Amino acid variations in hepatitis C virus p7 and sensitivity to antiviral combination therapy with amantadine in chronic hepatitis C

BACKGROUND: Formation of transmembrane ion channels by hepatitis C virus (HCV) p7 and abrogation of channel function by amantadine was demonstrated in vitro. The relevance of HCV p7 amino acid (aa) variations for response to antiviral therapy with amantadine is unknown. METHODS: HCV p7 was sequenced in 86 individuals who were infected with HCV genotype 1. Thirty-six of 86 patients received amantadine within an interferon-alpha (IFN-alpha)-based antiviral therapy. Helical wheel modelling for HCV p7 was performed. RESULTS: No significant correlation of overall aa variations within HCV p7 was observed with response to IFN-alpha-based therapy with amantadine in HCV genotype 1alpha/b infected patients. When analysis was restricted to non-conservative aa variations, a higher number of aa substitutions within complete HCV p7 and transmembrane helix 2 was associated with non-response in HCV-1b-infected patients receiving therapy with amantadine (P=0.015 and P=0.037, respectively), without amantadine (P=0.106 and P=0.118, respectively), and in the total cohort of HCV-1b-infected patients (P=0.00007 and P=0.011, respectively). Furthermore, substitution L20F was observed more often in non-responders than responders with HCV-1b infection and therapy with amantadine (P=0.099). By in silico modelling, aa 20 was located toward the p7 channel lumen. Substitution L20F may impair amantadine action by altering the shape of the ion channel pore. CONCLUSION: Substitution L20F within HCV p7 may be associated with non-response to combination therapy specifically with amantadine in HCV-1b-infected patients. Non-responders with HCV-1b infection showed higher numbers of non-conservative aa variations within HCV p7 than responders, irrespective of the application of amantadine.     

Changes in blood lactate and respiratory gas exchange measures in sports with discontinuous load profiles

This study compares two different sport events (orienteering = OTC; tennis = TEC) with discontinuous load profiles and different activity/recovery patterns by means of blood lactate (LA), heart rate (HR), and respiratory gas exchange measures (RGME) determined via a portable respiratory system. During the TEC, 20 tennis-ranked male subjects [age: 26.0 (3.7) years; height: 181.0 (5.7) cm; weight: 73.2 (6.8) kg; maximal oxygen consumption (V˙O₂max): 57.3 (5.1) ml·kg⁻¹·min⁻¹] played ten matches of 50 min. During the OTC, 11 male members of the Austrian National Team [age: 23.5 (3.9) years; height: 183.6 (6.8) cm; weight: 72.4 (3.9) kg; V˙O₂max: 67.9 (3.8) ml·kg⁻¹·min⁻¹] performed a simulated OTC (six sections; average length: 10.090 m). In both studies data from the maximal treadmill tests (TT) were used as reference values for the comparison of energy expenditure of OTC and TEC. During TEC, the average V˙O₂ was considerably lower [29.1 (5.6) ml^·kg^−1·min⁻¹] or 51.1 (10.9)% of VO₂max and 64.8.0 (13.3)% of V˙O₂ determined at the individual anaerobic threshold (IAT) on the TT. The short high-intensity periods (activity/recovery = 1/6) did not result in higher LA levels [average LA of games: 2.07 (0.9) mmol·l⁻¹]. The highest average V˙O₂ value for a whole game was 47.8 ml·kg^−1·min⁻¹ and may provide a reference for energy demands required to sustain high-intensity periods of tennis predominately via aerobic mechanism of energy delivery. During OTC, we found an average V˙O₂ of 56.4 (4.5) ml·kg⁻¹·min⁻¹ or 83.0 (3.8)% of V˙O₂max and 94.6 (5.2)% of V˙O₂ at IAT. In contrast to TEC, LA were relatively high [5.16 (1.5) mmol·l⁻¹) although the average V˙O₂ was significantly lower than V˙O₂ at IAT. Our data suggest that portable RGEM provides valuable information concerning the energy expenditure in sports that cannot be interpreted from LA or HR measures alone. Portable RGEM systems provide valuable assessment of under- or over-estimation of requirements of sports and assist in the optimization and interpretation of training in athletes. Electronic Publication     

Human extramedullary bone marrow in mice: a novel in vivo model of genetically controlled hematopoietic microenvironment

The interactions between hematopoietic cells and the bone marrow (BM) microenvironment play a critical role in normal and malignant hematopoiesis and drug resistance. These interactions within the BM niche are unique and could be important for developing new therapies. Here, we describe the development of extramedullary bone and bone marrow using human mesenchymal stromal cells and endothelial colony-forming cells implanted subcutaneously into immunodeficient mice. We demonstrate the engraftment of human normal and leukemic cells engraft into the human extramedullary bone marrow. When normal hematopoietic cells are engrafted into the model, only discrete areas of the BM are hypoxic, whereas leukemia engraftment results in widespread severe hypoxia, just as recently reported by us in human leukemias. Importantly, the hematopoietic cell engraftment could be altered by genetical manipulation of the bone marrow microenvironment: Extramedullary bone marrow in which hypoxia-inducible factor 1α was knocked down in mesenchymal stromal cells by lentiviral transfer of short hairpin RNA showed significant reduction (50% ± 6%; P = .0006) in human leukemic cell engraftment. These results highlight the potential of a novel in vivo model of human BM microenvironment that can be genetically modified. The model could be useful for the study of leukemia biology and for the development of novel therapeutic modalities aimed at modifying the hematopoietic microenvironment.     

Network compensation of cyclic GMP-dependent protein kinase II knockout in the hippocampus by Ca2+-permeable AMPA receptors

Gene knockout (KO) does not always result in phenotypic changes, possibly due to mechanisms of functional compensation. We have studied mice lacking cGMP-dependent kinase II (cGKII), which phosphorylates GluA1, a subunit of AMPA receptors (AMPARs), and promotes hippocampal long-term potentiation (LTP) through AMPAR trafficking. Acute cGKII inhibition significantly reduces LTP, whereas cGKII KO mice show no LTP impairment. Significantly, the closely related kinase, cGKI, does not compensate for cGKII KO. Here, we describe a previously unidentified pathway in the KO hippocampus that provides functional compensation for the LTP impairment observed when cGKII is acutely inhibited. We found that in cultured cGKII KO hippocampal neurons, cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors was decreased, reducing cytoplasmic Ca²⁺ signals. This led to a reduction of calcineurin activity, thereby stabilizing GluA1 phosphorylation and promoting synaptic expression of Ca²⁺-permeable AMPARs, which in turn induced a previously unidentified form of LTP as a compensatory response in the KO hippocampus. Calcineurin-dependent Ca²⁺-permeable AMPAR expression observed here is also used during activity-dependent homeostatic synaptic plasticity. Thus, a homeostatic mechanism used during activity reduction provides functional compensation for gene KO in the cGKII KO hippocampus.Some gene deletions yield no phenotypic changes because of functional compensation by closely related or duplicate genes (1). However, such duplicate gene activity may not be the main compensatory mechanism in mouse (2), although this possibility is still controversial (3). A second mechanism of compensation is provided by alternative metabolic pathways or regulatory networks (4). Although such compensatory mechanisms have been extensively studied, especially in yeast and nematode (1), the roles of metabolic and network compensatory pathways are not well understood in mouse.Long-term potentiation (LTP) and long-term depression (LTD) are long-lasting forms of synaptic plasticity that are thought to be the cellular basis for learning and memory and proper formation of neural circuits during development (5). NMDA receptor (NMDAR)-mediated synaptic plasticity is a generally agreed postsynaptic mechanism in the hippocampus (5). In particular, synaptic Ca²⁺ influx through NMDARs is critical for LTP and LTD through control of various protein kinases and phosphatases (6). LTP is in part dependent upon the activation of protein kinases, which phosphorylate target proteins (6). Several kinases are activated during the induction of LTP, including cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinases (cGKs) (6). In contrast, LTD results from activation of phosphatases that dephosphorylate target proteins (6), and calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase, is important for LTD expression (7). AMPA receptors (AMPARs) are postsynaptic glutamate receptors that mediate rapid excitatory transmission in the central nervous system (8). During LTP, activated kinases phosphorylate AMPARs, leading to synaptic trafficking of the receptors to increase synapse activity (5). For LTD, activation of postsynaptic phosphatases induces internalization of AMPARs from the synaptic membrane, thereby reducing synaptic strength (5). Therefore, both protein kinases and phosphatases control synaptic trafficking of AMPARs, underlying LTP and LTD.AMPARs are tetrameric ligand-gated ion channels that consist of a combinatorial assembly of four subunits (GluA1–4) (9). Studies of GluA1 knockout (KO) mice show that GluA1 is critical for LTP in the CA1 region of the hippocampus (10). GluA1 homomers, like all GluA2-lacking/GluA1-containing receptors, are sensitive to polyamine block and are Ca²⁺-permeable, whereas GluA2-containing AMPARs are Ca²⁺-impermeable (9). Moreover, GluA1 is the major subunit that is trafficked from recycling endosomes to the synaptic membrane in response to neuronal activity (11). Phosphorylation of GluA1 within its intracellular carboxyl-terminal domain (CTD) can regulate AMPAR membrane trafficking (12). Several CTD phosphorylations regulate trafficking (6). In particular, PKA and cGKII both phosphorylate serine 845 of GluA1, increasing the level of extrasynaptic receptors (13, 14). Therefore, activation of PKA and cGKII during LTP induction increases GluA1 phosphorylation, which enhances AMPAR activity at synapses. On the other hand, calcineurin dephosphorylates serine 845 of GluA1, which enables GluA1-containing AMPARs to be endocytosed from the plasma membrane during LTD (15, 16). This removes synaptic AMPARs, leading to reduction of receptor function during LTD. Taken together, the activity-dependent trafficking of synaptic GluA1 is regulated by the status of phosphorylation in the CTD, which provides a critical mechanism underlying LTP and LTD.Several studies have shown that acute inhibition of cGKII impairs hippocampal LTP (13, 17, 18). However, cGKII KO animals show apparently normal LTP in the hippocampus (19), suggesting that a form of functional compensation takes place in the KO hippocampus. Here, we show that cGKII KO reduces Ca²⁺ signals by decreasing cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors (IP3Rs), which in turn lowers calcineurin activity in hippocampal neurons, which stabilizes phosphorylation of GluA1 in homomeric, Ca²⁺-permeable AMPARs (CPARs). This elevates CPARs at the synapse as a previously unidentified compensatory mechanism for hippocampal LTP in cGKII-deficient animals that is alternative to the form of LTP expressed in WT.     

Das 8 %ige Capsaicin-Pflaster bei Phantomschmerz

Background

Post amputation pain presents a challenge for pain physicians and is often detrimental to the patient’s quality of life.

Patients and methods

A prospective 12-week non-interventional study (NIS) was conducted in Germany to obtain data on the effectiveness and safety of capsaicin 8?% cutaneous patches from real life use in patients with peripheral neuropathic pain. For the first time in a subgroup of amputees data on post amputation pain were collected. This article presents the results for patients who suffered from phantom limb pain (PLP), stump pain (SP) and combined phantom limb/stump pain (PLP/SP).

Results

The analyses included 21 patients with post amputation pain (PLP: n?=?10, SP: n?=?4, PLP/SP: n?=?7). The mean duration of pain (± standard deviation) was 12.8?±?13.0 years for PLP, 23.1?±?29.9 years for SP and 11.0?±?15.8 years for PLP/SP. A single treatment with capsaicin 8?% cutaneous patches significantly reduced the average pain intensity over the observational period of 12 weeks. The mean numeric pain rating scale (NPRS) baseline score changed by ??2.4 for PLP with a standard error of the mean (SEM) of 0.4 (median: ??2.9, Q1: ??3.5, Q3: ??1.0), ??1.7 for SP (SEM: 0.8, median: ??1.1, Q1: ??2.9, Q3: ??0.5) and ??1.5 for PLP/SP (SEM: 0.6, median: ??2.0, Q1: ??2.3, Q3: 0) during weeks 1–12. The 30 % responder rates (i.e. ≥?30?% reduction in pain, day 7/14 to week 12) were 70.0?% (PLP), 50.0?% (SP) and 28.6?% (PLP/SP). PLP and PLP/SP patients in particular, benefited from improvements in pain attacks, sleep duration and sleep quality and one patient (PLP/SP) reported an adverse drug reaction (increase of pain). Physicians rated the tolerability of the patch as very good or good in 90.5?% of patients. A poor tolerability was stated for none of the 21 amputees. Of the patients 80 % for PLP and 50?% for both SP and PLP/SP expressed the wish to receive retreatment with capsaicin 8?% patches.

Conclusion

Capsaicin 8?% cutaneous patches seem to be effective and safe for the treatment of post amputation pain, notably in patients suffering from phantom limb pain.     

Transient receptor potential channels as molecular substrates of receptor-mediated cation entry

Calcium is a versatile multitarget intracellular second messenger in eukaryotic cells. In addition to calcium release from intracellular stores and influx via voltage- or ligand-operated channels, agonist-induced calcium entry constitutes one of the main pathways by which cytosolic calcium is elevated. Receptor-stimulated currents are initiated in response to agonist binding to G-protein-coupled receptors and to receptor tyrosine kinases. Within the past few years our knowledge about the molecular identity of receptor-stimulated channels has expanded substantially. Drosophila melanogaster visual transduction channels associated with the transient receptor potential (trp) and the trp-like (trpl) mutant visual phenotypes were the first members of this category of channels to be identified at the molecular level. Since then an entire mammalian gene family of TRP homologues has been discovered by homology cloning. Only now are we beginning to fully understand the functional roles of TRP channels in mammalian cells. We review recent findings in TRP channel research and discuss the role of these proteins for receptor-activated cation entry.     

Collection of Wound Exudate From Human Digit Tip Amputations Does Not Impair Regenerative Healing: A Randomized Trial

The regrowth of amputated digit tips represents a unique regenerative healing in mammals with subcutaneous volume regrowth, restoration of dactylogram, and suppression of scar formation. Although factor analysis in amphibians and even in mice is easy to obtain, safety of harvesting biomaterial from human digit tip amputations for analysis has not yet been described.The aim of this study was to evaluate if recovering wound exudate does hamper clinical outcome or influence microbiologic or inflammation status.A predefined cohort of 18 patients with fresh digit tip amputations was randomly assigned to receive standard therapy (debridement, occlusive dressing) with (n = 9) or without (n = 9) collection of the whole wound exudate in every dressing change. Primary endpoint (lengthening) and secondary endpoints (regeneration of dactylogram, nail bed and bone healing, time to complete wound closure, scar formation, 2-point discrimination, microbiologic analysis, inflammatory factors interleukin (IL)-1α, tumor necrosis factor-α, IL-4, and IL-6) were determined by an independent, blinded observer.Patients’ characteristics showed no significant differences between the groups. All patients completed the study to the end of 3 months follow-up. Exudate collection did not influence primary and secondary endpoints. Furthermore, positive microbiologic findings as well as pus- and necrosis-like appearance neither impaired tissue restoration nor influenced inflammatory factor release.Here, the authors developed an easy and safe protocol for harvesting wound exudate from human digit tip amputations. For the first time, it was shown that harvesting does not impair regenerative healing. Using this method, further studies can be conducted to analyze regeneration associated factors in the human digit tip.DRKS.de Identifier: DRKS00006882 (UTN: U1111-1166-5723).