Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells.

Growth factor-induced signaling by receptor tyrosine kinases (RTKs) plays a central role in embryonic development and in pathogenesis and, hence, is tightly controlled by several regulatory proteins. Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered. Subsequently, four mammalian Sprouty homologues (Spry-1-4) have been identified. Here, we report the functional characterization of two of them, Spry-1 and -2, in endothelial cells. Overexpressed Spry-1 and -2 inhibit fibroblast growth factor- and vascular endothelial growth factor-induced proliferation and differentiation by repressing pathways leading to p42/44 mitogen-activating protein (MAP) kinase activation. In contrast, although epidermal growth factor-induced proliferation of endothelial cells was also inhibited by Spry-1 and -2, activation of p42/44 MAP kinase was not affected. Biochemical and immunofluorescence analysis of endogenous and overexpressed Spry-1 and -2 reveal that both Spry-1 and -2 are anchored to membranes by palmitoylation and associate with caveolin-1 in perinuclear and vesicular structures. They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane. The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.

CyBorD-DARA is potent initial induction for MM and enhances ADCP: initial results of the 16-BCNI-001/CTRIAL-IE 16-02 study.

Daratumumab (DARA) has shown impressive activity in combination with other agents for the treatment of multiple myeloma (MM). We conducted a phase 1b study to assess the safety and preliminary efficacy, as well as potential mechanisms of action, of DARA (16 mg/kg) in combination with a weekly schedule of subcutaneous bortezomib (1.3-1.5 mg/m2), cyclophosphamide (150-300 mg/m2), and dexamethasone (40 mg) (CyBorD DARA) as initial induction before autologous stem cell transplantation (ASCT). Eligible patients were ≤70 years of age with untreated MM requiring treatment and who lacked significant comorbidities. A total of 18 patients were enrolled. Their median age was 56 years (range, 32-66 years), and all patients had Eastern Cooperative Oncology Group performance status ≤1. The International Staging System stages were I, II, and III in 78%, 17%, and 6% of patients, respectively; 28% of patients had high-risk genetic features. There was no dose-limiting toxicity, and the incidence of grade 3 or 4 infection or neutropenia was <10%. On an intention-to-treat basis, 94% achieved ≥very good partial response with ≥complete response in 44% of patients. Among 14 of 15 patients who underwent ASCT and were evaluable for response, all 14 achieved at least very good partial response, with 8 (57%) of 14 achieving complete response. After ASCT, 10 (83%) of 12 patients in whom minimal residual disease analysis was possible were negative at a sensitivity of 10-5 (56% on intention-to-treat/whole study population) according to next-generation sequencing. Flow cytometry analysis of patient samples indicated CyBorD DARA induced activation of macrophage-mediated antibody-dependent cellular phagocytosis. This trial was registered at www.clinicaltrials.gov as #NCT02955810.

Guanidinium chloride molecular diffusion in aqueous and mixed water-ethanol solutions.

Solutions containing guanidinium chloride (GdmCl), or equivalently guanidine hydrochloride (GdnHCl), are commonly used to denature macromolecules such as proteins and DNA in, for example, microfluidics studies of protein unfolding. To design and study such applications, it is necessary to know the diffusion coefficients for GdmCl in the solution. To this end, we use molecular dynamics simulations to calculate the diffusion coefficients of GdmCl in water and in water-ethanol solutions, for which no direct experimental measurements exist. The fully atomistic simulations show that the guandinium cation Gdm (+) diffusion decreases as the concentration of both Gdm (+) and ethanol in the solution increases. The simulations are validated against available literature data, both transformed measured viscosity values and computed diffusion coefficients, and we show that a prudent choice of water model, namely TIP4P-Ew, gives calculated diffusion coefficients in good agreement with the transformed measured viscosity values. The calculated Gdm (+) diffusion behavior is explained as a dynamic mixture of free cation, stacked cation, and ion-paired species in solution, with weighted contributions to Gdm (+) diffusion from the stacked and paired states helping explain measured viscosity data in terms of atom-scale dynamics.

Adhesion molecule expression in acute and chronic exercise.

Adhesion molecules expressed on leukocytes and the vascular endothelial lining include the selectins, integrins, members of the immunoglobulin superfamily, and mucins. The changes in their expression that develop with acute and chronic exercise are briefly reviewed. Adhesion molecules are thought to modulate leukocyte trafficking, accounting for changes in the counts and possibly also the functional activity of various leukocyte subsets during and following an acute bout of physical activity. Some of the changes in the surface density of adhesion molecules can be explained through the action of epinephrine and other humoral factors on their expression, but an influence of sympathetic nerve terminals on cells sequestered in the spleen and liver, and an influx into the general circulation of leukocytes of differing phenotype also appear to be involved.

Low doses of melatonin and diurnal effects on thermoregulation and tolerance to uncompensable heat stress.

This study examined whether the reported hypothermic effect of melatonin ingestion increased tolerance to exercise at 40 degrees C, for trials conducted either in the morning or afternoon, while subjects were wearing protective clothing. Nine men performed four randomly ordered trials; two each in the morning (0930) and afternoon (1330) after the double-blind ingestion of either two placebo capsules or two 1-mg capsules of melatonin. Despite significant elevations in plasma melatonin to over 1,000 ng/ml 1 h after the ingestion of the first 1-mg dose, rectal temperature (T(re)) was unchanged before or during the heat-stress exposure. Also, all other indexes of temperature regulation and the heart rate response during the uncompensable heat stress were unaffected by the ingestion of melatonin. Initial T(re) was increased during the afternoon (37.1 +/- 0.2 degrees C), compared with the morning (36.8 +/- 0.2 degrees C) exposures, and these differences remained throughout the uncompensable heat stress, such that final T(re) was also increased for the afternoon (39.2 +/- 0.2 degrees C) vs. the morning (39.0 +/- 0.3 degrees C) trials. Tolerance times and heat storage were not different among the exposures at approximately 110 min and 16 kJ/kg, respectively. It was concluded that this low dose of melatonin had no impact on tolerance to uncompensable heat stress and that trials conducted in the early afternoon were associated with an increased T(re) tolerated at exhaustion that offset the circadian influence on resting T(re) and thus maintained tolerance times similar to those of trials conducted in the morning.

Contribution of exertional hyperthermia to sympathoadrenal-mediated lymphocyte subset redistribution.

The contribution of hyperthermia to the differential leukocytosis of exercise remains obscure. This study examined changes in circulating sympathoadrenal hormone concentrations and patterns of leukocyte and lymphocyte subset (CD3(+), CD4(+), CD8(+), CD19(+), CD3(-)16(+)/56(+)) redistribution during exercise, with and without a significant rise of rectal temperature (T(re)). Ten healthy men [age 26.9 +/- 5.7 (SD) yr, body mass 76.0 +/- 10.9 kg, body fat 13.9 +/- 4.6%, peak O(2) consumption: 48.0 +/- 12.4 ml x kg(-1) x min(-1)] exercised for 40 min (65% peak O(2) consumption) during water immersion at 39 or 18 degrees C. T(re) increased from 37.2 to 39.3 degrees C (P < 0.0001) after 40 min of exercise in 39 degrees C water but was held constant to an increment of 0.5 degrees C during exercise in 18 degrees C water. Application of this thermal clamp reduced exercise-associated increments of plasma epinephrine (Epi) and norepinephrine (NE) by >50% (P < 0.05) and abolished the postexercise increase in cortisol. Thermal clamping also reduced the exercise-induced leukocytosis and lymphocytosis. Multiple regression demonstrated that T(re) had no direct association with lymphocyte subset mobilization but was significantly (P < 0.0001) correlated with hormone levels. Epi was an important determinant of total leukocytes, lymphocytes, and CD3(+), CD4(+), CD8(+), and CD3(-)CD16(+)/56(+) subset redistribution. The relationship between NE and lymphocyte subsets was weaker than that with Epi, with the exception of CD3(-)CD16(+)/56(+) counts, which were positively (P < 0.0001) related to NE. Cortisol was negatively associated with leukocytes, CD14(+) monocytes, and CD19(+) B- and CD4(+) T-cell subsets but was positively related to granulocytes. We conclude that hyperthermia mediates exercise-induced immune cell redistribution to the extent that it causes sympathoadrenal activation, with alterations in circulating Epi, NE, and cortisol.

Interdigitating organic bilayers direct the short interlayer spacing in hybrid organic-inorganic layered vanadium oxide nanostructures.

Layered metal oxides provide a single-step route to sheathed superlattices of atomic layers of a variety of inorganic materials, where the interlayer spacing and overall layered structure forms the most critical feature in the nanomaterials' growth and application in electronics, health, and energy storage. We use a combination of computer simulations and experiments to describe the atomic-scale structure, dynamics and energetics of alkanethiol-intercalated layered vanadium oxide-based nanostructures. Molecular dynamics (MD) simulations identify the unusual substrate-constrained packing of the alkanethiol surfactant chains along each V(2)O(5) (010) face that combines with extensive interdigitation between chains on opposing faces to maximize three-dimensional packing in the interlayer regions. The findings are supported by high resolution electron microscopy analyses of synthesized alkanethiol-intercalated vanadium oxide nanostructures, and the preference for this new interdigitated model is clarified using a large set of MD simulations. This dependency stresses the importance of organic-inorganic interactions in layered material systems, the control of which is central to technological applications of flexible hybrid nanomaterials.

Molecular dynamics study of naturally occurring defects in self-assembled monolayer formation.

One of the major challenges for nanofabrication, in particular microcontact printing (mu-CP), is the control of molecular diffusion, or "ink spreading", for the creation of nanopatterns with minimized "smudging" at pattern boundaries. In this study, fully atomistic computer simulations were used to measure the impact of naturally occurring domain boundaries on the diffusion of excess alkanethiol ink molecules on printed alkanethiol self-assembled monolayers (SAM). A periodic unit cell containing approximately one million atoms and with a surface area of 56 nm x 55 nm was used to model a hexadecanethiol SAM on Au(111), featuring SAM domain boundaries and a range of concentrations of excess hexadecanethiol ink molecules diffusing on top. This model was simulated for a total of approximately 80 ns of molecular dynamics. The simulations reveal that domain boundaries impede the diffusion of excess ink molecules and can, in some cases, permanently trap excess inks. There is competition between ink spreading and ink trapping, with the ink/SAM interaction strongly dependent on both the ink concentration and the SAM orientation at domain boundaries. SAM defects thus provide potential diffusion barriers for the control of excess ink spreading, and simulations also illustrate atom-scale mechanisms for the repair of damaged areas of the SAM via self-healing. The ability of domain boundaries to trap excess ink molecules is accounted for using an accessible volume argument, and trapping is discussed in relation to experimental efforts to reduce molecular spreading on SAMs for the creation of ultrahigh resolution nanopatterns.

Quantification of ink diffusion in microcontact printing with self-assembled monolayers.

Spreading of ink outside the desired printed area is one of the major limitations of microcontact printing (micro-CP) with alkanethiol self-assembled monolayers (SAMs) on gold. We use molecular dynamics (MD) computer simulations to quantify the temperature and concentration dependence of hexadecanethiol (HDT) ink spreading on HDT SAMs, modeling 18 distinct printing conditions using periodic simulation cells of approximately 7 nm edge length and printing conditions ranging from 7 ink molecules per cell at 270 K to 42 ink molecules per cell at 371K. The computed alkanethiol ink diffusion rates on the SAM are of the same order of magnitude as bulk liquid alkanethiol diffusion rates at all but the lowest ink concentrations and highest temperatures, with up to 20-30 times increases in diffusion rates at the lowest concentration-highest temperature conditions. We show that although alkanethiol surfaces are autophobic, autophobicity is not enough to pin the ink solutions on the SAM, and so any overinking of the SAM will lead to spreading of the printed pattern. Comparison of experimental and calculated diffusion data supports an interpretation of pattern broadening as a mixture of spreading on fully and partially formed SAMs, and the calculated spreading rates establish some of the fundamental limitations of mu-CP in terms of stamp contact time and desired pattern width.

Melatonin has no effect on tolerance to uncompensable heat stress in man.

This study examined whether a 5 mg dose of melatonin induced a lower rectal temperature (Tre) response at rest in both a cool and hot environment while wearing normal military combat clothing, and then examined the influence of this response on tolerance to exercise in the heat while wearing protective clothing. Nine men performed four randomly ordered trials involving 2 h of rest at ambient temperatures of either 23 degrees C or 40 degrees C followed by exercise at an ambient temperature of 40 degrees C. The double-blind ingestion of placebo or melatonin occurred after 30 min of rest. The mean Tre during rest at 23 degrees C had decreased significantly from 36.8 (SD 0.1) degrees C to 36.7 (SD 0.2) degrees C at 90 min following the ingestion of the drug, whereas values during the placebo trial did not change. The lower Tre response during the melatonin trial remained during the first 50 min of exercise in the heat while wearing the protective clothing. Since the final mean Tre at the end of exercise also was significantly reduced for the melatonin [39.0 (SD 0.4) degrees C] compared with the placebo [mean 39.1 (SD 0.3) degrees C] trial, tolerance times approximated 95 min in both conditions. During rest at 40 degrees C, melatonin did not affect the mean Tre response which increased significantly during the last 90 min from 36.9 (SD 0.1) degrees C to 37.3 (SD 0.1) degrees C. This increase in Tre during the rest period prior to donning the protective clothing decreased tolerance time approximately 30 min compared with the trials that had involved rest at 23 degrees C. Total heat storage summated over the rest and exercise periods was not different among the trials at 15 kJ x kg(-1). It was concluded that the small decrease in Tre following the ingestion of 5 mg of melatonin at rest in a cool environment had no influence on subsequent tolerance during uncompensable heat stress.

Circulating levels of peripheral blood leucocytes and cytokines following competitive cycling.

The objective of the study was to determine if prolonged and strenuous cycling leads to a polarized cytokine response, and/or unique mobilization of circulating leucocyte populations. Resting venous blood samples were collected from 6 amateur cyclists, 24 hr before, and at 10-25 min and 150 min after completion of a 250-km road race (race time: 404 +/- 3.5 min). Total leucocyte counts were significantly elevated following competition. Cell counts of CD3+CD8bright+ lymphocytes were depressed by 50% 150 min after competition. A significant increase in CD4+ cells expressing the IL-2R alpha chain was evident 150 min after competition. IL-6 concentrations were greatly increased, both at 10-25 min and 150 min after competition. Resting TNF-a concentrations were approximately doubled at both time points after competition. Plasma levels of IFN-gamma, IL-10 and IL-12 were below detection thresholds at all time points. These results suggest that performance of a 6.5 h competitive cycle-race does not induce a Type-1 or Type-2-dominated cytokine response, but one that is typical of a proinflammatory cytokine response.

Indomethacin inhibits circulating PGE2 and reverses postexercise suppression of natural killer cell activity.

Natural killer (NK) cells are important in combating viral infections and cancer. NK cytolytic activity (NKCA) is often depressed during recovery from strenuous exercise. Lymphocyte subset redistribution and/or inhibition of NK cells via soluble mediators, such as prostaglandin (PG) E2 and cortisol, are suggested as mechanisms. Ten untrained (peak O2 consumption = 44.0 +/- 3.5 ml. kg-1. min-1) men completed at 2-wk intervals a resting control session and three randomized double-blind exercise trials after the oral administration of a placebo, the PG inhibitor indomethacin (75 mg/day for 5 days), or naltrexone (reported elsewhere). Circulating CD3(-)CD16(+)/56(+) NK cell counts, PGE2, cortisol, and NKCA were measured before, at 0.5-h intervals during, and at 2 and 24 h after a 2-h bout of cycle ergometer exercise (65% peak O2 consumption). During placebo and indomethacin conditions, exercise induced significant (P < 0.0001) elevations of NKCA (>100%) and circulating NK cell counts (>350%) compared with corresponding control values. With placebo treatment, total NKCA was suppressed (28%; P < 0.05) 2 h after exercise, and a postexercise elevation (36%; P = 0.02) of circulating PGE2 was negatively correlated (r = 0.475, P = 0.03) with K-562 tumor cell lysis. NK counts were unchanged in the postexercise period, but at this stage CD14(+) monocyte numbers were elevated (P < 0.0001). Indomethacin treatment eliminated the postexercise increase in PGE2 concentration and completely reversed the suppression of total and per CD16(+)56(+) NKCA 2 h after exercise. These data support the hypothesis that the postexercise reduction in NKCA reflects changes in circulating PGE2 rather than a differential lymphocyte redistribution.

Differential cell adhesion molecule expression and lymphocyte mobilisation during prolonged aerobic exercise.

This study was undertaken to determine the cell adhesion molecule profile of CD4+, CD8hi and CD56+ lymphocytes, which are mobilised to and from the peripheral blood during and after prolonged aerobic exercise. Ten healthy males (21-35 years old) were tested on two occasions, separated by at least 14 days. On the first occasion, subjects were examined in a rested state but did not exercise. On the second occasion, the same subjects were examined at the same time of day before, during and after 2 h of exercise at 65% of peak oxygen consumption. Blood samples obtained at rest (t0), during (at 0.5, 1, 1.5 and 2 h, t0.5, t1, t1.5 and t2, respectively) and after (at 4 and 24 h, t4 and t24, respectively) exercise were analysed by two-colour flow cytometry for CD4+, CD8hi and CD56+ cell surface expression, and density of CD62L, CD49d and CD11a. At t2, circulating concentrations of CD56+, CD8hi and CD4+ lymphocytes had increased (P < 0.05) by 330%, 105% and 30%, respectively. The majority of CD4+, CD8hi and CD56+ lymphocytes mobilised to the blood at t2 were CD62L- and CD11ahi, although populations of CD4+ and CD56+ cells that expressed CD62L+ and CD11alo were also mobilised. Changes in subset concentrations at t0.5 were positively associated (r = 0.63; P < 0.01) with their corresponding mean surface density of CD11a at t0. Our findings suggest that the differential mobilisation of lymphocytes during prolonged aerobic exercise is linked to the surface expression of CD11a (i.e. lymphocyte-function-associated antigen-1). However, mechanisms unrelated to CD11a expression also appear to be involved.

Naive and memory T cell subsets are differentially mobilized during physical stress.

This study examined the naive and memory phenotypic profiles of CD4+ and CD8hi T cells that were mobilized to the peripheral circulation during a combination of aerobic exercise and heat stress, determining expression of the adhesion molecules CD62L and CD11a on the recruited cells. Twelve recreationally active males (age 27.1 +/- 5.3 yr, height 1.77 +/- 0.08 m, mass 76.9 +/- 12.0 kg, VO2peak 43.9 +/- 6.7 mL x kg(-1) x min(-1)) completed a 40 min bout of cycle ergometry at 65 % of VO2peak while immersed to mid-chest in a water bath at 39 degrees C. Venous blood samples were collected before (T0), during (T40) and 30 min after (T70) exposure to combined exercise and heat stress. Specimens were analyzed by three-colour flow cytometry for CD4+ and CD8hi T cell expression of CD45RO, CD11a and CD62L. Some 80 % of the CD4+ T cells that were mobilized were of the CD45RO memory phenotype, with the numbers of CD11alo and CD62L+ cells increasing more than those of CD11ahi and CD62L- cells. For the CD8hi cells, there was a more equal recruitment of CD45RO- naive (43 %) and CD45RO+ memory (57 %) cells. The majority (84 %) of recruited CD8+ cells were CD11ahi; there was a trend to predominance of CD62L- cells (57 %) for the memory subset, but with almost equal recruitment of CD62L+/- for the naive subset. We conclude that the exercise + heat stress induced trend to an increase in CD4+ T cells is linked in some way to memory phenotype; it cannot be explained simply by a high density expression of CD11a and lack of the lymph node homing receptor (CD62L). Furthermore, although mobilization of CD8hi T cells is not linked to memory phenotype, a high density expression of CD11a and a lack of the lymph node homing receptor are important determinants of CD8hi T cell mobilization.

Cytokine induction during exertional hyperthermia is abolished by core temperature clamping: neuroendocrine regulatory mechanisms.

The immunomodulatory effects of physiological temperature change remain poorly understood and inter-relationships between changes in core temperature, stress hormones and cytokines during exertional hyperthermia are not well established. This experimental study was designed to examine how cytokine (tumour necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-12 and IL-1ra (receptor antagonist)) and hormone (epinephrine (Epi), norepinephrine (NE), growth hormone (GH) and cortisol (CORT)) responses are modified when the exercise-induced rise in core temperature is attenuated or exacerbated by immersion in a water bath. Ten men ((mean +/- SD) age: 26.9 +/- 5.7 years; height 1.75 +/- 0.07 m; body mass 76.0 +/- 10.9 kg; O(2 peak): 48.0 +/- 12.4 mL kg(-1) min(-1)) completed two 40-min cycle ergometer exercise trials at 65% O(2 peak) while immersed to mid-chest. Rectal temperature (T(re)) peaked at 39.1 +/- 0.03 and 37.5 +/- 0.13 degrees C during the hot (39 degrees C) and cold (18 degrees C) conditions, respectively. Blood samples were collected before, during (20- and 40-min) and after (30- and 120-min) exercise. Increases in circulating NE (>350%), Epi (>500%), GH (>900%), IL-12 (>150%) and TNF-alpha (>90%) were greatest after 40-min exercise in the heat. Substantial elevations of CORT (80%), IL-1ra (150%) and IL-6 (>400%) did not occur until after exercise was complete. Core temperature clamping decreased the rise in circulating stress hormone concentrations and abolished increases in plasma cytokine concentrations. These findings suggest that exercise-associated elevations of T(re) mediate increases of circulating stress hormones, which subsequently contribute to induction of circulating cytokine release.

beta-Endorphin and natural killer cell cytolytic activity during prolonged exercise. is there a connection?

This study was designed to test whether a single 50-mg dose of the opioid antagonist naltrexone hydrochloride, ingested 60 min before 2 h of moderate-intensity exercise (i.e., 65% peak O2 consumption), influenced the exercise-induced augmentation of peripheral blood natural killer cell cytolytic activity (NKCA). Ten healthy male subjects were tested on four occasions separated by intervals of at least 14 days. A rested-state control trial was followed by three double-blind exercise trials [placebo (P), naltrexone (N), and indomethacin] arranged according to a random block design. The indomethacin exercise trial is discussed elsewhere (S. G. Rhind, G. A. Gannon, P. N. Shek, and R. J. Shepherd. Med. Sci. Sports Exerc. 30: S20, 1998). For both the P and N trials, plasma levels of beta-endorphin were increased (P < 0.05) at 90 and 120 min of exercise but returned to resting (preexercise) levels 2 h postexercise. CD3(-)CD16(+)CD56(+) NK cell counts and NKCA were significantly (P < 0.05) elevated at each 30-min interval of exercise compared with correspondingly timed resting control values. However, there were no differences in NK cell counts or NKCA between P and N trials at any time point during the two trials. Changes in NKCA reflected mainly changes in NK cell count (r = 0.72; P < 0.001). The results do not support the hypothesis that the enhancement of NKCA during prolonged submaximal aerobic exercise is mediated by beta-endorphin.