A mechanistic understanding of core cognitive processes, such as working memory, is crucial to addressing psychiatric symptoms in brain disorders. We propose a combined psychophysical and biophysical account of two symptomatologically related diseases, both linked to hypofunctional NMDARs: schizophrenia and autoimmune anti-NMDAR encephalitis. We first quantified shared working memory alterations in a delayed-response task. In both patient groups, we report a markedly reduced influence of previous stimuli on working memory contents, despite preserved memory precision. We then simulated this finding with NMDAR-dependent synaptic alterations in a microcircuit model of prefrontal cortex. Changes in cortical excitation destabilized within-trial memory maintenance and could not account for disrupted serial dependence in working memory. Rather, a quantitative fit between data and simulations supports alterations of an NMDAR-dependent memory mechanism operating on longer timescales, such as short-term potentiation. Stein, Barbosa et al. show that anti-NMDAR encephalitis and schizophrenia are characterized by reduced serial dependence in spatial working memory. Cortical network simulations show that this can be parsimoniously explained by a reduction in NMDAR-dependent short-term synaptic potentiation in these diseases. In addition, hypofunction of NMDARs is linked to psychiatric disease, in particular schizophrenia6, and it possibly contributes to abnormal working memory function in patients with schizophrenia8. Indeed, reduced prefrontal NMDAR density characterizes this disease9. Yet, the specific neural alterations by which NMDAR hypofunction could lead to memory deficits in schizophrenia are still under debate8. Here, we studied working memory function in healthy controls, patients with schizophrenia, and patients recovering from anti-NMDAR encephalitis  subserves memory mechanisms at several timescales, including sustained working memory delay activity17 , as it is observed in schizophrenia and in studies using NMDAR antagonists 24, and alterations in NMDAR-regulated short-term synaptic potentiation25. In the modeling section of this study, we systematically test the potential of these candidate mechanisms for explaining our behavioral findings. We conclude that a reduction in short-term potentiation in a network model of working memory most parsimoniously reproduces the experimentally observed memory alterations in schizophrenia and anti-NMDAR encephalitis.We assessed memory alterations in a visuospatial delayed-response task Fig.  on two c26 (but see ref. 27). We calculated the circular standard deviation of bias-corrected response errors (“Methods”) as an inverse estimate of precision for each participant and delay. Correcting for biases as a systematic source of error allowed us to estimate memory precision independently of serial biases. For all groups, precision decreased equally with delay , “Methods”, Eq. ,  = 13.89, p = 1e−5). Moreover, biases differed between groups of participants (group × DoG(θd), F = 9.68, p = 0.0003), especially when comparing groups for different delay lengths (group × delay × DoG(θd), F = 8.45, p = 2e−5). Figure θd)): For healthy controls, initially repulsive biases became gradually more attractive with delay length  = 26.91, p = 6e−6; Supplementary Fig. p = 0.015). In contrast, no attractive bias emerged over delay in patients with schizophrenia  = 1.31, p = 0.30). Rather, a repulsive bias dominated all delay lengths in this group (DoG(θd), F = 9.07, p = 0.008). Post-hoc tests and between-group comparisons are reported in Fig. Serial dependence explained only a small fraction of single-trial errors in working memory , reflecd in Eq. , but it (θd), F2,9 = 9.68,θd),  as a covariate in our linear model ds”, Eq. ; Supplemds”, Eq. : For eacds”, Eq. .n = 12 out of 16 encephalitis patients, Supplementary Fig. θe as a function of CPZ and its one- and two-way interactions with delay and DoG(θd) in all subjects. On average, CPZ in patients with schizophrenia  explained a reduction of 1.06° in biases in the 3 s delay condition, and only a reduction of 0.08° in encephalitis patients . Residuals of the linear model, now free of linear and multiplicative effects of CPZ estimates, were fitted as a function of group, delay, DoG(θd), and their interactions. Supplementary Fig. We also controlled for potential effects of antipsychotic medication in chlorpromazine equivalents  in light of significant group differences in CPZ estimates . As expected, clinical symptoms improved in these patients , but again not significantly in the baseline session , although the direction of the effect was congruent between the two sessions. Moreover, patients with a stronger longitudinal normalization of biases improved more on the scale of positive symptoms (PANSS pos) in the follow-up session, when compared to the baseline session, r = −0.54, C.I. = , p = 0.04 .We did not find correlations between individuals’ bias estimates for 3 s delay trials and the severity of psychiatric symptoms for encephalitis or schizophrenia patients  memory traces that produce serial dependence18.Together, our experimental results show no differences in single-trial memory maintenance, but a strong reduction of delay-dependent biases in anti-NMDAR encephalitis that ameliorates with patients’ recovery, and a complete absence of attractive biases in patients with schizophrenia. These findings are not explained by ITI length, general response correlations between trials . This shift represents adaptation effects in sensory regions and is therefore not affected by local circuit alterations in prefrontal cortex.We modeled a local prefrontal circuit, composed of neurons selective to the locations presented in the spatial working memory task. We used a network of excitatory and inhibitory neurons recurrently connected through AMPAR-, NMDAR- and GABAcay Fig. . In our 5, NMDAR hypofunction would reduce the strength of STP at excitatory synapses and disrupt delay-dependent biases (hypothesis I: reduced STP). Also, we tested the explanatory potential of reduced NMDAR-mediated synaptic transmission. In particular, we tested cortical disinhibition27, caused by diminished NMDAR efficacy at inhibitory interneurons (hypothesis II: reduced gEI), and the hypofunction of NMDARs at recurrent excitatory synapses, leading to diminished delay activity33 (hypothesis III: reduced gEE). To assess each of these mechanisms, we independently varied STP strength, gEI and gEE, and we read out “behavioral responses” after 0, 1, and 3 s from population activity in our network simulations (“Methods”). Then, we fitted a linear model to measure bias strength in each condition  reduced the strength of serial dependence , as proposed by current theories of NMDAR hypofunction in schizophrenia27, cannot fully explain these behavioral findings. Instead, altered serial dependence is mechanistically accounted for by a disruption in slower dynamics, here specified as NMDAR-dependent associative STP (hypothesis I) that is triggered by sustained delay activity and influences memory representations in upcoming trials. Our results suggest that clinical reports of short-term memory alterations in schizophrenia and anti-NMDAR encephalitis could be understood in the light of reduced synaptic potentiation25. This is consistent with in vitro studies, which have demonstrated the dependence of STP on specific subunit components of the NMDAR4, and reduced STP in genetic mouse models of schizophrenia35. Importantly, our modeling is not incompatible with altered cortical excitatory or inhibitory tone as a result of hypofunctional NMDARs. Rather, it states the necessity of assuming alterations in a mechanism operating on longer timescales, such as STP. For instance, diminished STP alongside symmetric effects on both E-E and E-I synapses could maintain the excitation/inhibition balance and thus stable delay activity, while interrupting passive between-trial information maintenance.In this study, we assessed working memory alterations in two patient groups linked to NMDAR hypofunction, and hypothesized that their shared clinical and neurobiological features should be reflected in qualitatively similar behavioral patterns. In accordance with this reasoning, we found a drastic reduction of working memory serial dependence both in patients with anti-NMDAR encephalitis and schizophrenia, as compared to healthy controls. In contrast, we did not find memory maintenance deficits on timescales of a few seconds, suggesting that cognitive deficits in these patients3. Our results cannot address subunit specificity because anti-NMDAR encephalitis (and possibly schizophrenia9) is associated with hypofunction of the GluN1 subunit, which is contained in all NMDARs37. On the other hand, pharmacological studies in combination with neural recordings could reveal how trial-history representations are affected by the blockade of NMDARs38. In rodents, long-term pharmacological experiments during behavior could be complemented with in vitro studies to assess STP directly. Finally, pharmacological studies would clarify if the alterations in serial dependence occur as a result of acute NMDAR hypofunction or whether they depend on compensatory changes in STP that arise after early, acute phases of cortical excitation/inhibition imbalance in these diseases .Future studies should address the effects of pharmacological NMDAR blockade on serial dependence. These studies could unequivocally confirm the role of the NMDAR for trial-history effects in working memory, and at the same time allow to ask more specific questions: On the one hand, serial dependence effects under different NMDAR antagonists should vary according to how blocking specific NMDAR subunits modulates synaptic potentiation at different timescales39. Under this reasoning, we cannot exclude that distinct biological mechanisms in our two patient groups might lead to convergent patterns of working memory processing. For instance, our modeling shows that encephalitis patients’ biases could also be explained qualitatively by a reduced excitation-to-inhibition ratio in the memory circuit , and higher susceptibility to distractors44 in line with reported behavior in schizophrenia41, which was previously proposed to reflect an excessive excitation-to-inhibition ratio. In addition, some incongruences with previous findings might be explained by the acuteness of the patients’ condition, with more acute or psychotic stages being connected with patterns of disinhibition, and less acute stages with residual alterations in synaptic plasticity, but not cortical excitation. Alternatively, mechanisms not considered in our model could be at play. For instance, NMDAR dysfunction could negatively affect long-range connectivity47 between trial history-tracking areas38 and areas that hold current working memory contents , and in this way impede the integration of previous with current memories. Note, however, that recent combined experimental and theoretical work in primate and human prefrontal cortex shows how both past and current memories are jointly represented in prefrontal cortex, and how their interaction subserves serial dependence18.We showed how working memory in the two investigated diseases is altered in a parallel way, and how these alterations are parsimoniously explained by manipulating a single, NMDAR-dependent synaptic variable in our model. However, substantial neurobiological heterogeneity must underlie the differences in epidemiology and longitudinal development of schizophrenia and autoimmune anti-NMDAR encephalitisuit Fig. , consistdel Fig. . Reduced17. We found several indicators of clinical relevance for our finding. First, as anti-NMDAR encephalitis patients recovered, their biases normalized in the direction of healthy controls , n = 17 patients with schizophrenia or schizoaffective disorder , and n = 19 neurologically and psychiatrically healthy control participants (ctrl), all with normal or corrected vision. Behavioral data from n = 14 healthy controls has been included in a previous study18. Psychiatric diagnoses (or the absence thereof for controls) were confirmed using the Structured Clinical Interview for DSM IV (SCID-I)56. Patients diagnosed with anti-NMDAR encephalitis were recruited from different centers  at the moment of hospital discharge and completed the experiment around 5.5 months after disease onset . All patients fulfilled clinical diagnostic criteria of anti-NMDAR encephalitis with confirmation of CSF IgG antibodies against the GluN1 subunit of the NMDAR57. All subjects were tested in our laboratory for antibodies against NMDAR in serum36 and all healthy controls and patients with schizophrenia were seronegative. Anti-NMDAR encephalitis is known to have a prolonged process of recovery after the acute stage of the disease58, and patients in the prolonged recovery phase still suffer from cognitive deficits as has been previously described in cohorts with long follow-up12. All patients were sufficiently recovered to participate in the testing procedure. Controls and patients with schizophrenia were recruited from the Barcelona area and from Hospital Clínic , respectively. Patients with schizophrenia were tested 35.0 months after diagnosis  and were clinically stable at the time of testing. All participants  provided written informed consent and were monetarily compensated for their time and travel expenses, as reviewed and approved by the Research Ethics Committee of Hospital Clínic. All subjects were assessed for psychiatric symptoms and functionality through a battery of standard tests including the Spanish versions of the Positive and Negative Syndrome Scale (PANSS)59, the Young Mania Rating Scale (YMRS)60, the Hamilton Depression Rating Scale (HAM-D)61 and the Global Assessment of Functioning Scale (GAF)62. Finally, the dose of antipsychotic medication at the moment of testing was estimated as chlorpromazine equivalent 63. For a demographic and clinical overview of the populations, please refer to Supplementary Table We included Participants completed two 1.5 h sessions performing a visuospatial working memory task described in Fig. For stimulus presentation, we used Psychopy v3.1.5 on Python 2.7, running on a 17” HP ProBook laptop. Each trial began with the presentation of a central black fixation square on a gray background (0.5 × 0.5 cm) for 1.1 s. A single colored circle  was then presented during 0.25 s at one of 360 randomly chosen angular locations at a fixed radius of 4.5 cm from the center. The stimulus was followed by a randomly chosen delay of 0 , 1 , or 3 s  in which only the fixation dot remained visible . When the fixation dot changed to the stimulus’ color (probe), participants were asked to respond by making a mouse click at the remembered location (response). A white circle indicated the stimulus’ radial distance, so participants only had to remember the angular position. After the response, the cursor had to be moved back to the fixation dot to start a new trial (ITI). Participants were instructed to maintain fixation during the fixation period, stimulus presentation, and memory delay and were free to move their eyes during response and when returning the cursor to the fixation dot.n were measured as the angular distance between response and target. To exclude errors due to guessing or motor imprecision, we only analyzed responses within an angular distance of 1 radian and a radial distance of 2.25 cm from the stimulus. Further, we excluded trials in which the time of response initiation exceeded 3 s, and trials for which the time between the previous trial’s response probe and the current trial’s stimulus presentation exceeded 5 s. In total, 2.6 ± 4.2% , 4.8 ± 6.9% (enc) and 7.5 ± 9.6% (schz) of trials per participant were rejected (but only 0.1 ± 0.2% (ctrl), 0.4 ± 0.5% (enc) and 0.6 ± 0.7% (schz) of trials were excluded due to angular response errors).Response errors π/3 in steps of π/30. Positive mean folded errors should be interpreted as attraction towards the previous stimulus and negative mean folded errors as repulsion away from the previous location. In all figures including bias curves, s.e.m. are calculated across pooled trials from all subjects for each group and delay. For visualization, all values were transformed from radians to angular degrees.We then measured serial dependence as the error in the current trial as a function of the circular distance between the previous and the current trial’s target location. Figure n and subject m using linear mixed models that included the dummy-coded variables group  and delay , and a nonlinear function of previous-current stimulus distance 29. DoG and CPZgth (Eq. ) were asm subscripts):εn as main and interaction effects of group, delay, and DoG. In this model, we did not include random effects due to increased model complexity and resulting difficulties in model convergence. For extended linear models in Eqs. . Note that the normality assumption of residuals was not met , but with only slightly diverting kurtosis (Fisher) = 3.37 and skewness = 0.12 parameters. Due to the large number of trials , this should not compromise statistical inference68. Moreover, all effects of relevant task variables are visualized both in a model-based and model-free way to confirm their congruence.Baseline and follow-up sessions in encephalitis patients and controls were compared by:σ used in Eqs.  of predictions from 1000 cross-validation repetitions. σ was chosen so as to minimize the MSE obtained by the linear model, yielding σ = 0.8 , we , we σ usmentclass2pt{minimmentclass2pt{minimμ = 0 and To test whether a linear model with repulsive biases at high distances mentclass2pt{minimt-tests. Effect sizes for these comparisons were estimated as Cohen’s d, defined as s is the pooled standard deviation: 69 package). In the face of small, potentially non-normal samples, we confirmed significant results with bootstrap confidence intervals and p-values, leading to consistent results in all but one correlation . Confidence intervals of the mean . For sext, spike times were generated as a Poisson spike train of rate 1800 spikes s−1 .The kinetics of synaptic variables The slower and saturating NMDAR synaptic variables J is a Gaussian function (centered at μ = 0 with σ = 14.4 degrees) plus a constant, tuned so that J(0) = 1.63. As a result, neurons with similar preferred locations had 1.63 stronger weights than the average weight , but synapses depotentiated based on presynaptic activity3: at each presynaptic spikeThe associative nature of this rule was determined by a potentiation function that required synchronization within a specific temporal window Fig. :16\documn with stimulus s were slightly transformed to mimic a repulsive baseline bias away from previous stimulus locations, resulting from sensory aftereffects produced in lower-level cortical areas29: μ = 0 and σ = 0.8 radians, and We simulated 20,000 pairs of consecutive trials with independent randomized stimulus locations. Network inputs n − 1) for 0.25 s. After the input was removed, a delay of 1 s followed. A negative input to the whole network during 0.25 s simulated the response and removed stimulus-associated neural activity. After an ITI of 3 s, a second stimulus  was delivered at a random location for 0.25 s. The second delay duration was 3 s. To obtain behavioral readouts from the network, we counted each neuron’s spikes during three time windows of 0.25 ms: 0–0.25 s after stimulus offset (0 s delay condition), 0.75–1 s (1 s delay), and 2.75–3 s after stimulus offset (3 s delay). The behavioral response was determined as the angular direction of the population vector of spike counts.Simulations started with a stimulus presentation at 0°  separately as a function of delay and DoG. Circular standard deviations and serial dependence were then calculated from the network’s population vector responses analogous to human error analyses. In Fig. σ was determined in a cross-validation procedure for the baseline condition with P = 0.00022, gEE = 0.56 nS, and gEI = 0.424 nS, for values σ, we fitted the linear model described in Eq.  of predictions from 1000 cross-validation repetitions. σ was chosen to minimize the MSE of the linear model, yielding σ = 0.6 radians (Supplementary Fig. The value of hyperparameter d in Eq.  to 67% oFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


This waiting‐list randomized controlled trial examined the effectiveness of a self‐management mHealth app in improving fatigue and quality of life (QoL) in cancer patients and survivors.Persons with cancer‐related fatigue (CRF) were recruited across four English speaking countries, via social media, and randomized into intervention (n = 519) and control (n = 280) groups. Whereas the intervention group received immediate access to the Untire app, the control group received access only after 12‐weeks. Primary outcomes fatigue severity and interference, and secondary outcome QoL were assessed at baseline, 4, 8, and 12‐weeks. We ran generalized linear mixed models for all outcomes to determine the effects of app access (yes/no), over 12‐weeks, following the intention‐to‐treat principle.d = 0.40), fatigue interference (d = 0.35), and overall QoL on average (d = 0.32) (P's < .01), but not for overall QoL in the past week (P = .07). Sensitivity analyses indicated that participants with medium or high app use benefited most when compared with nonusers and control participants (P's ≤ .02). The intervention effect on fatigue interference was slightly stronger in younger participants (≤56 vs. >56). Effects did not depend on education and cancer status. Reliable change analyses indicated that significantly more people showed full recovery for fatigue in the intervention vs the control group (P's = .02).Compared with the control group, the intervention group showed significantly larger improvements in fatigue severity (The Untire app can be an effective mHealth solution for cancer patients and survivors with moderate to severe CRF. Details are provided in the protocol.3.2The Untire app study included cancer patients and survivors of ≥18 years, who experienced fatigue at moderate/severe levels . These campaigns match to female and male Facebook users who have shown interest in topics related to cancer, fatigue, and cancer survivorship. Based on participant characteristics of those who showed interest , comparable participants  were approached in subsequent advertisement campaigns.On the study landing page, interested persons were screened for eligibility.3.4Participants who completed the baseline were automatically randomized 2:1 by the online survey tool into the intervention and control groups. Since intervention participants received the incentive (app access) right away, we expected more dropout in the intervention group at 12‐weeks.3.5The Untire app intervention is based on evidence‐based methods for patients with CRF in clinical practice and comprises four modules , see 3.63.6.1The primary outcome was the change in fatigue severity and interference from baseline to 12‐weeks, assessed with the self‐report questionnaire FSI.3.6.2QoL during the past week15) as well as a self‐constructed item of QoL on average , on a 7‐point Likert‐scale ranging from 0 (very poor) to 7 (excellent). A higher score indicates a better QoL.The secondary outcome was change in QoL from baseline to 12 weeks, with the 1‐item QoL EORTC‐QLQ‐30 questionnaire  were collected as potential moderators.3.6.4Once participants activated their personal access code and gave consent to the privacy terms in the Untire app, participants were registered, and their in‐app activities were automatically stored anonymized (log data). Log data was used to determine the degree of app use, which was calculated as the number of days with ≥1 activity performed within the 12‐week intervention period.3.73.7.1We will present baseline characteristics  of participants who completed the baseline fatigue assessment. Alpha was set to .05.3.7.2Fatigue severity and interference were analyzed using generalized linear mixed models (GLMM) with four repeated measures  and two groups (intervention and control). Intention‐to‐treat (ITT) is used as the primary approach in all outcome analyses. Based on previous studies testing health‐related apps targeting behavior change,3.7.3QoL of the past week and overall QoL on average were analyzed by repeating the same GLMM analysis of the primary outcome using the ITT approach.3.7.4To gain insight into whether the hypothesized intervention effect varies among different groups of participants, potential moderating factors of the intervention effect on fatigue were explored by testing a three‐way interaction (time*condition*moderator) using GLMM. Variables with ≥30 participants per category were analyzed.3.7.5dif. After that, patients were classified into four categories based on their RCI, combining statistical and clinical significancedeterioration (negative change ≤ −1.96 SEdif), no change (>−1.96 and <1.96 SEdif), improvement (positive change ≥ 1.96), or recovery  tests.To determine validly whether the degree of change in fatigue was clinically meaningful, the reliable change index (RCI) was calculated.3.82 = 0.10 , using the simplest between/within‐group comparison (F tests ‐ RM‐ANOVA with within‐between interaction) in G*Power 3.1.Sample size calculations44.1P < .02).From the 3.060 persons who showed interest in the Untire app study, 1137 met the study criteria and gave informed consent, and 799 participants completed the primary outcome at baseline  = 4.55, P < .01, d = 0.40, and F = 4.10, P < .01, d = 0.35), see Table P's < .02; see Figure P's > .05).After 12‐weeks, participants in the intervention group showed significantly reduced levels of fatigue severity and interference as compared with participants in the control group (P = .04), whereas for fatigue severity, no statistically significant difference was found (P = .05), see Supporting Information 3. With regard to both fatigue severity and interference, significantly more people recovered in the intervention group vs the control group after 12‐weeks (P's = .02).The clinical relevance analysis for fatigue interference showed differences in the number of people who recovered, improved, not‐changed, and deteriorated, favoring the intervention group  = 2.34, P = .07, d = 0.15), whereas levels of QoL on average did improve significantly in the intervention group from T0 to T12 as compared with the control group  = 4.67, P < .01, d = 0.32), see Figure QoL during the past week (EORTC‐QLQ‐30) did not differ significantly between conditions after 12‐weeks  = 2.67, P = .046), with slightly stronger improvements in younger intervention participants (≤56 vs. >56). Education and cancer status did not moderate any of the intervention effects .The variables age, education, and cancer status were taken as moderators into account. The significant intervention effect on fatigue interference was moderated by age  can be effectively reduced with a self‐management app for cancer patients and survivors. While just a few web‐based/eHealth interventions targeted at reducing fatigue in cancer patients and survivors exist,5.1d = 0.35‐0.40) of the Untire intervention, which is larger than other self‐management eHealth interventions . Another drawback of online recruitment is the lack of access to hospital records, meaning that we cannot validate medical information . However, participants had nothing to gain by providing invalid medical information. Another limitation is that remotely conducted mHealth studies regularly show high attrition rates concerning study assessments and intervention participation . High dropout rates and decreasing app use may be common,We should be aware that the recruitment strategy may have influenced our study sample, and that our findings might be limited to middle‐aged female breast cancer patients who are using Facebook. However, our sample is likely to represent future users who might find the Untire app in a similar way, that is, through finding information on the internet or Facebook. This idea is supported by data from users who downloaded and used the Untire app after the study had been completed, which showed that most users were indeed middle‐aged and female.5.3Future research could explore the mechanisms behind the app  to understand the underlying processes determining the reduction of fatigue. To further validate the study results, also participants in non‐English speaking countries should be targeted. Besides online recruitment, also other pathways of introducing the app to the end‐user  could be explored concerning differences in sample characteristics, dropout, adherence, and effectiveness.6This study shows that a self‐management app can support cancer patients and survivors in managing their fatigue and QoL, and suggests that besides existing face‐to‐face therapy or therapist‐guided online interventions, also a low‐threshold mHealth app can be an effective treatment solution. The Untire app could present a scalable opportunity in supporting people worldwide experiencing disabling fatigue.The authors declare that they have no conflict of interest. The University Medical Center Groningen received funding from Tired of Cancer BV., the developer of the Untire app, to study its effectiveness independently. Independence is declared in a research agreement.The Untire app study has been approved by the Medical Ethical Committee of the University Medical Center Groningen (UMCG), the Netherlands. Hereafter, the study received either ethical approval or a waiver from authorized institutions in the four English‐speaking countries targeted . The trial has been registered on November 29, 2019 on the Netherlands Trial Registry (NL6642).Appendix S1: Supporting InformationClick here for additional data file.


The overall survival (OS) of patients diagnosed with stage II‐III colorectal cancer (CRC) can vary greatly, even between patients with the same tumor stage. We aimed to design a nomogram to predict OS in resected, stage II‐III CRC and stratify patients with CRC into different risk groups.Based on data from 873 patients with CRC, we used univariate Cox regression analysis to select the significant prognostic features, which were subjected to the least absolute shrinkage and selection operator (LASSO) regression algorithm for feature selection. Cross‐validation was used to confirm suitable tuning parameters (λ) for LASSO logistic regression. Then, the nomogram was used to estimate 3‐ and 5‐year OS based on the multivariable Cox regression model. The survival curves of the two groups were produced using the Kaplan‐Meier method. Risk group stratification was performed to assess the predictive capacity of the nomogram.P < .001).Preoperative mean platelet volume, preoperative platelet distribution width, monocytes, and postoperative adjuvant chemotherapy were identified as independent prognostic factors by LASSO regression and integrated for the construction of the nomogram. The nomogram provided good discrimination, with C‐indices of 0.67 and 0.69 for the training and validation sets, respectively. Calibration plots illustrated excellent agreement between the nomogram predictions and actual observations for 3‐ and 5‐year OS. Moreover, a significant difference in OS was shown between patients stratified into different risk groups (We constructed and validated an original predictive nomogram for OS in patients with CRC after surgery, facilitating physicians to appraise the individual survival of postoperative patients accurately and identify high‐risk patients who need more aggressive treatment and follow‐up strategies. Four points are allocated for preoperative mean platelet volume, preoperative platelet distribution width, monocytes, and postoperative adjuvant chemotherapy. Nomogram for predicting 3‐ and 5‐year probabilities of colorectal cancer patients was established. Draw a vertical straight line from the variable value to the axis labeled “Points”. Then calculate all variables’ points. The total points on the bottom scales that correspond to the 3‐ and 5‐year survival were showed apparently. This nomogram will provide more individualized prognoses, which can help clinicians and patients make informed treatment and management decisions.22.1Ethical approval for this retrospective analysis was obtained from our institutional review board. On the basis of the appointed inclusion and exclusion criteria, 873 patients with stage II‐III CRC who underwent surgical treatment at Guangxi Medical University Cancer Hospital between June 2004 and October 2018 were included.The inclusion criteria were as follows: (a) patients with CRC; (b) patients who underwent surgical resection of the primary tumor; (c) patients with II‐III pathological staging for CRC; The exclusion criteria were as follows: (a) patients who received antitumor therapy before surgery ; (b) patients who had other malignancies in the same period; (c) patients with severe liver disease and/or acute infection; (d) patients with incomplete follow‐up data whose prognosis was unknown.The medical records were obtained based on relevant demographic and clinical features. These features were divided into several categories, including general information, diagnosis, signs and symptoms, prior history, personal history, family history, auxiliary examination, imaging and endoscopy examination, pathological examination, and postoperative situation. General information, which was based on the patients’ identity cards, height, and weight were extracted from the hospital database. Diagnosis was based on the patients’ discharge diagnoses. Previous, personal, and family history were extracted from the hospital admission records, which were verified by a senior physician. Auxiliary examination was collected from tests performed on the first admission. Imaging and endoscopy examination were based on patients’ preoperative CT, MRI, and colonoscopy. Pathological examination included patients’ preoperative endoscopic biopsies and postoperative pathological reports. The names of 242 features in the study are listed in Table 2.2P‐value < .05 in the univariate analysis were selected. The selected features were then used in the least absolute shrinkage and selection operator (LASSO) regression algorithm. Dummy variables were created for categorical variables. Cross‐validation was used to confirm suitable tuning parameters (λ) for LASSO logistic regression. Finally, the most significant features were selected by LASSO.Features included in the nomogram were selected in two steps. First, univariate Cox regression analyses were conducted to screen for features significantly related to OS. Clinical features with a Construction of the Nomogram.P‐values < .05 by multivariate analysis were incorporated into nomograms that were constructed to predict the 3‐ and 5‐year OS rates.The most significant features selected by LASSO from the training dataset were used for multivariate Cox proportional hazards analyses; variables with 2.3The concordance index (C‐index) was calculated with the Cox regression model method. The value of the C‐index was between 0.5 and 1.0, which indicates the decent discriminatory capacity of the nomogram, whereas 0.5 indicates a random chance. The C‐indices of two models  were contrasted to assess the discrimination of the nomogram. The observed 3‐ and 5‐year OS were compared to the predicted 3‐ and 5‐year OS to further verify the predictive performance of the nomogram. We assessed the goodness‐of‐fit of the nomogram using calibration plots and a modified Nam‐D'Agostino test. The modified Nam‐D'Agostino test is an extension of the Hosmer‐Lemeshow goodness‐of‐fit test in survival data.2.4P‐values of the log‐rank test were recorded. The optimal segmentation threshold was determined when the minimum P‐value of the log‐rank test was obtained. Patients whose risk scores were higher than the optimal segmentation threshold were assigned to the high‐risk group, and the rest were assigned to the low‐risk group. The survival curves of the two groups were produced with the Kaplan‐Meier method.The risk scores of patients were calculated using the nomogram. To determine the optimal segmentation threshold for patient stratification, we repeatedly split the patients in the whole database into low‐risk and high‐risk groups based on each risk score. For each division, hypothesis testing was performed, and the hazard ratios (HR) and 2.5P‐values < .05 indicated statistical significance.R statistical software (version 3.4.0.) was used to perform the statistical analyses. LASSO regression analysis was operated with the “glmnet” package. 33.1In total, 873 eligible patients with integrated information were incorporated in the dataset and randomly divided into two independent cohorts at a ratio of 6.5:3.5 . There were 525 male patients and 348 female patients included in this study. The average age of these patients was 59 years. CT was performed an average of 6.2 days range, 3‐10) before surgery. Most patients had T3‐4 stages  and N0 stages  based on preoperative CT. The demographic and clinical features of the patients in the training and validation sets are listed in Table 0 before 3.2P = 5.23 × 10−11). Additionally, sex, pathological T‐staging, N‐staging, M‐staging, and pathological stage, the P‐values of which were all > .05, were included. All 64 features and P‐values are listed in Table λ for LASSO regression was 0.055 when the partial likelihood binomial deviance reached its minimum value , preoperative platelet distribution width , monocytes (P = .0086), and postoperative adjuvant chemotherapy (P = .0011) were independent predictors for OS in patients with CRC. Thus, these four variables were selected for the construction of the nomogram. Multivariate Cox proportional hazards analysis was performed using the four selected variables. As shown in Table P‐values for both the 3‐ and 5‐year survival probabilities . These results indicated that the nomogram had proper calibration for the training set.The six retained variables were used for multivariate Cox proportional hazards analysis. Among these, preoperative mean platelet volume . These results indicated that the nomogram had proper calibration for the validation set  , we repeatedly split the patients based on each risk score. As shown in Figure ) Figure B.4CRC is obviously diversified with regard to patient survival, even when all of the patients go through surgical resection and chemotherapy.Our primary data were complete and well‐organized. Through the LASSO regression algorithm, which effectively processed the demographic and clinical feature selection as a statistical method for high‐dimensional data, we distinguished preoperative MPV, preoperative PDW, monocytes, and postoperative adjuvant chemotherapy as independent prognostic factors. Many scholars have reported that MPV levels might serve as potential biomarkers for the diagnosis and early recognition of different stages of CRC.P‐values < .001, indicating satisfactory predictive performance.We consolidated the four selected characteristics into our nomogram by multivariate analysis of the training set, then estimated its performance by calibration and discrimination. Both clinicians and patients alike could conveniently calculate the individual survival probability using this point‐based nomogram. On the basis of the total points and defined threshold values, we stratified patients with CRC into high‐risk and low‐risk groups. With regard to the high‐risk group, clinicians could provide rational suggestions for additional individualized therapy and intensive follow‐up. Validation is an essential procedure in nomogram studies to avoid poor goodness‐of‐fit and determine the generalizability of the model.Although some nomograms have been developed to predict individual survival probabilities for patients with CRC, there are some unique points in our nomogram. The tradeoff between comprehensiveness and comprehensibility can be challenging to balance and is unavoidable in nomogram studies. However, the applicable target of our nomogram is relatively comprehensive, involving stage II‐III CRC. In addition, improved nomogram accuracy often comes at the cost of increased complexity. Our nomogram is concise, with only four predictive factors, but remains accurate. All the clinical parameters needed for our nomogram are available after surgical resection and routine pathologic examination, without adding any burden to patients.Though we successfully constructed and validated a nomogram to predict individual survival probability for patients with CRC after surgery resection, our study did have several limitations. First, our data were of limited size and from a single institution, which limit the generalizability and applicable scope of the nomogram. Secondly, there was no external validation from other institutions; single‐center data were used for external validation. Although the model still worked well in our internal cohort, which was intended for relatively strict validation, multi‐institutional external validation would provide more convincing evidence.5In summary, we constructed and validated an original predictive nomogram for the survival of patients with CRC after surgery, facilitating physicians to accurately evaluate the individual survival of postoperative patients and distinguish high‐risk patients who may need more aggressive treatment and follow‐up strategies.The author reports no conflict of interest in this work.Conceived and designed the experiments: WT, XH, JL, WY, XM; Performed the data collection: XH, JL, XM, GW, FJ, SC, CZ, WX, WT, WY, CL, ZL, LH, CT, JC, CW; Analyzed the data: XH, JL, XM, GW, WY, CL, ZL, LH, CT, JC, SC, CZ,CW; Contributed reagents/materials/analysis tools: XH, JL, XM, GW, FJ, SC, CZ, WT, WY, CL, ZL, LH, CT, JC, CW; Contributed to the writing of the manuscript: XH, JL, XM, WY, WT, CL, ZL, SC, CZ, LH, CT, JC, CW; All authors reviewed the manuscript.This study was approved by the Ethics and Human Subject Committee of Guangxi Medical University Cancer Hospital. Click here for additional data file. Click here for additional data file.


Cancer‐associated fibroblasts (CAFs) generated by bone marrow‐derived mesenchymal stem cells (BM‐MSCs) play an important role in cancer progression. In this study, we investigated the relationships of BM‐MSCs and CAFs in resected gastric cancers with the clinicopathological factors of patients.We analyzed 120 gastric cancer patients who underwent gastrectomy. Immunostaining was performed with an anti‐CD271 antibody (BM‐MSCs) and anti‐α‐smooth muscle actin (αSMA) antibody (CAFs). Staining intensity was used to divide patients into low and high expression groups. Observation sites in cancer tissues were invasive, central, and whole portions.Expression of αSMA was significantly related to depth of tumor invasion (T), lymph node metastasis (N), lymphatic invasion (ly), venous invasion (v), and stage. Expression of CD271 was significantly related to v, stage, stromal volume, and tumor infiltration pattern (INF). Overall survival (OS) of the high expression group was significantly lower than that of the low expression group for both αSMA and CD271. Multivariate analysis showed that N, αSMA (whole), and CD271 (invasive) were independent prognostic factors.Cancer‐associated fibroblasts and BM‐MSCs are related to the progression, invasion, and prognosis of gastric cancer and may be therapeutic targets of gastric cancer. We immunohistochemically examined bone marrow‐derived mesenchymal stem cells and cancer‐associated fibroblasts in gastric cancer tissues and obtained results indicating that they were associated with tumor infiltration and stroma, and closely related to prognosis. In worldwide cancer statistics, the number of gastric cancer cases ranks fifth, while the number of deaths ranks third.Mesenchymal stem cells (MSCs) are somatic stem cells derived from mesodermal tissue (mesenchyma), which are present in all tissues. Among tissue stem cells, although MSCs are considered to differentiate into various mesenchymal cell types such as bone, cartilage, muscle, fat, and nerves, MSCs originally exist in bone marrow, and MSCs that are isolated from bone marrow can reconstitute various mesenchymal tissues.Changes in cancer cells themselves along with microenvironmental changes in the cancer surrounding the stroma are critically involved the development and metastasis of cancer. The peritumoral microenvironment includes various stromal cell types, cancer‐associated fibroblasts (CAFs), endothelial cells, adipocytes, and immune cells.22.1This study was approved by the Institutional Review Board of Kurume University School of Medicine (ID: 19117) based on the Ethical Guidelines for Clinical Research of the Ministry of Health, Labour, and Welfare of Japan. Patients who underwent gastrectomy for gastric cancer at Kurume University Hospital agreed to donate surgically resected tumor specimens for research purposes. Written informed consent was obtained from all patients.A total of 120 cases out of 706 gastric cancer surgery patients at Kurume University Hospital from January 2005 to December 2010 were chosen randomly. Subsequently, intramucosal cancer, residual gastric cancer, multiple cancers, and post‐ESD cases were excluded. Finally, 88 males and 32 females with a median age of 71.5 years  were enrolled in the study. The median observation period was 51 months with the shortest observation period as 5 months and longest observation period as 128 months.2.2Immunohistochemical staining was performed using paraffin‐embedded sections, including the deepest part of the tumor in each case, with an anti‐CD271 (BM‐MSC marker) antibodyParaffin‐embedded tissue samples were sectioned at 4 μm thicknesses, mounted on PLATINUM PRO‐coated slides , and labeled with antibodies using a Bond‐III autostainer (Leica Microsystems): Antibodies against CD271  and αSMA  were used as primary antibodies. For CD271 staining, heat treatment was applied in Epitope Recovery Solution 2 (pH 9.0) for 30 minutes, followed by incubation with the anti‐CD271 antibody for 15 minutes. For αSMA staining, the antibody was applied without antigen recovery for 15 minutes. The automated system used for detection was a refined polymer detection system (Leica Microsystems) with horseradish peroxidase‐polymer as the secondary antibody, 3,3′‐Diaminobenzidine was used as the chromogen. The negative control for both αSMA and CD271 was phosphate‐buffered saline instead of primary antibody. The positive control for αSMA was the proper muscle layer of the stomach, and that for CD271 was melanoma tissue.2.3To evaluate myofibroblasts as CAFs, αSMA was stained in myofibroblasts of the tumor stroma, vascular smooth muscles, proper muscle layer, and mucosal muscle plate. CD271 was stained in the cytoplasm of cells in the tumor stroma as BM‐MSCs. We counted positively stained cells in all fields of one section containing the deepest part of the tumor. We examined positive cases to determine the immunoreactivity for αSMA or CD271 in the cancer stroma, and evaluated the staining intensity under 200× magnification. The staining intensity was scored as 0‐3 to examine the association with clinicopathological factors . Statistical significance of analytical data was evaluated using the chi‐squared test or 2.5Clinicopathological terms were used in compliance with the Japanese classification of gastric carcinoma 3rd English edition.33.1P = .003, .004, and .013, respectively); lymph node metastasis (N): central (P = .039); lymphatic invasion (ly): invasive and central ; venous invasion (v): center (P = .031); stage: central (P = .031)). However, no associations were found with age, sex, gross type, histology, stromal volume, or tumor infiltration pattern (INF) , no associations were found with age, sex, gross type, T factor, N factor, or ly : invasive, central, and whole , and whole portion , and strong correlation in the central portion .By Spearman rank correlation coefficient analysis, there were correlation between αSMA expression and CD271 expression in the invasive portion    rates of αSMA and CD271 high expression groups were significantly lower to those of the low expression groups in terms of invasive, central, and whole portions  Figure .3.4P = .0001), N factor (P = .0001), v (P = .0023), αSMA expression in the invasive, central, and whole portions, and CD271 expression in invasive, central, and whole portions  were significant prognostic factors. In the multivariate analysis, N factor , αSMA expression in the whole portion , and CD271 expression in the invasive portion  were independent prognostic factors (Table As factors involved in the prognosis of gastric cancer, according to univariate analysis, T factor (Therefore, the presence of CAFs in the whole tumor and BM‐MSCs in the invasive portion was considered to be involved in the prognosis of gastric cancer.4In this study, gastric cancer tissues were immunostained for αSMA and CD271 to examine CAFs and BM‐MSCs, respectively, with respect to gastric cancer stroma. CAFs were correlated with the T factor, Ly factor, V factor, N and stage, whereas BM‐MSCs were correlated with the V factor, stromal volume and INF. These observations suggested that αSMA was involved in tumor invasion and metastasis of CAFs because of its relationships with T and N, while CD271 was involved in the tumor stroma of BM‐MSCs because of its relationships with stromal volume and INF. Furthermore, BM‐MSCs in the invasive portion of the tumor and CAFs in the tumor stroma were poor independent prognostic factors.As tumors grow larger, they need to obtain oxygen and nutrition from newly developed vessels. CAFs generated from BM‐MSCs were recruited to the tumor after first accumulating in the peripheral blood.Epithelial‐mesenchymal transition was originally reported by Hay et al as a phenomenon observed early in ontogeny.Bone marrow‐MSCs are induced in tumor tissue by IL‐6, Wnt‐5a, and BMP4 expressed in the tumor tissue with further differentiation to CAFs promoted by TGF‐β1 and SDF‐1α.SenbaMoreover, it has been reported that the direct contact of BM‐MSCs with cancer cells induces EMT and stem cell recovery of cancer cells via the paracrine actions of TGF‐β derived from BM‐MSCs, along with the autocrine action of Wnt‐5a secreted from cancer cells.In this study, we immunohistochemically examined BM‐MSCs and CAFs in gastric cancer tissues and obtained results indicating that they were associated with tumor infiltration and stroma, and closely related to prognosis. In the future, we will investigate the relationship of BM‐MSCs and CAFs with EMT, CSC recovery, and expression of related cytokines to identify new biomarkers and therapeutic targets for gastric cancer. Recently, MSCs have been considered ideal gene career for therapy of malignant disease, because they are safe and can migrate tumors.Funding: This study was not funded.Conflicts of interest: The authors declare no conflicts of interests for this article.Author Contribution: The conception or design of the work, or acquisition, analysis or interpretation of data for the work: Taizan Minami, Keishiro Aoyagi, Taro Isobe, Yuya Tanaka, Hideaki Kaku. Drafting the work or revising it critically for important intellectual content: Taizan Minami, Keishiro Aoyagi. Analysis of histological findings or performing immunohistochemical staining: Taizan Minami, Keishiro Aoyagi, Akihiko Kawahara. Final approval of the version to be published; Keishiro Aoyagi, Naotaka Murakami, Fumihiko Fujita, Yoshito Akagi. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work: Yoshito Akagi.All procedures performed in studies involving human participants were in accordance with the ethics standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.Informed consent was obtained from all individual participants included in this study.


The impact of hepatitis C virus (HCV) clearance by direct‐acting antiviral agents (DAAs) on HCV‐related extrahepatic manifestations is not well known. We evaluated the effect of viral clearance on metabolic and renal parameters.In this prospective study, HCV patients who achieved a sustained virologic response (SVR) by DAAs were evaluated before, at the end, and 24 weeks after treatment for glycemic  and lipid  metabolism and renal function .P < 0.05). Regardless of hepatic fibrosis levels and BMI, a reduction in serum glucose (P = 0.001), HOMA‐IR (P < 0.001) and HOMA‐β (P < 0.001) and an increase in HOMA‐S (P < 0.001) values were observed at 24 weeks after HCV clearance as compared to pretreatment values; 32.4% of patients with impaired fasting glucose normalized serum glucose values and 44.6% of diabetics showed an improvement in glycemic control. In contrast, serum cholesterol (P < 0.001) and LDL cholesterol (P < 0.001) values were increased. Renal function was improved with about 10% reduction of serum creatinine values (P < 0.02) and an increase of eGFR (P < 0.001). A baseline eGFR of ≤60 mL/min/1.73 m2 was a negative predictor of renal function improvement. HCV clearance was an independent factor improving glucose metabolism and renal function.A total of 343 consecutive HCV patients were evaluated. At 24 weeks of post‐follow‐up, an increase in body mass index (BMI) was observed  infection who achieved sustained virologic response by direct‐acting antiviral agent (DAA) treatments were followed up for 24 weeks to evaluate the impact of viral clearance on metabolic and renal parameters. HCV clearance was independently associated with a significant reduction in serum glucose, HOMA‐IR, and HOMA‐β and an increase in HOMA‐S. Furthermore, an increase in serum cholesterol and low‐density lipoprotein cholesterol levels was observed. Renal function was improved with a significant decrease in serum creatinine levels and an increase in estimated glomerular filtration rate. Data show that HCV clearance by DAA induces significant changes in metabolic and renal parameters. Chronic hepatitis C virus (HCV) infection is currently one of leading causes of liver disease, affecting 71.1 million of patients worldwide.HCV‐infected patients showed also a higher risk of developing both chronic kidney disease (CKD) and a faster progression toward end‐stage renal disease (ESRD) compared with patients with no HCV infection.The recent introduction of direct‐acting antiviral agents (DAAs) has revolutionized the therapeutic approach. A sustained virologic response (SVR) is achieved in up to 99% of patients treated with DAAs. Considering the direct or indirect role of HCV on metabolic and renal changes, it is hypothesized that the clearance of HCV by DAA regimens may modify metabolic and renal parameters in these patients and that knowledge of such changes is essential for planning best follow‐up.Consequently, the aim of this study was to evaluate whether in chronic hepatitis C patients the viral clearance by DAAs influences the metabolic and renal function.In the current prospective study, from May 2016 to May 2018, all patients with chronic HCV infection referred to our hepatologic center who underwent DAA treatments were enrolled. Exclusion criteria were DAAs nonresponder, relapse, coinfection with hepatitis B virus or human immunodeficiency virus, and history of alcohol use (>3 drinks per day).Patients were evaluated before starting treatment, at the end of treatment, and 24 weeks after treatment discontinuation. Liver and kidney function tests, fasting blood glucose and insulin levels, blood counts, serum cholesterol, LDL and high‐density lipoprotein (HDL) cholesterol, and triglycerides levels were evaluated in all patients before, at the end and 24 weeks after treatment suspension.The diagnosis of T2DM and impaired fasting glucose (IFG) was done according to American Diabetes Association's guidelines.HOMA was used to evaluate IR and β‐cell function by the following formulas: HOMA‐IR = ([glycemia (mg/dL) × insulinemia (μU/mL)]/405); HOMA‐β% = (360 × fasting insulin μU/mL)/(fasting glucose mg/dL‐63); and HOMA‐S% = 1/(log(fasting insulin μU/mL) + log(fasting glucose mg/dL). The cutoff for the interpretation of HOMA‐IR data was obtained on a population of 130 healthy Caucasian subjects from the same geographical area and was equal to 2.60 (75th percentile of HOMA‐IR).2) and underwent abdominal ultrasound and transient elastography (TE) using Fibroscan  to assess the degree of liver fibrosis.Prior to the initiation of antiviral treatment, patients were evaluated for body mass index (BMI)  with 95% confidence interval (CI) for the univariate analysis and as adjusted HR (aHR) (95% CI) for the multivariate analysis. P values below 0.05 were considered statistically significant. All analyses were performed with the SPSS 24 software .Data are shown as median and interquartile range, in the case of continuous variables and number and percentage, for categorical variables. The normal/not normal distribution was preliminarily assessed through a Kolmogorov–Smirnov (K‐S) goodness‐of‐fit test. Continuous variables were assessed, in the case of normal distribution, by either a paired Three hundred forty‐nine patients with chronic HCV infection were treated with DAAs during a 2‐year period and evaluated for this study. Of these, 343 patients (98.3%) who achieved SVR were closely followed up for 24 weeks after the end of treatment, and data are reported in the present study. The characteristics of the study population are shown in Table n = 100) and group two with advanced hepatic fibrosis or liver cirrhosis . The cu‐off used to distinguish the two groups was 10 kPa of liver stiffness. Patients with liver cirrhosis (n = 164) were mostly classified as Child Pugh class A scores (91.5%). The most frequent genotype was genotype 1b , followed by genotype 2 . The DAA regimens used are shown in Table To discern the effect of liver fibrosis levels on results, the study population was divided in two subgroups according to the degree of liver fibrosis: group one included subjects with absence to moderate hepatic fibrosis . Conversely, total cholesterol levels were significantly lower in the F3–F4 group .As shown in Table Table In addition, in a control group of 104 patients with HCV (98 awaiting treatment with HCV plus 6 patients without response from this study), serum fasting glucose, cholesterol, and triglycerides levels were assessed during a 4‐month period. There were no significant changes in the values of fasting blood glucose (baseline: 98 mg/dL [91–119] and 4 months: 97.8 mg/dL [90–118]), serum cholesterol levels (baseline: 158 mg/dL 128–192] and 4 months: 156 mg/dL [126–194]), and triglycerides levels (baseline: 94 mg/dL [72–114] and 4 months: 93 mg/dL [74–116]). These data agree with those already published by our group in 65 HCV untreated patients evaluated during the same time period.8–192 andvs 10.2 ± 6.4, respectively; P < 0001).As shown in Table vs 78.4 ± 54.2, respectively, P < 0.001), whereas a significant reduction in HOMA‐β was observed .After HCV clearance, a significant increase in HOMA‐S was observed at the end of treatment and after 24 weeks of follow‐up with respect to baseline (102 ± 64.6 and 106 ± 66.2 P < 0.008).Table Among the 74 HCV patients with IFG before treatment, 32.4% normalized glucose values at the end of 24 weeks follow‐up.After elimination of HCV, 5.8% of the 74 diabetic patients had a reduction in blood glucose levels in the IFG range and their glycemic control no longer required any medication but only dietary regulation. In 38.8% of diabetic patients, an improvement in glycemic control was observed after HCV clearance, and these patients reduced the dose of antidiabetic drugs, particularly insulin by 20 to 36% of the dose used before HCV clearance.As shown in Table The effect of antiviral treatments with DAAs on renal function is shown in Table At the end of the follow‐up, the SVR was associated with a significant improvement in renal function in both F0–F2 and F3–F4 groups. There was a significant decrease in serum creatinine levels and a significant increase in eGFR, regardless of the degree of liver fibrosis (Table P < 0.012), while age was a negative predictive factor.Table vs F3–F4), and fasting glucose; fasting insulin; HOMA index; creatinine; eGFR; total, HDL, and LDL cholesterol; triglycerides, and BMI as independent values. Fixed effects were insulin, cholesterol, renal function, BMI, and triglycerides, while we used fasting glucose and HOMA index as random effect.A mixed model design was used to compare repeated longitudinal changes over time in between the subgroup. First, we computed generalized linear models with the best BIC and AIC, hence the most plausible ones. The linear model which best fitted to our data was that using fibrosis stage as dependent dichotomic variable  and in those with advanced fibrosis F3–F4). The data show that 89.6% of patients with IR before treatment had an improvement after HCV clearance, in 57.1% there was normalization of IR values and in 32.5% an improvement in IR levels. Furthermore, the data show an improvement in insulin sensitivity and a reduction of β‐cells pancreatic distress. Multivariate analysis shows that HCV clearance is an independent predictor of HOMA‐IR improvement  and without T2DM who demonstrated an improvement in blood glucose and IR levels, as well as an improvement in pancreatic β‐cell function and insulin sensitivity.Other studies,Regarding the impact of viral clearance on the lipid profile, our data show a significant increase in LDL, and consequently in total cholesterol, irrespective of the degree of hepatic fibrosis. This increase is already present at the end of the treatment and tends to persist during the subsequent follow‐up. Although there is also an increasing trend of triglycerides and a reduction in HDL, these changes appear to be statistically significant only in the group with absent to moderate hepatic fibrosis. According to our previous data,Changes in lipid profile are reported in several studiesThe explanation for this phenomenon is probably to be found in the close link between HCV and the host's lipoproteins. The latter appear to be essential in all phases of the life cycle of the virus.In our study, patients who eliminated HCV by DAAs achieved improvement in renal function at the end of the observation period, regardless of the degree of hepatic fibrosis. In fact, our patients had an improvement of serum creatinine and an increase of eGFR of about 10% compared to the baseline value. Multivariate analysis shows that HCV clearance is an independent predictor of eGFR improvement Table  and age As well documented in the literature, chronic HCV infection is associated with an increased risk of developing CKD.Few studies, mainly conducted in cryoglobulinemic patients, have investigated the impact of viral eradication achieved with DAAs on renal function showing an improvement in serum creatinine, proteinuria, and eGFR and a reduced risk of ESRD.Recently, the impact of DAA treatments on renal function has been evaluated in a retrospective study.2 and an older age are negative predictor of improvement of renal function after HCV clearance by DAAs  may improve after treatment with DAAs, while other causes may be less susceptible of improvement.In conclusion, HCV clearance by DAA treatments induces a significant improvement in glucose metabolism and renal function, while causing an increase in the serum LDL and total cholesterol levels which require careful future monitoring. The data underline the importance of HCV eradication not only to prevent the progression of liver disease but also to improve or prevent HCV‐related metabolic and renal extrahepatic manifestations that play an important role in the morbidity and mortality of these patients.


Postoperative pain after breast cancer surgery remains a major challenge in patient care. Local infiltration analgesia is a standard analgesic technique used for pain relief after surgery. Its application in patients who underwent mastectomy requires more clear elucidation. This study aimed to investigate the effect of ropivacaine infiltration of drainage exit site in ameliorating the postoperative pain after mastectomy.n = 37) or 10 ml normal saline (Group B) (n = 37). Pain scores were recorded in post-anesthesia care unit (PACU), at 6 h, 12 h, 24 h and 36 h after operation by using a visual analogue scale (VAS). Postoperative nausea and vomiting (PONV) incidence, postoperative analgesic and antiemetic requirements, the incidence of chronic pain, as well as the quality of recovery were recorded.A prospective randomized controlled study was conducted in 74 patients who were scheduled for unilateral mastectomy by standardized general anesthesia. Both intervention group and control group were given infiltration of the two entry points of drainage catheters with 10 ml 0.5% ropivacaine (Group A) (p < 0.0005), at 6 h (p < 0.0005), 12 h (p < 0.0005), and 24 h after surgery (p < 0.05) when compared to those in Group B. There were more postoperative analgesic requirements in Group B (p < 0.05). With regard to the quality of recovery, Group A was shown to be much superior over Group B (p < 0.05).The patients in Group A showed a significant reduction in postoperative pain in PACU .retrospectively registered in  Postoperative pain is one of the most common challenges in women following breast cancer surgeries, which impairs rehabilitation and increases the length of hospital stay. About 50% of patients who receive mastectomy might experience persistent postoperative pain , 2. SensAlthough multimodal analgesic strategies including opioids, acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs), peripheral regional techniques, patient-controlled modalities as well as local anesthetic techniques such as wound infiltration are available, postoperative pain still has been poorly managed. Analgesia-related side-effects such as nausea and vomiting, dizziness, constipation and itching are commonly observed, impairing patients’ satisfaction and delaying their discharge time , 8. PostThis prospective randomized controlled study aimed to investigate if infiltration of ropivacaine at the insertion sites of the two drain catheters in mastectomy would reduce postoperative acute pain, postoperative nausea and vomiting (PONV), and chronic pain.Chictr.org.cn registry system on 24 February 2020 (ChiCTR2000030139). This study was conducted in Cancer Hospital, Chinese Academy of Medical Sciences between September and November 2019, and has been approved by the institutional ethics committee . All patients were followed up until 3 months after discharge from the hospital.This prospective, randomized controlled trial was designed in adherence to the CONSORT guidelines and was registered in Patients who underwent unilateral mastectomy with axillary lymph node dissection (ALND) or sentinel lymph node biopsy (SLNB) were enrolled in this study. Written informed consent was obtained from patients. The patients aged 20 ~ 70 years, and with the American Society of Anesthesiologists physical status of I to III were included. Patients with the following conditions were excluded from the study: history of severe cardiovascular or pulmonary, hepatic, renal, neurologic, and psychiatric or metabolic diseases; history of allergy to any of the potential study medications; active drug abuse; intake of NSAIDs, opioids, or other analgesics in the 24 h before surgery; pregnancy; breastfeeding, active menstruation.Prior to study initiation, 80 sequentially numbered envelopes containing the allocation were prepared. The involved patients were randomly assigned to 10 ml 0.5% ropivacaine infiltration (Group A) or 10 ml normal saline infiltration (Group B) groups. A physician independent of the study randomly inserted 40 anesthesia strategies for each group into the envelopes. The random allocation sequence was generated using computer-generated random numbers. The researchers opened the envelope to determine as to which anesthesia strategy to implement before the induction of general anesthesia. All perioperative data were collected by an investigator who was blinded to the patient’s allocation, and was responsible for measuring the outcome.Standard general anesthesia was induced using sufentanil 0.3 ~ 0.6 μg/kg, propofol 1 ~ 2 mg/kg and cis-Atracurium 0.2 ~ 0.4 mg/kg in the two groups. After laryngeal mask airway insertion, the patients were mechanically ventilated to maintain the end-tidal carbon dioxide concentration at 35 ~ 45 mmHg with a fresh gas flow of 2 L/min 60% oxygen. Anesthesia was maintained by constant inhalation of 1.5 ~ 2.5% sevoflurane and constant infusion of remifentanil at a rate of 0.1 ~ 0.2 μg/kg/min. Sufentanil 0.1 μg/kg was added intraoperatively as required. At the end of the surgery all patients received 100 mg of flurbiprofen (an NSAID). Dexamethasone 8 mg (given after induction) and ondansetron 4 mg (given at the end of surgery) were used for prevention of postoperative nausea and vomiting (PONV). All surgical procedures were finished by the same surgical team with the same standardized technique. The two drainage catheters were placed by the surgeon before closing the surgical incision. Before the placement, the subcutaneous tissue of the two entry points of the catheters received local infiltration, which included intervention group (Group A) by 10 ml 0.5% ropivacaine and control group (Group B) by 10 ml normal saline (5 ml for each point). After operation, all patients were extubated and transferred to the post-anesthesia care unit (PACU). Flurbiprofen 100 mg was provided by intravenous injection daily to control the postoperative pain within 3 days after operation. If the pain visual analogue scale (VAS) was ≥3, 100 mg tramadol was administered as a rescue analgesic. For postoperative antiemetic treatment, metoclopramide was intravenously administrated if the nausea VAS was ≥5 or episodes of vomiting ≥2. All patients received standard postoperative therapies according to the pathological characteristics.The pain was immediately assessed after returning to PACU and at 6 h, 12 h, 24 h, and 36 h after operation using a VAS (0 = no pain to 10 = most severe pain). The incidence of PONV was recorded simultaneously, using a three-point ordinal scale , and nausea was evaluated by VAS. The number of patients who received postoperative analgesic or antiemetic drugs was recorded.The status of chronic pain in patients was collected using the breast cancer pain questionnaire, which was first developed by Gartner et al. . A Pain The quality of recovery including 40 questions (QoR-40), which is used as a measure of quality of recovery, was distributed to patients for collecting data 24 h after operation. This included five factors of emotional state, physical comfort, psychological support, physical independence, and pain. In all, the highest score was 200 while the lowest being 40 and the more score, the better the results.Also the remaining data, including the consumption of sufentanil during the surgery, types of surgical procedures, operation and anesthesia time, age, body mass index (BMI), history of smoking and PONV were collected,p < 0.05.The sample size was calculated based on our preliminary experiment that enrolled 10 cases in each group. The mean pain VAS at 12 h after the surgery was 1.2 ± 2.1 for Group A and 2.9 ± 2.2 for Group B. Using standard sample size calculation formula to achieve a power of 0.8 at α = 0.05, there should be at least 29 patients included in each group to detect a significant difference. Considering the possibility of data censored, a total of 40 patients in each group were recruited to guarantee the sample size. SPSS 23.0 for windows  was used for data analysis. Normally distributed continuous data were expressed as means (SD), and were analyzed using analysis of variance (ANOVA), independent-sample t-test or paired t-test. Nonparametric data were analyzed by Mann-Whitney and Wilcoxon text. Two-sided tests were performed to declare statistical significance at The random assignment of the participants into the two groups and analysis of the outcome was presented in Fig. 6. Although whether surgical drains increase opioid consumption after mastectomy has not been investigated, pain originating from the insertion sites of the two drainage catheters constitutes a major part of acute postoperative pain. In this study, we found that ropivacaine infiltration of the two drainage exit sites have significantly reduced the postoperative pain in PACU , at 6 h , 12 h , and 24 h after operation   .Surgical drains were reported to be associated with high postoperative opioid use after breast-conserving surgeryWe did not collect pain scores of restricted movement of the shoulder and values of arm abduction angle. Because the operated arm was tightly bound and forbidden to move by surgeons within 3 days after the operations, in case of wound dehiscence, subcutaneous effusion and hematoma.p < 0.05).The intervention showed no effect on the incidence of chronic pain within 3 months, and PBI in the two groups showed similar results Table . No signTo our knowledge, this is the first prospective randomized controlled study to reveal ropivacaine infiltration of the two drainage exit sites, which significantly reduced postoperative pain and analgesic requirements and improved the quality of recovery after mastectomy.Park’s study  reportedTo compare with the previous similar studies, in Table The incidence of PONV in the two groups remained similar. However, from 6 h to 12 h after operation, PONV occurred in 6 patients in Group A and 12 in Group B. This difference might due to the side-effects of higher tramadol requirement in Group B. At 3-months follow-up, no significant differences were discovered with regard to the incidence of chronic pain. The mechanism of chronic pain is complicated, and it is still poorly controlled. Although our intervention decreased the postoperative pain more effectively, no surgical, demographic, and psychosocial factors that influenced chronic pain after breast surgery considered . It has However, there are certain limitations in this study that require consideration. Firstly, the sample size is small, and so it is not sufficient to perform subgroup analysis. Secondly, follow-up of the patient’s pain after 3-months was not evaluated.In conclusion, ropivacaine infiltration of two drainage exit sites effectively decreased the degree of postoperative acute pain and analgesic requirements within 24 h, and meanwhile improved patients’ quality of recovery. Further large scale studies are warranted to study the outcomes in the future, and explore efficient approach that relieve pain after mastectomy in long run.


Intraventricular hemorrhage is a common cause of morbidity and mortality in premature infants. The rupture of the germinal zone into the ventricles entails loss of neural stem cells and disturbs the normal cytoarchitecture of the region, compromising late neurogliogenesis. Here we demonstrate that neural stem cells can be easily and robustly isolated from the hemorrhagic cerebrospinal fluid obtained during therapeutic neuroendoscopic lavage in preterm infants with severe intraventricular hemorrhage. Our analyses demonstrate that these neural stem cells, although similar to human fetal cell lines, display distinctive hallmarks related to their regional and developmental origin in the germinal zone of the ventral forebrain, the ganglionic eminences that give rise to interneurons and oligodendrocytes. These cells can be expanded, cryopreserved, and differentiated in vitro and in vivo in the brain of nude mice and show no sign of tumoral transformation 6 months after transplantation. This novel class of neural stem cells poses no ethical concerns, as the fluid is usually discarded, and could be useful for the development of an autologous therapy for preterm infants, aiming to restore late neurogliogenesis and attenuate neurocognitive deficits. Furthermore, these cells represent a valuable tool for the study of the final stages of human brain development and germinal zone biology. Germinal zone neural stem cells (Gz‐NSC) are isolated from the hemorrhagic cerebrospinal fluid of preterm infants with severe intraventricular hemorrhage. These cells express ventral and posterior forebrain markers, can be differentiated and do not cause tumors. Gz‐NSC represent a valuable tool for the development of new cell therapies and the study of human Gz biology. Intraventricular hemorrhage (IVH), occurring in 15% to 40% of preterm births, is frequently associated with long‐term neurological deficits. The rupture of the proliferative germinal zone in IVH disturbs late neuronal, ependymal, and glio‐genesis. Using a minimally invasive neuroendoscopic procedure, neural stem cells can be retrieved from the cerebrospinal fluid, which can be expanded, cryopreserved, and differentiated in vitro and in vivo, and are not tumorigenic. These cells display distinct hallmarks related to their origin in the germinal zone of the ventral forebrain and could be useful for the development of an autologous cell therapy aiming to attenuate neurocognitive sequelae.1Intraventricular hemorrhage (IVH) is a common complication of premature infants, occurring in 15% to 40% of preterm infants weighing less than 1500 g at birth and being particularly common in extremely low birthweight neonates.NSCs are the self‐renewing, multipotent cells that generate neuronal and glial cell populations during development. During brain development, primary NSCs located in the VZ have radial processes , divide symmetrically, are highly polarized, and express prominin‐1 (CD133).Here we demonstrate that a novel class of NSCs can be robustly isolated from the hemorrhagic CSF of preterm neonates during neuroendoscopic lavage. These NSCs that we named Gz‐NCS display distinctive features corresponding to their origin in the ventral forebrain. Gz‐NSCs could be useful to develop an autologous cell therapy aiming to reduce neurological disability in preterm infants and to further our understanding of human Gz biology.22.12O cm; the infusion rate is determined by gravity (1.8 m) so that there is no risk of inadvertently causing hypertension during the procedure. Additionally, the neurosurgeon constantly monitors the fontanel tension and can open or close the lines to stabilize the pressures. The CSF is collected in 50 mL syringes connected to the outflow channel to minimize exerting an excessive suction. Once the initial hypertension is corrected, continuous irrigation is established using warm, lactated Ringer solution  by passive inflow and outflow through the second channel (1.4 mm wide), which was also collected (lavage) in successive syringes until clear. Irrigation was stopped once the fluid within the ventricular system was clear, or at any time if hemodynamic instability appeared. Typically, 1000 to 2000 mL of Ringer solution were used and collected in 50 mL sterile syringes that were immediately closed to maintain sterility. The bleeding area was sealed with fibrinogen beads .CSF samples were obtained from eight preterm infants with grade IV IVH Table  at the H2.2g for 10 minutes. The cell pellet was resuspended in N2/B27 medium: Dulbecco's modified Eagle medium (DMEM)‐F12 (ThermoFisher Scientific #11530566), 0.1 mM nonessential amino acids (Sigma‐Aldrich #RNBG4911), 100 IU penicillin/100 μg/mL streptomycin (Sigma‐Aldrich #P0781), 2 μg/mL heparin (Rovi #641647), 1% N2 (ThermoFisher Scientific #11520536), 1X B27 (ThermoFisher Scientific #11530536), 20 ng/mL FGF (Miltenyi Biotec #130‐093‐564), 20 ng/mL EGF (Peprotech #AF‐100‐15), and 10 ng/mL LIF (Miltenyi Biotec #130‐108‐156). The cell suspension was seeded onto 20 μg/mL poly‐L‐ornithine (Sigma‐Aldrich # P4957)/20 μg/mL laminin from human placenta (Sigma‐Aldrich #L6274) (POL) or Matrigel (Corning #354277)‐coated plates. Medium was changed 24‐48 hours after seeding. Cells were seeded for expansion at 1 × 105 cells/mL in low binding flasks or at 12 000 cells/cm2 in Matrigel‐ or POL‐coated plates. Matrigel‐coated flasks were prepared by incubation with Matrigel diluted in cold DMEM‐F12 for 1 hour at room temperature according to manufacturer's instructions. For POL coating, flasks were incubated with 20 μg/mL poly‐L‐ornithine for 1 hour at 37°C or overnight at 4°C. Flasks were washed twice with distilled water and they were then further incubated with 20 μg/mL laminin for 2 hours at 37°C. Flasks were washed three times with phosphate buffered saline  before cell seeding. Cells were expanded for 3 (early) and 7‐10 (late) passages for characterization. Passage 7, which corresponds to 13 ± 1 accumulated population doublings, was considered “late passage” given that it will not be possible to extensively expand the cells in a clinical setting.CSF samples were transferred to appropriate tubes and centrifuged at 370Magnetic activated separation (MACS) was performed using the CD133 MicroBead kit (Miltenyi Biotec #130‐097‐049) following manufacturer's instructions.2.3Cells grown over Matrigel‐coated coverslips were fixed with 4% paraformaldehyde (SantaCruz Biotechnology #SC‐281692), permeabilized with 0.1% Triton X‐100 (Sigma‐Aldrich #T8787), blocked in PBS (ThermoFisher Scientific #A12856‐01) with 1% bovine serum albumin  for 30 minutes at 37°C and incubated with the primary antibody overnight at 4°C. Cells were subsequently incubated with the secondary antibody for 30 minutes at 37°C and mounted with ProLong Gold Antifade Mountant with 4′,6‐diamidino‐2‐phenylindole . Primary and secondary antibodies are listed in 2.4For CD133, CD24, CD34, CD45, PODXL, IL1RAP, and MHC detection, live cells were blocked in PBS with 1% BSA and incubated with conjugated antibodies for 15 minutes at 4°C. For TREK2, FZD5, and DLK1 analysis, cells were fixed with 3.7% formaldehyde (Sigma‐Aldrich #F8775), permeabilized with 0.1% Triton X‐100 (Sigma‐Aldrich #T8787), blocked in PBS with 1% BSA and incubated with the primary antibody for 30 minutes at 4°C. Cells were subsequently incubated with the secondary antibody for 30 minutes at 4°C. Antibodies are listed in Table 2.52.5.1Human fetal NSCs (n = 4) were derived from the forebrain of 15‐ to 22‐week‐old fetuses that had undergone spontaneous in utero death (miscarriage). Tissue procurement was approved by the Ethics Committee of the Institute “Casa Sollievo della Sofferenza” after receiving the mother's informed, written consent. These fetal NSC lines have been extensively characterized.PSC‐derived NSCs  were differentiated from embryoid bodies (EBs) in TeSR2 medium (Stemcell Technologies #05860) spiked with Rock inhibitor . After 7 days, EBs were plated on Matrigel and cultured in neural differentiation media. On day 10, 0.1 μM retinoic acid  was added to the medium. On day 15, neural tube‐like rosettes were mechanically detached and cultured in neural differentiation media with 20 ng/mL FGF (Miltenyi Biotec #130‐093‐564) and 20 ng/mL EGF (Peprotech #AF‐100‐15). Cells were expanded in suspension as neurospheres or in adhesion over Matrigel during 6 to 7 passages before RNA extraction for transcriptomic analysis.+ umbilical cord cells. These cells are available from the Spanish national repository, (BNLC) and the data regarding cell characterization can be downloaded at http://www.eng.isciii.es. WA09 (H9) were purchased from WiCell and the characterization data can be downloaded at www.wicell.org. L6‐iPS was generated by our group  were obtained from the Banc de Sang i Teixits, Barcelona. CD34+ cell purification was performed as previously described.+ cells were positively selected using Mini‐Macs immunomagnetic separation system . Purification efficiency was determined by flow cytometry analysis staining with CD34‐phycoerythrin  antibody.Umbilical cord blood samples (CD342.5.2http://amp.pharm.mssm.edu/Enrichr/).www.brainspan.org).RNA was extracted with RNeasy Mini kit (Qiagen #74104) following the instructions of the manufacturer and sent to the Genomics Unit of the Andalusian Center of Molecular Biology and Regenerative Medicine (CABIMER). RNA quality was analyzed by the Bioanalyzer 2100 (Agilent). All samples had RNA Integrity Number (RIN) higher than 9. cDNA was synthetized, labeled with biotin and hybridized with independent Human Clariom‐S Microarrays, (Affymetrix #902927) following Affymetrix protocol. Microarrays were scanned with Affymetrix GeneChip Scanner 3000 7G, and the obtained data were analyzed with the Affymetrix GeneChip Command Console 2.0 software. The microarray expression data set is publicly available at the GEO repository under the identifier GSE124361. Further analyses were performed using the Transcriptome Analysis Console  v4.0 software and R version 3.5.0.2.5.3First, 0.1 μg of RNA were used for cDNA synthesis using Oligo‐dT (Invitrogen #18418012), RNase OUT (Invitrogen #10777‐014) and SuperScriptII Retrotranscriptase (Invitrogen #18064‐014). PCR products were obtained using 5 ng of cDNA and Mytaq Red DNA Polymerase (Bioline #BIO‐21108) following the manufacturer's instructions. Oligonucleotides used for amplification are provided in Table 2.6Animal care and experimental procedures were conducted according to the current National and International Animal Ethics Guidelines and approved by the Italian Ministry of Health. Ten 7‐ to 8‐week‐old female athymic Nude‐Foxn1nu mice  received a single injection of 300 000 CD133+ cells in the striatum. Stereotaxic coordinates were determined using the Paxinos and Franklin atlas+ cells in those sections.Quantification of transplanted cells was done using Image J cell counter‐plugin on images acquired in a Nikon C2 confocal microscope and the NIS Elements 1.49 software. All cells expressing human nuclear antigen (huN) were counted in sections spanning the graft area, and the total number was calculated using Abercrombie correction2.7t test for comparisons between two samples. P < .05 was considered significant. Paired t‐tests or repeated measures (R) ANOVA were used to compare samples from the same individual at different stages. All statistical analyses were performed using the GraphPad Prism 8.01 software. Bioinformatic analyses were performed using the Affymetrix and R software, using t, ANOVA, and R‐ANOVA tests and selected thresholds, as indicated in the text and figure legends. A false discovery rate (FDR) <5% was established for significance.Data are presented as mean ± SEM. Significance was determined using two‐tailed Student's 33.1+ cells dropped drastically upon passaging. This case was excluded from further analyses given that IVH in full‐term neonates most often originates in the choroid plexus.Eight consecutive cases with a clinical and radiological diagnosis of grade IV IVH Table  underwen+CD34+ at passage 7, and 49.42 ± 10.65 of the cells were CD133+CD24+ at passage 7. Despite no significant differences, there was a trend to an increase of CD24 double positive cells with passages  and the iPSC used to obtain iPS‐NSCs. Principal component analysis (PCA) mapping and hierarchical clustering of global gene‐expression profiles showed that NSCs from CSF clustered together with fetal NSCs, being farther away from iPS‐derived NSCs .We next performed a transcriptomic analysis to study the differences and similarities between NSCs isolated from hemorrhagic CSF, fetal forebrain NSCs and NSCs derived from iPSCs. Given that CSF samples contained mostly blood cells we also included in the analysis hematopoietic stem cells . Quantification confirmed expression of these proteins by most cells Next, we used immunofluorescence to evaluate the expression at the protein level of typical radial glia markers such as SOX2, nestin, and brain lipid binding protein . Because OLIG2 is also expressed in neuronal progenitors, we examined the coexpression of OLIG2 and doublecortin (DCX). About 10% (9.9 ± 7) of the OLIG2 positive cells coexpressed DCX in these in vitro conditions, representing 1.5% of the total cells . CD133 has been used for the isolation of NSCs from normal brain tissues and CD133+ cells differentiate in vitro and in vivo into the three neuroectodermal lineages.+ and CD24+ double positive cells were variable and did not significantly change with sorting, although we observed that CD34+ cells tended to remain in the negative fraction . Therefore, in order to better define putative Gz‐NSC‐specific features and obtain a more homogenous population for future in vivo applications, we selected CD133n Figure .+‐sorted Gz‐NSCs  with no significant DEG between early and late passages  showed that the expression of genes implicated in neural and synaptic specific pathways was decreased  interneurons derived from the medial ganglionic eminence, suggesting that more differentiated cells are lost. KCNK10 (TREK2) expression was significantly decreased, but was still significantly higher than in fetal NSCs.Upon purification, there were significant changes relative to both early and late passage cells. Enrichment analysis of the common genes .www.brainspan.org),in vivo survival in nude mice in our experiment compared with published rates of 40% for iPS‐NSCs or 20% for fetal NSCs.In addition to conspicuous differences in the expression of regional transcription factors, we report the expression of novel Gz‐NSC markers that differentiate these cells from fetal  forebrain NSCs and were maintained after sorting for CD133+ Gz‐NSCs. Expression of the noncanonical Notch ligand DLK1, was also maintained in the sorted population. DLK1 is developmentally expressed in NSCs in the GEs and maybe involved in the regulation of the stem cell pool.KCNK10) a potassium channel that has been reported to be expressed by the ependymal cellsGz‐NSCs showed a strong signal at the cell surface for FZD5, the putative receptor for Wnt5A, which is involved in neural specification and highly expressed in the VZ+ cells was intriguing. We included HSC in the transcriptomic analysis to rule out a hematological origin of the CD133 population. In our data set, CD31 (PECAM1), CD53, CD37, as well as typical leukocyte markers, such as selectin‐L or myeloperoxidase, and erythrocyte markers like fetal hemoglobin (data not shown), were not expressed by Gz‐NSCs, suggesting that hematopoietic and endothelial stem cells were not present in this population. Indeed, although it is usually considered a marker of hematopoietic and endothelial progenitor cells,+ cells tended to remain in the negative fraction, suggesting that they may represent a more mature phenotype, perhaps in the ependymal lineage.There was a large variability in both CD24 and CD34 positive populations between samples but it will be necessary to increase the sample size in order to examine a possible correlation with developmental stage (age), weight, severity or any other individual parameter. While CD24 expression has been described in NSCs, the presence of CD34+ cells into the striatum of nude mice resulted in small neural grafts with no tumor formation or adverse reaction. Moreover, although survival in nude mice was very limited, a significant proportion of the grafted cells at 6 months expressed OLIG2, suggesting that Gz‐NSC treatment in an autologous setting could be a safe approach to restore oligodendrogenesis. Collectively our data demonstrate that Gz‐NSCs are a distinct population, which can be expanded, purified, and cryopreserved maintaining their ventral identity and a favorable safety profile. Therefore, we believe that Gz‐NSCs could be an optimal source to develop a cell therapy for preterm infants with IVH, for whom other cell therapies are already being tested.The shedding of neural progenitors and/or NSCs into the CSF of IVH and PHH patients had been previously suggested.5Here we describe a novel class of NSCs, the Gz‐NSCs that can be easily and robustly isolated from the CSF of preterm neonates with grade IV IVH undergoing neuroendoscopic lavage. We found that these cells, while being similar to fetal forebrain NSCs, have several distinctive hallmarks related to their regional and developmental origin in the ventral forebrain. Gz‐NSCs can be expanded and cryopreserved showing in vitro and in vivo differentiation potential, and pose no ethical concerns as the fluid is usually discarded. Thus, Gz‐NSCs could represent an optimal source for the development of an autologous cell therapy for infants with IVH, as well as a useful tool for studying the late stages of human neural development.B.F.M., E.G.M., R.S.P., and J.M. are authors of a patent application for the use of CSF‐NSCs (nº application European Patent Office: 200930943).The other authors indicated no potential conflicts of interest.B.F.‐M.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; C.R.‐V.: collection and assembly of data, data analysis and interpretation; D.F.: collection of data, data analysis and interpretation; J.A.‐A., M.Á.M., R.C.‐C., M.M.‐L., D.C.P., M.F.B., A.G.: collection of data; L.L.‐N.: assembly and analysis of data, administrative support; E.G.‐M.: conception and design, collection of data; J.M.‐R.: conception and design, provision of study material or patients, collection of data, data analysis and interpretation; R.S.‐P.: conception and design, financial support, assembly of data, data analysis and interpretation; All authors: revision and final approval of manuscript.Figure S1. Supporting informationClick here for additional data file.Figure S2. Supporting informationClick here for additional data file.Figure S3. Supporting informationClick here for additional data file.Figure S4. Supporting informationClick here for additional data file.Figure S5. Supporting informationClick here for additional data file.Figure S6. Supporting informationClick here for additional data file.Figure S7. Supporting informationClick here for additional data file.Video S1. Supporting informationClick here for additional data file.Table S1. AntibodiesClick here for additional data file.Table S2. Primers used for RT‐PCR.Click here for additional data file.Table S3. Flow cytometry results.Click here for additional data file.