Objective: To assess whether phosphorylated neurofilament heavy chain (pNfH) can discriminate different upper motor neuron (UMN) syndromes, namely, ALS, UMN-predominant ALS, primary lateral sclerosis (PLS) and hereditary spastic paraparesis (hSP) and to test the prognostic value of pNfH in UMN diseases. Methods: CSF and serum pNfH were measured in 143 patients presenting with signs of UMN and later diagnosed with classic/bulbar ALS, UMNp-ALS, hSP, and PLS. Between-group comparisons were drawn by ANOVA and receiver operating characteristic (ROC) analysis was performed. The prognostic value of pNfH was tested by the Cox regression model. Results: ALS and UMNp-ALS patients had higher CSF pNfH compared to PLS and hSP (p < 0.001). ROC analysis showed that CSF pNfH could differentiate ALS, UMNp-ALS included, from PLS and hSP , while serum did not perform as well. In multivariable survival analysis among the totality of UMN patients and classic/bulbar ALS, CSF pNfH independently predicted survival. Among UMNp-ALS patients, only the progression rate  and presence of multifocal fasciculations  were independent prognostic factors. Conclusions: CSF pNfH is significantly higher in classic and UMNp-ALS compared to UMN diseases with a better prognosis such as PLS and hSP. Its prognostic role is confirmed in classic and bulbar ALS, but not among UMNp, where clinical signs remained the only independent prognostic factors. Syndromes presenting with upper motor neuron (UMN) signs include diseases with very variable prognosis, ranging from “pure” UMN diseases  to UMN-predominant presentation of amyotrophic lateral sclerosis (ALS) . Recently, the diagnostic criteria for primary lateral sclerosis (PLS) were revised to facilitate an earlier diagnosis of the disease. Although the observation interval has been shortened, at least two years are still needed to define probable PLS . PLS is Among promising ALS biomarkers, neurofilament light chain (NfL) and phosphorylated neurofilament heavy chain (pNfH) have proved to discriminate well between cases and healthy controls . NeurofiIn the present study, we measured pNfH levels in CSF and serum of patients presenting with UMN signs, who were later diagnosed within a definite clinical group  with subsequent diagnostic confirmation after a long follow-up period (median: 85 months). This study was designed to investigate whether pNfH can be used as a complementary tool for differential diagnosis in patients presenting with UMN syndromes and to help reduce diagnostic delay in UMN syndromes. This retrospective study included patients who underwent lumbar puncture as part of their diagnostic work-up between 1 January 2007, and 1 January 2019. The Ethical Committee of Area Vasta Emilia Nord approved the study, which was performed according to the declaration of Helsinki. Among a total of 281 patients diagnosed with MND with UMN involvement during the study period, 143 patients underwent lumbar puncture during the diagnostic process and still had at least 0.5 mL of CSF and serum available when the present study was designed. All patients presented with signs of UMN syndrome as defined by a combination of hyperreflexia, spasticity, clonus, and pathological reflexes at the time of the diagnostic work-up. Patients were followed up with a multidisciplinary approach for a median of 85 months and finally diagnosed with definite or probable ALS —classic or bulbar phenotype—(95 patients) and UMNp ALS (20 patients). The phenotypic distinction among flail or respiratory forms was also taken into account in the analysis according to established criteria [As part of our systematized neurological assessment, for this study we considered several clinical signs of MN dysfunction that were detected at sampling time: fasciculations frequency and distribution, presence of Babinski and Hoffman signs as well as Achilles clonus , exaggerated masseter reflex, presence of frontal release signs , pseudobulbar affect (measured by CNS-lability score > 13)  and presAssessment of clinical progression was performed using the ALS Functional Rating Scale Revised (ALSFRS-R)   through Corticospinal involvement at brain MRI was assessed in 118 patients as the presence of corticospinal tract hyperintensity in FLAIR or diffusion tensor images; motor evoked potentials were assessed in 114 patients and the presence of prolongation of central conduction time was considered as a sign of corticospinal involvement. We did not include healthy controls (HC) since diagnostic difficulties are usually present when facing patients with variably distributed UMN signs and not with HC, and since pNfH have a known capacity to discriminate between MND and HC . CSF and serum samples were collected at the same time and underwent separate processing according to standard international procedures . CSF pNfH levels were determined using a CE-marked ELISA test with polyclonal capture and monoclonal detection antibodies , as previously reported ; serum pp < 0.001 both for CSF and serum pNfH). After cube root transformation, data appeared to be normally distributed, thus cube root-transformed pNfH (crt-pNfH) concentrations were used for graphical representation of the data and analysis of variance models.Density analysis of pNfH concentrations in CSF and serum revealed an extremely left-skewed, non-normal distribution  and ALS phenotypes  using ANOVA, with the Tukey–Kramer test for pairwise comparisons between groups. The influence of age, disease duration and sex on crt-pNfH within each group and overall was tested by ANCOVA. Correlations between crt-pNfH and quantitative variables  were assessed using Pearson’s correlation coefficient and linear regression models. Receiver operating characteristic (ROC) curves were used for a sensitivity analysis of pNfH cutoff values in CSF and serum for ALS versus other UMN diseases (PLS and hSP), for UMNp ALS versus PLS, for PLS versus hSP, and UMNp ALS versus hSP. Overall sensitivity and specificity were evaluated by areas under the curve (AUC). p < 0.1) using the stepwise backward method. HR were calculated for each variable with 95% CI. All statistical analysis was performed using the software STATA version 14.0 .Survival  was assessed first with univariate analysis using a Cox proportional hazards regression model, Kaplan–Meier curves, and log-rank test. The following variables were examined: crt-pNfH levels in serum and CSF, MND subtypes, age at onset, sex, diagnostic delay, time from onset to sampling, clinical MN signs, ALSFRS-R total score and subscores , and Ashworth scale score. A multivariable analysis of survival with a Cox proportional hazards regression model was then performed with the abovementioned variables, which reached a significance level (n = 20) apart from the other phenotypes of ALS, who were classified as following: 46 classic ALS, 20 bulbar ALS, 10 flail (including both flail arm and flail leg) and 3 respiratory ALS. Among the 13 hSP patients, 6 had a genetic confirmation. The median follow-up from sampling to the last visit (or date of death/tracheotomy) and the progression rate reflect the different prognoses of each group. There were obvious, different functional disability scores across the evaluated scales among groups. Clinical and instrumental signs of motor neuron impairment across different MND were considered with reference to the neurological examination at the time of sampling and are summarized in Median concentrations of pNfH in CSF and serum across the four diagnostic group are shown in p = 0.827 and p = 0.746 at one-way ANOVA, respectively). ALS patients had higher levels of crt-pNfH compared to hSP (p < 0.001 for both CSF and serum) and PLS, with the latter only reaching significance in CSF . Similar results were obtained when analyzing UMNp ALS with PLS and hSP . PLS patients had higher levels of crt-pNfH in CSF and serum compared to hSP (p = 0.030 and p = 0.013 in CSF and serum). The distribution of crt-pNfH in CSF and serum across ALS, UMNp ALS, PLS, and hSP is summarized in UMNp ALS patients and ALS patients with other clinical phenotypes  had similar levels of crt-pNfH in CSF and serum . The analysis was next repeated within diagnostic groups, confirming a significant effect of MND groups after correction for age, disease duration, and sex on CSF  and serum  concentrations of crt-pNfH.The ANCOVA excluded significant effects of sex or age at sampling on crt-pNfH concentrations, whereas disease duration affected crt-pNfH  and by serum pNfH with an AUC of 0.66 (CI: 0.52–0.80). The discriminatory performance of pNfH between ALS (including UMNp ALS) and hSP was more evident both in CSF  and serum . p = 0.007 for single fasciculations; p < 0.001 for single multifocal fasciculations; p = 0.005 for multifocal and continuous fasciculations with respect to patients without fasciculations, Patients with more frequent and widespread fasciculations had higher crt-pNfH, with more evident differences in CSF (p < 0.0001) and 0.24 , respectively. On the other hand, crt-pNfH did not correlate with any sign of UMN dysfunction, Penn UMN score included.These results were corroborated by logistic regression models where crt-pNfH in CSF and serum were input as the dependent variables and fasciculation grading as the independent variable, obtaining a coefficient of 0.09 . There was a correlation between concentrations of crt-pNfH in CSF and serum and diagnostic delay , progression rate at sampling , progression rate at last observation  and total ALSFRS-R score at sampling .The Spearman’s correlation coefficient for serum and CSF concentrations of crt-pNfH was 0.67 , time to generalization , presence of dementia , ALSFRS-R score at sampling , progression rate  and diagnostic group .When multivariate regression analysis was performed on the variables that were significant at univariate analysis, the following remained in the model: CSF concentration of pNfH , serum pNfH , time from onset to sampling , time to generalization , diagnostic delay , and progression rate, . All other variables, including age and site at onset, the presence of signs of upper or lower motor neuron, genetics, and cognitive/behavioral impairment did not affect survival. When multivariate regression analysis was performed, of the variables that were significant at univariate analysis, only CSF concentration of pNfH  and time to generalization  remained in the model.Then, separate survival analysis was carried out for ALS and UMNp ALS. Univariate analysis of survival through a Cox regression model among ALS patients showed the impact of the following variables: CSF concentration of pNfH , diagnostic delay , progression rate , presence of abundant fasciculations , and presence of FTD . When multivariate regression analysis was performed, only progression rate  and presence of abundant fasciculations  preserved their role in the model. Due to the low number of deaths among PLS and hSP, survival analysis was not performed in these diagnostic groups.Univariate analysis of survival through a Cox regression model among UMNp-ALS patients interestingly revealed the effect of the CSF concentration of pNfH . Furthermore, we confirmed a negative correlation between concentrations of pNfH in CSF and serum and diagnostic delay and a positive correlation between concentrations of pNfH in CSF and serum and progression rate at sampling ,21. Nfs,The significance of the relationship between neurofilament concentrations and motor neuron affection is not just a matter of pathobiology for ALS but it is also reinforced by the survival analysis. Indeed, whereas in multivariable survival analysis for ALS, pNfH sustains the model as a strong independent predictor of prognosis together with time to generalization, as also reported by Li and colleagues , in UMN-The main drawback of this study is the small sample size, particularly for the PLS and hSP groups, which may be addressed by larger multicentric studies to overcome the rarity of these conditions. Furthermore, the retrospective design and the consequent different times at which samples were collected and stored may have impacted measurements. Longitudinal clinical, neurophysiological and biological assessment would help in better understanding Nfs correlations with UMN and LMN burden of disease. Next, we recognize that the application of neurofilaments in the clinical practice of MND clinics is still limited by several factors, first and most important, the notion that these molecules are rather aspecific markers of neuroaxonal injury, and may be elevated in other degenerative conditions or for ancillary causes such as previous trauma . As alreIn conclusion, despite some statistical instability in the results due to the limitations of the sample size, our study supports the hypothesis that CSF pNfH may aid in the differential diagnosis of ALS, UMNp-ALS and PLS. This would represent a significant advance in the early diagnosis of PLS, which, despite the introduction of the most recent diagnostic criteria, still requires several years and clinical and neuropathological validation . In addiThe potential role of CSF pNfH as prognostic biomarkers for MND presenting with UMN signs merits further study with a larger population and longitudinal follow-up of PLS and hSP.Finally, the possibility of using analytical methods that are more sensitive than ELISA for serum pNfH could allow us to really evaluate the potential of this biomarker on a less invasive matrix that could also be collected longitudinally.


The higher side of PO and the apex side of scoliosis were interrelated , but the asymmetrical neurological upper extremity involvement was not. (4) Conclusions: severely impaired gross motor function, PO, vertebral rotation, and female sex were significantly related to the progression of scoliosis. The higher side of PO was opposite to the apex side of scoliosis. By identifying the factors that influence the progression of scoliosis, patients at high risk could be more actively intervened to minimize the severe complications.(1) Background: scoliosis is highly prevalent in children with neurological disorders, however, studies predicting the progression and affecting the direction of scoliosis have been insufficient. We investigated the factors associated with the progression and direction of scoliosis in children with neurological disorders. (2) Method: retrospectively, 518 whole spine radiographs from 116 patients were used for analysis. Factors affecting the progression of scoliosis over time were analyzed using linear mixed-effects model. Factors associated with the apex direction of the scoliosis were analyzed. (3) Results: pelvic obliquity (PO) ≥ 2.5°, gross motor function classification system level V, vertebral rotation, and female sex significantly affect the progression of scoliosis ( Scoliosis is defined as a structural alteration of the spine, in which the coronal plane curvature of the spine is greater than 10°. It is known that 25–74% of patients with cerebral palsy (CP) and 48–93% of patients with Duchenne muscular dystrophy have scoliosis . Th. Th8]. TPO was measured with the Maloney method, defined as the angle between the line perpendicular to the line connecting the bilateral iliac crests and the line between the center of T1 to S1 vertebral bodies in the AP radiographs B 10]. T. T10]. TLumbar lordosis was measured in the lateral radiographs by Cobb’s method, angle between S1 superior end plate and T12 inferior end plate C 11]..11].All AP radiographs were assessed vertebral rotational deformity by Nash–Moe method, defined as the percentage displacement of the convex pedicle in relation to the vertebral body width .p values less than 0.05 were considered statistically significant.Descriptive analysis was used for all variables, including median and interquartile range for continuous data and percentages for categorical data. Inter-rater reliability of radiographic indices was assessed by interclass correlation coefficients (ICCs) estimates, and their 95% confident intervals (CI) based on a mean-rating (k = 2), absolute-agreement, 2-way mixed effects model . A Chi-sThe patient’s demographics and clinical characteristics are shown in Inter-rater reliability of the measurements is described in p = 0.04, <0.001, <0.001, 0.005, respectively). Reduced lumbar lordosis (less than 18°) at or more than two years of age and the presence of epilepsy did not significantly contribute to the progression of scoliosis (p > 0.05 in both). A value that is two standard deviations lower than the average value of typically developing children aged 2–4 years reported in the previous study was used as the reference for reduced lumbar lordosis [lordosis .p > 0.05). However, in patients with PO ≥ 2.5°, there was a significant correlation between the higher side of the PO and the apex side of the scoliosis . The proportion of patients with apex side of scoliosis opposite to the high side of PO was 75.0% (43/58). Subgroup analysis was performed to investigate the correlation between the secondary curve apex and the higher side of PO. The secondary curve was at the lumbar level in six out of fifty-eight patients and at the thoracic level in ten patients. In the group with the secondary curve at the thoracic level, the higher side of PO was located on the opposite side of the major curve apex in eight out of ten patients (80.0%), but in the group with the secondary curve at the lumbar level, one out of six patients (16.7%), the higher side of PO was located on the opposite side of the major curve apex. Though it was difficult to confirm the statistical significance due to the small number of patients, the presence of secondary curve at the lumbar level tended to show additional associations with the higher side of PO.The effects of handedness and asymmetric upper limb function were analyzed in 82 patients with a Cobb angle of 10 degrees or more based on the final radiograph of each patient. Among them, the effect of PO was analyzed in 58 patients with 2.5 or more degrees of PO. The Chi-square test  revealedAmong spinal deformity, adolescent idiopathic scoliosis is the most common deformity, occurring in healthy children around puberty and progressing with skeletal growth. In patients with a curve of less than 30° after bone maturation, scoliosis rarely progresses, and in the case of 30° to 50°, an average of 10° to 15° progresses throughout lifetime . Saito eIn this study, we found that increased PO, severe motor dysfunction (GMFCS level V), vertebral rotation, and female sex were contributing to the progression of scoliosis with age. In general, an ambulatory function is known as a critical factor for scoliosis . SeveralPrevious researchers have investigated the association between coronal spine deformities and PO in scoliosis ,19. BiomVertebral rotation is an essential component of scoliosis from a three-dimensional perspective. Mohanty et al.  analyzedFemale sex is known to be a factor influencing the progression of the curve in adolescent idiopathic scoliosis . In the Previous studies have also suggested the relationship between the development of scoliosis and epilepsy ,24; howeA secondary analysis of the factors determining the apex side of scoliosis demonstrated that PO was a significant factor; however, handedness or asymmetric upper limb function had no significant effect on the direction of scoliosis. The current results were consistent with the previous reports that 89% of the patients with CP had the convexity of scoliosis on the opposite side of the high side of the PO . We addiA limitation of our study is that since all data were collected retrospectively, an unequal number of radiographs were collected for each subject at non-fixed time points. However, to compensate for this problem, we used the LMM to determine whether each variable significantly influenced the trend of changes in Cobb angle with time . Second,Various conservative treatments have been tried to prevent the progression of scoliosis in patients with neurological disorders. Physiotherapy and spinal orthoses are helpful for muscle balance, postural support, and prevention of contractures but are known to have a limited role in decreasing curve progression ,29. An iOur results indicated that PO, GMFCS Level V, vertebral rotation, and female sex were important factors affecting the progression of scoliosis. Underdevelopment of lumbar lordosis and epilepsy had no significant effect on the progression of scoliosis. In addition, there was a significant association with PO and scoliosis curve direction, with the higher side of PO was opposite to the apex side of scoliosis. However, asymmetric upper limb function or handedness did not affect the direction of scoliosis. The factors identified in our study can be used for further studies on the predictive model of scoliosis progression in patients with neurological disorders.


After additionally adjusting for sedation intensity, the HR became 1.04 . Thus, we concluded that excessive sedation occurs more frequently in critically ill patients treated with midazolam than in patients treated with lormetazepam. These findings require further investigation in prospective trials to assess if lormetazepam, due to its ability to maintain light sedation, might be favorable over other benzodiazepines for sedation in the ICU.The benzodiazepine, midazolam, is one of the most frequently used sedatives in intensive care medicine, but it has an unfavorable pharmacokinetic profile when continuously applied. As a consequence, patients are frequently prolonged and more deeply sedated than intended. Due to its distinct pharmacological features, including a cytochrome P450-independent metabolization, intravenous lormetazepam might be clinically advantageous compared to midazolam. In this retrospective cohort study, we compared patients who received either intravenous lormetazepam or midazolam with respect to their survival and sedation characteristics. The cohort included 3314 mechanically ventilated, critically ill patients that received one of the two drugs in a tertiary medical center in Germany between 2006 and 2018. A Cox proportional hazards model with mortality as outcome and APACHE II, age, gender, and admission mode as covariates revealed a hazard ratio of 1.75 [95% CI 1.46–2.09;  The use of sedatives is an essential element of intensive care medicine for stress and anxiety reduction as well as tolerance for invasive therapies such as mechanical ventilation ,2. HowevAs sedation intensity shows a dose-dependent effect on 180-days mortality , avoidinA receptor, are commonly used for sedation and anxiolysis in the ICU, but they have been criticized for causing prolonged deep sedation and delirium [Benzodiazepines, which act as positive allosteric modulators at the GABAdelirium . Due to delirium . Howeverdelirium ,10,11. Rdelirium . It was delirium ,14.In 2008, the benzodiazepine lormetazepam was repurposed as an intravenous formulation for sedation in Germany. The substance is metabolized and cleared independently from the cytochrome P450 system, and its metabolites are inactive .Since 2010, both substances have been available and could be administered for the same purposes in the ICU. The use of either midazolam or lormetazepam depends on the physicians’ decision and standard operating procedures. The concomitant use of both substances offers the opportunity to conduct a retrospective analysis comparing midazolam and lormetazepam in routine use.To our knowledge, midazolam and lormetazepam have not been compared with respect to sedation depth and mortality risk. We hypothesize that patients treated with lormetazepam are more likely to achieve a state of no or light sedation and, thus, are more likely to survive. The explorative analysis aims to generate hypotheses to assess lormetazepam in randomized controlled trials.We report a retrospective observational cohort study conducted at Charité—Universitätsmedizin Berlin, Germany. Ethical approval was obtained from the institutional review board of Charité—Universitätsmedizin Berlin, Germany (EA2/075/019). Informed consent was waived. The study was prospectively registered at ClinicalTrials.gov (NCT04408911).Routine data were collected from the hospital’s electronic health record  and ICU patient data management system . All identifiable information and case-specific numbers were removed before the analysis.Patients admitted to all medical and surgical ICUs at Charité—Universitätsmedizin Berlin, Germany, between 1 January, 2006 and 31 December, 2018 were screened for eligibility. We did not screen neuro-ICUs, because in neuro-critically ill patients, the indications for benzodiazepines differ from standard operating procedures of medical and surgical ICUs. Inclusion criteria were as follows: age ≥ 18 years; mechanical ventilation; patient received either midazolam or lormetazepam; and ICU length of stay ≥48 h. Exclusion criteria were: application of both, midazolam and lormetazepam, and application of only a single bolus of midazolam or lormetazepam during the entire ICU stay.Patients were grouped based on treatment with midazolam or lormetazepam for sedation, either as boluses or as continuous infusion. Both substances were administered according to current German clinical guidelines for the management of sedation, pain and delirium . One keyDuring the observation period, the German guideline underwent two updates, which did not change the indication spectrum of or recommendation for benzodiazepines . SedatioThe primary endpoint was in-hospital mortality. Secondary endpoints were incidence of delirium, duration of delirium, ICU length of stay, hospital length of stay, duration of mechanical ventilation, and sedation-specific characteristics. Screening of pain, agitation and delirium was performed according to the German Guidelines for the Management of Sedation, Pain and Delirium . Hence, Sedation intensity was quantified with the sedation index (SI), which was calculated as previously described . The SI Deeper sedation results in higher SIs. If not indicated differently, we used the SI for 48 h after commencement of sedation with either midazolam or lormetazepam. As the SI is a measure of sedation over time, it was only calculated if more than two RASS assessments per 24 h were available.n) and relative (%) frequencies. The midazolam and lormetazepam groups were compared regarding baseline characteristics, sedation characteristics, and outcome parameters using unpaired t-test, Mann–Whitney U-test, Fisher’s exact test, and χ2 test. To analyze the mortality risk between the midazolam group and lormetazepam group, we fitted multivariable logistic regression models that were adjusted for Acute Physiology and Chronic Health Evaluation (APACHE) II score, age, gender, admission due to medical reasons, and admission due to emergency surgery. We further analyzed the mortality using Kaplan–Meier plots for the midazolam group and lormetazepam group, and both groups stratified by sedation levels (light sedation and deep sedation). Kaplan–Meier curves were compared using log-rank tests. After checking the proportional hazard assumption, we fitted two Cox proportional hazards models with death as outcome. Model 1 included the use of midazolam (yes/no), APACHE II, age, gender, and admission mode as covariates. Model 2 additionally adjusted for a SI ≥ 1.5 in the first 48 h (yes/no). We defined statistical significance at p < 0.05. Analyses were carried out using SPSS (version 26) and R (version 4.0.5).For descriptive statistics, continuous variables were displayed as mean and standard deviation (SD) or as median and interquartile range (IQR). Categorical variables were displayed as absolute  indicating a higher severity of illness, and were more likely to have been admitted to the ICU due to medical reasons .Patients in the midazolam group had a significantly higher APACHE II score (19.6 (SD 9.6) vs. 23.6 (SD 9.8); ).In 84% of cases, lormetazepam was administered as a bolus, and in about one third of cases as a continuous infusion . In contTable 3p < 0.001; p < 0.001; In-hospital mortality was significantly higher in the midazolam group than in the lormetazepam group . Confirming the findings from the Kaplan–Meier analysis, the Cox proportional hazards model for the in-hospital mortality with APACHE II, age, gender, and admission mode as covariates revealed a hazard ratio (HR) of HR = 1.75  for the use of midazolam  and in the hospital (mean 49.7 days (SD 43.1) vs. 40.6 days (SD 36.8); p < 0.001) than in the lormetazepam group (p = 0.004).Patients in the midazolam group stayed significantly longer in the ICU (mean 31.5 days (SD 28.7) vs. 24 days (SD 23.1); am group . In addiIn this retrospective cohort study, we found that the intravenous benzodiazepine lormetazepam is associated with a lower in-hospital mortality when compared to midazolam in adult critically ill patients. The effect completely disappeared when correcting for sedation intensity. We are not aware of any study that showed an increased risk of mortality in critically ill patients treated with midazolam when compared to other substances. In our study, we observed an increased mortality risk in patients treated with midazolam in the univariate and multivariate analyses. However, this effect depended on sedation intensity; when the SI was added to the Cox proportional hazards model, the effect on the mortality risk disappeared. Patients being treated with midazolam seemed only more likely to have a fatal outcome because they were more deeply sedated. This does not come by surprise, as early deep sedation has been identified as a risk factor for mortality in critically ill patients in several studies ,6,7.To our knowledge, this is the first study comparing the repurposed benzodiazepine lormetazepam to a traditional benzodiazepine. Previous studies on benzodiazepines in critically ill patients have mostly compared midazolam and lorazepam to the non-benzodiazepines propofol or dexmedetomidine ,2. In thOur surprising findings question current guideline and expert recommendations that suggest to refrain from the general use of benzodiazepines in favor of non-benzodiazepines ,2,19,20.There is a potential explanation from experimental research that suggests that the detrimental effects of midazolam might be attributable to its pharmacokinetic properties and metabolization. For conversion to a water-soluble product, midazolam undergoes two phases of biotransformation, involving cytochrome P450-dependent hydroxylation and glucuronidation ,22,23. TOne has to consider several limitations when interpreting this study. Firstly, this is a monocentric, retrospective observational study that can only be considered hypothesis-generating. A change of sedation practice over the observation period or a difference in sedation practice between the groups might have impacted our results. To investigate if there was a change in sedation levels over time, we analyzed the mean SIs for each year of the observation period and detected a steady increase in mean SIs for both, the midazolam and lormetazepam group. This might be because we generally use fewer sedatives, but when we use them, there is a medical indication for a deeper sedation. In summary, there is a profound difference between sedation depths in the midazolam and the lormetazepam group that cannot be conclusively explained because of the uncontrolled, retrospective nature of our study. Moreover, the SI could not be calculated for about one quarter of patients in both groups due to too few documented RASS assessments within 48 h after commencement of sedation. This might have introduced a systematic bias in our analysis. In addition, we only analyzed midazolam and lormetazepam, neglecting other co-sedatives and other administered medications that might have acted as confounders by affecting SIs and mortality. In addition, dosages of sedatives were not available, but we are certain that substances were used for sedation purposes in general ICU patients according to the manufacturers’ dosages. Benzodiazepines were typically applied according to the German guidelines after an initial phase of propofol, which was never applied for more than seven days due to legal restrictions . Findings of this study should be confirmed in a randomized controlled trial. Furthermore, future studies comparing midazolam and lormetazepam should take the administration of alpha-2 agonists, analgesics, neuroleptics, and additional sedatives into consideration.In adult critically ill patients, decreased survival was associated with deeper sedation which is in line with previous studies. We found that in the midazolam compared to the lormetazepam group, deep sedation occurred more frequently. Differences in sedation depth and mortality between patients receiving lormetazepam and midazolam should be compared in a prospective, randomized controlled trial, which should have a particular focus on the ability of the substances to maintain a light-sedation regime in the ICU.


To evaluate the efficacy and safety of recombinant human serum albumin /granulocyte colony-stimulating factor (rHSA/G-CSF) in breast cancer following receipt of cytotoxic agents.The phase 1b trial assessed the pharmacokinetics, pharmacodynamics, and safety of dose-escalation, ranging from rHSA/G-CSF 1800 μg, 2100 μg, and 2400 μg. Randomized controlled phase 2b trial was further conducted to ensure the comparative efficacy and safety of rHSA/G-CSF 2400 μg and rhG-CSF 5 μg/kg. In multicenter, randomized, open-label, parallel, phase 2 study, participants treated with anthracycline-containing chemotherapy were assigned in a ratio 1:1:1 to receive double delivery of rHSA/G-CSF 1200 μg, 1500 μg, and continuous rhG-CSF 5 μg/kg.P = 0.71). Safety profiles were assessed to be acceptable ranging from rHSA/G-CSF 1800 μg to 2400 μg, while the double delivery of HSA/G-CSF 2400 μg failed to meet the noninferiority in comparison with rhG-CSF.Between December 16, 2014, to July 23, 2018, a total of 320 patients were enrolled, including 25 individuals in phase 1b trial, 80 patients in phase 2b trial, and 215 participants in phase 2 study. The mean duration of agranulocytosis during the first chemotherapeutic intermission was observed as 1.14 ± 1.35 days in rHSA/G-CSF 1500 μg, which was comparable with that of 1.07 ± 0.97 days obtained in rhG-CSF control , NCT03246009 (08/08/2017), NCT03251768  (08/07/2017).ClinicalTrials.gov identifiers: The online version contains supplementary material available at 10.1186/s12885-021-08093-z. Over the past decades, the application of cytotoxic agents has provided great benefits for almost all categories of malignancies in clinical practice , 2. HoweRecombinant granulocyte-colony stimulating factor (G-CSF) preparations have emerged as effective supportive therapies for chemotherapy-induced neutropenia, of which the clinical efficacy has been confirmed by numerous clinical trials, and generally recommended by guidelines –9. ThrouRecently, the pegylated G-CSF, as a covalent conjugate of rhG-CSF and polyethylene glycol (PEG), has been designed and developed to improve the systemic exposure to the drug delivery and further enhance the biological response to effective components. This novel structure of pegylation could weaken the proteases hydrolysis toward protein components and invariably prolong the half-life of the agent (~ 72 h) –18. HowePichia pastoris. Preclinical studies have suggested that the elimination half-life of rHSA/G-CSF (~ 38.6 h) is approximately over 10 times longer than that of rhG-CSF (~ 2.54 h). The double dose of rHSA/G-CSF administered at the interval of 96 h has comparable efficacy on the motivation of circulating neutrophils with standard rhG-CSF continuously administered once a day.Recombinant human serum albumin /granulocyte colony-stimulating factor (rHSA/G-CSF) was a novel biosimilar product of rhG-CSF, which was developed on the basis of a proprietary recombinant DNA process that was expressed in highly engineered yeast strain Herein, we conducted a study with three proportions of phase 1b trial, phase 2b trial, and phase 2 trial, to evaluate the safety, pharmacokinetics (PK), efficacy, and pharmacodynamics (PD) of rHSA/G-CSF in breast cancer patients receiving anthracycline-containing chemotherapy.This three-stage study comprised a dose-escalation study (phase 1b), a dose-expansion study (phase 2b), and a randomized controlled study (phase 2). A dose-escalation study was performed to assess the dose-dependent effect, observed in the aforementioned trial, and explored the safety, PK, PD of the growing dosage rHSA/G-CSF. Phase 2b trial involved an expanding cohort was to assess the comparative efficacy and safety of the regimens containing rHSA/G-CSF, based on the recommended dosage according to prior assessment, and the paralleling rhG-CSF. Patients enrolled in phase 2 trial were treated with successive increasing doses of rHSA/G-CSF or the paralleling controlled agent (rhG-CSF), of which the findings recommended the non-inferior dosage regimens of rHSA/G-CSF in terms of prevention from agranulocytosis following anthracycline-containing chemotherapy.This study was conducted in consistence with protocol, good clinical practice, and the Declaration of Helsinki. The protocol and corresponding amendments were approved by individualized review boards or ethics committees of participating institutions. All participants have provided written informed consent prior to enrollment. The experimental agent-rHSA/G-CSF-was provided by Tianjin SinoBiotech Ltd., a subsidiary of FortuneRock (China) Co., Ltd. The paralleling-controlled drug (Jisaixin) was provided by North China Pharmaceutical Jintan Biotechnology Co., Ltd. with 300 μg/tube.9/L, platelet ≥100 × 109/L at baseline; East Cooperative Oncology Group (ECOG) performance status of 0 or 1, and no bone marrow involvement. Patients were excluded if they received radiotherapy  within 4 weeks before enrollment; suffering the infectious disease in uncontrollability, and with axillary temperature ≥ 38 °C; durative diabetes mellitus, with the fasting serum level of glycose beyond the normal range; presenting the history of allergic to protein pharmaceutical preparations, and human chorionic gonadotropin testing positive in fertile women.Eligible patients were aged 18 to 65 years with histologically confirmed stage I-III after breast conservative or radical surgery or de novo stage IV breast cancer; treatment-naïve and scheduled to receive chemotherapy; an absolute neutrophil count (ANC) ≥ 1.5 × 109/L or ANC ≥ 5 × 109/L. The dosage was consecutively escalating if none of the patients at a given dose experienced dose-limiting toxicity (DLT). If two or more patients experienced a DLT, dose-escalation would cease for further enrollment at that dosage level. Any cohort in which one patient experienced DLT was expanded by up to six patients. If any one of the six patients exhibited evidence of a DLT, dose escalation was stopped and no further treatments were administered. Patients were considered as treatment failure if they presented with a grade 3–4 leukopenia or neutropenia on the 4th day following administration of rHSA/G-CSF. These patients would be withdrawn from the study and received standard supportive care of rhG-CSF, and the continuation or a failed termination of the study, for the patients allocated in the post dose, would be determined by investigators.Participants were enrolled in the phase 1b study  between May 23, 2016, to April 10, 2017, constituting two cohorts, in which rHSA/G-CSF was administrated on the single and double dosage in sequential dose-escalation design, and both the dosage regimens were on the basis of rHSA/G-CSF 1800 μg, 2100 μg, and 2400 μg to assess the dose-dependent effect and explore the safety, efficacy, PK, and PD. Based on the ‘3 + 3’ principle, eligible patients with leukopenia or neutropenia following anthracycline-containing chemotherapy were successively allocated initiated at the 1800 μg with subsequent doses studied in an ascending manner at an interval of 10 days. At a follow-up of 14 days, patients were observed for clinical and laboratory manifestations. The multi-dose study was based on the results of the single-dose study, in which patients in two dose cohorts were scheduled to receive repeatedly given doses of rHSA/G-CSF at an interval of 4 days until a presentation of white blood cell (WBC) ≥ 10 × 10The phase 2b trial was a multicenter, randomized, open-label, parallel study , of which breast cancer patients suitable for chemotherapy based on TEC or TE regimens were recruited between October 19, 2017, to July 23, 2018, and randomly allocated in a 1:1 ratio to two groups that were treated with either rHSA/G-CSF 2400 μg or rhG-CSF (Jisaixin) 5 μg/kg in the same pattern of administration on the chemotherapeutic intermissions.2 intravenously every 3 weeks, epirubicin (E) 75 mg/m2 intravenously every 3 weeks, with or without cyclophosphamide (C) 500 mg/m2 intravenously every 3 weeks. The experimental agent-rHSA/G-CSF-was given the first subcutaneous injection on the 3rd day after chemotherapy, followed by the second injection with an interval of 96 h., and the third administration was in the investigators’ choice depending on the dynamic monitored ANC.The phase 2 trial was a multicenter, randomized, open-label, paralleling-controlled study with the prospective registration on ClinicalTrials.gov . Eligible patients were enrolled between December 16, 2014, to December 23, 2015, and randomly assigned in a ratio of 1:1:1 to prophylactically receive twice rHSA/G-CSF 1200 μg, 1500 μg, and continuous rhG-CSF (Jisaixin) 5 μg/kg during the upfront two chemotherapeutic intermissions following treatment with docetaxel (T) 75 mg/m9/L after the initial peak following rHSA/G-CSF treatment, the dynamic ANC, the incidence and duration of FN, and the therapeutic course of prescribed antibiotics.The primary efficacy endpoint of phase 2/2b trials was the duration of agranulocytosis (DOA) during the first chemotherapeutic intermission. The secondary endpoints were the DOA after the second chemotherapeutic performance, the length of the period until ANC climbing up to 2 × 10The count of WBC and neutrophils were dynamically monitored, as stipulated by protocol, in the course of therapeutic administration. To weaken the effect from confounders, permitted concomitant medications were limited to scheduled chemotherapeutics, antiemetic agents or other premedication at the discretion of the physician. Prophylactic corticosteroids or non-narcotic analgesics were not permitted unless undisciplinable allergies or pains occurred. Do not use antibiotics prophylactically. Antibiotics only were used in the case of grade 4 neutropenia lasting more than 3 days, neutropenia fever, definite infection, or axillary temperature ≥ 38.0 °C, and infectious fever that cannot be ruled out.max), time to attain maximum plasma concentration (Tmax), area under plasma concentration-time curve (AUC), half-life (t1/2), apparent volume of distribution (Vz), clearance (CL), and mean residence time (MRT), the analysis of which performed by DAS 3.2.6 software using the non-compartmental method.Plasma and urine rHSA/G-CSF concentrations were determined using a validated sandwich enzyme-linked immunosorbent assay. In single dosage, blood samples were collected before rHSA/G-CSF delivery and at 1, 2, 6, 12, 24, 48, 72, 96, 120, 144, 192, and 240 h after administration. In double dosage, blood samples were collected before rHSA/G-CSF delivery of the first cycle and at 1, 2, 6, 12, 24, 48, 72, 96 after first administration, and before the second delivery and 1, 2, 6, 12, 24, 48, 72, 96, 120, 144, 192 and 240 h after the second administration. PK parameters included maximum plasma concentration (CPatients were followed for adverse events (AEs) in systemic symptoms, physical signs, complete blood count, biochemistry, urinalysis, and electrocardiogram at screening and scheduled time point post dose. Safety was assessed by a statistical description of frequency and severity of AEs, characterized as mild, moderate, or severe. Serious AEs (SAEs) were defined as the AEs that resulted in death, hospitalization, the prolonged period of hospitalization, persistent or significant disability or incapacity, congenital anomaly or birth defect, or AEs that were otherwise medically important.9/L, neutropenia was ANC ≤ 1.5 × 109/L, and leukopenia was WBC ≤ 3 × 109/L. FN was regarded as single oral temperature measurement > 38.3 °C (axilla temperature ≥ 38.0 °C), or oral temperature measurement > 38 °C lasting for at least 1 h, in company with neutropenia. DLT was referred to clinically significant AEs which were considered as associated with the experimental agent by investigators, including grade ≥ 2 hepatic or renal toxicity, grade ≥ 3 nonhematologic toxicity, and grade ≥ 4 hematologic toxicity lasting ≥7 days. All the classifications of AEs categories and grad were followed Common Terminology Criteria for Adverse Events (CTCAE) version 4.0.In this study, agranulocytosis or grade 4 neutropenia was defined as ANC < 0.5 × 10t test or the Wilcoxon rank sum test as appropriate. Statistical tests were two-sided with a significant level set at 0.05. All statistical analyses were performed using the SPSS software version 16.0.The analysis of variance and the Kruskal-Wallis test were used to test for divergence across groups. Pairwise differences were inspected using the A total of 29 patients in three research centers were enrolled into the phase 1b study, which included 13 patients in the single-dose proportion and 12 patients in the multi-dose part  subjects enrolled in single-dose part while 29 AEs were observed in 8 out of 12 (66.7%) participants involved in multi-dose cohorts. The most commonly reported AEs of any grade were increased alanine aminotransferase (ALT) (21.6%), anemia (18.9%), increased aspartic aminotransferase (AST) (10.8%), and just 3 treatment-related AEs were recorded including ostealgia and eye edema up to grade 2 . The statistical analysis of derived PK parameters indicated against dosage effect but the inherent variability from enrolled participants. No cycle-to-cycle accumulation (i.e. AUC (0-t), AUC (0-∞), P > 0.05) were shown in both the single-dose and multi-dose cohorts  and failed to be advantageous over rhG-CSF. This result was consistent with those from stratified analysis of chemotherapy protocol. However, the motivation of ANC tended to be moderate with a steady trend exhibited in the dynamic curve of neutrophils increased , while patients who received rHSA/G-CSF 1200 μg were suggested to suffer a prolonged DOA than those in both rhG-CSF cohort (P = 0.019) and rHSA/G-CSF 1500 μg (P = 0.046). The mean DOA after the second chemotherapeutic performance was 0.85 ± 1.35 days in rHSA/G-CSF 1200 μg, 0.50 ± 0.96 days in rHSA/G-CSF 1500 μg, and 0.44 ± 0.77 days in rhG-CSF, respectively, and the results indicated that no statistical differences were detected between rHSA/G-CSF 1500 μg and rhG-CSF as well as rHSA/G-CSF 1500 μg and rHSA/G-CSF 1200 μg. The incidences of febrile neutropenia were 23.9, 6.9, and 1.11% in rHSA/G-CSF 1200 μg, rHSA/G-CSF 1500 μg, and rhG-CSF group with statistical differences detected between rHSA/G-CSF 1500 μg and the other two cohorts, indicating that patients preventively receiving rHSA/G-CSF 1500 μg suffered the least risk of this detrimental complication following cytotoxic agents. However, no statistical differences of the durative period of febrile neutropenia in addition to the rate of antibiotic administration were demonstrated among the three groups. Besides, for the time before ANC ascending to 2 × 109/L from the first peak and the dynamic count of neutrophils, a steady trend in the growth of ANC was observed which revealed a moderate stimulus intensity for ANC provided by rHSA-G-CSF, although this recorded period of both rHSA/G-CSF 1200 μg and 1500 μg was prolonged than that of rhG-CSF  between the two-dose groups compared with the previous test .Safety profiles revealed that 36 AEs in total occurred in 25 (11.6%) patients including 10 AEs in 6 (8.5%) patients of 1200 μg group, 9 AEs in 8 (11.1%) patients of 1500 μg group, and 17 AEs in 11 (15.3%) patients of the control group. The leading AEs in 1200 μg and 1500 μg were ostealgia (21.1%), weak (15.8%), fever (15.8%), and anemia (15.8%), while the frequently occurring AEs in rhG-CSF group were backache (27.3%), fever (18.2%) and thrombocytopenia (9.1%). No statistical difference was suggested, and no treatment-related SAEs were observed in the three cohorts  , whereasThrough the initial assessment of the comparative phase 2 trial, the rHSA/G-CSF 1200 μg exhibited a prolonged DOA than rHSA/G-CSF 1500 μg, which revealed a dose effect on the stimulus motivation of neutrophils following anthracycline-containing chemotherapy. However, the following trials reported against this effect from regimens based on a higher dose at 2400 μg, which failed to provide a noninferiority response in terms of shortening DOA similar to 1500 μg in phase 2 study. This phenomenon indicated that the motivation of hematopoietic function might not synchronize with dose escalation of G-CSF agents, which was consistent with several studies conducted previously , 33. It Safety analysis revealed that rHSA/GCSF was well tolerated, and no serious toxicities were observed with the administration on breast cancer patients receiving myelosuppressive chemotherapy. The majority of AEs observed in this study were moderate and well-managed, even the foremost proportion of abnormalities from laboratory examinations were considered as far from treatment-related. On the basis of advantages in pharmacodynamic and pharmacokinetic assessments, patients treated with the delivery of rHSA-G-CSF were less vulnerable to transient leukemia symptoms, such as ostealgia and fever, which could contribute to superior treatment compliance. Of note, an approximate 20% incidence of thrombocytopenia occurred in the interventional group with consistency in the previous trials , 33. HowRandomized control trials comparing the pegylated G-CSF preparation and Balugrastim, which was designed and developed in the identical molecular structure, have reported a noninferiority efficacy and comparable safety profiles in breast cancer patients with the receipt of chemotherapy , 35. In In this study, rHSA/GCSF, as a novel preparation, was proven to be efficacious and well-tolerated in the course of prevention and management of agranulocytosis from breast cancer patients following upfront chemotherapy. With the comparable efficacy and safety profiles with rhG-CSF, this kind of injectable medication with both a less frequency and a lower expense of application could facilitate patients to receive the delivery of both chemotherapy and corresponding treatment for associated side effects in clinical practice.Additional file 1 : Table S1. Population demographics and baseline characteristics in this study. Table S2. Safety profiles of patients enrolled in dose-escalation study. Table S3. Pharmacokinetics parameters calculated from phase 1b trial. Table S4. Pharmacokinetics analysis from dose-escalation phase 1b trial. Table S5. Safety profiles of patients included in randomized studies


The cerebrospinal fluid tap test (CSF TT) is used for selecting shunt surgery candidates among patients with idiopathic normal pressure hydrocephalus (iNPH). We aimed to evaluate the predictive value of the CSF TT, by using the Hellström iNPH scale for shunted iNPH patients with a standardized method.One hundred and sixteen shunt-operated iNPH patients were retrospectively included in this study. The gait and balance domains in the iNPH scale were used as outcome measures for the CSF TT and the total iNPH scale score as the postoperative outcome. A positive response to CSF TT was defined as a change of ≥ 5 points in the gait domain and ≥ 16 points in the balance domain. Differences between CSF TT responders and non-responders, sensitivity, specificity, positive and negative predictive values, accuracy, and correlations between changes from baseline to post CSF TT and from baseline to the postoperative follow-up, were calculated.In the CSF TT there were 63.8% responders in the gait domain and correspondingly 44.3% in the balance domain. CSF TT responders had a significantly better postoperative outcome in the total scale score  and gait CSF TT responders improved more in gait (P ≤ 0.001) and balance CSF TT responders in balance (P ≤ 0.001). No differences between CSF TT gait or balance responders could be found in neuropsychological or urinary continence assessments postoperatively. The sensitivity and specificity of the CSF TT and the outcome of the total iNPH scale score postoperatively were 68.1% and 52.0% for gait and 47.8% and 68.0% for balance, respectively.The CSF TT, with the Hellström iNPH scale as the outcome measure, has clear limitations in predicting postoperative results. The gait domain may be used to predict outcomes for gait, but the balance domain is too insensitive. Idiopathic normal pressure hydrocephalus (iNPH) is a condition that usually has slowly progressive symptoms. In combination with a dilated cerebral ventricular system, the main symptoms are gait disturbance and poor balance, together with cognitive decline and urinary incontinence. The disease is caused by altered cerebrospinal fluid (CSF) dynamics, but the underlying pathophysiology is not fully understood . The onlThe cerebrospinal tap test (CSF TT), which involves a lumbar puncture (LP) with removal of 30–50 ml CSF and clinical evaluation before and after the LP, is commonly used to predict patients who could benefit from a shunt insertion –7. The CThe aim of this study was to evaluate the prognostic value of the CSF TT, using the Hellström iNPH scale  among shPatients with a diagnosis of possible and probable iNPH, based on the international guidelines of 2005  and treaA specialized team including neurologists, neurosurgeons, neuroradiologists, physiotherapists and occupational therapists assessed the patients preoperatively. The occupational therapist and physiotherapist assessments, accomplished within one week before the CSF TT, included all the measurements for gait, balance, neuropsychology and continence in the Hellström iNPH scale . A neuroApproximately 3 h after the LP, a physiotherapist reassessed the patients, using the measurements within the gait and balance domains in the iNPH scale . In addiThe Hellström iNPH scale  is constAccording to the instructions by Hellström et al. the results in each domain were converted to a 0–100 score, where 100 represents the performance of an age-matched healthy population. Each step in the ordinal balance scale was converted to 16 or 17 scores in the iNPH scale. A total score was calculated from the converted scores with a double weight from the gait domain and divided by the number of domain scores. If any domain had missing data, the total score was calculated with the available data .Normal distribution was tested with the Kolmogorov–Smirnov test. Normal distributed variables are presented with mean and Standard Deviations (SD), otherwise with median and interquartile range (IQR) or absolute and relative frequencies (%). In Table One hundred and sixteen individuals, 62 male and 54 female with a mean age of 75.4 ± 6.2 years were included in the analysis. Ninety-five patients were classified retrospectively from the patients´ charts by AE and FL as probable iNPH and 21 as possible iNPH according to the international guidelines [The gait domain CSF TT responders had significantly larger postoperative improvements from baseline in the total iNPH scale scores ; P ≤ 0.001. The gait domain CSF TT responders improved also significantly more in the gait domain scores postoperatively than the gait domain CSF TT non responders ; P ≤ 0.001 (Table The gait domain CSF TT non-responders had significant lower scores at baseline in the gait domain scores (P = 0.031), in the balance domain scores (P = 0.006), in the neuropsychology domain scores (P = 0.006) and in the total iNPH scale scores (P = 0.010) Table .When analyzing results from the subtests within the gait domain  there were no between-group differences for gait domain CSF TT responders versus gait domain CSF TT non-responders in changes from baseline to the postoperatively follow up. At baseline there were between group differences between the gait domain CSF TT responders and non-responders in the 10 m walk test  and scores in the ordinal gait scale (P = 0.011) Table .The balance domain CSF TT responders had significantly larger postoperative improvements from baseline in the total iNPH scale scores ; P = 0.012. The balance domain CSF TT responders improved significantly more in the balance domain postoperatively than the balance domain CSF TT non-responders ; P ≤ 0.001 . All other domains as well as for the total iNPH scale scores were equal at baseline Table .No significant between-group differences among CSF TT responders  compared to CSF TT non-responders were seen postoperatively in the neuropsychology or continence domains Table .The sensitivity using the gait domain as outcome in the CSF TT and the total iNPH scale as outcome postoperatively was 68.1% and the PPV 83.8%. The specificity was 52.0% and the NPV was 31.0% with an accuracy of 64.7%  individuals were true positive, 12 (10%) were false positive, 29 (25%) were false negative and 13 (11%) were true negative. With the outcome gait domain scores at the CSF TT and the same outcome (gait domain score) postoperatively, 61 of 116 (53%) were true positive, 13 (11%) were false positive, 24 (21%) were false negative and 18 (16%) were true negative  individuals had no change in the balance domain score in the CSF TT.Predicting a favorable postoperative result from the CSF TT, using the Hellström iNPH scale as an outcome parameter after shunt treatment in iNPH, has clear limitations. Gait domain CSF TT responders improved more in the gait domain and in the total iNPH scale scores from baseline to the postoperative follow-up versus the gait domain CSF TT non-responders. Both gait domain CSF TT responders and gait domain CSF TT non-responders improved significantly in all domains after surgery. The gait domain CSF TT responders performed significantly better in all domains except for continence at baseline Table . The gaiThe difference in outcome measures and timing for assessment, limits the comparability between studies . The choThe cut-off > 5 points in the total iNPH scale, is suggested by Hellström et al. to identify improvement in the postoperative outcome . The samWe used two different outcomes in the CSF TT, the gait domain and the balance domain in the iNPH scale. The iNPH scale is constructed to cover the most characteristic features in iNPH and to discriminate between patients with variations in symptoms and between patients and healthy individuals. The four domains of the iNPH scale are related but none of the domains could be excluded . In the The iNPH scale tolerates missing values but in our study there were few. When analyzing the separate subtests within the gait domain for CSF TT gait domain responders versus CSF TT gait domain non-responders there were somewhat surprisingly no differences in changes from baseline to the postoperative follow up for any of the subtests. In the converted gait domain score there was a significant difference with a larger improvement postoperatively in the CSF TT gait domain responders Table . The conOur results are consistent with previous research , 18, 19 In our study, with gait as outcome measure in the CSF TT and the total score as outcome postoperatively, the specificity was lower (52.0%) than in the Japanese study . Even wiThis was a retrospective study and a reflection of clinical routine, i.e. there was no blinding and both the patients and the assessors knew the conditions concerning the CSF TT assessment routines. Thus, a placebo effect could be, at least partially, an explanation. Gupta and Lang  describeAnother reason for a false positive result from the CSF TT could be a malfunctioning shunt at the follow-up. In our study, we had an accurate registration of shunt adjustments and complications. The shunt should be in a proper condition at the follow up. Three patients with positive outcome of gait in the CSF TT and negative outcome postoperatively hade shunt adjustments before the follow-up.The relatively long time from baseline to surgery (median 126.0 days) may have negatively influenced the results. Shunt treatment is emphasized to be performed as soon as possible after diagnosis according to the continuous progression of symptoms .In the European iNPH multicenter study, using the Hellström iNPH scale, a correlation between improvement after the CSF TT and outcome post-surgery was only found in the gait domain, with a low correlation (r = 0.22) . In our Patients within the CSF TT gait responder group in the present study had significantly higher baseline scores than the CSF TT gait non-responders in all iNPH scale domains (except for the continence domain) and in all subtests within the gait domain Table . In prevSeventy-eight percent of the operated participants improved on the total iNPH scale score after surgery, which is in line with the 75.0% improvement rate recently reported . The facDue to the high positive predictive value, there are those who support the use of the CSF TT as an additional test to detect shunt candidates. The low negative predictive value emphasizes the importance of not excluding patients from surgery , 10, 14.A limitation of this study is the retrospective design. We included patients over a period of 4 years and there were different examiners during that time. However, strengths of the study are that we used a standardized protocol and prospective inclusion. Using the robust iNPH scale, which tolerates missing values, is a strength and we had few missing data in the included material. The iNPH scale is valid when measuring outcome after surgery and we used the proposed cut-off level. The post CSF TT assessment was measured about three hours after the LP and we used no repetition. Virhammar et al.  have repUsing the gait domain in the Hellström iNPH scale for CSF TT can detect gait outcome after surgery but the negative predictive value is low. The balance domain is too insensitive to use as an assessment of the CSF TT. Clinical examination, accurate radiological assessment and careful consideration of other explanations to cover all aspects of iNPH are important parts in the evaluating process ultimately answering the question who would benefit from shunt surgery.


BR (39%), LGI1 (17%) and AMPAR (11%) antibodies. Results were reproduced in a separate cohort (B) of 54 AE patients with LGI1, GABABR or AMPAR antibodies in CSF which were missed in 30% by commercial IIFA. Patients with discordant GABABR antibody results  were less likely to develop full-blown clinical syndrome; no significant clinical differences were noted for the other antibodies. Overall, NSAb testing by commercial IIFA led to false negative results in a substantial number of patients, mainly those affected by anti-LG1, GABABR or AMPAR encephalitis. If these disorders are suspected and commercial IIFA is negative, more comprehensive antibody studies are recommended.Detection of neuronal surface antibodies (NSAb) is important for the diagnosis of autoimmune encephalitis (AE). Although most clinical laboratories use a commercial diagnostic kit  based on indirect immunofluorescence on transfected cells (IIFA), clinical experience suggests diagnostic limitations. Here, we assessed the performance of the commercial IIFA in serum and CSF samples of patients with suspected AE previously examined by rat brain immunohistochemistry (Cohort A). Of 6213 samples, 404 (6.5%) showed brain immunostaining suggestive of NSAb: 163 (40%) were positive by commercial IIFA and 241 (60%) were negative. When these 241 samples were re-assessed with in-house IIFA, 42 (18%) were positive: 21 (9%) had NSAb against antigens not included in the commercial IIFA and the other 21 (9%) had NSAb against antigens included in the commercial kit . False negative results occurred more frequently with CSF (29% vs 10% in serum) and predominantly affected GABA Detection of antibodies to neuronal surface proteins and synaptic receptors is important to establish a definitive diagnosis of autoimmune encephalitis . Well-chBR (n=23) antibodies confirmed by brain immunohistochemistry and in-house IIFA (Cohort B).We prospectively examined 6213 serum and CSF samples from patients referred to our diagnostic lab for detection of antibodies against neuronal surface antigens from October-2016 to October-2020 (Cohort A). Samples were screened with rat brain immunohistochemistry and results were examined by two independent observers. Samples showing positive immunostaining suggestive of a neuropil antibody were first studied with commercial IIFA  or CSF (1:2) overnight at 4°C. Biotinylated goat anti-human IgG  was added for 2 h, followed by incubation with the avidin–biotin immunoperoxidase complex  for 1 h. The reaction was developed with 0.05% diaminobenzidine .Tissue immunohistochemistry was performed as previously described . BrieflyB receptor , CASPR2, LGI1 , GABAA receptor , metabotropic glutamate receptors mGluR1, mGluR2, mGluR5, Ig-Like Domain-Containing Protein 5 (IgLON5) or Seizure 6-like protein 2 (SEZ6L2) as previously described .Samples that produced a neuropil immunostaining on rat brain immunohistochemistry were subsequently examined with two types of IIFAs: 1) the Autoimmune Encephalitis Mosaic 6 kit , following manufacturer’s instructions and recommended dilutions (undiluted CSF and 1:10 serum), to test IgG antibodies against N-methyl-D-aspartate (NMDA) receptor (GluN1), α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor , gamma-aminobutyric (GABA) B receptor (B1 and B2 subunits), contactin-associated protein-like 2 (CASPR2), leucine-rich glioma-inactivated protein 1 (LGI1) and dipeptidyl-peptidase 6 (DPPX), 2) in-house IIFAs in which HEK293 cells were transfected with DNA constructs to express the following antigens: NMDA receptor , AMPA receptor , GABAescribed –17. BrieThe study was approved by the ethics committee of Hospital Clínic of Barcelona. Patients’ samples were coded and clinical information was anonymized prior to analysis. Written inform consent was not required as the study was observational, and the detection of neuronal surface antibodies was requested as part of the routine diagnostic work-up.BR [6 sera/5 CSF] and 1 (1%) for DPPX [serum] antibodies (AR) (BR (1 serum/6 CSF), 2 AMPAR (2 CSF), and 1 NMDAR (serum)  for LGI1, 11% (2/18) for AMPAR, and 1.4% (1/69) for NMDAR antibodies. Among 41 patients with paired serum and CSF samples we obtained false negative results in 7 (17%): antibodies were not detected in one sample by the commercial IIFA in 5/15 patients with LGI  and 1/5 with AMPAR antibodies (in CSF), and not detected in serum and CSF in 1/3 patients with GABABR antibodies .In Cohort A, 404 (6.5%) of the 6213 samples  showed a positive staining on brain immunohistochemistry suggesting the presence of neuronal surface antibodies. These 404 samples were analyzed by the commercial IIFA and 163 (40%) resulted positive for IgG against one of the included antigens: 68 (42%) for NMDAR [27 sera/41 CSF], 52 (32%) for LGI1 [26 sera/26 CSF], 16 (10%) for AMPAR [11 sera/5 CSF], 15 (9%) for CASPR2 [9 sera/6 CSF], 11 (7%) for GABAtibodies Figure 1BR, LGI1 or AMPAR antibodies, confirmed by brain immunohistochemistry and in-house IIFA, and retested them by commercial IIFA (Cohort B). The commercial kit failed to detect antibodies in 16 (30%) samples: 4/12 (33%) with LGI1, 7/23 (30%) with GABABR, and 5/19 (26%) with AMPAR antibodies. Overall, we obtained a similar frequency of CSF false negative results in both cohorts: 5/51 (29%) from cohort A and 16/54 (30%) from cohort B.Considering that CSF samples gave more discordant results between commercial and in-house IIFAs and CSF antibody detection is crucial for the diagnosis of autoimmune encephalitis, we assessed 54 additional CSF samples of patients with encephalitis and GABABR and AMPAR antibodies with the commercial IIFA.As LGI1 cell transfection differed between the commercial kit and our in-house IIFA (co-transfected with ADAM23), we used a modified in-house GPI-LGI1 IIFA  . We testBR/LGI1/AMPAR antibodies in the CSF of Cohort A  and Cohort B (54 patients). BR antibody detection  developed less frequently the full-blown clinical encephalitis syndrome than those with a concordant detection (positive by both IIFAs). In the discordant group 3 patients had refractory epileptic seizures and 1 had chorea (4/11) when their samples were sent for study, whereas in the concordant group 1/21 had refractory seizures (p=0.011). Additionally, brain MRI from patients with discordant GABABR and AMPAR IIFA results were more often normal at the time antibodies were determined , LGI1 (17%) and AMPAR (11%). Similar results were obtained in a separate series of 54 CSF samples (Cohort B) from patients with LGI1, GABABR or AMPAR antibodies (detected by brain immunohistochemistry and in-house IIFA) showing that the commercial kit failed to detect 33% of LGI1, 30% of GABABR, and 26% of AMPAR antibodies.Our study shows that neuronal surface antibody detection using only the commercial IIFA has limitations in the diagnosis of autoimmune encephalitis. Among 241 samples that were positive by brain immunohistochemistry but negative by the commercial kit, we found 42 (18%) that had well-defined neuronal surface antibodies using our in-house IIFA. Half of these samples had antibodies against antigens not included in the commercial kit. However, the remaining 50% of samples were considered false negative results, as the in-house IIFA detected antibodies that should had been detected by the commercial IIFA. False negative results were more common in CSF (29% for CSF vs. 10% for serum), and particularly occurred for the antibodies against GABABR antibody detection in the serum of patients with autoimmune encephalitis. For example, a study assessing 256 patients with LGI1/CASPR2 antibodies with the same commercial kit found lower sensitivity in CSF than in serum testing. Among 196 patients with LGI antibodies the authors found commercial IIFA positivity in 63% of CSF samples (24 of 38), and recommended that serum should be tested for determination of LGI1 antibodies by IIFA  than in serum (29/30 positive). CSF results were slightly improved using in-house IIFA (18/20 positive) and with a modified assay co-transfecting the intracellular accessory protein of the B2 subunit of the GABA receptor potassium channel tetramerization domain-containing 16 (KCTD16) (20/20 positive) . When we re-tested the 11 CSF samples with discordant commercial/in-house LGI1 antibody results using GPI-LGI1 IIFA (cells transfected only with LGI1) all of them were found negative. An explanation could be that the recognition of some LGI1 target epitopes by CSF antibodies is improved when ADAM23  is co-expressed. The reasons for the disparity in the results of GABAositive) . AdditioBR or AMPAR antibodies had clinical features of the encephalitis typically associated with the corresponding antibody  regardless of the negative findings with the commercial kit  anti-LGI1 encephalitis is the most common encephalitis in adults, but up to 13% develop cognitive impairment without criteria of encephalitis ; 3) delaying or not giving appropriate immunotherapy may impact patients’ outcome; and 4) studies on incidence, prevalence and biology of encephalitis, and recommendations about diagnosis are frequently based on results using commercial kits, and not on the real data of the disease when antibodies are comprehensively tested and results validated. Future studies investigating autoimmune encephalitis should consider these limitations. In case of negative results with the commercial kit, we recommend to extend the study using brain immunohistochemistry and in-house IIFA.The main message of our study is that the commercial IIFA for autoimmune encephalitis leads to false negative results in a substantial number of patients, especially when CSF is used, and predominantly for LGI1, GABAphalitis . A falseThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study was approved by the ethics committee of Hospital Clínic of Barcelona. Patients’ samples were coded and clinical information was anonymized prior to analysis. Written inform consent was not required as the study was observational, and the detection of neuronal surface antibodies was requested as part of the routine diagnostic work-up. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.EM-H designed the study and wrote the manuscript in collaboration with RR-G, AS, JD, and FG. RR-G, GM-S, LN, LS, MG, and EM-H performed laboratory work and/or data analysis. All authors contributed to the article and approved the submitted version.Fondo Europeo de Desarrollo Regional (FEDER) – Instituto de Salud Carlos III ; Resident award “Josep Font” by Hospital Clínic Research, Innovation and Education Departments (MG); La Caixa Foundation (JD); Edmond J Safra Foundation (JD); Fundació CELLEX (JD) and Red Española de Esclerosis Múltiple RD16/0015/0002, RD16/0015/0003 (AS).JD receives royalties from Athena Diagnostics for the use of Ma2 as an autoantibody test and from Euroimmun for the use of NMDA, GABAB receptor, GABAA receptor, DPPX and IgLON5 as autoantibody tests. FG holds a patent for the use of IgLON5 as an autoantibody test.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The handling editor declared a past co-authorship with the authors FG and JD.


Research has demonstrated a positive effect of makeup on facial attractiveness . Makeup  Women in the United States are estimated to spend approximately $3,756 annually on their physical appearance and $225,360 during their lifetime . This hiSome have proposed that facial attractiveness is determined solely by culture; however, research suggests a biological basis for attractiveness as well . StudiesSexual dimorphism refers to feminine traits in female faces and masculine traits in male faces , and is Revealing a sexual dimorphism in facial coloration, In addition to its impact on facial contrast, makeup can also alter the apparent size of facial features . Research examining the impact of makeup on perceptions of eye size demonstrated that individually, eyeliner, mascara. and eye shadow make eyes appear larger, thus increasing the sexually dimorphic trait of larger eyes among females . ImportaThrough its positive effect on facial attractiveness, makeup has also been implicated in producing inflated social perceptions and more favorable social interactions. In a study directly examining how makeup affects ratings of attractiveness, competence, likeability, and trustworthiness, researchers presented participants with photos of female faces with minimal, moderate, or dramatic makeup . The resIn addition to its perceptual effects, facial attractiveness has also been found to significantly influence social interactions. Research on the interactions between mothers and their firstborn infants found that in comparison to mothers of less attractive infants, mothers of more attractive infants displayed greater affection and playfulness toward their infants . With reAlthough present research on facial attractiveness provides great insight on makeup’s enhancing effects, the methodology employed by most studies tests a relatively narrow set of conditions. Because many of the studies on makeup involve professional makeup artists, it is difficult to discern whether their application techniques accurately reflect those that typical women use on a day-to-day basis. Another limitation posed by several of the studies examining differential ratings of perceived competence and success is the focus on managerial and business-executive positions. While business positions are an important point of examination because of the particularly low occupation rates for females, we would argue that perceptions made in the academic setting, a much earlier point in a woman’s career, may be equally influential on their success. To address these issues, the current study seeks to advance the facial attractiveness literature through examination of the effects of makeup on facial attractiveness using different face stimuli than in previous studies (self-applied makeup in college-age participants) and examining the social implications of makeup use for women in a university setting. To accomplish the goals of the study, we collected facial stimuli through a process by which participants applied their own makeup. These stimuli were then used to evaluate the impact of makeup on perceived facial attractiveness, competence and sociosexuality.We tested attractiveness to determine whether self-applied light or heavy was rated as more attractive compared to wearing no makeup. We were interested in looking at this as previous data have been somewhat mixed . AlthougBased on previous research findings, we anticipate that the results of this project will replicate previous studies that have shown that makeup has a significant effect on perceived facial attractiveness, competence, and sociosexuality. Our more ecologically valid self-applied makeup application procedure may lead to results different from research using professional makeup artists to apply makeup. We also predict that varying levels of makeup will differ in their effects on the responses of participants across these different types of judgments.In this study we compared female faces with no makeup, self-applied light makeup, and self-applied heavy makeup. Participants rated faces on attractiveness, competence, and sociosexuality so we could measure a range of traits that have been found to relate to makeup use and attractiveness. The goal was to determine if self-applied makeup leads to similar findings compared to makeup applied by a professional makeup artist  or the eUndergraduate women were photographed with varying levels of makeup  across the span of two sessions. Each subject participated in two, 30-min data collection sessions. Sessions comprised of participants being photographed with no makeup first, then either light makeup or heavy makeup. Prior to attending each session, participants were asked to bring all necessary makeup supplies for applying their own makeup. All photographs were taken using a standardized procedure, holding constant the lighting and distance of the camera . Participants were asked to look directly at the camera with a neutral facial expression.In the first session, the primary researcher briefly explained the study to participants before providing them with the consent form and offering to answer any questions, should they arise. Following consent, participants were verbally asked if they currently had any makeup on or if they were using any beauty enhancement products , for the purposes of ensuring consistency among the facial stimuli collected. All participants were provided one face wipe to clean their face prior to being photographed, to ensure there was no residual makeup on their faces in the no makeup condition. The first photograph taken in the session was of participants with no makeup. Subsequently, participants were asked to apply what they would consider to be “light makeup,” or makeup that they would wear on a daily basis. After completing their makeup application participants were photographed once more.The second session followed the same procedure as the first: participants were asked if they were currently wearing makeup, and provided a face wipe to clean their face prior to being photographed. The first photograph taken was of participants with no makeup on. Subsequently, participants were asked to apply what they would consider to be “heavy makeup,” or makeup that they would wear on a night out or special occasion. After completing their makeup application, participants were photographed once more, then given a debriefing form that provided them with further information about the experiment and the contact information of the primary investigator. We split the makeup application phase into two separate sessions to avoid issues with applying then removing makeup. We were concerned that there would be residue left from the previous makeup application and that the rubbing required for removal might lead to skin irritation or discoloration.n = 17, 48.57%), while others identified as Asian , Pacific Islander , Biracial/Multiracial , White , Other , and one participant did not respond .Stimuli were reviewed for completeness and picture quality, and standardized using Adobe Photoshop  to ensure that no facial stimuli were significantly more or less attractive than any other stimuli. These participants were shown facial stimuli from the 35 different individual females and asked to rate the faces presented on facial attractiveness using a 1–7 Likert scale, with 1 being very unattractive and 7 being very attractive. Only the no makeup version of the faces was shown for this rating. Results from the rating study revealed that participant ratings for each of the facial stimuli were within two standard deviations of the overall mean attractiveness ratings . Given the absence of any minor or major outliers, all facial stimuli were used for the experiment.Following the collection of facial stimuli, the faces were independently rated by another group of participants (F) for the 35 faces in each of the three conditions. We found that the mean CF value was lowest for faces with no makeup (M = 0.213), light makeup faces had a slightly higher mean CF value (M = 0.227), and the heavy makeup faces had the highest mean CF value (M = 0.273). Paired t-tests indicated that the facial contrast value difference between the heavy makeup and no makeup was significant (t(102) = −4.26; p < 0.0001). The difference between the heavy makeup and the light makeup images was also statistically significant (t(102) = −3.23; p = 0.0016). However, the facial contrast value difference between the light makeup and no makeup images was not significant (t(102) = −1.02; p > 0.05).We quantitatively measured for contrast differences in the no makeup, light makeup, and heavy makeup conditions. Our measurement was based on that used by 1. The program was run on three 21-inch, 2013 iMacs .The computer-based experiment was created and administered using SuperLab 5 software2. Survey questions gathered demographic information and assessed positive and negative attitudes toward makeup use.The survey was administered through the online survey platform, Qualtricsn = 44, 64%) with a smaller number of males , one non-binary, and two participants who did not report their gender. Participants ranged in age from 18 to 53 with an average age of 19.97 (SD = 4.55). A third of participants identified as Hispanic/Latino , while another third identified as Asian/Pacific Islander . The remainder reported themselves as White/European , Biracial/Multiracial , Middle Eastern , and Black .The experiment was run several months after the face stimuli were collected, reducing the probability that participants would be familiar with individuals in the face stimulus set. In addition, both groups of participants were recruited primarily from sections of the introduction to psychology course, which are mostly first year students and include both majors and non-majors. Thus, the participants were very unlikely to have encountered the students from whom the faces were collected. A total of 69 students were recruited through the CSUF Psychology Department human subject pool. Individuals were awarded course credit for their participation. Participants were predominantly female , which includes eight response buttons; only seven buttons were used in this experiment. The participants were shown the labeled-response keypad specific to their condition, pressed any key to proceed, and then viewed a 500 ms fixation cross, followed by the face image, along with an image of the keypad with the forced choice responses labeled (1–7 and what each response corresponded to depending on condition); the keypad image appeared below the face image. Following Twenty-four participants in the facial attractiveness group were asked to rate the faces presented on facial attractiveness using a 1–7 Likert scale with 1 being very unattractive and 7 being very attractive.Twenty-two participants in the competence group were asked to rate the faces presented on perceived competence using a 1–7 Likert scale with 1 being very incompetent and 7 being very competent.Twenty-three participants in the sociosexuality group were asked to rate the faces presented on their sociosexuality. They rated how likely they believed the person would be to have casual sex, using a 1–7 Likert scale with 1 being very unlikely and 7 being very likely.Separate cross-classified multilevel models were constructed to determine the predictive value of Makeup Application  for Attractiveness, Competence, and Sociosexuality.2(6) = 1865.80, p < 0.001. Participants’ predicted Attractiveness ratings are equal to 3.53 + 0.25 (Makeup Application). Participants’ average Attractiveness ratings increased by 0.25 for each increase in Makeup Application  was created using the Heavy Makeup stimuli as the reference group. Thus, coefficients in the No Makeup and Light Makeup stimuli groups compared attractiveness ratings to those in the Heavy Makeup stimuli groups. The variability in attractiveness ratings across participants and stimuli as well as the variability in the effect of makeup on attractiveness ratings across participants and stimuli were included in the model as random effects. The item Makeup had a significant impact on the Attractiveness ratings of the participants, χlication . Differepost hoc analysis revealed that participant’s Attractiveness ratings were significantly higher for the heavy makeup application (M = 3.95) than for the light makeup application . Additionally, participant’s Attractiveness ratings were significantly higher for the light makeup  and heavy makeup applications  than the no makeup application  = 1161.42, p < 0.001. Participants’ predicted Competence ratings are equal to 4.17 + 0.08 (Makeup Application). Participants’ average Competence ratings increased by 0.08 for each increase in Makeup Application  was created using the Heavy Makeup stimuli as the reference group. Thus, coefficients in the No Makeup and Light Makeup stimuli groups compared competence ratings to those in the Heavy Makeup stimuli groups. The variability in competence ratings across participants and stimuli as well as the variability in the effect of makeup on competence ratings across participants and stimuli were included in the model as random effects. The item Makeup had a significant impact on the Competence ratings of the participants, χlication . Differepost hoc analysis revealed that participant’s Competence ratings were significantly higher for the light makeup  and heavy makeup applications  than for the no makeup application  = 828.89, p < 0.001. Participants’ predicted Sociosexuality ratings are equal to 3.87 + 0.52 (Makeup Application). Participants’ average Sociosexuality ratings increased by 0.52 for each increase in Makeup Application  was created using the Heavy Makeup stimuli as the reference group. Thus, coefficients in the No Makeup and Light Makeup stimuli groups compared sociosexuality ratings to those in the Heavy Makeup stimuli groups. The variability in sociosexuality ratings across participants and stimuli as well as the variability in the effect of makeup on sociosexuality ratings across participants and stimuli were included in the model as random effects. The item Makeup had a significant impact on the Sociosexuality ratings of the participants, χlication . Differepost hoc analysis revealed that participant’s Sociosexuality ratings were significantly higher for the heavy makeup application (M = 4.39) than for the light makeup application . Additionally, participant’s Sociosexuality ratings were significantly higher for the light makeup  and heavy makeup applications  than the no makeup application  than faces with no makeup and faces with heavy makeup received significantly higher sociosexuality ratings than both no makeup and light makeup faces. Overall, faces with heavy makeup were rated as the most likely to have “casual” sex with multiple partners. These results extend recent work, in which researchers found that faces with makeup were perceived as more sociosexual than the same faces without makeup . PreviouWe found support for our proposal that makeup would have significant effects on perceptions of facial attractiveness, competence, and sociosexuality. Ratings of facial attractiveness and sociosexuality were highest for faces with heavy makeup. Ratings of competence for faces with light makeup and heavy makeup were both higher than ratings for faces with no makeup, but there were no differences between faces with light makeup and heavy makeup. Our results suggest that self-applied heavy makeup will provide more positive results for attractiveness judgments compared to self-applied light makeup, a finding that is counter to the advice often given in popular media. It is usually suggested that “less is more” and that lighter makeup is more attractive . Our datThis study significantly expands our knowledge of how makeup use affects perceptions of others. Through advancing this literature, we are able to increase the societal understanding of why makeup influences social perception of women. A better understanding of these issues may help us increase well-being and success.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by California State University, Fullerton Institutional Review Board. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.EA developed the research idea, created the study, and collected the data, with help from JP. EA was also responsible for writing the manuscript, with input and revisions contributed by JP. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.