Verifying self-reports of smoking abstinence is challenging in studies that involve remote data collection. Resting heart rate (HR) decreases during smoking abstinence. This study assessed whether a decrease in resting HR measured using freely available smartphone apps could potentially be used to verify smoking abstinence.This study involved a repeated measures experimental design, with data collection in natural setting. Participants were 18 adult, daily smokers. They recorded resting HR in beats per minute (bpm) using freely available smartphone apps during five timepoints (two in the morning and three postnoon) on each of 3 days. The outcome measure was the mean of the postnoon HR recordings. The experimental condition for each of the 3 days  was as follows: (1) smoking as usual, (2) not smoking without nicotine replacement therapy (NRT), or (3) not smoking but using NRT. Abstinence was verified using expired-air carbon monoxide (CO) concentration.p = .001) in the not smoking without NRT condition and 10.4 bpm lower  in the not smoking with NRT condition. There was no statistically significant difference in HR between the two not smoking conditions (p = .39). Abstinence during not smoking days without and with NRT was CO-verified in 18/18 and in 16/18 cases, respectively.Compared with the smoking as usual condition, mean HR was 13.4 bpm lower (95% confidence interval [CI] = 5.4 to 21.4, Self-recording of resting HR in natural setting using smartphone apps shows a reliable decrease in response to smoking abstinence and may provide a basis for remote verification in smoking cessation studies.Remote verification of self-reported abstinence in smoking cessation studies remains challenging. Smoking abstinence has been shown to decrease resting HR under laboratory conditions. This study demonstrated that self-recording using freely available smartphone apps shows reliable decreases in resting HR during smoking abstinence and may provide a basis for inexpensive remote verification of smoking abstinence. It has long been known that resting heart rate (HR) declines substantially within a day of stopping smoking and the decrease persists indefinitely.2–5 Most smartphones can run free apps that use the phone’s camera to assess the changes in the color of the fingertip with each heartbeat to measure HR.6–8 The current study provided an initial assessment of whether a decrease in resting HR as measured using smartphones in people’s natural settings could provide a sufficiently reliable indicator of abstinence to be used to verify self-reports.In studies of smoking cessation, it is important to be able to verify self-reports of abstinence.1 These can, in principle, be used in smoking cessation studies of remote interventions, where there is no face-to-face contact, but there are major challenges to doing so. In the case of CO, study participants must be sent a CO monitor and learn how to use it. The cost of the monitors and practical difficulties in getting people to use them mean that this approach remains problematic.10 Saliva cotinine has been used successfully but the costs are high when one takes into consideration the cost of the assay and incentivizing people to engage with what some see as an unattractive procedure in providing body fluids.9The two most commonly used methods of verifying smoking abstinence are expired-air carbon monoxide (CO) concentration and cotinine concentration measured in saliva or urine.2–5 This is most likely because of the acute effect of nicotine on the cardiovascular system, an effect that reverses as the concentration of nicotine in body tissues falls to zero.12 The nicotine-induced increase in heart rate is subject to acute tolerance, such that HR increases rapidly on ingestion of nicotine from the first cigarette of the day, but does not increase substantially as tissue nicotine concentrations change during the day.13 This effect appears to be reliable and it may occur with all, or almost all smokers.15 If that is the case then HR should decrease substantially on days when smoking has not occurred and should increase again if smoking does occur. This makes decrease in HR a potentially useful marker for verifying self-reports of smoking abstinence. At the same time, using nicotine replacement therapy (NRT) may or may not increase HR to the same extent as smoking. Thus, NRT use may confound HR measurements if these are used to verify smoking abstinence.Resting HR decreases by an average of around 5–15 beats per minute (bpm) within a day of stopping smoking and remains at that level for at least a year and probably indefinitely.16As a starting point in assessing the value of app-based HR measurement, it is important to determine whether a reliable decrease can be observed from smoking to abstinent days. Given the above considerations, it is also important to assess whether use of nicotine replacement products during abstinence reduces the size of the HR decrease.Thus, the following research questions were addressed:Does resting HR, as assessed by a freely available smartphone app by participants in their natural settings decrease reliably on days when they are abstaining from smoking compared with days when they are smoking?Is this the case even if they are using a nicotine replacement product?This study involved a single-factor repeated-measures design. The study was approved by a Research Ethics Committee at UCL (CEHP/2013/508 and CEHP/2016/556).SD = 13.4); eight (44%) were female; their mean daily cigarette consumption was 12.2 (SD = 4.5) cigarettes per day. Participants used a single NRT product during their day of not smoking with NRT, and the patch was a common choice, but detailed information about NRT use was not recorded.The inclusion criteria for the study were: (1) ≥18 years old, (2) self-classified as healthy, (3) daily smoker of 5+ cigarettes per day, and (4) access to a suitable smartphone throughout the day. The recruitment was through posters, online, and by personal contact. Eighteen participants were recruited. Their mean age was 31.9 years (All participants provided written consent. They were paid £100 in cash or Amazon vouchers (according to their preference) for completing the experiment and up to £10 to pay for NRT products of their choice.17 manufactured by Bedfont Scientific Ltd : “Instant Heart Rate” or “Cardiio.” The participants were also provided with a personal CO monitor Ltd see . Partici Ltd see  and to sAll participants took part in three conditions on separate days: (1) smoking as usual, (2) not smoking, and (3) not smoking but using an NRT product of their choice. The sequence of the three conditions  was balanced across participants. Adherence to the requirements for abstinence in conditions (2) and (3) was assessed by self-measurement of expired-air CO concentrations. A reading of less than 10 parts per million was judged to confirm abstinence.On each of the three HR measurement days, participants were instructed to use the HR app to measure their HR (in bpm) five times at the following timepoints: (1) soon after waking (pre-9 am), (2) morning (10–12 pm), (3) lunchtime (12–3 pm), (4) afternoon (3–5 pm), and (5) evening (post-5 pm). Participants were instructed to remain seated for at least 5 minutes prior to each HR measurement. For each of the five measurement timepoints on a given day, they used the app to assess their resting HR by taking five consecutive readings and recorded them on the form.The HR data for each of the five timepoints were averaged to give five measurements for each of the three conditions. As participants may not have smoked yet in the morning, the three postnoon readings were used to calculate the mean HR for each condition.Participants were instructed in use of the expired-air CO monitors at the end of each condition day and recorded their CO readings on each of the three experimental days on a form provided.https://osf.io/8uy5p/). Means and 95% confidence intervals (CI) of mean postnoon HR were calculated for each of the three conditions. The differences between the three means were compared using the one-way analysis of variance (ANOVA) with repeated measures. Planned pairwise comparisons with Sidak correction were made between each of the conditions: (1) smoking as usual and not smoking with no nicotine product, (2) smoking as usual and not smoking but using a nicotine product, and (3) not smoking with no nicotine product and not smoking with a nicotine product. Given that HR data violated assumptions of normal distribution, in the sensitivity analysis we compared the means from the three conditions using Friedman’s test, which is a nonparametric equivalent test to repeated measures ANOVA.The data analysis plan was uploaded on Open Science Framework before the data were analyzed (Smoking abstinence during not smoking conditions without and with nicotine products was verified by CO in 18/18 (100%) and in 16/18 (88.9%) cases, respectively. Cases were analyzed on an intent to treat basis.F = 14.37, p < .001; see p < .001). Compared with the smoking as usual condition, mean HR was 13.4 bpm lower  in the not smoking without NRT condition and 10.4 bpm lower  in the not smoking with NRT condition. There was no statistically significant difference in HR between the two not smoking conditions (p = .39).A decrease in postnoon HR was observed in all cases from smoking as usual to not smoking without a nicotine product, and in 15 of 18 cases to not smoking with a nicotine product see . HR aver4 These results provide an initial indication that smartphone-assessed differences in HR may provide a basis for objective verification of smoking status.Measurement of HR using a smartphone app showed a reliable decrease on a day of smoking abstinence compared with when smoking as usual. The decrease in HR on not smoking days but when a nicotine product was used was still substantial and significant, but may be less reliable. The magnitude of the observed decrease in resting HR was in line with previous findings.15 However, the difference was not significant. This could be due to low power. Alternatively, nicotine on its own may not be solely responsible for the increase in HR during smoking. Finally, this could also be explained by the different kinetics of nicotine absorption or lower nicotine intake from nicotine products in comparison to cigarettes. Future studies should explore the impact on HR of different intake levels of nicotine products during the not smoking days.The afternoon HR on a not smoking day with nicotine products was on average 2.9 bpm higher than on a not smoking day without NRT, in line with past literature.18 It would also be important to collect data on the HR measuring app used by the participants, and to conduct a direct comparison of the readings from two or more of such apps. The time since last cigarette smoked before taking HR measurements should also be collected as a statistical control and to enable additional analyses of the latency effect between abstinence and changes in the HR.The next stage of the process of evaluating smartphone-assessed HR for verifying smoking cessation is to conduct a larger study to establish sensitivity and specificity at different thresholds of changes in HR for smokers who have maintained abstinence and who have smoked. This would allow a receiver operating characteristic (ROC) curve to be generated and a more definitive picture of the value of this method to emerge.If this method of verifying abstinence were to prove accurate, it would greatly improve our ability to assess abstinence on multiple occasions at a very low cost.With the advent of wearable devices that automatically measure HR, the opportunity to use HR to assess and motivate abstinence may be considerably enhanced. It would in theory be possible to link such devices, for example, a Fitbit, to a smoking cessation app through an application program interface with no effort required from smokers themselves. Such devices have accelerometers and so it should be possible to ensure that HR is only recorded when users are resting.In this exploratory study, it was found that smoking abstinence significantly reduced HR assessed using smartphone apps in natural settings, even when a nicotine product was used. This could open up possibilities for use of remotely assessed HR to verify smoking abstinence in cessation studies.https://academic.oup.com/ntr.A Contributorship Form detailing each author’s specific involvement with this content, as well as any supplementary data, are available online at ntaa021_suppl_Supplementary_MaterialsClick here for additional data file.ntaa021_suppl_Supplementary_Taxonomy_FormClick here for additional data file.


Recently, quantum anomalous Hall effect with spontaneous ferromagnetism was observed in twisted bilayer graphenes (TBG) near 3/4 filling. Importantly, it was observed that an extremely small current can switch the direction of the magnetization. This offers the prospect of realizing low energy dissipation magnetic memories. However, the mechanism of the current-driven magnetization switching is poorly understood as the charge currents in graphenes are generally believed to be non-magnetic. In this work, we demonstrate that in TBG, the twisting and substrate induced symmetry breaking allow an out of plane orbital magnetization to be generated by a charge current. Moreover, the large Berry curvatures of the flat bands give the Bloch electrons large orbital magnetic moments so that a small current can generate a large orbital magnetization. We further demonstrate how the charge current can switch the magnetization of the ferromagnetic TBG near 3/4 filling as observed in the experiments. The mechanism of current-driven magnetization switching in twisted bilayer graphene (TBG) is poorly understood. Here, He et al. show that a small current can generate a large orbital magnetization due to symmetry breaking by the twisting and substrate in TBG, leading to a giant orbital magnetoelectric effect. At small twist angles 2 and yields flat bands at a series of magic angles3, where electronic correlations become important. Recently, insulating4 and superconducting phases5, possibly driven by correlations at around 1/2 and 36 to understand the underlying insulating and superconducting mechanisms.A bilayer graphene with a twist angle 38. These observations are consistent with predictions24 that electron–electron interactions can give rise to ferromagnetism by lifting the spin and valley degeneracy, and that quantum anomalous Hall states will be obtained when bands with a total non-zero Chern number are filled. Strikingly, these experiments have also shown that the magnetization can be switched by driving very small DC currents (from 10 to 50 nA) through the samples38. The current needed for magnetization switching is several orders of magnitude smaller than those in state of the art spin-torque devices39. These observations strongly suggest the possibility of realizing ultralow power magnetic memory devices in TBG. However, it is not clear how a charge current can couple to the out-of-plane magnetization of the TBG, as the charge currents in graphene layers are generally believed to be non-magnetic.More recently, experiments have unveiled the topological properties brought about by the moiré potential, as the signatures of ferromagnetism and quantum anomalous Hall effect have been experimentally observed near the 3/4 filling41. However, the D38. Including substrate-induced sublattice symmetry breaking27 and strain34, the symmetry of the TBG is reduced to C40. Moreover, due to the large Berry curvature of the flat bands near the magic angle, the orbital magnetic moments carried by the Bloch electrons can be as large as tens of Bohr magnetons per electron even with very small strains. The lattice symmetry reduction and the large orbital magnetic moments of the electrons allow a large orbital magnetization to be induced by a small charge current. Near 3/4 filling when the Hall resistance 38), the bulk conducting channels which carry magnetization can couple to the bulk magnetization of the sample, allowing current-controlled magnetic switching.Here, we show that charge currents in TBG can induce very large orbital magnetization at general filling factors even when the sample is not ferromagnetic. We call this effect the giant orbital magnetoelectric effect. First, by symmetry analysis, we point out that due to twisting, the symmetry of bilayer graphene is reduced from D3Å is the carbon–carbon bond length42, the rotation matrix has the form Å42 and An isolated TBG can be described by coupling the top and bottom graphene layers with a twist angle 3 is present in the Methods section.An important effect of the moiré superlattice which originates from twisting is to fold the original Brillouin zone into the mini-Brillouin zones schematically shown in Fig. 19. At the mini-Brillouin zone corner 19. However, in the two recent experiments in which a ferromagnetic state has been seen, the TBG is coupled with a hBN cladding layer aligned to the TBG to less than 1°, which empirically appears necessary to support the ferromagnetism38. In our model, the hBN substrate affects the bottom graphene layer in two aspects: (1) it breaks the CFor an isolated TBG, the top and bottom graphene Hamiltonian along with the interlayer coupling respects the Dmentclass2pt{minim43, and 44. By combining the sublattice symmetry breaking and uniaxial strain effect from the hBN substrate, we are able to obtain the modified bottom graphene layer Hamiltonian as46. The staggered potential breaks the In this work, for simplicity we use a uniaxial strain tensor Denoting the interlayer coupling between the strained bottom layer graphene and the unstrained top layer graphene as The energy dispersion at each band for the TBG can then be directly obtained through diagonalizing the continuum Hamiltonian in Eq. . For an mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimWith the Hamiltonian in Eq. , we can 40 as demonstrated in the next section.For an isolated TBG, the Berry curvature and the magnetic moment are non-zero only at the Dirac point. In the absence of strain but in the presence of the staggered potential which is set to be entclass1pt{minimaentclass1pt{minima41, the general forms of the components of the magnetoelectric susceptibility tensor In quasi-two-dimensional materials with finite magnetoelectric response, the electric field induced magnetization can be described asmentclass2pt{minimmentclass2pt{minim48To calculate 49. For the conduction band To be specific, we apply a uniaxial strain with entclass1pt{minimaentclass1pt{minima51. The current-induced magnetization is anisotropic with respect to the direction of the current and it switches sign under reversal of the electric field. It is important to note that the current-induced magnetization discussed here can appear at a general filling factor even absent spontaneous ferromagnetism in the sample. This current-induced magnetization should be observable experimentally through optical Kerr effects as in the case of transition metal dichalcogenides52.Assuming an external electric field of 19. However, near the magic angles, the narrow band width at the Fermi level magnifies the role of interactions, and interaction-driven spontaneous symmetry breaking is observed experimentally38. Specifically, at 3/4 filling of the conduction band in hBN-aligned TBG with inter-graphene twist angle 37) a giant anomalous Hall effect of order 38, quantized anomalous Hall effect has been reported, in both cases at zero external magnetic field. Hysteresis in the Hall conductance under out-of-plane magnetic fields suggests spontaneous ferromagnetism with out-of-plane magnetization.TBG in the non-interacting limit possess valley and spin degeneracy for each flat band38 indicates that the spin and/or valley degeneracies are lifted, possibly by interactions27. As a result, there are four bands (which originated from the fourfold degenerate conduction band in Fig. The presence of net magnetization as revealed by anomalous Hall resistance37.At filling factor 3/4, if the three bands with lower energy are completely filled as depicted in Fig. To connect our theory with experiments, we note that the spontaneous ferromagnetism in TBG can be described by the Landau’s free energy density asy-direction and by calculating 37, the longitudinal resistance is measured to be 37. Since many of the details such as the strain, the band structure of the sample, the shifts of the polarized bands, etc. will affect the coercive current, the specific value of the coercive electric field calculated here can only be a rough estimation.To understand the coupling between the electric field and the magnetic field, we note that the total magnetization 55, twisted hBN-graphene heterostructure57, twisted transition metal dichacolgenides58, and gapped bilayer graphene59 with strain will exhibit similar magnetoelectric effects, although the magnitude of the magnetoelectric response will depend on the details of the materials. The current-induced orbital magnetization predicted can be tested by magneto-optical Kerr effect in experiments52.In the above sections, using a continuum model of TBG and incorporating the effects of sublattice symmetry breaking and strain, the magnetoelectric response was calculated. Here, we would like to emphasize that the analysis based on symmetry is very general. The exact form of the strain is not important. The breaking of the DRR38. The currents can flow between domains with different magnetization. As the symmetry of the problem is still CAnother important point is that in the experimental regime where current-induced magnetization switching is demonstrated, the Hall resistance 38 argued that even edge states which do not carry net magnetization can also switch the direction of the magnetic domains, giving an effect proportional to Our picture of current-induced magnetic switching does not apply directly to quantum anomalous Hall states with an insulating bulk when the current is carried by the edge states only. To obtain the current-induced magnetization, we assumed that the scattering time (n in Eq. . This asmentclasspt{minima61 studied previously. It is also interesting to note that the orbital magnetization can be strongly affected by strain. In this work, we only discussed the strain induced naturally by the hBN substrate. Experimentally, one can induce a much larger strain on the TBG artificially. In this case, the current-induced magnetization could be further enhanced. The orbital magnetization of some of the Bloch states in the Brillouin zone can even reach a hundred Bohr magnetons with moderate strain as shown in the Supplementary Fig. It is also worth noting that the current-induced magnetization in TBG is purely orbital in nature. It is different from the magnetoelectric effect induced by spin–orbit coupling in noncentrosymmetric materials3, the state at In the continuum model descriptionmentclass2pt{minimThe Hamiltonian for the TBG on a hBN substrate readsSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1


Because the cellular events that underlie these experimental findings are still poorly understood, we developed a simple computational model to investigate how β CaMKII regulates the direction of plasticity in cerebellar PCs. We present the first model of AMPA receptor phosphorylation that simulates the induction of long-term depression (LTD) and potentiation (LTP) at the PF-PC synapse. Our simulation results suggest that the balance of CaMKII-mediated phosphorylation and protein phosphatase 2B (PP2B)-mediated dephosphorylation of AMPA receptors can determine whether LTD or LTP occurs in cerebellar PCs. The model replicates experimental observations that indicate that β CaMKII controls the direction of plasticity at PF-PC synapses, and demonstrates that the binding of filamentous actin to CaMKII can enable the β isoform of the kinase to regulate bidirectional plasticity at these synapses.Calcium-calmodulin dependent protein kinase II (CaMKII) regulates many forms of synaptic plasticity, but little is known about its functional role during plasticity induction in the cerebellum. Experiments have indicated that the  The precision of the cerebellar anatomy has instigated the development of many theories that attempt to unravel cerebellar function. However, although it is known that the cerebellum contributes to motor learning and cognition, there is still no general agreement about its exact functional role7.Synaptic plasticity is an activity-dependent change in the strength of synaptic connections between pre and postsynaptic neurons. In cerebellar cortex, modifications in the strength of synaptic connections between parallel fibres (PFs) and Purkinje cells (PCs) such as long-term depression (LTD) and long-term potentiation (LTP) are thought to contribute to cerebellar learning13. In addition to undergoing LTD, PF synapses can also exhibit LTP. The strengthening of excitatory synapses between PFs and PCs by PF LTP is mediated by smaller calcium concentration increases that can result from the activation of PFs without any coincident CF input to the PC. LTP is necessary to balance LTD at cerebellar PF-PC synapses in order to prevent saturation and to allow reversal of motor learning14. A large number of experimental21 and several computer simulation studies27 have explored the biochemical pathways involved in PF LTD. Less is known about the mechanisms underlying PF LTP, but it has been shown that PF LTP occurs after dephoshorylation of AMPA receptors by protein phosphatases leads to the insertion of additional receptors into the postsynaptic membrane, while PF LTD is triggered by the internalization and removal of AMPA receptors after their phosphorylation by protein kinases21.PF LTD  is a process in which the strength of the PF-PC synapse is depressed by large increases in the postsynaptic calcium concentration in response to the coincident activation of PF and climbing fibre (CF) input onto the PC31. CaMKII is a critical mediator of the calcium signalling systems that underlie the induction of synaptic plasticity. Although significant progress has been made in understanding the role of CaMKII in synaptic plasticity in other brain areas, little is known about its functional role during plasticity induction in the cerebellum.Calcium-calmodulin dependent protein kinase II (CaMKII), which is one of the most abundant proteins in the brain, is a multifunctional enzyme that phosphorylates a wide range of substratesα CaMKII and β CaMKII, have been shown to mediate synaptic plasticity in the cerebellum, and therefore to be essential for cerebellar learning and memory formation33. Although β CaMKII is the predominant isoform of CaMKII in the cerebellum, the exact role of β CaMKII in cerebellar learning has yet to be established.Two CaMKII isoforms, Camk2b knockout mice, which have been genetically modified to prevent the expression of the gene encoding the β isoform of CaMKII, have addressed the role of β CaMKII in plasticity in cerebellar PCs. These studies demonstrated that the β CaMKII isoform regulates the direction of cerebellar plasticity at PF-PC synapses33. Stimulation protocols that induce LTD in wild-type mice result in LTP in knockout mice that lack β CaMKII, and vice versa . The simple model of AMPA receptor phosphorylation consists of six reactions: calcium-calmodulin (Ca4CaM)-dependent activation of CaMKII, CaMKII binding to F-actin, binding of calcium to calmodulin (CaM) to form Ca4CaM, PP2B activation by Ca4CaM, and AMPA receptor phosphorylation and dephosphorylation by, respectively, CaMKII and PP2B : WiAc, WbAc, WpAc, and WaAc. The CaMKII activation model developed by Dupont et al.35 was adapted here to express all kinase states in concentrations rather than fractions. Therefore, the Wi concentration may be computed from the mass conservation relationx] denotes the concentration of substance x, e.g. [Wtot] is the total concentration of CaMKII.In the simple model of Cab, Wp, and Wa states exhibit kinase activity, and can, therefore, phosphorylate CaMKII’s targets, including adjacent subunits in the CaMKII multimer. To be “ready” for phosphorylation, such adjacent subunits must be in the Wb state themselves. The overall phosphorylation rate associated with this process is indicated by Va, which is calculated using a phenomenological non-linear function of kinase subunits in the Wb, Wp and Wa forms as in Dupont et al.35.b, cp and ca are weighting factors proportional to the kinase activity of each active state.Subunits in the Wet al.35 includes an empirical cubic function (Ka) to model the neighbouring autophosphorylation, allowing the mathematical model to reproduce the experimental results in De Koninck and Schulman36. The equation for Ka iset al.35 adjusted to fit the experimental plots in De Koninck and Schulman36, and Ta is the total fraction of active subunits represented asThe model of Dupont 4CaM binds to Wi, the resulting Wb form can either be phosphorylated, release Ca4CaM, or bind to Ac. Wb is an active CaMKII subunit that can also phosphorylate AMPA receptors (AMPARs). The equation for Wb is therefore4CaM] denotes the concentration of Ca4CaM and [WbAMPAR] is the concentration of Wb bound to AMPARs.Once Cap subunit can release Ca4CaM, switching to the Wa state, or bind to Ac and form WpAc. As for Wb, Wp also phosphorylates AMPARs. The amount of Wp is calculated aspAMPAR] is the concentration of the complex of Wp bound to AMPARs.The Wa can bind to either Ca4CaM or Ac, and phosphorylate AMPARs as well. In our model, we also allow the CaMKII subunits in the Wa form to switch to the Wi state. CaMKII is therefore gradually inactivated after calcium stimulation stops. Thus,aAMPAR] is the amount of Wa trapped to AMPARs.W35, the model used here does not include a separate “trapped” state in which apo-CaM  is bound to CaMKII, mainly because dissociation of calcium and CaM cannot be distinguished experimentally  as two distinct processes. As a result, the values for the rate constants between the “trapped” CaM (Wt) and Wa states in Dupont et al.35 were adopted here as kap and kpa.Unlike the model by Dupont and collaboratorsiAc binds to Ca4CaM and changes to the active WbAc form, or dissociates from Ac and switches to the Wi state. Changes in WiAc concentration areWbAc can be phosphorylated by neighbouring active Ac-bound subunits: WbAc itself, WpAc or WaAc. The autophosphorylation process for Ac-bound CaMKII is analogous to the mechanism of autophosphorylation of the kinase unbound to Ac (Eqs. ac) is thereforeW Ac Eqs. , 3 and 4bAc can also switch to WiAc once Ca4CaM dissociates from this kinase subunit, or dissociate from Ac and change to Wb. Thus,W4CaM-bound form of CaMKII can release Ca4CaM and switch to the WaAc form, or dissociate from Ac and swap to the Wp state. The concentration of WpAc at each time step is expressed asaAc can bind to Ca4CaM and switch to WpAc, or dissociate from Ac and turn into Wa. The equation for WaAc isThe phosphorylated Ac- and Ca4CaM not only activates CaMKII, but can also bind to the inactive form of PP2B (PP2Bi). The phosphatase then gets activated and switches to the PP2Bac form. PP2Bac mediates the dephosphorylation of AMPA receptors. The temporal evolution of PP2Bi concentration isac concentration changes are expressed asacAMPARP] expresses the concentration of PP2Bac bound to phosphorylated AMPA receptors (AMPARPs).CaThe evolution of unphosphorylated AMPA receptors at each time step isMoreover, the equation that represents the evolution of AMPARPs is10, our simple model that simulates the bidirectional synaptic plasticity in Purkinje cells uses a calcium dynamics modelϕ(t) − κ([Ca] − [Camin]) describes the simple model of calcium dynamics we adopted, where ϕ(t) denotes calcium concentration increases at each time step whose values originate from an input table, κ is a term that reflects the calcium removal rate through diffusion, pumps, exchanges, and [Camin] is the basal calcium concentration.Standard protocols of long-term depression (LTD) and long-term potentiation (LTP) induction normally involve repetitive calcium stimuli (such as 1 Hz for 300 s). To generate calcium pulses with concentrations that reflect experimental dataϕ(t)) was used to stimulate the model to generate realistic amplitudes of calcium in response to parallel fibre (PF) alone and PF+ climbing fibre (CF) stimulations , a numerical tool for simulating dynamical systems, using the CVODE method with relative and absolute error tolerances of 10Camk2b knockout mice. We varied the amplitude of calcium pulses, which are used as input to our model, and calculated the average concentrations of all biochemical compounds during the simulated period for each level of calcium concentration. The goal of these simulations was to compare the predictions of our computational model with the proposed explanation of the experimental findings by Van Woerden and collaborators33, which is illustrated in schematic form in Fig. Using simulations of our mathematical model, we first investigated the dependence of kinase and phosphatase activities on the postsynaptic intracellular calcium concentration for wild-type mice and ac and PP2Bac for wild-type mice and Camk2b knockout mice as we increase the average concentration of calcium during the stimulation period. To create similar conditions as in a typical cerebellar plasticity induction protocol, which was also used by Van Woerden and colleagues33, the input to the model consisted of calcium pulses applied at a rate of 1 Hz for 300 s. The figure compares the resulting kinase and phosphatase concentrations averaged over the 300 s of calcium application with concentrations averaged over a longer time period of 6000 s in order to explore the continued behaviour of the system after the offset of calcium application.Figure et al.33 Low calcium concentrations, which would be expected to result from PF input alone, evoke larger average concentrations of PP2Bac than CaMKIIac in simulated wild-type mice. In contrast, for Camk2b knockout mice, in which the lack of β CaMKII prevents the binding of the CaMKII holoenzyme to F-actin, this relationship is reversed, with low calcium concentrations activating CaMKII more strongly than PP2B. Furthermore, high calcium concentrations, in the range of those expected to be caused by coincident PF and CF input, lead to higher concentrations of CaMKIIac than PP2Bac in wild-type mice, but larger phosphatase than kinase activities in Camk2b knockout mice  where the levels of unphosphorylated and phosphorylated AMPA receptors are similar for both types of mice. For these very low calcium concentrations, CaMKII is only weakly activated and the concentration of CaMKIIac rapidly drops after the calcium stimulation stops. In this case, the CaMKIIac decay is much faster than the deactivation of PP2B, favouring the dephosphorylation of AMPA receptors. In other respects, however, the results for 6000 s replicate those for 300 s, with increased (decreased) concentrations of unphosphorylated AMPA receptors for wild-type (knockout) mice at low calcium concentrations at around 0.2 μM, and decreased (increased) levels of unphosphorylated AMPA receptors for wild-type (knockout) mice for higher calcium concentrations (above 0.3 μM). Given that the level of unphosphorylated  AMPA receptors determines the strength of the PF-PC synapses, our simulations therefore replicate the experimental results by Van Woerden et al.33: in wild-type mice, LTP occurs at low calcium concentrations and LTD at high calcium concentrations, while knockout mice that lack β CaMKII exhibit LTD at low calcium concentrations and LTP at high calcium concentrations.The effect of CaMKII and PP2B on cerebellar synaptic plasticity is mediated by the phosphorylation and dephosphorylation of AMPA receptors, respectively, with AMPA receptor phosphorylation leading to the internalization and removal of AMPA receptors from the postsynaptic membrane and the induction of LTD. Thus, we also investigated the effect of the simulated P2B Fig. , Camk2b Camk2b knockout mice, PF input without CF input rapidly decreases the level of unphosphorylated AMPA receptors, indicating the induction of LTD rather than LTP  AMPA receptors in all cases  F-actin binding of CaMKII in wild-type mice, and the lack of F-actin binding in 27, none of these models has been set up to model the induction of LTP at these synapses. Moreover, despite experimental evidence for the involvement of CaMKII in PF LTD and LTP33, the CaMKII pathway has, so far, not been included in the signalling cascades of existing PF-PC synaptic plasticity models. The present simple model is the first model of intracellular signalling at PF-PC synapses that focusses on the role of CaMKII and that models the induction of both LTD and LTP in wildtype and Camk2b knockout mice. In order to concentrate on the role of β CaMKII in bidirectional synaptic plasticity, the model has been kept intentionally simple. In future work, however, the biological realism of the model could be incrementally extended by adding components of existing models of PF LTD. A comparison of results obtained from our simple model with a more complex one could then be used to identify the potential contribution of other biochemical species to regulating the sign reversal of synaptic plasticity in cerebellar PCs.Although there are many computational models that investigate the biochemical processes underlying LTD at PF-PC synapses41. As a complement to our deterministic model of intracellular signalling at the PF-PC synapse, a stochastic model of bidirectional plasticity at this synapse, which also accounts for spatial aspects of cellular signalling, should be developed. Results obtained from both deterministic and stochastic computational models should then be compared.Most modelling studies of intracellular signalling are based on the assumption that ionic and molecular concentrations can be described by mass action kinetics, and it is often assumed that reactions between chemical species occur in a single well-stirred compartment. As in the present model, this assumption leads to deterministic solutions of systems of ordinary differential equations. However, many signalling cascades involve very small numbers of molecules or ions, which are more faithfully represented by stochastic modelset al.33 that the binding of CaMKII to actin controls bidirectional plasticity at parallel fiber-Purkinje cell synapses can be implemented. As new and more precise experimental data become available, the parameter set of the model can be updated and the model retested. If the model predictions were falsified, this would require a re-evaluation of the model: either some of the remaining free parameters could be adjusted or new mechanisms would have to be added so that the model works.Some of the parameter values in our model could not be constrained by experimental data (see Table 24. Indeed, this is the case for the simple model proposed here. However, Antunes and De Schutter have recently suggested that the extent of synaptic plasticity should be measured as a reduction in the number of synaptic AMPA receptors, rather than the concentration of phosphorylated receptors26. A next step in this research could be the inclusion of the AMPA receptor trafficking modelled by Antunes and De Schutter into the model of bidirectional plasticity at PF-PC synapses. Moreover, several concentrations of biochemical compounds and kinetic rate constants are free parameters, due to the lack of experimental data. Additional experiments will lead to an incremental improvement of the computational model, which in turn might suggest new experiments that need to be conducted.Previous models of cerebellar LTD have measured synaptic plasticity by quantifying the concentrations or densities of phosphorylated and unphosphorylated AMPA receptors


IL36RN) but not elements of the IL-17/IL-23 axis. In human psoriatic response to dithranol, rapid decrease in expression of keratinocyte differentiation regulators , antimicrobial peptides , chemotactic factors for neutrophils  and neutrophilic infiltration was followed with much delay by reduction in T cell infiltration. Targeting keratinocytes rather than immune cells may be an alternative approach in particular for topical anti-psoriatic treatment, an area with high need for new drugs.Despite the introduction of biologics, topical dithranol  has remained one of the most effective anti-psoriatic agents. Serial biopsies from human psoriatic lesions and both the c-Jun/JunB and imiquimod psoriasis mouse model allowed us to study the therapeutic mechanism of this drug. Top differentially expressed genes in the early response to dithranol belonged to keratinocyte and epidermal differentiation pathways and IL-1 family members (i.e.  With the development and market introduction of biologics, much progress has been made in recent years in the systemic treatment of psoriasis. Currently, targeted therapy with antibodies against IL-17 or IL-23 exhibits high efficacy and allows complete or almost complete clinical clearance of psoriasis lesions in a high percentage of cases . HoweverWith the aim of unraveling dithranol’s therapeutic mechanisms and to possibly uncover new targets for topical treatment of psoriasis, we conducted a clinical trial and employed several mouse models including the c-Jun/JunB knockout model  and the As depicted in Dithranol slowly diminished CD3, CD4, FoxP3 and CD8 cell counts in the dermis as evidenced by unaffected cell numbers early on during the treatment course and significant reduction only at the follow-up visit (4–6 weeks after end of treatment) . The resKRT2, LCE1C, KRT73, LCE1A) and establishment of skin barrier . Furthermore, dithranol downregulated genes of antimicrobial peptides (AMPs) such as ß-defensin-2  and chemoattractants for neutrophils  , there was a significant upregulation of genes involved in keratinization and keratinocyte differentiation  .S100A7A, S100A12, DEFB103A) and genes involved in neutrophil-mediated inflammatory responses significantly diminished in their expression. With delay, dithranol also lowered expression of inflammatory response-related genes  only at the end of treatment (week 2–3) (At the end of treatment (week 2–3), dithranol had deregulated a total of 453 genes, with downregulation of 325 DEGs and upregulation of 128 genes. Compared to day 6, expression of various genes involved in keratinocyte differentiation decreased further upon dithranol treatment and other AMPs (e.g. eek 2–3) . Notablyeek 2–3) . Comparieek 2–3) . Top sigeek 2–3) . At folleek 2–3) .Lce6a, Lce1i, Lce1g, Lce1f), as well as hornerin (Hrnr)  in c-Jun/JunB psoriatic skin. To substantiate dithranol’s effect on keratinocytes, we employed the mouse-tail test, a traditional model to quantify the effect of topical anti-psoriatics on keratinocyte differentiation by measuring degree of orthokeratosis versus parakeratosis  and several AMPs )  . Gene onn (Hrnr) . Associaout mice . In totaelopment . Even moy RT-PCR . The groy RT-PCR  and havey RT-PCR , belongieratosis . We founeratosis , consisteratosis . Next, w Defb3)) . InteresIn contrast to the effects of dithranol in the c-Jun/JunB model and mouse-tail test, this agent had no therapeutic capacity in the immunologically mediated imiquimod (IMQ) mouse model, which is often referred to as a psoriatic-like skin inflammation model . Indeed,IL36A, IL36G and IL36RN) (IL36RN (murine Il1f5) and IL36B (Il1f8) was significantly reduced in dithranol-treated psoriatic c-Jun/JunB lesions compared to controls (IVL) and members of the serpin family  both in lesional skin of patients and c-Jun/JunB knockout mice (DEFB4A and DEFB4B) produced by keratinocytes (CXCL5 and CXCL8) , keratinocyte-neutrophil crosstalk and inhibits the IL-36 inflammatory loop in psoriasis, thus unraveling after over 100 years of use, the therapeutic mechanism of one of the most effective topical treatments of psoriasis. Dithranol significantly diminished mRNA expression of pro-psoriatic IL-1 family members ( IL36RN) . At day  IL36RN) . Expresscontrols  and amoncontrols , Table 7out mice . Moreoveinocytes  within 6d CXCL8) ; Table 2d CXCL8) .Surprisingly, there were no significant changes in overall T cell numbers including CD4+ and CD8+ T cells, as well as FoxP3 positivity indicative for regulatory T cells in the skin in the early phase (within 6 days) during dithranol treatment. Reflecting dithranol’s primary effect on the epidermal compartment, the reduction of T cell numbers in the epidermis preceded that in the dermis. Whereas dithranol had decreased T cell counts in the epidermis at week 2–3, an effect on T cell numbers in the dermis was only evident at the follow-up visit, 4–6 weeks after the end of dithranol treatment , in agreDEFB4 using qPCR analysis. However, based on their observations, they concluded that dithranol’s anti-psoriatic effects cannot be explained by direct effects on keratinocyte differentiation or cytokine expression , and neutrophils and their chemotactic factors (CXCL5 and CXCL8) or members of the serpin family (SERPINB7 and SERPINB13), are promising approaches. Such approaches may not only be helpful for chronic plaque-psoriasis, but also for pustular psoriasis, in which a vicious loop between AMPs such as cathelicidin (LL-37) and IL-36 signaling may drive psoriatic disease  treatment strategies in psoriasis. Not only targeting the IL-36 pathway, but also keratinocyte differentiation regulators (e.g. involucrin), keratinocyte-produced AMPs  within 2 weeks, and phototherapy within 4 weeks prior to study enrollment. None of the patients had received systemic treatment in the past prior to study enrollment. In total, 15 psoriasis patients  were enrolled. Mean duration of psoriasis had been 17.9 years (SD 11.5 years). Mean duration of dithranol treatment was 15.4 days (SD 3.6 days) and treatment was prematurely discontinued in two patients. Samples of normal skin from patients undergoing surgery for removal of benign skin lesions were available from 12 subjects  for control purposes. The samples were from lesion-adjacent, excised skin of patients who did not suffer from psoriasis, other inflammatory diseases or autoimmune diseases.Dithranol ointment was prepared in the hospital pharmacy with 2% salicylic acid and white vaseline as base. It was administered to the patients daily in increasing concentrations; dosage was adjusted individually to the level of skin irritation. Concentration was usually increased every other day  and mean treatment duration was 15.4 days.At each of the four visits see , i) PsorBiopsy samples were taken from the psoriasis patients before (day 0), during early treatment at first strong perilesional inflammation between day 4 and 9 (with most biopsies taken at day 6) at end of treatment (approximately after 2–3 weeks) and at a follow-up visit (4–6 weeks after end of therapy). Per patient, a total of five biopsy samples were taken on four study days by means of a punch cylinder (up to 5 mm) under local anesthesia. At the first visit before starting therapy, one biopsy sample was taken additionally from adjacent non-lesional skin, with a distance of at least 5 cm from the edge of psoriatic skin. One part of each biopsy was fixed in 4% neutral-buffered paraformaldehyde and used for histology and immunohistochemistry. The other part was stored in RNAlater solution  at −80°C until RNA extraction for further analysis.Human and murine samples fixed with paraformaldehyde were processed routinely, cut in 4 µm sections and stained with hematoxylin and eosin (HE). Five randomly selected fields per slide were investigated for histological analysis. Thickness of epidermis was measured from basal layer to stratum corneum using an Olympus BX41 microscope , cellSens software (Olympus Life Science Solutions) and 20x magnification. Semi-quantitative scoring  was performed at five randomly selected locations per slide and at 20x magnification.In the mouse-tail model, degree of orthokeratosis was analyzed as described by Antigen retrieval was performed using either EDTA-buffer , citrate-buffer  or trypsin (MPO). Primary antibodies used were: anti-human CD1a , anti-human CD3 , anti-human CD4 , anti-human CD8 , anti-human CK16 , anti-human FoxP3 , anti-human Ki-67  and anti-human MPO . Stainings were performed using the Dako REALTM Detection System, Peroxidase/AEC, rabbit/mouse  on the Dako Autostainer Link 48  according to the manufacturer’s instructions. For quantification of CD1a, CD3, CD4, FoxP3, CD8, and MPO staining, all positively stained cells with visible nucleus in five randomly selected fields (separately for epidermis and dermis) per slide were counted and results were averaged to obtain mean cell counts. To quantify Ki-67 and CK16 staining, area of positive staining was divided by epidermal area as follows: one representative image per slide was taken on an Olympus BX41 microscope  at 10x magnification using cellSens software (Olympus Life Science Solutions). TIFF images were imported into ImageJ program and pixels were converted to µm. Using the polygon sections tool, the outline of epidermis was framed, and the total area was measured. In addition, total area of particles (positively stained cells) within the epidermal area was assessed.Total RNA was extracted from frozen biopsies of psoriasis patients and control subjects. To facilitate homogenization, tissues were cut in 20 µm sections using a cryomicrotome and collected in precooled MagNA Lyser Green Beads tubes  and disruption of tissue was performed on the MagNA Lyser Instrument . After efficient homogenization, total RNA was extracted using the miRNeasy Mini Kit , according to the manufacturer’s instructions. To ensure complete DNA removal, on-column DNase digestion was performed and RNA was eluted in 15–20 µl RNase-free water. Mouse tissues were handled in the same way except that sufficient homogenization of samples was obtained without using a cryomicrotome.http://www.ncbi.nlm.nih.gov/geo/) with accession numbers GSE145126 and GSE145127.Isolated RNA was quality checked on a Bioanalyzer BA2100  using the RNA 6000 Nano LabChip according to manufacturer’s instructions and samples with RNA integrity numbers (RIN) between 5 to 8 were considered for analysis using Human GeneChip 2.0 ST arrays  for the human samples and mouse Clariom S Assay  for the mouse samples. For first and second strand cDNA synthesis 500 ng total RNA were used in the GeneChip WT PLUS Reagent Kit (ThermoFisher) according to manufacturer’s instructions. Fragmentation and terminal labelling was performed using the GeneChipTM WT Terminal Labeling Kit (ThermoFisher) and hybridization, wash and stain of arrays was performed on a GeneChip Fluidics 450 station using the GeneChip Hybridization, Wash and Stain Kit (ThermoFisher) according to manufacturer’s instructions. Arrays were scanned in a GeneChip GCS300 7G Scanner and analysed with the Affymetrix Expression Console Software 1.3.1 (ThermoFisher) for the human array and Transcriptome Analysis Console (TAC) 4.0.2 for the mouse arrays. Samples were processed at the Core Facility Molecular Biology at the Centre of Medical Research at the Medical University of Graz, Austria. Pre-processing of CEL-files for the human arrays was performed with Partek Genomics Suite version 6.6  using the robust multi-chip analysis (RMA) algorithm, which includes background adjustment, quantile normalisation and median polished probe summarisation. For statistical analysis, paired-sample t-tests were used and genes with p<0.05 and a fold change of at least 1.5 were considered to be significantly deregulated. All microarray data has been deposited at the public repository Gene Expression Omnibus (GEO)  with MicroArrayPipeline v1.0 Shiny app based on limma Bioconductor package for differential expression analysis and genes with p<0.05 and a fold change of at least 1.5 were considered to be significantly deregulated. Mouse and human DEGs were tested for potential overlap. Probeset IDs of early (day 6) as well as late (week 2–3) DEGs of human microarray were matched with DEGs of mouse microarray using NetAffxTM Expression Array Comparison Tool.For Nanostring analysis, a nCounter GX Custom codeset  were calculated using the ΔCt method. Primer sequences are listed in Per sample 2 µg of RNA was reverse transcribed into cDNA using iScript Reverse Transcription Supermix . Relative gene expression was determined using GoTag qPCR Master Mix  on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The following cycling conditions were used: Hot-start activation , denaturation for 40 cycles  and annealing/extension . Melting curve analysis was done to confirm amplification specificity. For each sample, qPCRs were run in triplicates. Cycle thresholds (Ct) were determined and relative mRNA expression to 6–9 week-old mice were kept with food and water ad libitum in the conventional animal facility at the Centre for Medical Research, Medical University of Graz or at the Medical University of Vienna, Austria. During experiments, all mice were monitored closely to ensure sufficient health status. BALB/c mice were purchased from Charles River . c-Jun/JunB knockout mice  were breFor all animal experiments, dithranol in different concentrations (dissolved in vaseline) and vehicle (vaseline cream only) was provided by the pharmacy of the Medical University of Graz, Austria. Aldara (IMQ) 5% cream  and tamoxifen  were purchased.2 of dorsal skin, both ears (treated and untreated as control) were collected.24 hr before starting an experiment, dorsal skin of BALB/c mice was shaved carefully. To induce psoriasis-like dermatitis, imiquimod (IMQ) cream was applied daily for five consecutive days on dorsal skin (approximately 40 mg) and right ear (approximately 20 mg). A daily topical dose of 62.5 mg of IMQ cream was not exceeded; translating into 3.125 mg of the active compound. Mice received IMQ cream in the morning and dithranol treatment in the afternoon, with a time gap of 8 hr. Dithranol was applied topically on dorsal skin (40 mg) and right ear (20 mg) and concentrations were increased every other day . Control mice were treated similarly with Vaseline cream. Double skin fold of dorsal skin and ear thickness was measured daily in triplicates before any application using a micrometer . 24 hr after the last topical treatment, mice were sacrificed and approximately 1 cmMice carrying floxed alleles for the JunB and c-Jun locus and expressing the K5-CreERT transgene (mixed background) received consecutive i.p. injections of tamoxifen (1 mg/day) for a period of 5 days, leading to deletion of both genes in the epidermis by inducible Cre-recombinase activity . One weeFor the mouse-tail model, 40 mg dithranol 1% was applied daily for 14 days to the surface of the proximal part of tails (approx. 2 cm in length starting 1 cm from the body). 24 hr after the last application, mice were sacrificed, and treated parts of tail skin were removed from the underlying cartilage. The mouse-tail test for psoriasis is a traditional model to quantify the effect of topical anti-psoriatics on keratinocyte differentiation by measuring degree of orthokeratosis versus parakeratosis .Statistical analyses for human and murine microarrays were performed as described in the specific sections. All other statistical analyses were determined using GraphPad Prism version 8 . Normality was determined by Shapiro-Wilk test and differences between two groups were assessed by Mann Whitney test, Wilcoxon test or T-test (paired or unpaired) as appropriate. For multiple comparisons, One-way ANOVA with Dunnett’s test or Tukey’s test was used for parametric data and Kruskal Wallis test with Dunn’s test was used for nonparametrical data as indicated in the specific sections. Significance was set at a p-value of ≤0.05. Each animal experiment was performed at least twice. For correlation analysis of clinical scores, as well as comparison of microarray and nanostring ratios, Pearson or Spearman correlation was used as indicated.A clinical study  in psoriatic patients treated with dithranol was completed in cooperation with the Department of Dermatology, Klagenfurt State Hospital. Clinical trial procedures were approved by the ethics committee of the federal state of Carinthia, Austria (protocol number A23/15) and all participants gave written informed consent in accordance with the principles of the Declaration of Helsinki. All mouse experiments were approved by the Austrian Government, Federal Ministry for Science and Research  and animal experiments performed in Vienna were additionally approved by the Animal Experimental Ethics Committee of the Medical University of Vienna. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The role of keratinocytes in the pathogenesis of psoriasis has gained a lot of interest recently and your work supplements the new insights from the therapeutic point of view. Dithranol by itself may still be a therapeutic niche, but the data regarding the mode of action can stimulate drug development along these lines.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Jos van der Meer as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Claus Johansen (Reviewer #3).Thank you for submitting your article "Dithranol targets keratinocytes, their crosstalk with neutrophils and inhibits the IL-36 inflammatory loop in psoriasis" 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.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional work is required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:Topic of interest but in major parts confirmatory to already published work and in other parts not conclusive. Please provide clarity about the data on AMPs in this manuscript and in the manuscript submitted to the other journal.Essential revisions:Statistical analysis (see reviewer 2). Interpretation of data (specific for dithranol or bystander effects (see reviewers 1 and 2). Interpretation of data regarding the mouse models used as there seem a number of inconsistencies (see reviewers 2 and 3).Interpretation of results in the light of the critique of all reviewers.Reviewer #1:The manuscript by Benezeder et al. addresses an interesting topic. The role of keratinocytes for psoriasis pathogenesis has been neglected for many years and the work presented here re-focuses on keratinocyte-mediated effects.Interestingly, the authors used dithranol as a model, which is challenging since this topical compound shows beneficial therapeutic effects but also induces dose- and individual-depended skin irritation. Although the methods employed are well established and state-of-the-art there are some points that need clarification.• Psoriasis is viewed as systemic disorder and to judge the overall beneficial effect of any topical therapy including dithranol it should be shown whether there are effects on systemic inflammation (e.g. vascular inflammation). The authors should mention this as limitation of their approach.• The most commonly used topical therapies are corticosteroids alone or in a fixed combination with a vitamin D analogue. The authors are encouraged to use such treatment as a control in order to prove that the observed effects are specific for dithranol.• The irritant potential of dithranol is helpful in cases of otherwise refractory alopecia areata. The authors should briefly mention this and discuss if their data may explain the effect as an outlook for further investigation.The reviewer is puzzled by the fact that he was asked to review a manuscript of the same group with the same first author with significant similarity to data that are described in this manuscript for another journal particularly about the effect of dithranol on antimicrobial peptides.The authors must make clear if there is an overlap of data and which of the data presented in the other manuscript are also contained in this one.Reviewer #2:Benezeder et al. present a paper on dithranol action on keratinocytes in psoriasis. The authors provide data on clinical outcome, reduction of epidermal thickness, cell proliferation and keratin 16 expression. All changes were observed at week 2-3 of treatment. By microarray analysis on day 6 and week 2-3 differentially expressed genes (DEGs) were identified, among these genes such encoding keratinocyte differentiation, skin barrier function and antimicrobial peptides (AMPs), but also IL-17 or IL-36. In addition, the authors used dithranol treatment in a c-Jun/JunB knockout mouse model. Again, microarray analysis was used to follow effects. The authors focused on genes belonging to keratinocyte function. Microarray data generated from the c-Jun/JunB knockout mouse model was compared to human microarray data. Dithranol was also applied to mouse tails. Here, they found increased thickening and orthokeratosis, increase in keratin 16 expression and AMPs. Finally, they used the imiquimod psoriasis-like mouse model where dithranol increased skin thickening. The authors conclude that the effects of dithranol are mainly mediated by keratinocytes and IL-36.Although this is an interesting study, there are some major caveats and conflicting results, which make it difficult to accept this paper.1) The main messages of this study – decrease of psoriatic epidermal thickening, reduction of Ki67 positivity, reduction of keratin 16 expression and AMPs by dithranol have been shown by previous reports from human skin .2) There is plenty of evidence demonstrating that psoriasis is an IL-17-driven immune disease. All treatments with a rapid clinical response will find a decrease in epidermal thickening, a normalization of keratinocyte differentiation and a decrease in immune cell infiltration as well as cytokine expression. From this perspective, one cannot conclude that the one or other treatment will work through keratinocytes and not influence the immune system. The changes in keratinocytes as observed in Figure 2  seem to be significant on week 2 to 3. A similar pattern with a decrease in epidermal thickening, keratin 16 and Ki67 expression is visible at week 2 of treatment when using monoclonal antibodies e.g. directed against IL-17. Similarly, antimicrobial peptides are regulated at early time points by immunobiologics .3) The mouse model used (c-Jun/JunB knockout) is somehow artificial and does not require T cells for developing epidermal changes. Therefore, the findings – although of interest – cannot be translated to the human situation.4) The Major concern is that most of the findings are descriptive. The authors performed many microarray analyses but studies on functional mechanisms confirming their hypothesis are missing.5) The increase in epidermal thickening, the changes in keratin 16 and AMPs found in the mouse tail experiment with dithranol contradict the results found in human psoriasis .6) IL-36 cytokines are mainly expressed by keratinocytes , but the authors describe a decrease at week 2 to 3. If dithranol targets primarily keratinocytes and inhibits IL-36, the effect should be visible at earlier time points.Reviewer #3:The manuscript by Benezeder et al. investigates the mode of action by which dithranol mediates its anti-psoriatic effects. Although dithranol has been used as topical treatment for psoriasis for many years the mechanism by which dithranol works is not that well described and therefore a better understanding of the mode of action of dithranol is needed and could pave the way for other topical drugs for psoriasis.The manuscript is interesting and well-written and the data are convincing.I have the following comments:1) Although the data in the manuscript clearly shows that the early mode of action of dithranol in psoriasis treatment is on keratinocyte-derived molecules and subsequently neutrophil infiltration to the skin the manuscript does not reveal the molecular mechanism behind this. Does dithranol use a membrane bound receptor, an intracellular receptor or does it inhibit specific transcription factors ? Have the authors investigated this or is there anything in the literature about this. The authors should discuss this more in the manuscript.2) Surprisingly, dithranol does not have an anti-psoriatic effect in the imiquimod-induced psoriasis model. In contrast, dithranol has an anti-psoriatic effect in the c-Jun/JunB mouse model. Were the mouse strains used in the two models the same? Were the mice similar in age and did the authors use females or males and was this the same in the two models? The authors write that dithranol rather worsened the psoriatic lesions in the IMQ model. And interestingly the authors write that "Dithranol treatment of healthy murine skin led to similar effects". Could this be the same effect as has been described in perilesional skin? Is the inflammatory effect of dithranol on perilesional skin known? If so the authors could examine if this is also what is seen in skin of IMQ-treated mice. These issues could be discussed in more detail.3) In the psoriasis patients treated with dithranol biopsies were taken from lesional skin over time. Did the authors take biopsies from the same "target" lesion throughout the study?4) In Table 1 the authors should in the legend include that the numbers in the table are based on data from 15 psoriatic patients. Summary:Topic of interest but in major parts confirmatory to already published work and in other parts not conclusive. Please provide clarity about the data on AMPs in this manuscript and in the manuscript submitted to the other journal.SERPINB7 and SERPINB13), anti-microbial peptides , chemotactic factors for neutrophils  and neutrophilic infiltration and was followed with much delay by reduction in T cell infiltration. These findings are novel and haven't been reported previously.We analyzed biopsy samples from 15 psoriasis patients at several time points, i.e., before, during, after dithranol therapy and at a follow-up visits and from a well-matched control group  and used three mouse models  to substantiate our findings on the effects of dithranol in diseased psoriatic skin. We are first by using genome-wide expression analysis to investigate the effects of dithranol and by doing so we were able to thoroughly dissect the mechanisms of dithranol in diseased psoriatic skin. As pointed out, psoriatic response to dithranol was characterized by a rapid decrease in expression of keratinocyte differentiation regulators  we report on dithranol's irritant effect  on healthy skin exclusively using healthy murine skin as well as xenografted healthy human skin. We want to point out that there is no overlap at all between the data and conclusions of the two manuscripts. However, we are happy to provide this letter on a confidential basis to Essential revisions:Statistical analysis (see reviewer 2). Interpretation of data (specific for dithranol or bystander effects (see reviewers 1 and 2). Interpretation of data regarding the mouse models used as there seem a number of inconsistencies (see reviewers 2 and 3).Interpretation of results in the light of the critique of all reviewers.We have responded to all these comments appropriately (see below), including a revision of statistics.Reviewer #1:The manuscript by Benezeder et al. addresses an interesting topic. The role of keratinocytes for psoriasis pathogenesis has been neglected for many years and the work presented here re-focuses on keratinocyte-mediated effects. Interestingly, the authors used dithranol as a model, which is challenging since this topical compound shows beneficial therapeutic effects but also induces dose- and individual-depended skin irritation.Although the methods employed are well established and state-of-the-art there are some points that need clarification.• Psoriasis is viewed as systemic disorder and to judge the overall beneficial effect of any topical therapy including dithranol it should be shown whether there are effects on systemic inflammation (e.g. vascular inflammation). The authors should mention this as limitation of their approach.We agree with the reviewer and have mentioned this limitation in the tenth paragraph of the Discussion.• The most commonly used topical therapies are corticosteroids alone or in a fixed combination with a vitamin D analogue. The authors are encouraged to use such treatment as a control in order to prove that the observed effects are specific for dithranol.Indeed, some downstream effects of dithranol seem to be overlapping with that of vitamin D3 analogues , whereas other effects such as keratinocyte-neutrophil crosstalk are differential and seem to be specific for dithranol. We discuss this now in the Discussion and provide a couple of references on papers investigating the molecular effects of calcipotriol and topical steroids on human psoriasis.• The irritant potential of dithranol is helpful in cases of otherwise refractory alopecia areata. The authors should briefly mention this and discuss if their data may explain the effect as an outlook for further investigation.We entirely agree with the reviewer on the effect of dithranol in alopecia areata  and have added a respective sentence to the eighth paragraph of the Discussion.The reviewer is puzzled by the fact that he was asked to review a manuscript of the same group with the same first author with significant similarity to data that are described in this manuscript for another journal particularly about the effect of dithranol on antimicrobial peptides.The authors must make clear if there is an overlap of data and which of the data presented in the other manuscript are also contained in this one.Please see response above to the Editor's comments.Reviewer #2:[…] Although this is an interesting study, there are some major caveats and conflicting results, which make it difficult to accept this paper.1) The main messages of this study – decrease of psoriatic epidermal thickening, reduction of Ki67 positivity, reduction of keratin 16 expression and AMPs by dithranol have been shown by previous reports from human skin .Please see response above to the Editor's comments. We would like to stress that we discuss the results of those previous studies by van de Kerkhof et al. and Eberle et al., 2017 now in more detail in our paper . In addition, we wish to point out that we are the first to show dithranol’s effect on a transcriptional level in a large patient cohort with multiple time points of biopsy sampling and in two psoriasis mouse models. Moreover, our main conclusion is that inhibition of keratinocyte-neutrophil crosstalk as well as IL-36 pathway is crucial for its anti-psoriatic effect. These findings are entirely novel.2) There is plenty of evidence demonstrating that psoriasis is an IL-17-driven immune disease. All treatments with a rapid clinical response will find a decrease in epidermal thickening, a normalization of keratinocyte differentiation and a decrease in immune cell infiltration as well as cytokine expression. From this perspective, one cannot conclude that the one or other treatment will work through keratinocytes and not influence the immune system. The changes in keratinocytes as observed in Figure 2  seem to be significant on week 2 to 3. A similar pattern with a decrease in epidermal thickening, keratin 16 and Ki67 expression is visible at week 2 of treatment when using monoclonal antibodies e.g. directed against IL-17. Similarly, antimicrobial peptides are regulated at early time points by immunobiologics .We agree with the reviewer, however, our data clearly indicate that dithranol treatment first acts on keratinocytes and later by delayed direct or indirect effects on the immune system. Indeed, certain biologics even of the latest generation have similar effects on the molecular level, which is fascinating, that a traditional topical agent like dithranol can show similar effects as a systemic antibody therapy. We believe that dithranol’s primary effect on keratinocytes is crucial for response to treatment, considering that differentially regulated genes in histological responders compared to non-responders belonged to pathways like keratinocyte differentiation, cornification and keratin filament formation (as mentioned in the third paragraph of the Discussion and Supplementary file 3).We thank the reviewer for hinting us to the work of Krueger et al., 2012 and 2019). We have slightly modified the last sentence of the Results section and added those references in the seventh paragraph of the Discussion section, together with a couple of respective sentences.3) The mouse model used (c-Jun/JunB knockout) is somehow artificial and does not require T cells for developing epidermal changes. Therefore, the findings – although of interest – cannot be translated to the human situation.We entirely agree that findings from mouse models cannot be directly translated to the human situation. However, in addition to the imiquimod model, a solely immunologically mediated psoriasis model, we specifically have chosen the c-Jun/JunB model that is based on keratinocytes rather than on the immune system. We had mentioned this in the manuscript but to make our purpose clearer, we have now modified the wording : "The therapeutic importance of reduction of IL-1 family members in human skin was substantiated by results generated in the keratinocyte-based c-Jun/JunB mouse psoriasis model."4) The Major concern is that most of the findings are descriptive. The authors performed many microarray analyses but studies on functional mechanisms confirming their hypothesis are missing.We would have loved to substantiate our findings on the functional level and have mentioned this shortage of the paper "What our work does not answer, is how dithranol exactly acts at the molecular level. Cell culture studies have shown that dithranol targets mitochondria.…". Anti-psoriatic effects of other topical treatment options have recently been linked to modulation of AhR  and AhR may play a role in pathogenesis of psoriasis . Thus, we made attempts to identify the receptor of dithranol by using aryl hydrocarbon receptor (AhR) knockout mice in order to substantiate our findings on the functional level. However, it appears that dithranol does not act via modulation of AhR, as we did not observe differences in the response of skin to dithranol comparing AhR knockout mice to controls. Certainly, future studies are indeed needed to identify dithranol’s yet unknown receptor. This all has now been worded in the Discussion.5) The increase in epidermal thickening, the changes in keratin 16 and AMPs found in the mouse tail experiment with dithranol contradict the results found in human psoriasis .Defb3 and S100a8 and S100a9 but decreased the pro-psoriatic antimicrobial peptide Camp/LL37 as well as Cxcl5, a chemotactic factor for neutrophils . We now have mentioned this in the Discussion, together with a couple of new references and stressed the limitations of the mouse tail-test (third paragraph).We agree with the reviewer, the effects of dithranol in the mouse-tail test seem to be contradictory. However, this test has limitations since its usefulness only refers to which degree an agent or treatment increases orthokeratosis as a measure of keratinocyte differentiation-inducing activity , as we have demonstrated it for dithranol. On the other hand, as the reviewer observed, besides upregulation of keratinocyte markers dithranol increased certain psoriasis-associated AMPs such as 6) IL-36 cytokines are mainly expressed by keratinocytes , but the authors describe a decrease at week 2 to 3. If dithranol targets primarily keratinocytes and inhibits IL-36, the effect should be visible at earlier time points.IL36A, IL36G and IL36RN) . At day 6, PASI had decreased by 33%, but at week 2-3 we saw a reduction of 58%. Most likely, dithranol’s effect on members of the IL-36 family might be linked to its strong clinical effect at week 2-3. The therapeutic importance of reduction of IL-1 family members in human skin was substantiated by results generated in the keratinocyte-based c-Jun/JunB mouse psoriasis model, where IL-36 family genes were significantly reduced in dithranol-treated psoriatic c-Jun/JunB lesions compared to controls. We have added a respective sentence on IL-36 in the first paragraph of the Discussion.We thank the reviewer for providing the reference of Wenzel et al. with regard to IL-36 expression in keratinocytes. Although we already saw a reduction of keratinocyte-related genes at day 6, a lot of these genes were only normalized at week 2-3, accompanied with a significantly diminished mRNA expression of pro-psoriatic IL-1 family members (Reviewer #3:[…] I have the following comments:1) Although the data in the manuscript clearly shows that the early mode of action of dithranol in psoriasis treatment is on keratinocyte-derived molecules and subsequently neutrophil infiltration to the skin the manuscript does not reveal the molecular mechanism behind this. Does dithranol use a membrane bound receptor, an intracellular receptor or does it inhibit specific transcription factors ? Have the authors investigated this or is there anything in the literature about this. The authors should discuss this more in the manuscript.We agree with the reviewer on that point and have discussed the limitations of our work now in more detail . What indeed our work does not answer, is how dithranol exactly acts at the molecular level, which we also mention in the ninth paragraph of the Discussion, paragraph 1. Little is known in the literature about dithranol's potential receptor that remains yet to be determined. Please see also our response in this regard to comment 4 of reviewer 2 about the aryl hydrocarbon receptor (AhR).2) Surprisingly, dithranol does not have an anti-psoriatic effect in the imiquimod-induced psoriasis model. In contrast, dithranol has an anti-psoriatic effect in the c-Jun/JunB mouse model. Were the mouse strains used in the two models the same? Were the mice similar in age and did the authors use females or males and was this the same in the two models? The authors write that dithranol rather worsened the psoriatic lesions in the IMQ model. And interestingly the authors write that "Dithranol treatment of healthy murine skin led to similar effects". Could this be the same effect as has been described in perilesional skin? Is the inflammatory effect of dithranol on perilesional skin known? If so the authors could examine if this is also what is seen in skin of IMQ-treated mice. These issues could be discussed in more detail.As stated in the manuscript , we used 6-9 week-old mice with different strains. BALB/c mice are commonly used for the Imiquimod model, as we did; the c-Jun/JunB knockout mice of our study have a mixed background, as described by Zenz et al., 2005. However, it is highly unlikely that the different mouse strains played any role in the outcome of the experiments since the two psoriasis models are per se completely different. Immunological aspects are important in the imiquimod model and molecular alterations dominate the c-Jun/JunB model. Concerning the effect of dithranol on healthy murine skin we have added a couple of sentences on dithranol’s irritant effect that may be rather a side effect without functional relevance in human psoriasis  but crucial for dithranol's action in alopecia areata (see response to the comment 3 of reviewer #1 above). We now have added a couple of respective sentences on alopecia areata in the Discussion.3) In the psoriasis patients treated with dithranol biopsies were taken from lesional skin over time. Did the authors take biopsies from the same "target" lesion throughout the study?We have now precisely outlined how marker lesions were defined, scored, treated and biopsy-sampled in the Materials and methods subsection under the new heading “Marker lesions and scoring”. This has also been illustrated in Figure 1A.4) In Table 1 the authors should in the legend include that the numbers in the table are based on data from 15 psoriatic patients.We thank the reviewer for mentioning this and have changed the heading of Table 1 accordingly.


HOXA9/HOXA10 and IRX1 genes and termed the two subgroups iALL-HOXA9 and iALL-IRX1. This expression pattern is critical as it suggests that there is a fundamental difference between the two subgroups. Investigation of the transcriptomes of the two subgroups reveals a more aggressive nature for the iALL-IRX1 group, which is further supported by the fact that patients within this group have a worse prognosis and are also diagnosed at a younger age. This could be reflective of a developmentally earlier cell of origin for iALL-IRX1. Our analysis further uncovered critical differences between the two groups that may have an impact on treatment strategies. In summary, after a detailed investigation into the transcriptional profiles of iALL-HOXA9 and iALL-IRX1 patients, we highlight the importance of acknowledging that these two subgroups are different and that this is of clinical importance.Infant t acute lymphoblastic leukemia is the most common leukemia in infant patients and has a highly aggressive nature. The patients have a dismal prognosis, which has not improved in more than a decade, suggesting that a better understanding of this disease is required. In the study described here, we analyzed two previously published RNA-sequencing data sets and gained further insights into the global transcriptomes of two known subgroups of this disease, which are characterized by the presence or absence of a homeobox gene expression signature. Specifically, we identified a remarkable mutually exclusive expression of the  HOXA9, and the other on separating the patients based on the expression of genes in the HOXA and IRX family of proteins high/IRXneg/low expression have a better prognosis than those with HOXA9low/IRXpos expression In the age of molecular medicine, transcriptional profiling of patients’ samples has become a vital component in improving our understanding of diseases. We now have the potential to dissect transcriptional variations among patients, identify unique components of their disease and provide customized treatment. This approach is particularly valuable for diseases that are rare and difficult to model as there is a scarcity of available information. A prominent example of such a disease is infant MLL-AF4-driven acute lymphoblastic leukemia (ALL). This devastating disease is known to arise in utero, and the patients have a poor prognosis p value ≤0.1. Library pcaExplorer was used for PCA analysis Raw reads were aligned with Kallisto (version 0.43.1) to GRCh38. The Bioconductor package Tximport was used to import transcript-level abundance, estimated counts, and transcript lengths (version 3.5) pink and green in blue) clustered closely with one of the infant clusters. Investigation into the genes driving the clustering revealed HOXA9, HOXA10 and IRX1, IRX2 to be among the top PC1 loadings—in opposite directions. This confirmed previous publications reporting that clustering of the patients was driven by genes of the HOXA and IRX families .IRX1 expression pattern could be indicative of iALL-IRX1 arising in a developmentally earlier cell type than iALL-HOXA9, which is supported by upregulation of genes such as PDGFRB and PDGFD in the iALL-IRX1 data set (www.exphem.org). Contrary to this, hematopoiesis-associated genes such as AFF1 (AF4), CD96, SPN, and PROM1 are upregulated in the iALL-HOXA9 set , which has recently been suggested to be a target for MLL-AF4+ patients, was specifically upregulated in the iALL-HOXA9 subgroup, with lower expression in the iALL-IRX1 group of patients C 24,25],2524,25]HOXA9. Therefore, it is important to replicate experiments in models and cell lines that mirror the iALL-IRX1 disease. We believe this would be critical when selecting therapeutic regimes for these patients, as exemplified by PROM1. We believe that future studies should therefore consider the HOXA9 and IRX1 expression status of infant ALL patients.One key point about the two subgroups of patients with MLL-AF4-driven ALL is that the majority of information we have gathered about this disease to date is derived from mouse models and cell lines, such as SEM, that express The authors declare no competing interests.


Repeat testing after adherence to a gluten-free diet for 1 year showed reduced titers for TG6 antibodies in 11/13 (85%), whereby 7 patients were now TG6 antibody-negative. Four patients seroconverted and tested positive for TG6 antibodies at one year, due to the ongoing exposure to gluten. We report another patient who presented with neurological manifestations  leading to the diagnosis of CD, who was intermittently adhering to a gluten-free diet. Serological testing at baseline showed him to be positive for antibodies to all 3 transglutaminases. Eleven years later, he developed DH. He also subsequently developed ataxia and peripheral neuropathy. Although TG3 and TG6 autoantibodies are linked to certain disease manifestations, TG2, TG3, and TG6 autoantibodies can be present across the spectrum of GRD patients and might develop years before onset of symptoms of extraintestinal manifestations. This is consistent with gluten-dependent adaptive immunity being a necessary but not sufficient pretext to organ-specific damage. TG6 antibodies appear to develop more frequently in patients where tolerance to gluten was broken but, either there was no development of the molecular state driving the tissue destruction at the level of the gut, or perhaps more likely, there was more resistance to developing this phenotype.Dermatitis herpetiformis (DH) is an extraintestinal manifestation of gluten sensitivity, in which an autoimmune response is directed against transglutaminase 3 (TG3), an epidermal transglutaminase. TG2 is the autoantigen in celiac disease (CD), defined by the presence of enteropathy, and TG6 is the autoantigen in neurological manifestations of gluten sensitivity. The interplay between B cell responses to these 3 transglutaminases in developing the clinical spectrum of disease manifestations is not completely understood. Also, the individual or combined diagnostic and predictive value of the respective autoantibodies is not fully explored. We examined the prevalence of TG6 antibodies in a cohort of patients with DH. TG6 positivity was found in 13/33 (39%), with IgA detected in 11 patients, IgG in 3, and both in 1. This was significantly higher compared to what is seen in the classic CD cases (14%) in a Finnish population. TG6 positive baseline samples constituted 60% of DH patients with no enteropathy ( Gluten-related disorders (GRD) are a group of immune-mediated diseases with diverse manifestations, triggered by the ingestion of gluten . EnteropThe identification of TG2 as the autoantigen in CD was an important step in our understanding of the pathophysiology of CD . AssessmThe potential interplay between B cell responses to these 3 transglutaminases in disease manifestation and individual or combined diagnostic and predictive utility of respective autoantibodies was not fully explored. Here, we examine the prevalence of TG6 antibodies in a well-characterized cohort of patients with DH, and we discuss the implications of the findings for interpretation of patient serology in the wider context of GRD. We also discuss an interesting clinical case that highlights the potential predictive value for these antibodies.Serology samples from 33 DH patients diagnosed with granular IgA deposits on skin immunofluorescence biopsy were collected at diagnosis, and then 6 months and one year after a gluten free-diet (GFD) at the Tampere University Hospital; 31 patients adhered to the GFD. All patients provided informed consent and the project was approved by the local ethics committee . The project adhered to the ethical principles for medical research, according to the Declaration of Helsinki. Small bowel biopsy was taken at diagnosis and was graded histologically as subtotal or partial villous atrophy with crypt hyperplasia and increased intraepithelial lymphocytes (24 patients), or normal mucosa (9 patients). Sera of a control group consisting of 27 patients with atopic dermatitis and 9 patients with psoriasis were also analyzed.TM 2 HB). All steps to reveal antibody binding were performed according to the published procedure. All serum samples were analyzed in duplicate, on wells containing antigen or only BSA, included on the same plate. The BSA-only background was subtracted from the values for antigen, and the units were calculated from a series of standards run in parallel, whereby a measurement >75 U/mL for IgA or >34 U/mL for IgG was considered to be positive. Standards were calibrated to be consistent with those used in commercial assay produced by Zedira (2nd generation assay). Results are given as the mean of two independent determinations.Determination of anti-TG6 IgA and IgG was done using our in-house ELISA assays. The methodology was described in detail elsewhere ,10. FullThe clinical characteristics of the cohort of patients with DH were already published . In brieIn the current study, TG6 antibody positivity was found in 13/33 (39%) of DH patients . Eleven n = 28, partial n = 3) showed a response in 11/13 (85%) patients (Repeat testing after adherence to a gluten-free diet (GFD) for 6 months and 1 year , six were dead and 25 were followed up to 2011 (mean 21 years), through questionnaire and hospital records. One death from Alzheimer disease was recorded but no other neurological conditions were found in any patient. Autoimmune diseases occurred in four, three had thyroid disease, and one had type 1 diabetes mellitus.A 41-years old man presented to neurology in 1997, with intractable headaches for the previous 2 years. He described them as severe, often unilateral and throbbing, lasting for several days and often associated with focal but transient neurological deficits . He also complained of memory difficulties and inability to concentrate. Brain MRI was done to rule out any ischemic cerebrovascular events. This showed white matter changes not typical of stroke or inflammation but of undetermined clinical significance . He had Serum from this patient was stored at the time of the diagnosis of gluten encephalopathy (1998), and was subsequently available for testing for TG2, TG3, and TG6 autoantibodies, when these serological tests became available ,13. The The significance of the serological presence of TG2, TG3, and TG6 antibodies amongst different populations of patients with gluten-related disorders needs clarification. Here, we report a high prevalence of circulating anti-TG6 autoantibodies in DH patients (39%), which was an unexpected finding. It supports the notion that GRD patients, independent of clinical presentation may produce any of these TG isozyme-specific antibodies, or indeed any combination thereof. However, we previously showed that the prevalence of circulating TG6 antibodies in CD patients presenting with ataxia in the UK was much higher than in classic CD patients (73% vs. 40%) . Using tThe TG6 antibody prevalence in these 3 groups  is analogous to what was observed in patients with DH. Circulating TG3 antibodies  were found in up to 87% of patients with DH but in only 24% and 11% of adult and pediatric classic CD patients, respectively . It is nSignificant advances in understanding of the events leading to autoantibody development were made over the last decade. DH patients were recently shown to have TG3-antibody secreting plasma cells at the level of the gut. Importantly, a gluten challenge of such patients revealed that gluten exposure drove rapid expansion of the TG3-specific plasma cell population in the lamina propria, and their frequency correlated with the serum titer of the corresponding autoantibodies . FurtherHow the development of adaptive immunity leads to extraintestinal manifestations remains a matter of debate, although some evidence that the autoantibodies themselves play a role in this has been put forth . PerivasOf interest is the potential significance of these antibodies when present in patients who do not seem, clinically at least, to have any obvious corresponding extraintestinal dysfunction. Indeed, our recent work demonstrated that the subgroup of patients with classical CD who are anti-TG6 antibody positive (40% of UK patients) already show a significant reduction in the volume of specific brain regions, when compared to those who are TG6 antibody negative . This fiMolecular etiology might also explain the observation that extraintestinal manifestations appear to be a late phenomenon. For example, patients with gluten ataxia and CD presenting with ataxia have a mean age at presentation of 53 years, as opposed to the mean age of 43 years, observed in patients presenting with the classic CD symptoms to gastroenterologists . The preThere were limitations to this study. Ideally, serological testing for all different GRD markers should take place without intermittent sample storage. Longitudinal analysis, as carried out here, is always a compromise between analysis of samples at the point of collection or at a later point when all linked samples for a cohort are available for simultaneous analysis; the latter approach was taken here. In our hands, there is no indication for sample storage (even long-term) affecting measurements of TG6 autoantibodies, as long as the samples were kept in sealed tubes for storage and repeated freeze–thawing cycles were avoided. Such sera are not easy to collect particularly from patients with DH who are becoming increasingly rare. The case described above is a single example of what we observed, and ideally, it should be followed up in a large cohort of patients who do not adhere to a GFD. However, such cases are not common and most patients with CD are likely to adhere to a GFD. Nevertheless, we believe that individual case reports can provide important insights.We demonstrated here for the first time that TG6 antibodies are prevalent in patients with DH, and occur at a much higher frequency than what is seen in patients with classical CD. It appears therefore that although linked to certain disease manifestations, TG2, TG3 and TG6 autoantibodies can be present across the spectrum of GRD patients and may develop years before the onset of symptoms of extraintestinal manifestations. Nevertheless, our findings suggest that these antibodies could have a predictive diagnostic value for the future development of specific extraintestinal manifestations. Such autoantibody positivity at diagnosis might further inform the decision to adopt a GFD, particularly for those patients that appear to be asymptomatic but are at risk of developing neurological dysfunction in the longer term.