Mitochondrial complex I activity in microglia sustains neuroinflammation.

Sustained smouldering, or low-grade activation, of myeloid cells is a common hallmark of several chronic neurological diseases, including multiple sclerosis1. Distinct metabolic and mitochondrial features guide the activation and the diverse functional states of myeloid cells2. However, how these metabolic features act to perpetuate inflammation of the central nervous system is unclear. Here, using a multiomics approach, we identify a molecular signature that sustains the activation of microglia through mitochondrial complex I activity driving reverse electron transport and the production of reactive oxygen species. Mechanistically, blocking complex I in pro-inflammatory microglia protects the central nervous system against neurotoxic damage and improves functional outcomes in an animal disease model in vivo. Complex I activity in microglia is a potential therapeutic target to foster neuroprotection in chronic inflammatory disorders of the central nervous system3.

Strategies for fighting mitochondrial diseases.

Mitochondrial diseases are extremely heterogeneous genetic conditions characterized by faulty oxidative phosphorylation (OXPHOS). OXPHOS deficiency can be the result of mutation in mtDNA genes, encoding either proteins (13 subunits of the mitochondrial complexes I, III, IV and V) or the tRNA and rRNA components of the in situ mtDNA translation. The remaining mitochondrial disease genes are in the nucleus, encoding proteins with a huge variety of functions, from structural subunits of the mitochondrial complexes, to factors involved in their formation and regulation, components of the mtDNA replication and expression machinery, biosynthetic enzymes for the biosynthesis or incorporation of prosthetic groups, components of the mitochondrial quality control and proteostasis, enzymes involved in the clearance of toxic compounds, factors involved in the formation of the lipid milieu, etc. These different functions represent potential targets for 'general' therapeutic interventions, as they may be adapted to a number of different mitochondrial conditions. This is in contrast with 'tailored', personalized therapeutic approaches, such as gene therapy, cell therapy and organ replacement, that can be useful only for individual conditions. This review will present the most recent concepts emerged from preclinical work and the attempts to translate them into the clinics. The common notion that mitochondrial disorders have no cure is currently challenged by a massive effort of scientists and clinicians, and we do expect that thanks to this intensive investigation work and tangible results for the development of strategies amenable to the treatment of patients with these tremendously difficult conditions are not so far away.

SURF1 knockout cloned pigs: Early onset of a severe lethal phenotype.

Leigh syndrome (LS) associated with cytochrome c oxidase (COX) deficiency is an early onset, fatal mitochondrial encephalopathy, leading to multiple neurological failure and eventually death, usually in the first decade of life. Mutations in SURF1, a nuclear gene encoding a mitochondrial protein involved in COX assembly, are among the most common causes of LS. LSSURF1 patients display severe, isolated COX deficiency in all tissues, including cultured fibroblasts and skeletal muscle. Recombinant, constitutive SURF1-/- mice show diffuse COX deficiency, but fail to recapitulate the severity of the human clinical phenotype. Pigs are an attractive alternative model for human diseases, because of their size, as well as metabolic, physiological and genetic similarity to humans. Here, we determined the complete sequence of the swine SURF1 gene, disrupted it in pig primary fibroblast cell lines using both TALENs and CRISPR/Cas9 genome editing systems, before finally generating SURF1-/- and SURF1-/+ pigs by Somatic Cell Nuclear Transfer (SCNT). SURF1-/- pigs were characterized by failure to thrive, muscle weakness and highly reduced life span with elevated perinatal mortality, compared to heterozygous SURF1-/+ and wild type littermates. Surprisingly, no obvious COX deficiency was detected in SURF1-/- tissues, although histochemical analysis revealed the presence of COX deficiency in jejunum villi and total mRNA sequencing (RNAseq) showed that several COX subunit-encoding genes were significantly down-regulated in SURF1-/- skeletal muscles. In addition, neuropathological findings, indicated a delay in central nervous system development of newborn SURF1-/- piglets. Our results suggest a broader role of sSURF1 in mitochondrial bioenergetics.

AAV9-based gene therapy partially ameliorates the clinical phenotype of a mouse model of Leigh syndrome.

Leigh syndrome (LS) is the most common infantile mitochondrial encephalopathy. No treatment is currently available for this condition. Mice lacking Ndufs4, encoding NADH: ubiquinone oxidoreductase iron-sulfur protein 4 (NDUFS4) recapitulates the main findings of complex I (cI)-related LS, including severe multisystemic cI deficiency and progressive neurodegeneration. In order to develop a gene therapy approach for LS, we used here an AAV2/9 vector carrying the human NDUFS4 coding sequence (hNDUFS4). We administered AAV2/9-hNDUFS4 by intravenous (IV) and/or intracerebroventricular (ICV) routes to either newborn or young Ndufs4-/- mice. We found that IV administration alone was only able to correct the cI deficiency in peripheral organs, whereas ICV administration partially corrected the deficiency in the brain. However, both treatments failed to improve the clinical phenotype or to prolong the lifespan of Ndufs4-/- mice. In contrast, combined IV and ICV treatments resulted, along with increased cI activity, in the amelioration of the rotarod performance and in a significant prolongation of the lifespan. Our results indicate that extraneurological organs have an important role in LS pathogenesis and provide an insight into current limitations of adeno-associated virus (AAV)-mediated gene therapy in multisystem disorders. These findings warrant future investigations to develop new vectors able to efficiently target multiple organs.

Mitochondrial reverse electron transport in myeloid cells perpetuates neuroinflammation.

Sustained smouldering, or low grade, activation of myeloid cells is a common hallmark of several chronic neurological diseases, including multiple sclerosis (MS) 1 . Distinct metabolic and mitochondrial features guide the activation and the diverse functional states of myeloid cells 2 . However, how these metabolic features act to perpetuate neuroinflammation is currently unknown. Using a multiomics approach, we identified a new molecular signature that perpetuates the activation of myeloid cells through mitochondrial complex II (CII) and I (CI) activity driving reverse electron transport (RET) and the production of reactive oxygen species (ROS). Blocking RET in pro-inflammatory myeloid cells protected the central nervous system (CNS) against neurotoxic damage and improved functional outcomes in animal disease models in vivo . Our data show that RET in myeloid cells is a potential new therapeutic target to foster neuroprotection in smouldering inflammatory CNS disorders 3 .

C-Raf antagonizes apoptosis induced by IFN-alpha in human lung cancer cells by phosphorylation and increase of the intracellular content of elongation factor 1A.

Interferon alpha (IFNalpha) induces both apoptosis and a counteracting epidermal growth factor Erk-dependent survival response in cancer cells. In this report, IFNalpha increased eukaryotic elongation factor 1A (eEF-1A) protein expression by inhibition of eEF-1A degradation via a proteasome-dependent pathway. The reduction of the expression level of eEF-1A by RNA interference enhanced the apoptosis induced by IFNalpha on the same cells. Moreover, IFNalpha induced the phosphorylation of both serine and threonine in eEF-1A. These effects were paralleled by an increased co-immunoprecipitation and colocalization of eEF-1A with C-Raf. The suppression of C-Raf kinase activity with the inhibitor BAY 43-9006 completely antagonized the increase of both eEF-1A phosphorylation and expression and of C-Raf/eEF-1A colocalization induced by IFNalpha and enhanced apoptosis and eEF-1A ubiquitination. Cell transfection with the mutated K48R ubiquitin increased EF-1A expression and desensitized tumor cells to the modulating effects of IFNalpha. The dynamic simulation of 3Dstructure of eEF-1A identified putative serine and threonine phosphorylation sites. In conclusion, the interaction between eEF-1A and C-Raf increases eEF-1A stability and induces a survival activity.

Integrated allosteric model of voltage gating of HCN channels.

Hyperpolarization-activated (pacemaker) channels are dually gated by negative voltage and intracellular cAMP. Kinetics of native cardiac f-channels are not compatible with HH gating, and require closed/open multistate models. We verified that members of the HCN channel family (mHCN1, hHCN2, hHCN4) also have properties not complying with HH gating, such as sigmoidal activation and deactivation, activation deviating from fixed power of an exponential, removal of activation "delay" by preconditioning hyperpolarization. Previous work on native channels has indicated that the shifting action of cAMP on the open probability (Po) curve can be accounted for by an allosteric model, whereby cAMP binds more favorably to open than closed channels. We therefore asked whether not only cAMP-dependent, but also voltage-dependent gating of hyperpolarization-activated channels could be explained by an allosteric model. We hypothesized that HCN channels are tetramers and that each subunit comprises a voltage sensor moving between "reluctant" and "willing" states, whereas voltage sensors are independently gated by voltage, channel closed/open transitions occur allosterically. These hypotheses led to a multistate scheme comprising five open and five closed channel states. We estimated model rate constants by fitting first activation delay curves and single exponential time constant curves, and then individual activation/deactivation traces. By simply using different sets of rate constants, the model accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the different kinetic properties of different isoforms. For example, faster kinetics of HCN1 relative to HCN2/HCN4 are attributable to higher HCN1 voltage sensors' rates and looser voltage-independent interactions between subunits in closed/open transitions. It also accounts for experimental evidence that reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus involves two processes: voltage sensor gating and allosteric opening/closing.

Antitumor therapeutic strategies based on the targeting of epidermal growth factor-induced survival pathways.

Cellular receptors for the Epidermal Growth Factor are considered important targets for the experimental treatment of human cancer. Monoclonal antibodies as well as small tyrosine kinase inhibitors have been developed and have undergone extensive evaluation in preclinical and clinical studies. Most of these studies have been conceived on the general idea that epidermal growth factor receptor (EGFR) plays a critical role on the growth and survival of human tumors. This assumption has been derived by the successful development of BCR/ABL tyrosine kinase inhibitors in human chronic myeloid leukemia as well as on the activity of antiCD20 monoclonal antibodies in lymphoproliferative disease and of anti HER2 agents in breast tumors overexpressing the targeted antigens. It is now becoming clear that factors regulating sensitivity to kinase inhibitors may differ from monoclonal antibodies and that the molecules targeted by interferring drugs must be prioritaire for growth and survival of those specific tumors in order to achieve valuable results. Recent evidence of major responses to the EGFR inhibitor Gefitinib in tumors harboring activating mutations in the EGFR appears on line with this concept. In this article we will discuss the significance of targeting the EGFR driven survival pathways. Specifically, we will afford the point of EGFR survival signalling prioritization by means of pharmacological treatment. Finally, we will address the role of profiling technologies and of novel computational system biology-based approaches for identification of innovative strategies for effective targeting of EGFR driven survival pathways.

Effects of dronedarone on acetylcholine-activated current in rabbit SAN cells.

1. The effect of the antiarrhythmic drug dronedarone on the Acetylcholine-activated K(+) current (I(K(ACh))) was investigated in single cells isolated from sinoatrial node (SAN) tissue of rabbit hearts. 2. Externally perfused dronedarone (0.001 - 1 microM) caused a potent, voltage independent block of I(K(ACh)). Fitting of the dose response curve of I(K(ACh)) block yielded an IC(50) value of 63 nM, a value over one order of magnitude lower than those reported for dronedarone block of other cardiac currents. 3. I(K(ACh)) block was not due to an inhibitory action of dronedarone on the muscarinic M2 receptor activation, since the drug was effective on I(K(ACh)) constitutively activated by intracellular perfusion with GTP-gammaS. 4. External cell perfusion with dronedarone inhibited the activity of I(K(ACh)) channels recorded from cell-attached patches by reducing the channel open probability (from 0.56 to 0.11) without modification of the single-channel conductance. 5. These data suggest that dronedarone blocks I(K(ACh)) channels either by disrupting the G-protein-mediated activation or by a direct inhibitory interaction with the channel protein.

Opioid-induced rigidity after intravenous fentanyl.

BACKGROUND: Large doses of intravenous opioids may cause severe rigidity and prevent spontaneous or controlled ventilation. The mechanism of this effect appears to be neuraxis dopamine antagonism. Rigidity after analgesic doses of fentanyl has not been reported previously. CASE: A pregnant woman receiving haloperidol for multiple psychiatric conditions presented for evaluation of vaginal bleeding. Intravenous fentanyl was administered to facilitate vaginal examination. Severe rigidity of the extremities and truncal region occurred, which prevented spontaneous or assisted ventilation. CONCLUSION: Opioid administration may result in rigidity and respiratory embarrassment. Decreased analgesic requirements in pregnancy and concomitant butyrophenone administration may predispose to opioid-induced rigidity.

Patient-controlled epidural analgesia during labor.

This study compared the safety, efficacy, local anesthetic usage, patient satisfaction, and anesthesia manpower demands of patient-controlled epidural analgesia and continuous epidural infusion during labor. After establishment of epidural analgesia, 88 parturients with vertex presentation were assigned randomly to receive either patient-controlled epidural analgesia or continuous epidural infusion, using 0.125% bupivacaine containing 1 microgram/mL of fentanyl. Inadequate analgesia was treated in both groups with a 10-mL "top-up" of 0.25% bupivacaine. Patients receiving patient-controlled epidural analgesia required significantly fewer supplemental top-up doses (36 versus 71%; P less than .05) and insignificantly less local anesthetic (13.6 +/- 0.6 versus 14.6 +/- 0.5 mL/hour; P = .10). The two groups did not differ in incidence of hypotension, high sensory blockade (above T8), mode of delivery, or patient satisfaction assessed by questionnaire. Use of local anesthetic solution was examined with respect to cervical dilatation and did not increase late in labor. Patients generally viewed infusion technology favorably. These findings suggest that patient-controlled epidural analgesia is safe and effective, reduces anesthesia manpower needs, and is well accepted.

Intrathecal lipophilic opioids as adjuncts to surgical spinal anesthesia.

BACKGROUND AND OBJECTIVES: Lipophilic opioids, especially fentanyl and sufentanil, are increasingly being administered intrathecally as adjuncts to spinal anesthesia. This review analyzes the efficacy of these opioids for subarachnoid anesthesia. METHODS: Medline search of the literature from 1980 to the present and a survey of recent meeting abstracts are reviewed. RESULTS: A significant number of citations regarding intrathecal lipophilic opioids as adjuncts to spinal anesthesia were found: 59 are cited in this review. Most clinical experience has been in obstetric surgery, but lipophilic spinal opioid administration is being used with greater frequency for other surgical procedures as well. The benefits include reduction of minimal alveolar concentration (MAC) when general anesthesia is combined with spinal anesthesia and enhancement of the quality of spinal anesthesia without prolongation of motor block. Intrathecal fentanyl and sufentanil allow clinicians to use smaller doses of spinal local anesthetic, yet still provide excellent anesthesia for surgical procedures. Furthermore, lipophilic opioid/local anesthetic combination permits more rapid motor recovery; short outpatient procedures are therefore more amenable to spinal anesthesia. Finally, the side-effect profiles of intrathecal lipophilic opioids are now well characterized and appear less troublesome than intrathecal morphine. CONCLUSIONS: The anesthesia-enhancing properties and side-effect profile of lipophilic opioids administered intrathecally suggest significant roles for these agents as adjuncts to spinal anesthesia for obstetric and outpatient procedures.

Maternal fever, neonatal sepsis evaluation, and epidural labor analgesia.

BACKGROUND AND OBJECTIVES: Numerous studies have found an association between epidural analgesia for labor and maternal fever (temperature > or =38 degrees C). Maternal fever often results in treatment with maternal or neonatal antibiotics, neonatal sepsis evaluation, and increased costs. METHODS: Medline was used to identify literature regarding the association between epidural labor analgesia and maternal fever/neonatal sepsis. Studies examining thermoregulation during pregnancy and/or epidural analgesia were also reviewed. RESULTS: There appears to be a strong association between epidural labor analgesia and maternal fever. The link between epidural labor analgesia and neonatal sepsis evaluation is less clear. The incidence of confirmed neonatal sepsis does not increase with maternal epidural analgesia. Causes of the association between epidural labor analgesia and maternal fever include selection bias, altered thermoregulation, and increased shivering or decreased sweating with epidural analgesia. CONCLUSIONS: Maternal epidural labor analgesia is associated with maternal fever and possibly increased neonatal sepsis evaluation. There is no proof the relationship is causal.

The effect of distance from injection site to the brainstem using spinal sufentanil.

BACKGROUND AND OBJECTIVES: Intrathecal (IT) sufentanil is commonly used in parturients to provide rapid onset of labor analgesia without motor block. This practice, although widely used, has been associated with severe respiratory depression in some patients. The mechanism of this respiratory depression is unclear, however, rapid cephalad movement and interaction with parenteral opioids are 2 frequently cited explanations for this complication. Because this complication has occurred only in women with heights between 150 and 157 cm, we elected to study the effect of the distance from injection site to the cisterna magna (CM) on peak brainstem cerebrospinal fluid (CSF) concentrations. METHODS: Ten adult ewes were injected with IT sufentanil (0.3 microg/kg) at a mean distance of either 71 cm (65 to 78 cm) from the brainstem (pelvic group) or 37 cm (34 to 42 cm) from the brainstem (thoracic group). CSF was then sampled at 5-minute intervals from the CM. RESULTS: Measurable CM concentrations of sufentanil were noted in the brainstem at 20 and 25 minutes, respectively, for the thoracic and the pelvic groups. Peak sufentanil concentrations from the thoracic group were nearly 10-fold higher (0.553 +/- 0.43 ng/mL) compared with the pelvic group (0.064 +/- 0.002 ng/mL) when measured in the CM (P =.023). CONCLUSIONS: Our results suggest that sufentanil migrates relatively large distances in the IT space. Injection site (distance from the brainstem) appears to be a prominent factor in determining brainstem concentrations and subsequent respiratory depression after spinal administration. Reg Anesth Pain Med 2001;26:306-309.

Spinal anesthesia versus intravenous sedation for transvaginal oocyte retrieval: reproductive outcome, side-effects and recovery profiles.

Transvaginal ultrasonically guided oocyte retrieval is commonly performed as part of in vitro fertilization efforts. The impact of anesthetic management on patient outcome from this procedure has not been well characterized. At our institution, patients are offered a choice of either heavy intravenous sedation or spinal anesthesia with minimal or no sedatives. In this pilot study, we retrospectively reviewed the anesthetic management, reproductive outcome and recovery room experience for all patients having oocyte retrieval during a 2-year interval (n = 95). Fifty-one oocyte retrievals were performed under spinal anesthesia, while 44 patients received solely intravenous sedatives. Both groups had similar reproductive outcomes. The intravenous sedation group required a significantly longer period until recovery room discharge criteria were met (P = 0.03), and were more likely to have postoperative emetic episodes (46% versus 6% in the spinal anesthesia group: P < 0.01). Two unplanned hospital admissions occurred in the intravenous sedation group: both were related to uncontrolled nausea and vomiting. We conclude that spinal anesthesia may have advantages over intravenous sedation for oocyte retrieval.

The use of remifentanil infusion to facilitate epidural catheter placement in a parturient: a case report with pharmacokinetic simulations.

We present a case in which remifentanil infusion was used to provide analgesia during epidural catheter placement in a parturient who was experiencing great difficulty staying motionless because of extremely painful uterine contractions. Remifentanil may provide certain advantages in this setting, including improved analgesia during the procedure, briefer residual maternal and fetal (or newborn) drug effects after the procedure, and greater technical ease of catheter placement because of decreased movement induced by pain. Pharmacokinetic simulation of the dose administered suggests that analgesic effect-site concentrations can be rapidly produced, and that these concentrations decline rapidly to clinically insignificant levels after the infusion.

Labor epidural catheter reactivation or spinal anesthesia for delayed postpartum tubal ligation: a cost comparison.

STUDY OBJECTIVES: To evaluate the costs and resource consumption associated with utilizing epidural catheters placed during labor versus spinal anesthesia for postpartum tubal ligation. To examine maternal demographics, anesthetic management variables, and time interval from delivery until surgery for association with epidural catheter reactivation success rate. DESIGN: Retrospective study. SETTING: University hospital labor and delivery center. PATIENTS: 120 consecutive postpartum patients with tubal ligations performed between June 1991 and December 1993. INTERVENTIONS: Postpartum women scheduled for tubal ligation with labor epidural catheters in place either had local anesthetic injected via the epidural catheter (n = 45) or had the catheter removed without reinjection and spinal anesthetic administered (n = 20). Patients with inadequate epidural anesthesia went on to receive spinal anesthesia. Women without a labor epidural catheter received spinal anesthesia (n = 55). MEASUREMENTS AND MAIN RESULTS: Adequate anesthesia for tubal ligation was achieved in 78% of women after reinjection of their epidural catheter. Operating room (OR) and anesthesia times were highest when epidural catheter reactivation was unsuccessful, intermediate when epidural catheter reactivation was successful, and lowest with initial spinal anesthesia (p < 0.05). The longer OR and anesthesia provider times associated with epidural catheter reactivation increased patient charges on average of $176 compared with the initial use of spinal anesthesia. CONCLUSIONS: Spinal anesthesia for postpartum tubal ligation was associated with lower anesthesia professional fees and OR charges compared with attempted reactivation of epidural catheters placed during labor. Anesthesiologists should weigh the cost advantages of spinal anesthesia against the small, but increased probability of headache after dural puncture.