T-lymphocyte control of HLA-DR blood monocyte differentiation into neo-fibroblasts. Further evidence of pluripotential secreting functions of HLA-DR monocytes, involving not only collagen but also uromodulin, amyloid-beta peptide, alpha-fetoprotein and carcinoembryonic antigen. Previous studies led us to demonstrate in pathological situations that the fibroblast, not the macrophage, was the terminal maturation step of the HLA-DR monocyte and that the entire process came under T-lymphocyte control. Fibrosis which developed under immunosuppressive treatment (cyclosporin) after organ transplantation is an illustration of these in vitro observations. The present in vitro study was undertaken in order to investigate whether or not this transformation process takes place under physiological conditions and if so, the nature of the T-lymphocyte control. We report that normal HLA-DR monocytes/macrophages are able to secrete type 1 collagen and to differentiate into neo-fibroblasts. However, contrarily to what happened in pathology, only a few neo-fibroblasts developed transiently. The addition of conditioned medium (CM) from activated T-lymphocytes greatly enhanced the transformation process. Counteracting this CM effect, cell-to-cell contact between neo-fibroblasts and T-cells resulted in the loss of fibroblastic shape. The 'end-result' macrophage engulfed numerous lymphocytes giving rise to a multinucleated cell. This giant cell no longer adhered to the slide and died. The question is raised as to whether the process observed in vitro is involved in vivo in tissue repair. We also report that HLA-DR monocytes and the neo-fibroblasts which derive from them are able to secrete, in addition to type 1 collagen, a variety of proteins such as uromodulin, amyloid-beta peptide, alpha-fetoprotein and carcinoembryonic antigen. In cystic fibrosis we previously reported a high level of uromodulin production by HLA-DR monocytes differentiating towards the fibroblastic phenotype. Pathologies characterized by excessive production of either alpha-feto-protein, carcinoembryonic antigen, beta-amyloid protein (Alzheimer's disease) should be investigated, taking into account the involvement of HLA-DR monocytes and their possible uncontrolled differentiation into neo-fibroblasts.


ConA induced changes in energy metabolism of rat thymocytes. The influence of ConA on the energy metabolism of quiescent rat thymocytes was investigated by measuring the effects of inhibitors of protein synthesis, proteolysis, RNA/DNA synthesis, Na+K(+)-ATPase, Ca(2+)-ATPase and mitochondrial ATP synthesis on respiration. Only about 50% of the coupled oxygen consumption of quiescent thymocytes could be assigned to specific processes using two different media. Under these conditions the oxygen is mainly used to drive mitochondrial proton leak and to provide ATP for protein synthesis and cation transport, whereas oxygen consumption to provide ATP for RNA/DNA synthesis and ATP-dependent proteolysis was not measurable. The mitogen ConA produced a persistent increase in oxygen consumption by about 30% within seconds. After stimulation more than 80% of respiration could be assigned to specific processes. The major oxygen consuming processes of ConA-stimulated thymocytes are mitochondrial proton leak, protein synthesis and Na+K(+)-ATPase with about 20% each of total oxygen consumption, while Ca(2+)-ATPase and RNA/DNA synthesis contribute about 10% each. Quiescent thymocytes resemble resting hepatocytes in that most of the oxygen consumption remains unexplained. In contrast, the pattern of energy metabolism in stimulated thymocytes is similar to that described for Ehrlich Ascites tumour cells and splenocytes, which may also be in an activated state. Most of the oxygen consumption is accounted for, so the unexplained process(es) in unstimulated cells shut(s) off on stimulation.


Rat bone marrow erythroid cell fractionation by counter current distribution in non-charge-sensitive two-phase systems. Counter-current distribution in non charge-sensitive aqueous poly(ethylene glycol)-dextran two phase systems allows the fractionation of rat bone marrow cells into two broad cell subpopulations with different distribution coefficients in a relatively short time. Morphological identification and enzymatic studies suggest that erythroid cells are mainly present in the subpopulation with the higher distribution coefficient. The distribution coefficient and, therefore, surface hydrophobicity of these cells, apparently increase in parallel with an increase in their degree of differentiation and maturation.


Lindane-induced modifications to membrane lipid structure: effect on membrane fluidity after subchronic treatment. Chronic lindane intoxication by injecting subcutaneously the toxicant, resulted in an altered lipid pattern in rat ventral prostate membranes. An increase of membrane fluidity was also observed using a fluorescence polarization technique. When in vitro experiments were carried out with both treated and untreated rats, an interesting lack of parallelism was found, which could indicate the development of a resistance to membrane disordering by lindane. The observed changes in cholesterol and phospholipid composition are also consistent with the hypothesis that lindane perturbs the lipid matrix of membranes, possibly inducing complex compensatory changes in the membrane lipid composition.


Kinetic properties and regulation of GABAA receptor channels. Single-channel recordings of GABAA receptor single-channel currents have been obtained from mouse spinal cord neurons in cell culture. Detailed kinetic analysis of single-channel main-conductance level currents has allowed development of a preliminary kinetic scheme which describes the gating of the GABAA receptor channel. The essential features of this kinetic scheme are presented in scheme 1 (see above). In this scheme, the GABAA receptor channel is envisioned to exist in multiple open and closed states. Properties can be broken into three main categories. First, the receptor can exist in a closed and nondesensitized set of states. In the kinetic model it is envisioned that there is an unbound (C13), a singly bound (C12), and two doubly bound (C11 and C10) closed states. The singly bound and doubly bound closed states are thought to open to three open states (O1, O2, O3). However, each of the open states opens to two distal closed states whose kinetic properties are similar for all three open states (C4-C9). Only one desensitized state (D0) has been incorporated into the model. While this characterization of desensitization is certainly incomplete, it is an initial step toward including the desensitization process which is clearly evident in whole-cell and single-channel recordings. This kinetic scheme should be considered only an initial working model. A number of features appear to be correct. First, all analyses of open time frequency histograms for GABA- and GABA agonist-induced single-channel openings have demonstrated the presence of at least three distinct open time constants. Furthermore, the concentration-dependent change in the relative frequency of occurrence of the three open states suggests that the open states occur from singly and doubly bound forms of the receptor. Second, the presence of two brief closed states adjacent to the open states appears fairly secure. However, it should be noted that the kinetic analysis primarily suggests that each open state opens to two brief adjacent closed states in a concentration-independent manner. While we have indicated that these two closed states are distal to the three open states, the actual assignment of the location of these states is unclear. Another interpretation of these data is that there is only a single distal closed state and that the proximal, extraburst closed states have very brief durations that are similar to each other. What appears clear, however, is that the open states can close to either brief closed state.(ABSTRACT TRUNCATED AT 400 WORDS)


Prebypass glucose-insulin-potassium infusion in elective nondiabetic coronary artery surgery patients. Perioperative GIK therapy has been advocated to ensure adequate energy substrate levels during cardiac surgery. However, hyperglycemia should be avoided because it may worsen neurologic outcome after cerebral ischemia. A prospective, randomized, clinical comparison was performed between two prebypass infusion regimens in 32 elective nondiabetic CABG patients. Sixteen patients (GIK group) received glucose, 0.6 g/kg/h, insulin, 0.12 U/kg/h, and KCl, 0.12 mmol/kg/h, from the induction of anesthesia to the start of CPB; while the remaining 16 patients (R group) received only Ringer's acetate. The pump prime was glucose free and a blood cardioplegia technique was used in both groups. No differences were found between the groups with regard to myocardial injury; the CK-MB enzyme fractions were elevated to a similar degree and the frequency of postoperative ECG changes were similar in both groups. Likewise, there were no differences in hemodynamic changes, need for inotropic support, arrhythmia frequency, or duration of ICU stay. The GIK patients had higher blood glucose (P < 0.05) and insulin levels (P < 0.01); blood glucose increased to 12.4 +/- 5.4 mmol/L (mean +/- SD) at cannulation, with a drop after starting bypass. Interindividual variation in GIK patients was great, with glucose values ranging between 20.1 mmol/L at cannulation to 2.0 mmol/L after starting CPB. A hyperglycemic response was seen in both groups during rewarming: 15.0 +/- 4.2 and 15.0 +/- 3.1 mmol/L in GIK and R patients, respectively. It is concluded that prebypass GIK infusion had no clinical benefits for elective CABG patients as compared to Ringer's acetate.(ABSTRACT TRUNCATED AT 250 WORDS)


Neuroanatomical labeling with biocytin: a review. Recent studies have shown that biocytin may have multiple applications in neuroanatomical studies. Biocytin may be injected into the brain by iontophoresis or by pressure injection methods, and localized in tissue sections using avidin-conjugated labels. It is taken up by neurons and rapidly transported down axons in an anterograde fashion. Axons are completely labeled in a Golgi-like manner and can be examined at both light and electron microscopic levels. Biocytin can also be used in retrograde tract tracing experiments, although in some cases it appears that fibers must be damaged to produce such labeling. Retrogradely labeled cells may be completely labeled, resembling neurons stained with the Golgi technique. Individual neurons can also be labeled in a Golgi-like manner by uptake of biocytin from the extracellular space. Thus, it appears that biocytin is an especially versatile marker for neuroanatomical investigations.


Colocalization of nitric oxide synthase and NADPH-diaphorase in rat adrenal gland. The distribution and colocalization of nitric oxide synthase (NOS) and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase was studied in the neuronal elements of the adrenal gland of the rat. Ganglion cells and many nerve fibres in the gland showed both NOS-immunoreactivity and NADPH-diaphorase staining. The adrenal cortical cells showed NADPH-diaphorase staining but were not immunoreactive for NOS. Positive labelling for both NADPH-diaphorase and NOS was found in bundles and in single fibres with varicosities, preferentially located around the noradrenaline (NA)-storing cells. Adrenaline (A)-storing cells and ganglion cells in the medulla, along with the cortical cells and blood vessels in the zona glomerulosa, received relatively fewer positive fibres.


Noradrenergic excitatory inputs to median preoptic neurones in rats. Extracellular single-unit activity was recorded from 21 median preoptic nucleus (MnPO) neurones, antidromically identified as projecting to the hypothalamic paraventricular nucleus (PVN), in urethane-anaesthetized male rats. Of these identified MnPO neurones, 14 displayed an excitatory response in neuronal excitability following electrical stimulation (5 Hz, 600 microA) of the A1 noradrenergic region of the ventrolateral medulla, while the remaining neurones were unresponsive. The excitatory response of MnPO neurones was blocked by microiontophoretically applied phentolamine, an alpha-adrenoceptor antagonist, but not by timolol, a beta-adrenoceptor antagonist. These results suggest that the A1 region acts to enhance the activity of MnPO neurones projecting to the PVN via an alpha-adrenoceptor mechanism.


P0 protein in normal, trembler heterozygous/homozygous mice during active PNS myelination. The quantification of P0 protein has been currently used to measure the myelination of the peripheral nervous system (PNS). Using immunological techniques, we show that, as early as postnatal day 8, the P0 content of mice homozygous and heterozygous for the Trembler mutation, represent respectively one tenth and half of the normal values. Thus, although the Trembler mutation is dominant, the myelination defect observed in heterozygous Trembler mice is more pronounced in the homozygous Trembler mice. This gene dosage effect should be taken into account when a molecular model of the Trembler mutation will be proposed.


Decrease in rat brain calcitonin binding sites and adenylyl-cyclase activity during ageing. In order to investigate the effects of ageing on the cerebral receptors for calcitonin (CT), we used an in vitro autoradiographic method to study the distribution of the binding sites for eel CT (eCT) in young and old rat brain. The inhibitory action of eCT on adenylyl-cyclase (AC) activity upon isolated brain cell membranes was also evaluated. The results show area-specific reduction of binding particularly in the hypothalamus and pons medulla of the old rat. The inhibitory action of eCT on AC activity was significantly reduced in the same areas, whereas in the striatum and mesencephalon no changes were observed. The parallel decrease of binding of eCT and of the inhibitory action of eCT on AC in ageing may represent a functional decline of neuronal activities during ageing.


Inhibition of nitric oxide synthase protects against hypoxic neuronal injury. We investigated the action of nitric oxide in hypoxic neuronal injury, using the hippocampal slice. Inhibition of nitric oxide synthase with the competitive inhibitor, NG-monomethyl-L-arginine, provided significant protection against hypoxia for population spike, EPSP and fiber volley responses, with an EC50 of 30 microM for protection of antidromic population spike amplitude. Confirming a stereo-specific site of action, this protection was reversed by the addition of L-arginine, but not D-arginine. These results indicate that nitric oxide generation may mediate acute CA1 neuronal injury during hypoxia, and that inhibition of nitric oxide synthase may be a useful neuroprotective strategy.


Nitric oxide synthase in VIP-containing vasodilator nerve fibres in the guinea-pig. We investigated the possibility of nitric oxide (NO), a powerful vasodilator agent, being synthesized by perivascular nerve fibres. Immunoreactivity and catalytic activity of the NO synthesizing enzyme, NO synthase (NOS), were demonstrated in perivascular nerve fibres of blood vessels receiving autonomic vasodilator innervation, but not of those innervated exclusively by vasoconstrictor nerve fibres. Double-labelling techniques allowed identification of NOS-containing nerve fibres as belonging to the vasoactive intestinal peptide (VIP)/acetylcholine-containing class whereas noradrenergic and substance P-containing perivascular fibres were devoid of NOS. We suggest that, in addition to its endothelial source, NO is a neuronal co-mediator of VIP/cholinergic vasodilation.


The AMPA antagonist, NBQX, protects against ischemia-induced loss of cerebellar Purkinje cells. We examined the effect of an AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole) antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline (NBQX), on rat cerebellar Purkinje cell loss and hippocampal pyramidal CA1 cell loss, after 10 minutes of global cerebral ischemia. NBQX was given intraperitoneally in a dose of 30 mg kg-1 at the end of ischemia, and 10 and 25 minutes later. Rats subjected to ischemia without post-ischemic administration of NBQX served as controls. Four days after ischemia the cerebellar Purkinje cell density was higher and the density of acidophilic (dead) Purkinje cells lower in the NBQX treated animals compared with the control animals (p = 0.01 and p less than 0.005 respectively). There was partial to total loss of pyramidal neurons in the CA1 region of the dorsal hippocampus in control animals, but no CA1 pyramidal neuron loss in the NBQX treated animals (p = 0.001).


A nitric oxide (NO) synthase inhibitor accelerates amygdala kindling. In response to NMDA receptor activation, hippocampal, striatal and cerebellar neurons synthesize nitric oxide (NO), which in turn elevates cGMP levels via guanylate cyclase. NO is increasingly being considered as a transsynaptic retrograde messenger, involved in neuronal plasticity. The effect of an inhibitor of NO synthase, L-NG-nitroarginine (NOArg), was studied on amygdala kindling and on kindled seizures in rats. NOArg increased kindling rate, particularly in its initial period, but did not modify seizure severity in previously kindled rats, although we have no definitive explanation for this effect. However, an enhanced post-synaptic excitability could be attributed to the blockade of the negative feed-back exerted by NO on the NMDA receptor.


Transport of RNA between nucleus and cytoplasm. The transport out of the nucleus of RNAs transcribed by RNA polymerase II (U snRNAs and mRNAs) has not been extensively studied. Basic questions, such as whether export requires association of the RNA with specific proteins, are not yet definitively answered. Nevertheless, recent progress in this area has been significant. Sequence or structural features of RNAs which are either required for export or which result in nuclear retention have been defined. These are presumed to interact with components of the transport machinery or with anchoring nuclear factors respectively. The unexplained dependence of the transport of certain mRNAs on either intervening sequences or for transcription from specific promoters suggests that RNAs may have to pass through different intranuclear compartments before export. Studies of the import of RNAs from the cytoplasm has revealed that different classes of nuclear localization signals exist, and protein components of viral RNPs that appear to determine the direction in which they move through the nuclear envelope have been identified.


RNA nucleocytoplasmic transport.

RNA nucleocytoplasmic transport. The transport of RNAs from the nucleus to the cytoplasm is an obligatory step in gene expression and may also be a target for regulation. The cellular machinery has the capacity to export a myriad of RNA transcripts, which differ significantly in sequence and structure. Recent work is providing the first glimpses into how RNA export occurs.


Differential effects of IL-3, GM-CSF and G-CSF in an adult with congenital neutropenia. Using a methylcellulose culture system, we studied the effects of recombinant human interleukin-3 (IL-3), recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), and recombinant human granulocyte colony-stimulating factor (G-CSF) on the growth of myeloid progenitor cells (CFU-C) from an adult patient with congenital neutropenia. The moderate clinical course and the maturation arrest at blast-promyelocyte stage in the marrow differentiated this patient from those described as having Kostmann-type congenital neutropenia. CFU-C growth in bone marrow cells from the patient responded to IL-3 normally in a dose-dependent manner. GM-CSF stimulated only macrophage colony formation in a dose-dependent manner comparable to that in normal subjects. Neither GM-CSF nor G-CSF stimulated any significant granulocyte colony formation. This evidence suggests that the hematopoietic progenitor cells in this patient had the potential for developing CFU-C with IL-3, and that the neutropenia in this patient could be a result of an intrinsic defect in myelopoiesis along a granulocytic pathway responsive to GM-CSF or G-CSF.


Trophic effect and modulation of ACTH-dependent stimulation of steroidogenesis by prolactin in guinea pig adrenal cortex. The effect of PRL in intact guinea pig on the incorporation of specific labelled precursors into DNA, RNA, proteins and corticosteroids in adrenocortical tissue of intact guinea pig at different levels of activation of the gland were studied. PRL (2 IU/100 g daily) did not cause any significant changes until 3 days of treatment. PRL administered alone for 3 days caused significant elevation of 3H-thymidine incorporation into DNA in adrenocortical slices and no significant changes in labelling of RNA, protein and corticosteroids. In contrast, PRL administered together with ACTH (0.5 IU/100 g) for 3 days did not affect DNA synthesis but caused increase of 3H-cholesterol incorporation into cortisol and cortisone additionally to the ACTH action. Labelling of 17-deoxycorticosteroids in adrenocortical slices from animals of ACTH+PRL group either did not change significantly (aldosterone) or decreased (corticosterone).


[Effects of parathormone on 45Ca2+ accumulation in neurosecretory cells and contents of vasopressin in blood after administration of parathyroidin in parathyroid gland insufficiency]. The functional state of hypothalamic-hypophyseal-neurosecretory complex was studied in parathyroidin injection and hypofunction of the parathyroid glands. In these conditions vasopressin levels in blood of the rats increased. The incubation of the hypothalamus with parathormone and 45Ca2+, as well as the injection of parathyroidin increased 45Ca2+ transfer to the neurosecretory cells and vasopressin release in the blood. Various mechanisms of similar influence of the parathormone and hypofunction of the parathyroid glands on a vasopressin level in blood are discussed.


[Effects of pesticide fluometuron (cotoran) on template synthesis of RNA]. The experiments on the investigation of pesticide fluometuron (cotoran) influence on nuclease sensitivity and template activity of rat liver chromatin were carried out. Cotoran was found to bind specifically with non-histone proteins of chromatin. It was shown that this pesticide considerably decreases template activity of chromatin and its sensitivity to the action of nucleases. It suggests, that certain conformation changes occur in chromatin upon the action of cotoran.


[Effects of flavobion on nucleic acids in tissues of rats irradiated with gamma rays]. Flavobion /SPOFA/ is a hepatoprotective preparation containing an effective constituent--the flavonoids, namely, silybin, silydianin and silychristin, collectively referred to as silymarin. Silymarin can influence certain metabolic processes including RNA synthesis and stabilize cell membranes. Suspension of this preparation was given p. o. by tube in a dose of 70 mg/kg, one hour before whole-body irradiation with a 5.7 Gy dose of gamma radiation (60Co). Animals were subjected to partial hepatectomy/by 30 min. after irradiation/and examined on hour 30 after operation. The changes in concentration and total content of RNA and DNA in the liver (both intact and regenerating), spleen and bone marrow were followed. We found that Flavobion administration influenced the concentration and total content of RNA and DNA in the spleen and bone marrow. However, in a target organ, liver, changes in nucleic acids were less pronounced.


[Effects of exogenous poly(A)+ mRNA on antigenic properties and growth of Krebs-2 tumor cells]. The treatment of Krebs-2 ascitic tumor cells (TC) with total RNA from the liver of Wistar rats (2 mg/ml) altered their antigenicity. As a result, the growth of treated TC in contrast with untreated TC was inhibited when transplanted i. m. to mice preimmunized with rat liver homogenate. Investigations of poly(A+)mRNA, rRNA and tRNA isolated from the same tissue established that the alteration of antigenicity is due to mRNA (8-24 micrograms/ml). In the cytotoxicity assay with antisera against rat Wistar antigens, the expression of rat antigens in TC treated mRNA was observed in the next cell generations.


[Hypotrypsinemia as a possible factor in the activation of motor function of the duodenum in pancreatic atrophy in rats]. The effect of pancreas atrophy on myoelectrical activity of the duodenum and serum trypsin, trypsin inhibitor, amylase, lipase activity has been described. The activation of the duodenum motor activity has been established. The changes in the motor activity were connected with trypsin and trypsin inhibitor activity of serum. The motor activity changes were compensated by trypsin therapy.


[Light-optical and ultrastructural immunohistochemistry of antigen H-4, a common marker of epithelial cells]. Epitope localization reacting with mice monoclonal antibodies (Mabs) H 4 was investigated using the specimen of epithelium of skin, human uterine cervix as well as in the culture of epithelium cells of guinea-pig duct deferent. Mice monoclonal antibodies against antigen H 4 obtained by the hybridoma method after immunization of mice with rat colon epithelium cytoskeletal fractions were used. Mabs H 4 were shown to react with antigen of intermediate filaments of all studied normal epithelial, cancer cells as well as culture epithelial cells. Mabs H 4 are supposed to be used as a unique immunohistochemical marker of epithelium cells under normal and malignant growth conditions.


[Identification, purification and physico-chemical properties of neurospecific alpha 1-globulin]. The partially purified and concentrated 500-600-folds protein fraction has been obtained from human brain extract. This protein fraction was used for the immunization of rabbits. The corresponding anti-sera have the potency to detect the brain specific alpha 1-globulin, which is not identical to known cytoplasmatic brain specific protein. These antisera were used for the control of the antigen purification procedure which included ion-exchange, affinity and hydropho'ic chromatography, gel-filtration and isochromatofocusing. The antigen, purified to the homogeneity, has the electrophoretic mobility of the alpha 1-globulins, M(r) = 110-10 kD, and isoelectric point at pH 2.9-3.1.


[Effects of concanavalin A on different variants of adhesive reactions of human neutrophils]. The influence of Con A on human neutrophils adhesion in interactions with sepharose 4B, C3b-sephadex G-25, DEAE-sephadex A-25, polymethylmethacrylate was studied. In concentrations 25 mg/ml and more Con A completely inhibited IgG-dependent adhesion but had no influence on other types of adhesion reactions. The effect was due to Con A interaction with neutrophils and could not be reproduced by Con A pretreatment of IgG-sepharose. Results are considered to be a manifestation of lectin-dependent modulation of cell receptors functions.


The protective effect of colchicine on bleomycin-induced pulmonary fibrosis in rats. With the purpose of evaluating the therapeutic effect of colchicine on lung fibrosis in rats, bleomycin A5 (BLM-A5) was injected intratracheally to produce a lung fibrosis model. The animals were then treated with colchicine, 50 micrograms/d i.m. One month later, collagen fiber deposition scores (stained by Masson trichrome) were significantly lower in the treated group than in the untreated group (P less than 0.01). This result was proved further by assaying total lung hydroxyproline content (P less than 0.05). Histopathologic findings showed that the proliferation of fibroblasts dropped slightly in the treated group as compared with the untreated group. From this study, we conclude that colchicine has certain antifibrotic effects and may be used in the treatment of pulmonary fibrosis.


In vivo effect of interleukin-1 alpha on hematopoiesis: role of colony-stimulating factor receptor modulation. To determine the mechanism(s) by which interleukin-1 (IL-1) promotes granulopoiesis in vivo, we examined the effect of in vivo administration of IL-1 alpha on colony-stimulating factor (CSF) receptor expression on bone marrow cells (BMCs) and whether this directly correlated with progenitor cell responsiveness. Administration of IL-1 alpha to mice induced the upregulation of both granulocyte-macrophage-CSF (GM-CSF) and IL-3 receptors, which reached a maximum 24 hours after IL-1 alpha injection on unfractionated BMCs. This upregulation was more pronounced on the progenitor-enriched cell population (lineage-negative [Lin(-)]). The enhanced GM-CSF and IL-3 receptor expression directly correlated with enhanced IL-3- or GM-CSF-induced growth of colony-forming unit-culture (CFU-c) or CFU-mixture (CFU-Mix; colonies containing macrophages, granulocytes, and erythroid cells). In addition, the absolute number of high proliferative potential-colony-forming cells (HPP-CFC) was increased fivefold. In contrast, granulocyte-CSF (G-CSF)-specific binding on unfractionated BMCs was rapidly (4 hours) reduced after IL-1 alpha administration and returned to control levels by 24 hours. This reduction correlated with IL-1 alpha-induced margination of mature granulocytes (RBC-8C5hi cells), which express high levels of G-CSF receptors. IL-1 alpha treatment did not affect G-CSF receptor expression on Lin- cells. Pretreatment of mice with anti-type I IL-1 receptor antibody blocked the IL-1 alpha-induced upregulation of GM-CSF and IL-3 receptor expression on BMCs. Taken together, as one possible mechanism, IL-1 alpha in vivo may stimulate the expression of functional GM-CSF and IL-3 receptors on BMCs indirectly, and, in concert with the induction of circulating CSF levels, may account for the ability of IL-1 alpha to stimulate hematopoiesis in vivo.


Characterization of primitive hematopoietic cells in normal human peripheral blood. The total number of clonogenic cells present in 5-week-old long-term cultures (LTC) initiated by seeding normal human marrow cells on competent adherent cell feeder layers allows for the quantitation of a more primitive hematopoietic input precursor cell type referred to as an LTC-initiating cell (LTC-IC). Previous studies have suggested that LTC-IC also circulate because production of clonogenic cells continues for many weeks when cells from the light-density (< 1.077 g/mL), T-cell-depleted fraction of normal blood are maintained on irradiated, marrow-derived feeder layers in LTC medium. We now show that the number of clonogenic cells present in such reconstructed LTC after 5 weeks is linearly related to the input number of peripheral blood (PB) cells over a wide range of cell concentrations, thereby permitting the quantitation of circulating LTC-IC by limiting dilution analysis. Using this approach, we have found the concentration of LTC-IC in the circulation of normal adults to be 2.9 +/- 0.5/mL. This is approximately 75-fold lower than the concentration of circulating clonogenic cells (ie, burst-forming units-erythroid plus colony-forming units [CFU] granulocyte-macrophage plus CFU-granulocyte, erythroid, monocyte, megakaryocyte) and represents a frequency of LTC-IC relative to all nucleated cells that is approximately 100-fold lower than that measured in normal marrow aspirate samples. Characterization studies showed most circulating LTC-IC to be small (low forward light scatter and side scatter), CD34+, Rh-123dull, HLA-DR-, and 4-hydroperoxycyclophosphamide-resistant cells, with differentiative and proliferative potentialities indistinguishable from LTC-IC in normal marrow. Isolation of the light-density, T-cell-depleted, CD34+, and either HLA-DR(low) or Rh-123(dull) fraction of normal blood yielded a highly enriched population of cells that were 0.5% to 1% LTC-IC (approximately 1,500-fold enriched beyond the light-density, T-cell-depletion step), a purity comparable to the most enriched populations of human marrow LTC-IC reported to date. However, purification of PB LTC-IC on the basis of these properties did not allow them to be physically separated from a substantial proportion (> 30%) of the clonogenic cells in the same samples, in contrast to previous findings for LTC-IC and clonogenic cells in marrow. These studies show the presence in the blood of normal adults of a relatively small but readily detectable population of functionally defined, primitive hematopoietic cells that share properties with marrow LTC-IC, a cell type thought to have in vivo reconstituting potential.(ABSTRACT TRUNCATED AT 400 WORDS)


Phenotypic heterogeneity of primitive leukemic hematopoietic cells in patients with chronic myeloid leukemia. The peripheral blood of chronic myeloid leukemia (CML) patients with chronic-phase disease and elevated white blood cell (WBC) counts typically contains markedly increased numbers of a variety of neoplastic pluripotent and lineage-restricted hematopoietic progenitors. These include cells detected in standard colony assays as well as their more primitive precursors. The latter are referred to as long-term culture-initiating cells (LTC-IC) because of their ability to generate clonogenic cell progeny detectable after a minimum of 5 weeks incubation on competent fibroblast feeder layers. In this study, we have investigated a number of the properties of the LTC-IC and clonogenic cells present in the blood of such CML patients with high WBC counts. This included an analysis of the light scattering properties of these progenitors, as well as their expression of CD34 and HLA-DR, Rhodamine-123 staining, and in vitro sensitivity to 4-hydroperoxycyclophosphamide. In the case of LTC-IC, the production of different types of lineage-restricted and multipotent progeny was also analyzed. Most of the circulating LTC-IC and clonogenic cells in the CML patients studied (on average approximately 70% and approximately 90%, respectively) showed features of proliferating or activated cells. This is in marked contrast to the majority of progenitors in the blood of normal individuals and most of the LTC-IC in normal marrow, all of which exhibit a phenotype expected of quiescent cells. Interestingly, a significant proportion of the circulating clonogenic cells and LTC-IC in the CML samples studied (on average approximately 10% and approximately 30%, respectively) appeared to be phenotypically similar to normal circulating progenitors, although their absolute numbers were indicative of a neoplastic origin. Both phenotypes of circulating CML clonogenic cells and LTC-IC could be obtained at approximately 10% to 20% purity by differential multiparameter sorting. These findings suggest that expansion of the Philadelphia chromosome-positive clone at the level of the earliest types of hematopoietic cells results from the activation of mechanisms that enable some, but not all, signals that block the cycling of normal stem cells to be bypassed or overcome. In addition, they provide strategies for purifying these primitive leukemic cells that should facilitate further analysis of the mechanisms underlying their abnormal proliferative behavior.


Reversible conformational changes induced in glycoprotein IIb-IIIa by a potent and selective peptidomimetic inhibitor. Platelet glycoprotein (GP) IIb-IIIa inhibitors may become useful antithrombotic agents. Ro 43-5054 is a low molecular weight, noncyclic, peptidomimetic inhibitor that is three orders of magnitude more potent than RGDS in inhibiting fibrinogen binding to purified GPIIb-IIIa and in preventing platelet aggregation. Comparisons of RGDS and Ro 43-5054 in cell adhesion assays showed that, in contrast to RGDS, Ro 43-5054 was highly selective GPIIb-IIIa inhibitor. Effects of RGDV and Ro 43-5054 on the conformation and activation state of GPIIb-IIIa were also examined. RGDV and Ro 43-5054 induced conformational changes in purified inactive GPIIb-IIIa as determined by binding of the monoclonal antibody D3GP3 (D3). These conformational alterations were not reversed after inhibitor removal, as indicated by the continued exposure of the D3 epitope and a newly acquired ability to bind fibrinogen. Similarly, RGDV and Ro 43-5054 induced conformational changes in GPIIb-IIIa on the intact platelet. However, after removal of the inhibitors, exposure of the D3 epitope was fully reversed and the platelets did not aggregate in the absence of agonist. Thus, while RGD(X) peptides and Ro 43-5054 transformed purified inactive GPIIb-IIIa into an irreversibly activated conformer, the effects of these inhibitors were reversible on the intact platelet. This suggests that factors present in the platelet membrane or cytoplasm may regulate in part the ability of the complex to shift between active and inactive conformers.


Enhanced expression of the tie receptor tyrosine kinase in endothelial cells during neovascularization. We have recently cloned a novel human receptor tyrosine kinase, tie, from human leukemia cells showing megakaryoblastoid differentiation. We report here that the 4.4-kb tie messenger RNA (mRNA) is present in all human fetal and mouse embryonic tissues. By in situ hybridization, the tie mRNA was localized to the endothelia of blood vessels and endocardium of 9.5- to 18.5-day mouse embryos. However, tie was not expressed by endothelial cells of developing hepatic sinusoids. Increased tie mRNA signal was seen in proliferating ovarial capillaries during hormone-induced superovulation. Only a weak tie signal was obtained from adult skin, except during wound healing, when the proliferating capillaries in the granulation tissue contained abundant tie RNA. These results suggest that tie may have a role in neovascularization.


p55 and p75 tumor necrosis factor receptors in patients with chronic lymphocytic leukemia. We studied the expression of the two tumor necrosis factor (TNF) receptors, p55 and p75, on B cells from patients with chronic lymphocytic leukemia (CLL), and the presence of soluble TNF receptors in serum. Expression of membrane-associated receptors was quantified by double labeling of peripheral blood mononuclear cells (PBMC) with monoclonal antibodies against CD19 and p55/p75 TNF receptors and flow cytometry. A high fraction of the CD19+ cells expressed the p55 receptor (44% +/- 34% [SD]) and p75 receptor (61% +/- 31%). In healthy controls, 0% to 1% of the CD19+ cells expressed the p55 receptor and 0% to 10% expressed the p75 receptor. Incubation of CD19+ cells with 10 ng/mL of TNF increased the incorporation of thymidine in 11 patients tested, and this was decreased to 65% (P < .05) by antibodies to the p55 receptor or the p75 receptor, and to 35% +/- 7% (P < .001) when both antibodies were combined. With an enzyme-linked immunoassay, we measured soluble TNF receptors in serum from CLL patients. The mean level of p55 receptors was increased to 12.9 +/- 8.9 ng/mL (P < .000001 v normal). The mean level of p75 receptors was increased to 13 +/- 24 ng/mL (P = .01 v normal). The membrane expression of the two receptors was positively correlated (r +/- 97, P < .01); however, there was no correlation between membrane expression and serum concentration of either receptor. Autologous serum containing high levels of soluble TNF receptors inhibited TNF-induced proliferation of CD19+ cells. In conclusion, we have demonstrated that neoplastic cells from patients with CLL have increased expression of p55 and p75 TNF receptors, and that both receptors mediate signal to proliferation. Furthermore, serum from CLL patients has elevated levels of soluble TNF receptors, which may counteract the proliferative effect of TNF.


Expression of the FMS/KIT-like gene FLT3 in human acute leukemias of the myeloid and lymphoid lineages. FLT3, a receptor belonging to the FMS/KIT family and localized to 13q12, could play a role in the biology of early hematopoietic progenitor cells. Because FMS and KIT are expressed in both normal progenitors and myeloid leukemias, we looked for FLT3 expression in fresh human leukemic cells using Northern blot analysis. High levels of FLT3 expression were detected in 92% of the cases of acute myeloid leukemia (AML) tested, ranging from the M1 to the M5 stages of differentiation assessed in the French-American-British classification. Immature (MO) AML cells, biphenotypic leukemias, and AML with megakaryocytic differentiation (M7 subtype) also expressed the FLT3 transcript. FLT3 was also expressed at high levels in acute lymphoid leukemias of T and B origins. Finally, it was not expressed in chronic myeloid leukemias in chronic phase, whereas it was expressed in most blast crisis samples. This pattern of expression of FLT3 contrasts with the expression of FMS and KIT restricted to myeloid leukemias, and suggests that the FLT3 product could play a role in the expansion of the leukemic blasts of both the myeloid and lymphoid lineages.


t(9;14)(p13;q32) denotes a subset of low-grade non-Hodgkin's lymphoma with plasmacytoid differentiation. In this series of 426 consecutively ascertained, karyotypically abnormal non-Hodgkin's lymphomas (NHLs) derived from 407 patients, a t(9;14)(p13;q32) was encountered in 7 cases; an additional case demonstrated t(9;14)(p1?3;q32). At the time of detection of t(9;14), four cases were small lymphocytic lymphomas with plasmacytoid features; in three of these the t(9;14) was the sole karyotypic abnormality. In two cases of large-cell NHL demonstrating t(9;14), retrospective review of prior lymph node biopsies showed the presence of a small lymphocytic lymphoma of the plasmacytoid subtype. The remaining two cases comprised a large-cell lymphoma of the brain and a follicular NHL. Thus, six of eight cases (75%) had an initial identical low-grade histology. Immunohistochemical analysis of six cases showed no reactivity with CD1, CD2, CD4, CD5, CD8, and CD10 and high reactivity with CD19 and CD20. All four lymphocytic lymphomas and one of the two large-cell NHLs showed cytoplasmic Ig, consistent with plasmacytoid differentiation. Of the eight cases in this series, six presented with or developed stage IV disease; all were characterized by a 6-month to 5-year clinical phase of indolent disease before treatment was instituted. All five patients with low-grade NHL at the time of cytogenetic analysis were alive with recurrent disease at 3-year median follow-up. The remaining three patients with large-cell diffuse histologies relapsed after intensive therapy and expired at a median of 3 years from diagnosis; two of these showed previous or metachronous small lymphocytic tumors. These results suggest a novel biologically distinct subset of NHL; a neoplasm of mature B lymphocytes with plasmacytoid differentiation, characterized by t(9;14); and an indolent presentation followed by gradual clinical progression of disease.


Epstein-Barr virus burden in Hodgkin's disease is related to latent membrane protein gene expression but not to active viral replication. The Epstein-Barr virus (EBV) has been increasingly detected in Hodgkin's disease (HD), but its role in pathogenesis remains uncertain. We analyzed 20 specimens of HD known to contain EBV DNA by a sensitive reverse transcriptase polymerase chain reaction (RT-PCR). The cases were assessed for the presence of RNA transcripts of the BNLF1 gene (coding for the viral latent membrane protein [LMP]) and the late replicative gene BLLF1 (coding for the principle envelope glycoprotein [gp220/350]). LMP RNA transcripts were found in 9 of 20 (45%) cases, mostly those containing many copies of viral DNA and of mixed cellularity (MC) histological subtype. Only one LMP RNA-positive case was also positive for RNA transcripts of the active replication gene BLLF1. Our results show that viral burden in HD is not primarily related to active viral replication, but is associated with LMP gene expression.


Thrombospondin mediates adherence of CD36+ sickle reticulocytes to endothelial cells. Initiation of vasocclusion in sickle disease pathophysiology may involve abnormal red blood cell (RBC) adhesivity to endothelium, a phenomenon influenced by both RBC and plasma factors. Using human umbilical vein endothelial cells and a gravity sedimentation adherence assay, we have examined thrombospondin (TSP) as a plasma factor in this adhesive event. The already-abnormal adherence of sickle RBCs in buffer/albumin is significantly augmented (P < .001) by the addition of TSP, with half-maximal effect at about 0.3 microgram/mL. This effect is abolished by antibodies to either TSP or glycoprotein (GP) IV (CD36), as well as peptides RGDS and CSVTCG. The even greater adherence (P < .005) of sickle RBCs in autologous platelet-rich plasma (without added TSP) is dramatically inhibited by alpha CD36 antibodies (OKM5 and alpha GPIV) and significantly diminished by alpha TSP, by peptides RGDS and CSVTCG, and by two antibodies to the vitronectin receptor (7E3 and LM609). Studies of density-separated subpopulations and of RBC adhesion to immobilized proteins, as well as analysis of sickle RBCs using fluorescence-activated cell sorting and single cell microfluorometry, show that TSP responsiveness is a feature of the immature sickle "stress" reticulocytes, which carry CD36 (and not GPIIbIIIa-like receptors) as the TSP-receptive moiety. The endothelial cell's participation in this phenomenon appears to be more complex, and the data are consistent with the notion that it involves TSP interaction with other plasma proteins and/or multiple receptor structures. Other potential adhesogenic proteins (plasma von Willebrand factor, vitronectin, fibrinogen, and fibronectin) neither exhibited an affinity for reticulocytes nor supported increased sickle RBC adherence when added to buffer/albumin in these assay systems. In aggregate, our results indicate that TSP may be the major promoter of RBC adhesivity in plasma, and they suggest that therapeutic benefit might derive from interference with sickle reticulocyte CD36, as achieved by antibodies and CSVTCG in these studies.


Cell lines produce factors that induce fetal hemoglobin in human BFUe-derived colonies. Established cell lines were screened for secretion of activities than can stimulate fetal hemoglobin (HbF) production in adult burst-forming unit-erythroid (BFUe) cultures. Conditioned media from four cell lines, a human teratocarcinoma, an osteosarcoma, a bladder cell carcinoma, and feline leukemia virus (FeLV) A-infected feline fibroblasts (FEF-A cells), consistently increased the relative production of fetal globin in BFUe-derived colonies. In vitro translation of RNA from these cells in Xenopus oocytes yielded products that increased the gamma to gamma+beta ratio in adult erythroid colonies. These results demonstrate that a variety of cell lines produce factors that stimulate the production of HbF in vitro. The genes of such factors could be isolated by expression cloning of cDNA from cell lines using the Xenopus oocyte system.


The generation of human natural killer cells from CD34+/DR- primitive progenitors in long-term bone marrow culture. We have adapted the stroma-dependent long-term bone marrow culture (LTBMC) system to study the development of human natural killer cells (NK) from the CD34+/HLA-DR- (CD34+/DR-) BM mononuclear cell (BMMNC) population. The CD34+/DR- population does not express any known antigens associated with myeloid or lymphoid lineage and has been shown by us and others to contain primitive hematopoietic progenitors capable of both self-renewal and differentiation to myeloid lineage. CD34+/DR- cells obtained from normal human BM by fluorescence-activated cell sorting were plated on allogeneic, irradiated BM stromal layers. After 5 weeks of culture in the presence of media containing recombinant interleukin-2 and human serum, 147- +/- 21-fold expansion of cells with the morphologic appearance of large granular lymphocytes was observed. Cultured cells (84.8% +/- 1.5%) expressed the characteristic CD56+/CD3- phenotype of NK. A proportion of CD56+/CD3- cells expressed other markers of lymphoid lineage that have been associated with mature NK, including CD2 (7.8% +/- 1.2%), CD7 (19.5% +/- 2.8), CD8 (3.1% +/- 1.0%), and CD16 (4.5% +/- 1.3%). The cultured cells did not express other antigens associated with T-lymphocyte (CD3, CD5, T-cell receptor [TCR] alpha/beta and TCR gamma/delta), B-lymphocyte (CD19), myeloid (MY8, CD33, and CD71), or monocytoid (CD14 and CD15) lineage and did not express the CD34 antigen associated with hematopoietic progenitors present on the starting population. This NK population was cytotoxic against both K562 (E:T 20:1; 79% +/- 1.9%) and Raji (E:T 20:1; 38% +/- 5.7%) target cell lines. The NK progenitor frequency in the CD34+/DR- cell population determined by limiting dilution of CD34+DR- on stromal layers followed by a functional chromium release assay against K562 targets was 1:169 +/- 50 CD34+/DR- cells. The data suggest that human LTBMC developed to study myeloid differentiation can be modified to study the origin and development of the NK and possibly other lymphoid lineages. Modified cultures show that cells with morphologic, phenotypic, and functional characteristics of NK can be derived from a population of BMMNC with the phenotype of primitive hematopoietic progenitors and without phenotypic evidence of lymphoid- or myeloid-lineage commitment. Further studies will address the cell of origin and the ontogeny of human NK and other lymphoid lineages.


Acute myeloid leukemia (AML) in Down's syndrome is highly responsive to chemotherapy: experience on Pediatric Oncology Group AML Study 8498. The treatment of acute myeloid leukemia (AML) in children with Down's syndrome (DS) has engendered considerable controversy. Because of the concerns for toxicity and increased rate of infections, treatment approaches varied considerably in the past with mixed results. However, experience on the recently completed Pediatric Oncology Group (POG) 8498 AML study suggests that DS children with AML constitute a distinct subgroup that responds well to therapy. Twelve of 285 children on POG 8498 (protocol for newly diagnosed AML) had DS. Children with DS and AML were predominantly male (9 of 12) and were quite younger at diagnosis (< 24 months in 10). The white blood cell count was less than 50 x 10(3)/microL in all 12 and French-American-British types M6 and M7 were frequent (5 of 12). An abnormal cytogenetic marker, in addition to constitutional trisomy 21, was present in 9 of 12 and involved chromosome 8 in 4 of 9. All cases studied (n = 5) were positive for myeloid cell surface markers (CD33, CD13, or CD11b) and, interestingly, were also positive for the CD7 antigen. Chemotherapy included daunorubicin, cytarabine (Ara-C), and 6-thioguanine for remission induction and featured high-dose Ara-C (3 g/m2 per dose) with or without L-asparaginase early in remission. Compared with children without DS, children with DS had a superior event-free survival (EFS at 4 years 100% v 28% +/- 6.2%; P = .003). The EFS remained superior even when compared with non-DS children less than 2 years of age with a white blood cell count less than 10 x 100,000/microL (100% v 48% +/- 17.3%; P = .01).


Stem cell factor directly stimulates the development of enriched granulocyte-macrophage colony-forming cells and promotes the effects of other colony-stimulating factors. The effects of the c-kit ligand (stem cell factor [SCF]) on the development of a highly enriched population of granulocyte-macrophage colony-forming cells (GM-CFC) were assessed. In soft agar assays, both in serum-containing and in serum-deprived cultures, SCF promoted the formation of colonies that contained predominantly granulocytic cells with some blast cells also present. The size of these colonies was far smaller than observed in the presence of interleukin-3 (IL-3). In serum-deprived conditions, no colonies were formed in the presence of macrophage colony-stimulating factor (M-CSF), but when M-CSF was combined with SCF, a marked change was noted in that large colonies were produced containing predominantly macrophages. When GM-CFC were cultured in the presence of IL-3 and SCF, colonies were formed that contained blast cells, granulocytes, and macrophages. A synergistic interaction was also seen using a combination of G-CSF plus SCF in either serum-containing or serum-deprived cultures. The addition of SCF to colony-forming assays markedly reduced the concentration of IL-3 or G-CSF required for optimal levels of colony formation. Furthermore, SCF was capable of promoting the survival of GM-CFC for several days, after which large colonies containing mature cells were formed upon the addition of a secondary growth factor such as G-CSF or IL-3. Thus, SCF can directly act on highly enriched committed progenitor cells in serum-deprived conditions to promote survival, proliferation, and development.


Induction of differentiation of human mast cells from bone marrow and peripheral blood mononuclear cells by recombinant human stem cell factor/kit-ligand in long-term culture. In the murine system, a number of cytokines (including interleukin-3 [IL-3], IL-4, and stem cell factor [SCF]) promote the growth of mast cells (MCs). However, so far little is known about factors controlling differentiation of human MCs. Recent data suggest that human MCs express receptors (R) for SCF. The aim of the present study was to investigate whether recombinant human (rh) SCF induces differentiation of human MCs from their precursor cells. For this purpose, bone marrow (BM; normal donors, n = 6) and peripheral blood (PB; normal donors, n = 11) mononuclear cells (MNC) were cultured in the presence of rhSCF, rhIL-3, rhIL-4, rhIL-9, recombinant human macrophage colony-stimulating factor (rhM-CSF), or control medium in long-term (8 weeks) suspension cultures. After 4 weeks, up to 5% of the MNC (BM and PB) cultured in the presence of rhSCF, but not in the presence of other cytokines, were found to exhibit the characteristics of MCs. These MCs expressed the YB5.B8-reactive domain of the SCF R as well as IgE R, as determined by combined toluidine blue/immunofluorescence staining. Myeloid antigens, likewise expressed on human basophils (ie, CD11b, CDw65, and Bsp-1), could not be detected on these cells. Furthermore, rhSCF, but not rhIL-3, rhIL-4, rhIL-9, or rhM-CSF, induced dose- and time-dependent increases in the formation of cellular tryptase (an MC-specific enzyme) (rhSCF [100 ng/mL], 1,308 +/- 679 ng/mL v control medium, 18 +/- 6 ng/mL tryptase on day 35 of PB cell cultures), as well as an increase in cellular histamine. After 6 to 8 weeks, when other mature hematopoietic cells decreased, MCs still could be detected in culture, with up to 40% of all cells being MCs. To test whether rhSCF also activates tissue MCs, we performed histamine release experiments (dispersed tissue; lung, n = 3; uterus, n = 3). SCF was found to enhance (by up to 3.4-fold) the capacity of the MCs to release histamine upon cross-linkage of IgE R with anti-IgE. Together, these observations suggest that rhSCF induces in vitro differentiation of human MCs from their BM and PB precursor cells in long-term culture and upregulates MC releasability.


Retinoic acid inhibits interleukin-6-induced macrophage differentiation and apoptosis in a murine hematopoietic cell line, Y6. We established a radiation-induced murine hematopoietic cell line, Y6, that could be induced to differentiate into macrophages by interleukin-6 (IL-6). IL-6 also induced growth inhibition and apoptosis in Y6 cells. Retinoic acid (RA) inhibited such effects of IL-6 on Y6 cells. The inhibitory effect of RA on the effects of IL-6 was not caused by the downregulation of the IL-6 receptor, because RA neither affected the expression of IL-6 receptor mRNA nor the expression of IL-6 receptor molecule on the cell surface. Furthermore, RA did not inhibit the IL-6-induced expression of junB mRNA, indicating that the expression of functionally active IL-6 receptor and the signal transduction pathway activating the junB gene are not inhibited by RA. IL-6-induced macrophage differentiation of Y6 cells was preceded by the downregulation of the c-myc gene, which was also prevented by RA. Because the inhibitory effect of RA on Y6 cells was reversible and seemed not to require de novo protein synthesis, the RA receptor by itself might be directly involved in the inhibition of the IL-6 signal transduction pathway. The results indicated that the IL-6 signal transduction pathways leading to the induction of macrophage differentiation and junB gene expression can be dissected by RA.


The antitumor activity of an anti-CD22 immunotoxin in SCID mice with disseminated Daudi lymphoma is enhanced by either an anti-CD19 antibody or an anti-CD19 immunotoxin. The antitumor activities of immunotoxins (ITs) constructed with deglycosylated ricin A chain (dgA) and either anti-CD19 (HD37) or anti-CD22 (RFB4) monoclonal antibodies were compared in SCID mice with disseminated human Daudi lymphoma (SCID/Daudi). As reported previously, after intravenous injection with Daudi cells, SCID mice develop disseminated lymphoma, which infiltrates the vertebral column and causes paralysis of the hind legs before death. The mean paralysis time (MPT) has been taken as an end point in this tumor model. We have previously reported that early treatment of SCID/Daudi mice with RFB4 coupled to dgA prolongs the MPT in a manner consistent with the killing of 4 logs of tumor cells. In the present study, we show that HD37-dgA kills 2 logs of tumor cells. The lower potency of the HD37-dgA is consistent with its lower IC50 on Daudi cells in vitro. We further show that the antitumor activity of a mixture of HD37-dgA and RFB4-dgA is significantly enhanced in SCID/Daudi mice and is consistent with the killing in excess of 5 logs of tumor cells. However, identical enhancement was observed when a mixture of the RFB4-dgA and the HD37 antibody was administered. In contrast, enhancement was not observed when mice were injected with a mixture of the RFB4 antibody and the HD37-dgA. The results indicate that a "cocktail" of HD37 antibody and RFB4-dgA immunotoxin can have significant antitumor activity in this mouse model of lymphoma and suggest that combinations of particular antibodies and ITs may have cooperative antitumor activity.


Potential of phenylalanine methylester as a bone marrow purging agent. Phenylalanine methylester (PME), a lysosomotropic compound can be used to deplete monocytes and myeloid cells from peripheral blood and bone marrow (BM). The potential of PME for purging leukemic cells from BM was investigated using U937 and HL-60 cell lines as models. Optimal purging conditions for U937 cells were determined using an MTT assay (3-4, 5-dimethylthiazol-2, 5-diphenyl tetrazolium biomide; Sigma). Elimination of U937 cells was time-, temperature-, and dose-dependent. PME activity was optimal at 37 degrees C for 45 minutes. Depletion of U937 was > 2.8 logs for 50 mmol/L PME. Compared with another purging agent, 100 micrograms/mL 4-hydroperoxycyclophosphamide had activity comparable to 40 mmol/L PME. HL-60 cells were even more sensitive to PME than U937 cells. To support observations made with the MTT assay, clonogenic assays were performed. PME, 50 mmol/L at 37 degrees C resulted in total depletion (> 5 logs) of U937 colonies. Progressive depletion of normal progenitor cells occurred when BM was incubated with PME at concentrations from 5 to 100 mmol/L. At 37 degrees C, 50 mmol/L PME reduced colony-forming units-granulocyte-macrophage and burst-forming units-erythroid (BFU-E) recovery by 98%. Recombinant human mast cell factor augmented BFU-E after PME treatment but had no effect on HL-60 or U937. These studies suggest that PME deserves further study as an agent for ex vivo marrow purging.


The insulin-like growth factor binding proteins (IGFBPs) in human breast cancer. Several lines of evidence suggest that IGFs are important regulators of breast cancer cell growth. Unlike other growth factors, the IGFs interact with specific binding proteins in all extracellular fluids. To date, six different IGFBPs have been cloned, although their exact physiological function is not understood. Experimental evidence accumulated over the past few years suggests that IGFBPs could function as modulators of IGF actions in a variety of systems. This includes breast cancer, since several groups have demonstrated the production of IGFBPs by human breast cancer cells. We have found that the pattern of expression of these binding proteins is heterogeneous and varies depending on the breast cancer cell ER status. We have also shown that estrogen is capable of regulating the expression of certain IGFBPs in MCF-7 cells. Specifically, estradiol enhanced the expression of IGFBP 2, 4, and 5, and decreased that of IGFBP-3 in this cell line. Since the IGFBPs can modulate IGF actions in different experimental systems, we and others have studied their potential role as inhibitors of IGF-induced mitogenesis in breast cancer cells. We have demonstrated that purified IGFBP-1 neutralized IGF-dependent growth of MCF-7 cells in a reversible manner. These results suggest that the IGFBPs might be used to inhibit IGF-mediated breast cancer proliferation.


The retinal cells generating the circadian small spikes in the Bulla optic nerve. A circadian rhythm in the frequency of compound action potentials (CAPs) in the optic nerve of the mollusc Bulla gouldiana is believed to be generated by the basal retinal neurons (BRNs) of the eye. Along with the CAPs, which are about 100 microV in amplitude, there are 10- to 40-microV impulses from an undetermined cell type in records from the optic nerve. These impulses, called "small spikes," are generated spontaneously in darkness and show a circadian rhythm in frequency that is about 12 hr out of phase with the CAP rhythm. To enable us to determine the origin of the small spikes, intracellular recordings were made from retinal cells while optic nerve activity was monitored. The cells were identified by their light responses and then injected with the fluorescent dye Lucifer Yellow CH or the tracer biocytin. It was found that the large photoreceptors of the distal retina generated graded depolarizations in response to light, and had axons in the optic nerve, but did not show impulses at the level of the photoreceptor layer. By contrast, the spiking retinal cells of the photoreceptor layer generated depolarizations and impulses in response to light. In addition, the spiking cells were found to be dye-coupled to a series of retinal cells approximately 7 microns in diameter, connected to a single axon in the optic nerve. Impulses from the spiking cells occurred spontaneously and correspond with the small spikes in the optic nerve. The BRNs appear to inhibit the retinal cells that generate the small spikes. Hyperpolarization of the BRNs, through constant-current injection, increased the number of small spikes in the optic nerve. Release from hyperpolarization led to a decrease in small spikes. This could explain how circadian changes in BRN membrane potential might modulate spontaneous firing of the spiking cells, resulting in the circadian rhythm in small-spike frequency.


The insulin-like growth factor family of ligands, receptors, and binding proteins. The insulin-like growth factors (IGFs) have important roles in normal cellular growth and development. The IGFs have also been implicated in regulation of tumor cell growth. Two ligands, IGF-I and IGF-II, have been identified that are expressed in both fetal and adult tissues. They interact with at least two specific cell surface receptors. The type I IGF receptor is homologous to the insulin receptor in structure and has tyrosine kinase activity. The type II receptor is identical to the mannose-6-phosphate receptor known to be important in the trafficking of lysosomal enzymes; its role in IGF signal transduction is not clear. Furthermore, a hybrid receptor composed of subunits from the insulin receptor and the type I IGF receptor have been identified. In addition to these receptors, six different IGF binding proteins have been identified, which modulate the activity of the IGFs in various ways. Thus, there is great potential for complex interactions between the family members that could ultimately regulate normal and neoplastic cell growth.


Synergism of chlorpyrifos against the German cockroach, Blattella germanica. Of fifteen compounds tested as synergists for chlorpyrifos against susceptible and resistant strains of Blattella germanica, the German cockroach, eleven were active against the resistant strain but only seven were synergistic against the susceptible strain. Overall, the most effective synergist was S,S,S-tributyl phosphorotrithioate (DEF) followed by phenyl saligenin cyclic phosphonate (PSCP) and two substituted N,N-dimethylcarbamates: SK-102 and SK-37. The most effective synergist for overcoming chlorpyrifos resistance was SK-37 which reduced the resistance ratio by 3.2-fold. Four other synergists which reduced chlorpyrifos resistance, in order of their effectiveness, were: SK-102 > PSCP > DEF > tridiphane. The potential usefulness of these synergists for German cockroach control is discussed.


Embryonic expression and functional analysis of a Xenopus activin receptor. We report the isolation and characterization of a Xenopus activin receptor (XAR1). The amino acid sequence of this protein shows extensive homology with a murine activin receptor. The mRNA is expressed maternally and is ubiquitously distributed during the early stages of embryogenesis. Consistent with a possible role in mesoderm induction and patterning, interference with the normal expression of the receptor by overexpression in the early embryo results in the formation of ectopic dorsal axial structures. During neurulation the XAR1 mRNA is expressed predominantly in the presumptive brain and spinal cord, suggesting an additional function for XAR1 in neurogenesis.


Effects of lithium chloride and retinoic acid on the expression of genes from the Xenopus laevis Hox 2 complex. We show that Xenopus laevis has a Hox 2 complex, and that this complex is strongly conserved with the mammalian one, both in structure and in the rules of spatial and temporal sequential expression of its genes during the early stages of development. Lithium chloride and retinoic acid, two reagents known to alter axial patterning of the body when applied to Xenopus embryos, produce, respectively, embryos with reduced posterior but exaggerated anterior structures and embryos with truncation of anterior structures. We report here on the effect of these reagents on the expression of Hox 2 genes in the Xenopus embryos. LiCl has a dramatic effect on Hox genes, suppressing the expression of these genes during gastrulation and early neurulation. However, later on expression of these genes reaches significant levels, suggesting the existence of two phases in the control of Hox gene expression. Retinoic acid increases the steady state level of transcripts from Hox genes with the greatest effect on Hox 2.7, the most anterior of the genes studied. This suggests that the results obtained in EC cells (Simeone et al., 1990, 1991) reflect what occurs in vivo. Neither LiCl nor RA change the sequential order of the onset of expression of the genes, showing that these reagents do not perturb the molecular mechanisms used to establish the sequential activation of the genes of the Hox complexes.


Coordinated induction of cell proliferation and syndecan expression in dental mesenchyme by epithelium: evidence for diffusible signals. Epithelial-mesenchymal interactions induce the expression of syndecan, a cell surface proteoglycan, and tenascin, an extracellular matrix glycoprotein in the mesenchymal component of many organ rudiments including the tooth. Experimental recombination cultures of early dental epithelium and mesenchyme were analysed by double immunostaining to compare the distribution of syndecan, tenascin, and proliferating cells (BrdU incorporation) in the induced dental mesenchyme. After 5-9 hr in culture expression of syndecan and tenascin as well as an increase in BrdU incorporation were evident in the mesenchymal cells adjacent to the epithelium and the positive area enlarged with time. Syndecan and tenascin were colocalized only partially in some explants. The expression of syndecan and tenascin in the recombinants correlates with their stage-dependent expression pattern during early tooth development in vivo (Vainio and Thesleff, 1992). The area of increased cell proliferation in the mesenchyme correlated closely with syndecan expression. In none of the explants was increased BrdU incorporation observed in syndecan negative areas. Epithelium induced also condensation of the mesenchymal cells. Induction and spread of the syndecan-positive zone in the dental mesenchyme required close and continuous contact with the epithelium. The mechanism by which the induction of syndecan expression spreads in the mesenchyme was studied in rat-mouse interspecies recombination cultures, using syndecan antibodies that recognize mouse but not rat syndecan. The rat mesenchyme and epithelium were first cultured in contact for 24 hr. Then the epithelium was removed and freshly dissected, "uninduced" mouse mesenchyme was placed in contact with different aspects of the rat mesenchyme. The rat mesenchymal cells that had located next to the epithelial tissue stimulated syndecan expression in adjacent mouse mesenchyme. The induction potential was gradually lost toward the periphery of the rat mesenchyme. Based on these findings we suggest that diffusible signal molecules mediate the spread of syndecan induction in the mesenchyme and that syndecan plays a role in the regulation of cell proliferation.


Immunocytochemical localization of cell adhesion molecules in the developing and mature olfactory system. The localization of Ca+(+)-independent cell adhesion molecules (CAMs) in the developing and mature olfactory epithelium and bulb is reviewed. The CAMs included in this article are the neural cell adhesion molecule (N-CAM), the 180 kD component of N-CAM (N-CAM 180), the embryonic form of N-CAM (E-N-CAM), L1 glycoproteins, J1 glycoproteins, and the adhesion molecule on glia (AMOG). In addition, the expression of the L2-HNK-1 carbohydrate epitope, shared by N-CAM, L1, J1 and myelin-associated glycoprotein (MAG) in the adult olfactory epithelium and bulb has also been documented. For the localization of these molecules at the light and electron microscopic levels, immunocytochemical techniques were used and are described in detail. During development and organogenesis, the olfactory system exhibits a pattern of CAM expression similar to the general pattern described for the developing nervous system. In the adult olfactory system, however, a significant retention of CAMs characteristic for developmental and morphogenetic processes, such as E-N-CAM, AMOG, as well as the high molecular weight components of J1 glycoproteins, can be observed. The retention of these embryonic features are most likely associated with the cell turnover and high plasticity of this system. Moreover, the predominance of N-CAM 180 with respect to other components of N-CAM, as well as the absence of the L2/HNK-1 carbohydrate epitope, are also particular traits of the primary olfactory system which could be associated with its exceptional properties.


Mapping of RNA polymerase on mammalian genes in cells and nuclei. The assembly of an RNA polymerase II initiation complex at a promoter is associated with the melting of the DNA template to allow the polymerase to read the DNA sequence and synthesize the corresponding RNA. Using the specific single-stranded modifying reagent KMnO4 and a new genomic sequencing technique, we have explored the melted regions of specific genes in genomic DNA of whole cells or of isolated nuclei. We have demonstrated for the first time in vivo the melting in the promoter proximal transcribed region that is associated with the presence of RNA polymerase II complexes. An interferon-inducible gene, ISG-54, exhibited KMnO4 sensitivity over approximately 300 nucleotides downstream of the RNA initiation site in interferon-treated cells when the gene was actively transcribed but not in untreated cells where the gene was not transcribed. The extent of KMnO4 modification was proportional to transcription levels. The KMnO4 sensitivity was retained when nuclei were isolated from induced cells but was lost if the engaged polymerases were further allowed to elongate the nascent RNA chains ("run-on"). The sensitivity to KMnO4 in isolated nuclei was retained if the run-on incubation was performed in the presence of alpha-amanitin, which blocks progress of engaged polymerases. A similar analysis identified an open sequence of only approximately 30 bases just downstream of the start site of the transthyretin (TTR) gene in nuclei isolated from mouse liver, a tissue where TTR is actively transcribed. This abrupt boundary of KMnO4 sensitivity, which was removed completely by allowing engaged polymerases to elongate RNA chains, suggests that most polymerases transcribing this gene paused at about position +20. The possibility of mapping at the nucleotide level the position of actively transcribing RNA polymerases in whole cells or isolated nuclei opens new prospects in the study of transcription initiation and elongation.


Escherichia coli ribonucleotide reductase expression is cell cycle regulated. The expression of the genes encoding ribonucleotide reductase in Escherichia coli was investigated in cultures synchronized by obtaining the smallest cells in a population after sucrose gradient centrifugation. Specific activity of ribonucleotide reductase and DNA initiation were found to increase in parallel, periodically as a function of the cell cycle. The expression of nrd was also determined in cells synchronized by periodic repeated doubling in a phosphate limited medium. Antibodies directed against the B2 subunit of ribonucleotide reductase were raised in a rabbit and purified. Immunoprecipitation of the B2 subunit and RNA-DNA dot blot hybridization assays were developed and employed to determine the expression of ribonucleotide reductase translational and transcriptional products during the cell cycle. Both of nrd-mRNA and B2 subunit expression were found to increase each generation at approximately the same time DNA synthesis was initiated and then to decrease back to the basal level shortly after DNA initiation. These results provided evidence of cell cycle dependent regulation of ribonucleotide reductase in E. coli. When the upstream regulatory region of nrd was fused to a promoterless lacZ gene on a single copy plasmid, lac-mRNA and beta-galactosidase were found to be synthesized in parallel to nrd expression from the chromosomal operon. When nrd sequences surrounding the promoter were removed from this construct, lac-mRNA and beta-galactosidase synthesis were no longer cell cycle regulated.


The effect of the immunophilin ligands rapamycin and FK506 on proliferation of mast cells and other hematopoietic cell lines. The immunosuppressive drugs FK506 and cyclosporin A have an identical spectrum of activities with respect to IgE receptor (Fc epsilon RI)-mediated exocytosis from mast cells and T cell receptor-mediated transcription of IL-2. These findings suggest a common step in receptor-mediated signal transduction leading to exocytosis and transcription and imply that immunosuppressive drugs target specific signal transduction pathways, rather than specific cell types. This hypothesis is supported by studies on the effect of rapamycin on IL-3 dependent proliferation of the rodent mast cell line PT18. Rapamycin inhibits proliferation of PT18 cells, achieving a plateau of 80% inhibition at 1 nM. This inhibition is prevented in a competitive manner by FK506, a structural analogue of rapamycin. Proliferation of rat basophilic leukemia cells and WEHI-3 cells was also inhibited, at doses comparable to those shown previously to inhibit IL-2-dependent proliferation of cytotoxic T lymphocyte line (CTLL) cells. In contrast, proliferation of A-431 cells, a epidermoid cell line, was not affected by rapamycin. DNA histograms indicate that complexes formed between the rapamycin-FK506-binding protein (FKBP) and rapamycin arrest-proliferating PT18 cells in the G0/G1-phase. It is concluded that FKBP-rapamycin complexes may inhibit proliferative signals emanating from IL-3 receptors, resulting in growth arrest of cytokine-dependent, hematopoietic cells.


Use of granulocyte colony-stimulating factor in the treatment of pancytopenia secondary to colchicine overdose. To report a case of pancytopenia following colchicine overdose and to discuss the use of granulocyte colony-stimulating factor (G-CSF) for treating this severe complication. A 19-year-old man developed pancytopenia four days after ingestion of approximately 50-60 0.6-mg colchicine tablets in a suicide attempt. His pancytopenia resolved after subcutaneous administration of one 300-micrograms dose of G-CSF. The patient recovered from his other multiorgan disturbances during his hospitalization and was discharged from the hospital with elevated liver enzyme concentrations. Colchicine overdose is rare, but can be fatal. The use of G-CSF appears to be beneficial in alleviating bone marrow depression in colchicine overdose situations.


Effects of dexamethasone on the production of insulin-like growth factor-I and insulin-like growth factor binding proteins in primary cultures of rat hepatocytes. The effects of dexamethasone (Dex) on insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-1 production were investigated in primary cultures of rat hepatocytes. Dex enhanced the secretion of IGFBP-1 as measured by ligand blot analysis but did not show any prominent effect on immunoreactive IGF-I secretion. EC50 of Dex on IGFBP-1 secretion was calculated to be 3 x 10(-8) M. The content of IGFBP-1 mRNA in the cells increased greatly in the presence of Dex but the IGF-I mRNA content did not change significantly under the same conditions. Insulin showed the opposite effect of Dex by decreasing the production of IGFBP-1 and the cellular content of IGFBP-1 mRNA. This effect of insulin was observed also with Dex in the medium. These results show that the gene expression of IGF-I and IGFBP-1 is differently regulated by glucocorticoids and insulin in primary cultures of rat hepatocytes. The results most possibly explain the in vivo effects of glucocorticoids and insulin in regulation of IGF-I and IGFBP-1 production by liver.


Murine beta 1,4-galactosyltransferase: round spermatid transcripts are characterized by an extended 5'-untranslated region. We have previously shown that the expression of the gene encoding murine beta 1,4-galactosyltransferase (beta 1,4-GT, UDP-galactose:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D galactosyltransferase, EC 2.4.1.38) is fundamentally different between somatic and male germ cells (Shaper et al., 1990b). In somatic cells, two transcripts of 3.9 kb and 4.1 kb are produced. In contrast, in spermatogonia only the 4.1 kb transcript is expressed. Maturation of spermatogonia to pachytene spermatocytes is accompanied by reduced expression of the 4.1 kb transcript to barely detectable levels. Continued differentiation to haploid round spermatids is coincident with renewed expression in which the 4.1 kb transcript is replaced by two truncated transcripts of 2.9 and 3.1 kb. In this study, we report the characterization of a full-length beta 1,4-GT cDNA clone from a murine round spermatid library that corresponds to the 2.9 kb transcript. This transcript encodes the same open reading frame as the 4.1 kb transcript, but utilizes alternative poly(A) signals embedded within the long 3'-untranslated region of the somatic transcript. Based on sequence analysis, together with primer extension and S1 nuclease protection experiments, both the 2.9 and the 3.1 kb round spermatid beta 1,4-GT transcripts are distinguished by the presence of an additional 5'-untranslated sequence of approximately 560 bp that is absent in premeiotic germ cells and somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Further characterization of the interaction between L-selectin and its endothelial ligands. L-Selectin is a lectin-like receptor on lymphocytes which mediates their attachment to high endothelial venules (HEV) within lymph nodes. Previous work has identified HEV-associated endothelial ligands for L-selectin as sialylated, fucosylated and sulphated glycoproteins of approximately 50 kDa and approximately 90 kDa (Sgp50 and Sgp90). The interaction of L-selectin with these ligands is carbohydrate directed, reflecting the involvement of its amino-terminal, calcium-type lectin domain. It has been reported, and we have confirmed, that anti-Ly22 blocks the adhesive function of L-selectin without reducing its binding to a carbohydrate- based ligand PPME (phosphomannan monoester core from Hansenula hostii). The epitope for this monoclonal antibody depends on the epidermal growth factor (EGF) domain of L-selectin. We demonstrate that anti-Ly22 inhibits the interaction of L-selectin with both of the Sgps, thus establishing that the interaction of L-selectin with HEV can be accounted for by the Sgps. Furthermore, the interaction of trypsin fragments of Sgp50 with L-selectin is inhibitable both by an antibody that maps to the lectin domain and by anti-Ly22. These findings raise the possibility that anti-Ly22 is affecting the function of the lectin domain of L-selectin rather than directly antagonizing the EGF domain. Toward a further characterization of L-selectin's carbohydrate specificity, we show that Sgp50 is partially inactivated by the linkage-specific Newcastle Disease virus sialidase (alpha 2,3 linkage). We additionally demonstrate that a sialyl Lewis x-related tetrasaccharide can interact with L-selectin, as has also been demonstrated for E-selectin and P-selectin.


The effect of low-density lipoprotein apheresis on plasma thrombomodulating factors. The authors have investigated the effects of double-membrane filtration with hollow fiber membranes made of different artificial materials and low-density lipoprotein (LDL)-adsorbent dextran sulfate cellulose (DS) columns on coagulation-related proteins and complement components to evaluate their hemocompatibility. All membrane filters showed similar sieving effects, which depended upon the molecular weights of the proteins. In the double-membrane filtration system, free protein S, protein C, antithrombin III, and alpha 1-antitrypsin were returned to the intracorporeal circulation, whereas alpha 2-macroglobulin and fibrinogen were almost completely removed from the circulation; C4b-binding protein (C4BP)-protein S complexes were also trapped by the second membrane, and in some instances were even blocked by the first membrane, if it was made of material that activated the classic pathway of the complement system. The DS column adsorbed C4BP-protein S complex, but free protein S was almost completely recovered in the eluate.


Effect of aprotinin on metastasis of Lewis lung tumor in mice. Kallikrein activity in human stomach tissue was measured and found to be about threefold higher in cancer tissue than in normal tissue. To clarify the physiological role of this tissue kallikrein, we investigated its effects on the spontaneous metastasis and tumor growth of Lewis tumors (3LL). Antiprotease, aprotinin, and gabexate mesilate (FOY) inhibited spontaneous metastasis but did not inhibit tumor growth, while tissue kallikrein and plasmin enhanced the spontaneous metastasis of 3LL. The results suggest that the inhibitory effects of aprotinin and FOY on metastasis are not only due to an inhibition of tumor cells released by tissue kallikrein, but that tissue kallikrein, a protease, also participates in metastasis. We thus conclude that aprotinin or FOY should be administered either before or immediately after operation to inhibit spontaneous metastasis.


Protease inhibitors in bronchoalveolar lavage fluid from neonates with special reference to secretory leukocyte protease inhibitor. An imbalance of proteolytic enzymes and protease inhibitors may contribute to the development of bronchopulmonary dysplasia. We studied secretory leukocyte protease inhibitor (not previously addressed), and alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 2-macroglobulin and elastase. Albumin was used as an internal reference. Infants with pneumonia had higher concentrations of secretory leukocyte protease inhibitor (p = 0.02) and elastase (p = 0.04) in bronchoalveolar lavage fluid than those with respiratory distress syndrome; those who also developed bronchopulmonary dysplasia had intermediate values. A decreased concentration of alpha 1-antitrypsin was found in the second and third postnatal weeks (p = 0.002). Further detailed studies of the balance between proteases and protease inhibitors and of the importance of pulmonary infections in the pathogenesis of bronchopulmonary dysplasia are suggested. Secretory leukocyte protease inhibitor is important both as an elastase inhibitor of bronchial mucus and as a marker of infection in the bronchi.


Identification of phosphotyrosine residues in peptides by high performance liquid chromatography on-line derivative spectroscopy. A method has been developed for the detection of phosphotyrosine residues in peptides based on reversed phase-high performance liquid chromatography (RP-HPLC) on-line spectral analysis. It has been found that tyrosine-containing peptides show a hypsochromic shift of the aromatic absorbance maximum when the tyrosine is phosphorylated. Subsequent second-order derivative spectra likewise reveal a hypsochromic shift of the corresponding minima of the phosphotyrosine residues compared to the unmodified tyrosine. This method allows mapping of tyrosine phosphorylation sites in proteins after cleavage into smaller peptides and separation and on-line spectral analysis of the latter by RP-HPLC. It furthermore provides a useful way for the characterization of synthetic phosphotyrosine-containing peptides.


Neurodevelopmental outcome in very-low-birth-weight infants with or without periventricular haemorrhage and/or leucomalacia. The aim of the study was to verify the predictive value of ultrasound performed in the neonatal period for short-term neurodevelopmental prognosis in 122 preterm very-low-birth-weight infants followed-up at 36 months. Neuromotor development was favourable in 53 (87%) subjects with normal ultrasound findings and in 21 (81%) subjects presenting uncomplicated haemorrhage. However, sensory and/or cognitive sequelae developed in 13% and 19% of the two groups, respectively. Outcome was unfavourable in 14 (50%) of 28 patients with ultrasound findings of complicated cerebral haemorrhages and in 5 (71%) of those (7) with ultrasound findings of parenchymal lesions without haemorrhage. Neonatal ultrasound examination seems to be fundamental in predicting neuromotor, but not cognitive, outcome in very-low-birth-weight infants.


Growth and motor performance in preterm children at 8 years of age. At 8 years of age 41 preterm and 24 term children were followed up in a long-term prospective study of 46 unselected infants born before 35 completed weeks of gestation and of 26 term infants. The two groups were comparable in physical growth and there was no significant difference between them in motor performance, as evaluated with the Test of Motor Impairment. Only minor motor problems were found (in 22% of the preterm and 17% of the term group). In the preterm group, motor impairment was correlated to birthweight. The preterm children had less developed postural control and manifested more compensatory movements during the balance tasks than the term children.


Somatostatin-gene expression in the postmortem adult and fetal human brain. We have examined the utility of in situ hybridization for detecting pre-prosomatostatin mRNA in postmortem human brain. In preliminary studies, Northern blot analysis using a rat model, which simulates the normal pattern of human post-mortem brain cooling, revealed retention of significant amounts of hybridizable somatostatin mRNA relative to control levels between 12 and 24 hours after death. mRNA extracted from postmortem fetal human brain specimens showed hybridization to cRNA probes directed against pre-prosomatostatin mRNA. We thus undertook in situ hybridization studies. Antisense RNA probes were hybridized to neurons that expressed pre-prosomatostatin in 10-microns sections of adult and fetal human brain. The distribution of pre-prosomatostatin mRNA-containing neurons was similar to that observed for somatostatin-like immunoreactivity; however, the in situ hybridization technique was a more sensitive marker of neuronal perikarya. Our results indicate that hybridization to pre-prosomatostatin mRNA is a useful method for localizing these peptidergic neurons in postmortem human brain tissue.


Toleration of amino acid substitutions within hepatitis B virus envelope protein epitopes established by peptide replacement set analysis. II. Region S(122-136). The envelope of the hepatitis B virus (HBV) consists of three related proteins, designated S-, M- and L-protein, all of which share a common 226-amino acid residue sequence, corresponding to the S-protein that is sufficient for eliciting protective immunity against HBV. HBV variants, resulting from point mutations leading to replacements of amino acids within the S(122-160) segment of S-protein, have been recently recognized. In order to assure the continued success of vaccination against HBV and the adequacy of diagnostic tests for HBV envelope antigens and antibodies, it is necessary to understand the impact of amino acid replacements on the immunological recognition of S-protein at both the B- and T-cell levels. Immunologically tolerated and forbidden amino acid replacements within the S(139-147) segment of S-protein have already been discerned. The impact of amino acid substitutions within the S(122-136) segment on the immunological recognition of S-protein is analyzed in this report. Such replacements do not appreciably affect the binding of rabbit and goat anti-S antibodies to replacement set peptides, while decreased murine antibody binding was observed with some peptides having substitutions at residues 122, 123 and 133. On the other hand, amino acid substitutions within the (126-136) region, except those distinguishing serological subtypes of HBV from each other, abrogated murine T-cell proliferative responses to the peptides, while substitutions at residues 122, 123 and 125 had a lesser effect. Some of the peptides with amino acid substitutions peculiar to the variants had diminished stimulatory activity for T-cells from individuals vaccinated against hepatitis B. Amino acid substitutions in both the S(139-147) and S(122-136) segments of S-protein may potentially result in variant viruses escaping immunological surveillance based on current hepatitis B vaccines.


Adjuvant cytostatic therapy of breast cancer as an important factor in the postponing of a relapse and longer survival period. This prospective clinical study shows the results of the adjuvant cytostatic therapy (ACT) in breast cancer applied to patients in the premenopausal age. Cyclophosphamide, methotrexate, 5-fluorouracil (CMF) group (70 patients): after operative and radiotherapeutic treatment the ACT is applied over the period of six months (six cycles). Control group (71 patients): only operative and radiotherapeutic treatment. Protocol of the ACT: cyclophosphamide, methotrexate, 5-fluorouracil (CMF) over 5 days with a 4-week break. Total 6 cycles. Control period: 10 years. Stratification of patients was made on the basis of the following risk factors: size of the tumour, number of positive lymph nodes of ipsilateral axilla, grade of the differentiation of the tumour, hormonal dependence of the tumour. Statistical method of analysis: actuary calculation, the Hi square test. The results show that the application of the ACT is statistically significant (P < 0.05) in regard to the disease-free interval. However, concerning the survival, the usefulness of its application is present but not statistically significant on the significance level of 5%. The usefulness of the ACT application as regards high risk factors (T3, T4 > or = 4 lymph nodes, grade of differentiation II, III, ER-PR-) is statistically significant (P < 0.05) both in regard to the DFI and survival. Regarding low risk factors the ACT application adversely influenced the results in the control group. This is probably the result of the ACT toxicity. The patients have a favourable prognosis in this subgroup in regard to the staging and biological nature of the tumour. The ACT in the premenopausal age of patients with high risk factors gives a significantly better results concerning the procrastination of relapse and the length of the survival period.


Photocoagulation of diabetic maculopathy. A comparative analysis of the efficiency of the two current techniques of the focal photocoagulation: the focal macular perifoveolar and the focal extramacular technique in the treatment of diabetic maculopathy was performed. The study included 92 insulin non-dependent diabetics with nonproliferative diabetic retinopathy, divided into two groups. The assessment of the results has been carried out on the basis of the changes in the visual acuity 12 months after the argon laser treatment. There has been a statistically significant difference in the effects of the focal macular perifoveolar treatment and the focal extramacular treatment on the visual acuity. It has been improved for two or more lines on the Snellen chart, to which difference has contributed the significantly lesser worsening of the visual acuity as a result of the focal macular perifoveolar treatment (p < 0.05). The mean visual acuity following the focal extramacular treatment has shown a statistically non-significant decrease (p < 0.05), practically with no change at all, but the mean visual acuity following the focal macular perifoveolar treatment has shown a statistically significant improvement (p < 0.01). It has been established that the focal macular perifoveolar treatment shows better results than the focal extramacular treatment in the treatment of diabetic maculopathy. This has been established by checking the visual acuity a year after the treatment had been completed. This leads to the conclusion that this treatment is preferable in everyday work.


The possible role of skin surface lipid in rosacea with epitheloid granulomas. The examination was carried out in 50 rosacea patients (30 females and 20 males) in order to establish the connexion between skin surface lipids, infestation with Demodex folliculorum and previous local corticosteroid treatment with the appearance of epitheloid granulomas. Our analysis showed three types of histological conditions: 1) chronic dermatitis of the rosacea type was noted in 15 patients (30%); 2) granulomas composed of epitheloid cells was found in 16 patients (32%). In three female patients of this group presence of caseous necrosis associated with epitheloid granuloma was noted, too; 3) prevalence of perifollicular absceses was found in 19 patients (38%). Demodex folliculorum was detected in 43 rosacea patients (86%), considerably more then in the control group. The normal value of the skin surface lipids was found in 9 female rosacea patients (30%) and in 9 female controls (43%), in 8 male rosacea patients (40%) and in 5 males controls (33%). Lower amounts of skin surface lipids were found in 19 female rosacea patients (63%) and in 9 female controls (43%), in 4 male rosacea patients (20%) and in 1 male control (7%). Higher amounts of skin surface lipids were found in 2 female rosacea patients (7%) and in 3 female controls (14%), in 8 male rosacea patients (40%) and in 9 male controls (60%). Lower quantities of lipids determined a higher incidence of Demodex folliculorum in rosacea patients. Demodex folliculorum were also more frequently detected in patients who had previously been treated with topical corticosteroids (even in 91.9%), what was often followed by epitheloid granulomas. The treatment with tetracycline yielded good or excellent results in 90% of all the patients.


Problems of geriatric pharmacotherapy.

Problems of geriatric pharmacotherapy. No one dies of old age by itself; death is always due to a disease of some kind. This means that safe and effective therapy for old people is a matter of great practical importance. From what has been said above it will be obvious that the effects of drugs in old people are similar to their effects in the young, but there may be differences in degree in their susceptibility to adverse reactions. The existing data are by no means abundant, sometimes contradictory and of limited evidential value. Looking at the picture as a whole, one cannot but agree with those authors who state that the available results are insufficient to substantiate the general opinion that old age in itself heightens the risk of all forms of pharmacotherapy. From the practical point of view it is nowadays essential that no drug should be prescribed for an elderly patient without strict scrutiny of the need for it, that such drugs should not be administered for any longer than is absolutely necessary, that treatment should normally be started with smaller doses than those usually given to younger adults and, lastly, that surveillance for possible side effects should be particularly close. The author states that it must not be forgotten that geriatric patients frequently consult specialists in addition to their family doctors and that they are perhaps overinclined to listen to advice on drug treatment from those about them. One of the duties of the family doctor is to check the assortment of drugs which his patient is consuming--often an alarming total--and to cut down their number to those which are really necessary.


Coronary vascularization in patient maintained on chronic hemodialysis. A patient, maintained on hemodialysis for 16 years because of chronic renal failure caused by chronic glomerulonephritis, who underwent surgical coronary revascularization, is presented. The authors conclude that preoperative hemodialysis, careful hydration of the patient, right management of cardiopulmonary bypass, use of hemodilution and aprotinin can contribute to escivating of hemofiltration and heterologue blood transfusion in uremic patients who undergo well timed coronary revascularization.