Effects of mannuronic acid (M2000) on gene expression profile of signal transducer and activator of transcription proteins (STATs) in rheumatoid arthritis patients.

Rheumatoid arthritis (RA), a form of inflammatory arthritis, is a chronic joint disease characterized by pain and inflammation that affects 0.5% to 1% of the population worldwide. The safety, efficacy, tolerability, and potency of ß-D-mannuronic acid (M2000) as a novel NSAID with immunosuppressive property has been reported by several in vitro studies, experimental models and clinical trials phase I/II and III in ankylosing spondylitis and rheumatoid arthritis (RA) patients This research is designed to study the therapeutic efficacy of oral administration of mannuronic acid in RA patients who had inadequate response to conventional drugs and to assess the effect of this drug on gene expression of the signal transducer and activator of transcription (STATs) protein (STAT1, STAT3, STAT4, and STAT6). The study has included 15 RA patients who had an insufficient response to the conventional therapy. The oral dose of mannuronic acid was 1000mg divided into two 500 mg doses per day for 3 months as an addition to conventional therapy. There were 15 healthy volunteer in the control group. Blood samples were collected from both groups, once from healthy controls and twice from RA patients before and after treatment by M2000. The peripheral blood mononuclear cells (PBMCs) were isolated to assess the gene expression level of STAT1, STAT3, STAT4, and STAT6 using the real-time PCR method. Results obtained in this study demonstrated a significant difference in the gene expression level of STAT1 between healthy controls and patients before treatment as well as a significant reduction in RA patients after treatment compared with the level before treatment. In addition, the gene expression level of STAT3 and STAT4 showed a significant reduction in RA patients after treatment compared to patients before treatment, while there was no significant difference between RA patients before treatment and the healthy control group for both molecules. On the other hand, there was no change in the gene expression level of STAT6 among all groups. The outcomes of this study confirmed that ß-D-mannuronic acid (M2000) has the ability to control the levels of STAT1, STAT3 and STAT4 in RA patients, and might be beneficial in the management and therapy of RA.

Circulating Helper T-Cell Subsets and Regulatory T Cells in Patients With Common Variable Immunodeficiency Without Known Monogenic Disease.

BACKGROUND: Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency (PID). It is characterized by heterogeneous clinical manifestations and defects in B cells and T cells. In the present study, we investigated helper T (TH) cell subsets and regulatory T (Treg) cells and their related cytokines and transcription factors in CVID patients with no definitive genetic diagnosis. METHODS: The study population comprised 13 CVID patients and 13 healthy controls. Mutation analysis was performed using whole exome sequencing in CVID patients to rule out monogenic PIDs. TH subsets and Treg were analyzed using flow cytometry. The expression of determinant cytokines (IFN-γ, IL-17, IL-22, and IL-10) and cell subset specific transcription factors was evaluated before and after stimulation. RESULTS: The main clinical presentations of these patients were infections only and lymphoproliferative phenotypes. No autoimmune or allergy phenotypes were recorded. The frequencies of CD4+ T cells, TH17, and Treg cells were significantly reduced in CVID patients; however, TH1, TH1-like TH17, and TH22 subsets were normal. After stimulation, expression of retinoic-acid-orphan-receptor-C (RORC), runtrelated transcription factor 1 (RUNX1), IL17, and IL10 was significantly lower in CVID patients than in the healthy controls. Moreover, the concentration of IL-17 and IL-10 in the cell culture supernatants of stimulated CD4+ T cells was lower in CVID patients than in healthy controls. CONCLUSIONS: Our findings demonstrate that the imbalance of TH17 and Tregs could be associated with infection and the lymphoproliferative phenotype in CVID patients without monogenic disorders.

Thin film growth by deposition of randomly shaped clusters.

We have investigated the growth of a porous thin surface film by deposition of randomly shaped clusters with different sizes over an initially flat linear substrate in (1 + 1) dimensions. In analogy with the ballistic deposition process, our approach results in aggregation of clusters with a porous bulk and a rough surface that obeys the Family-Vicsek dynamic scaling. The scaling exponents are calculated and found to agree with the ballistic deposition model. Moreover, the bulk porosity and its dependence on time and cluster size are also investigated. We have also studied the influence of the cluster size on the scaling exponents and the stationary porosity.

Transverse sarcomere splitting. A possible means of longitudinal growth in crab muscles.

Transversely split sarcomeres are seen in mouthpart muscles of the blue crab in the electron microscope. Sarcomeres split only at the H zone. Two new sarcomeres are formed by a Z disk which appears in the H zone of the splitting sarcomere. Splitting may involve breaking of the thick filaments in the H zone, elongation of these filaments, and formation of both new actin filaments and Z-disk materials, Sarcomere splitting would allow longitudinal growth of muscle cells without lengthening of sarcomeres and concomitant changes in contractile properties.

Three-dimensional ultrastructure of the crayfish neuromuscular apparatus.

The synapse-bearing nerve terminals of the opener muscle of the crayfish Procambarus were reconstructed using electron micrographs of regions which had been serially sectioned. The branching patterns of the terminals of excitatory and inhibitory axons and the locations and sizes of neuromuscular and axo-axonal synapses were studied. Excitatory and inhibitory synapses could be distinguished not only on the basis of differences in synaptic vesicles, but also by a difference in density of pre- and postsynaptic membranes. Synapses of both axons usually had one or more sharply localized presynaptic "dense bodies" around which synaptic vesicles appeared to cluster. Some synapses did not have the dense bodies. These structures may be involved in the physiological activity of the synapse. Excitatory axon terminals had more synapses, and a larger percentage of terminal surface area devoted to synaptic contacts, than inhibitory axon terminals. However, the largest synapses of the inhibitory axon exceeded in surface area those of the excitatory axon. Both axons had many side branches coming from the main terminal; often, the side branches were joined to the main terminal by narrow necks. A greater percentage of surface area was devoted to synapses in side branches than in the main terminal. Only a small fraction of total surface area was devoted to axo-axonal synapses, but these were often located at narrow necks or constrictions of the excitatory axon. This arrangement would result in effective blockage of spike invasion of regions of the terminal distal to the synapse, and would allow relatively few synapses to exert a powerful effect on transmitter release from the excitatory axon. A hypothesis to account for the development of the neuromuscular apparatus is presented, in which it is suggested that production of new synapses is more important than enlargement of old ones as a mechanism for allowing the axon to adjust transmitter output to the functional needs of the muscle.

Variation in terminal morphology and presynaptic inhibition at crustacean neuromuscular junctions.

Synaptic terminals of excitatory and inhibitory neurons supplying muscle fibers in leg muscles of crabs (Pachygrapsus crassipes and Hyas areneus) were investigated with light and electron microscopy. Terminals responsible for large excitatory postsynaptic potentials (EPSPs) at low frequencies of activation had a compact configuration with clusters of terminal boutons radiating from the main axon branch. Terminals responsible for small EPSPs had a more diffuse organization, with boutons often arranged in series along thin axon branches. Inhibitory neurons, when activated, produced both presynaptic and postsynaptic inhibitory effects, with the former being more potent at low frequencies of activation. Presynaptic inhibition was variable in magnitude but was generally strong in fibers with large EPSPs. Representative terminals from regions of strong and weak presynaptic inhibition were identified by activity-dependent uptake of horseradish peroxidase, serially sectioned, and reconstructed from electron micrographs. Both regions were found to contain axo-axonal synapses from inhibitory to excitatory terminals, with a larger number in the region of strong presynaptic inhibition. In addition, axo-axonal synapses were more uniformly distributed in the latter region. The number of inhibitory presynaptic dense bars (active zones) was somewhat higher in the region of weak inhibition, but larger individual dense bars occurred in the region of strong inhibition. Possible factors contributing to the differences in strength of inhibition include: (1) morphology and electrical properties of terminals; and (2) high probability of transmission at a relatively small number of inhibitory synapses during low frequency activation in the region of strong inhibition.

Benzodiazepine antagonist flumazenil reduces hippocampal epileptiform activity.

We examined the effects of the benzodiazepine antagonist, flumazenil, on epileptiform discharges evoked in the hippocampal CA1 region in vitro. Application of 100 nM flumazenil did not affect normal synaptic responses; however, flumazenil did depress epileptiform discharges induced by 8 mM [K+]o. Epileptiform discharges induced by the GABAA channel antagonist picrotoxin or by the K+ channel blocker 4-aminopyridine were unaffected. Application of the high-affinity, low-efficacy benzodiazepine partial inverse agonist, Ro 19-4603, blocked the anticonvulsant effect of flumazenil, indicating that this action of flumazenil is mediated at a benzodiazepine binding site located on the GABAA receptor. A likely explanation of the present results is that flumazenil antagonizes the action of an endogenous benzodiazepine inverse agonist, which is released during epileptiform discharges evoked in high K+ ACSF.

Amphetamine actions on pre- and postpubertal rat hippocampal dentate granule neurons.

Clinical evidence suggests different actions of amphetamine (AMPH) in children and adults. Using intracellular recording techniques, the actions of AMPH at 10 and 40 microM were investigated in granule neurons of hippocampal slices from pre- and postpubertal rats. AMPH (10-40 microM) caused depolarization of most postpubertal neurons, often with increased spontaneous activity, whereas most prepubertal neurons were hyperpolarized. In both age groups, AMPH caused increased neuronal excitability by reducing spike threshold, attenuating the postspike train afterhyperpolarization, reducing spike frequency adaptation, and potentiating excitatory postsynaptic potentials. Changes in cell input resistance were variable and Ca2+ currents were unaffected. AMPH actions took 10-15 min to appear and became maximal 30-55 min after application. The effects were reversible at 10 microM, but at 40 microM, prolonged washout for up to 2 h did not completely reverse these actions. The beta-adrenergic blocker, propranolol, partially blocked AMPH actions. The dopamine (D2) blocker, haloperidol, did not block AMPH actions. Mature neurons were also tested with 2.5 microM AMPH showing similar but more reversible effects as the higher concentrations. Depleting catecholamines by reserpine partly attenuated the effects of 40 microM AMPH in mature neurons. Perfusion of neurons with 10 and 20 microM cocaine did not produce effects similar to those of AMPH. It is suggested that AMPH produces its effects on granule neurons only in part through the release of norepinephrine. The involvement of other neurotransmitters and/or neuromodulators released by AMPH, or direct postsynaptic actions of AMPH are also possible.

Antiepileptic efficacy of topiramate: assessment in two in vitro seizure models.

The antiepileptic efficacy of topiramate (TPM) has been demonstrated in both whole animal seizure models and clinical trials; however, there is no consensus concerning its mechanism of action. We determined first whether the antiepileptic effect of TPM generalized to in vitro seizure models. Epileptiform discharges, recorded extracellularly, were evoked by repeated tetanic stimulation of Schaffer collaterals and layer III association fibers in entorhinal cortex/hippocampus and piriform cortex slices, respectively. TPM was applied at concentrations of 20 or 100 microM. Whole cell recordings were made from CA1 pyramidal neurons and the effect of TPM was assessed on a variety of intrinsic membrane properties including resting membrane potential, input resistance and postspike potentials. TPM (20 microM) was without effect in entorhinal cortex/hippocampus (N=6); however, 100 microM TPM decreased significantly the Coastline Burst Index from 358.3+/-65.8 to 225. 5+/-77.1 (N=4), the frequency of spontaneous epileptiform discharges to 44.6+/-21.8 (N=5) and the duration of primary afterdischarge (PAD) to 65.9+/-10.1 (N=10) percent of control. In contrast, phenytoin (50 microM, N=7; 100 microM, N=8) reduced PAD to 96.9+/-14. 8 and 86.5+/-17.3 percent of control, respectively. TPM (100 microM) did not reduce significantly the frequency of spontaneous discharges in piriform cortex (85.4+/-12.3 percent of control; N=5). TPM (100 microM) was without significant effect on intrinsic membrane properties in CA1 pyramidal neurons. Likely candidate mechanisms underlying the antiepileptic effect produced by TPM include enhancement of chloride-mediated GABA(A) currents and reduction of kainate and L-type calcium currents.

Common time-frequency analysis of local field potential and pyramidal cell activity in seizure-like events of the rat hippocampus.

To study cell-field dynamics, physiologists simultaneously record local field potentials and the activity of individual cells from animals performing cognitive tasks, during various brain states or under pathological conditions. However, apart from spike shape and spike timing analyses, few studies have focused on elucidating the common time-frequency structure of local field activity relative to surrounding cells across different periods of phenomena. We have used two algorithms, multi-window time frequency analysis and wavelet phase coherence (WPC), to study common intracellular-extracellular (I-E) spectral features in spontaneous seizure-like events (SLEs) from rat hippocampal slices in a low magnesium epilepsy model. Both algorithms were applied to 'pairs' of simultaneously observed I-E signals from slices in the CA1 hippocampal region. Analyses were performed over a frequency range of 1-100 Hz. I-E spectral commonality varied in frequency and time. Higher commonality was observed from 1 to 15 Hz, and lower commonality was observed in the 15-100 Hz frequency range. WPC was lower in the non-SLE region compared to SLE activity; however, there was no statistical difference in the 30-45 Hz band between SLE and non-SLE modes. This work provides evidence of strong commonality in various frequency bands of I-E SLEs in the rat hippocampus, not only during SLEs but also immediately before and after.

Transition to seizures in the isolated immature mouse hippocampus: a switch from dominant phasic inhibition to dominant phasic excitation.

The neural dynamics and mechanisms responsible for the transition from the interictal to the ictal state (seizures) are unresolved questions in epilepsy. It has been suggested that a shift from inhibitory to excitatory GABAergic drive can promote seizure generation. In this study, we utilized an experimental model of temporal lobe epilepsy which produces recurrent seizure-like events in the isolated immature mouse hippocampus (P8-16), perfused with low magnesium ACSF, to investigate the cellular dynamics of seizure transition. Whole-cell and perforated patch recordings from CA1 pyramidal cells and from fast- and non-fast-spiking interneurons in the CA1 stratum oriens hippocampal region showed a change in intracellular signal integration during the transition period, starting with dominant phasic inhibitory synaptic input, followed by dominant phasic excitation prior to a seizure. Efflux of bicarbonate ions through the GABA A receptor did not fully account for this excitation and GABAergic excitation via reversed IPSPs was also excluded as the prime mechanism generating the dominant excitation, since somatic and dendritic GABA A responses to externally applied muscimol remained hyperpolarizing throughout the transition period. In addition, abolishing EPSPs in a single neuron by intracellularly injected QX222, revealed that inhibitory synaptic drive was maintained throughout the entire transition period. We suggest that rather than a major shift from inhibitory to excitatory GABAergic drive prior to seizure onset, there is a change in the interaction between afferent synaptic inhibition, and afferent and intrinsic excitatory processes in pyramidal neurons and interneurons, with maintained inhibition and increasing, entrained 'overpowering' excitation during the transition to seizure.

Ultrastructural diversity in motor units of crustacean stomach muscles.

The physiological and ultrastructural properties of muscle fiber.s comprising three motor units in the gastric mill of blue crabs are described. In their contractile properties muscle fibers in all motor units are similar and resemble the slow type fibers in crustacean limb muscles. The majority of fibers generate large excitatory post-synaptic potentials which do not facilitate strongly. Structurally two types of fibers are found. The one type has long sarcomeres (greater than 6 mum), thin to thick myofilament ratios of 5-6:1 and diads located near the ends of the A-band. The other type has shorter sarcomeres (less than 6 mum), thin to thick myofilament ratios of 3:1 and diads located at mid sarcomere level. Both types of fibers occur within a single motor unit and this differs from the vertebrate situation. Furthermore, the finding of fibers with a low thin to thick myofilament ratio of 3:1 demonstrates that they are not exclusive to fast type crustacean muscle but also occur in slow stomach muscles.

Fast-axon synapses of a crab leg muscle.

Neuromuscular synapses of the "fast" excitatory axon supplying the main extensor muscle in the leg of the shore crab Pachygrapsus crassipes were studied with electrophysiological and electron-microscopic techniques. Electrical recording showed that many muscle fibers of the central region of the extensor muscle responded only to stimulation of the fast axon, and electron microscopy revealed many unitary subterminal axon branches. Maintained stimulation, even at a low frequency, resulted in depression of the excitatory junctional potentials (EJPs) set up by the fast axon but EJPs of different muscle fibers depressed at different rates, indicating some physiological heterogeneity among the fast-axon synapses. Focal recording at individual synaptic sites on the surfaces of the muscle fibers showed quantal contents ranging from 1.4 to 5.5 at different synapses; these values are relatively high in comparison with similar determinations made in the crayfish opener muscle. Synapse-bearing nerve terminals were generally relatively small in diameter and filiform, with many individual synaptic contact areas of uniform size averaging 0.6 micron2. All of the individual synapses had a presynaptic "dense body" at which synaptic vesicles clustered. If these structures represent release points for transmitter quanta, the initial high quantal content would have an ultrastructural basis. The mitochondial content of the nerve terminals, the synaptic vesicle population, and the specialized subsynaptic sarcoplasm were all much reduced in comparison with tonic axon synaptic regions in this and other crustaceans. The latter features may be correlated with the relatively infrequent use of this axon by the animal, and with rapid fatigue.

Cocaine actions on rat prefrontal cortical and hippocampal dentate granule neurons in vitro.

The electrophysiological actions of cocaine hydrochloride (COC) on medial prefrontal cortical (mpfc) and hippocampal dentate granule (DG) neurons were investigated in rat brain slices with intracellular recording techniques. The following parameters were measured: resting membrane potential (RMP), spike threshold, spike firing adaptation, postspike train afterhyperpolarization (AHP), excitatory postsynaptic potentials (EPSPs), and inhibitory postsynaptic potentials (IPSPs). In the mpfc, COC appeared to have both inhibitory and excitatory effects. In the majority of cells examined, the EPSP amplitude was attenuated by COC (200 nM-20 microM), whereas the amplitude of the postspike train afterhyperpolarization (AHP) was reduced (an excitatory effect). In DG neurons, 1 microM COC caused a small depolarization. COC potentiated the EPSPs at 1 microM but attenuated EPSPs and IPSPs at 10-100 microM. The amplitude of antidromically evoked EPSPs was also increased by 20 microM COC. At concentrations of 10 microM and greater, COC increased spike threshold. It is concluded that COC actions on mpfc and DG neurons are both excitatory and inhibitory and that these effects may be mediated by multiple neurotransmitters/modulators.

Lobster neuromuscular junctions treated with black widow spider venom: correlation between ultrastructure and physiology.

Black widow spider venom (BWSV) causes marked physiological and morphological alterations at the lobster neuromuscular junction. BWSV is also active at vertebrate neuromuscular junctions but the component which acts on the lobster preparation is different from the one which affects vertebrates. Following exposure to BWSV, lobster neuromuscular junctions showed elevated frequencies of spontaneous miniature synaptic potentials for 15-30 min. Nerve-evoked synaptic potentials became blocked during this period. Subsequently, spontaneous miniature potentials disappeared and less frequent 'giant' spontaneous potentials appeared. Ultrastructural examination of excitatory and inhibitory nerve terminals showed that both types were affected by venom treatment. In untreated terminals, synaptic vesicles were grouped near the dense specialized membranes of the synapses. Soon after venom treatment, the synaptic vesicles were dispersed throughout the terminals and many larger and elongated vesicular structures were apparent. At the time of appearance of 'giant' spontaneous potentials, few synaptic vesicles were seen in the terminals, but large irregular vacuoles were present. Many mitochondria within the nerve terminals were swollen or disrupted, while nearby muscle mitochondria remained normal in size and appearance. Very few presynaptic dense bodies ('active zones') were seen at synapses of affected terminals. The observations are consistent with the hypothesis that BWSV allows an abnormal amount of Ca2+ to enter the nerve terminals, causing the various physiological and morphological changes.

Excitatory synapses of blue crab gastric mill muscles.

Physiological and ultrastructural studies were made of neuromuscular synapses in stomach muscles, especially two gastric mill muscles of the blue crab innervated by neurons of the stomatogastric ganglion. These muscles depolarized and contracted with application of glutamate, but not acetylcholine, whereas the dorsal dilator muscles of the pyloric region depolarized and contracted in acetylcholine, but not in glutamate. Large excitatory postsynaptic potentials (EPSP's) of 5-20 mV were recorded in the gastric mill muscles. At low frequencies of activation, individual synapses released on average about 2 quanta of transmitter for each nerve impulse. Facilitation of EPSP's after a single nerve impulse could be detected for at least 10 s. Synapses were found on enlarged terminals of the motor axon; their contact areas ranged from 0.2 mum2 up to 3mum2. Both electron-lucent, round synaptic vesicles and dense-cored vesicles occurred near these synapses. A possible correlation between contact area of a synapse and output of transmitter, is discussed.

Coupling potentials in CA1 neurons during calcium-free-induced field burst activity.

Small amplitude depolarizations (fast prepotentials, spikelets) recorded in mammalian neurons are thought to represent either dendritic action potentials or presynaptic action potentials attenuated by gap junctions. We have used whole-cell recordings in an in vitro calcium-free model of epilepsy to record spikelets from CA1 neurons of the rat hippocampus. It was found that spikelet appearance was closely correlated with the occurrence of dye coupling between pyramidal neurons, indicating that both phenomena share a common substrate. Spikelets were characterized according to waveform (amplitude and shape) and temporal occurrence. Spikelet amplitudes were found to be invariant with neuronal membrane potential, and their pattern of occurrence was indistinguishable from patterns of action potential firing in these cells. Voltage and current recordings revealed a spikelet waveform that was usually biphasic, comprised of a rapid depolarization followed by a slower hyperpolarization. Numerical differentiation of spike bursts resulted in waveforms similar to recorded spikelet sequences, while numerical integration of spikelets yielded waveforms that were indistinguishable from action potentials. Modification of spikelet waveforms by the potassium channel blocker tetraethylammonium chloride suggests that spikelets may arise from both resistive and capacitive transmission of presynaptic action potentials. Intracellular alkalinization and acidification brought about by perfusion with NH4Cl caused changes in spikelet frequency, consistent with reported alterations of field burst activity in this model of epilepsy. These results suggest that spikelets result from gap junctional communication, and may be important determinants of neuronal activity during seizure-like activity.

The role of calcium homeostasis and calcium currents in ethanol actions on central mammalian neurons.

The ubiquitous role of calcium in ethanol actions measured electrophysiologically in central neurons is discussed. Acute ethanol administration to rat hippocampal neurons in vitro causes a hyperpolarization, increased AHPs, increased EPSPs and IPSPs, decreased modelled electronic interneuronal coupling, decreased high threshold Ca2+ currents, increased Ik, and increased synaptic GABAA currents. Alcohol withdrawal reverses some of these actions. Ca2+ is implicated in all of the above ethanol mediated effects.