Recombinant ADAMTS13 normalizes von Willebrand factor-cleaving activity in plasma of acquired TTP patients by overriding inhibitory antibodies

Severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 as observed in acquired thrombotic thrombocytopenic purpura (TTP) is caused by inhibitory and non-inhibitory autoantibodies directed against the protease. Current treatment with plasma exchange is considered to remove circulating antibodies and to concurrently replenish the deficient enzyme.

Does acupuncture needling induce analgesic effects comparable to diffuse noxious inhibitory controls?

Diffuse noxious inhibitory control (DNIC) is described as one possible mechanism of acupuncture analgesia. This study investigated the analgesic effect of acupuncture without stimulation compared to nonpenetrating sham acupuncture (NPSA) and cold-pressor-induced DNIC. Forty-five subjects received each of the three interventions in a randomized order. The analgesic effect was measured using pressure algometry at the second toe before and after each of the interventions. Pressure pain detection threshold (PPDT) rose from 299 kPa (SD 112 kPa) to 364 kPa (SD 144), 353 kPa (SD 135), and 467 kPa (SD 168) after acupuncture, NPSA, and DNIC test, respectively. There was no statistically significant difference between acupuncture and NPSA at any time, but a significantly higher increase of PPDT in the DNIC test compared to acupuncture and NPSA. PPDT decreased after the DNIC test, whereas it remained stable after acupuncture and NPSA. Acupuncture needling at low pain stimulus intensity showed a small analgesic effect which did not significantly differ from placebo response and was significantly less than a DNIC-like effect of a painful noninvasive stimulus.

Inhibitory regulation of dendritic activity in vivo

The spatiotemporal control of neuronal excitability is fundamental to the inhibitory process. We now have a wealth of information about the active dendritic properties of cortical neurons including axonally generated sodium action potentials as well as local sodium spikelets generated in the dendrites, calcium plateau spikes, and NMDA spikes. All of these events have been shown to be highly modified by the spatiotemporal pattern of nearby inhibitory input which can drastically change the output firing mode of the neuron. This means that particular populations of interneurons embedded in the neocortical microcircuitry can more precisely control pyramidal cell output than has previously been thought. Furthermore, the output of any given neuron tends to feed back onto inhibitory circuits making the resultant network activity further dependent on inhibition. Network activity is therefore ultimately governed by the subcellular microcircuitry of the cortex and it is impossible to ignore the subcompartmentalization of inhibitory influence at the neuronal level in order to understand its effects at the network level. In this article, we summarize the inhibitory circuits that have been shown so far to act on specific dendritic compartments in vivo.

Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF’s trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.

Apoptotic neutrophils release macrophage migration inhibitory factor upon stimulation with tumor necrosis factor-alpha

Macrophage migration inhibitory factor (MIF) is an important cytokine involved in the regulation of innate immunity and present at increased levels during inflammatory responses. Here we demonstrate that mature blood and tissue neutrophils constitutively express MIF as a cytosolic protein not associated with azurophil granules. Functionally active MIF, but not proteases stored in azurophil granules, was released from apoptotic neutrophils following short term tumor necrosis factor (TNF)-alpha stimulation in a caspase-dependent manner and prior to any detectable phagocytosis by monocyte-derived macrophages. Moreover, TNF-alpha-mediated MIF release was blocked by glyburide and propenicide, both inhibitors of ATP-binding cassette-type transporters, suggesting that this transporter system is activated during neutrophil apoptosis. Taken together, apoptotic mature neutrophils release MIF upon short term TNF-alpha stimulation. Therefore, apoptosis may not always occur without the induction of pro-inflammatory mechanisms.

Inhibitory control of the human dorsolateral prefrontal cortex during the anti-saccade paradigm--a transcranial magnetic stimulation study

In the anti-saccade paradigm, subjects are instructed not to make a reflexive saccade to an appearing lateral target but to make an intentional saccade to the opposite side instead. The inhibition of reflexive saccade triggering is under the control of the dorsolateral prefrontal cortex (DLPFC). The critical time interval at which this inhibition takes place during the paradigm, however, is not exactly known. In the present study, we used single-pulse transcranial magnetic stimulation (TMS) to interfere with DLPFC function in 15 healthy subjects. TMS was applied over the right DLPFC either 100 ms before the onset of the visual target (i.e. -100 ms), at target onset (i.e. 0 ms) or 100 ms after target onset (i.e. +100 ms). Stimulation 100 ms before target onset significantly increased the percentage of anti-saccade errors to both sides, while stimulation at, or after, target onset had no significant effect. All three stimulation conditions had no significant influence on saccade latency of correct or erroneous anti-saccades. These findings show that the critical time interval at which the DLPFC controls the suppression of a reflexive saccade in the anti-saccade paradigm is before target onset. In addition, the results suggest the view that the triggering of correct anti-saccades is not under direct control of the DLPFC.

Influence of inhibitory killer immunoglobulin-like receptors and their HLA-C ligands on resolving hepatitis C virus infection

An estimated 2%-3% of the world's population is chronically infected with hepatitis C virus (HCV) and this is a major cause of liver disease worldwide. Following acute infection, outcome is variable with acute HCV successfully resolved in some individuals (20%-30%), but in the majority of cases the virus is able to persist. Co-infection with human immunodeficiency virus has been associated with a negative impact on the course of HCV infection. The host's immune response is an important correlate of HCV infection outcome and disease progression. Natural killer (NK) cells provide a major component of the antiviral immune response by recognising and killing virally infected cells. NK cells modulate their activity through a combination of inhibitory and activatory receptors such as the killer immunoglobulin-like receptors (KIRs) that bind to human leukocyte antigen (HLA) Class I molecules. In this workshop component, we addressed the influence of KIR genotypes and their HLA ligands on resolving HCV infection and we discuss the implications of the results of the study of Lopez-Vazquez et al. on KIR and HCV disease progression.

Sub-inhibitory concentrations of vancomycin prevent quinolone-resistance in a penicillin-resistant isolate of Streptococcus pneumoniae

BACKGROUND: The continuous spread of penicillin-resistant pneumococci represents a permanent threat in the treatment of pneumococcal infections, especially when strains show additional resistance to quinolones. The main objective of this study was to determine a treatment modality impeding the emergence of quinolone resistance. RESULTS: Exposure of a penicillin-resistant pneumococcus to increasing concentrations of trovafloxacin or ciprofloxacin selected for mutants resistant to these drugs. In the presence of sub-inhibitory concentrations of vancomycin, development of trovafloxacin-resistance and high-level ciprofloxacin-resistance were prevented. CONCLUSIONS: Considering the risk of quinolone-resistance in pneumococci, the observation might be of clinical importance.

Medroxyprogesterone abrogates the inhibitory effects of estradiol on vascular smooth muscle cells by preventing estradiol metabolism

Sequential conversion of estradiol (E) to 2/4-hydroxyestradiols and 2-/4-methoxyestradiols (MEs) by CYP450s and catechol-O-methyltransferase, respectively, contributes to the inhibitory effects of E on smooth muscle cells (SMCs) via estrogen receptor-independent mechanisms. Because medroxyprogesterone (MPA) is a substrate for CYP450s, we hypothesized that MPA may abrogate the inhibitory effects of E by competing for CYP450s and inhibiting the formation of 2/4-hydroxyestradiols and MEs. To test this hypothesis, we investigated the effects of E on SMC number, DNA and collagen synthesis, and migration in the presence and absence of MPA. The inhibitory effects of E on cell number, DNA synthesis, collagen synthesis, and SMC migration were significantly abrogated by MPA. For example, E (0.1micromol/L) reduced cell number to 51+/-3.6% of control, and this inhibitory effect was attenuated to 87.5+/-2.9% by MPA (10 nmol/L). Treatment with MPA alone did not alter any SMC parameters, and the abrogatory effects of MPA were not blocked by RU486 (progesterone-receptor antagonist), nor did treatment of SMCs with MPA influence the expression of estrogen receptor-alpha or estrogen receptor-beta. In SMCs and microsomal preparations, MPA inhibited the sequential conversion of E to 2-2/4-hydroxyestradiol and 2-ME. Moreover, as compared with microsomes treated with E alone, 2-ME formation was inhibited when SMCs were incubated with microsomal extracts incubated with E plus MPA. Our findings suggest that the inhibitory actions of MPA on the metabolism of E to 2/4-hydroxyestradiols and MEs may negate the cardiovascular protective actions of estradiol in postmenopausal women receiving estradiol therapy combined with administration of MPA.

Cytochrome P450 inhibitory action of Echinacea preparations differs widely and co-varies with alkylamide content

Echinacea preparations are one of the best selling herbal medicinal products with a well established therapeutic use in the prophylaxis of upper respiratory tract infections. Their consumption is increasing, but information about their ability to inhibit cytochrome P450 enzymes (CYP) is fragmentary. The picture is further complicated by a lack of phytochemical characterization of previously tested preparations. Due to its well characterized immunomodulatory activity, the standardized Swiss registered Echinacea purpurea (L.) Moench Echinaforce extract was selected for detailed study. With the single baculovirus-expressed CYP isoforms 1A2, 2C19, 2D9 and 3A4, inhibitory actions were measured by monitoring fluorescent metabolites derived from enzyme substrates (supersome assay). The Echinaforce extract induced mild inhibition of all these isoforms, with CYP 3A4 being the most, and CYP 2D6 the least sensitive enzyme. To assess whether CYP inhibition might be a general feature of Echinacea preparations, an additional nine commercially available preparations were screened using CYP 3A4. All tested preparations were able to inhibit CYP 3A4, but inhibitory potencies (expressed as median inhibitory concentration, IC50) varied by a factor of 150. The alkylamides are thought to be responsible for the immunomodulatory activity of Echinacea, and so the concentration of 2E,4E,8Z,10E/Z-tetranoic acid isobutylamide (1) and total alkylamide content were determined in all preparations, and the latter was found to be associated with their CYP 3A4 inhibitory potency. The chemically pure alkylamides dodeca-2E,4E,8Z,10E/Z-tetranoic acid isobutylamide (1) and dodeca-2E,4E-dieonoic acid isobutylamide (2) showed inhibitory activity on CYP 2C19, 2D6 and 3A4. However, unlike the Echinaforce extract, the alkylamides did not induce CYP 1A2 inhibition. Thus, other, as yet unidentified constituents also contribute to the overall weak inhibitory effects seen with Echinacea preparations in-vitro.

Leukemia inhibitory factor favours neurogenic differentiation of long-term propagated human midbrain precursor cells

There is a lot of excitement about the potential use of multipotent neural stem cells for the treatment of neurodegenerative diseases. However, the strategy is compromised by the general loss of multipotency and ability to generate neurons after long-term in vitro propagation. In the present study, human embryonic (5 weeks post-conception) ventral mesencephalic (VM) precursor cells were propagated as neural tissue-spheres (NTS) in epidermal growth factor (EGF; 20 ng/ml) and fibroblast growth factor 2 (FGF2; 20 ng/ml). After more than 325 days, the NTS were transferred to media containing either EGF+FGF2, EGF+FGF2+heparin or leukemia inhibitory factor (LIF; 10 ng/ml)+FGF2+heparin. Cultures were subsequently propagated for more than 180 days with NTS analyzed at various time-points. Our data show for the first time that human VM neural precursor cells can be long-term propagated as NTS in the presence of EGF and FGF2. A positive effect of heparin was found only after 150 days of treatment. After switching into different media, only NTS exposed to LIF contained numerous cells positive for markers of newly formed neurons. Besides of demonstrating the ability of human VM NTS to be long-term propagated, our study also suggests that LIF favours neurogenic differentiation of human VM precursor cells.

The novel ATP-competitive inhibitor of the MET hepatocyte growth factor receptor EMD1214063 displays inhibitory activity against selected MET-mutated variants

The receptor tyrosine kinase MET is a prime target in clinical oncology due to its aberrant activation and involvement in the pathogenesis of a broad spectrum of malignancies. Similar to other targeted kinases, primary and secondary mutations seem to represent an important resistance mechanism to MET inhibitors. Here, we report the biologic activity of a novel MET inhibitor, EMD1214063, on cells that ectopically express the mutated MET variants M1268T, Y1248H, H1112Y, L1213V, H1112L, V1110I, V1206L, and V1238I. Our results demonstrate a dose-dependent decrease in MET autophosphorylation in response to EMD1214063 in five out of the eight cell lines (IC50 2-43nM). Blockade of MET by EMD1214063 was accompanied by a reduced activation of downstream effectors in cells expressing EMD1214063-sensitive mutants. In all sensitive mutant-expressing lines, EMD1214063 altered cell cycle distribution, primarily with an increase in G1 phase. EMD1214063 strongly influenced MET-driven biological functions, such as cellular morphology, MET-dependent cell motility and anchorage-independent growth. To assess the in vivo efficacy of EMD1214063, we used a xenograft tumor model in immunocompromised mice bearing NIH3T3 cells expressing sensitive and resistant MET mutated variants. Animals were randomized for the treatment with EMD1214063 (50mg/kg/day) or vehicle only. Remarkably, five days of EMD1214063 treatment resulted in a complete regression of the sensitive H1112L-derived tumors, while tumor growth remained unaffected in mice with L1213V tumors and in vehicle-treated animals. Collectively, the current data identifies EMD1214063 as a potent MET small molecule inhibitor with selective activity towards mutated MET variants.

Investigation of the inhibitory effects of the benzodiazepine derivative, 5-BDBD on P2X4 purinergic receptors by two complementary methods

BACKGROUND/AIMS ATP-gated P2X4 purinergic receptors (P2X4Rs) are cation channels with important roles in diverse cell types. To date, lack of specific inhibitors has hampered investigations on P2X4Rs. Recently, the benzodiazepine derivative, 5-BDBD has been proposed to selectively inhibit P2X4Rs. However, limited evidences are currently available on its inhibitory properties. Thus, we aimed to characterize the inhibitory effects of 5-BDBD on recombinant human P2X4Rs. METHODS We investigated ATP-induced intracellular Ca(2+) signals and whole cell ion currents in HEK 293 cells that were either transiently or stably transfected with hP2X4Rs. RESULTS Our data show that ATP (< 1 μM) stimulates P2X4R-mediated Ca(2+) influx while endogenously expressed P2Y receptors are not activated to any significant extent. Both 5-BDBD and TNP-ATP inhibit ATP-induced Ca(2+) signals and inward ion currents in a concentration-dependent manner. Application of two different concentrations of 5-BDBD causes a rightward shift in ATP dose-response curve. Since the magnitude of maximal stimulation does not change, these data suggest that 5-BDBD may competitively inhibit the P2X4Rs. CONCLUSIONS Our results demonstrate that application of submicromolar ATP concentrations allows reliable assessment of recombinant P2XR functions in HEK 293 cells. Furthermore, 5-BDBD and TNP-ATP have similar inhibitory potencies on the P2X4Rs although their mechanisms of actions are different.