Transfemoral prosthesis users’ high fall rate is related to increased injury risk, medical costs, and fear of falling. Better understanding how stumble conditions  affect transfemoral prosthesis users could provide insight into response deficiencies and inform fall prevention interventions.Six unilateral transfemoral prosthesis users experienced obstacle perturbations to their sound limb in early, mid, and late swing phase. Fall outcome, recovery strategy, and kinematics of each response were recorded to characterize (1) recoveries versus falls for transfemoral prosthesis users and (2) prosthesis user recoveries versus healthy adult recoveries.Out of 26 stumbles, 15 resulted in falls with five of six transfemoral prosthesis users falling at least once. By contrast, in a previously published study of seven healthy adults comprising 214 stumbles using the same experimental apparatus, no participants fell. The two oldest prosthesis users fell after every stumble, stumbles in mid swing resulted in the most falls, and prosthesis type was not related to strategy/fall outcomes. Prosthesis users who recovered used the elevating strategy in early swing, lowering strategy in late swing, and elevating or lowering/delayed lowering with hopping in mid swing, but exhibited increased contralateral (prosthetic-side) thigh abduction and trunk flexion relative to healthy controls. Falls occurred if the tripped (sound) limb did not reach ample thigh/knee flexion to sufficiently clear the obstacle in the elevating step, or if the prosthetic limb did not facilitate a successful step response after the initial sound-side elevating or lowering step. Such responses generally led to smaller step lengths, less anterior foot positioning, and more forward trunk flexion/flexion velocity in the resulting foot-strikes.Introducing training  and/or modifying assistive devices  may improve responses for transfemoral prosthesis users. Specifically, training or exoskeleton assistance could help facilitate sufficient thigh/knee flexion for elevating; training or prosthesis assistance could provide support-limb counteracting torques to aid in elevating; and training or prosthesis assistance could help initiate and safely complete prosthetic swing.The online version contains supplementary material available at 10.1186/s12984-022-01070-y. The transfemoral prosthesis user population experiences a substantially high fall rate and accompanying risk of injury, medical costs, and psychosocial effects. In a study of 17 transfemoral prosthesis users who reported falls after a four-week period, an average of 1.25 falls (averaging microprocessor and non-microprocessor knee results) were reported per person . FurtherWith higher fall risk comes increased injury incidence and medical costs. In studies of fall-related injury of lower-limb prosthesis users , approximately half of the prosthesis users who fell reported an injury that required medical care , 5. MundAside from physical and financial consequences, lower-limb prosthesis users experience psychosocial effects of this increased fall prevalence. Forty-seven percent of transfemoral prosthesis users reported a fear of falling , and 60%In order to develop interventions  to decrease fall likelihood, and thus alleviate the physical, monetary, and psychosocial burdens resulting from such falls, it is important to understand both the mechanisms that help prevent falls in healthy populations as well as the deficiencies in recoveries of transfemoral prosthesis users. While responses to trips/stumbles  in healthy adults have been well characterized, a comparable extent of research for the transfemoral prosthesis user population is lacking.Three primary responses have been identified and characterized as recovery strategies to swing-phase perturbations  for healthy adults: the elevating, lowering, and delayed lowering strategies , 10. ResFor healthy adults, such responses have been found to differ as a function of various factors, including (1) swing phase at which the perturbation occurs and (2) age of the participants. Regarding (1), recovery strategy selected depends on the dynamic state of the human body at the perturbation , factorsWhile these factors of (1) swing phase and (2) age likely also affect transfemoral prosthesis users’ stumble recovery responses, they may be additionally influenced by factors that are unique to their population, including: (3) knee prosthesis type, and (4) side tripped . Regarding (3), typically prescribed prosthetic knees have various joint designs and control schemes, aspects that affect their behavior after perturbations that are not under the participant’s direct control. A recent study highlighted different rates of falls for wearers of different prosthetic knee models . RegardiRelatively little research has been published examining how factors (1)–(4) affect stumble responses for transfemoral prosthesis users. In a retrospective study of lower-limb prosthesis users, walking was the most commonly reported activity at the time of a fall. Fifty-four percent of falls were attributed to a disruption of the prosthesis user’s base of support (as opposed to a disruption to their center of mass), and 22% were due to tripping specifically . HoweverTo date, two studies have introduced stumble perturbations to both limbs of transfemoral prosthesis users and report varying results related to factors (1)–(4) , 30. BotIn order to build upon these prior investigations, and specifically to better understand how different stumble conditions  affect responses, six transfemoral prosthesis users were recruited to undergo a series of stumble perturbations to both their sound and prosthetic limbs occurring in early, mid, and late swing phase. These experiments elicited substantially different responses depending on which limb was tripped , which warranted separate analyses and intervention suggestions. This paper presents the results from tripping the sound limb. Due to limitations in paper length, the results from tripping the prosthetic limb will be presented in a subsequent paper.Therefore the objective of this work is to examine the responses of transfemoral prosthesis users to sound-side stumble perturbations in order to gain insight into what contributes to falls or facilitates recovery for this population. The primary aim is to characterize their responses by reporting fall outcomes, recovery strategies, and relevant kinematics; the secondary aim is to further analyze these responses to investigate what differentiates recoveries versus falls for transfemoral prosthesis users, as well as what differentiates prosthesis user recoveries from healthy adult recoveries. The intent of this analysis is to provide insights into the deficiencies of transfemoral prosthesis user stumble recovery to better inform fall prevention interventions.Six unilateral transfemoral prosthesis users were recruited for the stumble recovery experiment. Participant details are tabulated in Table Participants walked on a treadmill at 0.8 m/s and were introduced to a series of obstacle perturbations, targeted to occur in early, mid, and late swing phase  for each limb . Specifically, a 35-lb steel block (obstacle) was positioned on a ramp apparatus in front of the treadmill and held in place by an electromagnet; when cued from an onboard targeting algorithm, the electromagnet switches off, releasing the obstacle down the ramp and onto the treadmill belt such that it contacts the participant’s foot at the experimenter-defined percentage of swing phase. The number of steps prior to perturbation, limb stumbled, and swing phase targeted were randomized. Randomization of trials, apparatus design and targeting algorithm, sensory occlusion techniques, and a distraction task were used to ensure the obstacle was unperceived by the participants prior to perturbation and to limit expectation of the stumble. The stumble perturbation system and experimental protocol employed are described in detail and validated experimentally in . Figure The goal was to capture the response to one early, one mid, and one late swing stumble for each limb for each participant. Recall that for this work sound-side stumble responses are presented, while prosthetic-side stumble responses will be reported separately. Depending on the participant’s comfort level, and due to the taxing nature of the protocol, the number of perturbations per session was capped and as such this protocol was completed in either one or two sessions . Note that the handrails were removed from the treadmill so they could not be used during recovery , but participants wore a full-body harness to prevent contact with the treadmill in the event of a fall.Each participant was given the same instructions prior to the stumble trials, namely: walk on the treadmill, perform serial sevens (counting backwards by seven) out loud and monitor visual feedback to stay centered on the treadmill; when the perturbation occurs, try to recover; the treadmill will be stopped to complete the trial when either (1) the participant falls into the harness or (2) the participant recovers and has returned to steady-state walking . Participants were given ample time to practice walking on the treadmill without perturbations prior to the stumble trials. All experimental protocols were approved by the Vanderbilt Institutional Review Board, and all participants gave their written informed consent.Ground reaction forces (GRFs) were recorded under each foot at a sampling rate of 1 kHz via a lateral split-belt, force-instrumented treadmill . Full-body kinematic data were collected via synchronized infrared motion capture  at a sampling rate of 200 Hz. Passive reflective markers were placed bilaterally over the lower limbs at the anterior/posterior superior iliac spines, medial/lateral femoral epicondyles and malleoli, posterior/medial/lateral calcaneus, 1st and 5th metatarsals, and navicular bone; the arms at the acromion, medial/lateral humeral epicondyles, medial/lateral styloid processes; and the torso at the left and right clavicle, sternum, and C7. Clusters of four markers were placed bilaterally on the thigh, shank, upper arm, and forearm. For the prosthetic-side lower limb, markers were placed on the estimated medial/lateral joint center of the prosthetic knee  and estimated medial/lateral malleoli and foot anatomical landmarks  of the prosthetic foot. GRF and motion capture data were filtered with a zero-phase, 3rd order, low-pass Butterworth filter with a cut-off frequency of 15 and 6 Hz, respectively. Inverse dynamics were computed using Visual3D  to estimate joint-level kinematics and kinetics for each trial.Several outcome metrics were computed to characterize each stumble response in order to address the primary aim. First, the swing percentage at which the perturbation occurred was estimated as the time from the preceding toe-off event to the instant of perturbation, relative to the average swing time from 20 strides during the same trial. The perturbation was determined as the instant at which the foot contacted the obstacle, which was identified via a transient peak in the anterior-posterior (AP) GRF measured by the treadmill, as in . Second,Several time-series kinematic trajectories were computed for further characterization of each response. Specifically, ipsilateral (tripped/sound-side) and contralateral (prosthetic-side) sagittal-plane thigh, knee, and ankle angle were computed to characterize and compare the lower-limb motion after each perturbation. Frontal-plane prosthetic-side thigh angle was also computed to characterize the thigh abduction involved in the subsequent step after the stumble. Additionally, sagittal-plane trunk angle  after the perturbation was computed, as trunk angle deviations have been reported to be indicators of increased likelihood of falling , 32. FinDiscrete summary metrics were also extracted in order to address the secondary aim . First, discrete metrics capturing key lower-limb dynamics during the response were extracted in order to identify lower-limb distinctions between recoveries and falls: peak ipsilateral (sound-side) thigh flexion, peak ipsilateral knee flexion, peak contralateral (prosthetic-side) thigh flexion, peak contralateral knee flexion, and contralateral knee angle at foot-strike. Second, the work of Grabiner et al. has previously identified several metrics that discriminate falls versus recoveries for various populations by capturing the body’s state at each of the response foot-strikes , 31–34. Data from a previous healthy adult stumble study , 24 wereFifteen out of the 26 stumbles to the sound side of the transfemoral prosthesis users resulted in falls. This is in stark contrast to the healthy participants, in which no falls occurred out of 214 total stumbles.Of the 26 transfemoral prosthesis user stumble responses, seven were elevating, nine were delayed lowering, and 10 were lowering. Four hopping strategies were used after lowering or delayed lowering strategies. No skipping strategies were observed. Five out of six participants fell at least once. Two participants fell after all perturbations, and three participants only fell after perturbations in mid swing. The breakdown of fall outcomes and strategies used for each participant are reported in Fig. Figures Discrete summary metrics from Figs. All four recoveries from early swing perturbations were accomplished by the elevating strategy, in which the tripped limb lifted up and over the obstacle in the same step, landing anterior to the obstacle. Refer to the legend of Fig. Two of the six participants fell consistently in response to early swing perturbations Fig. . Of noteLower-limb dynamics As shown in Fig. Foot-strike states As shown in Fig. All late swing recoveries were accomplished by the lowering strategy, in which the tripped limb was immediately lowered to the ground, terminating that step and initiating a step with the contralateral (prosthetic) limb, followed by another ipsilateral (sound-side) step to clear the obstacle. Refer to the legend of Fig. Two of the six participants fell consistently in response to late swing perturbations Fig. . Of noteLower-limb dynamics As shown in Fig. Foot-strike states As shown in Fig. Recoveries in mid swing were accomplished by two elevating strategies  and one lowering strategy with hopping (P6a). As shown in Fig. As shown in Fig. Five of the six participants fell during mid swing perturbations. P4 attempted a delayed lowering with hopping strategy, in which upon contacting the obstacle he initially elevated but ultimately terminated his step and lowered behind the obstacle, then subsequently hopped but experienced substantial trunk flexion just as he landed and loaded the harness with >50% bodyweight. P4 landed with his prosthetic-side thigh substantially more extended than P6, as evidenced by contralateral thigh angle at foot-strike Fig. J. P2 attLower-limb dynamics Transfemoral prosthesis users who recovered after mid swing stumbles either elevated or lowered with hopping. Thus kinematics that differentiate falls versus recoveries  foot-strike.As shown in Fig. Note that relative to HC, the prosthesis users who recovered with an elevating strategy used a comparable step length and foot positioning at first foot-strike, but landed with more trunk flexion and more forward trunk flexion velocity Figs.  and 12. Five of the six transfemoral prosthesis users fell at least once in this study, while none of the seven healthy control participants fell in , 24. TheThe following subsections provide an in-depth discussion of results with respect to the stumble conditions of (1) swing phase, (2) participant age, and (3) prosthesis type, as well as a commentary on arm motion. Finally, based on these observations, several interventions for improving recovery and mitigating falls for the transfemoral prosthesis user population are considered.Transfemoral prosthesis users who recovered in early swing P2, P3, P4, P6) used an elevating strategy, as HC did. This was accomplished via substantial sound-side thigh and knee flexion to complete the elevating step, and a successful initiation (thigh flexion) and completion (extended knee angle) of the next prosthetic-side step  either inadequately performed the elevating strategy or attempted a delayed lowering strategy but could not successfully initiate a contralateral (prosthetic-side) step. In the seven-participant healthy adult study, all 86 stumbles that occurred before 40% swing phase resulted in elevating strategies  , 24. In These early swing findings are inconsistent with the only other study of sound-side, early swing perturbations with transfemoral prosthesis users, in which majority lowering strategies and no falls were reported . This maCollectively these results suggest that properly performing the elevating strategy is key for recovering from early swing stumbles.Transfemoral prosthesis users who recovered in late swing P2, P3, P4, P6) used a lowering strategy, as HC did. This was accomplished via substantial thigh flexion and a safe landing configuration for the prosthetic-side step as well as substantial sound-side thigh and knee flexion in the subsequent step to clear the obstacle  all attempted a lowering strategy but could not initiate and/or complete the next (prosthetic-side) step. This was evidenced by the lack of prosthetic-side thigh flexion or a flexed knee at prosthetic-side foot-strike Fig. , which cThese late swing findings are mostly consistent with Shirota et al. , who alsCollectively these results suggest that properly initiating swing after the lowering step, landing in a safe  configuration on the prosthetic limb, and using sufficient thigh/knee flexion to clear the obstacle in the subsequent sound-side step are key for recovering from late swing stumbles.Transfemoral prosthesis users who recovered in mid swing  used an elevating or lowering with hopping strategy. The trends observed in early swing elevating strategies and late swing lowering strategies extend to mid swing regarding lower-limb dynamics Fig.  and footThese mid swing findings are mostly consistent with Shirota et al.  in termsCollectively these results suggest that either properly performing the elevating strategy or properly initiating prosthetic-side swing after the lowering step  is key for recovering from mid swing stumbles.It is notable that the two oldest participants in this case series (P1 and P5) were the only two participants to fall after every perturbation. These results are consistent with several studies of healthy (non-prosthesis user) older and younger adults, in which the older adults fell more , 41 whicPavol et al. 1999  reportedFour of the transfemoral prosthesis user participants wore an Ottobock C-Leg , which is a hydraulic-based microprocessor-controlled knee (MPK). Two participants wore hydraulic non-MPKs: P2 wore an Ottobock 3R80, a single-axis, rotary-hydraulic knee; P4 wore a Blatchford KX06, a four-bar, hydraulic knee. All six are hydraulic knees with stance control function . Stance resistance against flexion is initiated at heel-strike in the non-MPKs, while it is initiated during swing extension in the C-Leg. The C-Leg and 3R80 are both single-axis knees, while the KX06 employs 4-bar kinematics that increase toe clearance during swing phase and also enhance stability during stance phase.For early and late swing perturbations, there was no clear trend in prosthesis type contributing to falls versus recoveries. Recall that the two transfemoral prosthesis users who fell were also the oldest participants, which was discussed in the Considering Age subsection. However, the transfemoral prosthesis users who recovered (regardless of prosthesis type) used substantial thigh abduction during the subsequent prosthetic-side step Fig. , likely For mid swing perturbations, the participant who recovered using an elevating strategy wore a non-MPK (P4 with KX06). The participant who recovered using a lowering strategy wore an MPK (P6 with C-Leg). However, it is unclear whether the devices themselves helped facilitate recovery; especially in the case of P6, the C-Leg’s lack of stance release likely led to the hopping response after lowering that required substantial thigh abduction to facilitate swing and involved increased trunk flexion. Thus, the recoveries in mid swing are potentially attributed to the ability of the prosthesis user rather than the prosthesis itself. For the delayed lowering responses , the non-MPK users (P2 and P4) were able to initiate a subsequent step with their prosthetic side after the lowering step Fig. , while tThere was one instance of prosthetic knee buckling, in which P5 did not take a full prosthetic-side step after his delayed lowering step and landed with the knee substantially flexed Figs.  and 11.From these observations, for sound-side stumble recovery there does not seem to be a clear advantage of knee prosthesis type. Instead, both demonstrated similar deficits: namely, the inability to initiate swing and/or complete swing during the next step. All participants wore similar energy storage-and-return type prosthetic feet, and all showed similar deficits in terms of lack of active plantarflexion and less range of motion compared to HC. The effect of prosthetic foot model was beyond the scope of this work but could be investigated in future studies.Roos et al. 2008  concludeInterestingly, the transfemoral prosthesis users who fell after every perturbation were the two oldest participants, and they employed arm motion similar to those found in the older participants of Roos et al. 2008 ; describIn this case series, fall incidence was related to swing percentage  and age (the oldest prosthesis users fell after every stumble), but not with prosthesis type (MPK versus non-MPK), as discussed in the previous discussion subsections.Falls for this population can be attributed to two main deficiencies in the transfemoral prosthesis users’ responses after a stumble. First, falls occurred if the tripped (sound) limb did not reach ample thigh and knee flexion to sufficiently clear the obstacle in the elevating step Fig. A and C. Second, falls occurred if the prosthetic limb did not facilitate a successful step response Fig. F and H aBoth of these deficiencies in lower-limb dynamics  limb’s hip and knee flexors may improve success in elevating over the obstacle.  Training targeted at the prosthetic-side (support limb) hip flexors could also help by providing counteracting torques during the elevating step; however, transfemoral prosthesis users still may lack in the necessary knee or ankle torques due to their passive prostheses. Additionally, training targeted at the prosthetic-side hip could help initiate swing phase; however, the transfemoral prosthesis users who recovered in this study employed substantial thigh abduction to accomplish the step due to prosthesis constraints, likely necessitating external interventions discussed subsequently. Strength training has shown potential for improving responses for fall-prone populations ; future Second, a task-specific training protocol in the form of repeatedly introduced treadmill acceleration disturbances that require a stepping response may also improve recovery outcomes. In particular, the work of Grabiner et al. has shown that this task-specific training can improve the metrics at response foot-strikes linked to increased fall risk , 5251, 5First, a powered prosthesis could improve the sound limb elevating response by providing the necessary counteracting ankle and/or knee torques in the support limb, allowing for a more normative push-off to facilitate the elevating step and reduce the body’s forward angular momentum , 30.Second, a powered prosthesis could presumably sense a stumble and initiate powered swing phase for the subsequent step without requiring ballistic coordination with the thigh, as passive prostheses do. In other words, users could more easily initiate and complete swing phase in the following step, addressing one of the leading causes of falls among study participants. Additionally, powered prostheses can provide robust stance support even if the prosthesis lands with a flexed knee. Some energetically passive knees could provide robust stance support as well, although in general, any prosthesis that does so must implement this as a stumble-specific behavior, since otherwise doing so would interfere with stance knee yielding during slope or stair descent.Such improvements in the lower-limb deficiencies observed may improve the body’s state at prosthetic-side foot-strike .Despite this potential for improvement, to date there have been no prosthetic interventions developed or tested to address sound-side stumbles for transfemoral prosthesis users. Future work is needed to investigate the feasibility of such interventions.These suggestions may not only reduce falls but also generally improve responses for transfemoral prosthesis users who did not fall. For example, transfemoral prosthesis users still exhibited responses that indicated increased risk of falling (increased trunk flexion/flexion velocity) and compensation mechanisms (increased thigh abduction and arm motion) relative to healthy controls; thus, training and/or assistive device interventions may improve these metrics, ultimately helping to reduce the need to abduct and help control forward angular momentum to reduce these deficiencies.There are several limitations to this work. First, a sample size of more participants would have improved the confidence of the observations enumerated here. More non-MPK users would have improved the discussion on the effect of prosthesis type. Regardless, given the inherent heterogeneity of the prosthesis user population , a case series characterization such as that presented here is arguably more representative than one in which a singular averaged result is reported .Second, while substantial efforts were made experimentally to ensure the obstacle was not perceived prior to contacting the participant’s foot (see Methods and Results of ), the paThird, there is potential that requiring a cognitive task during the trials may alter balance outcomes; however, a recent stumble study found that performing Serial Sevens  did not alter the participants’ recovery response . FurtherFourth, rest was not standardized across participants; instead, participants were given the opportunity to rest as much as desired in between each stumble based on their own comfort and energy levels. This rest period was based on personal preference rather than physiological monitoring, so there is potential that fatigue could have affected responses. However, there were no observed trends in fall/recovery outcomes with order of trials  across participants.Finally, the authors note that there are other factors that may have played a role in responses: socket attachment, residual muscle condition, and physical fitness level. Though this type of analysis was beyond the scope of the present work, future studies investigating these factors are encouraged.This study presents a case series of sound-side stumble responses to obstacle perturbations in early, mid and late swing phase for six transfemoral prosthesis users of various ages and prosthesis types. Five out of six participants fell at least once—in contrast to a similar study of seven healthy participants in which none fell—highlighting the importance of studying sound-side stumbles and considering appropriate interventions. Stumbles elicited in mid swing resulted in the most falls (five out of six participants fell), suggesting a potential region of focus for intervention design. The two oldest participants were the only two to fall after every stumble, highlighting age-related differences in muscle strength and control and consequently ability to recover. Prosthesis type  did not relate to strategy or fall outcomes among the six participants studied; rather, both prosthesis types exhibited similar deficits regarding inability to initiate and/or complete swing phase.Strategies that resulted in recoveries for transfemoral prosthesis users matched those used by the healthy control participant; namely the elevating in early swing, the lowering in late swing, and either the elevating or the lowering/delayed lowering (with hopping) in mid swing. However, these recoveries still showed kinematic variations from the control participant, characterized by increased contralateral (prosthetic-side) thigh abduction, increased trunk flexion and flexion velocity, and exaggerated arm motions. Thigh abduction is likely a compensation performed to help initiate/complete swing phase on the prosthetic side. Trunk and arm responses are likely affected by the nature of the passive prosthetic support limb, which cannot generate the counteracting torques typically generated by healthy musculature. Those who fell inadequately performed these recovery strategies, evidenced by lower-limb deficiencies in executing the elevating step and/or in initiating and completing the prosthetic-side step, which led to a shorter step length, less anterior foot placement relative to COM, more trunk flexion, and more forward trunk flexion velocity at each foot-strike, metrics that have been previously reported as fall indicators.These observations suggest that interventions via training  and/or assistive devices  may address the main response deficiencies that led to falls for transfemoral prosthesis users. Specifically, training or exoskeleton assistance could help facilitate sufficient thigh/knee flexion for the elevating strategy; training or prosthesis assistance could provide counteracting torques in the support limb to facilitate elevating; and training or prosthesis assistance could help initiate and safely complete prosthetic swing.Additional file 1. A compilation video of each participant's stumble responses in early swing (< 40% swing phase). If a participant experienced more than one perturbation in early swing, it is identified by a lower-case letter which is used in the main text and figures. Refer to Fig. Additional file 2. A compilation video of each participant's stumble responses in mid swing (40-60% swing phase). If a participant experienced more than one perturbation in mid swing, it is identified by a lower-case letter which is used in the main text and figures. Refer to Fig. Additional file 3. A compilation video of each participant's stumble responses in late swing (> 60% swing phase). If a participant experienced more than one perturbation in late swing, it is identified by a lower-case letter which is used in the main text and figures. Refer to Fig.


Anterior clinoid process (ACP) mucoceles are an uncommon entity and an even rarer cause of visual impairment. We review the case of a 62-year-old female with a 2-year history of progressive right-sided monocular vision loss. Paranasal sinus computed tomography (CT) scan showed bilateral ACP pneumatization. A soft tissue density mass occupied the right ACP with bone expansion and compression of the right orbital canal. An endonasal approach was performed with total vision recovery. Dedicated images are necessary to diagnose ACP. On CT, the affected sinus will have bone erosions. On magnetic resonance imaging, the signal intensity is determined by its protein concentration and mobile water protons. ACP mucoceles’ accurate diagnosis determinates the treatment and surgical approach. Finally, the correct management selection will determine the retrieval of the visual ability. The anterior clinoid process (ACP) is a bony projection on the medial aspect of the posterior border of the lesser sphenoid wing. Its pneumatization is not a rare incidental finding, with a reported incidence of 4–29.3% . Its occBlockage of the drainage of a paranasal sinus may occur usually because of repeated inflammation or, in some rare cases, as a congenital variation . It may Due to its proximity to the optic nerve, ACP mucoceles may, although very rare, be a cause of orbital apex syndrome—compressive optic neuropathy . ComputeA 62-year-old woman with no medical background presented to the emergency department with a 2-year history of progressive right-sided monocular vision loss. The ophthalmological assessment evidenced a visual acuity of 20/200 −1 PH 20/100 + 2 in her right eye, relative left afferent pupillary defect and pale temporal neuroretinal ring with normal bilateral extraocular movements and normal vision of the left eye.At arrival, under the suspicion of a supraclinoid internal carotid aneurysm, an MRI angiography was performed, evidencing a rounded proteinaceous mass in the right ACP with compression of the optic nerve suggestive of an ACP mucocele . No vascA pre-surgical paranasal sinus CT scan showed bilateral pneumatization of ACP; the right one had thin eroded bony walls and was occupied by a soft tissue density lesion that compressed the orbital apex .An endoscopic endonasal approach with neuronavigation through an ethmoidal approach was performed 8 days after arrival by an experienced neurosurgeon. The expanding ACP was drilled, allowing the exposure of a soft, grayish lesion consistent with mucocele , which wThe post-operative course was uncomplicated, with the right eye visual acuity of 20/20 2 months after surgery. Follow-up CT showed a complete resolution of the anterior clinoid mucocele with no residual lesion.ACP mucoceles are an uncommon entity and a rare cause of visual impairment. Dedicated images are necessary to diagnose and treat them. On CT, the affected sinus will have rounded, eroded and thinned walls . The sigThe treatment of paranasal sinus mucocele aims to relieve the compressive effect on the surrounding structures and to prevent recurrences. Choosing the most appropriate surgical approach allows complete resection and minimal invasion with the least possible complications.Pterional and subtemporal approaches were classically used to achieve total decompression and resection. However, there was a high rate of complications and morbidity. Using an endoscopic endonasal approach with a microsurgical technique has shown good anatomic and functional results , 10, andIn this case, head images allowed an accurate diagnosis of a lesion in the right ACP with optical nerve compression, which was treated using a mini-invasive technique through an endoscopic trans-ethmoidal approach, with a total recovery of the visual deficit.


Tandem exon duplications play an important role in the evolution of eukaryoticgenes, providing a generic mechanism for adaptive regulation of proteinfunction. In recent studies, tandem exon duplications have been linked tomutually exclusive exon choice, a pattern of alternative splicing in which oneand only one exon from a group of tandemly arranged exons is included in themature transcript. Here, we revisit the problem of identifying tandem exonduplications in eukaryotic genomes using bioinformatic methods and show thattandemly duplicated exons are abundant not only in the coding parts, but alsoin the untranslated regions. We present a number of remarkable examples oftandem exon duplications, identify unannotated duplicated exons, and providestatistical support for their expression using large panels of RNA-seqexperiments. The major driving force of molecular evolution is mutation, a process thatintroduces changes to the genomic sequences that are transmissible throughgenerations. While the most frequent type of mutations are single-nucleotidepolymorphisms, which affect single bases, genomic duplications are anotherimportant type of DNA changes. A particular subtype are the so-called tandemgenomic duplications, which are represented by DNA sequences typically morethan 1 kb long, are immediately adjacent to each other, and have a high levelof sequence identity .ζ[ Tandem genomic duplications may affect entire genes, either protein-coding ornon-coding, or only gene parts. In the latter case, the duplication leads topropagation of only a portion of the gene sequence, thus affecting theexon-intron structure . The proζ , srp [ 1ζ , multidrζ  genes inζ  and tropζ . Perhapsζ ,20, 21.In 2002, a systematic study of common exon duplications and their role inalternative splicing reported that about 10% of animal genes contain tandemlyduplicated exons and discovered more than 2,000 unannotated candidate MXEs bysimilarity searches identifying homology to neighboring exons or within DNAadjacent to exons . HoweverGenome sequences and annotations The February 2009  assembly of the human genome wasdownloaded from the Genome Reference Consortium . GENCODEExon homology search The homology search was carried out using the EMBL-EBI’s exonerate toolto identify tandem exon duplication . The nucFiltering procedure for query-target pairs To identify unannotated tandem exon duplications, we filtered the table ofquery-target pairs using the bedtools intersect utility as follows. We removedthe query-target pairs in which the target sequence intersects at least oneannotated exon by more than 5% if its length. Additionally, we removed thequery-target pairs in which the target sequence intersects at least oneannotated interspersed repeat or low-complexity DNA sequence by more than 10%if its length, according to multiple repeats tracks from the UCSC Genomebrowser .RNA-seq data The RNA-seq data from 6,625 samples in the Genotype-Tissue Expression (GTEx)consortium v7 data were analyzed using the procedure described previously. Short rNucleotide increase ratio In order to detect exonic duplications, we used the largest to-date exonannotation datasets, including the GENCODE  and RefSAs expected, the NIR values decrease with increasing sequence identitythreshold .DespiteNext, we asked whether some genes are more prone to tandem exon duplicationsthan the others. To address this question, we computed the NIR values for eachannotated gene separately and plotted the NIR frequency distributions.The NIRThe difference in the propensity of tandem duplications in the genes with largeNIR compared to other genes could potentially arise from differences in theirexon lengths. To address this, we compared the NIR values in groups of exonsequally spaced in ten bins by length. We found that the NIR values decreaseapproximately fourfold as exon length increases from 20 to 220 nucleotides,thus indicating that longer exons do not contribute to larger NIR values.Indeed, the longer the target, the smaller the likelihood of finding a homologat 80% sequence identity cutoff should be. Additionally, the average exonlength for the top 200 genes with the largest NIR values did not differsignificantly from the average exon length in the population of all exons. Therefore, exon lengths do not significantlyaffect the propensity of tandem duplications. The Gene Ontology analysis of thetop 200 genes with large NIR values revealed a statistical enrichment of GOcategories related to cell adhesion and nervous system development , ion binding and receptor activities (molecular function), andmembrane localization (cellular compartment).To further investigate the structure of exonic duplications in these genes, wecreated a track hub for the UCSC Genome browser as a visualization tool for allquery-target pairs. As a positive control, we confirmed that our proceduresuccessfully identified clus ters of tandemly duplicated exons in the genesknown from the literature , 12,13 Obscurin (OBSCN) Obscurin (OBSCN) is a remarkable example of a human gene broadly affected bytandem exon duplications. It spans more than 150 kb and contains over 80 exons. The proOur analysis has shown that the vast majority of OBSCN exons are homologous toeach other and similar in length, being indicative of their origin in tandemduplication .The preUDP Glucuronosyltransferase Family 1 Member A (UGT1A) The human UGT1A gene encodes UDP Glucuronosyltransferase Family 1 Member Agroup of proteins, which is represented by thirteen unique alternate firstexons followed by four common exons. UGT1A is associated with diseasesincluding Gilbert syndrome  andCrigExamples of tandem exon duplications in D. melanogaster UTRs Two remarkable examples of tandem exon duplications in UTRs of D. melanogasterare the hydra and pip Expression support by RNA-seq data To assess the expression of tandem exon duplications using RNA-seq data, weconsidered query-target pairs in the human genes in which the target regiondoes not intersect any annotated exon or any annotated repeat element, andmerged the remaining targets using the bedtools merge program. This procedureyielded 4,027 intronic targets. Each of these targets was matched randomly to acontrol region of the same length that was located 30 nt upstream ordownstream.i = log10(1 +targeti) - log10(1 + controli), wheretargeti is the average target read coverage in the tissue i andcontroli is the average control read coverage in the tissue i.Tissues with an insufficient number of log FCi values  were excluded from further analysis.In a group of targets that showed at least 80% nucleotide sequence homology tothe query, we observed a significant positive departure of the log FCi metricfrom zero (Wilcoxon signed rank test), which remained significant in sometissues af ter Benjamini-Hochberg correction for multiple testing project  using on testing;e.g. Wh testing.These rFinally, we calculated the difference between average PhastCons  scores oAn interesting observation made in this work is that tandem exon duplicationsare prevalent not only in the coding regions, but also in the UTRs ofeukaryotic genes and, moreover, they seem to be associated with a mutuallyexclusive choice of tandemly duplicated initial and terminal exons. A recentstudy has shown that the regulatory mechanism underlying the mutually exclusivechoice of 3’ variable regions in D. melanogaster PGRP-LC pre-mRNAinvolves competing RNA structures .These RRecently, we have proposed an evolutionary mechanism for the generation ofcompeting RNA structures associated with mutually exclusive splicing viagenomic duplications that affect not only exons but also their adjacent intronswith stem-loop structures .AccordiTandem exon duplications are abundant not only in the coding parts, but also inthe untranslated regions of eukaryotic genes. It still remains an open questionwhether or not competing RNA structures are broadly involved in the regulationof mutually exclusive splicing of these exons, as well as whether they could begenerated as a byproduct of tandem genomic duplications.


Due to the negligence of the complex tumor immune microenvironment, traditional treatment for glioblastoma has reached its limitation and cannot achieve a satisfying outcome in the past decade. The emergence of immunotherapy based on the theory of cancer-immunity cycle has brought a new dawn to glioblastoma patients. However, the results of most phase II and phase III clinical trials are not optimistic due to the simple focus on T cells activation rather than other immune cells involved in anti-tumor immunity. NK cells play a critical role in both innate and adaptive immunity, having the ability to coordinate immune response in inflammation, autoimmune disease and cancer. They are expected to cooperate with T cells to maximize the anti-tumor immune effect and have great potential in treating glioblastoma. Here, we describe the traditional treatment methods and current immunotherapy strategies for glioblastoma. Then, we list a microenvironment map and discuss the reasons for glioblastoma inhibitory immunity from multiple perspectives. More importantly, we focus on the advantages of NK cells as potential immune regulatory cells and the ways to maximize their anti-tumor immune effect. Finally, our outlook on the directions and potential applications of NK cell-based therapy combining with the advance technologies is presented. This review depicts NK cell awakening as the precondition to unleash the cancer-immunity cycle against glioblastoma and elaborate this idea from biology to clinical treatment. Glioblastoma is one of the most aggressive and rapidly fatal tumor types in human brain.Traditional strategies have reached their limits in prolonging overall survival of glioblastoma patients.Current immunotherapies for glioblastoma did not show significant curative effect.NK cells activate the whole anti-cancer immunity and make up for single T cell activation.How are NK cells regulated in the glioblastoma microenvironment?What should be done to reprogram the dysfunctional NK cells infiltrated in glioblastoma?Lacking MHC I complex is one of the key features for NK cell activation and T cell inactivation, how can we achieve a balance for maximum anti-cancer immunity?6-methylguanine-DNA methyltransferase (MGMT), TMZ works more effectively on patients whose tumors harbor a methylated MGMT promoter. Radiotherapy can produce ionizing radiation and damage the DNA of cancer cells. The standard postoperative radiotherapy regimen for GBM in adults is 60 Gy divided into 30 fractions after surgery. In elderly GBM patients, hypofractionation with a 45 Gy dose (more than 2 Gy per fraction) is also recommended. Concurrent use of TMZ with radiotherapy for at least six cycles is the standard postoperative adjuvant therapy for GBM patients. Additionally, targeted therapy, as a treatment modality from the cellular molecular level, has also been widely researched in recent years. Usually, it directly kills cancer cells by targeting specific genes and is mainly applied in clinical recurrent GBM patients. The most commonly used targeted drug is bevacizumab (Bev), which is the antagonist of VEGF-A and approved by the FDA [Maximal safe resection is the top priority among glioblastoma (GBM) therapeutic schedules, removing tumor-occupying effect and helping to identify the cancer histopathology and molecular pathology which are the basis for precise postoperative adjuvant treatment. However, high invasiveness and inevitable residue of GBM always escape from simple surgical resection, and patients without postoperative adjuvant therapy suffer from tumor recurrence after a short period of time. Hence, postoperative adjuvant therapies are used to target residual tumor cells and delay tumor recurrence. Temozolomide (TMZ) is an alkylating agent with antitumor activity and exerts cytotoxic effects through the mismatch repair of methylated DNA . TMZ is In 2013, Chen and Mellman firstly raised the concept of “cancer-immunity cycle”, revealing a series of stepwise immunity events that must be initiated for effective killing of cancer cells . More reP = 0.76) [Drugs that reverse the activity of negative regulatory pathways limiting T cell activation are one of the most successful breakthroughs in malignancy treatment in the past decade. The common treatment targets include PD-L1 which are mostly expressed on a tumor cell, or PD-1 and CTLA-4 express on T cell. However, the overall therapeutic effect of immune-checkpoint blockade (ICB) in GBM is not optimistic, and the therapeutic effect varies greatly, which may be related to the relatively differential expression of PD-L1 in GBM and the individuals difference mutation load of tumor cells. More recently, results from a phase III clinical trial enrolling 369 patients with recurrent GBM showed that blocking PD-1 with nivolumab did not prolong median overall survival compared to bevacizumab  . SimilarP = 0.93) according to the result of Phase III clinical trial enrolling 741 patients [Since GBM rarely metastasize outside the central nervous system, it is a potential cancer treatment strategy to activate the immune surveillance function of the brain through strengthening the adaptive arm of the immune system, especially for vaccines. Presently, it is mainly divided into peptide vaccine and dendritic cell (DC) vaccine. The common immunogenic targets of the former one including epidermal growth factor receptor (EGFR) vIII, isocitrate dehydrogenase (IDH)-1 (R321H), interleukin (IL)-13Rα2 and gp100. Some vaccination approaches have reached phase III clinical development, but the treatment effect is not satisfactory. Rindopepimut, a vaccine targeting EGFRvIII, did not increase survival than the control group in patients with newly diagnosed GBM (ndGBM)  exposure. What’s more, CAR-T cells can be edited to have an activated phenotype. These characteristics can help to overcome immunologic suppression to some extent. It is especially critical for GBM, a highly heterogeneous tumor with a complex tumor microenvironment. Presently, initial clinical experiences of CAR-T therapy for GBM have only three target antigens  . Their eOncolytic virus therapy can not only directly kill tumor cells, but also activate antitumor immune effect by destroying tumor cells and releasing tumor related antigens. In addition, viruses usually activate macrophages through Toll-like receptors and the activated macrophages cells can promote the infiltration of T cells into tumors . HoweverGlioma tumor microenvironment (GME) is a complex and diversified network system, including tumor cells, immune cells, stromal cells, vascular endothelial cells and so on. In addition, with the advancement of tumor multi-omics research, the metabolic characteristics and tumor mechanism microenvironment of glioma have also attracted much attention. These are the main reasons leading to tumor immunosuppression and drug tolerance.The Nobel Prize in Physiology or Medicine in 2019 awarded the research of cellular sensation and adaptation to oxygen. Among them, hypoxia-inducible factor (HIF) plays a key role in orchestrating a metabolic switch that allows cells to survive in this environment. Hypoxia is a prominent feature in GBM due to the rapid growth of cancer cells and the unmatched angiogenesis and oxygen supply. Hypoxia always contributes to the malignant phenotype of GBM by reprogramming the cancer cells themselves and the tumor microenvironment . For gliGBM is infiltrated with amounts of suppressive immune cells such as microglia, macrophages, MDSCs and Tregs, and the immune cell composition differs based on the IDH mutation status, the telomerase reverse transcriptase (TERT) promoter mutation and transcriptional subtypes . The supAberrant metabolisms of glucose, lipid and amino acid are hallmarks of GBM, accompanying with the accumulation of amounts of metabolites. Lactate, reactive oxygen species (ROS) and lipid are three focused metabolites which affect malignant and normal cells to a great extent. Lactate metabolism depends on aerobic glycolysis (Warburg’s effect), indicating that cancer cells produce lactate after absorbing glucose as a substrate for oxidative phosphorylation, even under normoxia . The lacConsidering that GBMs are a lot stiffer than normal brain tissues, mechanical microenvironment is a specific property and becomes novel research target derived from the accumulation of extracellular matrix. Mechanical microenvironment contains physical components such as collagen and fibrin, and many intracellular signaling like integrin signaling. Mechanical microenvironment regulates the expressions of oncogenes and tumor suppressors, carcinogenesis, cancer progression and therapy resistances. Moreover, the immune system is found to promote the formation of mechanical microenvironment, especially the macrophages (CD68 and CD163 positive) infiltrated in cancer can secrete TGF-β, which mediates the production of collagen and activates LOX, leading to collagen crosslinking . Recent + T cells in an IFN-γ–dependent manner, inducing their activation and proliferation [+ NK cell was even able to directly take in and present certain antigens to CD8+ T cells as an APC [+ T cells by promoting DC activation [+ CTL [Cancer-immunity cycle firstly presents the progress of cancer elimination by immune system, containing details from antigen recognition to cancer cell killing. In the past decade, most immunotherapies directly aimed at homogeneously activating T cell-based adaptive immunity to eliminate cancer cells, while several recent strategies focus on maximum activation of the innate immune cells because they are frontline responders of cancer cells and capable of rapidly mediating a response . Howeverferation . Strikins an APC . After itivation . Addition [+ CTL . Recentln [+ CTL . In the n [+ CTL .Fig. 1NKNK cell is considered as the bridge between innate immune system and adaptive immune system, and it seems to play a promising role in next generation immunotherapy for GBM. Hence, emerging researches which focus on modulating NK cell function to improve anticancer immune effect have been promoted. A number of clinical rials of NK-cell based immunotherapies in glioma are currently active in Table Cytokines are crucial for NK cell differentiation, activation and immunological effect persistence, with their receptors expressed variously on NK cells depending on different states and functions. In GBM, immunosuppressive cytokines like TGF-β and IL-10, are dominant in tumor immune microenvironment (TIME), which inhibit NK cells directly or indirectly by inducing other immunosuppressive factors. Hence, a cluster of stimulatory cytokines are required to restore the attenuated NK cell function independently or cooperatively. IL-2 and IL-15 are the most widely studied cytokines in NK cells homeostasis and have many positive functional effects that enhance the antitumor response. IL-2 stimulate NK cells and CD8+ T cells via a dimeric IL-2R formed by IL-2Rβ (CD122) and IL-2Rγ (CD132) subunits, enhancing cell survival, proliferation, cytokine production and cytotoxic efficiency. HSP70/IL-2 treated NK cells cross the blood brain barrier and target tumor cells more effectively than untreated ones . AlthougNK cell activation is finely regulated by inhibitory receptors to avoid unnecessary or excessive immune responses. In cancer, these inhibitory receptors are often utilized to suppress NK cells function by cancer cells, leading to immune evasion . HLA-claAs CAR-T cell therapy being proved effective in hematological malignancies and some solid tumors, CAR-NK therapy emerged in recent years with potential advantages over CAR-T therapy, such as less CAR antigen loss induced immune escape, higher human safety and the possibility to be an off-the-shelf allogeneic therapeutic . EngineeIt is generally accepted that innate immune responses of NK cells would not be amplified when exposed to the same target. However, this dogma has been challenged in the past decade, increasing evidence proved that NK cells could acquire “memory-like (ML)” characteristic of elevated immune response upon a secondary stimuli . GeneralRepressed functional states of NK cells infiltrated in cancer have been studied to reprogram their activities impacted by TIME. Recent researches reveal that metabolic change is the key factor in immune cells fate, which affect their contribution in cancer progression –87. TumoPanax ginseng, Angelica sinensis, Radix Astragali, Ganoderma lucidum and Lentinus edodes [In addition to the above ways, novel immunomodulatory drugs have been paid much attention in cancer immunotherapy and several kinds of drugs have been put forward to activate NK cells, such as small molecular inhibitors & activators and Chinese herbal medicines (CHMs). Deletion of Rb (a tumor suppressor) and overexpression of mutated Ras (a proto-oncogene) are enough to confer resistance to NK cell-mediated cytotoxicity in glioma cells, Rb activator or Ras inhibitor may reprogram the immune evasion . Pharmacs edodes . In futuAnimal model has become a wildly used tool for immunotherapy efficiency validation in preclinical researches. Xenograft mouse model is one of the classic models in which human derived GBM cell line is engrafted in the immune-compromised mice. However, this model is not suitable for the study of T cells or NK cells due to deficiency of tumor microenvironment. While immune-competent mouse model can solve the problem of TME deficiency, but the injected mouse derived tumor cell lines cannot accurately reflect the real situation of human tumors. In patient-derived xenograft (PDX) model, tumor tissues which contain tumor cells, immune cells, stromal cells and vascular endothelial cells from patients are injected into nude mice, it not only solves the problem of lacking TME, but also reflects cancer individual features to a great extent. But this model is relatively time and financial consuming. Organoids are ex vivo three-dimensional (3D) models that are derived from human embryonic stem cells or patients derived tumor tissues, which have the ability to self-organize and can more realistic reflect both the structure and function of primary human organs . More reDue to their natural feature of scattered distribution, immune cells are more suitable for single-cell genomics, which present a potential to open up the way we explore immune cell heterogeneity and study their spatial organization, dynamics, clonal distribution, pathways, function and cross-talks . For exaThe clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR-Cas9) system was firstly discovered in prokaryotes to resist against viruses . Later, Current accomplishments have fueled interest in improving anti-tumor immunity for cancer therapy. However, these therapies focused on enhancing the T-cell immune response did not produce the same anti-tumor effect in GBM. In addition, limited by the specific antigen recognition process of T cells and the high heterogeneity of glioma cells, GBM patients with ideal response to immunotherapy at first almost end up with immune evasion due to specific antigen loss. Therefore, it is urgent to explore new core immune cells and create more effective and durable next generation immunotherapy regiments. NK cells are the most potent innate immune cells able to activate adaptive immunity and can mobilize a variety of immune cells to exert anti-tumor effect. Their complementary advantages with T cells are expected to achieve a breakthrough in GBM immunotherapy. Even though NK cells are sometimes found to be dysfunctional or tolerogenic in the TME, improved knowledge of how NK cells are regulated in this context may allow therapeutic exploitation in the near future. Overall, NK cells are promising in the field of GBM treatment. Reasonable combination with various new technologies and equipment and learning more about how we can optimally exploit specific NK cell subsets with specialized functions to orchestrate efficacious immune responses against cancer are expected to give better play to the antitumor effect of NK cells.


The liver plays crucial roles in systemic immunity and greatly contributes to the systemic defense mechanism. Antitumor immunity in the liver is especially critical for the defense against systemic tumor cell dissemination. To achieve effective defense against metastatic tumor cells, liver immune cells with powerful cytotoxic activities construct a potent defense mechanism. In the liver, as compared with other organs, there is a significantly more intense percentage of innate immune lymphocytes, such as natural killer (NK) and NKT cells. These characteristic lymphocytes survey the portal blood transferred to the liver from the alimentary tract and eliminate malignant cells with their robust cytotoxic ability. Additionally, with their active cytokine-producing capacity, these innate lymphocytes initiate immunological sequences by adaptive immune cells. Therefore, they are crucial contributors to systemic antitumor immunity. These attractive immune cells help conduct a fundamental investigation of tumor immunity and act as a target of clinical measures for cancer therapies. This review discusses the mechanisms of these innate lymphocytes regarding recognition and cytotoxicity against tumor cells and the possibility of clinical applications for therapeutic measures. The liver is the largest organ in the body and is currently recognized as an immune organ due to its characteristic vascular structure and numerous innate immune cells. The portal vein collects whole venous blood drained out from the alimentary tract, and the blood is filtered through the liver before entering the systemic vascular system, which means that external pathogens and malignant cells from the gut are processed in the liver. Therefore, the liver is the front line of the body’s defense mechanism, tackling various kinds of pathogens transferred by the bloodstream; for this purpose, a liver-specific immune network is constructed by characteristic immune cells.Antitumor immunity in the liver is an essential function that is exerted by innate immune lymphocytes, such as natural killer (NK) and NKT cells. Compared to other innate immune cells, such as Kupffer cells and neutrophils, NK and NKT cells have significant amounts of cell-killing effector materials such as perforin/granzyme. Therefore, their primary function is eliminating malignant cells and virus-infected cells. These cells are classified into large granular lymphoid cells according to their structure. In contrast to conventional lymphoid cells, they have granules in their intracellular space that offer distinct functions. In immune organs such as the spleen and lymphoid nodule, the number of NK and NKT cells represents a small and relatively rare cell population. In the liver, however, these two cell types comprise almost one-third of the total lymphoid cells and are a major cellular component of the immune system. Their functions have been discussed for decades, and their major function is antitumor or anti-viral immunity. NK and NKT cells can recognize damaged or abnormal hepatic cells or cancer cells transferred from the alimentary tract and eliminate them from the parenchymal space, which contributes to antitumor and anti-viral immunity.The mechanisms of effector functions against tumor cells go through several steps, such as the recognition and cytotoxicity of target tumor cells. This review focuses on the mechanisms of tumor cell elimination exerted by innate immune cells in the liver and possible therapeutic applications to human cancer immunotherapy.In 1975, NK cells were uncovered as lymphoid cells with natural cytotoxicity against tumor cell lines . Given tAs NK cells do not express antigen-specific receptors with a diverse repertoire that can detect various kinds of individual proteins; they do not recognize specific tumor cells. Instead, NK cells distinguish tumor cells with receptors that can detect irregular signals expressed on the target cells, and the receptors are classified into activating and suppressing functions.Among the activating receptors are the Fc receptors CD16 and CD62, which recognize specific antibodies attached to tumor cells. Through these Fc receptors, NK cells can exert efficient cytotoxic activity against antibody-coated tumor cells, and this process is called antibody-dependent cell-mediated cytotoxicity (ADCC) . The othThe normal MHC class I antigen, which is expressed on intact cells, inhibit effector function on NK cells, therefore, tumor cells lacking MHC class I are vigorously eliminated by NK cells . BecauseImmune cells with potential cytotoxic activity use several kinds of effector molecules to induce apoptosis on target cells. One of the most effective mechanisms is the perforin-granzyme pathway, in which effector lymphocytes form pores on a cellular surface with “perforin” and transfer serin protease “granzyme” to induce an apoptosis reaction against target cells . Ohkawa Recent research, however, has revealed a brand-new perforin-related cytotoxic mechanism. Among the granzyme protein family subclasses, granzyme B is the primary molecule for the induction of apoptosis, and the other protein, “granzyme A”, has different cytotoxic activity. Granzyme A can induce pyroptotic death against tumor cells with gasdermin B protein, which is expressed abundantly in epithelial cells in the gastrointestinal tract . PyroptoThe therapeutic applications of NK cells against cancer patients are divided into two strategies: one is to activate NK cells in the patients to recruit them into cancer focus and exert cytotoxicity against malignant cells, and the other is isolating NK cells from the patients and transferring them back after adequate stimulation in vitro. In line with this strategy, IL-2 cytokine therapy for activating intrinsic NK cells, combined with injection of ex vivo stimulated NK cells, was applied to clinical studies . HoweverThe antitumor activity of NK cells is regulated by balancing between stimulating and inhibiting receptors. The appropriate modulation of this balance can then efficiently up-regulate the antitumor defense. The molecular targeting therapy aiming to block the inhibitory NK receptor can up-regulate the antitumor effector function by NK cells. For example, “Monalizumab”, the monoclonal antibody targeting NKG2A, can augment NK cell cytotoxicity and improve the survival rate of the experimental mouse model . This prNK cells are directly activated by pathogen-associated molecular patterns (PAMPs) receptors, and PAMPs receptor agonists can activate NK cells and augment the antitumor cytotoxic effect. The toll-like receptor (TLR) 7/TLR8 agonist “Imiquimod” can exert an antitumor effect on MHC class I deficient tumor cells with the assistance of CD4 + T cells . This agThe elucidation of unusual T lymphocytes with invariant T cell receptors was a significant focus of immunology research from 1990 to 2000 . For theFor conventional T cells, the function of the T cell receptor is to detect a wide variety of specific antigens; consequently, a significant diversity of the receptor repertoire is assembled. However, the invariant T cell receptor of NKT cells suggests that they use TCR in a different manner, possibly for detecting a wide range of antigens, such as pattern recognition receptors for PAMPs and damage-associated molecular pattern molecules (DAMPs). One of the characteristic features of NKT cells is their strict dependence on the cluster of differentiation 1d (CD1d) molecule  37]. CD. CD37]. Among the lipid-related ligands that attach this invariant TCR, α-galactosylceramide  is the most potent NKT cell activator . The admUnlike in mice, the number of human iNKT cells with Vα24 Vβ8.2 TCR is much smaller than the other classical T cells. Despite their limited cell number, considerable evidence from different groups suggests the enormous contributions of NKT cells to antitumor immunity. The number and function of iNKT cells strictly correlates with the prognosis of various human cancer patients , includiThe activation of iNKT cells by α-GalCer exerts a potent antitumor effect upon metastatic tumor models in mice and is expected to be an efficient antitumor immune therapy . It is nWhile the limited cell number has hampered the application of iNKT cells in human clinical procedures, there are abundant T cells co-expressing NK cell-related antigen CD56 in the liver. Moreover, their cell number is closely correlated with the fibrosis stage and hepatocarcinogenesis of chronic hepatitis C . This reIn the current medical procedure, immune checkpoint inhibitors such as programmed cell death (PD)-1 antibodies demonstrate favorable clinical results. However, these checkpoint inhibitors have not provided significant remission effects in primary liver cancer, such as hepatocellular carcinoma and cholangiocarcinoma, suggesting that currently available inhibitors do not directly affect NK and NKT cell functions . HoweverNK and NKT cells are non-antigen specific innate lymphocytes that enormously contribute to antitumor immunity. They have significant surveillance functions and produce cytotoxic activity against tumor cells in various non-lymphoid organs. They particularly accumulate in crucial filtering organs such as the liver and lung and exert their functions to protect against systemic tumor cell dissemination in cancer patients. Considering their potential cytotoxic activity, NK and NKT cells are promising candidates for cancer immunotherapy. Understanding their physiological function can elucidate the mechanisms of antitumor immunity and contribute to the development of efficient cancer therapy strategies. Therefore, NK and NKT cells in the liver and the lung are attractive research targets to overcome the current refractory cancer status.


This study investigated the association between autism and self-reported eating problems and the influence of gender on the association, in a sample of adolescent and adult twins (N = 192). Autistic traits and autism diagnosis were associated with both total and specific eating problems, including selective eating and sensory sensitivity during mealtimes. Interaction effects indicated a stronger association between autistic traits and total eating problems in females, as well as more difficulties with eating in social contexts among autistic females. In within-pair analyses, where unmeasured confounders including genes and shared environment are implicitly controlled for, the association was lost within monozygotic pairs, which might further indicate a genetic influence on the relationship between autism and eating problems.The online version contains supplementary material available at 10.1007/s10803-021-05198-z. Second, to investigate potential gender-specific associations between autism and eating problems. Finally, to explore the influence of shared genetic and environmental factors on the relation between autism and eating problems using a co-twin control design and within-pair analysis. Based on previous research and clinical experience, we predicted that autistic traits and autism diagnosis would be associated with increased eating problems, and we predicted that these associations would be found particularly in females. We did not set up a specific hypothesis for the third aim as previous research related to this aim is scarce.The sample included 192 twins , comprising 63 monozygotic (MZ) and 33 dizygotic (DZ) twin pairs, who participated in the Roots of Autism and ADHD Twin Study in Sweden (RATSS)  . From one family two twin-pairs had participated and one pair was thus excluded (n = 2). Furthermore, we excluded twin pairs where one twin had not completed the SWEAA (n = 2), or where one or both twins had completely missing data on SRS-2 (n = 6) or the Wechsler Intelligence Scales (n = 4). Finally, twins where zygosity had not yet been determined (n = 8), or where one twin had a moderate ID (n = 2) were excluded, resulting in the final sample of 192 participants. Figure nd edition (ADOS-G or ADOS-2)  , B. Motor control (7 items), C. Purchase of food (3 items), D. Eating behavior (6 items), E. Mealtime surroundings (11 items), F. Social situation at mealtime (10 items), G. Other behavior associated with disturbed eating (8 items), and H. Hunger/satiety (2 items) and two single-item subscales, I. Simultaneous capacity and J. Pica. See Table Perception subscale which consists of seven items, the mean of the available six items was used.Eating problems common in autism were assessed using self-ratings on the SWEAA, which is a questionnaire developed based on a literature review of eating problems among autistic adolescents and adults Råstam,  as well /satiety  items anAll analyses were conducted in R within the Generalized Estimating Equations (GEE) framework with doubly robust standard errors (drgee package) . The subscale models were adjusted for the variables described above. We conducted follow-up analyses using autism diagnosis as exposure for SWEAA scores that showed a significant association with autistic traits to confirm the clinical relevance of the results. To address our second aim, the adjusted models included the interaction term autism (traits or diagnosis) by gender, to allow investigation of gender-specific effects of autistic traits and autism diagnosis on eating problems. Furthermore, follow-up analyses stratified by gender were conducted for models showing a significant interaction between autism and gender. As all follow-up analyses were conducted to further explore and validate the associations between autistic traits and eating problems, these models were not Bonferroni corrected, i.e. the significance level was set to p = 0.05.For our third aim, within-pair analyses were conducted using conditional linear regressions in order to investigate the influence of genetics, shared environment and non-shared environment on the association between autistic traits and total eating problems. These analyses implicitly control for all variables shared within twin pairs, including shared environment and genes (100% of segregating genes for monozygotic [MZ] pairs and on average 50% for dizygotic [DZ] pairs). The within-pair analyses were only adjusted for ADHD, internalizing conditions, and IQ, as age and gender are implicitly controlled for. A within-pair analysis was first run in the entire sample (96 twin pairs), and subsequently split in MZ- and DZ-twin pairs. A weaker association in MZ-pairs compared to DZ-pairs indicates a genetic influence on the association, while a lost association within both MZ- and DZ-pairs would indicate a shared environmental influence. An association that remains within MZ-pairs is consistent with autistic traits having a causal effect on eating problems, but could also indicate a common influence from non-shared environmental factors.2 = 0.073) with a power of 0.80 given an α of 0.05, using a multiple regression with six predictors in across-individuals analyses.Power calculations for this study are approximal, due to the sample consisting of twins and the sampling approach. More specifically, participants were not selected randomly, rather the sampling aimed to include a high proportion of pairs discordant and concordant for autism and ADHD because these are most informative for studying these conditions. A post-hoc power analysis using G*power 3.1 .See Table Social situation at mealtime in both the model using autistic traits as exposure and the autism diagnosis model. Having at least one internalizing condition was associated with more self-reported problems with motor control when autistic traits were used as exposure, and higher scores on Mealtime surroundings and Social situation at mealtime in the autism diagnosis models. In the diagnosis models only, higher age was associated with reduced scores in subscales Perception, Mealtime surroundings, Social situation at the mealtime, and simultaneous capacity. Also, in the autism diagnosis model, higher IQ was associated with lower scores on Mealtime surroundings.Regarding covariates, male gender was associated with a higher score on the subscale Mealtime surroundings subscale , but not males. Figure Mealtime surroundings for females and males respectively. For further exploration of this interaction effect, Mealtime surroundings was split into items focusing on eating in social contexts (six items including difficulties eating in school or with friends) and non-social items focusing on rituals and rigidity in mealtimes (five items). An interaction effect was only found for the cluster of social items, where autistic traits and autism diagnosis predicted increased scores for females but not males. These analyses are described in detail in the Supplementary Material. Supplementary Table 1 provides details on the follow-up analyses for females and males separately.An interaction effect between autistic traits and gender was found for total eating problems , and in the adjusted model . The adjusted association was significant within DZ-pairs  but not within MZ-pairs . ADHD, internalizing conditions and IQ were not associated with eating problems in the above within-pair models. Figure Perception, Mealtime surroundings, and Social situation at mealtime, see Supplementary Table 3). In contrast, the subscale Eating behaviors showed a significant association within both MZ- and DZ-pairs. For Simultaneous capacity, no association was found within MZ- or DZ-pairs.The SWEAA-subscales that showed associations with autistic traits across individuals were followed up in separate within-pair models. Within-pair associations for three of the SWEAA-subscales followed the same pattern as total eating problems, showing significant associations only in DZ-pairs  autism and self-reported difficulties with eating in social situations outside of the home was found specifically among females. The interaction effects suggest that autism and eating behavior are differentially associated among females and males. In the within-pair analyses, the association between autism and total eating problems remained significant in DZ-pairs but was lost within MZ-pairs, which might suggest a genetic influence on the association. However, the interpretation of these results should be made with caution, as confidence intervals of MZ- and DZ-twins were overlapping.Social situation at the mealtime) and, particularly among female participants, with inflexible routines and rituals in mealtimes . While autistic participants in the SWEAA validation study self-reported more difficulties with Simultaneous capacity, our data only supported an association between autistic traits and scores on this subscale. Unlike the validation study, we also saw associations between autism and subscales encompassing sensory issues (Perception) and selective eating (Eating behavior), reflecting recent findings in the study by Spek et al.  participants with higher IQ displayed less problems, when autism diagnosis was used as exposure. Internalizing conditions were also related to a higher SWEAA total score, suggesting that certain eating problems associated with autism also occur in anxiety and depression conditions.Our findings also indicate that ADHD is related to an increase in total eating problems beyond autism diagnosis. This association was however not found in the autistic traits model, potentially reflecting that participants with ADHD also have elevated autistic traits  , the items in Social situation at mealtime reflects challenges in adapting behavior to others  and preference to eat alone.While autistic traits and autism were related to difficulties in eating with others outside of the home exclusively in females, a main effect of gender was found for the The link between adolescent and adult autism and the various issues around eating and mealtimes suggested by our results is often not investigated in health care services encountering this population, increasing the risk of the eating problems seen in our study going unnoticed. For clinicians, our results emphasize the relevance of assessing eating problems in this group, but also in the wider population displaying elevated autistic traits but not diagnosed with autism. Our data further suggests that this might be of particular relevance when meeting females with elevated autistic traits. Further, the eating problems in people with high levels of autistic traits and/or diagnosed autism might not be in the form of anorexia or bulimia nervosa symptoms but rather concern other areas such as eating a limited diet or experiencing problems with sensory aspects of food or mealtimes, which psychiatry services might not be sufficiently familiar with. Individuals presenting with the eating problems described in our study might suffer from consequential detrimental effects on quality of life and functioning, including social participation, even if criteria for an ED are not fulfilled. Behavioral interventions for severe eating problems show promising results for adults in the general population , corresponding to a recent twin-study which reported a genetic influence on the link between autistic traits and sensory sensitivity . Similarly, the association between autism diagnosis and overall eating problems was significant only among DZ-pairs when restricting the sample to the 16 pairs discordant for autism. Thus, the within-pair analyses might indicate a genetic influence on the association between autism and eating problems. While previous research indicates a substantial genetic influence in both autism . Finally, as the SWEAA lacks items focusing on some of the main characteristics of EDs, such as fear of gaining weight, its ability to yield a valid measure of ED symptoms is limited. Also, while this measure has shown diagnostic validity in discriminating between autistic and neurotypical groups . While a larger sample would have increased the statistical power, the current sample of twins underwent thorough assessments, and the analyses used both parent-rated autistic traits and clinician assessment of autism diagnosis, reducing the risk of rater bias.Our results indicate that autistic traits and autism diagnosis are linked to increased eating problems among adolescents and adults, both in total eating problems and in specific problems including sensory sensitivity in mealtimes and selective eating. The link between autistic traits and total eating problems as well as specific problems with eating in social situations was especially pronounced among females, suggestive of a gender difference in the association between autism and eating behavior. Furthermore, our results point towards a possible genetic influence on the link between autism and eating problems. Our study adds to previous studies in emphasizing that eating problems are common also among autistic adolescents and adults, and provides further evidence of gender differences in eating issues associated with autism. Our findings highlight the clinical importance of assessing eating problems among autistic adolescents and adults, as well as their potentially detrimental social consequences, particularly for autistic females. Future research should investigate the impact of eating problems on quality of life, mental health and social functioning among autistic adults, as well as potential gender-related pathways suggested by our data, including whether eating problems among autistic females contribute to mental health issues or social isolation particularly.Supplementary file1 (DOCX 57 KB)Below is the link to the electronic supplementary material.


Anti-neural autoantibody-associated psychiatric disease is a novel field in immunopsychiatry that has been attracting attention thanks to its potentially positive therapeutic outcome and distinct prognosis compared with non-organic psychiatric disease. This review aims to describe recent novel technological developments for improving diagnostics in the field of autoantibody-related psychiatric disease.We screened for relevant articles in PubMed for this narrative article. We focused on research methods such as neuroimaging, immune cells and inflammation markers, and molecular biomarkers in human biofluids like serum and cerebrospinal fluid and plasma proteomics.We introduce several novel methods for investigating autoinflammation with the aim of optimizing therapies for autoantibody-associated psychiatric disease. We describe measuring the translocator protein 18kDa in activated microglia via positron emission tomography imaging, brain volumetric assessment, flow cell cytometry of cerebrospinal fluid and blood, and blood biological probes as well as psychopathological cues to help us gain insights into diagnosing inflammation and brain damage better in psychiatric patients presenting a suspected autoimmune etiology.Our short methodological review provides an overview of recent developments in the field of autoantibody-related immunopsychiatry. More research is needed to prove their usefulness in diagnosing and treating autoantibody-associated psychiatric disease and its subtypes. Immunopsychiatry is a novel and expanding subfield of psychoneuroimmunology that deals with the interaction between our immune system and psychiatric disorders. Neural autoantibodies in psychiatric disease are attracting more attention due to their broad spectrum and link to disease pathogenesis. Autoantibody-associated psychiatric disorders are of growing interest as the therapy is demanding and differs from standard psychopharmacological drug therapy. Psychiatric disorders associated with neural autoantibodies can be classified as autoimmune-mediated psychiatric disease when these neural autoantibodies occur in conjunction with specific clinical features, signs of central nervous system inflammation in neuroimaging, or in biomaterial probes such as cerebrospinal fluid (CSF) –4.via protein arrays and detected six different autoantibodies associated with specific psychopathological features. Anti-adaptor-related protein complex 3, beta 2 subunit (AP3B2) antibodies were identified in conjunction with persecutory delusions, whereas anti-tryptophan 2,3-dioxygenase (TDO2) antibodies were more frequently co-detected with hallucinations in patients. Affective symptoms such as anhedonia and dysphoria were related to anti-recombinant WAS protein homologously associated with actin, Golgi membranes and microtubules pseudogene 3 (WHAMMP3) antibodies. Cognitive dysfunction was found to be mainly associated with anti-olfactomedin 1 (OLFM1) antibodies, and sleep dysfunction (insomnia) in patients was associated with anti-Gamma-crystallin N (CRYGN) antibodies. These examples in Jernbom Falk´s study (2021) demonstrate that specific psychopathologic features might help deliver clues about which autoantibodies might be worth searching for. However, the likelihood of detecting autoantibodies is much higher if specific syndromes are present, ie, a psycho-organic syndrome accompanying cognitive dysfunction or paranoid-hallucinatory syndrome, as a recent cohort study showed that psychotic symptoms and cognitive impairment are the most relevant psychiatric conditions associated with autoantibodies  encephalitis is modulated by NMDAR autoantibodies . NMDAR iImmune cells are potential biomarkers in patients with anti-neural autoantibody-related cognitive impairment and epilepsy, as recent investigations of ours showed , 20, 21.via PET imaging (TSPO). TSPO imaging has not yet been adopted in the clinical routine although the evidence regarding the detection of glial neuroinflammation in psychiatric disease has been promising. Further research should be extended to study the interaction between neurotransmitters such as noradrenaline and acethylcholine and neuroinflammation via TSPO imaging in psychiatric and age-related, disease-associated anti-neural antibodies. TSPO imaging has proven useful in neuropsychiatric disease associated with autoantibodies such as lupus erythematosus, as (via the specific radioligand for TSPO named DPA (11C]DPA-713). Stronger DPA binding was shown in hippocampal and cerebellar regions in cognitively-impaired patients with neuropsychiatric lupus erythematosus  Our short review indicates that there are several innovative techniques (summarized in The author confirms being the sole contributor of this work and has approved it for publication.Funding is derived from the Open access fund of the University of Göttingen.The author declares 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.