Studies have reported elevated rates of dissociative symptoms and comorbid dissociative disorders in functional neurological disorder (FND); however, a comprehensive review is lacking.To systematically review the severity of dissociative symptoms and prevalence of comorbid dissociative disorders in FND and summarise their biological and clinical associations.We searched Embase, PsycInfo and MEDLINE up to June 2021, combining terms for FND and dissociation. Studies were eligible if reporting dissociative symptom scores or rates of comorbid dissociative disorder in FND samples. Risk of bias was appraised using modified Newcastle–Ottawa criteria. The findings were synthesised qualitatively and dissociative symptom scores were included in a meta-analysis (PROSPERO CRD42020173263).n = 3940; control n = 3073), most commonly prospective case–control studies (k = 54). Dissociative disorders were frequently comorbid in FND. Psychoform dissociation was elevated in FND compared with healthy  and neurological controls . Greater psychoform dissociation was observed in FND samples with seizure symptoms versus healthy controls  and FND samples with motor symptoms . Somatoform dissociation was elevated in FND versus healthy controls . Dissociation in FND was associated with more severe functional symptoms, worse quality of life and brain alterations.Seventy-five studies were eligible (FND Our findings highlight the potential clinical utility of assessing patients with FND for dissociative symptomatology. However, fewer studies investigated FND samples with motor symptoms and heterogeneity between studies and risk of bias were high. Rigorous investigation of the prevalence, features and mechanistic relevance of dissociation in FND is needed. Increasingly, subjective cognitive difficulties, dizziness and auditory problems are being recognised and classified as FND.2Functional neurological disorder (FND) is characterised by alterations in motor and/or sensory function that are not explained by or not compatible with identifiable neuropathology, according to DSM-5.3 Dissociation as a neurocognitive process has been theorised to underlie FND, reflecting its categorisation in ICD-11, where the official term is dissociative neurological symptom disorder;5 here, it is classified as a dissociative disorder, alongside several other dissociative disorders, including dissociative identity disorder (DID) and depersonalisation/derealisation disorder (DPDR).2 Nevertheless, the conceptualisation of FND as a dissociative disorder, with dissociation as a core underlying mechanism, is not universally accepted. There is a paucity of high-quality empirical evidence supporting the conclusion that dissociation is a causal process in the generation of FND symptoms. Furthermore, there is no reference to dissociation as a mechanism in the DSM-5 classification of FND and so there is currently a notable and unhelpful discrepancy between the two major classification systems. The critical need to resolve this discrepancy necessitates more rigorous examination of the prevalence and potential mechanistic role of dissociation in FND in future studies.Dissociative symptoms are frequently reported in FND. A recent meta-analysis demonstrated this, but was limited by inclusion of one singular measure of dissociation – thus a broader inclusion of dissociation measures is needed.9 Although dissociation is considered a natural response to certain triggers, such as sleep deprivation or extreme fear , we focus here specifically on pathological dissociation.11 Dissociative symptoms are seen in a variety of psychiatric disorders;15 symptoms can be chronic and severely disabling.8Dissociation can broadly be viewed as the loss of control or awareness of cognitive or physical processes that are normally readily controlled or engaged in conscious awareness. As a symptom it can manifest as memory disturbance, subjective disconnectedness from the self  or external stimuli , loss of bodily sensation and voluntary control, or altered sense of identity.16 however, recent evidence from network analyses of dissociative experiences has extended this to include absorption  and depersonalisation/derealisation.18 In compartmentalisation, individuals lose the ability to govern processes or actions over which they would normally have control; it is theorised to underpin FND-seizures and dissociative amnesia, for example.19 In detachment, individuals ‘detach’ from the ordinary sense of integration of self, body or external environment.19Dissociation has previously been partitioned into compartmentalisation and experiential detachment;21 Various symptom rating scales screen for these clusters of symptoms. 20 whereas the Dissociative Experiences Scale (DES) predominantly assesses psychoform dissociation.22 Little is currently known about the biological processes underpinning dissociation, and although several areas for further exploration have been highlighted recently,15 this is a crucial direction for future research to improve recognition, understanding and treatment of dissociative disorders.Dissociative symptoms can also be categorised as somatoform symptoms, which are sensory  or motor , or psychoform, which relate to mental experiences such as memory impairment or depersonalisation.25 Individuals with a dissociative disorder are more likely to report self-harm and attempted suicide relative to other psychiatric populations, and psychiatric in-patients with a history of attempted suicide reported more severe dissociative symptoms than those without.26 Pathological dissociation contributes heavily to healthcare spending; however, timely diagnosis and treatment can mitigate this cost.27 Therefore, conditions involving significant dissociative symptom burden merit careful review with regard to risk assessment and management.Dissociative symptoms contribute to morbidity in psychiatric populations, especially in those who have experienced trauma. The dissociative subtype of post-traumatic stress disorder (PTSD), for example, has been associated with more severe illness.It is possible that dissociation is a negative mediator or prognostic marker in the overall clinical presentation and morbidity of FND. If true, a case can be made for broadening the treatment lens through which FND is managed by incorporating assessment and management of dissociative symptomatology. However, not enough is known about the extent of the role that dissociation plays in FND.The purpose of this systematic review and meta-analysis was to summarise the available evidence pertaining to dissociation in FND, as a symptom, comorbid disorder and potential prognostic marker. The primary aim was to critically appraise and report on rates of dissociative symptoms and disorders among people with FND. The secondary aim was to examine whether dissociation varies in severity in different FND symptom subgroups. The third was to report on the available data pertaining to biological and clinical associations of elevated dissociation in FND.A protocol for this review was registered on PROSPERO on 24 April 2020 (ref CRD42020173263).https://doi.org/10.1192/bjo.2022.597.The following databases were searched using the Ovid platform on 29 March 2020: Embase, PsycInfo and MEDLINE. The searches were updated on 1 June 2021. Studies were eligible for inclusion if they reported on comorbid dissociative disorder diagnoses and/or severity of dissociative symptoms in FND populations. The search terms for dissociation and FND are shown in the Appendix. Further articles were identified by reviewing the references of relevant systematic reviews, in addition to studies published during the selection process. The search strategy, including Boolean operators, is shown in supplementary Box 1, available at studies that reported on dissociative symptoms, as measured by validated rating scales 31)studies reporting on comorbid dissociative disorder diagnoses based on ICD or DSM criteria studies written in English, French, Spanish or Swedishstudies published from 1 January 1980 or later, reflecting when DSM-III was released and the term ‘conversion disorder’ was adopted, replacing the outdated ‘hysteria’.The inclusion criteria were as follows:studies that explicitly stated that participants with FND had a comorbid major neurological diagnosis, e.g. FND-seizures with comorbid epilepsystudies in which participants with FND were included in mixed samples with additional psychiatric or physical health diagnoses, without presentation of disorder-specific dissociation datareviews and meta-analysesnewspaper articles, editorials, non-peer reviewed sources, conference abstracts and other grey literature.The exclusion criteria were:After removing duplicates, all titles and abstracts were screened by pairs of authors (M.C.C. and S.P. or A.S. and S.P.) and any that clearly did not meet the eligibility criteria were removed. The full texts of the remaining articles were then screened for eligibility by one author (M.C.C. or A.S.); reasons for study exclusion were documented.For each study, the following information was extracted and tabulated by one investigator (M.C.C. or A.S.), where available: FND sample , control group type , sample size, average age, gender ratio, dissociative symptom scales used and dissociative disorders diagnosed. Mean/median values for dissociation scales were recorded where available, in addition to measures of dispersion. Data from eligible studies that additionally used objective biological measures were also extracted, as were data on any clinical associations of dissociation in these studies.32 Discrepancies were discussed and resolved, with input from a third rater (S.P. or M.C.C.) where necessary. The criteria used are shown in supplementary Table 2.Studies were evaluated for quality and potential risk of bias by two independent raters (A.S. and M.R.-A.) using modified Newcastle–Ottawa criteria for case–control, cohort and cross-sectional studies.20 Studies that gave measures of central tendency as medians were excluded unless the authors also gave means and standard deviations. Studies that reported data from more than one dissociation measure or symptom subgroup were included in each measure group. Standard errors were converted to standard deviations according to Cochrane recommendations.33The available data on rates of comorbid dissociative disorders, dissociative symptom scores and the clinical and biological associations of dissociation were first tabulated and synthesised qualitatively. We then conducted a meta-analysis of dissociative symptom scale scores. In the first meta-analysis, studies were included if they provided ‘adequate data’  for at least one validated measure of dissociative symptoms and control comparison. Studies with missing data were not included in the meta-analysis. A combined ‘psychoform dissociation measure’ was created by pooling data from the DES, Dissociation Questionnaire (DIS-Q) and Clinician Administered Dissociative States Scale (CADSS). The DES data were used for studies that reported more than one of these scales. The Somatoform Dissociation Questionnaire (SDQ) was calculated separately because it measures a different construct of somatoform dissociation.34 to calculate heterogeneity variance τ2, Hartung–Knapp adjustments35 and Hedges’ g effect size metric, and were run on Windows 10 using the meta-R package36 with guidance.37 Funnel plots were generated to assess the risk of bias due to missing results. Meta regressions incorporating risk of bias categories were used to assess confidence in the body of evidence for each outcome.Meta-analyses calculating dissociation scores used a random-effects model, with restricted maximum likelihood estimation36 All meta-analyses are displayed in forest plots. Subgroup analyses were interpreted using Cochran's Q.382I heterogeneity statistics were interpreted using recommended levels.39We were able to explore the heterogeneity of our data by subgroup analyses because our meta-analyses contained more than ten studies. Subgroup analyses were conducted on studies that provided data for at least one FND group and a least one comparison group. The first subgroup analysis investigated dissociation scores by control group . The second investigated the effect of FND symptom (seizures versus motor). Subgroup analyses applied a mixed-effects model using the subgroup function of the meta-R package.The results of the study selection process are shown in the PRISMA flow diagram . Seventyk = 54). FND samples were predominantly recruited in out-patient settings (k = 52). Of the FND participants, the most prevalent symptom profile reported was FND-seizures (n = 2160). FND-unspecified and FND-motor were the next most reported symptom types, with 956 and 523 participants respectively. Fifty-four studies included control groups. The most common control groups were healthy samples (k = 28), followed by epilepsy samples (k = 13). The mean age of FND participants ranged from 20.5 to 50.4 years. The female:male gender ratio ranged from 1.1:1 to 19.5:1; all studies that had male and female participants reported a greater female: male gender ratio. There were five studies with an all-female FND group and one with an all-male FND group.Details for each study – participant characteristics, design, principal findings, key strengths and weaknesses – are shown in supplementary Table 3. The majority of the included studies were case–control prospective studies  criteria to rate the quality of all cohort and case–control studies.There were possible risks of bias identified across the studies; details of the key strengths and weaknesses of each study are shown in supplementary Tables 4–6. Examples of possible sources of bias observed in some studies include (but are not limited to) the following: small sample size; inadequate control for confounding variables ; lack of control group(s); not explicitly stating that gold-standard diagnostic measures were used or not stipulating that diagnosis was made by a psychiatrist or neurologist; non-consecutive recruitment of FND participants.41 the lowest was 8% in a group of 13 participants with FND-seizures.42 The most common comorbid dissociative disorder identified was dissociative disorder not otherwise specified (DDNOS); the least common was dissociative fugue. Diagnoses of dissociative amnesia, DPDR and DID were also reported in a proportion of participants in several studies.k = 5) did not present measures of central tendency and are detailed only in supplementary Table 3.82 FND-motor and FND-sensory samples endorsed lower DES scores in several studies.97 Participants without a comorbid dissociative disorder also reported lower DES scores.48 Conversely, participants with FND-seizures, mixed symptoms or comorbid dissociative disorders appeared to present with higher DES scores relative to the other FND subtypes.96 Although some of the highest scores on the DES were seen in FND-seizures samples (e.g.82), this was not a wholly consistent pattern as DES scores were low for this symptom type in some studies.97Thirty-nine studies presented DES scores; the mean DES score ranged from 4.71, in a sample of 17 individuals expressing multiple unspecified FND symptoms, to 86.6, in a sample of 17 with FND-seizures.107 Mean or median scores exceeded 30 in six separate samples: four with the FND-seizures subtype; two with a comorbid dissociative disorder; one with recorded prior suicide attempts; one with a history of sexual abuse; and one with multiple symptom types.91 A statistically significant elevation in DES scores was reported in FND samples relative to controls in 18 comparisons, with control groups including both clinical and non-clinical participants.94Scores of 30 or more suggest severe pathological psychoform dissociation; this score is typically seen in dissociative disorders.102 Five articles reported one or more control group scores, with seven control groups in total; the majority (four) were healthy control groups.102 DIS-Q scores were significantly greater in FND samples versus controls in four comparisons.102 However, DIS-Q scores varied without any clear trends across FND subtypes; in some cases, FND group DIS-Q scores were equal to or lower than control group scores.95Eight studies measured dissociation using the Dissociation Questionnaire (DIS-Q).83 Boesten and colleagues52 observed significantly higher TSI dissociation scores in their cohort of patients with previous trauma compared with the non-traumatised cohort (P = 0.03), whereas Myers et al83 did not observe an appreciable difference in TSI dissociation scores when comparing people with epilepsy with people with FND-seizures (P = 0.97).Two studies, both with participants with FND-seizures, used the Trauma Symptom Inventory (TSI); this includes a dissociation subscale.56 Depersonalisation as measured by this scale was significantly higher in the FND-seizures than the FND-motor group.Demartini and colleagues presented data for the Cambridge Depersonalisation Scale (CDS) in 20 individuals with FND-motor symptoms and 20 with FND-seizures.100 Most scoring systems measure trait dissociation, whereas the CADSS measures state dissociation.29 Total CADSS scores were significantly higher in the FND group relative to controls in three studies.100 In one study the FND group CADSS score increased following a laboratory-based dissociation induction procedure.89 In one study, total CADSS score was higher in the epilepsy than in the FND-seizures group.49Four studies presented state dissociation scores using the Clinician-Administered Dissociative States Scale (CADSS).90 The MDI has six subscales measuring dissociation .28 Pick et al90 included all subscales whereas Koreki and colleagues76 reported data for the depersonalisation subscale. Both studies investigated FND-seizures samples compared with healthy control groups; MDI scores for FND-seizures participants were significantly higher than for control groups in both studies. Pick et al reported statistically significant elevations in dissociation scores for all subscales. After controlling statistically for anxiety and depression, elevations remained significant for four subscales .90Two studies used the Multiscale Dissociation Inventory (MDI).63 Although the FND-seizures group had higher PAS scores than healthy controls, this difference was not statistically significant.Some studies did not report significantly elevated dissociation in FND samples. Goldstein et al measured dissociative tendencies using the Perceptual Alteration Scale (PAS) in 20 FND-seizures participants.71 The pure FND-seizures group endorsed lower WDS scores than the other groups.One study used the Wessex Dissociation Scale (WDS) in samples with pure FND-seizures, pure epilepsy, comorbid FND-seizures and epilepsy, ‘other non-epileptic events’ and unclear diagnoses.103 The FDS has included 16 new domains that are mainly FND symptoms and is thus not a pure psychoform measure. Walther and colleagues reported a significantly higher FDS score in FND-seizures participants with ongoing symptoms relative to those whose symptoms had abated.103 Irorutola et al observed a statistically significant elevation in FDS scores in FND-seizures participants relative to healthy controls.72Two studies used the German-version of the DES, the Fragebogen zu Dissoziativen Symptomen (FDS).113 Thirteen samples of FND participants had SDQ-20 scores ≥35.86 The highest scores were seen in an FND-motor sample, an FND-mixed sample, an FND-seizures sample  and a sample of individuals with unspecified FND symptoms.86 Relative to other FND samples, several FND-seizures samples endorsed lower SDQ-20 scores; nevertheless, they still scored higher than the control groups in these studies.106 However, this was not consistent, as four FND-seizures groups had scores above 35.80Twenty-four studies presented SDQ-20 scores . A score106 Of these, five studies controlled for anxiety, depression or other psychopathology.101 In eighteen studies, SDQ-20 scores were statistically higher for FND participants relative to control groups.106 Conversely, SDQ-20 scores in FND groups were similar to or lower than scores in some control group comparisons, including PTSD, complex regional pain syndrome, dissociative disorders, chronic pelvic pain and one healthy control group.96Twenty-two studies included control groups; some studies had multiple control groups, amounting to a total of thirty different control groups.103 Two studies reported significant associations between dissociation scores and severity of ictal symptoms.93 Pick and colleagues observed that MDI depersonalisation and derealisation were positively associated with ictal mental state symptoms, whereas MDI identity dissociation was correlated with cognitive ictal symptoms; all of these associations were significant.90 Reuber and colleagues observed a weak but significant association between DES score and severity of ictal symptoms; this association was no longer significant after incorporating other psychopathological scores into the analysis.93Several studies examined the relationship between dissociation and severity of clinical outcome in FND-seizures samples. Statistically significant positive associations between dissociation scores and frequency of seizures were reported in five studies using various measures of dissociation.114 (DERS).55 Emotion dysregulation was positively correlated with psychoform and somatoform dissociation in an unspecified FND sample.55 More severe alexithymia and emotion dysregulation was reported in a subset of FND-seizures participants who scored higher on the SDQ-20 than the less emotionally dysregulated subgroup.53 Elsewhere, greater alexithymia severity was seen to vary with higher DES and SDQ-20 scores in a mixed FND sample.75 Quality of life of people with FND-seizures was significantly associated with dissociative symptoms in two studies.115 Both measured quality of life using the 31-item Quality of Life in Epilepsy Inventory (QOLIE-31).116 Individuals with higher degrees of dissociation measured by the DES had worse QOLIE-31 scores than those with lower DES scores. Two studies reported an association between history of sexual abuse, severity of clinical presentation and dissociation scores.90Two studies measured emotion dysregulation in FND participants using the Difficulties in Emotion Regulation Scale101 DES scores were significantly higher in a sample with multiple FND symptoms who had attempted suicide than in a group who had not.68 Significant positive associations between severity of dissociation and various psychological features were reported in several samples of FND subtypes, including measures of alexithymia, emotion dysregulation, fearful attachment style and post-traumatic avoidance symptoms.117 Other associations included significant positive correlations between dissociation and number of FND symptoms, early onset of FND, in-patient treatment and rates of comorbid psychiatric illnesses.96One study found a significant association between psychological dissociation and psychopathology  in an FND-motor group.The subset of studies that used objective means to study biological associations are described in 1-weighted structural magnetic resonance imaging to compare participants with FND-motor symptoms and healthy controls.88 FND participants with higher SDQ-20 scores  had significantly reduced cortical thickness in the left caudal  anterior cingulate cortex (ACC) relative to controls; this association was not present in the complete FND cohort. Conversely, higher DES depersonalisation/derealisation subscores correlated with increased right lateral occipital cortical thickness in participants with FND.Perez and colleagues investigated cortical thickness in people with FND by use of T118 In the first study,118 participants with FND-seizures were compared with healthy controls during four separate fMRI phases, comprising pre- and post-resting states and two cognitive tasks. No significant differences were seen between the FND-seizures group and healthy controls during tasks. However, significant correlations were observed between functional connectivity values and DES, DIS-Q and SDQ-20 scores. Of note, in the FND-seizures cohort functional connectivity was significantly higher within the ACC and inferior frontal gyrus, which also correlated significantly with DES scores.Van der Kruijs and colleagues used functional magnetic resonance imaging (fMRI) in two studies to investigate alterations in functional connectivity in people with FND-seizures.102 Relative to controls, those with FND-seizures were observed to have increased co-activation of resting-state networks in frontoparietal , executive (cingulate and insular cortex), sensorimotor  and default mode  networks. There was a significant positive correlation between increased functional connectivity in these regions of interest in the FND-seizures group and all dissociation scores .In a subsequent study, participants with FND-seizures and healthy controls underwent resting-state fMRI.117 Xue and colleagues performed EEG with 15 participants with FND-seizures and 15 matched controls; all participants additionally completed the SDQ-20.106 The FND-seizures group had less linkage between frontal and posterior brain areas relative to controls; no significant associations between clustering coefficients and SDQ-20 scores were found.Two studies employed electroencephalography (EEG) to investigate brain connectivity in FND participants.117 Participants performed one of three emotionally arousing tasks during EEG recording and completed the SDQ-20. This protocol was repeated after a 4 week treatment period of physiotherapy and psychological therapy. Both groups had similar cortical regions of interest in response to unpleasant or neutral stimuli, as well as similar EEG representation during the emotion regulation task (referred to as the ‘regulation effect’). No significant change in these findings was observed after the treatment period; however, a significant association between the regulation effect and SDQ-20 score was seen in the FND group.Kienle et al investigated possible cortical indices of emotion regulation in a mixed FND sample and matched controls during an emotion regulation task.89 Skin conductance levels (SCL) represent baseline or tonic level of conductance of skin, whereas skin conductance response (SCR) represents a phasic change of skin conductance in response to a stimulus.119 Pick and colleagues measured SCL in 17 participants with mixed FND symptoms and matched controls throughout a mirror-gazing dissociation induction protocol.89 The FND group had greater average SCL than the controls at all measured points of the protocol; however, this group effect was not significant. No significant associations between dissociation (CADSS score) and SCL were noted.Skin conductance is used as a metric of autonomic response to stimuli, employed by two of our included studies.70 The DES was used to measure dissociative tendencies. SCR amplitude was significantly lower in the FND-seizures group than in controls in response to all images (P = 0.04); however, in response to negative images only there was no significant group difference. A significant negative correlation between DES score and SCR amplitude was observed in the FND-seizures group. A non-significant trend of increased SCR amplitude in response to strong-arousal negative images compared with low-arousal negative images was observed in the FND-seizures group but not in the control group.Herrero et al studied physiological, cognitive and behavioural emotional response to image stimuli in 34 female FND-seizures participants and 34 matched controls.82 Participants followed a daily protocol of actigraphy and recording of state dissociative symptoms , mood, number of FND-seizures and subjective sleep quality. The FND-seizures group reported more disturbed sleep overall than the control group; however, the only significantly worse parameter was sleep quality. Objectively, participants with FND-seizures had significantly worse sleep with respect to efficiency, awakenings and wakefulness after sleep. DES scores were significantly higher in the FND-seizures group relative to the control group. A multivariate linear mixed model did not find any association between sleep parameters (sleep time and number of awakenings) and state dissociation the following day.Mousa and colleagues investigated objective and subjective sleep complaints in a sample of 17 participants with FND-seizures and 20 age- and gender-matched healthy controls; the DES measured dissociative tendencies.100 Participants were asked to stand on a stabilometric platform under various conditions . Physicians administered the CADSS to assess state dissociation. The degree to which participants swayed on the platform under the different conditions was recorded. A greater radius of swaying was noted in the FND group relative to controls; this was more pronounced during the eyes-closed procedure. Distraction using the arithmetic task improved postural stability in the FND group. A significant correlation between dissociative symptoms and postural instability was observed, with higher dissociative scores associated with postural instability during the eyes-closed procedure. However, this was a total sample observation (participants with FND and controls), and this correlation became insignificant when examined in the two groups separately.Stins and colleagues investigated postural control in a sample of participants with FND and matched controls.k = 9, n = 659). A higher score indicates a greater level of somatoform dissociation. The variance between groups was substantial (I2 = 85.5%), indicating a substantial degree of heterogeneity between the studies. A funnel plot of data available for this meta-analysis is presented in supplementary Fig. 1. It shows asymmetry, which could indicate publication bias. However, this plot also includes a study  identified in sensitivity analyses as a potential outlier. When this study was removed, the prediction interval shifted to above zero , although heterogeneity between studies was only reduced to 75%. Supplementary Figs 2 and 3 shows the forest and funnel plots when Demartini et al is removed. In the original meta-analysis, all studies were case–control, five were at high risk of bias and four were at low risk of bias . A meta-regression showed that the studies’ risk of bias category is not a significant effect size predictor (P = 0.50). All studies included in all meta-analyses were case–control studies because of our data inclusion principles outlined in the Methods section above.k = 36, n = 3031). Overall, the test between control subgroups was not significant (Q(2) = 5.77, P = 0.056). The FND groups showed a significant effect towards increasing psychoform dissociation compared with healthy controls  and neurological controls . Psychiatric controls also showed an effect towards lower dissociation scores compared with the FND groups but heterogeneity was very high , suggesting that, across studies, the psychiatric samples were too dissimilar for interpretation at this stage. A funnel plot of data available for this meta-analysis is given in supplementary Fig. 4. It shows some smaller studies with large effect sizes, which might indicate some publication bias. All studies in this analysis were case–control, 21 data comparisons were from studies with high risk of bias, and the final 15 were from studies with low risk of bias. A meta-regression showed no significant effect of the studies’ risk of bias category (P = 0.97).k = 14, n = 799). Four valid data comparisons were available for healthy controls and FND-motor samples and ten for FND-seizures. The FND-seizures group showed a significant effect of increasing psychoform dissociation compared with the FND-motor group (Q(1) = 5.44, P = 0.020). As there were only four data points available for the FND-motor group, compared with ten for FND-seizures group, we checked results assuming common heterogeneity (τ2 = 0.071) and the effect remained significant (P = 0.020).g = 0.94, 95% CI 0.65–1.22). There was also an effect towards greater dissociation scores in the FND-motor group compared with healthy controls; however, this was a smaller effect compared with that for FND-seizures . Estimates of between-study heterogeneity ranged from 53.7% for FND-motor to 42% for FND-seizures and was 58.3% overall; therefore, we can be confident that each group represents the target population. A funnel plot of data available for this meta-analysis is given in supplementary Fig. 5. As above, it shows a study with a large effect size, despite large standard error.82 All studies included were case–control. Data comparisons were available from six studies at high risk of bias and eight at low risk of bias. A meta-regression showed no significant effect of the studies’ risk of bias category (P = 0.81).The effect of FND-seizures on increasing psychoform dissociation was significant compared with healthy controls, with a large effect size  – a subtype previously highlighted as being the most prevalent in epidemiological studies.122 When compared with prevalence rates reported for the general population, dissociative disorder rates are higher in FND groups. A review of dissociative disorder prevalence rates in community and clinical samples suggested an overall lifetime prevalence of 10%.123 Similarly, a recent meta-analysis of college student populations (n = 31 905) reported an overall prevalence of 11%.124 Prevalence of dissociative disorder might be higher in clinical populations; for example, Foote and colleagues observed an overall prevalence of 29% in 82 consecutive out-patient psychiatric clinic attendees, and Ross et al observed considerably higher lifetime prevalence in in-patients (28–44.5%).126 A separate study found that general psychiatric in-patients were more likely to have a comorbid dissociative disorder if they scored above 30 on the DES, with patients in this bracket having a prevalence of up to 80%.127 The majority of studies reviewed here reported prevalence rates within the clinical range observed by Ross et al, and in some cases considerably higher. However, two of the included studies45 had prevalence rates of 10% or lower, commensurate with the prevalence rate of dissociative disorders in the general population.124Fewer studies than expected commented on comorbid dissociative disorders in FND samples. In the nine studies that did, the proportion of participants with FND found to have comorbid dissociative disorder ranged considerably, from 8% to 80%.4 Given that dissociative disorders are prevalent in other psychiatric populations,126 dissociation as an explanatory mechanism for other mental disorders merits exploration. For FND, this proximity invites questions about treatment options and whether some people with FND would benefit from treatments that target dissociative disorders and symptoms, including those currently being evaluated in FND such as eye movement desensitisation and reprocessing (EMDR)131 and dynamic psychotherapies.133The prevalence of dissociative disorders seen in the samples with FND presented in this review supports the notion that FND and dissociative disorders might share similar mechanisms and aetiology , in addition to symptoms, supporting the classification of FND as a dissociative disorder in the ICD.Dissociative symptoms, as measured through validated scales, ranged widely in the FND samples. However, most studies reported elevated dissociative symptom scores in FND samples that were above clinical cut-off scores and/or the scores reported for comparison groups. Elevated scores were observed for both psychoform and somatoform manifestations of dissociation.3 In that meta-analysis, the mean DES score for FND was reported at 25.6, based on 20 studies, and dissociative disorders had a mean score of 38.9;3 11 studies from our review reported mean or median scores higher than this, and 4 reported scores above the mean score for dissociative disorders. We found 11 studies that used other psychoform dissociation scales  – the majority of FND groups scored higher on these dissociative measures when compared with controls.The DES was the most used measure of psychoform dissociation. DES scores have recently been meta-analysed in psychiatric populations.In our subgroup meta-analysis investigating psychoform dissociation in FND groups versus different control groups, a large effect towards higher dissociation scores was found in the FND groups compared with healthy controls. This effect was also observed in the comparison with neurological controls, but of a medium effect size. High heterogeneity prevents us from confirming an effect between FND groups and psychiatric controls. The last finding may have been affected by the amalgamation of multiple psychiatric populations into one group. Four out of eight studies included a mixed psychiatric control group, while the remaining four involved somatisation, eating disorder, chronic pain and depression.134 Using these as a reference, FND samples  endorsed mean or median SDQ-20 scores >30, supporting the view that compartmentalisation is a significant feature of FND. Our meta-analysis comparing SDQ scores in people with FND and healthy controls displayed high heterogeneity between studies , meaning it is likely that the FND samples contained important subgroup populations that would need to be studied before effect size estimates could be interpreted. However, subgroup analysis was not possible as there were fewer than ten studies.The SDQ-20 was the next most used dissociative symptom scale, and the only ‘pure’ measure of somatoform dissociation. Mean SDQ-20 scores in samples with dissociative disorder have been reviewed previously, observing mean scores >30 for FND samples, >43 for DDNOS and DPDR samples and >50 for DID samples. studies ; however66 for example, reported the highest DES scores in an FND-seizures sample (mean score 27) relative to FND-motor (mean 12) and FND-sensory samples (mean 7). Nearly all of the FND-motor sample DES scores were comparable to the aforementioned mean scores of non-clinical populations, or substantially lower.135 Only in Tezcan and colleagues’ study did an FND-motor group exhibit high psychoform dissociation (mean DES = 25).48 Demartini and colleagues directly compared FND-seizures and FND-motor groups and noted an inverse relationship between DES and SDQ-20 scores in the two groups .56 This observation was not totally consistent; some FND-seizures groups scored highly on the SDQ-20 , with no clear methodological differences that could account for this.Our results suggested that there might be different dissociative symptom profiles across FND subgroups, specifically elevated psychoform dissociation in FND-seizures and greater somatoform dissociation in FND-motor samples. Guz and colleagues,136 The authors argued therefore that compartmentalisation symptoms are more characteristic of FND-seizures, whereas detachment symptoms are less prominent. In our review, the highest DIS-Q score was also in an FND-seizures group.85 Perhaps, therefore, these results indicate that people with the FND-seizures subtype have a greater overall tendency to dissociate in general, with respect to both somatoform and psychoform symptoms. The discrepant findings noted here require further examination in additional studies using measures of both somatoform and psychoform dissociation in FND-seizures and FND-motor samples.Significantly higher SDQ-20 scores in an FND-seizures sample relative to epilepsy controls were reported in a study in which the DES and CDS were unable to differentiate participants with FND from controls.134A potential explanation of the observed trends is that FND motor symptoms are indeed comparable to so-called somatoform dissociative symptoms, whereas psychoform dissociative symptoms are more common in people with FND-seizures. Indeed, the SDQ-20 contains several items that map directly onto FND motor symptoms, whereas there is only one item regarding attacks resembling seizures. One of the developers of the SDQ-20 has already called for physical ‘conversion’ symptoms to be relabelled as somatoform dissociative symptoms.97 and our subgroup meta-analysis indicated that FND-seizures groups exhibited different levels of psychoform dissociation from FND-motor groups. This meta-analysis indicates the need for more FND-motor and healthy control comparisons. Importantly, future studies should examine closely the effect on SDQ-20 total scores of items pertaining to the particular FND symptoms experienced by each respondent. One methodological solution is to rerun analyses excluding items that resemble FND symptoms common in the sample under investigation.Several limitations affect the generalisability of the observations outlined above. The majority of studies that addressed FND subtypes focused on FND-seizures; very few studies specified the FND subtype and most involved people with FND endorsing multiple symptoms. There were only three studies presenting FND-sensory or ‘mixed’ (F44.6 and F44.7 respectively) subtype specific scores137 Reporting bias, and additionally recall bias, are also potential limitations of self-report scales, which may be mitigated by use of scales that are clinician-administered and/or state-based, such as the CADSS.Although the DES and SDQ-20 scales were the most commonly used dissociation scales, most studies did not administer both and thus did not screen for both types of dissociative symptoms. It has been suggested that rather than existing as a general attribute, dissociation represents a diverse and not necessarily connected cluster of symptoms; consequently, scales such as the MDI, which measure specific psychoform dissociative symptoms such as depersonalisation and identity dissociation, might be more valuable for assessing dissociative symptom profiles.The large values for measures of dispersion seen across the studies indicate that although some individuals with FND report elevated dissociative tendencies, there are some who appear less affected by dissociation. If specific FND presentations present with different types of dissociative symptoms, as suggested by our results, then omission of measures of psychoform and/or somatoform dissociation might result in falsely low or unrepresentative results. However, another interpretation is that dissociative symptoms, although common, are not a ubiquitous feature of FND. More studies that administer both psychoform and somatoform dissociative scales to people with various FND subtypes will help to establish more concretely whether the observed trends represent the population.15 recently systematically reviewed studies exploring potential biomarkers associated with pathological dissociation transdiagnostically, including dissociative disorders, FND, and affective, personality and stress-related disorders.15 Increased neural activity was observed in several brain regions in the dissociative groups relative to healthy or clinical control groups, in both resting-state and task-based functional neuroimaging studies. This was consistent in regions of the prefrontal cortex, insula and ACC – findings also reported by Drane et al in a previous review138 and in models of dissociation in specific disorders, including PTSD and DID.140Little is known about the biological processes underlying pathological dissociation. Roydeva & Reinders15 however, this process may be mediated by other comorbid disorders, such as depression.141 Further research is needed to better understand the neurobiological basis of dissociative symptoms and disorders.Trends in connectivity alterations included increased connectivity from amygdala seed regions to prefrontal cortex , precuneus and superior parietal cortex. In addition, structural imaging studies found evidence of volumetric reductions in several regions in the dissociative groups, including the hippocampus, thalamus and basal ganglia. Other trends included a negative correlation between tumour necrosis factor alpha and dissociative symptoms, and a positive correlation with interleukin 6, raising questions about an interaction between inflammation and dissociative symptoms;15 This is likely due to differing study inclusion criteria; we specified that our studies describe dissociative symptoms or comorbid disorders with validated measures in people with FND, whereas the aforementioned review accepted diagnosis of FND alone as a marker of dissociation. Despite this disparity, there were some common trends noted: three of the identified studies from our review shared consistent findings with some of the reports of altered neural circuitry described above.118 Volumetric and functional connectivity changes were observed within several brain regions, including the ACC – a region implicated in action planning, decision-making and empathy-related responses. The ACC is consistently highlighted as an area of interest in FND studies.143 Increased functional connectivity and neural activity within this cortical region, among several other related regions described earlier, has been advocated as a potential biomarker for pathological dissociation and for FND.144 Whether these demonstrable alterations in neural circuitry can be translated into an in vivo biomarker of disorder severity or response to treatment remains to be explored. Studies applying this method have shared promising results.146Our review found a noticeably smaller number of studies examining the relationships between biological factors and dissociative symptoms in people with FND when compared with the Roydeva & Reinders review.148 The first reported on cortical thinning in similar brain regions, using voxel-based morphometry and cortical thickness MRI techniques.147 Significant loss of grey matter volume in participants with FND-seizures was observed in primary motor and premotor cortices, the supplementary motor area and the ACC.147 Thickness alterations in these regions of interest were not associated with dissociation (on DES and SDQ scores); however, SDQ scores were significantly negatively associated with reduced cortical thickness in the left inferior frontal gyrus and the left central sulcus.147 The second, more recent, study contrasted similar brain regions of interest in subgroups of healthy participants and participants with depression and FND-seizures.148 Relative to healthy controls, both the depression and FND-seizures subgroups had noticeable thinning of the ACC and lateral orbitofrontal cortext on MRI. Although this was more pronounced in participants with depression, a significant positive relationship between SDQ scores and morphological changes in the right medial orbitofrontal cortext was observed.148Two relevant studies from Labate and colleagues could not be included in this review as they did not meet our inclusion criteria.89 These inconsistent findings are similar to those previously reported in Drane et al's review.138 One possible explanation is that in FND, interoceptive impairments lead to a discordance between subjective and autonomic responses. This is reflected further in other studies that have reported impaired interoceptive accuracy of people with FND.89 Owing to the limited number of studies found and the variety of objective measures, a meta-analysis was not possible for the biological associations of dissociation in FND.Other biological findings were more variable. Two studies measured skin conductance levels in people with FND, with inconsistent results.103 Quality of life in people with FND-seizures was also reportedly affected by degree of dissociation, even after controlling for a number of other factors.115 Other studies observed varying severity of presentation with level of dissociation in general FND populations. The concerning link between dissociative symptoms and suicide has been established previously in psychiatric samples.149 More longitudinal studies could therefore be helpful to further establish the link between dissociative symptoms and FND severity or quality of life.Several studies in this review offer compelling evidence in support of using dissociative tendencies, symptoms or comorbidity as a potential prognostic marker in FND. Many of these studies pertained to the FND-seizures subtype. Severity or frequency of seizure symptoms, and presence of ongoing seizures relative to remission states, were seen in people with FND who reported greater degrees of dissociative symptoms.75 Number of FND symptoms, earlier onset of symptoms and likelihood of symptom severity requiring in-patient management were also predicted, to a degree, by dissociative symptoms.46 Other findings included elevated general psychopathology and psychiatric comorbidity in FND participants with greater dissociative tendencies.101Several studies observed that alexithymia and emotion dysregulation were associated with dissociation severity in FND participants.99 It might be beneficial to measure psychoform or somatoform dissociative symptoms as an outcome for FND in future studies. In addition, high scores on dissociation measures in people with FND also reporting previous trauma might indicate the presence of post-traumatic symptoms,90 which would need to be addressed specifically in any effective clinical intervention. The associations between traumatic experiences, dissociative and post-traumatic symptoms in FND are important and understudied; although this unfortunately goes beyond the scope of this paper, it merits further exploration in future studies.Taken together, these findings have important clinical implications. Dissociation scales might provide a novel means of categorising FND severity, prognosis and guiding treatment. Reflecting this notion, some of the studies in this review adopted the SDQ-20 specifically as a marker of FND severity.Our review has several strengths. First, we have comprehensively summarised existing data on both dissociative disorders and dissociative symptoms across different FND symptom types, and our subgroup analyses have highlighted potentially important subgroup differences. We also included studies that used a range of validated dissociation measures, ensuring that our review captured as much of the existing data as possible and providing insights into the relative prevalence of different types of dissociative symptoms  in the FND samples. Our findings have also indicated several important directions for future research on biological mechanisms and clinical implications of dissociation in FND.We acknowledge some limitations in the methods used. Regarding the search strategy, terms for cognitive FND symptoms were not included because we designed the search strategy in 2019, before these symptoms were formally classified as FND in ICD-11. Overall, the meta-analyses indicated a general problem of large heterogeneity between studies, which limits the certainty with which some conclusions can be drawn. This is especially evident in the neurological and psychiatric control groups, which each subsumed several different clinical disorders. In addition to this, funnel plots all showed asymmetry within the data. There are cases of small studies reporting a large effect size and large standard error, which is a potential indictor of publication bias. However, this could also be reflective of the high heterogeneity and the presence of outliers. We were also unable to explore the influence of associated risk factors such as trauma on dissociation scores in the meta-analysis, which might be of value in future. Generally, there were only a small number of studies that investigated the relationship between dissociation scores and biological measures, with inconsistent findings on several measures. Finally, although we excluded any study that stated that participants with FND had comorbid neurological disorders, not all studies explicitly declared this , and therefore there is a possibility that some of the data included may have not been FND-specific.The findings presented here indicate that dissociation is a pervasive and clinically relevant feature in FND, also lending support to the view that dissociation is a possible underlying mechanism. Further research is needed to examine more rigorously the role of dissociative processes in the generation of FND symptoms, with methods designed to examine causal hypotheses. Improved understanding of the role of dissociation in FND will have implications for diagnosis and classification, with potential to facilitate resolution of the discrepancy between the ICD-11 and DSM-5 classifications.Despite the considerable literature reviewed here, few studies investigated the potentially differing degrees of psychoform and somatoform dissociation in different FND subtypes. Furthermore, few studies explicitly sought to examine the distinction between compartmentalisation and detachment subtypes of dissociation in FND subgroups. More studies should closely examine these different forms of dissociation, their underlying aetiological and neurobiological basis, and how they relate to the occurrence of core FND symptoms such as seizure, motor, cognitive and sensory symptoms.From a clinical perspective, this review highlighted several possible implications. To accurately capture all of the symptomatology and experiences of patients with FND, clinicians might consider screening for both somatoform and psychoform dissociative symptoms, as they are evidently overexpressed in this population and are associated with adverse clinical features and outcomes. For individuals with FND who report significant dissociation, a dedicated clinical interview to assess possible comorbid dissociative disorders would be another consideration, as would be a more detailed screening for past trauma and possible post-traumatic symptoms.A key research question is whether level of dissociative symptoms could constitute a prognostic indicator of FND severity. Lastly, there are some appreciable potential biomarkers of dissociation that, if explored further, could aid in understanding how this process occurs and potentially act as guides to monitoring treatment efficacy and/or developing novel treatments for the disorder.


Advanced imaging is key for visualizing the spatiotemporal regulation of immune signaling which is a complex process involving multiple players tightly regulated in space and time. Imaging techniques vary in their spatial resolution, spanning from nanometers to micrometers, and in their temporal resolution, ranging from microseconds to hours. In this review, we summarize state-of-the-art imaging methodologies and provide recent examples on how they helped to unravel the mysteries of immune signaling. Finally, we discuss the limitations of current technologies and share our insights on how to overcome these limitations to visualize immune signaling with unprecedented fidelity. Immune signaling processes occur over broad spatial and temporal scales. The spatial reorganization of immune signaling ranges from large micron-sized patterns, termed supramolecular activation clusters (SMACs) to signaling micro-clusters and nanoclusters below the resolution limit of conventional light microscopy ,2. The tThe triggering of the T-cell receptor (TCR) occurs within a few seconds of cell–cell contact , which iThe interface between a glass coverslip and immune cells presents an attractive platform for imaging signaling processes occurring at the plasma membrane. Total internal reflection fluorescence microscopy (TIRFM) can be applied to create an evanescent wave that penetrates 100–200 nm into the cell , enablinThese methods all attempt to model a cell–cell interface, typically by coating the coverslip with a supported lipid bilayer (SLB) that incorporates membrane proteins. However, this presents challenges as the physiological relevance of SLBs can be questioned due to unknown role of the glycocalyx , unexpecFluorescence images of an immunological synapse (IS) were first acquired using confocal microscopy in the late 90s ,19. CommWidefield fluorescence techniques utilize cameras to overcome scanning-induced speed limitations of confocal microscopy, but with reduced axial sectioning abilities. Structured illumination microscopy (SIM) is a widefield super-resolution microscopy technique that uses interference patterns to acquire multiple patterned fluorescence images from varying directions. These sequences then enable reconstruction of super-resolution images. This practically doubles resolution in all dimensions to achieve 125 nm and 350 nm resolutions in lateral and axial dimensions, respectively. In terms of immune signaling, most dynamic imaging studies to date have been performed in 2D . CombinaConfocal, SIM and epifluorescence microscopy are all prone to photobleaching as most of the sample is continuously exposed to laser excitation. To achieve gentler imaging, light-sheet microscopy  techniques have been developed, which selectively excite fluorophores within the focal plane of the sample. This combines the advantages of TIRFM and off-surface methods to enable 3D imaging with axial sectioning and high contrast due to reduced out-of-focus excitation. However, the difficulty in applying light-sheet microscopy to bioimaging has always involved the complexity of introducing the light sheet into the sample. Given the need for high numerical aperture objective lenses to visualize signaling processes, a variety of complex solutions have been developed including tilted objective lenses ,26, specA major improvement came with the development of lattice light-sheet microscopy (LLSM) that offered optical sectioning  over a large field-of-view (50 µm) ,32. ThisDynamic diffraction-limited 3D imaging of immune cells has mostly been solved with above introduced techniques, although there are needs in terms of imaging deeper into tissue and simplifying setups for increased accessability. While a great deal can be learned, understanding of immune signaling processes on the molecular scale requires nanoscale resolution, thus the field is in need to combine its efforts with super-resolution imaging techniques while maintaining the high temporal resolution offered by the dynamic 3D imaging techniques.Super-resolution imaging modalities paved the road to investigate immune signaling down to individual signal events initiated or mediated by single receptors, kinases, phosphatases, adaptor molecules or other contributing biomolecules. Nowadays commercial availability of super-resolution microscopes facilitates their active utilization to study cellular processes, such as immune signaling, on the nanometer scale. Common to all super-resolution modalities discussed in this section is the suppression of fluorescence emission from labels coupled to the biomolecule of interest to achieve spatial resolutions far below the classical resolution limit of light microscopy (∼250 nm).In stochastic and camera-based approaches like (direct) stochastic optical reconstruction microscopy ((d) STORM) ,39, flu.Recording thousands of frames for super-resolution imaging takes minutes to hours, rendering the observation of biomolecular dynamics challenging . TherefoIn contrast with these ‘single molecule localization microscopy' (SMLM) techniques, stimulated emission depletion (STED)  or grounOf note, long before the advent of super-resolution imaging, the high precision in localizing individual signals was utilized in single-particle or single-molecule tracking  to extract information about locations, kinetics, dynamics and interactions of individual molecule. The basic concept is to directly observe the motion of individual signals in live cell experiments and to extract the aforementioned information from trajectories generated from localized particles or molecules . High-spSuper-resolution techniques immensely contribute to our understanding of immune signaling by identifying spatiotemporal positions of individual key molecules on the nanometer-scale. However, the precise mode of action of these molecules within the signaling process needs to be addressed separately. One parameter gaining increasing attention in the field of immunology is the exertion of or response to mechanical forces .The structural plasticity of immune cells facilitated by cytoskeletal remodeling has been shown to exert a substantial amount of force on neighboring cells, on the extracellular matrix, and to the components of the IS. It changes the conformation of individual molecules, initiates cluster formation, and alters bond kinetics and biophysical properties such as membrane stiffness. Remodeling of the cytoskeleton, similar to cell spreading and motility, results in cycles of pushing and pulling ,73, whicWhile we reviewed techniques to probe mechanical forces elsewhere , the fieBesides the structural dynamics of IS formation , to trigTo detect forces on the single TCR–pMHC bond level, DNA-based tension sensors were instated, suggesting forces as high as 12 pN for ligand-bound TCRs  on glassCellular biophysical properties such as cellular deformability, stiffness, membrane fluidity, viscosity and tension differ for different cells, cellular states and diseases ,96. SuchImmune cells undergo extensive biophysical remodeling during their lifetime which is necessary for their function. A recent example by Lühr et al.  using enBiophysical properties of the IS attracted particular attention in the last decade. There is significant evidence suggesting a major role of biophysical properties of IS as regulating factors for signaling ,112. Owein vivo immune response was imaged in lymph nodes by light sheet dSTORM and SIM [While advanced imaging techniques reveal new important and complex details about the immune response, they come with technical, biological, and methodological limitations. In many applications mentioned before, the complexity of the biological systems was kept as low as possible to allow for high-contrast imaging. For example, imaging was performed on cells interacting with 2D surfaces to enable TIRFM, or simple cell–cell interactions in solution were observed using (lattice) light-sheet microscopy. Looking directly at immunological processes occurring in tissue, e.g. lymph nodes, demands new imaging strategies or modifications of existing techniques. Recently, the multiscale reorganization of TCRs during the  and SIM , demonst and SIM  or Lck [ and SIM . Softwar and SIM  or elabo and SIM , tempora and SIM  or using and SIM ) might oLimits are also set for the resolution of mechanical forces, where probes and methods sensing a high dynamic force range are still missing. Also, the ultimate aim of a force read-out for individual molecules within the whole IS will demand the combination of various imaging techniques, as was shown in first attempts by increasing the spatiotemporal resolution  or addreAnother major limitation to overcome is the handling and storage of large amounts of data generated during image acquisition. New concepts to increase the computational power for data analysis will have to be instated. The increasing complexity of microscopy systems and generated data will have to be matched by well trained and skilled operators and machine learning/artificial intelligence-based analysis algorithms, respectively.Immune signaling has been imaged at high speed , high reDynamic techniques (<1 s per image) are constantly pushing the spatial resolution limits. Parallelized RESOLFT, which relies on photoswitchable fluorescent proteins, now achieves 80 nm 3D resolution in entire cells at 1–2 frames per second . Light-sHigh-resolution SMLM techniques (<10 nm) have traditionally suffered from particularly slow imaging speed. However, improvements in analysis pipelines through deep-learning approaches ,130, optAnother aspect holding the field back is availability of state-of-the-art techniques and analysis tools. As mentioned earlier, it took ∼7 years to develop a turn-key commercial LLSM. Fortunately, the microscopy field has been embracing democratization of designs and low-cost solution to bring new techniques to the masses. 3D printed microscopes ,136 offeWhile dynamic imaging is key, there is much left to be learned from static approaches. Expansion microscopy has recently been used to map out receptors on T cells , which hDespite these technical developments, there is something inherently difficult about imaging cell–cell interactions and synapse formation and there are only a handful of studies out there taking advantage of the latest methodologies. This partially stems from the move into dynamic 3D super-resolution imaging being far from trivial. The relatively fast restructuring of the cell membrane and the reorganization of membrane proteins present a complex challenge involving multiple unanswered questions, even with the availability of high-speed high-resolution techniques. Where exactly is the membrane or glycocalyx? Where are the proteins in relation to the membrane? Is the cell currently signaling? Is the cell under stress? Furthermore, the basic definition of detecting signaling is itself challenging: does it correspond to calcium flux, expression of certain proteins or formation of signaling complexes? Complementary labeling approaches have the potential to define where the interactions occur, which can provide a helpful cue , but theAdvanced imaging is key for visualizing the spatiotemporal regulation of immune signaling.We summarize state-of-the-art imaging methodologies and provide recent examples on how they helped to unravel the mysteries of immune signaling.We discuss the limitations of current technologies and share insights on how to overcome these limitations to visualize immune signaling with unprecedented details.


Giardia duodenalis is an important zoonotic parasite that causes economic losses to animal husbandry and threatens public health. In the present study, a total of 1466 fresh fecal samples were collected from sheep (n = 797), goats (n = 561) and beef cattle (n = 108) in Southwest Inner Mongolia, China. Giardia duodenalis was initially screened via nested polymerase chain reaction (PCR) targeting the β-giardin (bg) gene, and bg-positive samples were subjected to PCR amplification targeting the glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) genes. A total of 4.0% of samples (58/1466) were positive for G. duodenalis, with a prevalence of 3.4% in sheep, 3.7% in goats and 5.2% in beef cattle. Three G. duodenalis assemblages  were identified, with E as the prevalent assemblage. Four and one novel assemblage E sequences were obtained for the gdh and tpi loci, respectively and four assemblage E multilocus genotypes (MLG) were obtained. This study demonstrates high genetic variations in G. duodenalis assemblage E, and provides baseline data for preventing and controlling G. duodenalis infection in livestock in Inner Mongolia. Giardia duodenalis  is one of the most common intestinal pathogens in both humans and animals [Giardiasis are diarrhea, abdominal pain and weight loss [G. duodenalis, with an individual prevalence ranging from 0 to 73% [G. duodenalis infection is commonly asymptomatic, many reports of Giardiasis in calves, goats and lambs show decreased weight gain and impairment in feed efficiency, causing significant economic losses to the farm [ animals . The symght loss , 10, 30.0 to 73% , 17. Altthe farm , 29.Giardia duodenalis has a complex assemblage with a classification that is based on sequence analyses. The genetic locus of small subunit rRNA (SSU rRNA) [bg) [gdh) [tpi) is commonly used for PCR to characterize G. duodenalis [bg, gdh, and tpi is widely used for identifying genetic variations in G. duodenalis [SU rRNA) , beta-giNA) [bg) , glutamag) [gdh) , and triodenalis . Multiloodenalis , 8.G. duodenalis have been identified based on genetic analysis and specific hosts [Thus far, eight assemblages (A–H) of ic hosts . Assemblic hosts , 24. Preic hosts , 33.Giardia duodenalis is widely distributed in sheep, goats, and cattle  in China [G. duodenalis, in sheep and Bactrian camels [G. duodenalis in livestock. The objectives of this study were to further investigate and expand the prevalence information on G. duodenalis in ruminants in Southwest Inner Mongolia, China.in China . Inner Mn camels , 34, 37.Following the Chinese Laboratory Animal Administration Act of 1988, the research protocol was reviewed and approved by the Research Ethics Committee of Henan Agricultural University . Appropriate permission from farmers was obtained before collecting fecal samples, and no animals were harmed.n = 797), goats (n = 561), and beef cattle (n = 108), respectively , strictly following the specifications of the manufacturer. All the extracted DNA samples were stored at −20 °C.Giardia duodenalis was initially screened via nested PCR amplification targeting the bg [gdh [tpi [bg is 511 bp, gdh is 520 bp, tpi is 530 bp).g the bg  gene, an bg [gdh  and tpi gdh [tpi  genes. Ahttp://www.clustal.org/). Samples were amplified at the bg, gdh and tpi loci to form MLGs to further reveal genetic diversity. The same nomenclature system as in previous reports was used in naming G. duodenalis assemblage E subtypes at each genetic locus. Undesignated subtype sequences previously published and novel subtype sequences identified in this study were named accordingly as E36–E40 at the bg locus, E45–E52 at the gdh locus, and E32 at the tpi locus [Positive PCR amplicons with the target band were sequenced by SinoGenoMax . Bidirectional sequencing was chosen to ensure the veracity of sequences. The sequences in this study aligned with reference sequences from GenBank using ClustalX 2.1 .Phylogenetic analysis was conducted using the maximum composite likelihood model, and bootstrap values were calculated by analyzing 1000 replicates and the other chosen default parameters in MEGA 7.0 software  were calculated using Crosstab in SPSS, version 24.0 . A Pearson’s chi-squared test was used for comparisons between two groups, and OL456202, OL456203, OL456204 and OL456206 for the gdh gene, and OL456207 for the tpi gene.The representative nucleotide sequences were submitted to the GenBank at the National Center for Biotechnology Information under accession numbers: G. duodenalis-positive fecal samples were identified by the nested PCR analysis based on the bg gene, with 3.4% (27/797) in sheep, 3.7% (21/561) in goats and 9.2% (10/108) in beef cattle. The infection rates in winter were significantly higher than in summer  and autumn .A total of 58 (4.0%) G. duodenalis infection between pastoral and captive sheep . The G. duodenalis infection rate was significantly different between different age groups of beef cattle . There were no significant differences in prevalence of G. duodenalis among different age groups of sheep  and goats   .bg sequences, 17 gdh sequences and 6 tpi sequences were obtained. Three kinds of assemblages were identified, including G. duodenalis assemblage A (n = 1), assemblage E (n = 56), and a mix of assemblages B and E (n = 1). Additionally, 4 samples were simultaneously amplified at all three intra-assemblage variation genetic loci , forming 4 novel assemblage E MLGs (MLG-E1 to MLG-E4). The MLG-E2 and MLG-E4 sequences were obtained from sheep; the MLG-E1 sequences were obtained from goats, and the MLG-E3 sequences were obtained from beef cattle (Table 4).A total of 58 G. duodenalis bg-sequences, gdh-sequences and tpi-sequences, three phylogenetic trees were constructed to evaluate the genetic relationships of the G. duodenalis isolates. The results showed that G. duodenalis isolates from this study were clustered within the G. duodenalis assemblage E, and high genetic diversity was observed in the assemblage E subtypes  [tpi locus. Based on the SSU rRNA gene, G. duodenalis was detected in livestock in the United Kingdom (34.3%) and sheep in Inner Mongolia, China (64.1%) [This study presented (10.95%) , cattle (10.95%) , beef ca(10.95%) , Tibetan(10.95%) . However(10.95%) , and she(10.95%) , which wa, China 4.1% [6,  (64.1%) .SSU rRNA, bg and tpi loci have frequently been used to detect G. duodenalis. In this study, G. duodenalis in fecal samples was detected by nested-PCR of the bg locus, and only 29.3% and 10.3% bg-positive samples were amplified based on the gdh and tpi loci, which were similar to previous studies [G. duodenalis infection rate in this study and that in other studies which used the bg locus may be partially attributed to the state of feces, age group, sample size, detection methods and climate.The  studies , 21, 22.G. duodenalis infection rate was significantly different between different age groups of beef cattle (p < 0.001). Previous studies showed a higher prevalence in sheep, goats and cattle before weaning, and G. duodenalis infection is inversely associated with animal age [G. duodenalis infection rates in winter were significantly higher than in summer and autumn (p < 0.01), and the same phenomenon was reported in dairy calves in Norway and pigs in Denmark [All samples in this study were collected from non-diarrhea livestock in the age groups of seven months and older. The imal age , 17, 35. Denmark , 23; how Denmark .Giardia duodenalis assemblages A, B and E were identified, and G. duodenalis assemblage E was the dominant assemblage found in this study, which is consistent with previous reports [Giardia duodenalis assemblages A and E were identified as the two most common assemblages in sheep, goats and cattle, with assemblage B reported occasionally [G. duodenalis assemblages in this study were also reported in humans, companion animals and wildlife [ reports , 7, 25. sionally , 36. A fsionally , 20, 31.wildlife , and morbg locus, eight published assemblage E subtypes were found in sheep, goats and beef cattle, and the bg-positive samples were analyzed by the multilocus genotyping tool with high resolution (gdh and tpi) to further reveal the genetic variations in G. duodenalis. A total of four and one novel assemblage E subtypes were found at the gdh and tpi loci, respectively and the analysis yielded four novel MLGs of assemblage E. A high degree of genetic diversity in G. duodenalis assemblage E has been reported in livestock, which was probably a cause of the high occurrence rate of G. duodenalis in Tibetan sheep and yaks [G. duodenalis assemblage E subtypes  were found in sheep, goats and beef cattle simultaneously, which may indicate a potential occurrence of cross-species transmission. Cross-species transmission of G. duodenalis assemblage E subtypes was also found in Tibetan sheep and yaks [High genetic diversity was observed in the assemblage E subtypes. At the and yaks , 32. In and yaks , black-band yaks .G. duodenalis is a common parasite in sheep, goats and beef cattle in Inner Mongolia, and the infection rate is related to the season, and age of beef cattle. Based on molecular analysis, three G. duodenalis assemblages  were found and assemblage E was predominant. Novel subtypes found in this study show further genetic diversity of G. duodenalis assemblage E. This study provides baseline data for preventing and controlling G. duodenalis infection in livestock.The results of this study show that


The concentration of uric acid in blood is sex-, age- and diet-dependent and is maintained close to its maximal solubility, indicating that it plays some important role. Indeed, it has been demonstrated that, at physiological concentrations, uric acid is a powerful antioxidant and is a scavenger of singlet oxygen and radicals. At high intracellular concentration, uric acid has been demonstrated to act as a pro-oxidant molecule. Recently, uric acid has been reported to affect the properties of several proteins involved in metabolic regulation and signaling, and the relationship between uric acid and cancer has been extensively investigated. In this review, we present the most recent results on the positive and negative effects played by uric acid in cancer and some new findings and hypotheses about the implication of this metabolite in the pathogenesis of several diseases such as metabolic syndrome, diabetes, and inflammation, thus favoring the development of cancer.Homo sapiens and great apes, although exposing these species to the potentially adverse effects of uric acid, appears to be associated with evolutionary advantages.Uric acid is the final product of purine catabolism in man and apes. The serum concentration of uric acid is sex-, age- and diet-dependent and is maintained close to its maximal solubility, indicating that it plays some important role. Indeed, it has been demonstrated that, at physiological concentrations, uric acid is a powerful antioxidant, while at high intracellular concentrations, it is a pro-oxidant molecule. In this review, we describe the possible causes of uric acid accumulation or depletion and some of the metabolic and regulatory pathways it may impact. Particular attention has been given to fructose, which, because of the complex correlation between carbohydrate and nucleotide metabolism, causes uric acid accumulation. We also present recent results on the positive and negative effects played by uric acid in cancer and some new findings and hypotheses about the implication of this metabolite in a variety of signaling pathways, which can play a role in the pathogenesis of diseases such as metabolic syndrome, diabetes, and inflammation, thus favoring the development of cancer. The loss of uricase in  Homo sapiens and great apes and the associated evolutionary advantages.Uric acid (UA) is the final product of purine catabolism in humans and great apes ,2. Its cSUA homeostasis is maintained by an accurate balance between its income (either endogenous production or ingestion in diet) and its excretion. Any alteration of this system can lead to either hypouricemia or hyperuricemia. It is well known that the assumption of purine rich foods , fructose, coffee, and alcohol increases SUA . De OlivThe enzyme responsible for the endogenous production of UA is XOR, which catalyzes the last two irreversible steps of purine catabolism in uricotelic animals, the oxidation of hypoxanthine to xanthine and of xanthine to UA. XOR is a homodimeric metalloflavoprotein with a molecular mass of approximately 300 kDa in which each subunit is composed of three domains: the 20 kDa N-terminal domain, with two identical iron–sulfur clusters, the 40 kDa intermediate domain with a FAD cofactor and the 85 kDa C-terminal domain with a molybdopterin cofactor containing a molybdenum atom. The C-terminal domain expresses nitrite reductase, xanthine dehydrogenase (XDH) and xanthine oxidase (XO) activities, while NADH oxidase activity resides in the FAD domain ,29. TherThe complex mechanisms of UA excretion have been thoroughly studied, and a plethora of transporters have been identified. For extensive and detailed coverage, the reader is referred to the numerous reviews on this subject ,42,43,44A relationship between defects in the structure and function of several urate transporters and pathologies has been described, particularly regarding hypertension and vascular diseases , and alsIt has long been established that a load of fructose is accompanied by an increase in UA. In fact, as described below, fructose has been demonstrated to stimulate both the purine de novo synthesis and the breakdown of adenylic compounds. For this reason, in several published articles, UA is defined as a fructose metabolite. Indeed, none of its component atoms derive directly from fructose. To avoid any confusion, it would be better to point out that the increase in UA concentration, which follows a load of fructose, is a consequence of the complex array of correlation between carbohydrate and nucleotide metabolism .Raivio et al.  investigm for fructose of the C and A isoforms differ markedly, at approximately 0.5 and 8 mM, respectively. While KHK-A is widely expressed at low levels, KHK-C is selectively expressed in key metabolic tissues, such as liver, small intestine, and kidney . Al. Al71]. Overall, fructose has been reported as an alternative energy source, exploited for tumor growth. Its utilization appears preferential under low oxygen conditions  and acceLipid accumulation in the liver (hepatic steatosis) might lead to steatohepatitis and, finally, to cirrhosis . The perHepatic steatosis is associated with a reduction in AMP-activated protein kinase (AMPK) activity. AMPK-dependent phosphorylation of transcription factors such as sterol regulatory element-binding protein (SREBP)-1c, SREBP-2 and ChREBP regulate lipogenesis in liver. The phosphorylation of these factors leads to the inhibition of the transcription of genes coding for fatty acid synthase, acetyl-CoA carboxylase (ACC1) and stearoyl-CoA desaturase. AMPK can phosphorylate ACC1 at Ser79, resulting in its inactivation and reduction in lipogenesis . In addiIt has been demonstrated that AMPK is inhibited when AMPD is activated in the HepG2 cell line, and that silencing of AMPK results in an increase in AMPD activity . LanaspaCicerchi et al.  have alsWang et al.  suggesteThe molecular mechanisms underlying activation and/or inhibition of AMPK by UA are still unknown, but the experiments described above indicate that the modification of AMPK activity, including the use of XOR inhibitors, could be useful either to delay hepatic steatosis that could ultimately lead to cancer or to alleviate chemotherapeutic-induced toxicity.UA has been described as an endogenous danger signal released by necrotic cells able to activate adaptive immune responses . Indeed,The activation of intracellular inflammasome is usually triggered by bacterial proteins such as flagellin, but also by other endogenous signals. Not only UA crystals, but also other large particulate elements , can induce inflammation through NLRP3 inflammasome , suggestIn summary, the pro-inflammatory action of UA is mediated by both intracellular and extracellular receptors involved in innate immunity.The activation of the mechanism of innate immunity by UA crystals is involved in the pathogenesis of gout and explains most of the symptoms present in patients .The formation of UA crystals is commonly caused by an increase in circulating UA, usually due to a defect in its excretion and/or reabsorption  and alsoThe activation of the inflammatory mechanism and neoplastic transformation are accompanied in many cases by activation of the mTOR pathway. In fact, the protein kinase mTOR forms a complex with other proteins, some of which are involved in its functional connection with the amount and nature of ingested food ,120. WitIndeed, UA priming causes a decrease in autophagic mechanism in HeLa cells . AutophaOverall, most of the reports indicate that UA activates the AKT/mTOR pathway, leading to an increase in all the anabolic pathways including lipid, protein and nucleotide synthesis and inhibiting autophagy, thus favoring cell proliferation. Furthermore, it increases inflammatory mechanisms, lipid circulation and storage, generating a metabolic disarrangement that, if constantly present, strongly increases the risk of all site cancer, as demonstrated by several reports ,130.Excess of UA has always been considered a danger signal, being correlated to an incorrect diet, defective transport systems, or a surplus of ATP hydrolysis not compensated by oxidative phosphorylation , and thuUA is the final product of the catabolism of purine nucleotides in humans and apes, whereas all other mammals further process this metabolite to produce allantoin . This isThe relationship between high UA and cancer has been suggested in many papers, although a causal connection is difficult to demonstrate, due to several confounding factors such as diet, alcohol consumption, and underlying co-morbidity disorders. Furthermore, hyperuricemia, rather than being an independent risk factor for cancer, could be the result of cancer-related cell death . NeverthIn conclusion, the maintenance of a correct balance of SUA appears to be necessary to decrease the risk of severe diseases, including cancer. Many factors contribute to SUA homeostasis: transport systems, XOR activity, and eating behavior. In modern society, the excessive intake of fructose, present in industrial food and beverages, appears to represent a real danger for public health.


Human T cell leukemia virus-1 (HTLV-1) is the causative agent of a severe cancer of the lymphoid lineage that develops in 3-5% of infected individuals after many years. HTLV-1 infection may also induce a serious inflammatory pathology of the nervous system designated HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Two virus-encoded proteins, the viral transactivator Tax-1 and the HTLV-1 basic leucine-zipper factor HBZ, are strongly involved in the oncogenic process. Tax-1 is involved in initial phases of the oncogenic process. Conversely, HBZ seems to be involved in maintenance of the neoplastic state as witnessed by the generation of leukemic/lymphomatous phenotype in HBZ transgenic mice and the persistent expression of HBZ in all phases of the oncogenic process. Nevertheless, the intimate molecular and cellular mechanism mediated by the two viral proteins, particularly HBZ, in oncogenesis still remain elusive. An important step toward the complete comprehension of HBZ-associated oncogenicity is the clarification of the anatomical correlates of HBZ during the various phases of HTLV-1 infection to development of HTLV-1-associated inflammatory pathology and ultimately to the establishment of leukemia. In this review, I will summarize recent studies that have established for the first time a temporal and unidirectional expression of HBZ, beginning with an exclusive cytoplasmic localization in infected asymptomatic individuals and in HAM/TSP patients and ending to a progressive cytoplasmic-to-nuclear transition in leukemic cells. These results are framed within the present knowledge of HTLV-1 infection and the future lines of research that may shed new light on the complex mechanism of HTLV-1- mediated oncogenesis. The human T cell leukemia/lymphoma virus-type 1 (HTLV-1), the first described human retrovirus . HTLV-1 via its bZIP domain. This results in strong inhibition of the CREB-2/Tax-1 interaction instrumental for the activation of HTLV-1 LTR (HBZ contains a bZIP domain in addition to an activation (N-terminus) and a central domain . HBZ is LV-1 LTR .disheveled-associating protein with a high frequency of Leucine residues (DAPLE)  . On the  (DAPLE) .tax gene, discrete bursts of Tax-1 expression in a minority of cells take place and may favor the growth of the ATL population . Pr. Pr26). The above summarized studies, although mostly phenotypic in nature, did have an important impact on the previously assumed idea that HBZ was a nuclear protein and, as such, would exert its biological functions in the nucleus only. The change in paradigm generated by our results thus modified the perspective in the evolution of HTLV-1-associated pathology, strongly discriminating the HTLV-1-associated chronic inflammatory pathologies, such as HAM/TSP, from the cancer state such as ATL. Nevertheless, the unambiguous and exclusive cytoplasmic expression found in asymptomatic carriers, mostly representing early stages of HTLV-1 infection, compared with the nuclear localization in ATL cell lines, representing a very late effect of HTVL-1 infection, suggested that development of ATL could be accompanied by, and/or be associated to, a unidirectional cytoplasmic-to-nuclear transition of HBZ. If this were the case, we should have been able to find leukemic patients with a dual cytoplasmic and nuclear localization of the viral protein. In collaboration with the group of Olivier Hermine we thus investigated fresh leukemic cells of an extended number of both acute and chronic ATL patients, by immunofluorescence and confocal microscopy. With our surprise, we found that all patients analyzed, irrespective of the acute or chronic disease state, expressed HBZ in the cytoplasm and in the nucleus . Indeed,The other very interesting finding related to the study of fresh leukemic cells consisted in the evidence that treatment of the cells with LMB did not result in increased nuclear retention of HBZ. While in the case of AC and HAM/TSP a similar finding simply indicated that HBZ was not freely shuttling between cytoplasm and nucleus, in the case of leukemic cells cytoplasmic HBZ did not freely shuttle to the nucleus in presence, however, of a distinctive proportion of HBZ molecules already present in the nucleus. This suggests that the progressive nuclear translocation of HBZ takes place under a very strict control. The molecular mechanism(s) at the basis of the HBZ retention in the cytoplasm during the history of HTLV-1 infection from AS to the development of ATL are presently unknown. Oncogenic transformation is accompanied and caused mostly by accumulation of mutations that affect cell homeostasis and control of cell growth –37. If tIt may be conceivable that the process of cytoplasmic retention of HBZ would gradually diminish because of defects in the regulation of expression of gene (s) encoding still unknown docking factor(s). Lowering the amount of these molecules would allow HBZ to escape the interaction with them and freely migrate into the nucleus Figure 1Another interesting point of our recent study was related to the expression of spliced versus unspliced HBZ mRNA in the fresh leukemic cells of ATL patients. The unprecedented HBZ cytoplasmic localization in ATL cells was paralleled by a more abundant expression of spliced vs unspliced form of HBZ mRNA , similarResearch on the function of HTLV-1-encoded HBZ is rapidly evolving and certainly will witness important developments in the future. Many aspect of the HBZ biology are still incompletely clarified. Among them, two are of particular relevance in my opinion. First, the elucidation of the functional correlates of HBZ in the different subcellular compartments in which it localizes. As highlighted in this report, the unidirectional cytoplasmic-to-nuclear transition of HBZ protein, marking the passage from infection to development of leukemic state, underlines the importance of assessing the biological role of cytoplasmic HBZ in ATL. Importantly, the interaction of HBZ with host factors in the cytoplasm and in the nucleus which, I anticipate, will be more complex than previously envisaged, should better clarify the role of HBZ in the onset and persistence of the oncogenic process. In this direction, the study of the HBZ interactome initiated by several groups, including ours, is unveiling unexpected interactions with a large array of proteins involved, for example, in RNA splicing and stability , mechaniSecond, the full clarification of the role of HBZ mRNA in the HTLV-1 associated diseases. It has been previously suggested that HBZ mRNA plays a role in supporting proliferation of ATL cells . The ratRA conceived and wrote the paper. The author confirms being the sole contributor of this work and has approved it for publication.http://www.hepavac.eu; and in part by Associazione Italiana per la Ricerca sul Cancro (AIRC) 2021, Grant no. 26195 “Novel anti-glioblastoma therapeutic vaccines based on optimal activation of tumor-specific CD4+ T helper cells”.This work was supported by The Institutional Grant, University of Insubria, to RA; in part by European Community FP7 Grant no. 602893 “Cancer Vaccine Development for Hepatocellular Carcinoma-HepaVAC” 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. 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