The phosphoinositide 3-kinase-dependent activation of Btk is required for optimal eicosanoid production and generation of reactive oxygen species in antigen-stimulated mast cells

Activated mast cells are a major source of the eicosanoids PGD(2) and leukotriene C(4) (LTC(4)), which contribute to allergic responses. These eicosanoids are produced following the ERK1/2-dependent activation of cytosolic phospholipase A(2), thus liberating arachidonic acid, which is subsequently metabolized by the actions of 5-lipoxygenase and cyclooxygenase to form LTC(4) and PGD(2), respectively. These pathways also generate reactive oxygen species (ROS), which have been proposed to contribute to FcepsilonRI-mediated signaling in mast cells. In this study, we demonstrate that, in addition to ERK1/2-dependent pathways, ERK1/2-independent pathways also regulate FcepsilonRI-mediated eicosanoid and ROS production in mast cells. A role for the Tec kinase Btk in the ERK1/2-independent regulatory pathway was revealed by the significantly attenuated FcepsilonRI-dependent PGD(2), LTC(4), and ROS production in bone marrow-derived mast cells of Btk(-/-) mice. The FcepsilonRI-dependent activation of Btk and eicosanoid and ROS generation in bone marrow-derived mast cells and human mast cells were similarly blocked by the PI3K inhibitors, Wortmannin and LY294002, indicating that Btk-regulated eicosanoid and ROS production occurs downstream of PI3K. In contrast to ERK1/2, the PI3K/Btk pathway does not regulate cytosolic phospholipase A(2) phosphorylation but rather appears to regulate the generation of ROS, LTC(4), and PGD(2) by contributing to the necessary Ca(2+) signal for the production of these molecules. These data demonstrate that strategies to decrease mast cell production of ROS and eicosanoids would have to target both ERK1/2- and PI3K/Btk-dependent pathways

Update on treatment of follicular non-Hodgkin’s lymphoma: focus on potential of bortezomib

Follicular lymphoma is predominantly managed as a chronic disease, with intermittent chemo/immunotherapy reserved for symptomatic progression. It is considered incurable with conventional treatments, and current therapeutic options are associated with significant toxicities that are especially limiting in older patients. Bortezomib (PS-341; Velcade®), a first-in-class drug targeting the proteolytic core subunit of the 26S proteasome, has emerged as a therapeutic alternative in follicular lymphoma, with promising preclinical data and efficacy in patients with other hematological malignancies. Several clinical trials were conducted with bortezomib for the treatment of non-Hodgkin’s lymphoma. As a single agent, overall responses in follicular lymphoma varied greatly (16%–41%), with weekly bortezomib showing less neurotoxicity than twice-weekly regimens, but with concern about decreased responses. Combination with rituximab was projected to improve the efficacy of bortezomib, but this resulted in increased toxicities and questionable added benefit. Although the largest Phase III study in follicular lymphoma of bortezomib plus rituximab versus rituximab alone demonstrated a significant progression-free survival difference, the absolute difference was small (12.8 months versus 11 months). Combining bortezomib with established regimens, such as rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), rituximab, cyclophosphamide, vincristine, and prednisone (R-CVP), or rituximab-bendamustine also did not show definite benefit, and many of these studies did not meet their primary endpoint when bortezomib failed to improve responses or survival to the degree anticipated. In a disease where the goal of treatment is palliative and affected patients often have other medical and treatment-related comorbidities, decisions regarding therapies which carry risks of additional toxicities must be considered carefully. Conclusive evidence of the ability of bortezomib to improve patient outcomes meaningfully and to justify the added toxicity is lacking, but limitations in cross-trial comparisons are recognized. Large randomized trials and investigations of combinations with promising novel targeted agents will aid in determining the role of bortezomib, if any, in the future treatment of follicular lymphoma

Ovarian vein thrombosis in a polytrauma patient

A young mother presented to a Major Trauma Centre (MTC) following a road traffic collision. Her admission computed tomography (CT) traumagram demonstrated liver and renal lacerations, spinal and pelvic fractures with no abnormalities of the ovarian veins. Her inpatient course was uncomplicated other than a sustained, isolated raised c-reactive protein (CRP). CT abdomen one week after injury demonstrated stable solid organ injuries and the additional, unexpected finding of a right ovarian vein thrombosis (OVT). A pragmatic approach was taken towards the management of the OVT given the haemorrhagic risk from her traumatic injuries. A multi-disciplinary, consultant-led plan was made to slowly increase enoxaparin to a therapeutic dose under close surveillance and to then switch to warfarin following an outpatient consultation with a consultant haematologist. A magnetic resonance venogram was performed after 3 months of anticoagulation and this demonstrated complete resolution of the OVT and normal appearances of the ovary

Antigen and Thapsigargin Promote Influx of Ca2+ in Rat Basophilic RBL-2H3 Cells by Ostensibly Similar Mechanisms That Allow Filling of Inositol 1,4,5-Trisphosphate-Sensitive and Mitochondrial Ca2+ Stores

In single, Fura 2-loaded RBL-2H3 cells, antigen and thapsigargin depleted the same intracellular pool of Ca2+ in the absence of external Ca2+; provision of external Ca2+ induced immediate increases in levels of free Ca2+ ([Ca2+](i)). These increases were dependent on the presence of external Ca2+ and, presumably, on influx of Ca2+ across the cell membrane. Both stimulants enhanced intracellular accumulation of 45Ca2+ through ostensibly similar mechanisms because accumulation was blocked to similar extents by various multivalent cations or by depolarization with K+. Because thapsigargin blocked reuptake of Ca2+ into inositol 1,4,5-trisphosphate sensitive stores, uptake occurred independently of the refilling of these stores. Uptake was dependent instead on sequestration of 45Ca2+ in a pool of high capacity that was insensitive to thapsigargin, caffeine, GTP and inositol 1,4,5-trisphosphate but sensitive to ionomycin and mitochondrial inhibitors. The existence of an inositol 1,4,5-trisphosphate-insensitive pool was also apparent in permeabilized cells; at 0.1 μM [Ca2+](i), uptake of 45Ca2+ was largely confined (\u3e 80%) to the inositol 1,4,5-trisphosphate-sensitive pool, but at 2 μM [Ca2+](i) uptake was largely (\u3e 60%) into the inositol 1,4,5-trisphosphate-insensitive pool. Provision of mitochondrial inhibitors along with thapsigargin to block uptake into both pools, did not impair the thapsigargin-induced increase in [Ca2+](i) or influx of Ca2+, as indicated by changes in Fura 2 fluorescence, but did block the intracellular accumulation of 45Ca2+. The studies illustrate the utility of simultaneous measurements of [Ca2+](i) and 45Ca2+ uptake for a full accounting of Ca2+ homoeostasis as exemplified by the ability to distinguish between influx and mitochondrial uptake of Ca2+

Regulation of Reactive Oxygen Species and the Antioxidant Protein DJ-1 in Mastocytosis

Neoplastic accumulation of mast cells in systemic mastocytosis (SM) associates with activating mutations in the receptor tyrosine kinase KIT. Constitutive activation of tyrosine kinase oncogenes has been linked to imbalances in oxidant/antioxidant mechanisms in other myeloproliferative disorders. However, the impact of KIT mutations on the redox status in SM and the potential therapeutic implications are not well understood. Here, we examined the regulation of reactive oxygen species (ROS) and of the antioxidant protein DJ-1 (PARK-7), which increases with cancer progression and acts to lessen oxidative damage to malignant cells, in relationship with SM severity. ROS levels were increased in both indolent (ISM) and aggressive variants of the disease (ASM). However, while DJ-1 levels were reduced in ISM with lower mast cell burden, they rose in ISM with higher mast cell burden and were significantly elevated in patients with ASM. Studies on mast cell lines revealed that activating KIT mutations induced constant ROS production and consequent DJ-1 oxidation and degradation that could explain the reduced levels of DJ-1 in the ISM population, while IL-6, a cytokine that increases with disease severity, caused a counteracting transcriptional induction of DJ-1 which would protect malignant mast cells from oxidative damage. A mouse model of mastocytosis recapitulated the biphasic changes in DJ-1 and the escalating IL-6, ROS and DJ-1 levels as mast cells accumulate, findings which were reversed with anti-IL-6 receptor blocking antibody. Our findings provide evidence of increased ROS and a biphasic regulation of the antioxidant DJ-1 in variants of SM and implicate IL-6 in DJ-1 induction and expansion of mast cells with KIT mutations. We propose consideration of IL-6 blockade as a potential adjunctive therapy in the treatment of patients with advanced mastocytosis, as it would reduce DJ-1 levels making mutation-positive mast cells vulnerable to oxidative damage

Melatonin and sleep responses following exercise in elite female athletes

To determine the melatonin concentrations and subsequent sleep indices of elite netball athletes following a training day when compared to a control day. Ten elite female netball athletes (mean ± SD; age = 23 ± 6 yrs) provided saliva samples PRE (17:15h) and POST (22:00h) a training session, and a day with no training (CONTROL). Sleep monitoring was performed using wrist actigraphy to assess total time in bed (TTB), total sleep time (TST), sleep efficiency (SE) and sleep latency (SL). Melatonin levels were significantly lower (p < 0.05), both PRE and POST the training condition (6.2 and 17.6 pg/mL, respectively) when compared to the CONTROL (14.8 and 24.3 pg/mL, respectively). There were no significant differences observed between conditions for any of the sleep variables. However, a small reduction in TST could be observed following the training session condition compared to the CONTROL condition. The scheduling of netball training in the evening is shown to suppress salivary melatonin levels. This may have an influence on subsequent sleep following night-time exercise

The Efficacy of Wrestling-Style Compression Suits to Improve Maximum Isometric Force and Movement Velocity in Well-Trained Male Rugby Athletes

Purpose: The prevalence of compression garment (CG) use is increasing with athletes striving to take advantage of the purported benefits to recovery and performance. Here, we investigated the effect of CG on muscle force and movement velocity performance in athletes. Methods: Ten well-trained male rugby athletes wore a wrestling-style CG suit applying 13–31 mmHg of compressive pressure during a training circuit in a repeated-measures crossover design. Force and velocity data were collected during a 5-s isometric mid-thigh pull (IMTP) and repeated countermovement jump (CMJ), respectively; and time to complete a 5-m horizontal loaded sled push was also measured. Results: IMTP peak force was enhanced in the CG condition by 139 ± 142 N (effect size [ES] = 0.36). Differences in CMJ peak velocity (ES = 0.08) and loaded sled-push sprint time between the conditions were trivial (ES = −0.01). A qualitative assessment of the effects of CG wear suggested that the likelihood of harm was unlikely in the CMJ and sled push, while a beneficial effect in the CMJ was possible, but not likely. Half of the athletes perceived a functional benefit in the IMTP and CMJ exercises. Conclusion: Consistent with other literature, there was no substantial effect of wearing a CG suit on CMJ and sprint performance. The improvement in peak force generation capability in an IMTP may be of benefit to rugby athletes involved in scrummaging or lineout lifting. The mechanism behind the improved force transmission is unclear, but may involve alterations in neuromuscular recruitment and proprioceptive feedback

Phase Variable O Antigen Biosynthetic Genes Control Expression of the Major Protective Antigen and Bacteriophage Receptor in Vibrio cholerae O1

The Vibrio cholerae lipopolysaccharide O1 antigen is a major target of bacteriophages and the human immune system and is of critical importance for vaccine design. We used an O1-specific lytic bacteriophage as a tool to probe the capacity of V. cholerae to alter its O1 antigen and identified a novel mechanism by which this organism can modulate O antigen expression and exhibit intra-strain heterogeneity. We identified two phase variable genes required for O1 antigen biosynthesis, manA and wbeL. manA resides outside of the previously recognized O1 antigen biosynthetic locus, and encodes for a phosphomannose isomerase critical for the initial step in O1 antigen biosynthesis. We determined that manA and wbeL phase variants are attenuated for virulence, providing functional evidence to further support the critical role of the O1 antigen for infectivity. We provide the first report of phase variation modulating O1 antigen expression in V. cholerae, and show that the maintenance of these phase variable loci is an important means by which this facultative pathogen can generate the diverse subpopulations of cells needed for infecting the host intestinal tract and for escaping predation by an O1-specific phage

Radar backscatter measurements from Arctic sea ice during the fall freeze-up

Radar backscatter measurements from sea ice during the fall freeze-up were performed by the United States Coast Guard Icebreaker Polar Star as a part of the International Arctic Ocean Expedition (IAOE'91) from Aug. to Sep. 1991. The U.S. portion of the experiment took place on board the Polar Star and was referred to as TRAPOLEX '91 (Transpolar expedition) by some investigators. Before prematurely aborting its mission because of mechanical failure of her port shaft, the Polar Star reached 84 deg 57 min N latitude at 35 deg E longitude. The ship was in the ice (greater than 50 percent coverage) from 14 Aug. until 3 Sep. and was operational for all but 6 days due to two instances of mechanical problems with the port shaft. The second was fatal to the ship's participation in the expedition. During the expedition, radar backscatter was measured at C-band under a variety of conditions. These included measurements from young ice types as well as from multiyear and first-/second-year sea ice during the fall freeze-up. The sea ice types were determined by measurement of the ice properties at several of the stations and by visual inspection on others. Radar backscatter measurements were performed over a large portion of the ship's transit into the Arctic ice pack. These were accompanied by in situ sea ice property characterization by the U.S. Army Cold Regions Research and Engineering Laboratory (CRREL) at several stations and, when snow was present, its properties were documented by The Microwave Group, Ottawa River (MWG)