Slow Time Course Research Articles

Evaluation Of Bcma As a Therapeutic Target In Multiple Myeloma Using An Antibody-Drug Conjugate

B-cell maturation antigen (BCMA) is upregulated at the terminal stages of plasma cell (PC) differentiation, and is expressed on normal and malignant PC. Apart from low levels of mRNA detected on dendritic cells, expression appears absent on other tissues, indicating the potential as a target for novel antibody therapeutics for multiple myeloma (MM). We generated a humanised anti-BCMA antibody, modified for Fc-enhanced function, and conjugated to mcMMAF (anti-BCMA antibody drug conjugate, ADC). Flow cytometric studies on human myeloma cell lines (HMCL) showed rapid internalisation of anti-BCMA antibody by flow cytometry and confocal microscopy. The internalised antibody was transported to the lysosome and was nearly undetectable by confocal microscopy after 6 hours, indicating the suitability of BCMA as a target for an antibody drug conjugate (ADC). BCMA expression reached original surface levels by 6 hours post antibody treatment, thus maintaining the cell as a target for effector mediated killing. Evaluation of BCMA expression on HMCL revealed variable surface expression (1/11 high, 5/11 moderate, 5/11 low). Tumour cell killing by the ADC was expression level, dose and time dependent. The highest expressing HMCL, H929, showed significant killing (60% at 100ng/mL) at 24 hours, and up to 90% after 2 days. Cells expressing moderate levels of BCMA required incubation for up to 4 days to show maximal levels of cell death, suggesting the importance of continued internalisation of the antibody/antigen complexes over this period. ARH77 cells were transduced to express varying levels of BCMA, and killing at 3 days (200ng/ml) was directly proportional to level of surface expression (NT 0% killing, Low BCMA 75% killing, High BCMA 90%). We studied surface antigen levels in a cohort of patients to ascertain the need for patient selection. Like the HMCL, patient CD138+ plasma cells (PC) displayed a range of expression. Of 67 patients tested, CD138+ PC from 12 expressed high levels, 52 expressed intermediate, and 3 had low/negative surface BCMA as determined by MFI ratio of specific antibody to isotype control. Non-CD138+ cells from the bone marrow (BM) were negative for BCMA. Immuno-histochemistry (IHC) on paraffin-embedded BM sections, using a murine antibody and dual staining with anti-Blimp1 to identify tumour cells, revealed both membrane and diffuse, or punctate, cytoplasmic staining. Expression levels varied, from high uniform, to heterogeneous and patchy, to uniform low level. In no patient were the tumour cells entirely negative for BCMA. There was broad correlation between FACS analysis and IHC, thus patients were divided into high, moderate and low expressing groups. Examination of patient and disease characteristics revealed no correlation between BCMA expression and disease stage, response to last treatment, time from diagnosis, isotype, CD56 expression, or cyclin D-type, but there was a trend towards higher BCMA levels in tumours with adverse genetics (90% of patients with adverse genetics had high/moderate levels cf 64% of patients with standard CGN (p=0.06, Fisher’s exact test, 2-tailed). CD138+ cells in LPL (n=3) were positive for BCMA, but CD20+ lymphocytes were negative. Serum BCMA levels in MM patients (175.6±242.6ng/mL; mean±SD, n=34) were higher than in normal subjects (9.28±1.9ng/mL; n=38) but no correlation with bone marrow plasmacytosis or surface BCMA was noted. Levels appeared similar between new diagnosis (147.6±190.8ng/mL; mean±SD, n=8) and relapsed disease (184.3±259.1ng/mL; n=26). We tested ADC activity on primary tumour cells in whole BM cultures, enumerating viable CD138+ cells by flow cytometry. As with the HMCL, ADC mediated cytotoxicity was expression level, dose and time dependent, with a slower time course than with HMCL, perhaps reflecting the slower turn-over of these cells. In samples expressing moderate levels of BCMA, ADC-mediated cytotoxicity increased from 23.1±2.9% (mean±SEM, n=6) at 1-2 days to 48.3±5.1% at 4 days, and 61.2±6.2% by 6-7 days. Optimal doses of ADC ranged from 500ng-1ug/ml. In summary, these preclinical data in MM support the potential utility of an anti-BCMA ADC across the whole MM population, perhaps with particular efficacy in patients with adverse genetics, for whom an unmet need remains. Disclosures: Yong: GSK: Research Funding. Germaschewski:GSK: Employment. Mayes:GlaxoSmithKline: Employment. Sully:GlaxoSmithKline: Employment. Seestaller-Wehr:GlaxoSmithKline: Employment. Fieles:GlaxoSmithKline: Employment. Tunstead:GlaxoSmithKline: Employment. McCahon:GlaxoSmithKline: Employment. Craigen:GlaxoSmithKline: Employment.

Rebuttal from Scott K. Powers, Ashley J. Smuder, David Fuller and Sanford Levine

Drs Sieck and Mantilla have presented an eloquent argument that diaphragm muscle fibre atrophy following unilateral phrenicotomy, tetrodotoxin (TTX) nerve blockade or high cervical spinal hemisection injury does not result from muscle inactivity alone. Nonetheless, we retain our position that when diaphragmatic contractile work is diminished during prolonged mechanical ventilation (MV), the ensuing diaphragm fibre atrophy occurs primarily due to decreased diaphragm contractile activity. In our response to Dr Sieck and Mantilla's position, we highlight several fundamental differences between the unilateral denervation model of diaphragm inactivity and the reduced diaphragmatic contractile work that occurs during prolonged MV. A fundamental difference between these two models of diaphragm inactivity is that prolonged MV results in atrophy of all diaphragm muscle fibre types whereas unilateral phrenic denervation promotes a transient hypertrophy of diaphragmatic type I and IIa fibres followed by atrophy of type IIx and IIb fibres. This is a unique physiological response as muscle hypertrophy does not occur in any other model of skeletal muscle inactivity. Another major difference is that diaphragm movement patterns differ markedly between the unilateral phrenic denervation model of diaphragm inactivity and MV. During MV, both hemidiaphragms move synchronously as the lungs inflate and empty. In contrast, following unilateral phrenic denervation, the contralateral hemidiaphragm contracts and shortens, thus eliciting small fibre length changes in the paralysed hemidiaphragm (Zhan et al. 1995). Since muscle fibre size is a highly regulated process controlled by mechanical signals, neurotrophic influences, and paracrine signalling which regulate muscle protein turnover (Rennie et al. 2004), the possibility exists that distortion of the paralysed diaphragm elicits an anabolic signalling response to promote hypertrophy. Importantly, not all studies report diaphragmatic hypertrophy following denervation-induced inactivity. Indeed, a study from Dr Sieck's laboratory revealed that unilateral denervation of the rat diaphragm results in a net loss of muscle protein after 5 days (Argadine et al. 2009). It follows that this deficit of muscle protein should translate into diaphragm fibre atrophy but unfortunately, this study did not report diaphragm fibre size. Further, in direct contrast to the rat data, hemidiaphragm paralysis in humans results in atrophy of diaphragm slow muscle fibres with atrophy of fast fibres occurring at a slower time course (Welvaart et al. 2011). Interestingly, these results agree with the Sieck–Mantilla initial theoretical argument that inactivity-induced diaphragm muscle fibre atrophy should occur most rapidly in slow fibres. In conclusion, based on our work and the work of others, we maintain the position that during prolonged MV, when diaphragm work against external loads is greatly diminished, the ensuing diaphragm atrophy is primarily due to decreased diaphragm contractile work.

55-OR

Aim The apparent clinical and research utility of soluble HLA (sHLA) underscores the need to further investigate immunological effects triggered by injections into mice. Developing reliable methods for immune response monitoring is crucial for individualizing therapies aimed at extending organ survival and improving patient health. In this study, we examined the humoral immune response elicited by immunization of sHLA into mice. Methods The method involved five steps: (1) production/purification of injectable grade sHLA; (2) immunization of 9 female BALB/c mice with 25, 50, and 150 μg of sHLA-A*02:01; (4) injections at day 0, 21, 42, 63; (5) blood samples taken at day 0, 21, 42, 63, 72; (3) monitoring immunized mice for HLA-specific antibody production by an in-house, 120-allele Luminex-based single specificity solid phase assay. Results Our results indicated that all mice immunized with sHLA-A*02:01 triggered an immune response. Titration analysis allowed appropriate measurements of semi-quantitative relationships between samples at different stages. It was noted that mice with 150 μg injection doses were capable to elicit high antibody titers as early as 21 days. Mice at lower injection doses had a slower time course. The average immune response distribution between loci was found to be highest in A alleles followed by C and weakest in B alleles. Overall, data indicate the generation of antibodies of broad specificity rather than against allogeneic determinants. Conclusions Low variability between Balb/c mice is explained by the fact that all mice have an identical genetic makeup. These findings underscore the potential importance of polymorphisms in the MHC locus on immune responses. Furthermore, this data may allow increasing accuracy and efficiency in the determination of immunological responses that could help monitor occurrences observed in humans. In addition, injectable sHLA may serve as quality reagents in the creation of therapeutic/diagnostic anti-HLA antibodies or to induce tolerance.

Indistinguishable Synaptic Pharmacodynamics of the N-Methyl-d-Aspartate Receptor Channel Blockers Memantine and Ketamine

Memantine and ketamine, voltage- and activation-dependent channel blockers of N-methyl-d-aspartate (NMDA) receptors (NMDARs), have enjoyed a recent resurgence in clinical interest. Steady-state pharmacodynamic differences between these blockers have been reported, but it is unclear whether the compounds differentially affect dynamic physiologic signaling. In this study, we explored nonequilibrium conditions relevant to synaptic transmission in hippocampal networks in dissociated culture and hippocampal slices. Equimolar memantine and ketamine had indistinguishable effects on the following measures: steady-state NMDA currents, NMDAR excitatory postsynaptic current (EPSC) decay kinetics, progressive EPSC inhibition during repetitive stimulation, and extrasynaptic NMDAR inhibition. Therapeutic drug efficacy and tolerability of memantine have been attributed to fast kinetics and strong voltage dependence. However, pulse depolarization in drug presence revealed a surprisingly slow and similar time course of equilibration for the two compounds, although memantine produced a more prominent fast component (62% versus 48%) of re-equilibration. Simulations predicted that low gating efficacy underlies the slow voltage-dependent relief from block. This prediction was empirically supported by faster voltage-dependent blocker re-equilibration with several experimental manipulations of gating efficacy. Excitatory postsynaptic potential-like voltage commands produced drug differences only with large, prolonged depolarizations unlikely to be attained physiologically. In fact, we found no difference between drugs on measures of spontaneous network activity or acute effects on plasticity in hippocampal slices. Despite indistinguishable synaptic pharmacodynamics, ketamine provided significantly greater neuroprotection from damage induced by oxygen glucose deprivation, consistent with the idea that under extreme depolarizing conditions, the biophysical difference between drugs becomes detectable. We conclude that despite subtle differences in voltage dependence, during physiologic activity, blocker pharmacodynamics are largely indistinguishable and largely voltage independent.

Thalamocortical relationships and network synchronization in a new genetic model “in mirror” for absence epilepsy

Electroencephalographic generalized spike and wave discharges (SWD), the hallmark of human absence seizures, are generated in thalamocortical networks. However, the potential alterations in these networks in terms of the efficacy of the reciprocal synaptic activities between the cortex and the thalamus are not known in this pathology. Here, the efficacy of these reciprocal connections is assessed in vitro in thalamocortical slices obtained from BS/Orl mice, which is a new genetic model of absence epilepsy. These mice show spontaneous SWD, and their features can be compared to that of BR/Orl mice, which are free of SWD. In addition, since gap junctions may modulate the efficacy of these connections, their implications in pharmacologically-induced epileptiform discharges were studied in the same slices.The thalamus and neocortex were independently stimulated and the electrically-evoked responses in both structures were recorded from the same slice. The synaptic efficacy of thalamocortical and corticothalamic connections were assessed by measuring the dynamic range of synaptic field potential changes in response to increasing stimulation strengths. The connection efficacy was weaker in epileptic mice however, this decrease in efficacy was more pronounced in thalamocortical afferents, thus introducing an imbalance in the reciprocal connections between the cortex and thalamus. However, short-term facilitation of the thalamocortical responses were increased in epileptic mice compared to non-epileptic animals. These features may favor occurrence of rhythmical activities in thalamocortical networks. In addition, carbenoxolone (a gap junction blocker) decreased the cumulative duration of 4-aminopyridine-induced ictal-like activities, with a slower time course in epileptic mice. However, the 4-aminopyridine-induced GABA-dependent negative potentials, which appeared to trigger the ictal-like activities, remained.Our results show that the balance of the reciprocal connections between the thalamus and cortex is altered in favor of the corticothalamic connections in epileptic mice, and suggest that gap junctions mediate a stronger cortical synchronization in this strain.

The glycolytic enzyme aldolase C is up-regulated in rat forebrain microsomes and in the cerebrospinal fluid after repetitive fluoxetine treatment

The antidepressant drug fluoxetine is widely used for the treatment of a broad range of psychiatric disorders. Its mechanism of action is thought to involve cellular adaptations that are induced with a slow time course after initiation of treatment. To gain insight into the signaling pathways underlying such changes, the expression levels of proteins in a microsomal sub-fraction enriched in intracellular membranes from the rat forebrain was analyzed after two weeks of treatment with fluoxetine. Proteins were separated by two-dimensional gel electrophoresis, and the differentially regulated protein spots were identified by mass spectrometry. Protein network analysis suggested that most of the identified proteins could potentially be regulated by the insulin family of proteins. Among them, Fructose-bisphosphate aldolase C (AldoC), a glycolytic/gluconeogenic enzyme primarily expressed in forebrain astrocytes, was up-regulated 7.6-fold. An immunohistochemical analysis of the dorsal hippocampus revealed a robust decrease (43±2%) in the co-localization of AldoC and the astrocyte marker GFAP and a diffuse staining pattern, compatible with AldoC secretion into the extracellular space. Consistently, AldoC, contained in an exosome-like fraction in astrocyte conditioned medium, increased significantly in the cerebrospinal fluid. Our findings strongly favor a non-canonic signaling role for AldoC in cellular adaptations induced by repetitive fluoxetine treatment.

Acute Ischemia Activates Distinct Ion Currents in Endothelial Cells and Pericytes of Capillaries in Guinea Pig Cochlear Lateral Wall

Capillary blood flow in spiral ligament (SL) and strial vascularis (SV) of the cochlear lateral wall are critical for cochlear health and to meet the high energy‐demand of auditory transduction, but the physio‐pathology of the local capillary cells remains poorly understood. Using acutely isolated capillary segments and whole‐cell recording techniques, we characterized the main membrane currents and the responses to acute ischemic treatment (AIT, hypercapnic pH 6.2, pO2=0 mmHg) of the endothelial cells (EC) and pericytes (PC) in the SL and SV. We found: 1) In physiological solutions, the PCs, but not ECs, showed a [K+]o‐regulated and Ba2+‐sensitive inward rectifier current (Kir). 2) ECs, but PCs rarely, showed a significant outward rectifier current, which was inhibited by 1 mM 4‐AP, 100 μM niflumic acid (NFA) but not TEA. 3) When recorded in situ of a vessel segment, both types of cells showed variable electrical coupling that was blocked by 30 μM 18β‐glycyrrhetinic acid or 100 μM 2‐APB. 4) AIT (3 – 24 min) caused a reversible and partially repeatable outward current at holding potential −20 mV in isolated ECs and PCs. The AIT‐activated net current had I‐V curves and kinetics which mostly resemble to those of Ca2+−activated Cl−channels: strong outward rectification with quick or slow time course to reach steady‐state within 0.5 – 2 s voltage steps. This AIT‐induced current was inhibited by niflumic acid and 1 mM TEA but not 4‐AP. 5) AIT (>;3 min) also inhibited gap junction coupling current. We conclude that the capillary EC and PC in the SV and SL express different K+‐channels, suggesting a role of PCs as the K+‐sensor for blood flow regulation, and the acute ischemia activates chloride channels and BK channels likely via elevation of cytosolic Ca2+ in both ECs and PCs. Supported by NIH grants DC004716 (ZGJ) and DC005983, DC 00105 (ALN).

I Ks channels open slowly because KCNE1 accessory subunits slow the movement of S4 voltage sensors in KCNQ1 pore-forming subunits

Human I(Ks) channels activate slowly with the onset of cardiac action potentials to repolarize the myocardium. I(Ks) channels are composed of KCNQ1 (Q1) pore-forming subunits that carry S4 voltage-sensor segments and KCNE1 (E1) accessory subunits. Together, Q1 and E1 subunits recapitulate the conductive and kinetic properties of I(Ks). How E1 modulates Q1 has been unclear. Investigators have variously posited that E1 slows the movement of S4 segments, slows opening and closing of the conduction pore, or modifies both aspects of electromechanical coupling. Here, we show that Q1 gating current can be resolved in the absence of E1, but not in its presence, consistent with slowed movement of the voltage sensor. E1 was directly demonstrated to slow S4 movement with a fluorescent probe on the Q1 voltage sensor. Direct correlation of the kinetics of S4 motion and ionic current indicated that slowing of sensor movement by E1 was both necessary and sufficient to determine the slow-activation time course of I(Ks).

Reply to A. Levy et al

In their letter, Levy et al provide thoughtful comments with respect to some of RECIST’s limitations and highlight recent developments regarding techniques toevaluatenovel targetedcompounds.LikeLevyetal, we too celebrate adaptations of response methodologies in the evaluation of anticancer therapies. Examples include incorporation of tumor morphology by computed tomography (CT) in colorectal cancer, tumoral enhancement in arterial CT phases for hepatocellular carcinoma, metabolic response by [F]fluorodeoxyglucose positron emission tomography (PET) for GI stromal tumors, and even modifying criteria to allow for agents that may have a slower time course to action than traditional anticancerdrugs(eg, immune-relatedresponsecriteria). Additionally, as Levy et al point out, adaptations that incorporate information regarding tumor dynamics such as tumor growth rate or the nonprogression index can also add valuable information when assessing tumor response to treatment. Despite the fact that these and other modifications are becoming available, our data suggest that the traditional RECIST assessment of change in tumor size is still generally meaningful when assessing tumor response, even in the current era of targeted agents. As we reported, more than 90% of patients included in our study had treatment regimens that included only targeted agents without traditional cytotoxic drugs. Additionally, our data were generated by analyzing a wide variety of tumor types across trials of agents with diverse mechanisms of action, but modified response methodologies have often been studied in limited settings only. Although specialized tumor response evaluation methodologies can be difficult to implement in a uniform fashion across global cancer treatment sites, traditional methods of evaluating change in tumor size by CT or magnetic resonance imaging are relatively straightforward to implement and have been in use by the global community for more than 30 years. Certainly no single tumor response methodology is better than others in all cases. There are trade-offs between using a generally applicable methodology versus measures that are more specific to individual settings. RECIST’s benefits are that it is straightforward and supported by many years of global experience. Our data suggest that incorporating RECIST evaluation of change in tumor size into early-phase clinical trials of novel agents as a key end point is reasonable and can provide critical information in assessing a novel drug’s potential efficacy. As we report, this is particularly true when tumor size changes are evaluated on a continuous scale rather than by RECIST’s complete response/partial response/stable disease/progressive disease response categorizations. In many cases, RECIST data may be well supplemented by a second, concurrently employed methodology that is appropriate for the situation such as [F]fluorodeoxyglucose PET, fluoro-l-thymidine PET, dynamic contrast-enhanced magnetic resonance imaging, magnetic resonance spectroscopy, modified RECIST criteria as referenced by Levy et al, and others. We look forward to the further refinement of RECIST and other tumor response methodologies, which will ultimately help our patients with cancer.

Rod and cone pathway signaling and interaction under mesopic illumination

This study investigates the time-course and post-receptoral pathway signaling of photoreceptor interactions when the rod (R) and three cone (L, M, S) photoreceptor classes contribute to mesopic vision. A four-primary photostimulator independently controls photoreceptor activity in human observers. The first experiment defines the temporal adaptation response of receptoral (L-, S-cone, rod) and post-receptoral (LMS, LMSR, +L-M) signaling and interactions. Here we show that nonopponent cone-cone interactions (L-cone, LMS, LMSR) have monophasic temporal response patterns whereas opponent signals (+L-M, S-cone) show biphasic response patterns with slower recovery. By comparison, rod-cone interactions with nonopponent signals have faster adaptation responses and reduced sensitivity loss whereas opponent rod-cone interactions are small or absent. Additionally, the rod-rod interaction differs from these interaction types and acts to increase rod sensitivity due to temporal summation but with a slower time course. The second experiment shows that the temporal profile of the rod signal alters the relative rod contributions to the three primary post-receptoral pathways. We demonstrate that rod signals generate luminance (+L+M) signals mediated via the MC pathway with all rod temporal profiles and chromatic signals (L/L+M, S/L+M) in both the PC and KC pathways with durations >75 ms. Thus, we propose that the change in relative weighting of rod signals within the post-receptoral pathways contributes to the sensitivity and temporal response of rod and cone pathway signaling and interactions.

What Determines the Kinetics of the Slow Afterhyperpolarization (sAHP) in Neurons?

What Determines the Kinetics of the Slow Afterhyperpolarization (sAHP) in Neurons?

Mechanism of TRPM7 Channel Inhibition by 2-Aminoethyl Diphenyl Borinate (2-APB)

2-APB is a widely used chemical compound in ion channel research. It inhibits numerous channels that include inositol 1,4,5-trisphosphate receptors, store-operated calcium channels, connexins and TRP family cation channels TRPC3, TRPC5, TRPC6, TRPM2, TRPM3 and TRPM7. Native and overexpressed TRPM7 channels are inhibited by 2-APB with IC50-s in the 70-170 μM range. A characteristic of TRPM7 channels is their inhibition by intracellular Mg2+ and acidic pH. using patch-clamp electrophysiology, we recorded native TRPM7 channel activity in Jurkat T lymphocytes and tested 2-APB at 10-300 μM for its ability to inhibit TRPM7 currents. When internal HEPES buffer concentration was low (1 mM), 100-300 μM 2-APB inhibited 60-70% of TRPM7 current with a slow time course. This inhibition was voltage-independent and reversible. Increasing the pH buffering capacity of internal solution to 140 mM HEPES abolished the inhibitory 2-APB effect. Simply making the internal recording solution alkaline, greatly diminished 2-APB inhibition. using single-cell pHi imaging with the fluorescent pH indicator BCECF, we found that at concentrations of 100 μM and higher, 2-APB potently acidifies the cytoplasm. A similar 2-APB-induced acidification was also observed in HEK293 cells, often used for TRPM7 channel overexpression. In contrast to 2-APB, its analog 2,2-diphenyltetrahydrofuran (DPTHF) did not produce cytosolic acidification, when applied at concentrations of 150-300 μM. We also found that Kv1.3 channels expressed in Jurkat T cells are modulated by 2-APB: the apparent voltage dependence of these channels was reversibly altered by the compound. Interestingly, this 2-APB effect on Kv1.3 channels did not depend on cytosolic pH. In summary, these experiments suggest that inhibition of TRPM7 channels by 2-APB is not direct but rather, can be explained by cytoplasmic acidification and resulting channel inhibition.

Shear stress-induced improvement of red blood cell deformability

Classically, it is known that red blood cell (RBC) deformability is determined by the geometric and material properties of these cells. Experimental evidence accumulated during the last decade has introduced the concept of active regulation of RBC deformability. This regulation is mainly related to altered associations between membrane skeletal proteins and integral proteins, with the latter serving to anchor the skeleton to the lipid matrix. It has been hypothesized that shear stress induces alterations of RBC deformability: the current study investigated the dynamics of the transient improvement in deformability induced by shear stress at physiologically-relevant levels. RBC were exposed to various levels of shear stress (SS) in a Couette type shearing system that is part of an ektacytometer, thus permitting the changes in RBC deformability during the application of SS to be monitored. Initial studies showed that there is an increase in deformability of the RBC subjected to SS in the range of 5-20 Pa, with kinetics characterized by time constants of a few seconds. Such improvement in deformability, expressed by an elongation index (EI), was faster with higher levels of SS and hence yielded shorter time constants: absolute values of EI increased by 3-8% of the starting level. Upon the removal of the shear stress, this response by RBC was reversible with a slower time course compared to the increase in EI during application of SS. Increased calcium concentration in the RBC suspending medium prevented the improvement of deformability. It is suggested that the improvement of RBC deformability by shear forces may have significant effects on blood flow dynamics, at least in tissues supplied by blood vessels with impaired vasomotor reserve, and may therefore serve as a compensating mechanism for the maintenance of adequate microcirculatory perfusion.

Reelin promotes microtubule dynamics in processes of developing neurons

The extracellular matrix protein reelin controls radial migration and layer formation of cortical neurons, in part by modulation of cytoskeletal dynamics. A stabilizing effect of reelin on the actin cytoskeleton has been described recently. However, it is poorly understood how reelin modulates microtubule dynamics. Here, we provide evidence that reelin increases microtubule assembly. This effect is mediated, at least in part, by promoting microtubule plus end dynamics in processes of developing neurons. Thus, we treated primary neuronal cultures with nocodazole to disrupt microtubules. After nocodazole washout, we found microtubule reassembly to be accelerated in the presence of reelin. Moreover, we show that reelin treatment promoted the formation of microtubule plus end binding protein 3 (EB3) comets in developing dendrites, and that EB3 immunostaining in the developing wild-type neocortex is most intense in the reelin-rich marginal zone where leading processes of radially migrating neurons project to. This characteristic EB3 staining pattern was absent in reeler. Also reassembly of nocodazole-dispersed dendritic Golgi apparati, which are closely associated to microtubules, was accelerated by reelin treatment, though with a substantially slower time course when compared to microtubule reassembly. In support of our in vitro results, we found that the subcellular distribution of α-tubulin and acetylated tubulin in reeler cortical sections differed from wild-type and from mice lacking the very low density lipoprotein receptor (VLDLR), known to bind reelin. Taken together, our results suggest that reelin promotes microtubule assembly, at least in part, by increasing microtubule plus end dynamics.

Pharmacokinetic-Pharmacodynamic Assessment of the Interrelationships Between Tesaglitazar Exposure and Renal Function in Patients With Type 2 Diabetes Mellitus

The effects of tesaglitazar on renal function (assessed as urinary clearance of 125I-sodium iothalamate or estimated by the modification of diet in renal disease formula) were studied in a 24-week open-label trial in type 2 diabetes mellitus patients randomized to daily doses of either tesaglitazar 2 mg or pioglitazone 45 mg. The aim of the analysis was to develop a population pharmacokinetic-pharmacodynamic model that could simultaneously describe the interrelationship between tesaglitazar exposure and reduction in renal function over time in patients with type 2 diabetes mellitus. The pharmacokinetic-pharmacodynamic model could adequately describe the interplay between tesaglitazar and glomerular filtration rate. A one-compartment model in which the apparent clearance was influenced by glomerular filtration rate characterized the pharmacokinetics of tesaglitazar. An indirect-response model was used for the slow time course of change in glomerular filtration rate, which decreased from 100 to 78 mL/min/1.73m(2) after 12 weeks of treatment. All tesaglitazar-treated patients had a reduction in glomerular filtration rate, and available demographic variables could not explain differences in response. Patients treated with an angiotensin converting enzyme inhibitor were more sensitive to tesaglitazar and had larger glomerular filtration rate decrease compared to nontreated patients. Approximately 8 weeks after discontinuing treatment, mean glomerular filtration rate had returned towards baseline. The model and data give valuable insights into the dynamic changes in glomerular filtration rate over time.

Combined application of brain-derived neurotrophic factor and neurotrophin-3 and its impact on spiral ganglion neuron firing properties and hyperpolarization-activated currents

Neurotrophins provide an effective tool for the rescue and regeneration of spiral ganglion neurons (SGNs) following sensorineural hearing loss. However, these nerve growth factors are also potent modulators of ion channel activity and expression, and in the peripheral auditory system brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) have previously been shown to alter the firing properties of auditory neurons and differentially regulate the expression of some potassium channels in vitro. In this study we examined the activity of the hyperpolarization-mediated mixed-cation current (Ih) in early post-natal cultured rat SGNs following exposure to combined BDNF and NT3. Whole-cell patch-clamp recordings made after 1 or 2 days in vitro revealed no change in the firing adaptation of neurons in the presence of BDNF and NT3. Resting membrane potentials were also maintained, but spike latency and firing threshold was subject to regulation by both neurotrophins and time in vitro. Current clamp recordings revealed an activity profile consistent with activation of the hyperpolarization-activated current. Rapid membrane hyperpolarization was followed by a voltage- and time-dependent depolarizing voltage sag. In voltage clamp, membrane hyperpolarization evoked a slowly-activating inward current that was reversibly blocked with cesium and inhibited by ZD7288. The amplitude and current density of Ih was significantly larger in BDNF and NT3 supplemented cultures, but this did not translate to a significant alteration in voltage sag magnitude. Neurotrophins provided at 50 ng/ml produced a hyperpolarizing shift in the voltage-dependence and slower time course of Ih activation compared to SGNs in control groups or cultured with 10 ng/ml BDNF and NT3. Our results indicate that combined BDNF and NT3 increase the activity of hyperpolarization-activated currents and that the voltage-dependence and activation kinetics of Ih in SGNs are sensitive to changes in neurotrophin concentration. In addition, BDNF and NT3 applied together induce a decrease in firing threshold, but does not generate a shift in firing adaptation.

Cross-talk between Insulin and Wnt Signaling in Preadipocytes: ROLE OF WNT CO-RECEPTOR LOW DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN-5 (LRP5)

Disturbed Wnt signaling has been implicated in numerous diseases, including type 2 diabetes and the metabolic syndrome. In the present study, we have investigated cross-talk between insulin and Wnt signaling pathways using preadipocytes with and without knockdown of the Wnt co-receptors LRP5 and LRP6 and with and without knock-out of insulin and IGF-1 receptors. We find that Wnt stimulation leads to phosphorylation of insulin signaling key mediators, including Akt, GSK3β, and ERK1/2, although with a lower fold stimulation and slower time course than observed for insulin. These Wnt effects are insulin/IGF-1 receptor-dependent and are lost in insulin/IGF-1 receptor double knock-out cells. Conversely, in LRP5 knockdown preadipocytes, insulin-induced phosphorylation of IRS1, Akt, GSK3β, and ERK1/2 is highly reduced. This effect is specific to insulin, as compared with IGF-1, stimulation and appears to be due to an inducible interaction between LRP5 and the insulin receptor as demonstrated by co-immunoprecipitation. These data demonstrate that Wnt and insulin signaling pathways exhibit cross-talk at multiple levels. Wnt induces phosphorylation of Akt, ERK1/2, and GSK3β, and this is dependent on insulin/IGF-1 receptors. Insulin signaling also involves the Wnt co-receptor LRP5, which has a positive effect on insulin signaling. Thus, altered Wnt and LRP5 activity can serve as modifiers of insulin action and insulin resistance in the pathophysiology of diabetes and metabolic syndrome.

Macaque V1 representations in natural and reduced visual contexts: spatial and temporal properties and influence of saccadic eye movements

Vision in natural situations is different from the paradigms generally used to study vision in the laboratory. In natural vision, stimuli usually appear in a receptive field as the result of saccadic eye movements rather than suddenly flashing into view. The stimuli themselves are rich with meaningful and recognizable objects rather than simple abstract patterns. In this study we examined the sensitivity of neurons in macaque area V1 to saccades and to complex background contexts. Using a variety of visual conditions, we find that natural visual response patterns are unique. Compared with standard laboratory situations, in more natural vision V1 responses have longer latency, slower time course, delayed orientation selectivity, higher peak selectivity, and lower amplitude. Furthermore, the influences of saccades and background type (complex picture vs. uniform gray) interact to give a distinctive, and presumably more natural, response pattern. While in most of the experiments natural images were used as background, we find that similar synthetic unnatural background stimuli produce nearly identical responses (i.e., complexity matters more than "naturalness"). These findings have important implications for our understanding of vision in more natural situations. They suggest that with the saccades used to explore complex images, visual context ("surround effects") would have a far greater effect on perception than in standard experiments with stimuli flashed on a uniform background. Perceptual thresholds for contrast and orientation should also be significantly different in more natural situations.

Reorganization of Oscillatory Activity in Human Parietal Cortex during Spatial Updating

Single-neuron recordings have shown that the posterior parietal cortex (PPC) processes spatial information in many frames of reference, including gaze-centered, head-centered, body-centered, and intermediate coding frames. At the population level, rhythmic neuronal synchronization may provide a mechanism by which PPC could selectively emphasize the task-relevant reference frame in spatial processing. Using magnetoencephalography, we tested this hypothesis by studying the modulations in oscillatory activity in a spatial updating task. Human subjects had to remember the location of a target, briefly flashed left or right of central fixation. Next, they refixated and then, after a further memory delay, made a saccade to the memorized target location. We observed gamma-band (>40 Hz) synchronization and alpha-band (8-12 Hz) desychronization in contralateral occipital and parietal areas, both showing updating in a gaze-centered reference frame but with fast and slow time courses, respectively. Furthermore, after updating, ipsilateral areas showed less alpha desynchronization when they had been contralateral to the target before updating. Taken together, our results suggest that power in the gamma band is instantly reorganized to encode task-relevant visuomotor space in a gaze-centered reference frame, while power in the alpha band reflects a regulatory mechanism actively facilitating the gating of the saccade target and inhibiting the original stimulus representation.

Modulation of voltage-gated sodium channels hyperpolarizes the voltage threshold for activation in spinal motoneurones

Previous work has shown that motoneurone excitability is enhanced by a hyperpolarization of the membrane potential at which an action potential is initiated (V(th)) at the onset, and throughout brainstem-evoked fictive locomotion in the adult decerebrate cat and neonatal rat. Modeling work has suggested the modulation of Na(+) conductance as a putative mechanism underlying this state-dependent change in excitability. This study sought to determine whether modulation of voltage-gated sodium channels could induce V(th) hyperpolarization. Whole-cell patch-clamp recordings were made from antidromically identified lumbar spinal motoneurones in an isolated neonatal rat spinal cord preparation. Recordings were made with and without the bath application of veratridine, a plant alkaloid neurotoxin that acts as a sodium channel modulator. As seen in HEK 293 cells expressing Nav1.2 channels, veratridine-modified channels demonstrated a hyperpolarizing shift in their voltage-dependence of activation and a slowing of inactivation that resulted in an enhanced inward current in response to voltage ramp stimulations. In the native rat motoneurones, veratridine-modified sodium channels induced a hyperpolarization of V(th) in all 29 neonatal rat motoneurones examined (mean hyperpolarization: -6.6 ± 4.3 mV). V(th) hyperpolarization was not due to the effects on Ca(2+) and/or K(+) channels as blockade of these currents did not alter V(th). Veratridine also significantly increased the amplitude of persistent inward currents (PICs; mean increase: 72.5 ± 98.5 pA) evoked in response to slow depolarizing current ramps. However, the enhancement of the PIC amplitude had a slower time course than the hyperpolarization of V(th), and the PIC onset voltage could be either depolarized or hyperpolarized, suggesting that PIC facilitation did not mediate the V(th) hyperpolarization. We therefore suggest that central neuronal circuitry in mammals could affect V(th) in a mechanism similar to that of veratridine, by inducing a negative shift in the activation voltage of sodium channels. Furthermore, this shift appears to be independent of the enhancement of PICs.