We developed a physiological assay to monitor lysosomal Ca2+ store refilling using specific activators of lysosomal Ca2+ channels to repeatedly induce lysosomal Ca2+ release. In contrast to the prevailing view that lysosomal acidification drives Ca2+ into the lysosome, inhibiting the V-ATPase H+ pump did not prevent Ca2+ refilling. Instead, pharmacological depletion or chelation of Endoplasmic Reticulum (ER) Ca2+ prevented lysosomal Ca2+ stores from refilling. More specifically, antagonists of ER IP3 receptors (IP3Rs) rapidly and completely blocked Ca2+ refilling of lysosomes, but not in cells lacking IP3Rs. Furthermore, reducing ER Ca2+ or blocking IP3Rs caused a dramatic LSD-like lysosome storage phenotype. By closely apposing each other, the ER may serve as a direct and primary source of Ca2+for the lysosome.Impaired homeostasis of lysosomal CaDOI:http://dx.doi.org/10.7554/eLife.15887.001 A reduction in [Ca2+]Ly is believed to be the primary pathogenic cause for some LSDs and common neurodegenerative diseases , as well as lysosomal Ca2+ sensors such as the C2 domain–containing synaptotagmin VII, are also required for these functions  . Like ths (LSDs) , lysosom4–0.6 mM , which idiseases . Using te fusion , lysosome fusion . Consistunctions . Whereasher LSDs .2+ concentration gradient across the lysosomal membrane is established and maintained is poorly understood. The most well understood Ca2+ store in the cell is the ER. Upon store depletion, the luminal sensor protein STIM1 oligomerizes to activate the highly Ca2+-selective ORAI/CRAC channels on the plasma membrane, refilling the ER Ca2+ store via the SERCA pump  Ca2+ external solution produced robust lysosomal Ca2+ release measured by GCaMP3 fluorescence  completely blocked the ML-SA1 response  external solution  cytosol . Furthermore, disruption of Golgi function using Brefeldin-A also had no effect on refilling P2 is a lysosome-specific phosphoinositide that regulates multiple lysosomal channels and transporters including ML1  ethylenediamine (TPEN), a membrane-permeable metal chelator with a low affinity for Ca2+ (2+-binding affinity in the acidic pH (pHLY = 4.6) suggests that chelation of lysosomal Ca2+ would be minimal. Acute application of TPEN completely blocked lysosomal Ca2+ refilling   . As expe release . Interes3T cells . Furtheroverslip . Collect2+ stores was previously suggested , both of which are expressed in HEK cells  cells  also slowly (up to 10 min) reduced lysosomal Ca2+ release, suggesting that constitutive lysosomal Ca2+ release under resting conditions may gradually deplete lysosome Ca2+ stores if refilling is prevented  cells . Acute arevented . Consist release , further2+ refilling  concentrations of ryanodine  DT40 chicken B cells  that werefilling , Xesto hefilling . In addiefilling . These rhibitors .2+, we employed two intraluminal Ca2+ indicators Fura-Dextran and Oregon 488 BAPTA-1 dextran (OG-BAPTA-dextran) (2+ ([Ca2+]LY) changes, but preferentially only when the intra-lysosomal pH (pHL) remains constant below pH 5.0  . After bdextran)  after endextran) . Due to w pH 5.0  , representing a high affinity uptake system. Our work, however, suggest that a low affinity uptake mechanism is more likely. Hence either a low affinity Ca2+ transporter or rectifying Ca2+ channel might suffice. A putative VDAC-like channel in the lysosome, resembling mitochondrial VDAC channels  Ca2+ transporter , free [Ca2+]Ly is generally agreed to be in the high μM range (100–500 μM)  increase in cytosolic Ca2+ (2+ dyes by affecting their chromophore fluorescence and Ca2+-binding affinity (Kd)   . Therefo buffers . Ca2+ buutral pH . Hence ilic Ca2+ . Additioity (Kd) . Because0–40 nM) . Moreoverescence  are inco2+ chelation is important for understanding the limits of this lysosome-specific pharmacological tool. GPN can certainly be used in conjunction with other tools to examine lysosome specificity, but caution is necessary with its use for Ca2+ store measurement, as a component of the signal observed in Fura-2 loaded cells, although small, is a result of the membrane permeabilization that causes a decrease in cytosolic pH. Similarly, reagents like Baf-A and NAADP that are used to mobilize lysosomal Ca2+ also release H+ into the cytosol   . Therefo2+ uptake studies on isolated lysosomes  (2+ reduces lysosome storage (versus prolonged treatment), many assumed-to-be ER-specific reagents may indirectly affect lysosome Ca2+ stores. This may impact the interpretations of a large body of literature on Ca2+ signaling. Although we demonstrated a central role of IP3Rs in lysosomal Ca2+ refilling, other ER Ca2+ channels may also participate under certain conditions, as seen in the IP3R TKO cells.Our results not only provide an explanation for the reported sensitivity of the Cag agents , but arem the ER . The unes (LSDs) , and how storage . In addi2+ release in cholesterol transport  as described previously .Standard Western blotting protocols were used. HEK293T cells were treated every 4 hr for 24 hr with IP3R antagonists 2-APB and Xestospongin-C, ER Ca2. HEK293T cells, Tet-On HEK293 cells stably expressing GCaMP3-ML1 (HEK-GCaMP3-ML1 cells), Cos-7 cells, and human fibroblasts were cultured in DMEM F12 (Invitrogen) supplemented with 10% (vol/vol) FBS or Tet-free FBS. DT40 cells were kept in suspension in RPMI 1640 (Invitrogen) supplemented with 450 µL β-mercaptoethanol, 2 mM L-glutamine, 10% FBS, and 1% chicken serum  were purchased from ATCC and cultured following standard culture protocols. DT40-WT and IP3R-TKO cells were a generous gift from Dr. Darren Boehning . Human fibroblasts were obtained from the Cornell Institute for Medical Research . HEK 293 cells stably expressing GCaMP3-ML1 (HEK-GCaMP3-ML1 cells) were generated using the Flip-In T-Rex 293 cell line (Invitrogen). All these cells were neither authenticated nor tested for mycoplasma contamination. HEK293 cells are on the list of frequently misidentified or cross-contaminated cell lines. All cells were cultured in a 37°C incubator with 5% COen serum . We noteHuman fibroblasts and DT40 cells were transiently transfected using the Invitrogen Neon electroporation kit . HEK293T cells, HEK-GCaMP3-ML1 cells, and Cos-7 cells were transfected using Lipofectamine 2000 (Invitrogen). All cells were used for experiments 24–48 hr after transfection.Live imaging of cells was performed on a heated and humidified stage using a Spinning Disc Confocal Imaging System. The system includes an Olympus IX81 inverted microscope, a 100X Oil objective NA 1.49 , a CSU-X1 scanner (Yokogawa), an iXon EM-CCD camera (Andor). MetaMorph Advanced Imaging acquisition software v.7.7.8.0 (Molecular Devices) was used to acquire and analyze all images. LysoTracker  was dissolved in culture medium and loaded into cells for 30 min before imaging. MitoTracker was dissolved in culture medium and loaded into cells for 15 min before imaging (25 nM). Coverslips were washed 3 times with Tyrode’s and imaged in Tyrode’s.470) using a EasyRatio Pro system (PTI). Cells were bathed in Tyrode’s solution containing 145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM Glucose, and 20 mM Hepes (pH 7.4). Lysosomal Ca2+ release was measured in a zero Ca2+ solution containing 145 mM NaCl, 5 mM KCl, 3 mM MgCl2, 10 mM glucose, 1 mM EGTA, and 20 mM HEPES (pH 7.4). Ca2+ concentration in the nominally free Ca2+ solution is estimated to be 1–10 μM. With 1 mM EGTA, the free Ca2+ concentration is estimated to be <10 nM based on the Maxchelator software (http://maxchelator.stanford.edu/). Experiments were carried out 0.5 to 6 hr after plating. Because baseline may drift during the entire course of the experiment (up to 20 min), we typically set F0 based on the value that is closest to the baseline.GCaMP3-ML1 expression was induced in Tet-On HEK-GCaMP3-ML1 cells 20-24h prior to experiments using 0.0 1µg/mL doxycycline. GCaMP3-ML1 fluorescence was monitored at an excitation wavelength of 470 nm (F340/F380) was used to calculate changes in intracellular [Ca2+]. All experiments were carried out 1.5 to 6 hr after plating.Cells were loaded with Fura-2 (3 µM) and Plurionic-F127 (3 µM) in the culture medium at 37°C for 60 min. Florescence was recorded using the EasyRatio Pro system (PTI) at two different wavelengths (340 and 380 nm) and the ratio (FKd) affinities of OG-BAPTA-dextran were determined using KCl-based solutions  adjusted to different pH . By varying the amount of added Ca2+ (X= 0- 10 mM), solutions with different pH and free [Ca2+] were made based on the Maxchelator software (http://maxchelator.stanford.edu/). OG-BAPTA-dextran (5 μg/ml) fluorescence for each solution was obtained to plot the calibration curve (min and Fmax) were determined by perfusing the cells with 0 or 10 mM Ca2+ external solutions, respectively. Lysosomal [Ca2+] at different pH were determined using the following calibration equation: [Ca2+] = Kd × (F-Fmin)/ (Fmax-F).Cells were loaded with Oregon Green 488 BAPTA-1 dextran (100 μg/ml) at 37°C in the culture medium for 4–12 hr, and then pulsed/chased for additional 4–16 hr. Fluorescence imaging was performed at 37°C. In vitro calcium-binding  that contained 10 μM nigericin and 10 μM monensin  internal solution or a 'low-pH' solution  were placed close to 'puff' the vacuoles. Images were captured using a CCD camera connected to the fluorescence microscope.GCaMP3-ML1-positive vacuoles were isolated from vacuolin-1-treated HEK-GCaMP3-ML1 cells, as described previously . Briefly2+ solutions for experiments. 2-APB, ATP, Con-A, CPA, Doxycycline, DHBP, TG, TPEN were from Sigma; GPN and U73122 were from Santa Cruz; Ryanodine was from Abcam; LysoTracker, Fura-2, Mitotracker, Plurionic F-127, and Fura-Dextran were from Invitrogen; Baf-A was from LC Laboratories; ML-SA1 was from Chembridge; and Xestospongin-C was from Cayman Chemical, AG Scientific, and Enzo; Oregon Green 488 BAPTA-1 dextran was from life technologies. ML-SI compounds were identified from a Ca2+-imaging-based highthroughput screening conducted at NIH/NCATS Chemical Genomics Center . ML-SI compounds are available upon request.All reagents were dissolved and stored in DMSO or water and then diluted in Tyrode’s and 0 CaData are presented as mean ± SEM. All statistical analyses were conducted using GraphPad Prism. Paired t-tests were used to compare the average of three or more experiments between treated and untreated conditions. A value of p<0.05 was considered statistically significant. In the cases only individual traces were shown, the traces are representative from at least 30–40 cells, or from at least independent repeats. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by David Clapham as the Reviewing Editor and Eve Marder as the Senior Editor. One of the three reviewers, Murali Prakriya, has agreed to share his identity.Thank you for submitting your article "The Endoplasmic Reticulum, Not the pH Gradient in the Lysosome, Are the Source of Calcium to Lysosomes" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:2+ refilling. Using various manipulations designed to inhibit the proton pump (Baf-A or Con-A) and several Ca2+ imaging tools that directly and indirectly monitor lysosomal Ca2+, the authors show that the proton pump by is likely not responsible for refilling of the lysosomal Ca2+ store. Rather the authors report that manipulations that deplete ER Ca2+ stores also affect refilling of the lysosomal compartment, suggesting a functional and possibly physical coupling of the ER and lysosomal Ca2+ stores. The authors include new results using OG-BAPTA-dextran showing that depletion of ER Ca2+ stores by thapsigargin, or inhibition of IP3Rs by the membrane permeable Xestospongin inhibited the lysosomal Ca2+ content. An interesting point is that the previously postulated H+-dependent Ca2+ transporter must operate with cytosolic free Ca2+ level of 100 nM and therefore be extremely high affinity. The present mechanism, although not identified, would be a low affinity uptake mechanism.Garrity et al. challenge the previously held view that lysosomal V-ATPase driven-acidification provides the driving force for lysosomal CaOverall the data represent quite a challenging and intriguing new concept. Although most of the data appear convincing, some major open questions remain to be addressed. We feel these can be remedied within a month by additional experiments.Essential revisions:2+]. In the Christensen studies, inhibiting V-ATPase with Baf (similar to the treatment used in this paper) increased luminal pH to ~ 7.0 (where the Ca2+ imaging dye should work well) and drastically decreased [Ca2+]lys from ~ mM to μM. Although you added new OG-BAPTA imaging, it needs calibration. You should show the data measuring the pH and [Ca2+]. The bar graphs (2+ change and how they may (or may not) correlate with the Ca2+ changes reported by the GCaMP ML1 indicator shown in the majority of the data. Example traces of the lysosomal Ca2+ indicator in response to lysosomal and ER Ca2+ store depletion should be shown.1) You need at least one convincing experiment showing that inhibiting the V-ATPase increases pH (needs to be measured) but doesn't affect store [Car graphs  do not p2+]lumen measurements were unreliable because of the pH-sensitivity of the dyes. In the Christensen et al. studies, however, the pH of each lysosome was monitored and used to calibrate the Kd of the dye. The Christensen studies (using lysosomes that do not overexpress any Ca-releasing channel) suggest that [Ca2+]lumen faithfully follows [pH]lumen. Other studies using 45Ca uptake measurement also support the importance of pH gradient . However, the pH sensitivity of the indicators used in the present paper were not tested in situ. We suggest you test the pH sensitivity of your Ca2+ indicator in situ by placing a small mouth pipette electrode containing low pH solution+ x [Ca2+] next to tagged channel indicator and measure its sensitivity to Ca2+ changes. Please provide more detail in the Discussion why you think the previously published measurements were erroneous.2) The authors contend that previous . In the Christensen studies, inhibiting V-ATPase with Baf (similar to the treatment used in this paper) increased luminal pH to ~ 7.0 (where the Ca2+ imaging dye should work well) and drastically decreased [Ca2+]lys from ~ mM to μM. Although you added new OG-BAPTA imaging, it needs calibration. You should show the data measuring the pH and [Ca2+].1) You need at least one convincing experiment showing that inhibiting the V-ATPase increases pH (needs to be measured) but doesn't affect store  ([Ca2+]Ly), which was ~ 360 μM under the resting condition, dropped quickly to ~ 100 μM upon ML-SA1 application (see Kd is ~300 μM (2+ ranging from 30 (0.1 Kd) to 3,000 (10 Kd) μM [3]. At the resting pHL of 4.8 in the HEK cells, OG-BAPTA- dextran’s working range is 130-1,300 μM (Kd ~ 130 μM). Overall, all these values are in rough agreement with previous measurements .We performed the experiments as suggested by the reviewers. First, we monitored lysosomal pH under different conditions using Oregon green 488-dextran, a commonly- used luminal pH indicator   are often not separately-considered since these two parameters are interrelated. Whereas the total [Ca2+]Ly is reported to be in the low mM range (5-10 mM), free [Ca2+]Ly is generally agreed to be in the high μM range  [3]. Therefore, the lysosome lumen must contain a substantial amount of Ca2+ buffers [3]. Ca2+ buffers in the acidic compartments and ER are known to bind Ca2+ much better at neutral pH (H+ and Ca2+ compete with each other for Ca2+ buffers) [4]. Hence increasing pHL from 4.8 to 7.0 may effectively reduce free [Ca2+]Ly without necessarily triggering lysosomal Ca2+ release and affecting total [Ca2+]Ly.First, a change in lysosomal pH may cause a significant change in luminal Ca2+ dyes by affecting their chromophore fluorescence and Ca2+-binding affinity (Kd) [3]. Because Kd is dropped more than 1,000 times when pHL is increased from 4.8 to 7.0 . In the current study, upon ML-SA1 application to deplete the stores, free [Ca2+]Ly dropped from 360 to 100 μM , which is not suitable for measuring Ca2+ over 10 μM.Second, lysosomal pH may act on luminal Ca8 to 7.0 , perfecto 100 μM . Hence, 2+ homeostasis, for instance, via membrane fusion and fission between compartments containing different amounts of Ca2+, H+, and their buffers.Third, prolonged lysosomal pH manipulations may also indirectly affect lysosomal Ca2+ release, the accompanied increase in cytoplasmic Ca2+ was rather small (20-40 nM) . This minor increase is more consistent with the interpretation that other pH-dependent processes contribute to the changes of OG-BAPTA fluorescence upon Baf-A1 and NH4Cl application. In addition, the instantaneous changes (following pH increase and decrease) of Ca2+ probe fluorescence  internal solution or a “low pH” solution  close to the vacuoles to produce a “puffing” effect. We showed that GCaMP3- ML1 responded to both Ca2+ and pH , and included a discussion in the Results.4Cl markedly increased lysosomal pH from 4.8 to 7.0 , preventing accurate determinations of [Ca2+]LY under these pH manipulations”.Results: “In contrast, treatment of cells with Baf-A1 or NH8 to 7.0 . Such latran see  and lumi2+]Ly . However, in addition to triggering lysosomal Ca2+ release, as proposed by Christensen et al. , lysosomal pH elevation is also known to affect [Ca2+]Ly or its measurement via several other mechanisms. Whereas the total [Ca2+]Ly is reported to be in the low mM range (5-10 mM), free [Ca2+]Ly is generally agreed to be in the high μM range (100-500 μM) . Therefore, lysosome lumen must contain substantial amount of Ca2+ buffers . Ca2+ buffers in the acidic compartments and ER are known to bind Ca2+ much better at neutral pH . Hence increasing pHLfrom 4.8 to 7.0 may effectively reduce free [Ca2+]Ly without necessarily triggering lysosomal Ca2+ release and affecting total [Ca2+]Ly. Consistent with such interpretation, a compelling study recently demonstrated that in secretory granules and the ER, increasing luminal pH changed the Ca2+ buffering capacity of both Ca2+ containing compartments intraluminally to reduce free [Ca2+], while causing a minimal (20 nM) increase in cytosolic Ca2+ . Additionally, lysosomal pH may act on luminal Ca2+ dyes by affecting their chromophore fluorescence and Ca2+-binding affinity (Kd) . Because Kd is dropped more than 1,000 times when pHLis increased from 4.8 to 7.0, perfect calibration is near impossible. Furthermore, prolonged lysosomal pH manipulations may also indirectly affect lysosomal Ca2+ homeostasis, for instance, via membrane fusion and fission between compartments containing different amounts of Ca2+, H+, and their buffers. Finally, although elevating lysosomal pH may trigger lysosomal Ca2+ release, the accompanied increase in cytoplasmic Ca2+ was rather small (20- 40 nM) . Moreover, the instantaneous changes (following pH increase and decrease) of Ca2+ probe fluorescence  are inconsistent with the slow rates of Ca2+ leak and re-uptake demonstrated in the current study”.Discussion:“Lysosomal pH gradient is thought to be essential for the maintenance of high free [Ca3) A triple IP3R knockout cell line was used to assess the proposed role of IP3Rs in refilling the lysosomal store. However, in contrast to the authors' theory, this does not have a lysosomal Ca2+ defect. The authors attribute this to an unknown compensatory mechanism. Please examine whether the Xestospongin abolishes ML1-SA1 responses in the triple IP3R KO cells and show the kinetics of the response. If the responses in the IP3R KO are not affected, and the kinetics now differ, the results would provide an additional layer of confidence for the proposed role of IP3Rs in lysosomal refilling.2+ refilling still occurred in IP3R TKO cells, the refilling kinetics were much slower than WT cells  Johnson, D.E., et al., The position of lysosomes within the cell determines their luminal pH. J Cell Biol, 2016. 212(6): p. 677-92.2) DiCiccio, J.E. and B.E. Steinberg, Lysosomal pH and analysis of the counter ion pathways that support acidification. J Gen Physiol, 2011. 137(4): p. 385-90.3) Morgan, A.J., L.C. Davis, and A. Galione, Imaging approaches to measuring lysosomal calcium. Methods Cell Biol, 2015. 126: p. 159-95.4) Dickson, E.J., et al., Orai-STIM-mediated Ca2+ release from secretory granules revealed by a targeted Ca2+ and pH probe. Proceedings of the National Academy of Sciences of the United States of America, 2012. 109(51): p. E3539-48.5) Christensen, K.A., J.T. Myers, and J.A. Swanson, pH-dependent regulation of lysosomal calcium in macrophages. Journal of cell science, 2002. 115(Pt 3): p. 599-607.


Calcium signalling relies on the flux of calcium ions across membranes yet how signals in different compartments are related remains unclear. In particular, similar calcium signals on both sides of the nuclear envelope have been reported and attributed to passive diffusion through nuclear pores. However, observed differing cytosolic and nucleosolic calcium signatures suggest that the signalling machinery in these compartments can act independently.We adapt the fire-diffuse-fire model to investigate the generation of perinuclear calcium oscillations. We demonstrate that autonomous spatio-temporal calcium patterns are still possible in the presence of nuclear and cytosolic coupling via nuclear pores. The presence or absence of this autonomy is dependent upon the strength of the coupling and the maximum firing rate of an individual calcium channel. In all cases, coupling through the nuclear pores enables robust signalling with respect to changes in the diffusion constant.We show that contradictory interpretations of experimental data with respect to the autonomy of nuclear calcium oscillations can be reconciled within one model, with different observations being a consequence of varying nuclear pore permeabilities for calcium and refractory conditions of channels. Furthermore, our results provide an explanation for why calcium oscillations on both sides of the nuclear envelope may be beneficial for sustained perinuclear signaling.The online version of this article (doi:10.1186/s12918-016-0289-9) contains supplementary material, which is available to authorized users. The intμm2 [2+ spreads from a channel, C, firing once on the cytosolic side of the NE to a point on the nucleosolic side of the NE, PN, Fig. C to PN, that are larger than Ca2+ micro-domains formed near firing calcium channels. We measured the maximum amount of Ca2+ that reaches PN relative to the release quantity from C.Different species and cellular stages can have different pore numbers and distributions –56, withμm2 . We accoμm2 , 57 by v2+ transmission increases with pore density before saturating at relatively low pore densities of about 2 pores /μm2. The addition of pores does not increase the transmission further. To understand the meaning of this saturation, we asked what would happen if PN was on the same side as C. If there were no pores present then, as illustrated in the grey region of Fig. 2+ measured at PN would be twice the value measured before. This happens if channel C had released the same amount of Ca2+ as before. The circled values in the grey region of Fig. PN when C releases half as much Ca2+. For the cases where the diffusion constants on either side of the NE are equal, we see that these circled values coincide with the saturation values. This illustrates that when we have a saturation pore density half the Ca2+ released by a channel is passing through the pores to the other side of the NE. This is equivalent to diffusion of a substance in a half space, compared to diffusion without the barrier, in a full space. It confirms that as far as diffusing Ca2+ is concerned, beyond a certain pore density the NE provides no barrier to passage between the two sides. We refer to this as the NE becoming transparent.We observe in Fig. 2+ back into the NE. This limits the maximum amount of Ca2+ that can be transmitted for a smaller diffusion constant D . This led us to look more carefully into the actual contribution of individual pores to transmission. The spatial distribution of pores determines the characteristics of the Ca2+ diffusion paths between calcium release channels, which will be relevant for the CICR activation.Even with a large pore diameter of 0.029 2+ concentration that results from Ca2+ diffusing from a given pore  and cytosolic (black lines) sides of the NE. We consider a pore density of 2 pores/ μm2 so the results in Fig. In this section we show that under certain conditions distinct oscillation frequencies can occur in the different compartments despite the coupling of channel activation that occurs in the presence of pores. We considered a nucleus with 24 randomly placed channels on each side of the nucleus, and studied the generation of signals in the two compartments. In Fig. 4D, which permits cytosolic Ca2+ oscillations in the absence of pores. We demonstrate this by shutting the channels on one side of the NE after 15 seconds, to see the effects on the other side. Inactivating the nucleosolic channels leaves cytosolic calcium release unaffected, Fig. 42+ oscillations can not be sustained without cytosolic influx. As illustrated in Fig. 4The cytosolic side of the NE is taken as having a larger diffusion constant, 2+ it enters a refractory period during which it will not fire  have similar amplitudes, since Ca2+ microdomains with very different amplitudes and spatial extent can result in the same global Ca2+ level, as the comparison of the spatial profiles represented in Fig. 4Figure tes Fig. a1 and b1tes Fig. a2 and b2tes Fig. a1 and b1chniques . However2+ patterns [2+ oscillations. Here we show that within one framework we can obtain 1) oscillations on only one side of the NE for different diffusion constants and little flux through the nuclear pores, 2) synchronised Ca2+ signals in cases where the nuclear pores transmit sufficient Ca2+ between different sides of the NE if the channels have the same refractory period, 3) different oscillations when the NE becomes transparent to Ca2+and when different conditions on either side of the NE lead to different refractory periods. Different conditions could be caused by different buffering capacity for Ca2+ [2+ oscillations, which may be important for sustaining oscillations over longer periods in varying conditions.Experimental data shows a variety of similarities but also differences between cytosolic and nuclear Capatterns –68. In pfor Ca2+ . Further2+ oscillation patterns to be maintained, even when the oscillations are coupled via diffusion through the nuclear pores. This coupling may be essential to sustain oscillations. This result reveals a new role for pores, as elements that increase the robustness of the channels’ activation by CICR, against variations in the diffusion coefficient or buffering conditions of the inner and outer nuclear membranes. Therefore, instead of the Ca2+ flowing from the cytosol through pores being a significant fraction of the nuclear Ca2+ transients, it may simply provide access to an alternative path of diffusion in the fire-diffuse-fire process.We demonstrated that free diffusion through the NE allows different cytosolic and nuclear CaOur models reveal that the permeability of the nuclear envelope is determined not only by the number and size of pores, but also by their distribution and the characteristics of the environments they connect, such that a large number of pores does not necessarily translate into a larger permeability.2+ binding, which is supported by the identification in some systems of IP3R channels on both sides of the NE. Our results are valid also for indirect forms of calcium-induced calcium release, e.g., if Ca2+ activates a cation channel that regulates a voltage-gated calcium channel [2+ oscillations patterns offers a clue to the channels activation mechanism.We have assumed that channel gating depends on Ca channel , 70. Nev channel , 41, or 2+ signals observed. While we know channels enter a refractory period during signalling, what mechanism controls the length of the refractory period during biological signalling processes is still unclear [2+ signatures.The refractory period has an important influence on the overall Ca unclear . Given t2+ signatures [Next steps include extending our model to a three-dimensional implementation to investigate the role of morphology in determining different nuclear Cagnatures , as wellgnatures .2+ is reduced by the uptake into the nuclear envelope (NE) store, which we modelled as a uniform pump rate across the NE that depends linearly on the Ca2+ concentration. On the surface of a NE without pores, the Ca2+ concentration c=c evolves with time as: We adapt the fire-diffuse-fire model  with pumD is the diffusion constant and ks is the rate at which Ca2+ is pumped back into the store . For comparability of units, we have assumed that the relevant domain for the concentration, c, is a thin layer, however, we highlight the fact that all the calculations are performed on a surface by using the units M ∗ to indicate that it’s is not strictly per volume.where ri on the same side of the NE. The calcium release channels are treated as point sources, which are expressed as δ-functions. We explore the saltatory regime [i=1,…,N and k=1,2,… We consider the interaction between N channels located at positions y regime  of the Fσ is the amount released by a channel in a single firing event. To determine the firing times according to the CICR mechanism, we assume that the channel will fire if the local Ca2+ concentration, computed at the channels’ coordinates, is greater than a given threshold concentration, cth.where 2+ is released from multiple stores such as the ER, mitochondria or possibly the nucleoplasmic reticulum, and propagates in complicated geometries and crowded environments. However, to model the influence of pores on nuclear Ca2+ signalling, we note that the Ca2+ spreading from non-perinuclear regions would arrive at the nucleus in negligible proportions [t the Ca2+ concentration at a location rj at the surface is: Caportions  and can co and ci are the Ca2+ concentrations on the outer and inner sides of the nuclear envelope. Equation 2+ to move across the surface of the sphere representing the nucleus. The position r is expressed in spherical coordinates as r=, where θ is the polar angle, ϕ the azimuthal angle and R is the radius of the nucleus. In this case, the shortest distance Δrij between a source ri and any point rj is the length of the great circle arc that connects them, given by where N=24 channels >cth. So we find the time t that satisfies: For a given activation threshold σ have been made, the condition for the critical surface for propagation in terms of the parameters ks and D becomes Once choices of the average inter-channel distance σ, the activation requirement is fulfilled when the uptake rate is not too large, or the diffusion constant not too small. A graphical representation of the parameter choices for which traveling wave solutions exist appears in Additional file D is larger. Faster pumps increase the risk of propagation failure unless D is large enough.For a given D as the parameter to distinguish the Ca2+ propagation abilities on the inner and outer nuclear membrane, but the analogous results are expected for other choices that lead to either propagation failure or a Ca2+ wave. Throughout the paper and for simplicity, we have assumed a larger diffusion constant D on the cytosolic side of the NE but analogous conclusions hold if we reverse the diffusion constant ratio between different sides of the NE.Characteristics of the signals, such as frequency or amplitude, are affected by various interchangeable parameters. Our illustrative examples use the diffusion coefficient cth=0.0425 μM*, amount released by a single release event σ=2.0×10−20 mol.Unless otherwise stated in the figure legend, the results refer to the following parameters throughout the paper: firing threshold We couple the inner and outer nuclear membranes through the inclusion of pores, which we treat as point sources . This adnp nuclear pores at positions rk, k=1,…,np, having a flux through them of Ωk(t). We assume the pores are either closed, in which case Ωk(t)=0, or open allowing Ca2+ to passively diffuse through them according.where the summation is over all the Δt). We assume that beyond their geometry, the pores pose no additional obstacle to diffusion. The thickness of the nuclear envelope, Δm, the diffusion constant for Ca2+ in the pore, DP, and its diameter, pd, determine the size of the pulse, given by According to Fick’s law of diffusion, the flux across pores will depend on the calcium concentration gradient between the nucleus and the cytosol, which can be approximated by a difference formula at each time step −ci).We had, Eq. ed by Eq., for theΩ can therefore take negative and positive values depending on the relative concentration on the inner and outer nuclear membranes. The overall Ca2+ concentration, At each time step, the flux ci/ois the contribution from the channels on the same side of the NE, as before  channels’ refractory period as long as two conditions are fulfilled. First, coupling must be strong enough to allow the channels to fire at the maximum rate imposed by their refractory period. Secondly, nuclear channels should fire in unison, so their joint contribution to global Ca2+ levels is much larger than the pores contribution. If these conditions are met, nuclear and cytosolic Ca2+ will oscillate with clearly independent frequencies, see Fig. Then, the observed nuclear Ca2+ release. One way to couple and try to synchronise nuclear and cytosolic refractory periods is to make their duration depend solely on the rate of decrease of the actual local Ca2+ concentration. In case pores were able to homogenise calcium concentrations on either side of the NE in between firings, the nuclear and cytosolic channels could then fire simultaneously. Otherwise, cytosolic and nucleosolic channels would still fire at different times, but their firings times would not be autonomous, see Additional file For completeness, we have further explored some situations that promote simultaneous nuclear/cytosolic Ca2+ oscillations, which is the object of our interest.Note on parameters: When considering a realistic number of pores, the simulation of Eq. α of the threshold as α=0.15. There, we were considering the Ca2+ levels at an isolated channel, as explained in this subsection. The important point is that the actual local Ca2+ levels, which include the contribution of other channels and pores, do not drop below the activation threshold, at least for some channels. Otherwise the continuation of the CICR mechanism would require some external stimulus.In the main text, we choose the fraction 2+ away from the surface is an important consideration.By restricting ourselves to a 2 dimensional model of the surfaces, we can dramatically increase the efficiency of our simulation. Since the key components involved in perinuclear signalling, the pumps, channels and pores, are localised to the nuclear membrane, the inner and outer nuclear surfaces are the most important regions to consider when modelling signal generation. However, diffusion of CaD, as D increases, and then it fails to propagate when D is large enough. This counterintuitive effect is due to the finite nature of the calcium source in the continuum limit.Extending the FDF model, Pando et al.  investigD would not be observed even for unrealistically large D. For the pores sources, the regime is not so clear: pores are more closely spaced than channels and release calcium until the concentration gradient vanishes. However, the ability for pores to mediate efficient signalling should remain. This ability depends on a diffusion path from the channels in the surface with larger D, via pores, to channels on the other side with smaller D. The first leg of the path  is not likely to fail due to the saltatory regime for the calcium source. The second leg , isn’t likely to fail either even if sources - pores - would operate in the continuum limit, because D is small in this surface.For diffusion spreading from calcium channels in the saltatory regime Eq. D is. However, they don’t count the effect of the pumping rate as a sink, and as we saw (Eq. D. This is not likely to change in a 3D context.Moreover, even if we had considered a continuum wave limit, it is not clear whether a similar effect would be acting in our system. In Pando et al. , diffusi saw Eq. , pumps aAdditionally, propagation into a cell or a nucleus would not make calcium vanish into an infinite space. Particularly on the inner nuclear membrane where paths between two points through the bulk are shorter than paths along the surface.2+ signals can be modulated by the morphology of the nucleus [To model the complex geometry of the perinuclear signalling process is beyond the scope of this work. In biological systems, diffusion is typically anisotropic, with diffusion coefficients in the bulk and cellular membranes varying significantly. Additionally, cellular environments are crowded and we don’t expect free diffusion over long distances. Nevertheless, the extension of our model to a 3D geometry would be a very important future development. As the results of Pando et al.  showed,  nucleus .3, inositol 1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptors; NE, nuclear envelopeCICR, calcium-induced calcium release; FDF, fire-diffuse-fire model; IP


Knowledge translation (KT) supports use of evidence in healthcare decision making but is not widely practiced. Mentoring is a promising means of developing KT capacity. The purpose of this scoping systematic review was to identify essential components of mentoring that could be adapted for KT mentorship.Key social sciences and management databases were searched from January 2002 to December 2011 inclusive. Empirical research in non-healthcare settings that examined mentorship design and impact for improving job-specific knowledge and skill were eligible. Members of the study team independently selected eligible studies, and extracted and summarized data.Of 2,101 search results, 293 were retrieved and 13 studies were eligible for review. All but one reported improvements in knowledge, skill, or behavior. Mentoring program components included combining preliminary workshop-based training with individual mentoring provided either in person or remotely; training of mentors; and periodic mentoring for at least an hour over a minimum period of six months. Barriers included the need for infrastructure for recruitment, matching, and training; lack of clarity in mentoring goals; and limited satisfaction with mentors and their availability. Findings were analyzed against a conceptual framework of factors that influence mentoring design and impact to identify issues warranting further research.This study identified key mentoring components that could be adapted for KT mentorship. Overall, few studies were identified. Thus further research should explore whether and how mentoring should be tailored to baseline knowledge or skill and individual KT needs; evaluate newly developed or existing KT mentorship programs based on the factors identified here; and examine whether and how KT mentorship develops KT capacity. The conceptual framework could be used to develop or evaluate KT mentoring programs. Despite widespread focus on quality in the healthcare sector, performance reports highlight the need to improve the organization, delivery, and outcomes of healthcare services and programs . KnowledKT capacity is needed to better equip those interested or engaged in KT practice or science. Decision makers, including clinicians, managers and policy makers, have professed that they are unfamiliar with, or confused about the concept and practice of KT, which may be why KT is not widely practiced ,6. In CaClearly, both researchers and decision makers require knowledge and skill to engage in KT practice or science. Recognizing this need, the United States National Institutes of Health and Veterans Health Administration developed a five-day training institute for postdoctoral fellows to learn about dissemination and implementation . It was Mentorship is an interactive, facilitative process meant to promote learning and development that is based on educational and social learning theories . MentoriIn healthcare, mentoring has largely been studied in the context of academic medicine where physician trainees or junior physician investigators are mentored by those more experienced for career and personal development . The benResearch funders, educators, investigators, and decision makers worldwide have identified the need to improve the quality of healthcare by building capacity for KT. Training programs for KT practice and science are few. Mentorship is a strategy that enhances access to knowledge which could be used alone, or in conjunction with KT training programs to develop KT capacity. However, limited empirical research has examined its application in healthcare, particularly outside of academic mentorship for physicians or nurses. Mentorship has been widely used in disciplines other than healthcare. The purpose of this research was to review literature in management and social sciences and identify essential components of mentoring programs that could be adapted for KT mentorship. More specifically, this review described the quantity and quality of research employing a variety of designs on mentorship, and examined the effectiveness of mentorship when used to develop job-related knowledge and skills among professionals in non-healthcare settings.post hoc based on increasing familiarity with the literature [We conducted a scoping review of published research in the social sciences and management disciplines . While sterature . A scopiterature . A protoThere is no single theory or model that describes mentorship processes, outcomes, and influencing factors. However, a useful framework for this study was developed by Karcher, who reviewed educational and psychology literature to outline elements of mentoring program design that might influence outcomes  . These eBusiness Source Premier (BSP) and Social Sciences Citation Index (SSCI) were searched from January 2002 to December 2011 inclusive for empirical studies focusing on mentorship features and outcomes  were eligible for review  and measures of impact so pooling of data was not possible, nor would it be an option for future research, further limiting the inferences that can be drawn from this review. However, a strength of this study was use of a conceptual framework by which to analyze data, and identify gaps in knowledge and recommendations for future research, which is a key purpose for a scoping review.KT training programs mentioned earlier ,21 alreaThough only two of the eligible studies assessed barriers, several were identified, including the need for considerable infrastructure and resources to support activities such as recruitment, matching, and preliminary training events; lack of clarity in mentoring goals; and limited confidence in, and satisfaction with mentors and their availability. These barriers have also been identified by others, though in reference to academic mentoring ,67. FurtSeveral issues included in the conceptual framework were not addressed by eligible studies. This included stages of mentoring; and feedback and ongoing support. Ongoing studies that evaluate mentorship for developing KT capacity should explore whether and how complementary activities or mentoring content should be tailored according to baseline knowledge or skill, individual KT needs, and maturity of the mentoring relationship that reflects stages of mentoring. Such studies could also examine whether periodic interaction or other type of support may be needed following the formal period of mentorship to reinforce learning. No eligible studies examined mentor characteristics or outcomes. Given that the mentor characteristics may strongly influence the formation, function, and success of mentoring , furtherKey components of mentoring programs that may form the basis of KT mentorship include combining preliminary workshop-based training with individual mentoring provided either in person or remotely; training of mentors; and periodic mentoring for at least an hour over a minimum period of six months. Further research is needed to address identified gaps includes developing and implementing, or evaluating existing KT mentorship programs based on the factors identified here, and examining whether and how KT mentorship develops KT capacity.


Peripheral blood mononuclear cells (PBMC) concentrates collected by apheresis are frequently used as starting material for cellular therapies, but the cell of interest must often be isolated prior to initiating manufacturing.The results of enriching 59 clinical PBMC concentrates for monocytes or lymphocytes from patients with solid tumors or multiple myeloma using a commercial closed system semi-automated counter-flow elutriation instrument  were evaluated for quality and consistency. Elutriated monocytes (n = 35) were used to manufacture autologous dendritic cells and elutriated lymphocytes (n = 24) were used manufacture autologous T cell therapies. Elutriated monocytes with >10% neutrophils were subjected to density gradient sedimentation to reduce neutrophil contamination and elutriated lymphocytes to RBC lysis.Elutriation separated the PBMC concentrates into 5 fractions. Almost all of the lymphocytes, platelets and red cells were found in fractions 1 and 2; in contrast, most of the monocytes, 88.6 ± 43.0%, and neutrophils, 74.8 ± 64.3%, were in fraction 5. In addition, elutriation of 6 PBMCs resulted in relatively large quantities of monocytes in fractions 1 or 2. These 6 PBMCs contained greater quantities of monocytes than the other 53 PBMCs. Among fraction 5 isolates 38 of 59 contained >10% neutrophils. High neutrophil content of fraction 5 was associated with greater quantities of neutrophils in the PBMC concentrate. Following density gradient separation the neutrophil counts fell to 3.6 ± 3.4% . Following red cell lysis of the elutriated lymphocyte fraction the lymphocyte recovery was 86.7 ± 24.0% and 34.3 ± 37.4% of red blood cells remained.Elutriation was consistent and effective for isolating monocytes and lymphocytes from PBMC concentrates for manufacturing clinical cell therapies, but further processing is often required. Peripheral blood mononuclear cells (PBMC) concentrates collected by apheresis are rich in lymphocytes, monocytes and NK cells and they are a common source of starting material for the manufacture of cell and gene therapies. PBMC concentrates are used as a source of monocytes for the manufacture of dendritic cells (DCs), lymphocytes for cytokine stimulated and genetically engineered T cell therapies and natural killer (NK) cells for autologous and allogeneic immunotherapy. They also contain red blood cells (RBCs), platelets and neutrophils and in order to consistently produce high quality products, it is often necessary to isolate monocytes, lymphocytes or NK cells from PBMC concentrates.When PBMCs are used as a source of autologous monocytes to manufacture DCs for cancer or antiviral immunotherapy the manufacturing process requires that monocytes are enriched from the PBMC concentrates. The monocytes are then incubated with agents such as GM-CSF and IL-4 for 3 to 7 days to differentiate them into immature DCs and with further culture on to mature DCs ,2. A varAnother option for the isolation of monocytes from PBMC concentrates is continuous counter-flow elutriation. Elutriation uses centrifugal force against a continuously increasing fluid flow to separate cells based on size and to a lesser extent density. An automated continuous counter-flow elutriation instrument is available that has been used primarily to isolate monocytes from PBMC concentrates, the Elutra system. This instrument makes use of a closed system sterile plastic disposable kit that allows for the sterile processing of the cells –11. The PBMC concentrates are also used as a source of allogeneic lymphocytes from hematopoietic stem cell (HSC) transplant donors to treat patients with disease relapse following HSC transplantation; furthermore autologous PBMCs are used as a source of lymphocytes for adoptive immunotherapy of cancer. Aliquots of unprocessed PBMC concentrates can often be used for donor lymphocyte infusions, but for adoptive immunotherapy, lymphocytes generally must be free from contaminating RBCs and neutrophils. Immunomagnetic selection with CD3, CD4 or CD8 monoclonal antibodies conjugated to magnetic particles can also be used to select or deplete T cells or specific subsets of T cells –15. We hWhile elutriation has been used to enrich for and separate monocytes ,16 and lSubjects were enrolled in IRB-approved protocols that made use of mature DCs to treat adults with prostate or other advanced cancers or rapamycin-generated Th1/Tc1 lymphocytes ,19 to trPBMC concentrates were collected using a blood cell separator  by processing 8 to 12 liters of whole blood.9 cells but not more than 20×109 cells. The program for the collection of monocytes collects cells in 5 fractions. The chamber rotation speed was maintained at 2400 RPMs for fractions 1 through 4 and the media flow rate was maintained at 60 ml/min for fraction 1, 120 ml/min for fraction 2, 122 ml/min for fraction 3 and 124 ml/min for fraction 4. Fraction 5 was the cells remaining in the chamber and they were collected with the rotor turned off.The PBMC concentrates were subject to elutriation using a semi-automatic counter-flow elutriation instrument  using a user defined profile. Our procedure required that the quantity of white blood cells loaded into the Elutra be at least 5×108 cells per mL. Density gradient separation was performed by placing 40 mL of leukocyte suspension in a 50 ml conical tube and underlying cells with 10 mL of Ficoll/Hypaque . After centrifugation at 835×g for 25 minutes the mononuclear cells were removed, washed  and resuspended in HBSS.The leukocytes were pelleted by centrifugation (350×g) for 10 minutes and resuspended in Hank’s Balance Salt Solution (HBSS)  at a concentration of 1-2×10After pelleting the cells, they were incubated for 7 to 10 minutes with ACK Lysing Buffer  and then washed and resuspended in 0.9% saline  with 0.3% trisodium citrate .Blood counts were measured using automated hematology analyzer (Cell-Dyn 3700).The values shown are mean ± 1 standard deviation unless otherwise indicated. Groups were compared using t-tests .A total of 59 autologous elutriated PBMC concentrates were studied which were collected from 51 subjects: PBSCs were collected from 6 subjects twice and 1 subject three times. Elutriated monocytes from 35 PBMC concentrates were used to manufacture autologous DCs for immunotherapy of cancer and elutriated lymphocytes from 24 PBMC concentrates were used to manufacture rapamycin-generated Th1/Tc1 lymphocytes cells for adoptive cell therapy following autologous transplantation of patients with multiple myeloma. 35 subjects were male and 16 were female and 42 were White, 7 were African American, 1 was mixed and for 1 the race was not known. The median age of the subjects was 59.3 years and ranged from 35.7 to 77.3 years.Analysis of all 59 elutriated PBMC concentrates found that the majority of leukocytes were found in fractions 1, 2 and 5  and monocytes  but similar quantities of neutrophils, lymphocytes, platelets and RBCs.The separation of neutrophils and lymphocytes was more consistent than that of monocytes. For all 59 elutriated PBMC concentrates fractions 1 through 4 contained less than 1×10Our clinical DC manufacturing protocols use fraction 5 as a source of starting material for manufacturing DCs. While the majority of leukocytes in the 5 fraction were monocytes 68.1 ± 22.4%), a large quantity of neutrophils were present (24.8 ± 23.1%). Our protocols require that monocytes used to manufacture DCs contain 10% or less neutrophils. Among the 59 PBMC concentrates in this study, 35 were used to manufacture DCs and only 12 of the 35 fraction 5′s (34%) contained 10% or less neutrophils. The 23 fraction 5′s with >10% neutrophils were subjected to density gradient separation over ficoll to separate the monocytes and neutrophils. Following ficoll density gradient separation all 23 products contained less than 10% neutrophils and few monocytes were lost   and the other 31 products were from patients with either multiple myeloma or various advanced solid cancers. In general, the health of the patients with prostate cancer was better than that of the other patients. To determine if the condition of people donating PBMC concentrates influenced the effectiveness of the counter-flow elutriation process we compared the composition of the cells loaded into the elutriation instrument and the elutriated fractions among these two groups. Fraction 5 from patients with multiple myeloma and advanced cancers had significantly more monocytes and lymphocytes but similar number of neutrophils Table . This isWe used an automated counter-flow elutriation instrument to enrich monocytes from PBMC concentrates to manufacture clinical DC products and clinical rapamycin-generated Th1/Tc1 lymphocytes for autologous adoptive immunotherapy. When PBMC concentrates from patients with prostate cancer, multiple myeloma and other advanced malignancies were processed with the elutriation instrument, lymphocytes were easily, consistently and cost effectively separated from monocytes. Almost all monocytes were found in fraction 5 and almost all lymphocytes were present in fractions 1 and 2. However, almost all neutrophils were also found in fraction 5 with monocytes and almost all RBCs were in fractions 1 and 2 with lymphocytes. These results are similar to those of other reports which evaluated the same instrument to isolate monocytes from small numbers of PBMC concentrates collected from healthy subjects ,11,20–239 cells but there were no upper limits on the quantity of monocytes loaded.We found that the separation of monocytes was less consistent than that of lymphocytes and neutrophils. For 10% of the PBMC concentrates elutriation resulted in the distribution of relatively large quantities of monocytes into fractions 1 or 2. This was not a failure to separate monocytes since significant quantities of monocytes were also found in fraction 5. The fact that PBMCs concentrates with large quantities of monocytes in fractions 1 or 2 contained larger quantities of white blood cells and monocytes suggest that loading too many monocytes into the elutriation instrument may result in suboptimal monocyte separation. Our elutriation protocol limited the number of white blood cells loaded to ≤ 20×10While counter-flow elutriation was consistent and effective at separating lymphocytes and monocytes, the elutriated cells of interest commonly require further processing before they could be placed into culture. In order to ensure consistent manufacturing of DCs from monocytes, our manufacturing protocols require that the monocytes used for starting material contain less than or equal to 10% neutrophils. We found that only about one-third of the products met these criteria, but the quantity of neutrophils among the monocytes could be reduced greatly by density gradient separation with little loss of monocytes. We investigated factors that might influence the neutrophil content of the elutriated monocytes and found that the portion of neutrophils in the monocyte rich fraction 5 was dependent on the quantity of neutrophils loaded into elutriator at the onset of the process; this result is similar to the findings of Chen et al. . If we wWhen the counter-flow elutriator was used to enrich lymphocytes from PBMC concentrates, we used the lymphocyte rich fractions 1 or 2 or only fraction 2, depending on the quantity of lymphocyte required. However, RBCs were found in fractions 1 and 2 with fraction 1 containing approximately 3 times more RBCs than fraction 2. In order to obtain RBC-depleted lymphocytes, the desired fractions were treated with ACK lyse solution to lyse contaminating RBCs. This process reduced the number of RBCs approximately 3-fold with only a small reduction of lymphocyte quantity. In most cases the isolated lymphocytes were cryopreserved prior to manufacturing the rapamycin-generated Th1/Tc1 lymphocytes. This freeze/thaw cycle resulted in further lysis and depletion of RBCs (data not shown).While both density gradient separation and RBC lysis were effective at increasing the purity of the elutriated monocytes and lymphocytes, they required the use of additional reagents and staff time. In addition, both density gradient separation and lysis procedures were semi-open manual processes which increased the risk of microbial contamination of the final product. Ideally, it would be best to avoid further processing of monocytes and lymphocytes isolated by counter-flow elutriation by collecting PBMC concentrates that have very low quantities of neutrophils and RBCs, but this was not always possible. The quantities of neutrophils and RBCs contaminating PBSC concentrates collected by apheresis is dependent on several factors including the donors neutrophil count, RBC characteristics, duration of the apheresis procedure and characteristics of the blood cell separator. All blood cell separators collect some neutrophils and RBCs with the lymphocytes and monocytes ,26 and tElutriation is effective for isolating monocytes from PBMC concentrates for DC manufacture, but the elutriated monocyte fraction often required further processing to reduce the neutrophil content. Elutriation was also effective at obtaining lymphocytes from PBSC concentrates, but all lymphocytes fractions contained RBCs and an RBC lysis step was also required prior to initiating cultures. The separation of monocytes was less consistent that of lymphocytes and inefficient separation was associated with loading the elutriator with large quantities of monocytes.


However, RANK deletion had no positive impact on slow-twitch Sol muscles. In addition, denervating RANKmko EDL muscles induced an increase in the total calcium concentration ([CaT]), which was associated with a surprising decrease in SERCA activity. Interestingly, the levels of STIM-1, which mediates Ca2+ influx following the depletion of SR Ca2+ stores, were markedly higher in denervated RANKmko EDL muscles. We speculated that extracellular Ca2+ influx mediated by STIM-1 may be important for the increase in [CaT] and the gain of force in denervated RANKmko EDL muscles. Overall, these findings showed for the first time that the RANKL/RANK interaction plays a role in denervation-induced muscle atrophy and dysfunction.The bone remodeling and homeostasis are mainly controlled by the receptor-activator of nuclear factor kB (RANK), its ligand RANKL, and the soluble decoy receptor osteoprotegerin (OPG) pathway. While there is a strong association between osteoporosis and skeletal muscle dysfunction, the functional relevance of a particular biological pathway that synchronously regulates bone and skeletal muscle physiopathology remains elusive. Our recent article published in the American Journal of Physiology (Cell Physiology) showed that RANK is also expressed in fully differentiated C2C12 myotubes and skeletal muscles. We used the Cre-Lox approach to inactivate muscle RANK (RANK RANK, the receptor-activator of RANKL, and the OPG triad (RANK/RANKL/OPG) play important roles in fine-tuning the activity of these two bone cell types. RANKL is mainly expressed by bone marrow mesenchymal stromal cells and osteoblasts –6. The binding of RANKL to RANK on osteoclast precursors trimerizes its receptor and induces osteoclast differentiation and activation, resulting in bone resorption. RANK knockout impairs osteoclastogenesis and induces osteopetrosis, while the overproduction of RANKL induces osteoporosis , 8. OPG, the third protagonist, is mainly produced by bone marrow mesenchymal stromal cells and osteoblasts and exerts an inhibitory effect on the osteoclastic process. OPG has a high affinity for RANKL and inhibits the RANKL/RANK interaction and subsequent bone degradation. In addition to RANKL, OPG serves as a low affinity decoy receptor for TRAIL. Pre-clinical studies have suggested that this interaction increases cell survival by blocking the apoptotic effects of the RANKL/RANK interaction , 10. The fact that the overexpression of OPG or an exogenous OPG treatment  in mice results in osteopetrosis and that OPG-null mice are osteoporotic are testimony to the physiological importance of OPG .Bone remodeling is an essential process for maintaining bone homeostasis throughout the life of an individual. It is under the control of local and systemic factors that orchestrate osteoblast/osteoclast activation , 13. While bones and skeletal muscles are closely related and are mechanically linked, the possibility of dynamic molecular cross-talk between these tissues and a common signaling pathway that can efficiently control them has been underappreciated to date. Since the RANK/RANKL/OPG pathway is the most important cytokine network involved in bone biology and diseases, we postulated that this bone pathway is also involved in muscle atrophy and dysfunction. We first showed by Western blotting and confocal microscopy that C2C12 myotubes and fully differentiated skeletal muscles express RANK on the membranes of fast and slow-twitch myofibers . Based on the protective effect of RANK deletion on bone mass , we next postulated that selective muscle RANK deletion preserves muscle mass and function and favors a fast-twitch phenotype following denervation.Clinical studies have clearly shown that there is an association between osteoporosis and muscle atrophy and that the worsening of these conditions happens in tandem . These two muscles encompass the two extremes of the phenotypic spectrum of the skeletal muscle contractile apparatus. Type I fibers play an important role in maintaining the body in an upright position, while type IIb and IIx fibers are responsive during movement and physical activity. Type IIa fibers are a hybrid of type I and type IIb fibers and can perform short or prolonged exercises. Specific exercises, immobilization, unloading, denervation, muscle diseases, or glucocorticoid treatments affect all four muscle fiber phenotypes to different degrees. For instance, decreases in mechanical loading and neuromuscular activity favor muscle atrophy and a conversion of muscle fiber phenotypes from slow to fast . Functional muscle overload causes a gain in muscle mass while prolonged exercise leads to the transformation of pre-existing fast-twitch muscle fibers to a slow-twitch oxidative phenotype. Type IIb fibers, the most powerful fibers, are converted to oxidative phenotype fibers (type I or IIa) and disappear through a necrotic process during myopathies, aging, and glucocorticoid treatments , 19. Intracellular/cytosolic Ca2+ ([Ca2+]i) fluctuations play a crucial role in the maintenance and adaptation of muscle phenotypes. The increase in [Ca2+]i is caused by a Ca2+ influx from the extracellular space and/or Ca2+ release from [Ca2+]i stores. The SR is the major releasable [Ca2+]i store. Ca2+ channels and pumps maintain a steep Ca2+ concentration gradient between different cell compartments. Generally speaking, the spatial pattern of increased free [Ca2+]i at rest induces the activation of calmodulin and downstream Ca2+ decoders, namely calcineurin and CaMKII . These two decoders modify gene transcription by activating transcription factors such as NFATc1 and MEF2 and transcription modulators such as HDAC and PGC-1α, promoting the slow myofiber phenotype   is an estimate of [Ca2+]SR content . We used Cre-Lox technology to inactivate muscle RANK and found that the selective deletion of muscle RANK (RANKmko) decreases [CaT] in fast-twitch EDL muscles but not in Sol muscles, when compared to RANKflox/flox (RANKf/f) muscles. However, sciatic denervation markedly increased [CaT] in RANKmko EDL muscles. In addition, we discovered that denervated RANKmko EDL muscles are smaller, more fatigable, and surprisingly stronger than RANKf/f EDL muscles. Since force production is usually proportional to muscle size, we hypothesized that the increase in [CaT], i.e., [Ca2+]SR at rest, would favor Ca2+ release, increasing free [Ca2+]i and thereby the specific force of denervated RANKmko EDL muscles. The phenotypic profiles, which are consistent with these observations, showed a lower proportion of slow-twitch type I and fast-twitch type IIx fibers and a higher proportion of fast-twitch type IIa/IIb fibers in RANKmko EDL muscles relative to RANKf/f EDL muscles. However, muscle fatigue was significant and could not be explained by the modest changes in myofiber type in sham and denervated RANKmko EDL muscles. In this context, it would be highly relevant to determine whether muscle RANK deletion influences mitochondria Ca2+ uptake, another important regulator of [Ca2+]i and energy production. Our results showed for the first time that muscle RANK is an important regulator of Ca2+ storage, muscle phenotype, and muscle fatigue in normal and pathological conditions.Skeletal muscles are primarily composed of four muscle fiber types: type I fibers (slow and oxidative), type IIa fibers (fast and oxidative), and type IIx and IIb fibers (fast and glycolytic). The function largely dictates the phenotype and composition of each skeletal muscle. For example, the Sol muscle, a postural muscle, is largely composed of slow oxidative type I fibers while the EDL muscle, a phasic muscle, is almost exclusively composed of fast-twitch fibers enotype  . Interes. This results in the rapid efflux of large amounts of Ca2+ into the cytoplasm and the binding of Ca2+ to troponin C, causing the formation of cross-bridges between actin and myosin, the shortening of the sarcomere, and force development . To avoid permanent contraction and allow muscle relaxation, Ca2+ is in part pumped back into the SR by an ATP-dependent Ca2+ pump called SERCA. Ca2+ reuptake into the SR is mainly controlled by the fast-twitch SERCA-1a and slow-twitch SERCA-2a isoforms in myofibers. The movement of Ca2+ is reduced when PLN is associated with SERCA. Under β-adrenergic stimulation, like the stress hormones adrenaline and noradrenaline, or sympathomimetic drugs, the phosphorylation-dependent dissociation of PLN by PKA increases SERCA activity and Ca2+ movement, enhancing skeletal muscle and heart functions  demonstrated that STIM-1+/+ myotubes maintain full Ca2+ stores whereas STIM-1−/− myotubes fail to refill SR Ca2+ stores following chronic stimulations . Interestingly, we showed that denervated RANKmko EDL muscles have a markedly higher STIM-1 content. Since the increase in [CaT] and the decrease in SERCA activity in denervated RANKmko EDL muscles are somewhat irreconcilable, we proposed that STIM-1 and Orai-1 compensate for the lack of SERCA activity, refilling SR Ca2+ stores in the absence of muscle RANK.Muscle contraction involves the depolarization of the transverse-tubular system and activation of DHPRs, which in turn open RYR1 receptors adjacent to the SR membrane unctions . Alterna2+]i oscillations . SERCA is essential for [Ca2+]i oscillations and plays a critical role in osteoclastogenesis . It has recently been reported that SERCA-2a heterozygote mice (SERCA2a+/−) present defects in osteoclast differentiation and the suppression of RANKL-induced Ca2+ oscillations . Tmem64, a positive modulator of osteoclast differentiation, regulates RANKL-induced Ca2+ signaling via SERCA2a . In addition to SERCA, Ca2+ oscillations may partly derive from extracellular Ca2+. Gadolinium, a specific SOCE blocker, abolishes RANKL-induced Ca2+ oscillations  while the knockdown of STIM-1 significantly reduces the frequency of RANKL-induced Ca2+ oscillations, indicating that SOCE and RANK communicate. TRP channels are also candidates for the Ca2+ influx involved in RANKL-induced Ca2+ oscillations . Chamoux et al. (2010) showed that RANKL induces a significant increase in [Ca2+]i from outside, presumably through the TRPV-5 Ca2+ channel. While an elaborate approach has been proposed to elucidate how RANKL/RANK induces Ca2+ oscillations in bone cells, the importance of this pathway in skeletal muscle is extremely limited. Our work reported in the American Journal of Physiology (Cell Physiology) was, to our knowledge, the first attempt to assign a regulatory role for RANKL/RANK interactions on SERCA activity in bone and muscle cells . The identification of downstream effectors of muscle RANK will be an important step in determining precisely how the RANKL/RANK interaction modulates SERCA activity. Bioinformatic approaches may eventually be required to improve our understanding of the cell signaling network and to determine how the RANKL/RANK interaction regulates SERCA activity and possibly other Ca2+ channels in skeletal muscles.In bone, RANKL/RANK interactions activate TRAF-6, which then induces the activation of downstream signaling molecules and [Ca that OPG-Fc (OPG fused to immunoglobulin Fc) completely restores the function of dystrophic EDL muscles and significantly improves the function of Sol and diaphragm muscles. OPG treatments also provide significant protection against eccentric-contraction-induced muscle damage. As observed with OPG-treated dystrophic mice, muscle RANK deletion preferentially protects fast-twitch fibers. It is important to note that fast-twitch fibers are essential for brief and powerful contractions. They are predominantly affected directly or indirectly in aging and in several forms of muscle disease and chronic illness, including diabetes, congestive heart failure, renal failure, chronic heart disease, and chronic obstructive pulmonary disease. Our results clearly show that the role of the RANK/RANKL/OPG pathway extends well beyond bone remodeling and osteoporosis and that skeletal muscles and other tissues may share the RANK/RANKL/OPG pathway as a common denominator.We convincingly show in another publication in the American Journal of Pathology (2015)


In order to explore signaling pathway, triggered by these compounds, we assessed interleukin-2 (IL-2) mRNA expression and extracellular signal-regulated kinase-1/2 (ERK1/ERK2) phosphorylation. Finally, we determined increases in free intracellular Ca2+ concentrations, [Ca2+]i, by employing Fura-2/AM in rat lymphocytes.TheP. glaucum grains in polyphenols was estimated to be 1660 µg gallic acid equivalents (GAE)/g. Lipids represented 4.5 %, and more than 72% of the fatty acids belonged to unsaturated family. Our investigation showed that both lipid and phenolic compounds inhibited mitogen-induced T-cell proliferation. Compared with phenolic compounds, lipids exerted weaker effects on ERK-1/ERK2 phosphorylation and Ca2+ signaling in mitogen-activated T-cells.The composition of P. glaucum  could be contributed by its phenolic and lipid contents.We conclude that the immunomodulatory effects of  Current recommendations from international health and nutritional organizations, like Food and Drug Administration (FDA), include an increase in the consumption of high-bran cereals because of their potential benefits on human health . MilletsMillet grains have been shown to exert beneficial effects in health and disease –6. Lee eIn vitro and in vivo studies have shown that fatty acids modulate a number of lymphocyte functions i and mitogen-activated protein kinase (MAPK) phosphorylation, are the part of early events of T-cell activation, it was thought worthwhile to elucidate the effect of PM lipids and phenolic extracts on Ca2+ and MAPK signaling in T-cells.Pearl millet (PM), i.e., n Africa , and it n Africa . PM contPennisetum glaucum, obtained from the region of Ouled Aïssa , were used in this study. Wistar rats were obtained from Janvier Elevage . RPMI 1640 medium and L-glutamine were purchased from Lonza Verviers SPRL . Fura-2 AM was procured from Life Technologies (France). Anti-Phospho-p44/42 mitogen-activated protein kinase  and anti-p44/42 MAPK (Erk1/2) were obtained from Cell Signaling (France). All other chemicals were purchased from Sigma (USA).Grains of pearl millet, The general guidelines for the care and use of laboratory animals, recommended by the council of European Economic Communities, were followed. The experimental protocol was approved by the Regional Ethical Committee (Dijon).The plant was recognized by a botanist  of the Herbarium Center of the Faculty of Pharmacy (Tlemcen) which contained the voucher specimen (PM 1681). PM phenolic extracts were obtained according to the method of Liyana-Pathirana and Shahidi  with sliTotal phenolic contents in plant extracts were determined by Folin-Ciocalteu method  as descrPhenolic extract (20 μl) was analyzed by HPLC  with reverse phase Zorbax Eclipse XDB-C18 column (4.6 × 100 mm) and a diode array UV-detector (operating at 280 nm). The gradient mobile phase was composed of two solvents: A and B. Solvent A was methanol and solvent B was 0.1 % formic acid (v/v). Phenolic acid separation was achieved using a 35 min linear solvent gradient at a flow rate of 0.4 ml/min, as follows: 0 min 90 % B, 5 min 80 % B, 10 min 70 % B, 15 min 50 % B, 20 min 30 % B, 25 min 10 % B, 30 min 50 % B, 35 min 90 % B. PM phenolic compounds were identified with reference to retention time of authentic standards and quantified on the basis of their peak area. Standard phenolic compounds used were: p-coumaric, chlorogenic, ferulic, gallic, trans-hydroxycinnamic, syringic acid, ellagic acids, quercetin and apigenin.Lipids were extracted according to the method of Hara and Radin . In ordeIn brief, 0.1 ml of hexane, containing lipid extract, was transferred into screw cap tubes and dried under nitrogen, then 1 ml of methanolic NaOH (0.5 N) was added into tubes, and heated at 80 °C during 20 min. After cooling at 4 °C, 2 ml of boron trifluoride-methanol solution (BF3) were added, and the methylation was performed at 80 °C for 20 min. After cooling-down in ice, 2 ml of NaCl (35 %) plus 2 ml of hexane were added into the tubes. After vigorous agitation and centrifugation (1200 g/5 min), the upper phase containing fatty acid methyl esters was transferred into new tubes, and analyzed by gas − liquid chromatography (GLC).GLC was performed in a Packard Model 417 gas–liquid chromatograph, equipped with a flame ionization detector and a 30-m capillary gas column coated with carbowax 20 M. The analysis conditions were as follows: oven temperature was 85 °C/1 min, increased to 150 °C at 30 °C/min, then increased at 4 °C/min to 210 °C. Helium was used as carrier gas, with a flow rate of 0.4 ml/min. Analysis of fatty acid peaks was achieved with reference to the internal standards  by using DELSI ENICA 31 . The fatty acid levels were expressed as g/100 g of total fatty acids.2, to remove the macrophages by adherence. T lymphocytes were isolated by panning i, were calculated by using the following equation: [Ca2+]i = Kd × (R-Rmin)/(Fmax-F)(Sf2/Sb2). A value of 224 nM for Kd was added into the calculations. Rmax value was obtained by the addition of ionomycin (5 μM) and Rmin value was obtained by the addition of MnCl2 (2 mM), Triton X-100 (0.1 %) and EGTA (24 mM).Splenic T-cells (2 × 102+), CaCl2 was replaced by 1 mM EGTA in the buffer i in 0 % Ca2+ medium as compared to that induced in 100 % Ca2+ medium.Figure PGL Fig. . In orde2+]i even in the absence of external calcium, it was thought worthwhile to examine the nature of intracellular stores involved in this rise in [Ca2+]i. We used thapsigargin, known to induce increase in [Ca2+]i by inhibiting the endoplasmic reticulum Ca2+-ATPase [2+]i in these cells.As the polyphenolic and lipidic extracts were able to induce increases in i activates via calcineurin induces IL-2 gene expression i in T-cells. In the presence of 100 % Ca2+, PGPC and PGL induced high increases in [Ca2+]i, suggesting that Ca2+ influx plays a major role in the increase in [Ca2+]i evoked by theses extracts. The kinetic study of PGL and PGPC-induced Ca2+ mobilization showed that these compounds produced a sustained increase in [Ca2+]i. We also noted that PGPC induced stronger and more important increases in Ca2+ (around 6-fold), compared to PGL. These sustained [Ca2+]i increases are correlated with the immunosuppressive effects of PM extracts, as reported for the prickly pear phenolic compounds [We examined the actions of PGL and PGPC on the increases in [Caompounds .2+ pool mobilized by lipids and phenolic compounds, thapsigargin, an inhibitor of Ca2+-ATPase of endoplasmic reticulum [2+ peak, and vice versa, suggesting that PGL and PGPC did not seem to act on Ca2+-ATPase.To ascertain the nature of the intracellular Caeticulum , was emp2+ concentration and ii) the inhibition of IL-2 mRNA expression and MAP kinase phosphorylation. Our results argue for the use of millet diet as dietary supplements for treatment of diseases associated with a sustained activation of the immune system such as autoimmune diseases.We can state that both PM polyphenols and lipids exhibited an immunosuppressive effects. The PM polyphenols seem to be more active than lipids. Two molecular mechanisms seem to be involved in the immunosuppressive activity of PM extracts: i) the sustained increases in intracellular free Ca


Approximately 20% of patients with acute myeloid leukaemia (AML) have a mutation in FMS-like-tyrosine-kinase-3 (FLT3). FLT3 is a trans-membrane receptor with a tyrosine kinase domain which, when activated, initiates a cascade of phosphorylated proteins including the SRC family of kinases. Recently our group and others have shown that pharmacologic inhibition and genetic knockdown of Bruton’s tyrosine kinase (BTK) blocks AML blast proliferation, leukaemic cell adhesion to bone marrow stromal cells as well as migration of AML blasts. The anti-proliferative effects of BTK inhibition in human AML are mediated via inhibition of downstream NF-κB pro-survival signalling however the upstream drivers of BTK activation in human AML have yet to be fully characterised. Here we place the FLT3-ITD upstream of BTK in AML and show that the BTK inhibitor ibrutinib inhibits the survival and proliferation of FLT3-ITD primary AML blasts and AML cell lines. Furthermore ibrutinib inhibits the activation of downstream kinases including MAPK, AKT and STAT5. In addition we show that BTK RNAi inhibits proliferation of FLT3-ITD AML cells. Finally we report that ibrutinib reverses the cyto-protective role of BMSC on FLT3-ITD AML survival. These results argue for the evaluation of ibrutinib in patients with FLT3-ITD mutated AML. Acute myeloid leukaemia (AML) is primarily a disease of the elderly with a median age at diagnosis of 72 years. Many elderly patients tolerate current intensive cytotoxic chemotherapy regimens poorly and therefore treating the older less fit patient with AML presently remains challengingDrug targeting of pro-tumoral tyrosine kinases has resulted in considerable progress in outcomes for patients with chronic myeloid leukaemia3568910Various receptor tyrosine kinase mutations have been identified in AML patients1213Bruton’s tyrosine kinase (BTK) is a non-receptor tyrosine kinase which is functionally important in a spectrum of benign and malignant haematopoietic cells of both the lymphoid and myeloid compartments181920219In this study we place BTK activation downstream of mutated FLT3 in primary AML cells and furthermore show how inhibition of BTK (by ibrutinib and RNA interference) targets FLT3 mutated AML cells by inhibiting cell survival. We also report how ibrutinib synergises in combination with daunorubicin, and how ibrutinib functions in part by reducing the cyto-protection provided to FLT3-ITD AML cells by bone marrow stromal cells. Here we provide a biologic rationale for the targeting of BTK in FLT3 mutated AML.Anti-phosphorylated and total FLT3, AKT, BTK, STAT5 and MAPK antibodies were purchased from Cell Signalling Technology . Anti-CD34-PE, anti-CD90-FITC, anti-CD73-PE, anti-CD105-APC antibodies were purchased from Miltenyi Biotec . Ibrutinib was obtained from Selleck Chemicals. All other reagents were obtained from Sigma-Aldrich , unless indicated.The AML-derived cell lines were obtained from the European Collection of Cell Cultures and DMSZ where they are authenticated by DNA-fingerprinting. In the laboratory they are used at low passage number for a maximum of 6 months post-resuscitation, testing regularly for Mycoplasma infection. AML blasts were obtained from patients’ bone marrow or blood following informed consent and under approval from the UK National Research Ethics Service (LRECref07/H0310/146). Moreover, all aspects of the study were carried out in accordance with the approved guidelines. For primary cell isolation, heparinized blood was collected from patients and human peripheral blood mononuclear cells (PBMCs) isolated by Histopaque density gradient centrifugation. AML samples that were less than 80% blasts were purified using the CD34 positive selection kit. FLT3-ITD analysis was performed by the haemato-oncology diagnostic service  as previously described and 5 patients with FLT3-ITD and 6 with no FLT3 mutation were selected for this study14Human bone marrow stromal cells (BMSC) were isolated from bone marrow aspirates of AML patients. Mononuclear cells were collected by gradient centrifugation and plated in growth medium containing RPMI and 20% FBS and 1% l-glut. The non-adherent cells were removed after 2 days. When 60%–80% confluent, adherent cells were trypsinised and expanded for 3–5 weeks. BMSCs were checked for positive expression of CD105, CD73, and CD90 and the lack of expression of CD45 and CD34 by flow cytometry as previously describedSodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses were performed as described previously. Briefly, whole cell lysates were extracted and sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation performedCells were treated with different doses of ibrutinib then viable numbers measured with CellTiter-Glo . Flow cytometry for measuring apoptosis was performed on the Accuri C6 flow cytometer . Samples were collected and stained with annexin- V and propidium Iodide (PI) , followed by detection. For the AML-BMSC co-cultures AML cell viability was measured using flow cytometry. After exclusion of BMSC by electronic gating using negative for CD45 expression the extent of AML cells apoptosis was measured using annexin-V.6 cells using the Amaxa Nucleofactor Kit II. Control siRNA and BTK siRNA 1–3 were purchased from Invitrogen were transfected in at a concentration of 50 nm. For all gene expression experiments the cells were incubated for 24 hours post transfection before RNA extraction. For the Western blot and cell viability assays AML cell were incubated for 48 hours post transfection.AML cell line transfections were carried out with 1 × 106 cells by use of the Nucleic acid PrepStation from Applied Biosystems, according to the manufacturer’s instructions. Reverse transcription was performed using the RNA PCR core kit . Real-time PCR primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and BTK were purchased from Invitrogen. Relative quantitative real-time PCR used SYBR green technology  on cDNA generated from the reverse transcription of purified RNA. After pre-amplification (95 °C for 2 minutes), the PCRs were amplified for 45 cycles (95 °C for 15 seconds and 60 °C for 10 seconds and 72 °C for 10 seconds) on a 384-well LightCycler 480 . Each mRNA expression was normalized against GAPDH mRNA expression via use of the standard curve method.Total RNA was extracted from 1 × 10Mann-Whitney test was performed to assess statistical significance from treated sample compared to controls. Results with P < 0.05 were considered statistically significant (*). Results represent the median and in some instances mean ± SD of 3 independent experiments. For Western blotting, data are representative images of 3 independent experiments.We have previously shown that ibrutinib inhibits factor induced AML blast proliferation and downstream AKT and MAPK signalling pathwaysTo determine if ibrutinib inhibits downstream signalling in FLT3-ITD mutated primary AML blasts, we compared AML with FLT3-ITD to AML without FLT3-ITD in response to increasing concentrations of ibrutinib for 3 h. We next examined the effect of ibrutinib in combination with front line AML chemotherapy daunorubicin by using increasing concentrations of daunorubicin, alone and then in combination with Ibrutinib at 500 nM. We and others have shown that BMSC provide a level of protection for AML blast cells from chemotherapy treatments91423It has been reported that ibrutinib has other kinase targetsThe FLT3-ITD mutation is present in circa 20% of patients with AML121328in vitro and in vivo. The FLT3 inhibitor AC220 (quizartinib) has shown good efficacy in in vitro studies3334To date a number of FLT3 inhibitors have been assessed BTK is a non-receptor tyrosine kinase that belongs to the Tec kinase family and plays an important role in both benign and malignant cells of the haematopoietic system93738394145548in vitro that ibrutinib permits dose reduction of daunorubicin to achieve a given rate of cytotoxicity. As daunorubicin toxicity presents a clinical challenge in the treatment of older less fit patients with AML ibrutinib may allow protocols in the future to dose reduce daunorubicin while maintaining clinical efficacy but improving tolerability. This may result in effective but reduced dose daunorubicin containing treatment regimens being offered with curative intent to patients not currently deemed fit enough for a standard daunorubicin/cytarabine intensive schedule.In this study we demonstrate BTK is activated downstream of the FLT3-ITD in AML and that BTK inhibition targets FLT3-ITD induced survival and proliferation pathways including AKT, STAT5 and MAPK. In addition when we used the FLT3 inhibitor AC220 in combination with ibrutinib there was no additive effect on AML survival, thus supporting the hypothesis that these two inhibitors are working on the same survival pathway/s in FLT3-ITD AML. Furthermore we have shown that treatment of FLT3-ITD AML with ibrutinib or BTK knockdown in combination with daunorubicin increases tumour apoptosis over daunorubicin alone. We show Resistance to FLT3 inhibitors can occur through mutations at the drug binding site in the target molecule31In summary this study links a common genetic mutation found in AML to a specific clinically available downstream kinase (BTK) inhibitor, ibrutinib. Furthermore we have identified the potential efficacy of combination therapy with daunorubicin.How to cite this article: Pillinger, G. et al. Targeting BTK for the treatment of FLT3-ITD mutated acute myeloid leukemia. Sci. Rep. 5, 12949; doi: 10.1038/srep12949 (2015).