful indicator for disease activity in patients with eosinophilic pneumonia of unknown etiology.


Leukoencephalopathy induced by tegafur: serial studies of somatosensory evoked potentials and cerebrospinal fluid. A case of leukoencephalopathy induced by tegafur, an antineoplastic derivative of 5-FU, is reported. The patient received 600 mg of tegafur p.o. for 16 days before excision of rectal cancer. After the operation, gait disturbance and mental abnormalities appeared. He became akinetic and mute within a few days following readministration of tegafur. Serial studies of brain CT, somatosensory evoked potentials (SEP) were made, and myelin basic proteins (MBP) in the cerebrospinal fluid were measured. The level of MBP was about twice the normal value and the central conduction time (CCT) of SEP was prolonged at admission. The value of MBP and CCT improved with recovery from akinetic mutism.


Silent partners. Patients' views about choice and decision making in a day unit. The profile of day case surgery has been gaining prominence in the last decade. Improvements in surgical techniques have allowed a greater range and number of surgical procedures to be performed on a day case basis. Day case surgery also offers considerable efficiency benefits, with lower cost per case than the equivalent in patient treatment. Consequently, the number of surgical day cases has been increasing rapidly although considerable variation still exists between districts. A further advantage of day case surgery arises from its compatibility with the consumerism inherent in the health service reforms. The flexibility and speed of day case surgery permits a response to consumers' demands for a service that meets their needs rather than the hospitals. It offers a reduction in waiting times and the choice to avoid the arduous and often traumatic experience of being admitted to hospital. Bowling, in a short review of the research evidence, suggests that day case surgery generally meets with consistently high levels of patient satisfaction. The most welcome aspect of the recent health service reform is, perhaps, the emphasis on patients' rights and individual choice. Nurses, who are often the closest to the patients, have frequently drawn attention to the need for partnership, and an informed and participative relationship with the patient. While the Department of Health's genuine commitment to patient choice and participation may be little more than skin deep, in the case of day case surgery it appears that consumption and the improvements in efficiency desired by the government may be compatible with nurses' aspirations for greater patient choice.


[Integrated traditional and Western medicine treatment of severe viral myocarditis]. 33 patients divided into 2 groups for treatment. Group A (18 cases) early use of adrenal cortico-steroid treatment in combination with traditional Chinese medicine; group B (15 cases): early use of adrenal cortico-steroid treatment is combination with interferon, then compared the therapeutic effects of these 2 groups. The results showed that, in these 2 groups, the sinus-atrial block, atrial ventricular block, ventricular tachycardia, acute pump failure as well as the EKG changes were relieved and improved to the same degree. In group A, after therapy, the parameters of OKT3 (%) was elevated from 51 +/- 15.22 to 55.23 +/- 8.53; OKT4(%) was elevated from 30.06 +/- 11.47 to 47.32 +/- 10.87; ratio of OKT4/OKT8 was elevated from 1.11 to 1.53; all of their P values were < 0.01. NK cell activity was elevated from 8.36 +/- 3.75 to 13.08 +/- 5.77, P value < 0.05. In group B, the parameters of OKT3 (%) was elevated from 50.91 +/- 8.12 to 56.75 +/- 8.29; OKT4 (%) was elevated from 32. 55 +/- 6.78 to 45.13 +/- 10.85; ratio of OKT4/OKT8 was elevated from 1.00 +/- 0.21 to 1.37 +/- 0.16; P value < 0.05, < 0.05 and < 0.01 respectively. NK cell activity (%) was elevated from 8.20 +/- 3.08 to 14. 0.3 +/- 4.89, P value also < 0.01. Hence, not only in group A but also in group B, after therapy, the parameters of OKT3,4,8, OKT4/OKT8 and NK cell activity (%) were all improved. But there was no significant statistical difference between group A and B.(ABSTRACT TRUNCATED AT 250 WORDS)


Cytokeratin filaments of the liver of BALB/c mice as a sensitive marker of liver damage. Computer-aided characterization with the image analysing system "IBAS". The cytoskeleton forms a complex structural network which is of major importance for both structural integrity and physiologic functions of the cell. The aim of the present investigation was to investigate whether hepatotoxic effects of nitrosamine and colchicine can be detected through changes in the cytokeratin filament system which is an important component of the cytoskeleton. Groups of 10 male and 10 female newborn BALB/c mice were treated with either 25 micrograms dimethylnitrosamine (DMN) injected intraperitoneally on the day of birth; or with a daily subcutaneous injection of 0.12 micrograms/kg b.w. colchicine starting at ten weeks of age. A third group of mice served as untreated controls. All animals were held under standard laboratory conditions until necropsy at 16 weeks of age. The group injected with DMN received 0.05% phenobarbital with their drinking water after weaning. The histologic changes in the liver tissue were characterized by light microscopy. Changes of the cytokeratin filaments were visualized by the indirect immunofluorescent technique and quantified by using the image analysing system "IBAS". The cytokeratin filaments in the DMN group were markedly increased in amount and had a variety of morphological alterations. These effects could be measured quantitatively and did not indicate any sex-dependent behavior. The colchicine group did not display any structural changes in the cytokeratin filaments but sex-dependent changes in the amount of keratin material was revealed by image analysis.(ABSTRACT TRUNCATED AT 250 WORDS)


RNA kinetics at the wound margins.

RNA kinetics at the wound margins. The in vitro incorporation rate of 3H cytidine was determined by autoradiography in vital specimens taken from cut wounds in the outer ear of rats and evaluated by means of a Quantimet 920. Seven rats in each group were allowed to survive for the same periods of time between 0 and 60 hours. The following findings were observed: after 10 hours survival time the cytidine incorporation rate in the basal cells increased significantly, while remaining unchanged in all other skin layers. The relationship between the higher RNA synthesis rate and the increase in DNA synthesis--particularly in the basal cell layer--is discussed.


Cell membranes as barriers for antisense constructions. The results of studies on interaction of oligonucleotides and polynucleotides with cell membranes are reviewed. Oligonucleotides and polynucleotides bind to lipid membranes in the presence of divalent cations that may result in spontaneous encapsulation of nucleic acids and transfer of the formed vesicles to the other side of the membrane. Oligonucleotides can enter eukaryotic cells and interact with cellular RNA and DNA. On the surface of eukaryotic cells, there are proteins capable of binding to nucleic acids that may be involved in oligonucleotide uptake. Oligonucleotides bind to cellular CD4 receptors. Efficient delivery into cells can be achieved by conjugation of oligonucleotides to lipophilic groups or by encapsulation into membrane carriers.


Prospects of vaccination in autoimmune diseases. Over the past several years, intensive efforts have been directed to define the pathogenic mechanisms and explore the potential immunotherapy of autoimmune diseases. The advances of molecular genetics and cellular immunology have now permitted select experimental immunotherapy using synthetic peptides, T cell receptor idiotypes and monoclonal antibodies. There remain many obstacles for success. First, the T cell receptor usage of autoreactive T cells in human autoimmune diseases are more heterogeneous than animal models. Clearly, there may not be an oligoclonal derivation of T cells in humans with autoimmune diseases. We foresee that the following studies might contribute to further understand pathogenic mechanisms and design feasible immunotherapy: (1) detailed characterizations of self-antigen specific T cell clones; (2) definition of interactions between MHC molecules and T cell receptors at the molecular level; (3) identification and elution of relevant disease inducing self-peptides complexed with MHC molecules and (4) identification of relevant genes that predispose to autoimmunity.


Establishment of a sensitive radioimmunoassay for the detection of human IgE-binding factor (soluble CD23). A monoclonal antibody (mAb) specific to low-affinity receptor for IgE (FceRII/CD23) was established by the fusion of spleen cells of BALB/c mice immunized with the FceRII+ human B lymphoblastoid cell line (RPMI 8866) with mouse myeloma P3U1. Four mAbs, 10/3 (IgG1), 11/4 (IgG1), 12/2 (IgG2b) and 15/6 (IgM), almost completely inhibited the IgE binding to FceRII+ cells but not to FceRII- cells. More directly, they were demonstrated to react only with 43-kD component/FceRII of the cell lysate of RPMI 8866 cells by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. Since they have a different epitope specificity, a solid-phase radioimmunoassay (RIA) for the measurement of IgE-binding factor (IgE-BF) was established. It was found that the RIA with the use of 10/3 and 125I-labeled 11/4 or 12/2 gave good results in the detection of IgE-BF derived from B cells and monocytes as well as of T-cell-derived IgE-BF. More importantly, serum IgE-BF was also quantitatively measured by this RIA. Although increased serum levels of IgE-BF were observed in atopic patients, serum IgE-BF was decreased rather than increased in patients with very high serum IgE. This phenomenon may be explained by the decreased ability of the patients' B cells to spontaneously release IgE-BF in vitro.


Quantitative studies of immunofluorescent staining. VII. Quantitative reference standard slide for standardization of fluorescence microscopes. We evaluated a quantitative reference standard (QRS) slide with 10-microns beads with different concentrations of Coulter Electronics green dye No. 1. Microfluorospectrophotometric readings of the QRS slides provided quantitative comparisons between the sensitivity of the different fluorescence microscopes. The visual comparisons between beads with a range of intensities of fluorescence of Coulter Electronics green dye No. 1 indicated that the end points vary with different optical systems as do the end points in standard indirect immunofluorescent titrations. Fluorescent emission wavelength of QRS slide beads gave the same peak as fluorescein-labeled beads; both differed from the peak of orange beads of an 'Optical Standard' slide. Since shelf life studies show no changes in fluorescence intensity of beads over a period of 27 months or longer, QRS slide beads can afford a device for standardization of fluorescence microscopes used for immunofluorescent tests.


Mouse IL-3 induces histamine release from mouse peritoneal mast cells. In this study, we have attempted to determine whether mouse peritoneal mast cells released histamine in response to IL-3. Recombinant mouse (m)IL-3 induced histamine release from mouse peritoneal mast cells in a dose-dependent fashion. Histamine release did not occur in the absence of phosphatidyl serine (PS), and was dependent on PS concentrations. The release was 14.3 +/- 3.8 and 43.5 +/- 11.5% (mean +/- SEM, n = 5) at 1 nM IL-3 in the presence of 10 and 20 micrograms/ml of PS. Calcium was required for the response, and in the absence of calcium, significant histamine release was not observed. The kinetics were slower than those of anti-IgE-induced response. IL-3-induced histamine release reached a peak within 15 min, while that by anti-IgE reached 80% of the maximum in 3 min. Lower concentrations of IL-3, which failed to directly induce histamine release, did not enhance anti-IgE-induced histamine release. Other cytokines, including mIL-4, mIL-5, m-granulocyte-macrophage colony-stimulating factor, human (h)IL-1 alpha, hIL-1 beta and hIL-8, neither induced histamine release nor enhanced anti-IgE induced histamine release. IL-4 had no capacity to enhance IL-3-induced histamine release. These results suggest that locally produced IL-3 might modulate mast cell-related inflammation through histamine release from mast cells.


Factors affecting the growth and maintenance of human skin mast cells in cell culture. The isolation and kinetics of survival of human mast cells from newborn and adult skin is described. Recombinant human interleukins and conditioned medium from several human cell lines were tested for their ability to maintain mast cells in vitro. Growth medium supplemented with IL-2, IL-4 and conditioned medium from a mixed lymphocyte culture enhanced mast cell survival resulting in a 30-fold increase in survival (relative to that obtained with non-supplemented medium) at 7 days, and a 15-fold increase at 15 days. Cell survival for time periods longer than 21 days was not observed. Inclusion of cAMP, agents that elevate cAMP, insulin, and epidermal growth factor in supplemented growth medium prevented the enhanced survival by 40-70%. Incorporation of bromodeoxyuridine (BrdU) into mast cells in 3-day cultures demonstrated that 15% of the mast cell population was capable of proliferation. At 21 days, no incorporation of BrdU could be detected. After 3 days in culture mast cells released 16% of their histamine stores in response to A23187 and 10% in response to anti-human IgE. Electron microscopy of cultured cells at 3 days revealed cells with both intact and empty mast cell granules. These results demonstrate that human skin mast cells proliferate in response to cytokines and release histamine when stimulated with classical secretagogues. Since human skin mast cells retain these basic properties in vitro, they may be useful in further functional studies involving their proliferation and secretion.


Spontaneous in vitro generation of histamine releasing factor from mononuclear cells of patients with hydatidosis. We have shown that Echinococcus granulosus infection can induce an enhanced sensitivity of basophils to IgE-dependent stimuli. To determine whether a cytokine, histamine releasing factor, could account for it, we evaluated 35 patients with active hydatidosis, 5 patients with past E. granulosus infection, and 20 normal volunteers. The spontaneous production of a factor in vitro that provoked the release of histamine from basophils was observed by mononuclear cells from patients with active disease, but not by those from subjects with past infection or from normal individuals. The histamine releasing factor was found to activate basophils through surface-bound IgE. It is concluded that E. granulosus infection induces both generation of histamine releasing factor and production of IgE that can bind this cytokine.


Effect of capsaicin as a neuropeptide-releasing substance on sneezing reflex in a type I allergic animal model. To elucidate the effect of capsaicin-sensitive nerve fibers, which are known to contain substance P (SP) and other sensory neuropeptides, on the sneezing reflex, we have investigated the effect of capsaicin on this reflex provoked in guinea pigs passively sensitized with anaphylactic antibody followed by specific antigen challenge. It has already been established that histamine released from mast cells is a reliable inducer of the sneezing reflex in type I allergy. Our experimental results indicated that the frequency of sneezing provoked by antigen challenge as well as histamine application was significantly reduced by pretreatment with capsaicin in a dose-dependent fashion. SP is considered to be one of the main neurotransmitters in sensory nerves. When the amount of SP in animal nasal mucosa was measured 12 h after capsaicin treatment, a marked reduction was noted. However, the histamine content in the nasal mucosa was not changed by capsaicin treatment. These data suggest that neuropeptides, especially SP, which are released or depleted from sensory nerves by capsaicin treatment, probably play an important role as neurotransmitters of the stimulant histamine in the development of sneezing in type I allergy.


Response of mast cells against filarial antigens from experimentally infected Mastomys natelensis with Brugia malayi. The role of antigens of Brugia malayi adult worms in induction of histamine release from mast cells was studied. Both peritoneal and lung mast cells were passively sensitized using immune serum collected from Mastomys natelensis on different days after infection with B. malayi. A significant release of histamine both with crude worm and 60-kD antigens was shown. However, the role of the 43-kD antigen in histamine release was comparable to that of control. When the sera were heat inactivated, the histamine release was minimal, thus indicating the heat-labile nature of the antibodies. Furthermore, the responses of peritoneal and lung mast cells to filarial antigens were similar.


Intratracheal application of Sephadex in rats leads to massive pulmonary eosinophilia without bronchial hyperreactivity to acetylcholine. Fourteen Brown-Norway rats were pretreated with physiological saline (n = 7) or 500 micrograms Sephadex (n = 7) intratracheally. 24 h later, a bronchial provocation test was performed under pentobarbital anaesthesia using increasing doses of acetylcholine aerosol and the degree of bronchospasm was measured using a modified Konzett-Rössler method. Subsequently, leucocyte counts were determined in the bronchoalveolar lavage fluid (BALF), BALF cells were differentiated, and the chemiluminescence of the BALF leucocytes were measured. Finally, the lungs were removed and histologically examined. The cell count in the BALF was significantly (p less than 0.05) increased in the animals pretreated with Sephadex compared to those in the saline group (mean value +/- SEM: 0.38 +/- 0.07 vs. 0.15 +/- 0.02 x 10(6)/ml). This difference was also reflected in the chemiluminescence measurements (2.51 +/- 0.53 vs. 0.20 +/- 0.03 x 10(6) counts/0.5 ml). In the Sephadex-treated animals there was also a significant increase in the absolute number of neutrophil (0.040 +/- 0.010 vs. 0.011 +/- 0.002 x 10(6)/ml) and, in particular, eosinophil granulocytes (0.188 +/- 0.055 vs. 0.003 +/- 0.001 x 10(6)/ml) in the total leucocytes of the BALF. Lung histology showed massive perialveolar and peribronchial oedema and granulomatous infiltrates, primarily with eosinophils, after intratracheal application of Sephadex; these findings were not observed in the saline group. None of these changes in the rats pretreated with Sephadex manifested themselves in increased bronchial reactivity to acetylcholine aerosol. It is uncertain if the Sephadex-induced increase in the eosinophil count is accompanied by an activation of this cell population, which appears to be of importance for the occurrence of bronchial hyperreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)


[Active valvular infective endocarditis: problems of surgical treatment]. The authors analyze the results of surgical treatment of patients suffering from active endocarditis with impairment of the mitral, aortal and tricuspid valves and their combinations in 242 patients operated on during 1969 to 1989. The total hospital mortality is at the level of world statistics and constitutes 15.3% (37 patients died). The main cause of the hospital mortality was acute progressive heart failure (48.6%). The long-term results of the surgery were examined in 202 patients. By the 5th observation year the total survival was 73.2%, the stability of good results 83.6%. The defined grades of the activity of infectious process play an important role in the diagnosis and treatment of patients with active valvular infectious endocarditis.


[Evaluation of right ventricular myocardial excursions in patients with mitral valve stenosis without pulmonary hypertension]. As many as 23 patients with mitral stenosis were examined intraoperatively, by evaluating of intracardiac hemodynamics and right ventricle myocardial excursions. Before mitral stenosis was corrected, 56% of patients had demonstrated myocardial excursion disorders. After the correction the number of patients with myocardial dysfunction halved. The transition of patients belonging to the group with abnormal myocardial excursions to the group with normal excursions depends on the scope of surgical intervention and pharmacological support. After the correction of mitral stenosis cardiogenic and pulmonogenic complications may mostly be seen in the patients' group showing abnormal myocardial excursions. It is concluded that the symptom of abnormal right ventricle myocardial excursions is of prognostic significance with respect to the development of complications in the early postoperative period.


[Quantitative relationship between ischemic heart disease and parameters of energy metabolism]. The relationship between coronary heart disease, postischemic work recovery and tissue ATP levels as well as mitochondrial respiration rates were studied. Respiration of mitochondria was assessed without their isolation by using a novel method applying skinned fibers in physiological saline. The maximal mitochondrial respiration rates were unchanged during 35 min of normothermic ischemia in St. Thomas Hospital cardioplegic solution in the subsequent 30 min aerobic reperfusion period. A reversible increase in the basal respiration and a decrease in creatine-stimulated oxygen uptake were observed. Thus, the combined determination of mitochondrial respiration in situ in skinned cardiac fibers and tissue ATP may be a useful approach to studies of the pathogenesis of cardiac diseases.