Adult macrophage activation syndrome-haemophagocytic lymphohistiocytosis: 'of plasma exchange and immunosuppressive escalation strategies' - a single centre reflection.

OBJECTIVE: In the context of systemic autoimmunity, that is systemic lupus erythematosus (SLE) or adult-onset Still's disease (AOSD), secondary haemophagocytic lymphohistiocytosis (HLH; also referred to as macrophage activation syndrome (MAS) or more recently MAS-HLH) is a rare and potentially life-threatening complication. Pathophysiological hallmarks are aberrant macrophage and T cell hyperactivation and a systemic cytokine flare, which generate a sepsis-like, tissue-damaging, cytopenic phenotype. Unfortunately, for adult MAS-HLH we lack standardized treatment protocols that go beyond high-dose corticosteroids. Consequently, outcome data are scarce on steroid refractory cases. Aside from protocols based on treatment with calcineurin inhibitors, etoposide, cyclophosphamide and anti-IL-1, favourable outcomes have been reported with the use of intravenous immunoglobulin (IvIG) and plasma exchange (PE). METHODS: Here we report a retrospective series of steroid refractory MAS-HLH, the associated therapeutic regimes and outcomes. RESULTS: In this single-centre experience, 6/8 steroid refractory patients survived (median follow-up: 54.4 (interquartile range: 23.3-113.3) weeks). All were initially treated with PE, which induced partial response in 5/8 patients. Yet, all patients required escalation of immunosuppressive therapies. One case of MAS-HLH in new-onset AOSD had to be escalated to etoposide, whereas most SLE-associated MAS-HLH patients responded well to cyclophosphamide. Relapses occurred in 2/8 cases. CONCLUSION: Together, early use of PE is at most a supportive measure, not a promising monotherapy of adult MAS-HLH. In refractory cases, conventional cytoreductive therapies (i.e. cyclophosphamide and etoposide) constitute potent and reliable rescue approaches, whereas IvIG, anti-thymoglobulin, and biologic agents appear to be less effective.

Yarrowia lipolytica Pex20p, Saccharomyces cerevisiae Pex18p/Pex21p and mammalian Pex5pL fulfil a common function in the early steps of the peroxisomal PTS2 import pathway.

Import of peroxisomal matrix proteins is essential for peroxisome biogenesis. Genetic and biochemical studies using a variety of different model systems have led to the discovery of 23 PEX genes required for this process. Although it is generally believed that, in contrast to mitochondria and chloroplasts, translocation of proteins into peroxisomes involves a receptor cycle, there are reported differences of an evolutionary conservation of this cycle either with respect to the components or the steps involved in different organisms. We show here that the early steps of protein import into peroxisomes exhibit a greater similarity than was thought previously to be the case. Pex20p of Yarrowia lipolytica, Pex18p and Pex21p of Saccharomyces cerevisiae and mammalian Pex5pL fulfil a common function in the PTS2 pathway of their respective organisms. These non-orthologous proteins possess a conserved sequence region that most likely represents a common PTS2-receptor binding site and di-aromatic pentapeptide motifs that could be involved in binding of the putative docking proteins. We propose that not necessarily the same proteins but functional modules of them are conserved in the early steps of peroxisomal protein import.

Intercellular coupling in frog heart muscle. Electrophysiological and morphological aspects.

Passive electrical parameters of bullfrog atrial trabeculae were measured in a single gap arrangement. Attention was focussed on the resistance of internal longitudinal pathway. The influence of external Ca2+ depletion was tested using EGTA as chelating agent. Morphometry of trabeculae, fine structure of junctional complexes, and distribution of membrane-bound Ca were investigated by light and electron microscopic methods. The specific internal resistance to longitudinal current flow was 523 omega cm with normal Ringer as perfusing fluid and 1140 omega cm in EGTA-containing solution. These values are considered to represent the sum of myoplasmic and junctional resistivity. Morphometrical studies indicated an interstitial space of 12%, a mean cell length of 358 micron, and a mean cell diameter of 3.2 micron. In freeze-fractured preparations junctional structures were observed in the form of "atypical gap junctions" consisting of 10 nm particles arranged in a circular or linear array. The number of gap junctions was estimated to range between 20 and 50/cell which is equivalent to a junctional area of 0.01 or 0.03% of total surface area. A mean number of 55 particles/gap junction was calculated. After 20 min of exposure to EGTA the majority of junctional complexes were converted to clusters; the number of particles/gap junction was not significantly altered. The fluorescent dye CTC was used as a probe for membrane-bound Ca of isolated living cells. In normal Ringer a strong fluorescence was seen at the cell surface and in different intracellular compartments. With EGTA both superficial and internal fluorescence disappeared completely. From a combination of electrical and morphometrical data the resistance of intercellular junctions was calculated. Under normal conditions the specific resistance of junctional membrane amounted to 0.4 omega cm2 and the resistance of an individual connection was of the order of 10(11) omega. With EGTA, the respective values were increased by about 230%. The mechanism underlying this depression of junctional conductance is not clear. It seems not related to a rise of cytoplasmic free Ca2+. The EGTA-induced increase in internal resistance was reflected by a decrease of the length constant of a bundle. The nature of "atypical gap junctions" and their relation to tight junctions are discussed. It is concluded that the junctions observed in frog atrial muscle are analogous to gap junctions of insect or mammalian cells in spite of the different size and arrangement of the particles. A theoretical model is presented for the electrical behaviour of a bundle in a single gap arrangement.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of lidoflazine on membrane currents and contraction in voltage-clamped frog atrial fibers.

The effect of lidoflazine on action potential, membrane currents and contraction of frog atrial fibers was tested using the double sucrose voltage clamp technique. Lidoflazine was found to decrease the sodium conductivity of the heart cell membrane, probably by blocking the sodium channels. The availability of the sodium system at resting potential was slightly enlarged by lidoflazine and the recovery from inactivation was prolonged in most of the preparations tested. A small decrease of the slow inward current and a reduction of phasic and tonic tension was observed. The outward current at higher depolarizations was increased by lidoflazine resulting in a shortened action potential duration. The data suggest that lidoflazine's antifibrillatory properties are less pronounced than those of classical antiarrhythmic agents; but the slight antifibrillatory and negative inotropic effect might be helpful in the treatment of angina pectoris.

Transient tension responses of voltage-clamped frog atrial muscle related to sudden changes in external Ca or Na.

Frog atrial muscle strips were placed in a double sucrose gap chamber and perfusion of the central node was arranged as to allow rapid changes of external Ca or Na concentration during long-lasting (15 s) depolarizing clamps. When the superfusing fluid was suddenly switched, the intercellular space inside the fibre bundle equilibrated with a time constant in the order of 1 s. A fast change of perfusate during clamp was followed by a delayed change of tonic (sustained) contraction. When [Ca]0 was increased from 0.25 to 4 mM, tension rose in a sigmoid manner and the level reached at the end of the clamp was almost identical with the steady control in 4 mM-Ca-Ringer. A similar tension increase occurred upon a reduction of [Na]0 from 100 to 25% of normal. At a given depolarization time course and height of the tension responses followed the ratio of [Ca]0/[Na]20. Transient tension responses are interpreted in terms of a sudden perturbation of a transmembrane Ca-Na exchange system leading to a depression of Ca pumping activity.

Calcium-sodium antagonism on the frog's heart: a voltage-clamp study.

1. In double sucrose-gap voltage-clamped frog atrial fibres the influence of [Ca]o and [Na]o on membrane current and contraction was investigated. 2. The slow (secondary) inward current varied with [Ca]o but was almost insensitive to changes in [Na]o. In contrast, the phasic (transient) contraction initiated by the slow inward current was affected by both [Ca]o and [Na]o. 3. With moderate changes of [Ca]o and [Na]o from normal, the strength of phasic contraction at a given depolarization followed the [Ca]o/[Na]2o ratio approximately. This was best seen at membrane potentials near zero level. 4. Under the same conditions, tonic (sustained) contractions associated with prolonged depolarizations were strictly correlated to the [Ca]o/[Na]2o ratio at any potential. No interrelation between tonic tension and steady-state current was found. 5. With extensive changes in [Ca]o and [Na]o, the sensitivity of both phasic and tonic tension to the [Ca]o/[Na]2o ratio declined, the negative effect of [Na]o becoming smaller than was expected from this ratio. 6. In Na-free choline-Ringer, a strong contracture developed followed by a spontaneous relaxation. Starting from the relaxed state, application of depolarizing clamps gave rise to phasic contractions with a very slow relaxation while tonic contractions were apparently lacking. 7. The results are interpreted in terms of an energy-dependent carrier mechanism exchanging one Ca for two Na ions across the cell membrane. The model implies a strong asymmetry in the rate constants governing the chemical reactions on both sides of the membrane. The system is thought to operate close to equilibrium at any potential, thereby determining the steady level of myoplasmic Ca. The equilibrium itself is considered to shift upon depolarization. Assuming that [Na]i is constant, the steady level of [Ca]i is expected to be proportional to the [Ca]o/[Na]2o ratio, the scale factor being a function of membrane potential. 8. The carrier model suggests the occurrence of a depolarization-induced inward transfer of Ca which might be involved in the generation of tonic contractions. 9. The apparent lack of tonic contractions in the absence of external Na ions may be explained by a suppression of carrier-mediated Ca influx normally occurring upon depolarization. 10. The antagonistic effects of [Ca]o and [Na]o on phasic contraction are understood as being due to alterations of the Ca pumping system rather than changes in slow inward current.

Effects of prenylamine on cardiac membrane currents and contractility.

The influence of prenylamine on the electrical and mechanical activity of frog atrial muscle fibers has been studied under voltage clamp conditions. At a concentration of 10-4 M, prenylamine blocks the action potential without much affecting the resting potential. The drug depresses the peak transient sodium conductance with a dissociation constant of 1.7 times 10-5 M and on a one-to-one stoichiometric basis. The curve relating peak sodium conductance to membrane potential is slightly shifted in the direction of hyperpolarization. The time to peak sodium current and the rate of sodium inactivation are not significantly altered. With 2 times 10-5 M prenylamine, the steady-state sodium inactivation curve is shifted by 5 mV to more negative membrane potentials but the decreased availability of the sodium system at the resting level is not sufficient to account for the reduction of sodium current. Recovery from sodium inactivation upon repolarization is distinctly slowed. The slow (secondary) inward current carried by calcium and/or sodium and the steady-state outward current are also depressed by prenylamine. The phasic (twitch-like) contraction related to the slow inward current is slightly decreased. The tonic (sustained) contraction associated with long-lasting depolarizations is increased and the time course of relaxation is retarded by prenylamine.

Membrane current and contraction in frog atrial fibres.

1. Membrane current and mechanical activity were recorded from short segments of frog atrial muscle strips using a double sucrose gap voltage clamp arrangement. Experiments were performed at 4-7 degrees C. Two types of contraction were observed dependent upon the duration of the clamp.2. Short-lasting depolarizations caused a flow of Ca inward current, I(Ca), and development of a phasic contraction. Time to peak tension approximated 400 msec. Both I(Ca) and contraction, as functions of membrane potential, had a threshold of about - 40 mV and were maximal at inside positive potentials in normal Ringer fluid. Peak tension decreased at strong depolarizations.3. The minimum time of depolarization required for initiation of a phasic contraction was 40-70 msec. The time necessary for full activation of contraction was 200-300 msec and comparable to the period of time covered by the flow of I(Ca).4. There was no marked change in peak tension upon repetitive depolarization to the same membrane potential.5. Restoration of (phasic) contractility after a preceding contraction was strongly dependent on the level of membrane potential between conditioning and test pulse. Restoration was half complete at potentials around - 45 mV.6. Long-lasting depolarizations generated tonic (sustained) contractions superimposed on the phasic (transient) ones. Threshold potential for initiation of tonic contractions was usually positive to the threshold of phasic contractions. The time taken to attain the final level of tension ranged between 0.7 and 3 sec. Plateau tension, as a function of membrane potential, increased with increasing depolarization and reached a flat maximum at about + 50 mV in normal Ringer fluid.7. At membrane potentials near zero level, plateau tension developed by the tonic mechanism was about twice peak tension due to phasic contraction.8. Removal of Ca ions from the external medium resulted in an almost complete abolition of phasic contraction within 1-2 min and a gradual decrease of tonic contraction during the first 10 min. Application of a ;Ca inhibitor' to normal Ringer fluid caused a strong reduction of both I(Ca) and phasic contraction without affecting tonic contractions.9. It is concluded that phasic contractions are directly activated by the flow of I(Ca). Generation of tonic contractions may be attributed to a Ca transfer mechanism different from I(Ca) or a release of Ca from intracellular stores.

Electrical activity and metabolism in cardiac tissue: An experimental and theoretical study.

(1) Effects of the metabolic inhibitor 2,4-dinitrophenol (DNP) on electrical activity in frog atria were studied by means of the sucrose-gap technique and in tracer experiments. (2) Voltage-clamp studies of ionic membrane currents showed a suppression by DNP of peak Na inward current without marked changes in the kinetics of the Na-carrying system and an increase of steady state outward current to three to five times its normal value. In(42)K tracer experiments, DNP increased K resting efflux by about 10% and decreased K influx by 25 to 30%. (3) The depression of Na inward current is regarded as being caused by a partial block of Na channels and an increase of internal Na concentration after inhibition of active Na extrusion. (4) The strong rise in outward current is probably not caused by a K current since K efflux fails to show a correspondingly large change. As a possible explanation for current and flux changes, an electrogenic K pump is discussed. (5) A mathematical model of a carrier system transporting a single ion species is described. The system is designed as a direct "potential" pump. Uphill transport requires an asymmetry of the rate constants governing the cyclic formation and breakdown of carrier-ion complex. The asymmetry is brought about by an input of metabolic energy. Reduction of energy input decreases the asymmetry and induces a carrier-mediated downhill ion movement, with corresponding changes in membrane current and ion fluxes. (6) A model of electrogenic K inward transport is calculated that approximately accounts for the steady state current and the K flux changes experimentally observed after inhibition.