Multilineage glycosylphosphatidylinositol anchor-deficient haematopoiesis in untreated aplastic anaemia.

Aplastic anaemia and paroxysmal nocturnal haemoglobinuria (PNH) are closely related disorders. In PNH, haematopoietic stem cells that harbour PIGA mutations give rise to blood elements that are unable to synthesize glycosylphosphatidylinositol (GPI) anchors. Because the GPI anchor is the receptor for the channel-forming protein aerolysin, PNH cells do not bind the toxin and are unaffected by concentrations that lyse normal cells. Exploiting these biological differences, we have developed two novel aerolysin-based assays to detect small populations of PNH cells. CD59 populations as small as 0.004% of total red cells could be detected when cells were pretreated with aerolysin to enrich the PNH population. All PNH patients displayed CD59-deficient erythrocytes, but no myelodysplastic syndrome (MDS) patient or control had detectable PNH cells before or after enrichment in aerolysin. Only one aplastic anaemia patient had detectable PNH red cells before exposure to aerolysin. However, 14 (61%) had detectable PNH cells after enrichment in aerolysin. The inactive fluorescent proaerolysin variant (FLAER) that binds the GPI anchors of a number of proteins on normal cells was used to detect a global GPI anchor deficit on granulocytes. Flow cytometry with FLAER showed that 12 out of 18 (67%) aplastic anaemia patients had FLAER-negative granulocytes, but none of the MDS patients or normal control subjects had GPI anchor-deficient cells. These studies demonstrate that aerolysin-based assays can reveal previously undetectable multilineage PNH cells in patients with untreated aplastic anaemia. Thus, clonality appears to be an early feature of aplastic anaemia.

Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin.

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation in the gene PIGA, which encodes an enzyme essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. The PIGA mutation results in absence or marked deficiency of more than a dozen proteins on PNH blood cells. Current flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of specific GPI anchor proteins, such as CD59. However, because no single GPI anchor protein is always expressed in all cell lineages, no one monoclonal antibody can be used with confidence to diagnose PNH. We describe a new diagnostic test for PNH, based on the ability of a fluorescently labeled inactive variant of the protein aerolysin (FLAER) to bind selectively to GPI anchors. We compared GPI anchor protein expression in 8 patients with PNH using FLAER and anti-CD59. In all cases, FLAER detected similar or higher proportions of PNH monocytes and granulocytes compared with anti-CD59. Because of the increased sensitivity of detection, FLAER could detect small abnormal granulocyte populations in patients to a level of about 0.5%; samples from healthy control subjects contained substantially fewer FLAER-negative cells. FLAER gives a more accurate assessment of the GPI anchor deficit in PNH.

Resistance of paroxysmal nocturnal hemoglobinuria cells to the glycosylphosphatidylinositol-binding toxin aerolysin.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder caused by a somatic mutation of the PIGA gene. The product of this gene is required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; therefore, the phenotypic hallmark of PNH cells is an absence or marked deficiency of all GPI-anchored proteins. Aerolysin is a toxin secreted by the bacterial pathogen Aeromonas hydrophila and is capable of killing target cells by forming channels in their membranes after binding to GPI-anchored receptors. We found that PNH blood cells (erythrocytes, lymphocytes, and granulocytes), but not blood cells from normals or other hematologic disorders, are resistant to the cytotoxic effects of aerolysin. The percentage of lysis of PNH cells after aerolysin exposure paralleled the percentage of CD59(+) cells in the samples measured by flow cytometry. The kinetics of red blood cell lysis correlated with the type of PNH erythrocytes. PNH type III cells were completely resistant to aerolysin, whereas PNH type II cells displayed intermediate sensitivity. Importantly, the use of aerolysin allowed us to detect PNH populations that could not be detected by standard flow cytometry. Resistance of PNH cells to aerolysin allows for a simple, inexpensive assay for PNH that is sensitive and specific. Aerolysin should also be useful in studying PNH biology.

Manganese superoxide dismutase protects nNOS neurons from NMDA and nitric oxide-mediated neurotoxicity.

Neuronal nitric oxide synthase (nNOS) neurons kill adjacent neurons through the action of NMDA-glutamate receptor activation, although they remain relatively resistant to the toxic effects of NMDA and NO. The molecular basis of the resistance of nNOS neurons to toxic insults is unknown. To begin to understand the molecular mechanisms of the resistance of nNOS neurons, we developed a pheochromacytoma-derived cell line (PC12) that is resistant to the toxic effects of NO. We found through serial analysis of gene expression (SAGE) that manganese superoxide dismutase (MnSOD) is enriched in the NO-resistant PC12 cell-derived line (PC12-R). Antisense MnSOD renders PC12-R cells sensitive to NO toxicity and increases the sensitivity to NO in the parental, NO-sensitive PC12 line (PC12-S). Adenoviral transfer of MnSOD protects PC12-S cells against NO toxicity. We extended these studies to cortical cultures and showed that MnSOD is enriched in nNOS neurons and that antisense MnSOD renders nNOS neurons susceptible to NMDA neurotoxicity, although it has little effect on the overall susceptibility of cortical neurons to NMDA toxicity. Overexpression of MnSOD provides dramatic protection against NMDA and NO toxicity in cortical cultures, but not against kainate or AMPA neurotoxicity. Furthermore, nNOS neurons from MnSOD -/- mice are markedly sensitive to NMDA toxicity. Adenoviral transfer of MnSOD to MnSOD-/- cultures restores resistance of nNOS neurons to NMDA toxicity. Thus, MnSOD is a major protective protein that appears to be essential for the resistance of nNOS neurons in cortical cultures to NMDA mediated neurotoxicity.

[Antibodies to neuromediators in allergic diseases (pollinosis)]. / Antitela k neiromediatoram pri allergicheskikh zabolevaniiakh (pollinoze).

Sera from 33 pollinosis patients aged 16-33 have been examined for autoantibodies to neuromediators serotonin and catecholamine, serum samples from 21 healthy subjects served controls. Antibodies to dopamine and norepinephrine (43.0 +/- 4.0, 35.0 +/- 0.0 rel. units) have been detected in 81.8% and 69.7% of the pollinosis patients, respectively. Serotonin antibodies (54.6 +/- 6.0 rel. units) occurred in the patients 3.8 times more frequently than in healthy subjects. Antibodies to neuromediators in quantities within 12.0 rel. units were recorded in 9.5-38.0% of the controls. It is suggested that induction of neuromediator antibodies synthesis may represent a compensatory mechanism of neuromediator metabolism control in pollinosis.

[Choline acetyltransferase in the nerve plexuses of the basilar artery of the cat brain]. / Kholinatsetiltransferaza v nervnykh spleteniiakh osnovnoi arterii mozga koshki.

Localization of choline-acetyltranspherase (CHAT 2. 3. 1. 6) and acetylcholinesterase (ACHE 3. 1. 1. 7) in the neuronal plexus of the main artery in the cat brain has been studied. It has been stated that CHAT is mainly revealed in preterminal and terminal fibres of the plexus, while AXE--along the whole length of the fibre. ACHE activity is higher than that of Chat.

[Comparative characteristics of the cholin- and adrenergic innervation of the arteries of the cerebral dura in several mammals and man]. / Sravnitel'naia kharakteristika kholin- i adrenergicheskoi innervatsii arterii tverdoi mozgovoi obolochki u nekotorykh mlekopitaiushchikh i u cheloveka.

Cholinergic and adrenergic neuronal apparatus of the nasal (anterior), medial and caudal (posterior) meningeal arteries was studied in rats, guinea pigs, rabbits, cats, dogs, pigs, cows and in man by means of histochemical methods of Koelle and Falck. Dural arteries in the above mentioned animals and man were stated to be surrounded with a paravasal and two adventitial plexus--superficial and profound. The greatest concentration of cholinergic and adrenergic conductors was observed on the medial meningeal arteries. The least density of the conductors had the nasal arteries. The mammal and human caudal arteries occupy intermediate position by their number of cholinergic and adrenergic conductors per area unit. A definite increase in concentration of cholinergic and adrenergic nerves was observed in the successive line of the animals: rat--guinea pig--rabbit--cat--dog--pig--cow. The greatest density of cholinergic and adrenergic nerve fibres was observed in human meningeal arteries.

[Effect of adrenomimetics and sympatholytics on the adrenergic neural fibers and mast cells of the dura mater]. / Vliianie adrenomimetikov i simpatolitikov na adrenergicheskie nervnye bolokna i tuchnye kletki tverdoi obolochki mozga.

The effect of noradrenaline, dophamine and rausedyle on the adrenergic nervous apparatus and on the labrocytes of the dura mater in white rats was studied by means of Falk's and Glenner's methods and under light electron microscopy. The adrenergic nerve fibres and labrocytes are stated to respond to the injection of adrenomimetic and sympathomimetic drugs with increase or decrease of deposited monoamines on the background of corresponding clinical and vessel phenomena. The data obtained makes it possible to consider the adrenergic nerve fibres and labrocytes to be a single adrenergic apparatus of the dura mater.

[Choline acetyltransferase in the neural plexus of the main artery of cat's brain]. / Kholinatsetiltransferaza v nervnykh spleteniiakh osnovnoi arterii mozga koshki

Methods of Burt (1961, 1970) and Kása (1970) were used in order to study localization of choline acetyltransferase (ChAT 2.3.1.6) and method of Koelle to study the activity of acetylcholinesterase (AChE 3.1.1.7) in nerve plexuses of the cat's brain major artery. It has been established that ChAT is detected mainly in preterminal and terminal fibres of the plexus while AChE is detected along the total length of the fibre. The activity of AChE is higher than that of ChAT.

[Cholin- and adrenergic components of the vegetative innervation of the dura mater]. / Kholin- i adrenergicheskii komponenty vegetativnoi innervatsii tverdoi mozgovoi obolochki

Cholin- and adrenergic nerves in the fornix and base of the dura mater of rats, cats and dogs have been studied by methods of Kelle, Falck and Glenner. It has been established that the dura mater has a developed cholin- and adrenergic nervous apparatus innervating arteries, veins and the connective tissue of the mater. The concentration of nerve fibres is always greater on a meningea media and its daughter branches. The statistical processing of the data obtained has shown that the maximum quantity of nerve fibres is in cats, less in dogs and still less in rats. It has been established that in the dura fornix of cats and dogs there are more cholinergic nerve fibres than in the base. In rats there is no such difference. The amount of fibres with monoaminoxidase approximately corresponds to the amount of conductors with noradrenaline.