Axonal projections of Renshaw cells in the thoracic spinal cord

Renshaw cells are widely distributed in all segments of the spinal cord, but detailed morphological studies of these cells and their axonal branching patterns have only been made for lumbosacral segments. For these, a characteristic distribution of terminals was reported, including extensive collateralization within 1-2 mm of the soma, but then more restricted collaterals given off at intervals from the funicular axon. Previous authors have suggested that the projections close to the soma serve inhibition of motoneurons (known to be greatest for the motor nuclei providing the Renshaw cell excitation) but that the distant projections serve mainly the inhibition of other neurons. However, in thoracic segments, inhibition of motoneurons is known to occur over two to three segments (20-40 mm) from the presumed somatic locations of the Renshaw cells. Here, we report the first detailed morphological study of Renshaw cell axons outside the lumbosacral segments, which investigated whether this different distribution of motoneuron inhibition is reflected in a different pattern of Renshaw cell terminations. Four Renshaw cells in T7 or T8 segments were intracellularly labeled with neurobiotin in anesthetized cats and their axons traced for distances ≥6 mm from the somata. The only morphological difference detected within this distance in comparison with Renshaw cells in the lumbosacral cord was a minimal taper in the funicular axons, where in the lumbosacral cord this is pronounced. Patterns of termination were virtually identical to those in the lumbosacral segments, so we conclude that these patterns are unrelated to the pattern of motoneuronal inhibition

Motoneurone synchronization for intercostal and abdominal muscles: interneurone influences in two different species

The contribution of branched-axon monosynaptic inputs in the generation of short-term synchronization of motoneurones remains uncertain. Here, synchronization was measured for intercostal and abdominal motoneurones supplying the lower thorax and upper abdomen, mostly showing expiratory discharges. Synchronization in the anaesthetized cat, where the motoneurones receive a strong direct descending drive, is compared with that in anaesthetized or decerebrate rats, where the direct descending drive is much weaker. In the cat, some examples could be explained by branched-axon monosynaptic inputs, but many others could not, by virtue of peaks in cross-correlation histograms whose widths (relatively wide) and timing indicated common inputs with more complex linkages, e.g., disynaptic excitatory. In contrast, in the rat, correlations for pairs of internal intercostal nerves were dominated by very narrow peaks, indicative of branched-axon monosynaptic inputs. However, the presence of activity in both inspiration and expiration in many of the nerves allowed additional synchronization measurements between internal and external intercostal nerves. Time courses of synchronization for these often consisted of combinations of peaks and troughs, which have never been previously described for motoneurone synchronization and which we interpret as indicating combinations of inputs, excitation of one group of motoneurones being common with either excitation or inhibition of the other. Significant species differences in the circuits controlling the motoneurones are indicated, but in both cases, the roles of spinal interneurones are emphasised. The results demonstrate the potential of motoneurone synchronization for investigating inhibition and have important general implications for the interpretation of neural connectivity measurements by cross-correlation

The role of BRAF V600 mutation in melanoma

BRAF is a serine/threonine protein kinase activating the MAP kinase/ERK-signaling pathway. About 50 % of melanomas harbors activating BRAF mutations (over 90 % V600E). BRAFV600E has been implicated in different mechanisms underlying melanomagenesis, most of which due to the deregulated activation of the downstream MEK/ERK effectors. The first selective inhibitor of mutant BRAF, vemurafenib, after highly encouraging results of the phase I and II trial, was compared to dacarbazine in a phase III trial in treatment-naïve patients (BRIM-3). The study results showed a relative reduction of 63 % in risk of death and 74 % in risk of tumor progression. Considering all trials so far completed, median overall survival reached approximately 16 months for vemurafenib compared to less than 10 months for dacarbazine treatment. Vemurafenib has been extensively tested on melanoma patients expressing the BRAFV600E mutated form; it has been demonstrated to be also effective in inhibiting melanomas carrying the V600K mutation. In 2011, both FDA and EMA therefore approved vemurafenib for metastatic melanoma carrying BRAFV600 mutations. Some findings suggest that continuation of vemurafenib treatment is potentially beneficial after local therapy in a subset of patients with disease progression (PD). Among who continued vemurafenib >30 days after local therapy of PD lesion(s), a median overall survival was not reached, with a median follow-up of 15.5 months from initiation of BRAF inhibitor therapy. For patients who did not continue treatment, median overall survival from the time of disease progression was 1.4 months. A clinical phase I/II trial is evaluating the safety, tolerability and efficacy of vemurafenib in combination with the CTLA-4 inhibitor mAb ipilimumab. In the BRIM-7 trial vemurafenib is tested in association with GDC-0973, a potent and highly selective inhibitor of MEK1/2. Preliminary data seem to indicate that an additional inhibitor of mutated BRAF, GSK2118436, might be also active on a wider range of BRAF mutations (V600E-K-D-R); actually, treatment with such a compound is under evaluation in a phase III study among stage III-IV melanoma patients positive for BRAF mutations. Overall, BRAF inhibitors were well tolerated; common adverse events are arthralgia, rash, fatigue, alopecia, keratoacanthoma or cutaneous squamous-cell carcinoma, photosensitivity, nausea, and diarrhea, with some variants between different inhibitors

Regulatory T cell frequency in patients with melanoma with different disease stage and course, and modulating effects of high-dose interferon-α 2b treatment

<p>Abstract</p> <p>Background</p> <p>High-dose interferon-alpha 2b (IFN-α 2b) is the only approved systemic therapy in the United States for the adjuvant treatment of melanoma. The study objective was to explore the immunomodulatory mechanism of action for IFN-α 2b by measuring serum regulatory T cell (Treg), serum transforming growth factor-β (TGF-β), interleukin (IL)-10, and autoantibody levels in patients with melanoma treated with the induction phase of the high-dose IFN-α 2b regimen.</p> <p>Methods</p> <p>Patients with melanoma received IFN-α 2b administered intravenously (20 MU/m²each day from day 1 to day 5 for 4 consecutive weeks). Serum Treg levels were measured as whole lymphocytes in CD4⁺cells using flow cytometry while TGF-β, IL-10, and autoantibody levels were measured using enzyme-linked immunosorbent assays.</p> <p>Results</p> <p>Twenty-two patients with melanoma received IFN-α 2b treatment and were evaluated for Treg levels. Before treatment, Treg levels were significantly higher in patients with melanoma when compared with data from 20 healthy subjects (<it>P </it>= 0.001; Mann-Whitney test). Although a trend for reduction of Treg levels following IFN-α 2b treatment was observed (average decrease 0.29% per week), statistical significance was not achieved. Subgroup analyses indicated higher baseline Treg levels for stage III versus IV disease (<it>P </it>= 0.082), early recurrence versus no recurrence (<it>P </it>= 0.017), deceased versus surviving patients (<it>P = </it>0.021), and preoperative neoadjuvant versus postoperative adjuvant treatment groups (not significant). No significant effects were observed on the levels of TGF-β, IL-10, and autoantibodies in patients with melanoma treated with IFN-α 2b.</p> <p>Conclusions</p> <p>Patients with melanoma in this study showed increased basal levels of Treg that may be relevant to their disease and its progression. Treg levels shifted in patients with melanoma treated with IFN-α 2b, although no firm conclusions regarding the role of Tregs as a marker of treatment response or outcome can be made at present.</p

Thick primary melanoma has a heterogeneous tumor biology: an institutional series

<p>Abstract</p> <p>Background</p> <p>Thick melanomas (TM) ≥4 mm have a high risk for nodal and distant metastases. Optimal surgical management, prognostic significance of sentinel node biopsy (SLNB), and benefits of interferon (IFN) for these patients are unclear. As a continuum of increasing tumor thickness is placed into a single TM group, differences in biologic and clinical behavior may be lost. The purpose of this study was to better characterize the diverse biology in TM, including the value of increasing thickness and nodal status information, potentially identifying high risk TM subgroups that may warrant more aggressive treatment/follow up.</p> <p>Methods</p> <p>155 consecutive TM patients treated at a single institution between 1971 and 2007 were retrospectively reviewed. Patient, disease and treatment features were analyzed with respect to disease-free (DFS) and overall survival (OS).</p> <p>Results</p> <p>Median patient age was 66 years and 68% of patients were men. The trunk was the most common TM location (35%), followed by the head and neck (29%) and lower extremities (20%). Median thickness was 6 mm and 61% were ulcerated. 6% patients had stage IV disease, 12% had clinical nodal metastases. Clinically negative lymph node basins were treated by observation (22 patients - 15.4%), elective lymph node dissection (ELND) (24 patients - 17.6%) or SLNB (91 patients - 67%). 75% of ELND's and 53% of SLNB's were positive. Completion node dissection was performed in 38 SLNB+ patients and 22% had additional positive nodes. 17% of the study patients received IFN. At median follow up of 26 months, 5 year DFS and OS were 42% and 43.6%. For SLNB positive vs negative, median DFS were 22 vs 111 months (p = 0.006) and median OS were 41 vs 111 months (p = 0.006). When stratified by tumor thickness ≤ vs > 6 mm, 5 year DFS was 58.3% vs 20% (p < 0.0001) and OS was 62% vs 20% (P < 0.0001). IFN had no impact on DFS or OS (p = 0.98 and 0.8 respectively).</p> <p>Conclusion</p> <p>Within the high risk group of patients with TM, cases with tumor thickness > 6 mm or a positive SLNB had a significantly worse DFS and OS (p < .0001, <.0001 and .006, .006).</p

Expression of Drug Targets in Patients Treated with Sorafenib, Carboplatin and Paclitaxel

Introduction: Sorafenib, a multitarget kinase inhibitor, targets members of the mitogen-activated protein kinase (MAPK) pathway and VEGFR kinases. Here we assessed the association between expression of sorafenib targets and biomarkers of taxane sensitivity and response to therapy in pre-treatment tumors from patients enrolled in ECOG 2603, a phase III comparing sorafenib, carboplatin and paclitaxel (SCP) to carboplatin, paclitaxel and placebo (CP). Methods: Using a method of automated quantitative analysis (AQUA) of in situ protein expression, we quantified expression of VEGF-R2, VEGF-R1, VEGF-R3, FGF-R1, PDGF-Rβ, c-Kit, B-Raf, C-Raf, MEK1, ERK1/2, STMN1, MAP2, EB1 and Bcl-2 in pretreatment specimens from 263 patients. Results: An association was found between high FGF-R1 and VEGF-R1 and increased progression-free survival (PFS) and overall survival (OS) in our combined cohort (SCP and CP arms). Expression of FGF-R1 and VEGF-R1 was higher in patients who responded to therapy ((CR+PR) vs. (SD+PD+ un-evaluable)). Conclusions: In light of the absence of treatment effect associated with sorafenib, the association found between FGF-R1 and VEGF-R1 expression and OS, PFS and response might reflect a predictive biomarker signature for carboplatin/paclitaxel-based therapy. Seeing that carboplatin and pacitaxel are now widely used for this disease, corroboration in another cohort might enable us to improve the therapeutic ratio of this regimen. © 2013 Jilaveanu et al

Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium

In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca 2ϩ . Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca 2ϩ and in a scratch repair assay. The results confirmed the functional expression of P2Y4 receptors and excluded nonexpressed receptors/channels (P2X 1, P2X3, P2X6, P2Y6, P2Y11, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X 2, P2X4, P2Y1, P2Y2, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/ antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca 2ϩ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/ channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting. calcium; purinergic; transient receptor potential channel; urothelium THERE HAS BEEN a growing appreciation that rather than a simple passive barrier, the urothelium plays a more active role in the urinary tract. After physical or other damage, the urothelium will self-repair by switching from a mitotically quiescent to a highly regenerative state More intriguingly, the urothelium has been reported to possess sensory neuronal-like properties and to respond to mechanical and chemical stimulation through the release of transient mediators (4). Various mediators have been implicated, including ATP, nitric oxide, acetylcholine, and substance P (1, 7, 11). These short-lived mediators are considered to actuate suburothelial afferent neurons involved in the regulation of sensory perception and pain, but the urothelium is itself widely reported to express an array of receptors and channels that may respond in an autocrine/paracrine fashion to released mediators. These include purinergic P2X and P2Y (8, 24, 27), transient receptor potential (TRPV1, TRPV2, TRPV4, and TRPM8), acetylcholine (nicotinic and muscarinic), tachykinin, nerve growth factor, endothelin, sphingosine-1-phosphate, and bradykinin (3, 9, 15, 17) receptors. The outcome of such signaling is incompletely understood as it may play a bidirectional feedback role in modulating the neuronal signal and/or effect changes in urothelial homeostasis, such as barrier repair. It has also been suggested that abnormal expression of receptors and/or mediator release by the urothelium may be involved in dysfunctional diseases of the bladder, including idiopathic detrusor instability and interstitial cystitis Despite the literature reporting expression of these channels and receptors by the urothelium, consensus is confounded by contradictions in experimental approaches, including the species, specificity of reagents, and the nature of the tissue preparation (for a review, see Ref. 30). There has been limited characterization of these receptor/mediator signaling pathways in the human urothelium, where functional TRPV1 (10) and an autocrine-activated P2Y receptor pathway (19, 26) have been reported. Ultimately, this conflict and the lack of consensus are hindrances to the development of selective drugs. To attribute expression and function to specific tissue compartments, the present study was designed to define the functional expression of purinergic and transient receptors in the isolated human urothelium and stromal cells in situ and in vitro. A preliminary investigation revealed a lack of specificity of commercially available antibodies. For this reason, our rationalized experimental approach was to identify candidate receptors based on mRNA expression followed by confirmatory functional experiments to measure changes in intracellular Ca 2ϩ using specific agonists/antagonists. Finally, to examine whether receptor activation plays a role in urothelial homeostasis, we examined the effect of receptor activation on human urothelial scratch wound repair in vitro

An in silico model of the ubiquitin-proteasome system that incorporates normal homeostasis and age-related decline

BACKGROUND: The ubiquitin-proteasome system is responsible for homeostatic degradation of intact protein substrates as well as the elimination of damaged or misfolded proteins that might otherwise aggregate. During ageing there is a decline in proteasome activity and an increase in aggregated proteins. Many neurodegenerative diseases are characterised by the presence of distinctive ubiquitin-positive inclusion bodies in affected regions of the brain. These inclusions consist of insoluble, unfolded, ubiquitinated polypeptides that fail to be targeted and degraded by the proteasome. We are using a systems biology approach to try and determine the primary event in the decline in proteolytic capacity with age and whether there is in fact a vicious cycle of inhibition, with accumulating aggregates further inhibiting proteolysis, prompting accumulation of aggregates and so on. A stochastic model of the ubiquitin-proteasome system has been developed using the Systems Biology Mark-up Language (SBML). Simulations are carried out on the BASIS (Biology of Ageing e-Science Integration and Simulation) system and the model output is compared to experimental data wherein levels of ubiquitin and ubiquitinated substrates are monitored in cultured cells under various conditions. The model can be used to predict the effects of different experimental procedures such as inhibition of the proteasome or shutting down the enzyme cascade responsible for ubiquitin conjugation. RESULTS: The model output shows good agreement with experimental data under a number of different conditions. However, our model predicts that monomeric ubiquitin pools are always depleted under conditions of proteasome inhibition, whereas experimental data show that monomeric pools were depleted in IMR-90 cells but not in ts20 cells, suggesting that cell lines vary in their ability to replenish ubiquitin pools and there is the need to incorporate ubiquitin turnover into the model. Sensitivity analysis of the model revealed which parameters have an important effect on protein turnover and aggregation kinetics. CONCLUSION: We have developed a model of the ubiquitin-proteasome system using an iterative approach of model building and validation against experimental data. Using SBML to encode the model ensures that it can be easily modified and extended as more data become available. Important aspects to be included in subsequent models are details of ubiquitin turnover, models of autophagy, the inclusion of a pool of short-lived proteins and further details of the aggregation process

Anti-CTLA4 monoclonal antibodies: the past and the future in clinical application

Recently, two studies using ipilimumab, an anti-CTLA-4 monoclonal antibody (mab) demonstrated improvements in overall survival in the treatment of advanced melanoma. These studies utilized two different schedules of treatment in different patient categories (first and second line of treatment). However, the results were quite similar despite of different dosage used and the combination with dacarbazine in the first line treatment. We reviewed the result of randomized phase II-III clinical studies testing anti-CTLA-4 antibodies (ipilimumab and tremelimumab) for the treatment of melanoma to focus on practical or scientific questions related to the broad utilization of these products in the clinics. These analyses raised some considerations about the future of these compounds, their potential application, dosage, the importance of the schedule (induction/manteinance compared to induction alone) and their role as adjuvants. Anti-CTLA-4 antibody therapy represents the start of a new era in the treatment of advanced melanoma but we are on the steep slope of the learning curve toward the optimization of their utilization either a single agents or in combination

The Population Impact of a Large School-Based Influenza Vaccination Campaign

The optimal vaccination strategy to mitigate the impact of influenza epidemics is unclear. In 2005, a countywide school-based influenza vaccination campaign was launched in Knox County, Tennessee (population 385,899). Approximately 41% and 48% of eligible county children aged 5-17 years were immunized with live attenuated influenza vaccine before the 2005-2006 and 2006-2007 influenza seasons, respectively. We sought to determine the population impact of this campaign.Laboratory-confirmed influenza data defined influenza seasons. We calculated the incidence of medically attended acute respiratory illness attributable to influenza in Knox and Knox-surrounding counties (concurrent controls) during consecutive seasons (5 precampaign and 2 campaign seasons) using negative binomial regression and rate difference methods. Age-stratified analyses compared the incidence of emergency department (ED) visits and hospitalizations attributable to influenza.During precampaign seasons, estimated ED visit rates attributable to influenza were 12.39 (95% CI: 10.34-14.44) per 1000 Knox children aged 5-17 years and similar in Knox-surrounding counties. During the campaign seasons, annual Knox influenza-associated ED visit rates declined relative to rates in Knox-surrounding counties: rate ratios 0.55 (95% CI: 0.27-0.83) and 0.70 (95% CI: 0.56-0.84) for the first and second campaign seasons, respectively. Overall, there were about 35% or 4.86 per 1000 fewer influenza-associated ED visits among Knox County children aged 5-17 years attributable to the campaign. No significant declines in Knox compared to surrounding counties were detected for influenza associated ED visits in children aged <5 years, all adults combined or selected adult age subgroups, although power for these analyses was limited. Alternate rate-difference analyses yielded consistent results.Vaccination of approximately 45% of Knox school-aged children with influenza vaccine was associated with a 35% annual reduction (4.86 per 1000) in ED visit rates attributable to influenza. Higher vaccination coverage and/or larger studies would be needed to determine whether similar interventions have indirect benefits in other age groups