Infectious bronchitis virus (IBV) is a gammacoronavirus that causes a highly contagious disease in chickens and seriously endangers the poultry industry. A diversity of serotypes and genotypes of IBV have been identified worldwide, and the currently available vaccines do not cross-protect. In the present study, an efficient reverse genetics technology based on Beaudette-p65 has been used to construct a recombinant IBV, rIBV-Beaudette-KC(S1), by replacing the nucleotides 21,704–22,411 with the corresponding sequence from an isolate of QX-like genotype KC strain. Continuous passage of this recombinant virus in chicken embryos resulted in the accumulation of two point mutations (G21556C and C22077T) in the S1 region. Further studies showed that the T248S (G21556C) substitution may be essential for the adaptation of the recombinant virus to cell culture. Immunization of chicks with the recombinant IBV elicited strong antibody responses and showed high cross-protection against challenges with virulent M41 and a QX-like genotype IBV. This study reveals the potential of developing rIBV-Beau-KC(S1) as a cell-based vaccine with a broad protective immunity against two different genotypes of IBV. Coronaviruses are important pathogens of humans and animals and cause mainly enteric or respiratory diseases with clinical manifestations ranging from mild, self-limiting to severe and even life-threatening symptoms . Vero cell-adapted IBV Beaudette strain (Beaudette-p65), obtained by adapting the egg-adapted Beaudette strain (ATCC VR-22) to Vero cells as previously described  supplemented with 10% fetal bovine serum (FBS) in the presence of penicillin (100 units/mL) and streptomycin (100 g/ mL) in a 5 % COOne-day-old SPF chickens were purchased from Xinxing Dahuanong Poultry Egg Co., Ltd.  for the immune efficiency assay. The study did not involve endangered or protected species and was approved by the Animal Experiments Committee of Zhaoqing Dahuanong Biopharmaceutical Co., Ltd.According to the manufacturer's protocol, total RNA was extracted from 200 μL of allantoic fluid of IBV KC strain using the Viral RNA Isolation Kit , and was reverse transcribed into cDNA using FastKing RT SuperMix kit . The S1 region of the KC strain was amplified with forward primer PR-S1F and reverse primer PR-S1R , and cloCoronavirus N gene transcript plays an accessory role to promote the recovery of the rescued virus  according to the manufacturer's instructions with certain modifications. Briefly, 10 μL of the transcription reaction mixture with 2 μL of template, 5 μL of 2 × CAP/NTP, 1 μL of 10 × buffer, and 1 μL of T7 RNApol enzyme was incubated at 37°C for 30 min, and then 1 μL of GTP was added and further incubated at 37°C for 150 min. The transcripts were extracted with phenol/chloroform.The full-length cDNA and N transcripts were generated The electroporation procedure was performed as previously reported with certain modifications  in culture cells. In brief, 1 mL of allantoic fluid was inoculated into Vero cells in T25 plates with 9 mL of FBS-free DMEM. Vero cells were harvested and lysed by three freeze–thaw cycles at 72 h post-inoculation. Subsequently, 1 mL of virus stock was again inoculated into Vero cells in T25 plates with 9 mL of FBS-free DMEM for 72 h and repeated up to nine passages. CPE was observed in each passage. Besides, RT-PCR, Western blot, and immunofluorescence assay (IFA) were also performed to confirm the cultivability of rIBV-Beau-KC(S1).Immunofluorescence assay was carried out as previously reported with certain modifications  and its parental strain Beaudette-p65 in cells , the two viruses infected the cells in six-well plates. At 12 or 24 h post-infection, IFA was used to detect if the cells were infected. Meanwhile, the infected cells were harvested at 0, 12, 24, 36, and 48 h post-infection (hpi) for real-time qPCR (RT-qPCR) assay. In brief, total RNA from the infected cells was extracted using the Viral RNA Isolation Kit  according to the manufacturer's instructions. Reverse transcription was conducted at 42°C for 15 min using the FastKing-RT SuperMix Kit , and 9 μL of 10-fold diluted cDNA was analyzed by Taqman RT-qPCR to quantitatively detect the viral load. All qPCR reactions were carried out in triplicate, and the IBV copies were calculated based on a standard curve of a positive plasmid. Specific primers and probes of RT-qPCR are listed in 50 and TCID50 values of rIBV-Beau-KC(S1), 10-fold serial dilutions of each viral stock were inoculated into 9-day-old SPF embryonated chicken embryos (ECEs) or Vero cells. For each dilution, 0.2 mL of the virus suspension was injected into each egg or cells in 96-well plates. Five eggs or plates were used for each dilution. The EID50 and TCID50 values were calculated by the method of Reed and Muench (To determine the EIDd Muench .n = 15/group) were prime-immunized with a dose of 105 EID50 rIBV-Beau-KC(S1) live vaccine by nasal-ocular route, and were boost-immunized intramuscularly with a dose of 105 TCID50 rIBV-Beau-KC(S1)-inactivated vaccine at 14 days. The same dose of Beaudette-p65 and PBS was used as control.One-day-old SPF chickens  of IBV M41 strain at 84 dpi. The challenged chickens were then observed daily for clinical symptoms, such as tracheal rales, wheezing, nasal discharge, or death for 7 days. The surviving chickens in each group were euthanized at 7 days post-challenge, and necropsies were performed immediately after postmortem. Kidney tissues and tracheal tissues from the two challenged groups were collected and fixed in 10% neutral buffered formalin for further histopathological analysis, respectively.At 35 days post-immunization (dpi), six chickens were randomly taken from each group to three isolators under negative pressure, and challenged with ~10Six serum samples were randomly taken from each group of chickens at 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, and 84 dpi. Total serum immunoglobulin G (IgG) specific for IBV was measured by indirect enzyme-linked immunosorbent assay (ELISA)  with certain modifications. Serum samples were 50-fold diluted and then detected by ELISA, and a positive sample was defined as absorbance of sample/positive control > 0.2. The positive serum samples that were 50-fold diluted were further 2-fold serially diluted, and endpoint titers were defined as the highest reciprocal serum dilution that yielded an absorbance >0.2.5.47 EID50 and 106.01 EID50, respectively. The mixture was incubated at room temperature for 1 h, and each mixture was inoculated into the group of five 9-day-old SPF ECEs. Endpoints were calculated by the Kärber or the Reed and Muench methods, and the results were expressed as a neutralization index (NI) that represented the log10 difference in the titers of the virus/negative serum mixtures and that of the virus/antiserum mixtures. NI ≥ 1.5 indicated a positive neutralization activity of the immunized serum.The serum neutralization antibody against IBV was determined by the alpha method with certain modifications  according to the manufacturer's instructions.Six anticoagulant peripheral blood samples were randomly taken from each group of chickens at 14 dpi and 28 dpi for flow cytometric analysis to detect the levels of CD4P-values .The data were analyzed using the GraphPad software package, and a two-way analysis of variance (ANOVA) was used to analyze significant differences between the indicated samples and the respective control samples. Significance levels are presented by the in vitro transcription to generate the full-length and N transcripts  was constructed by replacing the S1 region from nucleotides 21,704-22,411 of Beaudette-p65 with the corresponding region from an isolate of QX-like genotype KC strain . The S1 nscripts .in vitro transcribed full-length and N transcripts and cultured up to 96 h post-electroporation. As no obvious CPE was observed, cells were harvested and lysed by three freeze–thaw cycles. Total lysates were subsequently inoculated into the allantoic cavity of 9-day-old embryonated eggs for continuous blind passages to p10 (Recovery of rIBV-Beau-KC(S1) from the cloned cDNA was performed according to a protocol that was successfully used for the recovery of a HiBiT-tagged recombinant IBV  were found in p8 and p10, respectively. It was noted that double peaks of G and C were detected in the G21556C position in p6, p8, and p10, suggesting the presence of both quasispecies in these passages .As the initially rescued rIBV-Beau-KC(S1) appears to be unable to grow in culture cells, it would be interesting to see if this recombinant virus could be adapted to cell culture, and if so, the amino acid residue(s) responsible would be determined. For this purpose, the continuous passage of p4 and p10 in the Vero cells was carried out initially by inoculating 1 mL of the corresponding allantoic fluid into the Vero cells cultured in T25 plates with 9 mL of FBS-free DMEM for 72 h, and harvested as virus stocks by three freeze–thaw cycles. Subsequently, 1 mL each of these virus stocks was used to infect Vero cells in T25 plates, and the process was repeated up to nine passages. The initial virus stocks harvested from the infected Vero cells were labeled as rIBV-Beau-KC(S1)-p4V1 and rIBV-Beau-KC(S1)-p10V1, and the virus stocks harvested from the last passage were labeled as rIBV-Beau-KC(S1)-p4V9 and rIBV-Beau-KC(S1)-p10V9, and so on .At 36 h post-infection, a typical CPE of the Vero cell-adapted IBV, that is, the formation of giant syncytium cells, was observed in cells infected with rIBV-Beau-KC(S1)-p10V6, which carries G21556C and C22077T mutations, but no CPE was observed in the cells infected with rIBV-Beau-KC(S1)-p4V9 carrying the C22077T mutation only . Both RTThe initial occurrence of the G21556C point mutation in rIBV-Beau-KC(S1) was investigated by cloning RT-PCR products covering the replacement region from rIBV-Beau-KC(S1)-p4, rIBV-Beau-KC(S1)-p10, and rIBV-Beau-KC(S1)-p10V9, respectively, to pMD-19T, and 24-32 single colonies from each plate were picked for sequencing. The results showed that all p4 clones had a G at position 21,556 (G21556), and all p10V9 clones contained a C at the same position (C21556). However, mixed clones were found in the 30 clones from p10, with a G/C ratio of 4:30 . ImmunofThe growth properties of this recombinant virus in culture cells were first studied by infecting Vero, H1299, HeLa, and DF1 cells with Beaudette-p65 and rIBV-Beau-KC(S1)-p10V9, respectively. The infected cells were stained with anti-IBV N monoclonal antibody at 12 or 24 hpi, confirming that both Beaudette-p65 and rIBV-Beau-KC(S1)-p10V9 can efficiently replicate in all the four cell lines .50 and TCID50 values of rIBV-Beau-KC(S1)-p10V9 in the chicken embryos were determined. As shown in 50 and TCID50 values of rIBV-Beau-KC(S1)-p10V9 reached 106.41 and 105.95, respectively, with no statistically significant difference from the maximum EID50 (106.63) and TCID50 (105.99) values of Beaudette-p65 (The EIDette-p65 .The growth curves of Beaudette-p65 and rIBV-Beau-KC(S1)-p10V9 in Vero, H1299, HeLa, and DF1 cells were then determined by infecting the four cell lines with Beaudette-p65 and rIBV-Beau-KC(S1)-p10V9, respectively, at an indicated MOI. The two viruses showed similar growth characteristics, reaching peaks at 24 hpi in Vero, HeLa, and DF1 cells, and at 12 hpi in H1299 cells .The influence of G21556C point mutation on each step of the rIBV-Beau-KC(S1) replication cycle was then studied. The binding activity of the virus to Vero cells was assessed by adding Beaudette-p65, rIBV-Beau-KC(S1)-p4, and rIBV-Beau-KC(S1)-p10V9 to Vero cells and incubating at 4°C for 2 h. After removal of the unbound viruses by washing the cells with PBS for three times, the RNA copy numbers of the bound viruses were determined by RT-qPCR. As shown in To assess their entry efficiency, the unbound viruses were removed by digestion with 0.25% trypsin. The cells were then incubated at 37°C for 2 h after thorough washing, and the viral RNA copy numbers were determined. The results showed that the RNA copy number of rIBV-Beau-KC(S1)-p10V9 was similar to that of Beaudette-p65, but rIBV-Beau-KC(S1)-p4 can hardly be detected . FurtherTo evaluate the immunogenicity of rIBV-Beau-KC(S1), 1-day-old SPF chickens were immunized with rIBV-Beau-KC(S1) and rIBV-siMutBeau, respectively, and exsanguinated, according to the process listed in The determination of CD4+ and CD8+ cells by flow cytometric analysis and indirect ELISA showed that very similar levels of both cell types were stimulated in chicks immunized with rIBV-Beau-KC(S1), rIBV-siBeau vaccine, and control groups . ConsistThe protective efficacy of rIBV-Beau-KC(S1) against challenges with virulent strains of QX-like genotype (CK/CH/JS/TAHY) and Mass genotype (M41) was evaluated by infection of five immunized chickens in each group at 35 and 84 dpi, respectively .5.5 EID50 of CK/CH/JS/TAHY, chickens in the rIBV-Beau-KC(S1)-immunized group showed no obvious clinical symptoms, whereas two (33.33%) in the control group and one (16.67%) in the rIBV-siMutBeau-immunized group showed mild symptoms, including poor spirit and fluffy feathers. At 7 days post-challenge, three chickens in each experimental group were randomly euthanized and necropsied, showing no obvious macroscopic lesions in kidneys  of M41, chickens in the rIBV-Beau-KC(S1)-immunized group had no clinical symptoms, but all chickens (100%) in the control group had obvious tracheal rales within 5 days post-challenge, and two chickens (22.22%) in the rIBV-siMutBeau-immunized group also had slight tracheal rales at 2 days post-challenge  glycoprotein is a homo-trimer and the main immunogenic protein that induces neutralizing antibodies  largely determines the host and tissue range of a coronavirus. Specific protein receptors for many alpha- and betacoronaviruses have been identified  gene derived from either H120, M41, 4/91, or QX, recombinant IBVs were constructed by chimerizing the S genes (or S1 region) and rescued  containing a chimeric S1. Continuous passage of rIBV-Beau-KC(S1) in chicken embryos resulted in the accumulation of two point mutations, in which the S248T (G21556C) substitution was confirmed to play a crucial role in determining the cell adaptability of IBV. Immunization of chicks with rIBV-Beau-KC(S1) elicited higher antibody responses than did rIBV-siMutBeau and showed better cross-protection against challenges with M41 and CK/CH/JS/TAHY (QX-like genotype), revealing the potential of developing rIBV-Beau-KC(S1) as a cell-based vaccine with broad-spectrum protection against two different genotypes of IBV.The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by the Animal Experiments Committee of Zhaoqing Dahuanong Biopharmaceutical Co., Ltd.RC and DL designed and organized this study. XT did all the experimental work. CX, RC, DL, and XT analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was partially supported by the Provincial Scientific Research Institutions Key Areas R&D Plans (grant number 2021B0707010009), the National Natural Science Foundation of China , and the Zhaoqing Xijiang Innovative Team Foundation of China (grant number P20211154-0202).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Conversely, incorporating slower Ca2+ removal or a fractional increase in type I fibre area emulated expected changes; the former was required to simulate slowing of the twitch measured experimentally. Slower Ca2+ removal more than compensated for force loss arising from a large reduction in Ca2+ sensitivity or moderate reduction in Ca2+ release, producing realistic age-related shifts in the force-frequency relationship. Consistent with empirical data, reductions in free Ca2+ concentration and Ca2+ sensitivity reduced maximum tetanic force only slightly, even when acting in concert, suggesting a modest contribution to lower specific force. Lower tendon stiffness and slower intrinsic shortening speed slowed and prolonged force development in a compliance-dependent manner without affecting force decay. This work demonstrates the advantages of muscle modelling for exploring sources of variation and identifying mechanisms underpinning the altered contractile properties of aged muscle.Age-related alterations of skeletal muscle are numerous and present inconsistently, and the effect of their interaction on contractile performance can be nonintuitive. Hill-type muscle models predict muscle force according to well-characterised contractile phenomena. Coupled with simple, yet reasonably realistic activation dynamics, such models consist of parameters that are meaningfully linked to fundamental aspects of muscle excitation and contraction. We aimed to illustrate the utility of a muscle model for elucidating relevant mechanisms and predicting changes in output by simulating the individual and combined effects on isometric force of several known ageing-related adaptations. Simulating literature-informed reductions in free Ca Statistical significance was set at P < 0.05 and effect sizes were calculated as eta squared (η2).Plantar flexion twitch torque was measured in 10 young  and 18 older  healthy human adult males. An analysis of the experimental data obtained from young adults and a detailed description of the experimental protocol used for both young and older adults have been published previously  are released transiently at a constant rate in response to each stimulus and subsequently removed at a rate dependent on the Ca2+ concentration  and τ1 and τ2 are the time constants for the rise and fall of Ca2+, respectively , isi is the interstimulus interval and r is the time constant for the recovery of relative Ca2+ release  are indices of cooperative activation and the Ca2+ sensitivity of force, respectively , giving a normalised stiffness [kSEE (Lichtwark and Wilson LSEE) and L0 were set at 300 —relates closely to the fixed-end compliance, which represents CE strain against the stretch of the SEE during a maximum tetanic contraction , consistent with experimental observations for the human plantar flexors  measured for human medial gastrocnemius fascicles in vivo  of lower free Ca2+ availability on the time course  and amplitude of the twitch, submaximal force and the force-frequency relationship, and maximum force.To our knowledge, there exists only one study of the effect of age on SR Ca2+ uptake may be lower in advanced age, but it is not a universal observation. The effect of age on SR Ca2+ uptake rate has been predominantly studied in rodent muscle and using a variety of muscle preparations. To our knowledge, only a single study has been performed on human muscle, specifically, crude homogenates form the vastus lateralis muscle  without detecting a significant age effect . Accordingly, we incorporated a reduction in pCa50 of 0.05 or 0.10 pCa units and described the qualitative and quantitative effect on the time course and amplitude of a twitch, submaximal force and the force-frequency relationship, maximum force, and relative force summation.Lower CanH—is unaltered in advanced age  to be lower for type II fibres —represented by the Hill coefficient, 2+ concentrations is practically insensitive to shifts in Ca2+ sensitivity. A deficit in maximum force generation may only arise when Ca2+ concentration and Ca2+ sensitivity decrease concomitantly. Accordingly, we reduced the instantaneous Ca2+ concentration by 30 or 50% whilst lowering pCa50 by 0.05 or 0.10 pCa units, consistent with our previous manipulations of Ca2+ concentration and Ca2+ sensitivity. We described the qualitative effect of concurrent reductions in Ca2+ concentration and Ca2+ sensitivity on submaximal force and the force-frequency relationship, and quantified the effect on maximal force.Because the force-pCa relationship is sigmoidal in form, force at near-maximal Ca2+ transients whether impaired Ca2+ release and slowed Ca2+ uptake occur in parallel and the extent to which they may offset one another  in concert. Reductions of equal amount would not alter steady-state Ca2+ in the model relative to the control condition. We described the qualitative effect of lower Ca2+ release and slower Ca2+ uptake on submaximal force and the force-frequency relationship.It is unclear from recordings of intracellular Ca2+ uptake will increase free Ca2+ concentration, thereby increasing force, whereas lower Ca2+ sensitivity will act to decrease force, we examined the effect of these two adaptations acting concomitantly. Specifically, we incorporated both a 30% reduction in the rate constant for Ca2+ removal (43% increase in τ2) and a 0.1 pCa unit reduction in Ca2+ sensitivity (pCa50) and described the qualitative effect on the force-frequency relationship.On the basis that slower CaVmax  does not appear to be altered in aged fibres that do not exhibit a reduction in Vmax . We described the qualitative effect on the time course and amplitude of a twitch and brief tetanic contraction. We did not incorporate reductions in a/P0 because the model was unable to simulate the twitch measured experimentally with a more realistic value for muscle at physiological temperatures [model: 0.10; mouse soleus: 0.18  0.15 pCa units greater and nH 40% lower  parameter—analogous to muscle PCSA—to allow twitch force to be lower in advanced age, which we observed experimentally. We assumed that the deficit in twitch force is owing, at least in part, to muscle atrophy . Substantial force loss was also evident during sustained stimulation at submaximal frequencies, as shown by the marked rightward shift of the force-frequency relationship  and prolonged the contraction time and half-relaxation time by 25% and 38%, respectively . Lowering Ca2+ sensitivity by 0.10 pCa units resulted in additional force attenuation and an even briefer contraction. Reduced Ca2+ sensitivity also decreased force during stimulation at submaximal stimulation frequencies (< 50 Hz) such that the force-frequency relationship was shifted rightward  expressed relative to twitch force —was higher following the reduction in Ca2+ sensitivity, though there remained a deficit in tetanic force.Reducing the CanH, or cooperativity, from 3 to 2 to reduce the slope of the force-pCa relationship increased twitch force by 11% and prolonged the twitch contraction time and half-relaxation time by 26 and 44%, respectively . Lowering cooperativity also reduced the slope of the force-frequency relationship such that force was slightly greater at low frequencies but considerably lower at moderate and high frequencies of stimulation . Despite twitch force being 20% (0.04 P0) greater for a nH of 2 compared to a nH of 4, peak force during the brief tetanic contraction  was 12% (0.06 P0) lower for the former compared to the latter  still exceeded 92% during 200 Hz stimulation  or 0.10 pCa (Act = 82%) units , reducing Vmax by 30% reduced twitch force by 12% and increased twitch contraction time by 13%; twitch half-relaxation time was practically unaltered . A similar effect was observed for a brief tetanic contraction  following a 30% reduction in Vmax . Increasing a/P0 produced qualitatively similar results for twitch and tetanic contractions as increasing Vmax .Reducing SEE stiffness by 30% prolonged twitch contraction time by 13% and attenuated twitch force by 9%. Conversely, increasing SEE stiffness by 50% abbreviated twitch rise time by 14% and increased twitch force by 9% Fig. e. The atble Fig. f, inset.Vmax by 50%, from 6 to 3 L0·s−1, had a more modest effect on peak twitch force when SEE stiffness was adjusted to give normalised SEE deformations of less than 8% , twitch force was 13% (0.05 P0) lower  by 26% (0.06 P0) when normalised SEE deformation was 30% [i.e., 3.33 P0·L0−1 . The slowing effect increased to 37 and 33% when type I fibre area was increased to 0.80. Both adjustments of type I fibre fractional area increased force at submaximal stimulation frequencies  to exceed that of the reduction in twitch force (18%) despite additional force loss from reductions in Vmax and SEE stiffness . The plantar flexor twitch of older men was of a lower amplitude (18%) and exhibited a prolonged contraction time 25%) and prolonged half-relaxation time (29%) compared to young adult men . Our findings are supported by experimental observations from studies of dantrolene exposure  during maximum tetanic stimulation was greatly overestimated in the model. This assertion would imply that maximum tetanic stimulation does not induce saturating CaP0 can require a near two-fold increase in stimulation frequency and free Ca2+ concentration  arose when free Ca2+ and force from intact single fibres , suggesting that force rise during a twitch of a single fibre may not be appreciably affected by a slower Vmax. The current work supports the involvement of slower intrinsic shortening speed and higher SEE compliance as factors contributing to prolonged and slower force rise in advanced age, although consideration may need to be given to the muscle preparation. Elevated tendon stiffness, conversely, would likely act to offset factors causing force development to be slowed or twitch force to be attenuated.In addition to activation dynamics, force development is regulated by the intrinsic speed of shortening and the stiffness of the SEE being acted upon by the CE Hill . IncorpoP0·L0−1) and 53% (5 vs. 2.33 P0·L0−1) reductions in normalised SEE stiffness reduced twitch force by 16 and 29%, respectively. Cat soleus twitch force was reduced by 35–40% , Vmax (20%), and SEE stiffness (6%). Multiple solutions were possible, and it is likely that the slowing of twitch speed could have been emulated with parameter adjustments more consistent with an increase in type I fibre content. An increase in Ca2+ sensitivity and reduction in cooperativity would likely lessen the required reduction in Ca2+ uptake rate. We speculate because we imposed parameter limits that were consistent with the directionality of impairment reported in the literature.The age-related prolonging of force rise and decay during a twitch observed for the plantar flexors in this study was within the range of slowing reported by others for the same muscle group  without greatly influencing maximum tetanic force, even when acting in concert (< 10%). These predictions are at odds with experimental observations of the effect of age on indices of isometric contraction speed , and suggest that reduced Ca2+ sensitivity and impaired SR Ca2+ release may contribute only modestly to the reduction in specific force in advanced age (depending on Ca2+ saturation during tetanic stimulation). Conversely, simulations that incorporated slowed Ca2+ removal or a greater fractional area of type I fibres prolonged the rise and decay of twitch force and shifted the force-frequency relationship leftward. These predictions are consistent with the characteristic fast-to-slow shift in contractile performance associated with ageing. Slowed and prolonged force development, and lower twitch force, also resulted from imposing a slower intrinsic shortening speed and lower SEE stiffness but occurred without a concomitant slowing of force decay or elevation of submaximal force. As such, these properties alone did not produce the characteristic slowing of contraction speed. The effect of Vmax depended on SEE stiffness, and empirical observations do not always support a pronounced delay in peak force from added compliance, possibly because of additional factors related to active shortening not captured in the model. Simulating the slower, weaker twitch observed experimentally for the plantar flexors of older men required significant slowing of Ca2+ uptake (~ 25%) and could be coupled with an appreciable reduction in force-generating capacity . Slowed Ca2+ removal, when acting in concert, negated the depressive effects of moderate and large reductions in Ca2+ release and Ca2+ sensitivity, respectively.Age-related reductions in Ca2+ uptake and a greater fractional area of type I fibres seem to be suitable mechanisms for explaining the slower isometric contraction speed exhibited by aged muscle. Simulations incorporating these adaptations with a degree of impairment reported in the literature generated realistic age-related changes in contractile behaviour. Ageing-appropriate adjustments to these parameters also emulated the age-related slowing of twitch speed observed experimentally. In general, the model simulations were well-supported by empirical observations. We propose that this model or similar models might be effective in determining a meaningful impairment threshold or identifying the factors contributing to altered contractile properties.Whole muscle contractile performance in advanced age is characterised by slowed isometric contraction speed. This work provides support for the involvement of multiple mechanisms, although these adaptations do not necessarily affect the same aspects of contraction speed. As such, identifying the most important adaptations should be aided by characterising an array of isometric contractile properties in advanced age. Both slower CaAdaptation of muscle function and structure in advanced age is inconsistent and wide-ranging, thus, careful consideration should be given to the strength of evidence implicating the presence of a particular adaptation . With the multifaced nature of impairment in mind, we adjusted multiple model parameters concurrently and have illustrated the importance of considering interaction effects. Inconsistent reports regarding the effect of age on contractile performance may relate to variation in the disruption of function and structure. We believe this work underscores the utility of simple, yet physiologically-grounded and parameter rich Hill-type muscle models for studying conditions that involve a multitude of adaptations . Such models also hold great value in being able to predict functional performance  when used in musculoskeletal simulations (e.g. Song and Geyer


Second, we obtained the relationship between the number of events and the foci size experimentally measured by the γ-H2AX focus formation assay. Third, using this relationship, we evaluated the degree of DSB complexity induced after photon irradiation for various X-ray spectra using the foci size, and the experimental DSB complexity was compared to the results estimated by the well-verified DNA damage estimation model in the PHITS code. The number of events in a water cube was found to be proportional to foci size, suggesting that the number of events intrinsically related to DSB complexity at the DNA scale. The developed method was applicable to focus data measured for various X-ray spectral situations . This method would contribute to a precise understanding of the early biological impacts of photon irradiation by means of the γ-H2AX focus formation assay.Complex DNA double-strand break (DSB), which is defined as a DSB coupled with additional strand breaks within 10 bp in this study, induced after ionizing radiation or X-rays, is recognized as fatal damage which can induce cell death with a certain probability. In general, a DSB site inside the nucleus of live cells can be experimentally detected using the γ-H2AX focus formation assay. DSB complexity is believed to be detected by analyzing the focus size using such an assay. However, the relationship between focus size and DSB complexity remains uncertain. In this study, using Monte Carlo (MC) track-structure simulation codes, i.e., an in-house WLTrack code and a Particle and Heavy Ion Transport code System (PHITS), we developed an analytical method for qualifying the DSB complexity induced by photon irradiation from the microscopic image of γ-H2AX foci. First, assuming that events  potentially induce DNA strand breaks, we scored the number of events in a water cube (5.03 × 5.03 × 5.03 nm When the human body is exposed to photon beams, such as X-rays and γ-rays, high-energy photoelectrons are generated after interactions with human tissues (mainly composed of liquid water) . Such phWhen quantifying DSB yields in vitro, immunofluorescence staining for phosphorylated histone H2AX (γ-H2AX)  enables etsmode) [To clarify this relationship, we focused on a Monte Carlo (MC) track-structure simulation, which is traditionally used as an effective tool for investigating the mechanisms of DNA damage induction . To dateetsmode) ,28 and aetsmode)  as we coetsmode) ,18,29,30In this study, using electron track structure codes (PHITS and WLTrack), we developed an analytical method for quantifying the yield of a complex DSB coupled with additional strand breaks see  from theWe detected DSB sites in the cell nucleus 30 min after X-ray irradiation using the γ-H2AX focus formation assay. The X-ray energy spectrum used in this study was categorized as diagnostic X-rays  and therapeutic X-rays . First, to evaluate the spatial patterns of foci generation in the cell nucleus, we measured the distance between two foci sites in the cases of the diagnostic kVp and therapeutic MV X-rays. Note that we scored the foci distance within a circle with a 5 μm diameter in the cell nucleus see . As showWe then calculated the theoretical distribution of the distance between the two points generated randomly in the circle , which iThe development of a super-resolution localization microscope  enabled Based on the results shown in Electrons and photons are categorized as low linear energy transfer (LET) radiation. In the case of high-LET radiation (such as heavy ions), Nakajima et al. and Antonelli et al. showed that the γ-H2AX foci area caused by inducing closely localized individual DSBs instead of clustered DNA damage was larger than that for low-LET radiation ,17, wher3, which is equivalent to that of the sampling site in a previous study [As shown in us study .3) shows good agreement with the distribution of focus size. In the preliminary test, we calculated the number of events per cube for various cube sizes. Among the various sizes, the nm size of 5.03 × 5.03 × 5.03 nm3, which is the same volume as that in the sampling site used in the previous study by Garty et al. [−2 in the experimental distribution. Note that this conversion coefficient of horizontal axis from foci size to the number of events was determined to match both distributions by the least-square method, and there is a correlation between these distributions (R2 = 0.982). The standard deviation calculated by the error propagation was 0.268. From the good agreement of the distributions . From this result, the developed method was found to be applicable to focus data measured for various X-ray spectral situations .Using the conversion coefficient determined above, we converted the threshold value for DSB complexity based on the cluster size to that based on the foci area. This threshold value was determined to reproduce the fraction of complex DSBs for 35 kVp X-ray calculated using the PHITS code. Based on this threshold, we estimated the fraction of isolated (simple) or complex DSBs using a γ-H2AX focus formation assay for various X-ray spectra. The DSB complexities evaluated by these methods were compared to the results estimated using the DNA damage estimation model in the PHITS code. The relative yields of simple and complex DSBs for various X-ray spectra are shown in From the simulation standpoint, the possibility to induce a DSB from a linkage ) is 0.00124 (DSB per linage). Considering this, DSBs can be induced at the track end of secondary electrons, and there are very few cases in which two or more DSBs occur simultaneously along an electron track. These facts suggest that the large foci reflect not piled up some foci composed of isolated DSBs see A, but a However, the yields of total DSBs for 35–150 kVp X-rays were higher than those for 6 MV-linac X-rays, indicating that diagnostic X-rays exhibit higher biological impacts than therapeutic X-rays. This tendency agrees well with what has been reported in previous studies ,13. ConsIn this study, we focused on photon irradiation (as well as electron irradiation) and developed an analytical method for quantifying the DSB complexity using the γ-H2AX foci area. When discussing complex DNA lesions, detecting co-localized DSBs and non-DSBs  , it is n2 incubator. The cells were seeded onto ϕ12-mm glass-based dishes .We used a mammalian cell line, Chinese hamster lung fibroblast V79-379A, obtained from JCRB Cell Bank, Osaka, Japan (IFO50082). V79-379A cells were maintained in Minimum Essential Medium Eagle  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin  at 37 °C in a humidified 95% air and a 5% CO2, and the cell culture dishes were filled with the cell culture medium. An absorbed dose of 1.0 Gy was delivered to the cells for all types of X-rays. Each experiment was performed at room temperature.We used various types of X-ray spectra: 35, 40, 50, 60, 60, 80, 100, 120, and 150 kVp with 1.6 mm Be and 1.0 mm Al filtration  and 6 MV-linac . The mean X-ray energy for each X-ray spectra is also summarized in Thirty minutes after irradiation, cells were fixed with 4% paraformaldehyde for 10 min. After rinsing with phosphate-buffered saline (PBS), the cells were permeabilized in ice-cold 0.2% Triton X-100 in PBS for 5 min and blocked with a solution of 1% bovine serum albumin (BSA) in PBS for 30 min. A primary antibody, γ-H2AX  diluted at 1:400 with 1% BSA in PBS, was then fed and stored overnight at 4 °C. After rinsing thrice with 1% BSA in PBS, Alexa Fluor 594-conjugated goat anti-rabbit , diluted at 1:250 with 1% BSA in PBS, was added and kept for 2 h. After rinsing thrice with 1% BSA in PBS, the cells were incubated with 1 μg/mL DAPI  for 15 min. After rinsing once with methanol, the γ-H2AX foci were observed using a High Standard all-in-one fluorescent microscope .First, we measured the distance between two γ-H2AX foci within a 5.0 μm diameter sphere in the cell nucleus. Second, we measured the area of the γ-H2AX foci. Third, we counted the number of γ-H2AX foci per cell nucleus to evaluate the dependency of X-ray energy on the relative biological effectiveness. All measurements were performed using ImageJ ,37.The experimental distances between two γ-H2AX foci were compared to the theoretical distance between two points generated randomly in the circle as follows :(1)fx,r=jx randomly using the input data f (Equation (1)). Second, we calculated the minimum distance between two foci which were piled up by each other xmin by the foci area randomly extracted from the experimental data. In this calculation, the foci were assumed to be circular, and the distance data were subtracted when the distance of the foci was shorter than the minimum distance.The theoretical distribution was corrected by subtracting the frequency of the distances whose foci were piled up with the adjacent focus . First, etsmode [etsmode has been well validated in previous studies [To estimate the yield of DSBs and the content of the complex form induced by photon irradiation, we used two Monte Carlo simulation codes: an in-house code for electron WLTrack  and the etsmode ,28 in PH studies ,18 for s3), having a volume equivalent to that of the sampling cylinder used in a previous study [To evaluate the relationship between the foci area and inelastic event-cluster size, the local density of ionization and excitation was estimated based on the WLTrack code . Assuminus study , were raetsmode was measured [Nlink was scored. As previously reported [Nlink per energy deposition of an electron track passing in liquid water Edep is proportional to the DSB induction, we calculated the yield of DSBs (YDSB) as follows: kDSB is the proportionality constant (keV/Gy/Da). This DNA damage estimation model was in good agreement with experimental data and other simulations from a previous study [Ncl) within a sampling site with a 10 bp radius , namely, 2 ≤ Ncl < 14 for simple DSB, 14 ≤ Ncl < 26 for DSB+, and 26 ≤ Ncl < 38 for DSB++. Note that DSB+ is the DSB coupled with an SB within 10 bp and DSB++ is the DSB coupled with two SBs within 10 bp [The DSB complexity estimated using the γ-H2AX focus formation assay and WLTrack was compared with that calculated using PHITS. The yield of the DSBs was estimated using the analytical code in PHITS version 3.27 . In thismeasured ,18. To ereported ,18, assuus study . Additioin 10 bp .For the X-ray source data, each X-ray spectrum calculated using the present formula was used as the input . Then, tetsmode. We estimated the fraction of simple or complex DSBs for irradiations with 35–150 kVp X-rays and 6 MV X-rays. The yield of DSBs was calculated with a large number of electrons to make the uncertainties less than a few percent in general.The photons were transported by the EGS mode  in PHITSIn this study, we developed an analytical method for quantifying the DSB complexity induced by photon irradiation from microscopic images of γ-H2AX foci. Assuming that ionizations and excitations can induce DNA strand breaks (SBs), we scored the number of events in a water cube and the pair of events within a 10 bp separation along the electron track in order to estimate the yields of DSBs and the complexity. We then obtained the relationship between the events per cube (at a DSB site) and the focus area detected using a γ-H2AX focus formation assay. Using this relationship, we successfully reproduced the yields and fractions of complex DSBs induced by photon irradiation for various X-ray spectra. The developed method was found to be applicable to the foci data measured for various X-ray spectral situations.To obtain the relationship between the foci area and the number of events at a DSB site, we used track-structure codes, i.e., in-house WLTrack codes for electrons. Results of the γ-H2AX focus formation assay  showed that the DSB complexity was independent of the X-ray energy. However, the number of DSBs per nucleus was different between diagnostic kVp X-rays and therapeutic 6 MV X-rays. These results indicate the total DSB yield  to evaluate the biological impacts of photon irradiation. The present technique of evaluating DNA damage complexity would be beneficial for easily quantifying complex DSBs, but is limited only to photon and electron irradiation. In the case of low-LET irradiation, it is sufficient to analyze the spatial pattern of physical events . When ev


The present narrative review provides a comprehensive update of the current knowledge on urticaria, both in adult and pediatric populations, and on the safety and efficacy of fexofenadine hydrochloride (HCl) as a treatment option.A literature search was conducted on Embase and Medline.Clinical studies published in English and published between 1999 and 2020 were selected.1-antihistamines are the preferred choice of treatment across several guidelines worldwide. Herein, the efficacy and safety of fexofenadine HCl, a representative second-generation H1-antihistamine approved for the treatment of urticaria, is discussed. The occurrence of urticaria manifestations in COVID-19 patients is also briefly presented.Although the exact pathogenesis of urticaria is not fully understood, multiple pathways of mast cell activation are discussed to explain the existence of phenotypically different clinical manifestations of urticaria. An overview of the worldwide prevalence of chronic urticaria, including disease burden and patient’s quality of life is provided. The impact of urticaria on patient’s life differs on the basis of whether its form is acute or chronic, but pharmacological approaches are most often needed to control the disabling symptoms. A summary of the current management of urticaria recommended by different guidelines across countries  is presented. Non-sedating, second-generation HThe burden of acute and chronic urticaria is high for patients. Second generation anti-histamines such as fexofenadine HCl can help managing the symptoms. Urticaria and angioedema are frequent conditions among the general population requiring medical consultation, diagnostic testing, and implementation of preventive and pharmacological approaches to control the disabling symptoms suffered by the patient . AlthougThe present article provides an updated review of the current knowledge on urticaria with a focus on the efficacy and safety of fexofenadine hydrochloride (HCl). A literature search was conducted on Embase and Medline. Eligible articles included information on urticaria prevalence, classification, pathogenesis, etiology, disease burden, impact on patient’s quality of life (QoL), diagnostic approach and practical management, and clinical studies on the safety and/or efficacy of fexofenadine HCl. The search was limited to articles published in English between 1999 and 2020 and a total of 180 publications were reviewed. Following the outbreak of the coronavirus disease 2019 (COVID-19) and the occurrence of urticaria manifestations in COVID-19 patients, available studies on this matter are also briefly discussed.Urticaria is a common disease worldwide that can occur at any age. An estimated 20% of adults have experienced an episode of acute urticaria at least once in their life , with upUrticaria is defined as the occurrence of wheals (hives), angioedema, or both. Wheals are erythematous and superficial edema of the skin associated with severe itching (pruritus) or burning sensation which can be generalized over the entire body or localized to specific areas . Wheals Urticaria and angioedema should not be confused with other skin manifestations such as auto-inflammatory diseases, urticarial vasculitis, bradykinin-mediated adverse events, drug reactions, anaphylaxis, urticaria pigmentosa, ectoparasitosis, contact dermatitis, and autoimmune bullous diseases .The classification of urticaria, valid for both adult and pediatric populations, is based on the duration and cause of symptoms. There are several subtypes of urticaria, a summary of which and corresponding etiology is presented in Table Acute urticaria, while more prevalent in females, is less common in adults and is predominately considered a childhood affliction . Acute uChronic urticaria is defined by recurrent wheals, with or without angioedema, that persist for six weeks or longer . The ideSpontaneous urticaria accounts for approximately 50–75% of chronic urticaria cases , and is Chronic inducible urticaria accounts for approximately 20–35% of cases and is commonly induced by cold, heat, dermographism, pressure, vibration, sunlight or water . RegardlAlthough the exact pathogenesis of urticaria is still poorly understood, investigators agree that urticaria is a mast cell-driven disease resulting from the dysregulation of mast cells and basophils, followed by the release of inflammatory mediators and the stimulation of signaling pathways responsible for the formation of wheals and angioedema , 10. TheFor example, all mast cells express high-affinity IgE receptors (FcεRI) and are therefore involved in IgE-dependent allergic reactions. When an allergen binds to IgE causing receptor FcεRI cross-linking, the interaction result in mast cell degranulation  . Mast ceADGRE2 gene, has been identified as the basis of autosomal dominant vibratory urticaria. This substitution results in gain of function in ADGRE2 which consequently sensitizes mast cells and results in IgE-independent, vibration-induced degranulation [Furthermore, antibodies targeting the eosinophilic receptor CD23 may induce the release of major basic protein (MBP) and eosinophil cationic proteins (ECP), which also activate mast cells . Besidesnulation .Fig. 2MoRegardless of the initial factor stimulating mast cell activation, three pathways are consecutively activated. Histamine is released within minutes from mast cell degranulation, together with other preformed inflammatory mediators such as tumor necrosis factor (TNF)-α, IL-1, IL-4, IL-6, and IL-8 [The role of infection as a causative factor in multiple forms of urticaria is well documented, thus is not surprising that in the last year reports of different dermatologic manifestations associated with COVID-19 have progressively grown . A heterThe disease burden is measured by disability-adjusted life years (DALYs). The 2016 updates from the Global Burden of Disease Study revealed that the global weighted average of DALY rate adjusted for the differences in age distribution among the population (age-standardized DALY) for urticaria is 55.5/100,000 in the general population, 47.4/100,000 in men and 62.5/100,000 in women . Among sThe economic burden of chronic urticaria is also considerable. Mean yearly direct and indirect costs of chronic urticaria in the United States of North America have been estimated to be $244 million, with medication costs accounting for 62.5%, and work absenteeism for 15.7% of the total cost . The chrThe analysis by age group from the Global Burden of Disease Study showed that children aged 1–9 years old have the highest burden of disease among the pediatric population . EpidemiCurrent guidelines for urticaria management recommend a diagnostic work-up that focuses on the examination of clinical signs and assessment of symptoms associated with urticaria . Since uThe ultimate aim of urticaria treatment is complete symptom control . Althoug1-antihistamines are the cornerstone of first-line treatment [Second-generation non-sedating H1-antihistamines varies depending on local regulation and approved medicines. The European Academy of Allergology and Clinical Immunology, the Global Allergy and Asthma European Network, the European Dermatology Forum and the World Allergy Organization (EAACI/GA2LEN/ EDF/WAO) 2018 guidelines recommend the use of omalizumab, a monoclonal anti-IgE antibody, for the treatment of unresponsive chronic urticaria [2LEN/ EDF/WAO 2018 guidelines due to lack of evidence [Patients unresponsive to antihistamines should be referred to a clinical specialist with expertise in the evaluation and management of urticaria and/or angioedema. The treatment of patients unresponsive to second-generation Hrticaria . Ciclosp1-antihistamines alone has been proven to be inadequate to stop mast cell histamine degranulation in COVID-19 patients, but it can reduce the severity of urticaria in some patients [The classical recommended approach for the management of urticaria with first-line Hpatients , 59. Ant1R–H4R) are important mediators of the immune response and allergic inflammation; the H1-receptor drives cellular migration, nociception, and vasodilatation [1-antihistamines act as inverse agonists by stabilizing the H1-receptor in its inactive conformation, therefore preventing normal functioning [1-antihistamines down-regulate allergic inflammation via direct or indirect down-regulation of antigen presentation, expression of pro-inflammatory cytokines and cell adhesion molecules, and chemotaxis of inflammatory effector cells [1-antihistamines are categorized as first- and second-generation antihistamines. First generation H1-antihistamines are less selective than second-generation as interaction with other types of receptors has been extensively reported . Because of this limited receptor selectivity, adverse effects such as paradoxical excitation, irritability, hyperactivity and hallucinations, constipation, dry mouth, urinary retention, and tachycardia have been reported [1-antihistamines can cross the blood–brain barrier and act on H1-receptors in the central nervous system which can interfere with histaminergic neurotransmission, thereby causing drowsiness, sedation, somnolence, fatigue, and headache [1-antihistamines have been associated with impairment of cognitive function, memory, and psychomotor performance. Global guidelines strongly recommend against the use of first generation H1-antihistamines due to their adverse effects; their use is only recommended in countries where second-generation H1-antihistamines are not available (mainly South-East Asian countries) or when maximum doses of second-generation antihistamines are ineffective. However, when the use of first-generation antihistamines is necessary, possible benefits and risks should be thoroughly discussed with the patient before making any decision [1-antihistamines were developed to be devoid of such adverse effects, some of them are selective inverse agonists for H1-receptor which do not cross the blood brain barrier, resulting in minimal or non-sedating effect [1-antihistamines include acrivastine, bilastine, cetirizine, desloratadine, fexofenadine HCl, levocetirizine, loratadine, and mizolastine, although several of these agents are not available in all countries [1-antihistamines. It is beyond the scope of this review to discuss all second-generation antihistamines, and fexofenadine HCl is presented as representative of the effectiveness of this class of medicines for the treatment of urticaria. Fexofenadine is a non-sedating second generation commercialized for 25 years in more than 100 countries around the world [Histamine and its four receptors  twice a day is available for children older than 6 months .1–receptors is inhibition of the histamine-induced wheal and flare via skin tests (histamine test). This technique provides objective data about the onset, potency and duration of action of antihistamines, although its predictive value on drug effectiveness needs to be confirmed [The most common method used to assess blockade of histamine Honfirmed .1-antihistamines most used in the local clinical practice  [1-antihistamines with slightly different methodologies confirmed similar results regarding the efficacy of fexofenadine HCl in the suppression of the induced wheal [When directly compared with placebo, fexofenadine HCl 60, 120, 180 or 240 mg suppressed histamine-induced wheal and flare reactions. A recent meta-analysis performed by Huang et al., found that antihistamine effect of fexofenadine HCl was significantly higher than that of placebo , and non-inferior to other second-generation antihistamines  based on the analysis of five studies on healthy subjects . In a reatadine) . Two houatadine) . Fexofenatadine) . Other sed wheal , 68.Well-designed clinical trials have shown the efficacy of fexofenadine in patients suffering from urticaria. In a multicenter, double-blind study conducted by Paul et al., the reduction of mean daily total symptom score (TSS) of pruritus and wheals was found to be dosage-dependent and statistically significant compared with placebo for fexofenadine HCl 180 mg (p = 0.0041) and 240 mg once-a-day (p = 0.0008), but not for fexofenadine HCl 60 or 120 mg once-a-day . HoweverUp-dosing of antihistamines up to fourfold the licensed dose is recommended by the major clinical guidelines for the treatment of urticaria if control is not achieved, although the indication is based on the expert’s opinion. A recent systematic review assessing the results of 14 studies, of which six were placebo-controlled randomized trials, found that increasing doses of fexofenadine HCl  resulted in better control of urticaria symptoms with minor treatment-related adverse events (headache) , 74. TabOverall, fexofenadine HCl is well-tolerated and discontinuation due to adverse effects generally occurs in less than 5% of patients .In adults, second-generation antihistamines are not considered to be cardiotoxic  . In heal1-antihistamines have been investigated for safety: cetirizine, levocetirizine, loratadine, fexofenadine HCl, desloratadine, and rupatadine [In children, only a few Hpatadine , 87. A spatadine .Fexofenadine HCl is considered well tolerated in adults and children, and safe to take if necessary during pregnancy or while lactating, however it is recommended to follow the fexofenadine prescribing information , 64. TabThe burden of acute and chronic urticaria is high, in terms of patient quality of life and disability-adjusted life years. The aim of treatment for urticaria is that of full control of the associated wheals and angioedema, and second generation anti-histamines such as fexofenadine HCl have been shown to significantly reduce these symptoms whilst being well-tolerated. Recently, urticaria has been reported as a manifestation of COVID-19, however further analyses are needed to fully establish the link.


Measurable residual disease (MRD) is emerging as an important prognostic and predictive biomarker in acute myeloid leukemia (AML). However, its use is currently hampered by the disparity and lack of harmonization between the available MRD methodologies. In addition, the current assessment of MRD in AML focuses only on the quantification of the residual leukemic burden, without addressing the parallel alterations of the antineoplastic immune response that can critically affect the course and outcome of AML, often despite MRD persistence. Incorporating parameters of immune competence provides more consistency with the biological concept of MRD and may lead to higher accuracy. Multiparameter flow cytometry (MFC) is a highly efficacious and sensitive technology for the thorough and synchronous investigation of the kinetics of both antitumor immunity and the leukemic clone. MFC-based MRD provides the platform for the development of a composite leukemia- and immune-based biomarker which can outcompete the current MRD assessment.Acute myeloid leukemia (AML) is a heterogeneous group of clonal myeloid disorders characterized by intrinsic molecular variability. Pretreatment cytogenetic and mutational profiles only partially inform prognosis in AML, whereas relapse is driven by residual leukemic clones and mere morphological evaluation is insensitive for relapse prediction. Measurable residual disease (MRD), an independent post-diagnostic prognosticator, has recently been introduced by the European Leukemia Net as a new outcome definition. However, MRD techniques are not yet standardized, thus precluding its use as a surrogate endpoint for survival in clinical trials and MRD-guided strategies in real-life clinical practice. AML resistance and relapse involve a complex interplay between clonal and immune cells, which facilitates the evasion of the leukemic clone and which is not taken into account when merely quantifying the residual leukemia. Multiparameter flow cytometry (MFC) offers the possibility of capturing an overall picture of the above interactions at the single cell level and can simultaneously assess the competence of anticancer immune response and the levels of residual clonal cells. In this review, we focus on the current status of MFC-based MRD in diverse AML treatment settings and introduce a novel perspective of combined immune and leukemia cell profiling for MRD assessment in AML. AML par−4 to one in 10−6 of total leukocytes compared to one in 20 in standard morphology [Complete remission (CR) achievement is a necessity for the cure and prolonged survival of AML patients. However, as morphological remission has long been known to be insensitive for relapse prediction and approximately 50% patients who achieve CR eventually relapse, the notion of “measurable residual disease” negativity (MRD-negative CR) has recently been introduced by the ELN as a new outcome definition ,8. MRD irphology . The addrphology ,10,11; hThe investigation and use of cell surface antigens to differentiate normal hematopoietic cells from leukemic blasts started decades ago. Baker et al., in 1974, introduced a rabbit-derived antiserum that was non-reactive with normal cells but highly reactive with leukemic blasts from patients with ALL and AML, presenting evidence that antigenic differences existed between leukemic and non-leukemic cells . Greaves−3 to 10−5 depending on the method, the number of cells analyzed, the design of the panel, the instrument settings, the number and type of antibodies used, and a clinically validated cut-off point for MRD positivity [The main advantages of MFC-MRD in AML, namely the high applicability >90%), short turnaround time, relatively high sensitivity, and the possibility to discriminate between living and dead cells ,12,18,190%, shortsitivity ,8,11. ThThe pediatric protocol AML02 investigated the use of targeted chemotherapy and alloSCT, and a better clinical outcome was reported compared to previous studies. In this study, risk categories were based on the genetic/cytogenetic profile of the patients and MRD findings. FCM-MRD was applied after the first cycle of chemotherapy and treatment was intensified by gemtuzumabozogamicin in patients with high levels of MRD . In the Regarding adult patients, in the study by Liu et al. , MFC-MRDThe QUAZAR AML-001 Maintenance Trial is the first large prospective, double-blind, placebo-controlled randomized trial with long-term longitudinal assessment of MRD in patients with AML in remission. In this trial, administration of the oral formulation of azacytidine (CC-486) as maintenance was associated with significantly longer overall and relapse-free survival than placebo among older patients with AML who were in remission after chemotherapy. In both treatment arms, MRD-positive status (≥0.1%) after induction ± consolidation was associated with significantly shorter OS and RFS compared to MRD-negative status .The prognostic value of MRD assessment after intensive chemotherapy and allogeneic hematopoietic stem cell transplantation (SCT) has been widely studied. Lower intensity therapy (LIT) has been introduced for the treatment of older or so-called “unfit/frail” patients and, apart from the improved tolerability, it has resulted in longer overall survival compared to conventional care regimens The MFC-MRD-based prediction of prognosis in older patients with AML who are not eligible to receive intensive chemotherapy was investigated in many clinical trials . Maiti eThe combination of venetoclax plus hypomethylating agents seems to be an effective treatment regimen leading to improvements in complete remission rates and overall survival. In a single center phase II study in patients with newly diagnosed (ND) AML older than 60 years of age, secondary AML (sAML), and relapsed or refractory (R/R) AML, the administration of azacytidine with oral venetoclax was highly efficacious and safe ,41. The The PETHEMA-FLUGAZA phase III trial in elderly AML patients reported that MFC-MRD status was the only independent predictor of relapse-free survival in 72 patients who achieved CR either by the combination of low-dose cytarabine plus fludarabine or azacytidine at standard doses. However, even patients with MRD negativity exhibited substantial genetic abnormalities in CD34+ progenitors, indicative of the inability of the current MRD threshold to detect the existence of very low levels of clonal cells that can drive relapse .In a meta-analysis of large-scale AML studies reported by Short et al., 2020, patients with MRD negativity had improved rates of overall survival  and disease-free survival  at 5 years versus patients with MRD positivity . PatientIn the GIMEMA adult AML1310 trial , patientThe National Cancer Research Institute’s AML17 trial is currently the largest study to investigate the key elements impacting MRD in AML. A total of 1874 adults < 60 years of age with AML were enrolled and treated with standard daunorubicin plus cytarabine based induction treatment followed by risk-adapted chemotherapy consolidation, with or without alloSCT. Patients with MRD positivity (defined as ≥0.1% by the MFC assay) after cycle 1 had similar 5-year overall survival as patients who only achieved a partial response , emphasizing the poor outcomes associated with persistent MRD . Other sOf note, the combination of MFC-MRD with mutational analysis using NGS or digital PCR increased the sensitivity for the diagnosis significantly . The firAs MRD is becoming an invaluable outcome predictor for AML, the need for standardization and improvement of sensitivity and specificity of the current methods is imperative. Crucial limitations still exist, as reflected by the fact that the false-negative rates of MFC-MRD in AML vary from 13–30% and that up to 70% of patients with low/negative MRD, based on the 0.1% cut-off, will finally relapse ,47,48.Beyond the technological and methodological limitations of MRD evaluation, there are other issues pertaining to the biological concept of MRD. Merely quantitative measurement of the residual leukemic cells, although established in several clinical settings ,49, has An even more important issue is the fact that the mere measurement of residual leukemic burden does not take into account tumor–immune system interactions. Oncogenesis is not merely a cell-intrinsic process but involves complex interactions of the malignant clone with neighboring non-clonal cells . The patThe pivotal role of crosstalk between AML and immune cells in disease relapse after an initial remission is long known, but three recent studies provide intriguing data that arouse further concerns about the validity of conventional MFC-MRD. By analyzing primary AML samples, Christofer et al. demonstrated that relapse after allogeneic hematopoietic stem cell transplantation  is accompanied by a theoretically reversible epigenetic dysregulation of immune pathways, which may be important in immune evasion of the leukemic clone . Almost + and CD8+ effector subsets expanded further post-treatment [+ T cells of high risk MDS patients is strongly associated with response and outcome to azacytidine, potentially representing immune-mediated antileukemic activity of the latter [+CD4–CD8– double-negative T cells [Though both targeted and conventional therapies can induce immune cell priming by several mechanisms, including immunogenic cell death and eradication of the immunosuppressive tumor milieu, the increasing use of low-intensity therapies in younger patients poses another challenge. In a phase II study combining azacytidine and nivolumab in relapsed/refractory (R/R) AML, the pretherapeutic size of the bone marrow T cell pool predicted response to treatment, whereas two CD4reatment . In addie latter . Similar T cells . Collect T cells .Two studies of multiple myeloma reported that the concomitant measurement of immune cell subsets by flow cytometry considerably improves the prognostic power of MRD in diverse disease settings ,69. Howe+ AML patients bearing the NPM1 mutation [Despite its unequivocal value in treatment guidance, MRD is not yet used routinely and has not been incorporated in the treatment algorithms relating to AML. Establishing MRD negativity as a surrogate endpoint for survival will accelerate drug selection and approval, whereas in everyday practice this will spare patients unnecessary and potentially harmful treatment approaches. However, the current methods of MRD assessment are solely focused on measuring the size of the residual leukemic clones without integrating the intrinsic genetic variability of the clonal cells and/or any metric of immune competence, thus overlooking the parallel alterations of the immune milieu . This famutation  potentiaFlow cytometry remains the key tool for the comprehensive characterization of both myeloid and immune cells and its versatility is ideal for the synchronous study of these two pivotal players in the pathobiology of AML relapse. As flow cytometric technologies are rapidly advancing, and the combined analysis of immunophenotypic, spatial, and morphological traits is becoming a reality, the parallel study  of the k


Extramedullary presentation of acute myeloid leukemia is a rare event that can occur at diagnosis or at relapse, together or independent of bone marrow involvement. This event is usually unamenable to standard therapies. Due to the rarity of this disorder, most of the literature comprises small retrospective studies and case reports. The introduction in clinical practice of newly approved drugs for acute myeloid leukemia will pave the way for new treatment approaches for this rare disease. This report will present a review of acute myeloid leukemia with extramedullary manifestations responding to FLT3-directed target therapy.FLT3, as well known, represent the most common genomic alteration in acute myeloid leukemia (AML), identified in approximately one-third of newly diagnosed adult patients. In recent years, this has represented an important therapeutic target. Drugs such as midostaurin, gilteritinib, and sorafenib, either alone in association with conventional chemotherapy, play a pivotal role in AML therapy with the mutated FLT3 gene. A current challenge lies in treating forms of AML with extramedullary localization. Here, we describe the general features of myeloid sarcoma and the ability of a targeted drug, i.e., gilteritinib, approved for relapsed or refractory disease, to induce remission of these extramedullary leukemic localizations in AML patients with FLT3 mutation, analyzing how in the literature, there is an important development of cases describing this promising potential for care.FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase family member. Mutations in  FLT3 and its inhibitory drugs) promise to revolutionize the therapeutic workup of this patient setting.Acute myeloid leukemias (AML) are still an open challenge today. As known, these are diseases involving the bone marrow, with the presence of immature cellular elements. However, these neoplastic cells are capable of homing in tissues other than bone marrow through mechanisms not yet fully explained. Although a more appropriate name for this manifestation would be “extramedullary acute myeloid leukemia tumor”, the condition is commonly known as myeloid sarcoma (MS). This term is strictly considered by World Health Organization for the cases with no concurrent bone marrow (BM) involvement. A more appropriate name for contemporary manifestation in the BM and another site should be referred to as “extramedullary acute myeloid leukemia tumor”. This is a reality that every hematologist must deal with. In a retrospective analysis of 346 AML patients, extramedullary involvement had an incidence of 11% and was found to significantly complicate prognosis and therapeutic strategies [The cancer cells of acute myeloid leukemia can sometimes aggregate in extramedullary sites. This extramedullary tumor, known as myeloid sarcoma , is defined by the World Health Organization as a tumor mass consisting of myeloid blasts with or without maturation occurring at an anatomic site other than BM. MS  is composed of cancer cells in various stages of maturation: myeloblasts, monoblasts, and, rarely, megakaryocytic or erythroid precursors . The patIn contrast to this hypothesis, however, a large-scale study showed that the occurrence of CD56-positive leukemic blasts was similar in patients with and without e-AML or MS ,8. e-AML18Fluorodeoxy-glucose positron emission tomography/CT (FDG-PET/CT) is the gold standard imaging modality to evaluate the presence of e-AML and monitor the response to therapy over time [Diagnosis starts with the patient’s medical history and clinical suspicion, detailed with instrumental examinations. Among them, computed tomography (CT) is the first imaging modality. CT scans display mild to marked homogeneous post-contrast enhancement. In addition, magnetic resonance imaging (MRI) provides images of the central nervous system (CNS). ver time ,12.For this reason, FDG-PET/CT is among the mandatory exams of the NCCN for the evaluation of extramedullary localizations of AML. However, a biopsy of the lesion is essential to establish a diagnosis with certainty. A condition of severe thrombocytopenia  can make biopsy difficult for many suspected organs, particularly those involving the CNS. In such situations, the regression of the lesion following therapy for AML establishes diagnosis with reasonable certainty. The evaluation of the histological section typically reveals infiltrating myeloid cells at various stages of maturation, with either monocytic or granulocytic maturation, in the same way as in AML. Diagnosis is confirmed using immunohistochemistry, an antibody panel commonly used in AML. Specifically, staining for myeloperoxidase (MPO-expressed in 66% to 96% of MS) is used to help differentiate lesions from lymphoma . The othOther techniques that can confirm or improve diagnostic capacity are immunohistochemistry, flow cytometry, and fluorescence in situ hybridization (FISH). A percentage of cases ranging from 25% to 47% are initially mislabeled as melanoma, thymoma, Ewing sarcoma, or malignant lymphoproliferative disorders  or poorly differentiated carcinoma ,16,17.e-AML could also be confused with a blastic plasmacytoid dendritic cell neoplasm (BPDCN), a rare and aggressive hematologic malignancy of the BM and blood that can affect organs such as lymph nodes, skin, spleen, and CNS. Skin lesions are characteristic of this disease; therefore, they must be distinguished from e-AML .NCCN guidelines recommend that patients affected by extramedullary disease should be considered for a screening lumbar puncture evaluation, followed by an empiric dose of intrathecal chemotherapy .-Extramedullary involvement with concurrent newly diagnosed AML;-Extramedullary relapse of AML, including in the post bone marrow transplant setting;-Blast phase/transformation of a myeloproliferative neoplasm or chronic myelomonocytic leukemia;-Isolated MS in association with a normal bone marrow biopsy and blood film, as well as the absence of any history of myeloid neoplasia.The European Society for Hematology (ESH) classifies MS into four subgroups : -ExtramExtramedullary leukemic involvement associated with concurrent AML or at relapse occurs more frequently in monoblastic leukemia with translocations involving 11q23, acute myelomonocytic leukemia with eosinophilia, inv(16) p13;q22) or t  ,21, and  or t. For localized forms of the disease, one therapeutic possibility to relieve symptoms may be represented by surgical decompression. MS appears to be sensitive to ionizing radiation, so involved-field radiotherapy (RT) should be considered for all patients with isolated MS refractory to systemic therapy [Several factors influence the choice of best therapy for e-AML. The choice depends on the involved organ, the synchronous presence of medullary involvement of the AML, and any previous lines of treatment for the underlying disease ; furthermore, as already discussed, the choice of therapy is contingent upon the presence of targetable abnormalities  decitabine, appear to have been able to induce complete remission after 2–4 cycles of therapy in few selected clinical cases [IDH2 or IDH1 mutations, occurring in 30% and 15% of cases, respectively [On the other hand, for patients not eligible for intensive care, hypomethylating agents, e.g., azacitidine (HMA) and deoxyribonucleic acid methyltransferase inhibitor  inhibitors, i.e., venetoclax. In this context, there is no solid scientific evidence yet. Sporadic case reports include e-AML patients treated with venetoclax both in the course of disease refractory to the first line of therapy and in the extramedullary localization of AML arising after HSCT ,32. FurtRegarding the immunotherapeutic approach, success is poor. A proportion of 10% of leukemic cells of the MS overexpress PD-1 ,35, and FLT-3) could play a pivotal role in the study and knowledge of human neoplastic pathologies. The FLT3 gene, belonging to the class III receptor tyrosine kinase family, encodes a human protein of 993 amino acids and is expressed as a receptor in the placenta, gonads, and brain. FLT3 receptor activation by its ligand (FL) leads to rapid receptor autophosphorylation and induces the activation of several intermediate signal-transduction mediators, determining cell proliferation and expansion [Since its discovery by two independent groups in 1991 ,41, the xpansion ,44,45,46FLT3 in developing multipotent stem and B cells.These data demonstrate an essential role for FLT3 has played a cardinal role in the study and management of AML since 1996, when Yokota et al., using reverse transcriptase-polymerase chain reaction (RT-PCR), found, in 5 AML patient samples (17%), a different transcript with a primer combination that could amplify the transmembrane (TM) domain through the juxtamembrane (JM) domain (FLT3-ITD mutation). These repeat sequences disrupted the JM domain’s autoinhibitory activity, resulting in constitutive tyrosine kinase activation [FLT3, a component of the tyrosine kinase domain (FLT3-TKD mutation) [Furthermore, tivation . Subsequutation) ,49,50.FLT3 mutations are found in approximately 30% of de novo diagnosed AML cases and are divided between ITDs (about 25%) and point mutations in the TKD (7–10%) [Larger-scale evaluations have shown that  (7–10%) .FLT3-TKD mutation, the prognostic impact is not yet well defined. Despite being based on small samples of patients, the literature data do not confirm a relevant correlation between the presence of this mutation and clinical outcome [FLT3-ITD. The higher frequency of incidence of this mutation has allowed us to develop reliable prognosis prospects and make the most appropriate therapeutic decisions. Risk stratification guidelines for AML have been drawn up by several organizations, including the World Health Organization (WHO), the National Comprehensive Cancer Network (NCCN), and the European Leukemia Network (ELN) [FLT3-ITD is a genetic alteration that identifies a well-defined subgroup of patients. The NCCN guidelines classify patients with these mutations as having poor prognoses [FLT3 mutation plays a prominent role, specifically as an adverse prognostic indicator of disease. The concept of allelic ratio (AR) represents the number of ITD-mutated alleles compared with the number of the wild-type alleles and therefore is not only influenced by the amount of leukemia versus normal cells in the tested sample but also by the percentages of cells with 0, 1, or 2 mutated alleles. Numerous studies have concluded that a high ratio (AR > 0.5) is associated with worse prognoses and patient outcomes. This concept made it possible to stratify patients into three risk classes  depending on the presence or absence of the nucleophosmin member 1 (NPM1) gene mutation and to favorably mediate the prognostically adverse effect of FLT3-ITD [FLT3-ITD gene mutation-positive AML, stratifying them, according to ELN indications, into high and low AR, depending on the presence or absence of the NPM1 mutation. They found that FLT3-ITDlow AR was not associated with favorable outcomes , 41.3%). Furthermore, only patients who underwent allogeneic stem cell transplantation  in the first complete remission (CR1) had a more favorable outcome than those who did not  p = 0.013; OS p = 0.003). It was also underlined by multivariate analysis that allo-HSCT in CR1 is the only prognostic factor capable of giving a better OS and progression-free survival (PFS), demonstrating that prognosis was unfavorable in NPM1-mutated AML with FLT3-ITDlow AR when allo-HSCT was not carried out in CR1 [FLT3-ITD) or the presence, at relapse, of an allelic burden higher than that at diagnosis, capable of negatively influencing the prognosis, unlike the diagnosis, where there is a greater probability of facing a polyclonal disease [FLT3-ITD mutations are newly detected at relapse more often than FLT3-TKD mutations  [Over the years, studies have highlighted the crucial role of these mutations in the context of newly diagnosed AML, as well as their ability to actively characterize the prognosis and, above all, their potential as a therapeutic target. For the  outcome . On the rk (ELN) ,52, specrognoses . The samFLT3-ITD . In contt in CR1 . Regardi disease ,58,59,60ctively) , with a ctively) . FLT3 D835 mutations were identified in the 20 cases examined, whereas FLT3-ITD mutations were identified in three of the 20 cases (15%). Contrary to what happens in FLT3-mutated AML, their data were too few to identify a reliable prognostic significance [FLT3-ITD gene would favor the infiltration of leukemic cells into the visceral organs, simultaneously reducing the BM homing of leukemic cells by deregulating CXCR4 signaling [In e-AML, the study of molecular markers is crucial for categorizing the disease and guiding clinicians to the best therapeutic choice for patients. Ali Ansari-Lari, M. et al. analyzed 24 e-AML specimens from 20 patients in a study protocol. Clinical information was available for 15 out of 20 patients. e-AML was diagnosed in the setting of AML in nine cases (60%), CML in three cases (20%), and precursor B (Pre-B) ALL in one patient (6.7%), with appearance ranging between 3 months and 21 years after a leukemia diagnosis. In three instances, the e-AML was diagnosed concurrently with leukemia . However, six patients had no leukemia bone marrow involvement (it should be emphasized that three cases did not have a concurrent bone marrow aspirate or biopsy). From a molecular point of view, no ificance . Mutatioignaling . In the ignaling .FLT3 mutations occur in a significant subgroup of patients with e-AML. However, as already discussed, if FLT3-ITD mutations appear to be associated with increased relapse risk, adverse disease-free survival, and overall survival, drawing a definitive prevalence is not possible due to the limited sample size of analyzed patients. This evidence explains how FLT3-ITD mutation plays a leading role because, in recent years, the so-called tyrosine kinase inhibitors (TKIs) have been developed to the point that they can bind to this therapeutic target and inhibit its action. Among these, midostaurin has been approved since 2017 in association with chemotherapy with cytarabine and daunorubicin (“7 + 3” scheme) for induction of remission in patients with newly diagnosed AML and in combination with high doses of cytarabine as consolidation therapy. A pivotal drug study (RATIFY) demonstrated improved event-free survival  and OS  compared with 7 + 3 chemotherapy alone in all FLT3 mutation subtypes , albeit with different results observed among the single analyzed groups [FLT3 gene mutation (ADMIRAL study). In this setting, 371 patients were randomly treated, receive gilteritinib or any other salvage chemotherapy . Patients randomized to the gilteritinib arm had significantly longer survival than those in the chemotherapy arm, with a median OS of 9.3 months versus 5.6 months, respectively. This was also confirmed by a higher rate of complete remission with complete (21.1% for gilteritinib arm versus 10.5% for chemotherapy arm) or partial (13% for gilteritinib arm versus 4.8% for chemotherapy arm) hematological recovery [As already mentioned, d groups . Other Irecovery . Concernrecovery .FLT3-ITD diagnostic assay can identify cases of patients suffering from AML with a poor prognosis and guide clinicians’ therapeutic choices, providing patients the chance to improve their survival thanks to the use of FLT3-ITD targeted therapies [For all these reasons, a rapid herapies ,53,68.FLT3-mutated AML with extramedullary involvement, gilteritinib is the inhibitor drug with the best response. In this work, we focused on collecting the few clinical cases reported in the literature demonstrating the therapeutic success of gilteritinib in MS  expression and GVHD onset. He was treated with donor lymphocyte infusion and immunosuppressive therapy. On day + 400, a right supraclavicular mass was observed simultaneously with the appearance of AML blasts in bone marrow (6.9%). Histological examination of the tumor lesion identified many cells with atypical nuclei and scant cytoplasm that were immunohistologically positive for CD68, MPO, and lysozyme; slightly positive for CD117; and negative for CD34 and terminal deoxynucleotidyl transferase. Extramedullary and medullary cells harbored FLT3-ITD mutation. Therapy with 120 mg/day of gilteritinib was abruptly begun, achieving a bone marrow and MS complete response to treatment, allowing the patient to undergo a second allo-HSCT, achieving a new complete molecular remission, maintained even 150 days after the cell infusion.In another case , a 38-yeNPM1 and FLT3-ITD-mutated therapy-related AML was treated by induction and consolidation therapy with CPX-351, obtaining a complete response with positive MRD. Nevertheless, he suddenly began to report burning leg pain and weakening of the right hand and leg muscles, associated with the absence of osteotendinous leg reflexes. A meningeal relapse of AML was confirmed upon examination of cerebrospinal fluid and magnetic resonance imaging, demonstrating two meningeal implants of myeloid sarcoma. The disease progressed despite a brief second response to treatment with medicated lumbar puncture and FLA-Ida chemotherapy. For this reason, it was treated with gilteritinib. After 3 months of therapy, although only a partial response to the bone marrow evaluation of the condition was obtained, the meningeal localization of the disease progressively decreased until it completely disappeared, demonstrating for the first time the potential biological effect of these drugs within the central nervous system.Another patient  with NPMFLT3-ITD mutated AML involving the skin and treated with induction therapy based on cytarabine and idarubicin is described [FLT3-ITD mutation, the patient began treatment with oral gilteritinib, with rapid regression of the tumor, complete disappearance of the iris component, and a significant reduction in the size of the ciliochoroidal tumor accompanied by an improvement in visual acuity. Despite the excellent response of the extramedullary part of the AML, the patient died due to the progression of the medullary AML.Localizations of e-AML in infrequent organs are possible. The case of a 60-year-old patient with escribed , with thNPM1-mutated and FLT3 wild-type AML and treated with “7 + 3” cytarabine/anthracycline induction and four cycles of high-dose cytarabine consolidation, obtaining complete remission (CR). After a first relapse of the disease, she was enrolled in a clinical trial and underwent HSCT. Three months post-transplant, she had disease relapse, with a new appearance of FLT3-ITD mutation at a low mutant-to-wild type allelic ratio of 0.27. A new CR was achieved with two cycles of sorafenib (a first-generation TKI inhibitor) and azacitidine. Sorafenib was continued on maintenance therapy. Sixteen months later, she developed a new vaginal mass and bilateral breast masses, with 18fluorodeoxy-glucose avidity on positron emission tomography/computed tomography (PET/CT). Biopsy of the lesion confirmed MS. Marrow evaluation was normal. NGS testing of MS revealed FLT3-ITD mutation. She began treatment with 120 mg of gilteritinib daily, and after 1 month of therapy, a repeat PET/CT showed a fourth CR, and the patient was able to undergo a second life-saving HSCT.Lastly, a 47-year-old female  was diagDespite the increase in cases of extramedullary localizations of leukemic cells found at various times during AML and described in the literature, there are still too many limitations and uncertainties to conclude with certainty. Specifically, there is a lack of knowledge comparing the various FLT3 inhibitors, where only gilteritinib manages to have more critical data to support its use, with its efficacy probably due to its mechanisms of action. Another explanation could lie in the potential of the drug to penetrate into the extramedullary environment, whereas in the context of the bone marrow, the medullary niches in these hematological malignancies could guarantee an environment of survival and escape of cancer cells from standard drugs. For these reasons, more data and evidence from real-life settings are needed. FLT3 mutations. However, as already seen, the shorter duration of responses and the poor results obtained at the time of relapse with salvage therapies have led to a significant reduction in overall survival for patients with FLT3-ITD mutations [FLT3 inhibitors has dramatically changed the therapeutic prospects and the possibility of survival of this subset of patients, both in association with traditional chemotherapy and in monotherapy [FLT3 clone with a higher AR than the diagnosis, when there is a greater probability of facing a polyclonal disease. For this reason, newly diagnosed FLT3-mutated AML might be less likely to respond clinically to highly selective FLT3 inhibition [FLT3-ITD627E mutation [Historically, the response to induction therapies in bone marrow or extramedullary forms of AML has been similar between patients with and without utations . Althougotherapy , the shootherapy . Intrinshibition . Secondlmutation ,77.FLT3-ITD [FLT3-ITD mutated AML cells. Gilteritinib could also exert its mechanism of action, inhibiting this target [The same primary intrinsic mechanism could involve the upregulation of the Bcl-2 protein, the expression levels of which were not reduced after dephosphorylation of FLT3 and its downstream target, STAT5, in patient samples with FLT3-ITD . Genomics target . On the s target ,81.FLT3-ITD mutation, which leads to an increase in chemotaxis by blocking the downregulation of Rho-associated kinase via the CXCL12–CXCR4 signaling axis [-1 protein (a member of PIM family of serine/threonine kinases) and the phosphorylation of CXCR4, enabling its expression [FLT3-ITD mutated AML, can induce TKD mutations, against which they are ineffective [FLT3-mutated AML in relapse. In this context, an explanation of the potential of gilteritinib to act in the extramedullary localizations of the disease can be hypothesized by evaluating the estimates of the volume of distribution of this drug, both central and peripheral. They were found to be 1092 L and 1100 L, respectively. This finding indicates a wide distribution of extraplasmic gilteritinib, which could mean widespread tissue diffusion.The same phenomenon happens with the secretion of fibroblast growth factor 2 (FGF2) . Extensiing axis , both thpression . The onsffective . Among tFor this reason, gilteritinib could have greater efficacy in MS compared to the ability of blast cell clearance in bone marrow, where the inflammatory survival niche allows for avoidance of the response. The last mechanism of the ineffectiveness of the drug could be related to its mode of transport. The binding of gilteritinib to plasma proteins in vivo is approximately 90% in humans, binding mainly to albumin. For this reason, albumin could have a reduced penetrance within bone marrow or, particularly in debilitated patients, albumin deficiency could be an interesting clinical indicator/predictor of drug response. Further studies are needed to confirm these hypotheses, especially in the field of MS, where the complexity of diagnosis, clinical characteristics of the patient, and the rarity of the disease still require more significant support.The gold-standard therapy for extramedullary acute myeloid leukemia and MS is still an open challenge, as there are no clear indications as to which treatment is suitable for each patient. Indeed, the advent of molecular biology studies and progress in the field of target therapy have opened up new landscapes. In this context, gilteritinib could represent a valid therapeutic option for patients with extramedullary involvement associated with FLT3-mutated AML at relapse, especially for its efficacy on the extramedullary component. New preclinical and clinical data continue to be rapidly generated, intending to guarantee the best successful targeted therapy for this uncommon type of hematological disease.


Editorial on the Research TopicThe complexity of urticariaGoméz et al. discuss the urgency in improving the diagnostic criteria, and the importance of identifying and managing properly the disease to reduce its burden.Urticaria is a common condition that presents with transient, pruritic wheals, angioedema or both. It often leads to a reduced quality of life and significant socioeconomic burden. Although the definition is clear, characterizing the lesions may be challenging for the health-care provider and an incorrect diagnosis may directly impact in treatment outcomes. Matos et al. propose two interesting diagnostic algorithms and a practical review of acute and chronic urticaria differential diagnosis. Not limited to skin lesions, the authors also review the main features and differences of histaminergic and bradykinin-mediated angioedema, highlighting the importance of an early diagnosis of hereditary angioedema.Distinguishing between wheals and urticarial lesions is the first step to provide a correct diagnosis. As important as the characterization of the lesions is the presence of systemic symptoms such as fever, malaise, and arthralgia. Ensina et al. provide a comprehensive review on main aspects, similarities and differences regarding definitions, mechanisms, causes, diagnosis and treatment of acute urticaria and anaphylaxis.Furthermore, according to the international EAACI/GA²LEN/EuroGuiDerm/APAAACI guideline the prevalence of acute urticaria in a lifetime is around 20% . The undKocatürk et al. review the reported effects of sex hormones and pregnancy-specific immunological changes on urticaria, the impact of pregnancy on urticaria, and current information and guidance on the management of urticaria during pregnancy and lactation.Chronic urticaria is not only more common but is also more severe in females, and although it is a relatively common disease, there is not enough information about the effects of hormonal conditions on the disease. Publications from the PREG-CU project showed that urticaria gets better during pregnancy in half of women while one third of the patients experience symptoms' worsening and 10% reported visits to emergency departments due to urticaria exacerbations . Almost Papapostolou et al. overview current data on comorbidities of CU, and furthermore comment on the potential linked pathways underlying these diseases. In the era of tailored made intervention, CU patients should be recognized and treated as a multimorbid group with treatment interventions targeting the comorbidities and the urticaria management per se until the complete unravelling of the underlying pathophysiology of chronic urticaria.The prevalent fear of patients suffering from chronic urticaria and a major concern of treating physicians is the possible co-existence of other severe diseases, especially malignancies and systemic autoimmune disorders. Accordingly, extensive laboratory testing is being performed in many cases in contrary to current evidence and international guidelines . AutoimmAltrichter et al. reported a marked average diagnostic delay of almost 3 years. Diagnostic provocations and/or laboratory tests were performed in a small minority of patients. Despite several physician contacts 90% of the patients stated to have an uncontrolled disease, resulting in a strong impact on their everyday activities, sleep, and QoL.Despite the fact, that chronic urticaria is a common disease, some inducible forms of chronic urticaria are rare and their diagnosis is often delayed. In a survey conducted in german speaking countries Garcia-Gomez et al. 123 patients were followed upon their treatment response. The percentage of patients with controlled CSU at 3 months on omalizumab treatment was 80% and at 6 months 87% respectively, while the safety profile was almost excellent. On the other hand, omalizumab is a comparatively high-cost medicine and access to this treatment can be challenging. Ridge et al. report a dramatic reduction in unplanned healthcare interactions at primary care and emergency departments in Ireland when patients are treated with omalizumab; thus, the increased cost of omalizumab may at least partly counter-balanced by the reduced use of health system resources.Omalizumab is recommended as second-line therapy in chronic spontaneous urticaria. In a Colombian study conducted by Petrelli et al. report their experience on 6 patients with refractory normocomplementemic urticarial vasculitis successfully treated with omalizumab suggesting that this biological therapy may be a safe and effective therapeutic option in urticarial vasculitis.Urticarial vasculitis is a small-vessel leukocytoclastic vasculitis characterized by different clinical manifestations ranging from long-lasting urticarial lesions to severe and potentially life-threatening multi-organ involvement. In conclusion, the scientific works of this Research Topic cover different aspects of urticaria and provides state-of-the-art knowledge in the field of urticaria that could be implemented in both recearch projects and clinical practice.