To combine Intravoxel Incoherent Motions (IVIM) imaging and diffusion kurtosis imaging (DKI) which can aid in the quantification of different biological inspirations including cellularity, vascularity, and microstructural heterogeneity to preoperatively grade rectal cancer.  A total of 58 rectal patients were included into this prospective study. MRI was performed with a 3T scanner. Different combinations of IVIM-derived and DKI-derived parameters were performed to grade rectal cancer. Pearson correlation coefficients were applied to evaluate the correlations. Binary logistic regression models were established via integrating different DWI parameters for screening the most sensitive parameter. Receiver operating characteristic analysis was performed for evaluating the diagnostic performance. p < 0.05). The better discrimination between high- and low-grade rectal cancer was achieved with the combination of different DWI-derived parameters. Similarly, ROC analysis suggested the combination of D (true diffusion coefficient), f (perfusion fraction), and Kapp (apparent kurtosis coefficient) yielded the best diagnostic performance . According to the result of binary logistic analysis, cellularity-related D was the most sensitive predictor (odds ratio: 9.350 ± 2.239) for grading rectal cancer.  For individual DWI-derived parameters, all parameters except the pseudodiffusion coefficient displayed the capability of grading rectal cancer ( The combination of IVIM and DKI holds great potential in accurately grading rectal cancer as IVIM and DKI can provide the quantification of different biological inspirations including cellularity, vascularity, and microstructural heterogeneity. It has been reported that there were around 0.7 million new cases of rectal cancer, accounting for approximately 40% of 1.8 million new colorectal cancer cases in 2018. Moreover, rectal cancer has posed a huge threat to human health because of high mortality rates (∼0.2 million deaths in 2018) . CurrentKapp) indirectly represents the microstructural complexity in comparison to IVIM-derived f (perfusion fraction) that can quantify the vascularity [Due to the clinical significance of providing different biological inspirations, Diffusion-Weighted Magnetic Resonance Imaging (DW-MRI) has shown tremendous clinical potential. During the past few decades, much effort has been made to propose novel DWI models such as intravoxel incoherent motion (IVIM) , diffusicularity . Hence, cularity –12. Howecularity . Li perfcularity . MoreoveThe approval from a local institute review board was obtained for this prospective research. Written informed consent was obtained from all patients. A total number of 69 patients were recruited into this prospective research between December 2018 and August 2019. The inclusion criteria and exclusion criteria were established according to a previous research [Endoscopic biopsy-proven rectal cancerMore than one week of interval between biopsy and MRIInclusion criteria:Poor quality of DKI or IVIM images caused by artifactsPatients who underwent surgery before the MRI examinationTime interval between MR examination and surgery of more than 2 weeksPreoperative neoadjuvant chemoradiotherapyInaccessible clinical pathology results of histopathologic gradeExclusion criteria:2; matrix, 336 × 432; slice thickness, 6 mm; intersection gap, 1.2 mm; and number of slices, 25), dynamic three-dimensional T1 weighted gradient echo (GRE) sequence , and oblique axial Single Shot- Echo Planar Imaging sequence termed as SS-EPI . It should be noted that both DKI and IVIM were based on the abovementioned SS-EPI sequence with different selections of b values for subsequent postprocessing.All MRI measurements were performed with a 3T whole-body scanner  with a twelve-channel coil. The MRI protocol mainly included a T2-weighted Fast Spin Echo sequence termed as FSE T2WI .S0 and Sb are, respectively, the signal intensity when a b value of 0 s/mm2 and other b values are applied. f is the perfusion volume fraction, D (unit: ×10−9 m2/s) is the true diffusion coefficient representing pure diffusion, and D∗ (unit: ×10−9 m2/s) is the pseudodiffusion coefficient representing perfusion related diffusion (incoherent microcirculation within the voxel). Moreover, the fitting of IVIM was based on the images of b values of 0, 10, 20, 30, 50, 80, 100, 150, 200, 400, 600, and 800 s/mm2.The quantitative pixelwise parameters derived from IVIM were obtained through the previously-reported fitting model :(1)SbS0=Sb and S0 are identical to those in IVIM. Dapp (unit: ×10−9 m2/s) and Kapp (unitless) are, respectively, the apparent diffusion coefficient and apparent kurtosis coefficient representing the degree to which molecular motion deviated from the Gaussian diffusion. Additionally, the fitting of DKI was based on the images of b values of 0, 800, 1500, and 2000 s/mm2.The quantitative pixelwise parameters derived from the DKI were obtained through the previously-reported fitting model :(2)lnSb=D map with T2WI images as the references. Necrosis, cyst, and haemorrhage were carefully excluded. In this way, the whole tumor was incorporated into the VOI. Then, the outlined regions were automatically copied to other parametric maps including f map, D∗ map for IVIM, and Dapp and Kapp map for DKI. Finally, the pixel-based average values for each parameter were acquired by means of the whole tumor averaging approach reported before [Two radiologists (ZJ.G and CM.X), with 8 and 25 years' experience of gastrointestinal imaging were asked to draw the Volumes of Interest (VOIs) along the tumor border, which meant that all DWI-derived parameters were measured based on the whole-lesion method, and the entire tumor was maximally included into the VOI. The definitions of each VOI were based on the consensus of the abovementioned two radiologists. Before drawing VOIs, radiologists were blinded to the results of histopathological examination. For each slice within tumor, freehand regions were drawn along the border of the low signal of the tumor on the d before .μm slices and, then, stained with hematoxylin-eosin (H&E). Histological grading was performed according to the WHO criteria [All pathological examinations were concluded by an experienced pathologist with more than 5 years' experience. Surgical specimens of rectal cancer were routinely prepared into 5 criteria . Rectal t-test was applied to see whether there existed significant differences between different groups (low grade (G1-2) and high grade (G3)). Moreover, one-way ANOVA and the Tukey post hoc test were performed for multiple comparison of quantitative diffusion parameters among the groups of G1, G2, and G3 rectal cancer. The Pearson correlation test was performed to assess the correlation coefficients abbreviated as r between the parameters. Binary logistic regression analysis was performed to establish the diagnostic model with the combination of different parameters including D (cellularity), f (vascularity), and Kapp  of IVIM and DKI which showed significant differences between low-grade and high-grade rectal cancer groups for subsequent ROC analysis. D∗ was excluded because it did not display a significant difference between the low-grade rectal cancer and high-grade rectal cancer. Although Dapp also displayed a significant difference between the low-grade and high-grade rectal cancer, it was excluded because of the following issues: (1) Dapp possesses the same biological insight of cellularity as D. (2) Eliminating the influence of perfusion, D is better at characterizing the true diffusion restriction resulted from cellularity [D and f (cellularity and vascularity), D and Kapp , and f and Kapp , as well as D and f and Kapp , were established via logistic regression. Besides introducing three variables , binary logistic regression analysis was also performed to evaluate which DWI-derived biological insight among cellularity, vascularity, and microstructure was the most sensitive for predicting high-grade rectal cancer by comparing the standardized regression coefficients (β) and the odds ratio (OR) of different parameters. The OR was calculated according to the following formula: OR=exp(|β|). In order to obtain the standardized regression coefficients of binary logistic regression analysis, D, f, and Kapp were, firstly, standardized as the Z score to eliminate the effect of dimension and quantity of data. It is worthwhile to be noted that, for obtaining more intuitive comparison, the odds ratio of f and D was defined as the ratio of the positive  probability after the variable decreased by one standard unit to the probability before the change, which was different from the standard definition. The definition of the odds ratio of Kapp was the same as the standard definition. The ROC (receiver operating characteristic curve) analysis was performed to evaluate the diagnostic performance of parameters showing significant differences between low-grade and high-grade rectal cancer groups together with their combinations by comparing the AUCs (area under curve). All parameters in this research were statistically analyzed with statistical tools including SPSS software , Medcalc , R version 3.6.1 (R Core Development Team), and RStudio . It was regarded as having a statistical significance when the p value was less than 0.05.Firstly, the Kolmogorov–Smirnov test was performed for analyzing normality. According to the result of the Kolmogorov–Smirnov test, independent Student's lularity . As a reBetween December 2018 and August 2019, a total of 69 patients were initially recruited into this prospective study. Three patients were excluded because of poor quality of MR images. Two patients were excluded because they underwent surgery before the MR examination. In addition, two patients were excluded because of the preoperative neoadjuvant chemoradiotherapy. Four patients were excluded as the pathological results of histopathologic grade were inaccessible. Ultimately, 58 patients  were included for subsequent analysis. Of the 58 patients, 11 were classified as WHO G1, 29 were classified as WHO G2, and 18 patients were classified as WHO G3. The clinical data of 58 patients are presented in Representative MR images of a patient with WHO G1 rectal cancer and a patient with WHO G3 rectal cancer are displayed in D and Dapp (cellularity), D∗ and f (vascularity), and Kapp  are presented in D∗ showed the capability of discriminating between rectal patients of different grades with significant difference (p < 0.05). As the histopathologic grade increased, D : 1.362 ± 0.117 and high grade (G3): 1.105 ± 0.103. Unit: ×10−9 m2/s), Dapp , and f  decreased, but Kapp  increased. Detailed comparisons and significance levels are presented in f showed a weaker capability of grading rectal cancer as it not only displayed no significant difference between G1 and G2 (pG1 vs G2 = 0.079) but also smaller difference  between the subgroups compared to other parameters.The results of directly grading rectal cancer patients by individual DWI parameters including D and f, D and Kapp, f and Kapp, and D and f and Kapp. The results in the first row of D), vascularity (f), and microstructural complexity (Kapp) were simultaneously integrated with small data overlap.The first row of D, f, and Kapp. The standardized regression coefficients (β) of different DWI-derived biological inspirations are presented in βcellularity = −2.235 ± 0.806, βvascularity = −0.081 ± 0.527, and βmicrostructural complexity = 1.238 ± 0.905). The odds ratios (OR) of different DWI-derived biological insight are presented in cellularity = 9.350 ± 2.239, ORvascularity = 1.084 ± 1.694, and ORmicrostructural complexity = 3.440 ± 2.472). The OR of microstructural complexity was 3.440, indicating that when Kapp increased by one standard unit, the probability of high-grade rectal cancer was 3.440 times as high as before. The odds ratio of cellularity was 9.350, indicating when D decreased by one standard unit, the probability of high-grade rectal cancer was 9.350 times as high as before. It should be noted that the definition of ORcellularity and ORvascularity was different from the standard definition of OR. Detailed definitions can be found in the Section 2.A binary logistic regression model was established by introducing three variables: D (cellularity) & f (vascularity) & Kapp  = 0.953, AUC D (cellularity) &Kapp  = 0.951, AUC D (cellularity) & f (vascularity) = 0.946, AUC D (cellularity) = 0.912, AUC Kapp  = 0.910, AUC f (vascularity) & Kapp  = 0.901, AUCDapp (cellularity) = 0.901, and AUC f (vascularity) = 0.843. All the abovementioned AUCs were significantly different from the AUC of 0.500 (p < 0.001).r, which are presented in D or Dapp were significantly and positively correlated with f  but negatively correlated with Kapp . Besides, f was negatively correlated with Kapp . Interestingly, only f and Kapp showed significant correlations with D∗ .The binary correlations between different DWI-derived biological inspirations were evaluated via Pearson correlation coefficients abbreviated as f, and D for tumor grading, staging, and so on, important highlights of this research are the followings: (1) combining DWI models according to their biological inspirations will benefit the accurate grading of rectal cancer via more comprehensive tumor characterization. (2) The combination of DKI and IVIM was enough to provide three main DWI-derived biological inspirations, which can avoid the disadvantages caused by excessive DWI models such as long scan time, difficult postprocessing and manipulation, and low patient compliance. (3) The results of the present research suggest that all three DWI-derived biological inspirations are tightly correlated with each other, which further proved the necessity of combining these DWI-derived inspirations together. (4) The results indicate that DWI-derived cellularity was the most sensitive for grading rectal cancer followed by microstructural complexity and vascularity.In comparison to plenty of previous studies that simultaneously combined many DWI models but focused more on comparing the clinical effectiveness of different parameters that were calculated from various DWI models such as ADC, D∗ all had the capability of distinguishing the high-grade from low-grade rectal cancer. The biological basis is as follows: (1) The rapid proliferation of cancer cells leads to an increase in nuclear-to-cytoplasmic ratio and to a decrease in extracellular space, which ultimately results in an increase in the degree of diffusion restriction reflected by a decrease of diffusion coefficient (Dapp and D) [Kapp) [f decreased as histopathological grade increased was because of the following reasons: the vascular systems will be severely destroyed together with the macromanifestations such as intratumoral bleeding as tumor proliferates, which resulted in the decrease of f representing the perfusion fraction [D and Dapp) and microstructural complexity-related parameter (Kapp), as well as vascularity-related parameters (f) and microstructural complexity-related parameter (Kapp). Moreover, cellularity-related parameters (D and Dapp) significantly and positively correlated with the vascularity-related parameter (f). Interestingly, IVIM-derived D∗ displayed a negative correlation with f but a positive correlation with Kapp, which can be explained by the following points: (1) according to IVIM theory, D∗ is influenced by the several factors that can be expressed as the following equation: D∗=(l × v)/6, where  l represents the capillary length and v represents the average velocity of blood in the capillary. (2) As mentioned above, the poor structure of lumenized vessels dominates in high-grade rectal cancer. However, in order to meet the rapidly growing need of oxygen and nutrients for the tumor cells, the average velocity of blood in the capillary will increase compensatively. Therefore, D∗ was significantly positively correlated with Kapp but negatively with f. Correlations among DWI-derived cellularity, vascularity, and microstructural complexity further proved the necessity of integrating these inspirations together to achieve a more comprehensive tumor characterization. In addition, the vascularity-related f showed the weaker diagnostic power compared to cellularity- and microstructural complexity-related parameters. These results can be explained as follows: (1) D∗ is very vulnerable to the effects of a low signal to noise ratio. (2) The vascularity variation is not as sensitive as cellularity and microstructural complexity during carcinogenesis. (3) Several studies proposed that f is not accurate for diagnosing tumor. [For individual parameters, individual DWI-derived biological insight-based parameters except p and D) . (2) Dif) [Kapp) . (3) f dfraction . Based og tumor.  When difD was the most sensitive for predicting high-grade rectal cancer, followed by microstructural complexity quantified by DKI-derived Kapp and vascularity quantified by IVIM-derived f. The abovementioned results are consistent with the ROC analysis results. The reasons may be the following: (1) Among cellularity, vascularity, and microstructural complexity, the cellularity variation is the most pronounced. Similar to the results of our study, Fujima demonstrated that D (25th percentile) served as the most powerful indicator for predicting the treatment outcome of nasal or sinonasal squamous cell carcinoma patients. [D value obtained from IVIM showed a higher value in discriminating distant metastasis compared to the parameters derived from DKI [f, representing the vascularity, is not accurate enough, always leading to mixed results [Kapp is the most valuable diagnostic marker for grading glioma in comparison to the other parameters [Kapp is much more effective in grading and evaluating the proliferation of diffuse astrocytic tumors [b values is significant for accurate fitting of the diffusion model, which further determines the calculation of parameters. (3) Data analysis can also contribute to the variance of conclusion. For instance, data analysis of hybrid IVIM-DKI is quite different from separate analysis of DKI and IVIM [The binary logistic regression analysis results demonstrated that cellularity quantified by IVIM-derived atients.  It was rfrom DKI . (2) Som results , 20, 23.rameters . Similarc tumors . We beliand IVIM . (4) OthThere were several limitations in the present study. First, the sample size of patients was not large enough, and the number of patients was unbalanced in different histological grades. Particularly, the number of patients with WHO G1 rectal cancer is not enough. Second, the diagnosis was only applied, referring to the WHO criteria in the present study. However, other grading standards such as the poorly differentiated clusters called PDCs-based grading were not referred to further evaluate the diagnostic performance. Third, merely, the logistic regression was introduced to explore the integration of different DWI inspirations. Other complex statistical models should be introduced in the following work. Fourth, too large tumor may make it difficult to determine the tumor boundary and ultimately affect the accurate calculation of DWI-derived parameters.In conclusion, this research indicates that greater diagnostic performance for grading rectal cancer can be obtained through integrating the DWI-derived cellularity, vascularity, and microstructural complexity quantified by parameters that were sourced from DKI and IVIM. Furthermore, because there were tight correlations among all DWI-derived biological inspirations, the integration of different biological inspirations of DWI holds great potential in achieving a more comprehensive tumor characterization, which will be meaningful for many other clinical applications including cancer treatment evaluation, tumor detection, and tumor recurrence prediction.


Objective: The burden of comorbidity among young patients with hemorrhagic stroke is high. This study examines the effect of comorbidity on the length of stay (LoS) and mortality among immobile hemorrhagic stroke patients younger than 50 years.Materials and Methods: A retrospective post-hoc analysis study design was used. A total of 767 younger adults (mean age 41.64 ± 8.16 years) hospitalized for hemorrhagic stroke between November 2015 and July 2017 were evaluated. All-cause mortality was recorded for 90 days after admission regardless of whether death occurred before or after discharge. Comorbidity was assessed using the Charlson Comorbidity Index (CCI), categorized as low (0–1) and high (≥2). A multiple linear regression model was applied to examine the association between CCI and LoS. Survival was evaluated with Kaplan–Meier and Cox regression analyses.Results: The mean CCI of all patients was 1.25 (SD ± 0.79) and median was 1.0 . The average LoS for patients with a low CCI was 17.73 (± 11.59) days compared with 19.49 (± 15.21) days in those with a high CCI (p = 0.142). The mortality rate was 6.0% . After controlling for confounders, high CCI was not associated with longer LoS , whereas high CCI was associated with greater likelihood of death than a low CCI .Conclusions: High global comorbidity was associated with increased short-term mortality but not with LoS. Measures to manage comorbidities aimed at reducing negative clinical impacts of stroke among younger adults are warranted. Stroke is a major cause of death worldwide . In ChinPrevious studies have suggested an upward trend of stroke in younger people and a declining incidence of stroke in older adults –9. StrokIn addition, China has 7.5 million stroke survivors who suffer greatly through comorbidity . It was This study was derived from a research project from the National Health and Family Planning Commission of the People's Republic of China, which aimed to develop a standardized nursing intervention model (SNIM) among immobile stroke patients. As one of the main gauges of this study, the Charlson Comorbidity Index (CCI) addresses multiple comorbidities by creating a weighted total score based on the presence of various conditions, and is commonly used in outcome and mortality studies , 22, 24.Study subjects enrolled were the participants of the SNIM, a prospective study conducted in 25 general hospitals covering five provinces and one municipality city in China . Further information about the SNIM study is published elsewhere , acute ischemic stroke , and transient ischemic attack   . AccordiAll participants in SNIM, except for those who died or relinquished medical treatment, underwent a 90-day follow-up after enrollment in the study. All-cause mortality was recorded for 90 days including in-hospital deaths. Survival time was regarded as the duration from the date of admission to the date of death. Dates of death were collected from death certificates.Comorbidity was assessed using the CCI. The index contained 19 comorbid conditions and was adjusted specifically for stroke evaluation , 23, 27.N = 767) because our aim was to evaluate young immobile hemorrhagic stroke patients with comorbidities, with the study outcomes focusing on mortality and LoS. Immobility was defined as when the patient's basic physiological needs were carried out in bed, except for active or passive bedside sitting/standing/wheelchair use for examination. LoS was defined as the period from the date of admission to the date of discharge or date of death. All-cause mortality was recorded for 90 days after the date of admission regardless of whether death occurred before or after discharge. Therefore, we opted to exclude participants in accordance with the following criteria: older than 50 years , AIS  cases (n = 623), TIA cases (n = 57), and discharged within 24 h of admission (n = 14).In this study we considered a sample of participants with hemorrhagic stroke who were younger than 50 years , experience of surgery, duration of immobility, level of hospital, payment type, and complications that occurred during hospital stay including pressure injuries, pneumonia, deep vein thrombosis (DVT), and urinary tract infections.We assessed 19 diagnoses for each patient according to the CCI: “history of myocardial infarction (weight = 1),” “congestive heart failure (weight = 1),” “cerebrovascular disease ,” “peripheral disease ,” “chronic pulmonary disease (weight = 1),” “connective tissue disease (weight = 1),” “peptic ulcer disease (weight = 1),” “dementia (weight = 1),” “mild liver disease ,” “diabetes without end-organ damage ,” “moderate or severe renal disease (weight = 2),” “diabetes with end-organ damage ,” “hemiplegia (weight = 2),” “tumor without metastasis ,” “leukemia ,” “lymphoma (weight = 2),” “moderate or severe liver disease (weight = 3),” “metastatic solid tumor (weight = 6),” and “AIDS ” , 28.2 test for proportions and Student's t-test for means. We used Kaplan–Meier survival curves and the log-rank test to compare mortality between the two groups. Sociodemographic and clinical parameters were adjusted by a Cox proportional hazards model. A multiple linear regression model was applied to evaluate the relationship between CCI and LoS while controlling for the above covariates listed. In all analyses, CCI was categorized as low (0–1) and high (≥2) comorbidity .We employed descriptive statistics to summarize patients' characteristics. Variables were compared across patients in the two CCI categories using the χorbidity . A p < 0A total of 767 immobile hemorrhagic stroke patients younger than 50 years were hospitalized between November 2015 and July 2017. The mean age of the patients was 41.64 (standard deviation [SD] ± 8.16) years. The mean CCI of the patients was 1.25 (SD ± 0.79) and the median was 1.0 . The percentage of patients with high CCI score (≥2) was 16.17%. Forty-six patients died, and the average LoS was 18.01 (SD ± 12.26) days. p = 0.142). As shown in p = 0.413).p = 0.002)  (p = 0.002) in the crude estimate analysis. In the Cox proportional hazards model, a high CCI score (≥2) was associated with an even greater likelihood of death compared with a low CCI score (0–1) after adjusting for confounders .Overall, death occurred in 6.0% of the study population. The number of patients with a low (0–1) and a high (≥2) CCI score who died were 31 (4.82%) and 15 (12.10%), respectively (= 0.002) . Patient 0.0016) . As showTo our knowledge, this is the first study from China to focus on the effect of comorbidity on LoS and short-term mortality among immobile patients younger than 50 years of age with hemorrhagic stroke. The results indicated that a high CCI score (≥2) could be an effective predictor for higher risk of death compared with a low CCI score, although the present study did not show a significant association between increased LoS and a high CCI score. These findings highlight the essential clinical impact of comorbid conditions among a diverse population of younger patients from a developing country with a universal healthcare system.No significant associations were found between LoS and comorbidities in this study, suggesting that this predictor of clinical outcome has less influence on LoS among the younger hemorrhagic stroke population. Contrary to our results, Richard et al.  analyzedIn the present study we found a low mortality rate of 6.0%, which is lower than the previous studies reporting case fatality rates ranging from 7.7 to 9.9% among younger hemorrhagic and ischemic stroke patients , 31. HowPrevious studies have reported an association between comorbidity and an increased rate of adverse clinical outcomes, particularly regarding mortality , 35, 36.Comorbidities can hinder the young patient's recovery from a stroke . TherefoOur study has some strengths. This study firstly demonstrated the prognostic value of comorbidity using the CCI for short-term survival among immobile hemorrhagic stroke patients younger than 50 years in China, whereby our findings reinforce the importance of monitoring chronic comorbidities. Besides this, in the future we will address long-term mortality, allowing us to better delineate a stroke-management scenario that to date has been lacking in the developing world.post-hoc analysis, whereby the data were prospectively collected for the SNIM research project and later were used retrospectively as part of our study design. Secondly, there is a lack of other relevant clinical data, including clinical severity of stroke (NIHSS), premorbid Rankin scale, volume of intracerebral bleeding, laboratory data, and the details of causes of death, because these features were not recorded in the first place, which is an inherent drawback of a retrospective analysis of a prospectively collected database. The data about the etiologic classification of patients with intracerebral hemorrhage, such as hypertension, and oral anticoagulants is limited. In addition, no affirmative criteria were available to determine the severity of comorbidities, which may potentially influence the clinical outcome of the participants. Prospective studies with more sophisticated evaluations are required to confirm our findings.As for limitations, firstly this study is a retrospective Among hemorrhagic stroke patients younger than 50 years of age, patients in the high-CCI group (CCI ≥2) were associated with increased short-term mortality but not with LoS. More emphasis should be placed on comorbidities as part of wider strategies to reduce the negative clinical impact in young hemorrhagic stroke patients.The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.The studies involving human participants were reviewed and approved by the ethics committee of Peking Union Medical College Hospital. Patients' records and information were anonymized and de-identified prior to the analysis. The patients/participants provided their written informed consent to participate in this study.XWu: study concept and design. XWu and HL: analysis and interpretation of data. HL: edited the manuscript and drafted the tables. JC and JJia: critical review of the manuscript for important intellectual content. CZ, BS, JJin, YL, XWe, and SC: recruited patients, collected data, and edited the manuscript. All authors critically reviewed and approved the manuscript before it was submitted.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Murina cyclotis from Guangxi province, China, was sequenced and analyzed. The genome is a circular molecule of 16,463 bp length, containing 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a control region. Most of the genes were encoded on the H-strand, except for 8 tRNA and ND6 genes. Phylogenetic trees of the complete mitochondrial genome were constructed using RAxML. Our result suggests that M. cyclotis is closely related to M. leucogaster from Korea. The complete mitochondrial genome sequence of M. cyclotis will be helpful for future taxonomic and phylogenetic studies on Murina.In this study, the complete mitochondrial genome of a male individual of  Murina is high in Asian region, with 39 recognized species. The current number of species listed is twice the number of species listed until 2005  do not support a close relationship between them (Eger and Lim The species diversity in genus M. cyclotis (GZHU 16335) collected from Diding country, Guangxi Province (23°06′51.6″N 105°58′12.6″E), China, was sequenced. Complete genomic DNA was extracted from liver tissue using MiniBEST Universal Genomic DNA Extraction Kit  and was sequenced using Illumina Hiseq X.In this study, complete mitochondrial genome (MK747248) of a male individual Murina mitochondrial genomes  and T–– , which may be modified by the polyadenylation after transcription  do not represent phylogenetic assemblages. Given the fact that a multi-lineages and cryptic species were observed within M. cyclotis using DNA barcoding technique (Francis and Eger M. cyclotis is provided, and it can benefit future phylogenetic and evolutionary studies on this genus and taxon.The phylogenetic relationships of lionidae . Such pa


Influenza virus-associated encephalopathy (IAE) can lead to neurological sequela and mortality among children. Therefore, instant recognition and therapeutic intervention for IAE are crucial. In some clinical subtypes of IAE, steroid pulse therapy might be beneficial, especially when it is administered in the early phase. However, early identification of patients who may benefit from steroid pulse therapy is sometimes difficult. We aimed to assess the effectiveness of early steroid pulse therapy among children with IAE.In this retrospective observational study, we used a national database that covers half of the acute care inpatients across Japan to identify inpatients aged ≤ 18 years with a diagnosis of IAE between July 2010 and March 2017. Unfavorable outcome was defined as a composite outcome of sequela including Japan Coma Scale ≥ 10 at discharge, requiring tracheostomy, mechanical ventilation, enteral tube feeding, rehabilitation at discharge, or in-hospital death. Propensity score matching was performed to compare unfavorable outcome and in-hospital mortality between patients with and without steroid pulse therapy within 2 days of admission.P = 0.16) or in-hospital mortality .Among 692 patients included in the study, the mean age was 5.8 years, and 55.8% were male. The overall in-hospital mortality was 1.3%, and the proportion of the unfavorable outcome was 15.0%. We observed no significant difference in the unfavorable outcome between matched patients (168 patients in each group) with and without early steroid pulse therapy (13.7% vs 8.3%; We did not observe the effectiveness of early steroid pulse therapy on patient outcomes among children with IAE in our study population including all clinical subtypes of IAE. Further studies considering severity of illness are warranted to determine whether steroid pulse therapy is beneficial, especially for specific clinical subtypes of IAE. Acute encephalopathy, one of the most serious complications among children with influenza virus infection, can lead to neurological sequela and mortality . NeuroloIAE has been reported worldwide –7, but mSome previous studies reported that only “early” steroid pulse therapy could be beneficial among children with IAE , 17, whiIn this study, we aimed to assess the effectiveness of early steroid pulse therapy on patient outcomes among children with all subtypes of IAE using a nationwide database in Japan.This was a retrospective observational study using a nationwide database.All data were retrospectively abstracted from the Diagnosis Procedure Combination database in Japan. This database is a national inpatient database that includes administrative claims and discharge abstract data from more than 1000 participating acute care hospitals across Japan. All academic hospitals are obliged to contribute to the database, and community hospitals participate voluntarily. Approximately 50% of all acute-care inpatient data of all ages in Japan are included in the database. The details of this database were described in a previous study .Patients aged ≤ 18 years who were admitted to hospitals with a diagnosis of IAE and discharged between July 2010 and March 2017 were included in the study. Diagnosis of IAE was identified according to documented diagnosis of influenza virus infection and encephalopathy during the same hospitalization. Diagnosis of influenza virus infection was determined according to the International Classification of Diseases 10th Revision (ICD-10) codes of J09 (influenza due to identified zoonotic or pandemic influenza virus), J10 , and J11 , as well as a manually documented diagnosis of influenza virus infection. Diagnosis of encephalopathy was determined according to diagnostic codes including virus-associated encephalopathy (ICD-10 code: G948), acute encephalopathy (G934), and delirium due to other medical condition (F058). When a patient was admitted twice or more to the same hospital with a diagnosis of IAE during the study period, only the first admission with a diagnosis of IAE was included.We excluded the following patients: (1) those transferred from other hospitals because their therapeutic interventions at the initial hospital were unknown, (2) those transferred to other hospitals within 7 days of admission because their outcomes were difficult to determine, (3) those who died within 2 days of admission to avoid immortal time bias, and (4) those aged < 1 month at admission to avoid including unproved congenital diseases such as metabolic disorders.Unfavorable outcome was defined as a composite outcome of neurologic sequela and death during the hospital stay including (1) the Japan Coma Scale (JCS) ≥ 10 at discharge, (2) requiring tracheostomy during hospital stay, (3) requiring mechanical ventilation at discharge, (4) requiring enteral tube feeding at discharge, (5) requiring rehabilitation at discharge, and (6) in-hospital death. These unfavorable outcomes did not include those that already existed before admission with IAE.JCS comprises four categories: alert, 1-digit (awake without stimulation), 2-digit (awake with stimulation), and 3-digit codes (non-awake with stimulation) . Each caAnti-influenza agents included peramivir hydrate, oseltamivir phosphate, laninamivir octanoate hydrate, and zanamivir hydrate. Intravenous antiepileptic drugs included thiopental, thiamylal, propofol, midazolam, diazepam, fosphenytoin, phenytoin, phenobarbital, and levetiracetam. Vasoactive agents included adrenaline, noradrenaline, dopamine, and dobutamine. Steroid pulse therapy was generally administered for 3 consecutive days and comprised 30 mg/kg bodyweight of intravenous methylprednisolone per day . We defiWe collected patient demographic data such as age, sex, body weight at admission, and data on the hospitals they were admitted to. Congenital anomaly was detected according to the ICD-10 codes Q00–99, and epilepsy was detected according to the ICD-10 codes G40 and G41. Epilepsy did not include patients diagnosed after the incidence of IAE. We collected the JCS at admission and at discharge, tracheostomy, mechanical ventilation, enteral tube feeding, and rehabilitation during the hospital stay. Patients with missing data were excluded. We also collected the in-hospital mortality rate of patients, date of admission and discharge, and therapeutic interventions described above.t test. Propensity score matching was performed with 1:1 nearest-neighbor matching without replacement using a caliper width of 0.2 of the pooled standard deviation of the logit of the propensity scores between patients with and without steroid pulse therapy [P values were < 0.05. All analyses were conducted using Stata version 15.0 .Categorical variables were evaluated using Fisher’s exact test. Continuous variables were evaluated using the Student’s  therapy . The fol therapy . We thenThis study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline  Supplem.During the study period, 821 patients aged ≤ 18 years were admitted to hospital with a diagnosis of IAE and discharged. Among these, 106 patients were transferred from other hospitals, 22 patients were transferred to another hospital within 7 days of admission, and 3 patients died within 2 days of admission. Two patients were transferred from other hospitals and were transferred to other hospitals. There was no neonatal patient aged < 1 month. Consequently, 692 patients were included in the analysis , 5.5–6.1), 386 (55.8%) patients were male, 8 (1.2%) patients had a congenital anomaly, and 115 (16.6%) patients had preexisting epilepsy at admission. These 692 patients were cared for in 261 hospitals across Japan. The mean length of stay was 13.6 days . The overall in-hospital mortality was 15 out of 821 (1.8%) among all children with IAE identified in the database and 9 out of 692 (1.3%) among patients included in the analysis.Table P < 0.001). There were no missing data in the database regarding the outcome variables included in Table Among 692 patients included in the analysis, the number of patients with each unfavorable outcome is shown in Table P = 0.16) or in-hospital mortality .Table N = 649), the proportions of the composite unfavorable outcomes were 26 out of 166 (15.7%) and 17 out of 166 (10.2%) in patients with and without early steroid pulse therapy, respectively (P = 0.19). In this sensitivity analysis, in-hospital mortalities were 0 out of 166 (0%) and 2 out of 166 (1.2%) in patients with and without early steroid pulse therapy, respectively (P = 0.50).When performing a post hoc sensitivity analysis by propensity score matching for patients with an ICD-10 code of G948  or G934  , and 55.8% were male, similar to previous studies . AdditioAmong IAE as a whole, in-hospital mortality was as high as 31.8% in a questionnaire study from 1998 to 1999 ; thereafIAE includes variety of clinical manifestations such as AESD, ANE, and MERS , 8. In pN = 17; influenza related, N = 5), and observed a lower incidence of unfavorable outcomes among patients who received early steroids: 5 out of 12 patients who received early steroids (within 24 h) had unfavorable outcomes, and 5 out of 5 patients who received steroids after 24 h or who did not receive steroids had unfavorable outcomes (P = 0.04) [In our study population including all clinical subtypes of IAE, after adjusting for the factors in Table  = 0.04) , 17. TheThe present study had some limitations. First, this was a retrospective study, and all the data relied on past records in the database. Additionally, we only assessed the Japanese database; therefore, the generalizability of the results is limited. Second, we excluded patients who were transferred from and to other hospitals. Third, data on laboratory results such as higher creatinine , 19, lacIn our retrospective observational study, we did not observe a significant difference in the proportions of unfavorable outcomes or in-hospital mortality between the patients with and without early steroid pulse therapy within 2 days of admission among children with IAE as a whole. However, further studies considering severity of illness are warranted to determine whether steroid pulse therapy is beneficial especially for specific clinical subtypes of IAE.Additional file 1.


TM and the Quality of Life Pediatric Inventory 4.0-PedsQLTM. Lower scores indicated worse symptomatology or QoL. Thirty-seven CF children were included. A FC level above 250 µg/g was associated with worse gastrointestinal symptoms and QoL scores. The FC level was inversely correlated with several gastrointestinal scores assessed by children . Several QoL scores were correlated with gastrointestinal scores. The FC level was weakly associated with clinical parameters. Some gastrointestinal and QoL scores were related to disease severity associated parameters. In CF, the FC level, biomarker previously related to intestinal inflammation and dysbiosis, was associated with worse digestive symptoms and QoL scores.In cystic fibrosis (CF), cystic fibrosis transmembrane regulator (CFTR) dysfunction leads to digestive disorders that promote intestinal inflammation and dysbiosis enhancing gastrointestinal symptoms. In pancreatic insufficiency CF patients, both intestinal inflammation and dysbiosis, are associated with an increase in the fecal calprotectin (FC) level. However, associations between the FC level, gastrointestinal symptoms, and quality of life (QoL) remain poorly studied. We aimed to assess such associations in pancreatic insufficiency CF children. The FC level was measured in pancreatic insufficiency CF children’s stool samples. Children and their parents completed two questionnaires: The Gastrointestinal Symptoms Scales 3.0-PedsQL Cystic fibrosis (CF) is an autosomal recessive disorder leading to a dysfunctional cystic fibrosis transmembrane regulator (CFTR) affecting various organs ,2. In reIn the digestive tract, CFTR dysfunction leads to a dehydration and acidification of intestinal mucus, impaired integrity of the intestinal barrier and to a decrease in transit time, innate immunity and activity of digestive and antibacterial enzymes ,2,3. AllIn IBD, an increased FC level is associated with worse gastrointestinal (GI) symptomatology and quality of life (QoL) . By contThus, we aimed to assess the association between the FC level and GI symptoms and QoL scores determined using validated questionnaires. The secondary objectives were (i) to compare GI symptoms and QoL scores according several clinical parameters; (ii) to assess the association between the FC level and clinical parameters and (iii) to assess the association between GI symptoms and QoL scores in CF.This observational, prospective, and bi-centric pilot study involved CF children between 4 to 18 years of age, having exocrine pancreatic insufficiency  followed-up from November 2015 to May 2016 at CF reference centers of Bordeaux and Toulouse, France. Patients previously diagnosed with IBD or celiac disease; patients with diagnosis of an ongoing acute infection; patients previously treated with probiotics or CFTR modulator were excluded. The study was approved by the regional ethical committee “CPP Sud-Ouest et Outremer III” (DC 2015/129). Informed consent was obtained from study participants and their parents.Pseudomonas aeruginosa and Staphylococcus aureus), cystic fibrosis-related diabetes (CFRD) and treatments (pancreatic enzymes replacement therapy (PERT), ursodeoxycholic acid, inhaled antibiotics (ATB), and oral ATB prophylaxis, IV ATB). We also assessed clinical data  expressed as Z-scores) and the last lung function testing results , forced vital capacity (ppFVC), forced mid-expiratory flow between 25% and 75% of forced vital capacity (ppFEF25–75), lung total capacity (ppLTC), residual volume (ppRV), residual volume capacity (ppRCF), and resistance of airways (ppRaw)) measured within the past month before inclusion and obtained from patient’s medical file. The FC level was determined using ELISA fCAL® kit  on children stool sample collected the day of inclusion. Since we previously demonstrated that CF children with FC level 250 µg/g of stool were associated with an increased dysbiosis [At inclusion visit, we assessed patients’ CFTR mutation, past history , number of hospitalizations, number of exacerbations and number of intravenous (IV) antibiotic cures within the year before inclusion), the presence of chronic colonization , “Stomach Discomfort When Eating” (5 items), “Food and Drink Limits” (6 items), “Trouble Swallowing” (3 items), “Heart Burn and Reflux” (4 items), “Nausea and Vomiting” (4 items), “Gas and Bloating” (7 items), “Constipation” (14 items), “Blood in Poop” (2 items), and “Diarrhea” (7 items)) [TM was composed of 23 items grouped into 4 dimensional scales , “Emotional Functioning” (5 items), “Social Functioning” (5 items), and “School Functioning” (5 items)) [At inclusion visit, CF children, using self-assessments, and their parents, using heterogeneous assessment, also completed a French version of both the Gastrointestinal Symptoms Scales 3.0-PedsQLTM  and the TM [TM . The Gas items)) . The Qua items)) . Each it items)) ,25. Scor items)) ,25.p value under 0.05 was considered significant. Two-way mixed absolute single measure intraclass correlations were computed to express test-retest reliability between children and parents. Values indicate of poor (<0.5), moderate (0.5–0.75), good (0.75–0.9), or excellent (>0.9) reliability [We used Graphpad Prism 5  for the analyses. Results were expressed as median with 95% confidence interval (95% CI) or with minimum and maximum  for continuous variables or in absolute values and percentage (n/N (%)) for categorical variables. Comparison between groups was performed using the Mann–Whitney test for unpaired nonparametric variables. Scores obtained from children and those obtained from their parents were compared using the Wilcoxon’s test. The Fisher’s exact test was used for categorical variables. Correlation coefficients were assessed using univariate Spearman’s test. A iability .Staphylococcus aureus with a preserved lung function despite a decreased FEF25–75 and without severe event in past history or severe comorbidities  were not significantly different between the two groups . Among the 37 CF children included, 5 were assigned to the High FC group ) and 32 were assigned to the Low FC group ). Characteristics of the two groups were presented in o groups .TM and the Quality of Life Pediatric Inventory 4.0-PedsQLTM, scores obtained from children and their parents were correlated  or from their parents   . Howeverchildren .Pseudomonas aeruginosa or Staphylococcus aureus, hospitalization for exacerbation or recurrent IV ATB treatment with a year), low lung function or treatment   . To notehylaxis) .There was no significant correlation between FC and clinical parameters  or lung function testing results . There wPseudomonas aeruginosa were associated with lower results in few Gastrointestinal Symptoms Scales 3.0-PedsQLTM scores    or in QuM scores . No otheM scores .Correlations between GI symptoms and QoL scores are presented in Taken together, these results demonstrated for the first time that, in CF children with pancreatic insufficiency, (1) the FC level was associated with GI symptom scores, (2) a high FC level was associated to a worse QoL score and (3) that QoL scores were correlated with digestive symptoms. We also demonstrated that clinical parameters, lung function results, or treatment were moderately associated with QoL scores, poorly with gastrointestinal scores and not associated with the FC level, in CF children.Pseudomonas aeruginosa chronic colonization) were associated with significant differences in GI symptoms and linked to the disease natural evolution and severity, except for gender.In CF, GI disorders can be related to pancreatic insufficiency, liver injury, dehydration, and acidification of the intestinal mucus, inappropriate lower esophageal sphincter relaxation, dysbiosis, intestinal inflammation leading to various digestive symptoms . Thus, GPseudomonas aeruginosa, recurrent ATB (prophylaxis or regular intravenous treatment), or ursodeoxycholic acid treatment that indicated hepatic injury) and worse quality of life. By contrast with the study of Gee et al. including CF children and adults [Improvement of QoL is a major objective in CF and QoL assessment represents a widely accepted clinical endpoint in Clinical trials ,32. Thusd adults , we demoAs previously described for abdominal pain in CF , we highOur study had several limitations. Firstly, the number of patients included in the study was low which limits the number of patient with a FC level above 250 µg/g of stool. However the number of patients included is comparable with most other studies investigating fecal calprotectin in CF children ,11,16,41Pseudomonas aeruginosa chronic colonization were overrepresented despite a statistical significance with the low FC group and Z-score Weight et Z-score were lower. Since these elements may have introduce bias in the analyses, we demonstrated that most of scores which have differences according clinical outcomes were different compared to those having differences according FC Level. Moreover, our results were supported by a correlation analysis performed with the entire population. This threshold was chosen because we previously demonstrated that patients with a FC level above 250 µg/g were associated with microbiota disturbance close to that observed in IBD [Secondly the choice of an elevated FC level limited the number of participants in the High FC group and possibly generated a type I statistical error. However, the number of CF children in the High FC group was limited by a lower prevalence of patients with a FC level above 250 µg/g than described in the literature ,42. It id in IBD . In addid in IBD  and it cd in IBD ,44,45. TThirdly, the FC level can be affected by several parameters such as treatments. Indeed, the FC level is decreased by probiotics ,5,14,18,In cystic fibrosis, abdominal symptoms are common and need more attention from clinicians because often overshadowed by respiratory symptoms. These abdominal symptoms may be related to various digestive organs disorder but can be the sign of dysbiosis or intestinal inflammation. Our results show that in CF children, an increase in fecal calprotectin, a biomarker known to be related to dysbiosis and intestinal inflammation, is associated with worse GI symptoms and impaired QoL. Measurement of the FC level could help the clinician to better discriminate the origin  of gastrointestinal manifestation and impaired quality of life in CF, and thus allow to optimize or adapt the treatment.


To explore the mechanism of calcium-sensing receptors (CaSRs) during the development of nephrolithiasis.3, an agonist of CaSR) and NPS 2390 (an antagonist of CaSR) were added. Oxidative stress (OS) and calcium oxalate crystals in the kidney were observed. CaSR expression and the expression of extracellular signal-regulated protein kinase (ERK), OPN, and KIM-1 were determined by western blotting. In addition, renal tubular epithelial cells were isolated from the kidney to observe phosphatidylserine (PS) ectropion using flow cytometric analysis. Various biochemical parameters were assessed in serum and urine at the end of the experiment.Wistar rats were treated with ethylene glycol to induce calcium oxalate crystallization, and gadolinium chloride . Compared with calcium oxalate treatment alone, the above deleterious changes were further significantly confirmed by GdCl3 but were reversed by NPS-2390. However, urine calcium excretion was decreased after ethylene glycol treatment but was significantly reduced by NPS 2390 and increased by GdCl3 (p < .05).Calcium oxalate increased OS, crystal adhesion, PS ectropion, and the expression of CaSR and ERK, OPN, and KIM-1 The results suggest that CaSR might play significant roles in the induction of nephrolithiasis in rats by regulating reactive oxygen species (ROS) and PS ectropion and the composition of urine, OPN, KIM-1, and ERK expression. Numerous studies have confirmed that the incidence and prevalence of nephrolithiasis are increasing remarkably . As a co2+, trivalent cations, antibiotics, amino acids, and other external stimuli, such as ionic strength and pH [CaSR is a candidate gene to explain the individual predisposition to calcium stones [CaSR, a dimeric G protein-coupled receptor, plays an important role in calcium homeostasis. The CaSR regulates parathyroid hormone release and calcium reabsorption in the parathyroid glands and kidneys at the highest levels ,7. CaSR h and pH . The polh and pH . Studiesm stones . A publim stones . In our m stones .Growing studies have indicated that calcium oxalate crystal deposition in the kidneys is associated with renal epithelial injury since Randall emphasized the importance of renal damage in stone formation . Many stPhosphatidylcholines, choline phospholipids, and sphingomyelins are concentrated in the exoplasmic (outer) leaflet, while aminophospholipids, PSs, and most phosphatidylethanolamines are predominantly located on the cytoplasmic (inner) leaflet . Membran2O2 in renal calculus rats [One study showed that oxalic acid-induced ROS production could lead to PS externalization on the renal epithelial cell membrane, while negatively charged PS exposed to the cell surface might act as anion molecules mediating crystal adhesion . Treatmelus rats . In addilus rats ,20. In mlus rats .The present study was designed to examine the possible effects of CaSR in a calcium oxalate nephrolithic model and to provide direct pharmacological evidence to determine whether CaSR is involved in this process.3 (product number 43977-0) was obtained from Sigma-Aldrich . Anti-OPN (ab36125), anti-phospho-ERK (ab21396), and rabbit polyclonal antibodies against KIM-1 were obtained from Abcam . Antibodies against the calcium-sensing receptor  and glyceraldehyde 3-phosphate dehydrogenase  were purchased from Santa Cruz Biotechnology, Inc. . Fluorescein isothiocyanate (FITC)-labeled annexin V (annexin V-FITC) was obtained from BD Pharmingen . Ethylenediaminetetraacetic acid (EDTA) was obtained from Sigma-Aldrich Co., Ltd. . An ECL chemiluminescence kit (WBKLS0500) was obtained from Millipore Corporation .Quinoxaline-2-carboxylic acid adamantan-1-ylamide  was obtained from Tocris Bioscience , GdCl3 was administered daily by injection for 4 weeks. Group D: animals were exposed to 1% EG in their drinking water, and a 0.20 g/kg dose of NPS-2390 was administered daily by injection for 4 weeks [All of the rat experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of Lanzhou University. Male Wistar rats, aged 6–8 weeks, were purchased from Lanzhou University Medical Laboratory Animal Center . Rats were housed in polypropylene cages and had access to food and water. The rats were divided into four equal groups. Group A: the intact control group was given tap water as their drinking water and fed a standard diet. Group B: the rats were exposed to 1% EG in their drinking water for 4 weeks to induce nephrolithiasis. Group C: animals were exposed to 1% EG in their drinking water, and an 8.67 mg/kg dose of GdCl 4 weeks .On day 28, the rats were placed in metabolic cages for 24-h urine sample collection before sacrifice. The urine sample was either stored at −80 °C or analyzed immediately. Urinary pH, calcium, uric acid, and magnesium levels were determined by an automated analyzer . Oxalate in urine samples was analyzed by direct precipitation followed by titration. A Citric Acid Enzyme Bioanalysis Kit  was used to measure citrate levels.Approximately, 3 mL of blood samples was collected from the jugular vein into heparinized syringes and was immediately placed on ice. Whole samples were transferred to serum separator tubes and centrifuged at 3000 rpm for 10 min to isolate the plasma, which was then subjected to SOD, MDA, and renal function analysis .The animals were sacrificed under anesthesia; both kidneys were removed and washed with cold 0.9% NaCl. The left kidney was cut in half; one half was stored for tissue microbiological studies, primary culture and identification of Wistar rat renal tubular epithelial cells, and the other half was fixed in 10% formalin, embedded in paraffin, cut into 5-µm-thick sections, and stained for histopathology (hematoxylin and eosin). The right kidney was cut in half; one half was minced with scissors and then homogenized in 0.9% NaCl using a glass homogenizer. The homogenate was centrifuged at 2000 rpm for 10 min in a refrigerated centrifuge to remove cell debris. Tissue MDA and SOD levels were determined in the supernatant using commercially available kits . The other half of the kidney was immediately placed in liquid nitrogen and stored at −80 °C for western blotting analyses.As mentioned earlier, crystal deposition in the kidney was assessed using the following score: no deposition = 0; crystal deposition at the tip of the nipple = 1 point; crystal deposition at the cortical medullary junction = 2 points; and crystal deposition in the cortex = 3 points. If crystal deposition is observed at multiple sites, the points are combined to provide a total score for each pathological section. Pathological changes were semiquantified according to the damage area: 0–3, 0 = invisible lesions; 1 = tubulointerstitial inflammatory infiltration in the lesion area/20%, slight tubule dilatation; 2 = tubulointerstitial inflammatory infiltration in the lesion area/40%, tubules significantly expanded; 3 = tubulointerstitial inflammatory infiltration in the lesion area/40%, tubules severely dilated. For further observation of the crystals, transparent tissue portions of the crystals (without HE staining) were also observed under a laser-scanning confocal microscope .−1, 10−3, and 10−5) before the tissue homogenates were cultured on plates, and the bacterial count was performed after the dilution factor was corrected. Standard microbiology techniques were carried out. Urinary infection was defined as more than 105 colony-forming units per mL (cfu/mL) in urine. Renal tissue ≥105 cfu/g suggests pyelonephritis.On day 28, urine from separate metabolic cages was also assessed for microbial variation. Half of the left kidney was homogenized separately in sterile saline (5 mL). The homogenate was diluted . Seventy-two hours for the first fluid change, after every two-day fluid change.Identification of Wistar rat renal tubular epithelial cells: morphology: Inverted microscopy revealed that the cells were larger in volume, appeared as multilateral cobblestones, had strong transparency and refractive sex and were closely connected. Immunocytochemistry: After passage for one generation, renal tubular epithelial cells were seeded in coverslips in a 24-well cell culture plate, and the cover glass was removed when the cells were approximately full. Rinse twice in PBS, fix in 4% cold paraformaldehyde for 30 min. After two PBS rinses, add 3% H2O2 deionized water and incubate for 10 min to prevent peroxidase, cytokeratin 18 primary antibody (1:400) at 4 °C overnight (the negative control uses PBS instead of the primary antibody). The secondary antibody (ready-to-use) was incubated at 37 °C. Incubate for 1.5 h, then develop DAB color, rinse with tap water, hematoxylin counterstain, and remove water, transparent, coverslip. The results were observed under an ordinary light microscope. Detection of renal tubular epithelial cells by immunocytochemical staining: heterosexually expressed cytokeratin 18.2. Samples were analyzed though a FACS Calibur flow cytometer  equipped with an argon-ion laser emitting at 488 nm. The fluorescence channels and light scatters were set on a log scale. Data from 5000 events were collected and analyzed using Cell Quest Pro software.Annexin V-FITC was used as a marker for the detection of phosphatidylserine (PS). Negative controls for annexin V binding were stained with annexin V-FITC in the presence of 2.5 mM EDTA instead of 2.5 mM CaClg for 5 min) at 4 °C, supernatants were collected. The protein concentration was measured using a Bradford Protein Assay Kit . Then, samples were heated at 95 °C in 2.5% mercaptoethanol and Laemmli sample buffer , and a total of 25 µg of protein was loaded per lane onto 4–20% Mini-PROTEAN TGX Precast gels . Following separation at 130 V, the proteins were transferred onto trans-blot turbo transfer pack membranes . PVDF blocking reagent was used to block the membranes for 1 h at room temperature. After washing with Tris-buffered saline with Tween 20  three times, the membranes were incubated with antigen-specific antibodies at 4 °C overnight. Then, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Finally, the immunoreactive bands were visualized with enhanced chemiluminescence western blotting reagent.The kidney tissues were homogenized in 1 mL lysis buffer mixed with 10 μL phenylmethanesulfonylfluoride (100 mM) and NaF (100 mM) for 10 s on ice. After centrifugation . However, urinary calcium was significantly reduced in group D compared with group A and group B (p < .05).As shown in 3 administration in group C but was increased in group D compared with group B (p < .05). The urinary citrate concentration was also lower in group B than in group A, and after treatment with GdCl3 in group C, the urinary citrate concentration decreased significantly, but NPS2390 treatment group D showed a significant increase in the urinary citrate concentration. Group B had three times higher values of blood urea and two times higher values of serum creatinine than group A, and treatment with NPS2390 nearly reduced the two indices to normal concentrations. However, treatment with GdCl3 further impaired renal function compared to group B (p < .05).Hyperoxaluria was evident in group B on the 28th day and was subsequently reduced by GdClp < .05). There were fewer crystal deposits in group D than in group B (p < .05). HE staining of renal tissues from group A revealed regular histology with normal tubules with a single epithelial lining along the margin and did not reveal any crystal deposition . As show group B  . Furthermore, after treatment with NPS-2390, the remaining renal tubular epithelial cells in group B did not express much PS exposure, which was in clear contrast with group C.Phosphatidylserine exposure has been suggested as an important event in kidney stones, which frequently concurs with morphological alterations from cells into tissue. In p < .05 compared with group B), indicating that NPS-2390 could protect the urinary tract from bacterial infection. However, the UTI rates were 80% (eight of 10) in group C (p>.05 compared with group B). The incidence of pyelonephritis  in groups A, B, C, and D was consistent with the UTI rate.Damage to the kidney leads to crystal deposition, and bacterial attack might develop, leading to full crystal deposition in the kidney. As shown in p < .05)  . The evap < .05).However, when CaSR expression was decreased by NPS-2390, SOD activity was restored, and MDA and levels were reduced compared with group B (p < .05).To clarify the protein involved in nephrolithiasis formation in our rat model, western blot analysis was carried out on total protein extracted from rat kidneys. First, more than twofold changes in CaSR expression in group B compared with group A were detected . UpregulTo further validate the stone-related protein expression changes, a semiquantitative western blot of ERK, KIM-1, and OPN expression was carried out with the four groups of animals.p < .05) increased ERK, KIM-1, and OPN expression in group B compared to normal animals. Treatment with NPS2390 produced a significant (p < .05) reduction in ERK, KIM-1, and OPN levels compared to group B. However, the elevated levels of ERK, KIM-1, and OPN were significantly further increased by treatment with GdCl3 in group C (p < .05) (Ethylene glycol treatment significantly (p < .05) .2+ homeostasis, including parathyroid cells, thyroidal calcitonin-secreting C-cells [CaSR has been found to be expressed in all key tissues involved in extracellular Ca C-cells , kidney  C-cells , and int C-cells .There is growing evidence that CaSR may play a role in increasing the propensity for kidney stones. Our previous studies have shown that CaSR is present in HK-2 cells and rat kidneys, and it is worth noting that CaSR upregulation may increase intracellular MAPK signaling, which may lead to crystal retention and ultimately damage renal function . Studies3, agonist of CaSR) and NPS 2390 (antagonists of CaSR) to explore the effects of CaSR on nephrolithiasis in rats. The expression of CaSR was significantly increased after ethylene glycol treatment for 28 days and was further significantly increased when cotreatment with the CaSR agonist GdCl3 but was attenuated by NPS-2390. Except for many cations , polyamines, and some antibiotics can activate CaSR [In the present study, we used gadolinium chloride . This finding indicates that PS exposure on the renal cell membrane surface increased under the action of calcium oxalate crystals. GdCl3 further enhanced PS exposure, while NPS-2390 decreased it (p < .05), which suggests that CaSR helps to enhance PS exposure.Cao et al. found that high oxalate promoted PS redistribution and crystal adhesion increase in renal epithelial cells and supported the idea that oxalate toxicity may lead to nephrolithiasis by altering the properties of renal epithelial cell membranes . PS expoProteus mirabilis [E. coli, not P. mirabilis, is the most common bacterium found in the urine and stone matrix of stone patients [E. coli plays a pathogenic role in the formation of nephrolithiasis or whether it is confined to the stone matrix. Interestingly, previous studies have indicated that E. coli could promote the growth and aggregation of calcium oxalate crystals [Urinary tract infection is reported to be associated with renal calculi, especially struvite, which is the result of being infected by urea-splitting bacteria, such as irabilis ,32. Howepatients . Howevercrystals ,35, both3) and antagonist (NPS 2390) CaSR. We found increases in the levels of OPN, ERK, and KIM-1 after treatment with calcium oxalate, which is consistent with a previous study [3 but were reversed by NPS-2390. OPN is not only an effective target molecule for the treatment of inflammation [In this study, we evaluated the effects of CaSR on stone-related proteins in rats exposed to ethylene glycol using agonist (GdClus study . Our resammation  but alsoammation . Studiesammation . A studyammation  studied ammation . ERK medist NPS 20 CaSR. Wammation , and cel3 or attenuated by the CaSR inhibitor NPS-2390, suggesting that calcium oxalate mediates OS though CaSR. CaSR promote crystal adherence, but it indirectly promote crystal adherence. The upregulation of the CaSR happened first, subsequently excessive amounts of crystals could trigger the production of ROS though CaSR in kidney tissues. ROS production leads to renal epithelial injury, which provide sites for crystal adherence and eventual deposition within the kidney.It has been reported that OS caused by calcium oxalate is the initial trigger for the vicious cycle of nephrolithiasis ,42. In tFor stone formation, OS could lead to renal injury and dysfunction. These renal pathological changes are accompanied by crystal retention. Crystal nucleation, aggregation, and retention are promoted by lethal epithelial cellular injury. The crystal nucleation, aggregation, and retention, in turn, further damages the kidneys. Dysfunctional cells and sublethal injury may generate ineffective crystallization inhibitors and localized areas of supersaturation in the interstitium. In our study, compared with group B, increased calcium oxalate-induced OS in group C may lead to more crystal retention and dysfunction. However, decreased OS in group D could inhibit this process.3 compared with group B; one possible explanation for this finding is that CaSR increased free calcium, which binds to oxalate and thus reduces urinary oxalate. In addition, the main risk factor for recurrence of calcium oxalate stones is thought to be urinary citrate deficiency. Urinary citrate inhibits the formation of stones by inhibiting nucleation and growth [In addition, we found that rats with nephrolithiasis showed low urinary calcium and citrate and high urinary oxalate compared to control animals. Oxalate is a more important promoter of kidney stones and exhibits 15-fold greater efficacy on urinary calcium oxalate saturation than calcium . Notablyd growth . Our resThe findings of the present study may provide novel biomarkers for the diagnosis, prognosis, and clinical trials of calcium oxalate nephrolithiasis.The results suggest that CaSR might play significant roles in the induction of nephrolithiasis in rats through ROS production and regulation of PS ectropion, stone-related protein expression, and composition of urine in urine. These findings may serve as potential therapeutic and drug targets.


Rituximab is increasingly used as off-label therapy in multiple sclerosis (MS). More data are needed on safety and efficacy of rituximab, particularly in cohorts of de novo patients and patients in early therapy escalation.To investigate the safety and efficacy of off-label treatment with rituximab in an MS-cohort of predominantly de novo patients or as therapy escalation.We retrieved safety and efficacy data from the Norwegian MS-registry and biobank for all MS-patients treated with rituximab at Haukeland University Hospital, Bergen, Norway, during a four year period.In the 365 MS-patients (320 relapsing-remitting MS (RRMS), 23 secondary progressive MS (SPMS), and 22 primary progressive MS (PPMS)), the overall annualized relapse rate (ARR) was 0.03 and annualized drug discontinuation rate (ADDR) was 0.05. NEDA-3 was achived in 79% of patients with available data (n=351). Sixty-one patients experienced infusion-related adverse events of which two were serious (CTCAE grade 3–4). Eighteen patients experienced serious non-infusion related adverse events, of which 16 were infections. Infections , hypogammaglobulinemia  and neutropenia  were the most common non-infusion-related adverse events.Rituximab was a safe and highly efficient disease modifying therapy in this cohort of MS-patients; however, infections and neutropenia need to be monitored. At Haukeland University Hospital, Western Norway, rituximab has been the preferred therapeutic option for highly active MS in treatment naive patients since 2016, as well as an equivalent option for therapy switch or escalation in MS. Hence, rituximab has become the most commonly used MS-immunomodulatory therapy at our hospital. Based on the experience from this treatment approach, we conducted a retrospective cohort study to describe safety and efficacy of rituximab therapy in MS patients at Haukeland University Hospital during 2015–2019.This study was a single-centre, retrospective cohort study of all MS-patients who initiated rituximab therapy from 01.01.15 until 01.01.19 and consented to registration in the National MS registry at the Department of Neurology, Haukeland University Hospital, Bergen, Norway. Patients were identified through the Norwegian MS registry and cross-checked with the Department's list of patients treated with rituximab. From 2016 rituximab became the drug of choice for highly active disease both in therapy escalation and in treatment naïve patients, i.e. patients with two or more of the following characteristics: young age, severe function loss or extensive MRI-lesions. Observation time was set from the date of treatment initiation (at latest 01.01.19) until 01.07.19 if rituximab was not discontinued. If patients discontinued rituximab, observation time was set until six months after the latest rituximab infusion, or the date of initiation of a new disease modifying therapy (DMT).Data were retrieved from the Norwegian MS Registry and biobank and included age, sex, date of disease onset, number of relapses in total and during the last two years prior to rituximab therapy, previous treatment, and reason for initiation or switch to rituximab. Efficacy recording included date of any relapse or new magnetic resonance imaging (MRI) T1 gadolinium henhancing (T1Gd+) lesion, or new T2 lesion, and change in Kurtzke Expanded Disability Status Scale (EDSS) from baseline to latest observation, at least six months from rituximab initiation. Safety recording included date and reason for rituximab discontinuation, and adverse events.The majority of patients received a single infusion of rituximab 1000 mg intravenously (i.v.) at initiation, followed by 500 mg i.v. every 6 months. Patients who started rituximab treatment between 01.01.2015-31.12.2016 normally received a dose of total 2000 mg i.v. at initiation  followed by 500 mg i.v. every six months .MRI scans were performed on 1.5 T or 3 T scanners. The most recent MRI before rituximab initiation was defined as the baseline MRI scan. A re-baseline scan was planned 6 months after rituximab treatment initiation, followed by a new MRI scan at 12 months, and further every 12 months, or if clinically indicated. Brain MRI scan was performed routinely, and a spinal MRI scan was performed if clinically indicated. An EDSS was planned as part of clinical evaluation in the outpatient clinic at latest six months after rituximab initiation, and at regular follow-up every 6–12 months. Laboratory tests including hemoglobin (Hb), white blood cells with differential count, platelet count, ESR (erythrocyte sedimentation rate), CRP (C-reactive protein), liver function parameters (GGT (Gamma-Glutamyl Transferase), ALT , ALP ), kidney function (creatinine), IgG and IgM (immunoglobuline G and M) were performed every 6 months.Efficacy outcomes included (i) annual relapse rate, (ii) number of patients experiencing new MRI-disease activity during observation, defined as new T2 or T1Gd+ lesions, and (iii) disability progression, measured as change in EDSS (0.5 points or more), and (iv) proportion of patients with no evidence of disease activity (NEDA-3). NEDA-3 was defined as a composite score comprising absence of clinical relapses and disability progression, in addition to no new MRI disease activity (new T1Gd+ or new/enlarging T2-lesions) on MRI examinations for the given period. Treatment outcomes were evaluated for the period before the re-baseline MRI (i.e. the first six months of therapy) and after the re-baseline MRI (i.e. >six months of therapy). Safety outcomes included all reported adverse events, including infusion-related adverse events, except mild infections (Common Terminology Criteria for adverse events (CTCAE) grade 1), which were not included due to suspected incomplete registration in the registry and patient records.Survival  was estimated with Kaplan–Meier survival curves. Analyses were performed in SPSS version 24 . Descriptive data were described as means, medians and percentages.All data were retrieved from the Norwegian MS Registry and biobank, where participants have given a prospective informed consent. The study was approved of by Regional Committee for Medical Research Ethics, Western Norway (REK no 87388).A total of 365 MS-patients  who initiated disease modifying treatment in Haukeland University Hospital, Department of neurology with rituximab during the predefined study period, and consented to registration in the Norwegian MS registry, were included. One patient  was identified through the Department's list of patients but had refused consent to the registry and was not included. About one third of the patients  were treatment naïve, either newly diagnosed ) or never received DMT . The rest were switched from different interferon beta preparations , glatiramer acetate , teriflunomide , dimethyl fumarate , natalizumab , alemtuzumab , fingolimod , other  or haematopoetic stem cell transplantation (HSCT) , or did at the moment not receive DMT . Treatment failure was the most common reason for swithching from another DMT .In patients receiving no treatment (n = 42), disease progression was the most common reason for initiating rituximab .Relapse rate, frequency of new MRI lesions, EDSS change and drug discontinuation rate were calculated and are all displayed in Observation time for patients in the study was <12 months in 74 patients, and ≥12 months in 291 patients. In 133 patients, observation time was ≥24 months.The overall annualized relapse rate (ARR) in this cohort was 0.03 . During the first six months of treatment, fourteen patients experienced a relapse of which two had a further relapse. After six months of treatment or more, seven patients experienced a relapse and two had a further relapse. When excluding observation time and early relapses <6 months of treatment, with the intention of looking at treatment effect after 6 months, ARR was calculated to 0.02. In two patients, repeated relapses or a relapse after six months of treatment was the reason for drug discontinuation. In patients switching to rituximab after HSCT (n=2), new relapses occurred in one.ARR for the subgroup receiving an initial dosage of 2000 mg (1000 mg + 1000 mg) was 0.05 (n = 44), and 0.02 for an initial dosage of 1000 mg (n = 321).ARR in the subgroup of newly diagnosed patients was 0.02 (n = 104), and 0.03 in other patients (n = 219) during study observation time.One or more control MRIs during rituximab treatment were available for 358 patients (98%). New or enhanced MRI-lesions occurred in 58 patients . When excluding MRI events before six months of treatment, 17 patients experienced new or enhanced MRI lesions. In nine of these, no early MRI scan (<6 months) after rituximab initiation was available.Two patients discontinued rituximab due to treatment failure; both experienced relapses and more than one MRI event. EDSS was available at baseline and after at least 6 months of treatment for 352 patients (96%). In 316 (89.8%) patients EDSS was unchanged between their first and last available score. In RRMS patients, EDSS improved in 18 (5.7%), and worsened in ten (3.2%) patients. In the progressive disease subgroup, EDSS improved in one (3.4%) and worsened in seven (16.7%) patients. In progressive disease courses, observation time was less than a year in 13 patients, 1-2 years in 32 patients, and ≥ 2 years in 13 patients. NEDA- 3 was available for calculation in 351 patients. 279 of 351 (79%) patients with available data fulfilled the criteria for NEDA-3  throughout their observation time in this study.Treatment was discontinued in 33 (9%) patients, resulting in an annual drug discontinuation rate (ADDR) of 0.05 . The most frequent reason for discontinuation was adverse events . In two patients (0.5%), discontinuation was due to treatment failure . Six patients (1.1%) requested rituximab discontinuation in order to receive HSCT abroad. Safety data results are displayed in Infusion-related adverse events (CTCAE grade 1-5) were registered in 61 patients (16.7%). All were mild (CTCAE grade 1-2) except 2 cases (CTCAE grade 3): One patient reported a rash and a unilateral, mild facial paresis 30-60 min after infusion, which resolved spontaneousely within hospital admission the next 60 minutes. One patient experienced an acute generalized rash and dyspnoea during infusion, which resolved after peroral antihistamine. Both were admitted to hospital. Rituximab was continued with no further serious infusion-related adverse events.Among non-infusion-related adverse events, 18 (5.0%) were serious (CTCAE grade 3–4). The majority (n = 16) were infections; Urosepsis (n = 3), diverticulitis (n = 2), neutropenic fever (n = 2), pneumonia (n = 3), pansinusitis (n = 1), cholecystitis (n = 1), spondylodiscitis (n = 1), neoerlichiosis (tick-borne infection) (n = 1), enteritis (n = 1) and proctitis (n = 1). The rate of infections, excluding mild cases not requiring any medical treatment or doctor's visit (CTCAE grade 1), was 17.4% in SPMS, 8.7% in RRMS and 9.1% in PPMS patients.One patient died during the study observation time; cause of death was suicide, considered not to be related to rituximab treatment. Two patients were diagnosed with malignant disease  during observation time, both were diagnosed after rituximab initiation, and both discontinued rituximab treatment. No case of progressive multifocal leukoencephalopathy (PML) was recorded.9/L) during rituximab treatment with no other known cause was recorded in 16 (4.3%) patients. 6 patients experienced moderate or severe neutropenia  and expressed the following characteristics: All six patients had neutrophil count and leucocyte counts within normal range at baseline (within 2 weeks before rituximab initiation). One had experienced neutropenia earlier, which resolved. Five out of six were < 40 years of age. 1 patient had PPMS and EDSS > 6, the remaining had RRMS with EDSS 0-2. None had relevant comorbidities. Earlier treatment included HSCT (n = 1) and dimetylfumarat (n = 2), the remaining were treatment naïve (n = 3). Total number of rituximab infusions were 2–4; 1 patient had received an initial dose of 2000 mg rituximab. Two neutropene patients were admitted to hospital with febrile neutropenia, both were treated with i.v. antibiotics. One was admitted to hospital with a suspected fungal infection and received treatment with G-CSF (Granulocyte Colony Stimulating Factor). Three patients had no symptoms and neutrophile count normalized within 1-4 weeks. All patients recovered fully, but rituximab was discontinued in two cases due to recurrent neutropenia and high age, respectively.Neutropenia (count < 1.5 × 10In patients with neutropenia (n = 16), 5 patients were registered with any infection as an adverse event. In patients without neutropenina (n=349), 31 patients were registered with an infection.In patients receiving an initial dose of rituximab of 2000 mg, 3/44 (6.8%) were registered with any infection (CTCAE grade 2–5) versus 31/321 (9.7%) in patients receiving regular dose (1000 mg) at rituximab initiation, and 2/44 (4.5%) were registered with neutropenia versus 14/321 (4.4%) in patiens receiving regular dose at initiation. 6/44 (13.6%) patients who received 2000 mg rituximab at initiation were registered with hypogammaglobulinemia (low IgG) at any time during observation, compared to 13/321 (4.0%) in the group who received a regular dose at initiation. We present one of the largest cohorts of MS-patients treated with rituximab to date, utilizing real world data from the Norwegian MS Registry.12 including a larger cohort study of similar design and study population.10 Another large, recent multicentric observational study reported similar results; one difference to notice is the higher proportion of treatment naive patients in our cohort, pointing to the differences in therapeutic approach and patient selection in reported off-label MS-treatment with rituximab. Our cohort includes a relatively high proportion (28.5%) of newly diagnosed patients starting rituximab as their first choice DMT compared to many studies based on real-world data. Efficacy data are in line with results from several studies of similar designThe overall ARR of 0.03 was low, and so was the ARR of 0.02 calculated in >6 months of treatment, when assuming a transient initial periode of suboptimal treatment effect. The calculated ARR for patients receiving a higher starting dose of rituximab was 0.05; this subgroup of patients also started treatment earlier (2015-2016) while rituximab was not concidered a first choice treatment, and could possibly represent a subgroup with more persistent disease activity in the cohort. The total number of patients with new MRI lesions was lowafter initiation of rituximab, and after >6 months of treatment. A large proportion of patients starting rituximab had a recent history of disease activity , which strengthens the efficacy results and indicate low inflammatory disease activity during rituximab treatment, further underlined by the low proportion of patients discontinuing treatment, and a very low proportion of patients discontinuing rituximab due to new disease activity. EDSS was mainly unchanged during rituximab treatment. Notably, the baseline EDSS score might have been transiently worsened in some patients at rituximab initiation due to recent relapses, which could lead to overestimation of improvement in EDSS during treatment. EDSS was recorded during regular clinical follow up and a validation of change in EDSS score was not demanded in the study design. 14 For progressive disease courses, the observation time was relatively short, and the number of patients (n = 45) was low, which limits further conclusions.We recorded a clinically significant number of patients in progressive disease courses registered with EDSS progression during rituximab treatment . This is in line with previous studies reporting that disability progression may be reduced, but not completely stopped, during rituximab or ocrelizumab treatment in progressive MS disease courses.16 Among serious adverse events, infections was the most common, corroborating a recent report from a Swedish MS-population.17 Moderate and severe neutropenia (count <1.0 x 10 9) was seen in 1.6% of patients in this cohort and may represent a serious risk to the patient's health. As neutrophil counts were registered only at baseline and every 6 months, we could not estimate the onset time of neutropenia or the full extent. Late onset neutropenia is a well-known side effect of rituximab treatment18 but has only been documented in few MS-patients treated with rituximab in retrospective studies.20 Our results indicate that neutropenia may be underreported if not monitored. One of the main goals was to evaluate safety of rituximab treatment in MS. The rates of adverse events and the main categories of side effects in this cohort were comparable to those seen in populations of rheumatological and other autoimmune diseases.The present study has several strengths. As a single-centre study, the treatment- and observation protocol was standardized, and all safety data and clinical records were fully available. Thus, serious adverse events should be well monitored and recorded, including laboratory monitoring and all hospitalizations, though recording of mild adverse events offer challenges. The dosing regimen in this cohort was largely homogenous, only a few patients (12.1%) received a higher initial dosing, and almost 90% of the patients receiving the regular maintenance dose regimen with no postponed doses. The study population included a relatively high proportion of treatment naive patients, contributing novel data for this subgroup. An important limitation of the present study is the retrospective, uncontrolled design, without adjustments for possibly confounding factors. The relatively short observation time, especially in progressive disease, is another limitation, and a control group would have strengthened the study.Our data indicate that rituximab is a safe and highly effective treatment option in MS, both as first-line and escalation therapy. The extent of treatment with rituximab is still limited, most likely because it is off-label therapy, and because of the lack of phase III treatment study documentation. Systematic safety documentation is therefore important. Infections, including serious infections, seem to represent an important safety issue during rituximab therapy in MS, and the frequency of both mild and severe neutropenia could be underestimated as there is a lack of knowledge about onset time, and patients might be asymptomatic. Further studies should focus on treatment naive patients, and also how to prevent infections through improved screening or adjusted dosing or dosing intervals.


Previous studies show large variations in physical activity (PA) levels among adolescents. However, the number of studies is limited and even fewer studies have assessed PA in adolescents by accelerometer devices. This study aimed to describe accelerometer-measured PA levels in adolescents in a population-based cohort in Northern Norway.In 611 students aged 16–17 years attending the Fit Futures Study, PA was measured by Actigraph GT3X for seven consecutive days. PA was expressed as total PA volume , time spent in intensity zones, steps per day, and fulfilment of WHO recommendation (i.e. accumulation of 60 min or more of at least moderate intensity PA per day). Potential correlates of PA such as sex, socioeconomic status, study program, self-perceived health, and PA variations by weekday versus weekend were also examined.p < 0.05). PA levels differed with study program and increased with better self-perceived health, but were not associated with socioeconomic status. Both boys and girls were more active on weekdays than weekends  versus 299 (SD 178) CPM, p < 0.05).16% of the girls and 25% of the boys fulfilled current WHO-recommendations. Total PA volume (CPM) was higher in boys than in girls (353 (SD 130) versus 326 (SD 114) CPM, In this cohort of adolescents, less than 25% of 16–17-year-old boys and girls fulfilled the WHO recommendations. The levels of physical activity in 16–17-year-old adolescents are similar to previous data reported in adults. Insufficient physical activity (PA) is one of the leading risk factors for mortality globally , 2, and There are different ways to measure PA, with different strengths and weaknesses. Questionnaires gives an insight in what kind of activity (behaviour) and can include types of activities not recognized by accelerometers. However, accelerometers are objective indicators of body movement (acceleration) and yield more precise measures of intensity, frequency and duration . Most poThe use of devices such as accelerometers to measure PA is increasing, providing more accurate data on PA levels and patterns . Device-The Fit Futures Study (TFF) is a population-based cohort study of adolescents in Northern Norway and part of the population-based Tromsø Study , 25. We The participants signed a written informed consent. Participants younger than 16 years of age signed with written permission from guardians and those aged 16 and above signed at the study site. The Regional Committee for Medical and Health Ethics has approved the study (2012/1663/REK nord).2 (underweight), 18–24.9 kg/m2 , 25–29.9 kg/m2 (overweight) and ≥ 30 kg/m2 (obese). Study program  was registered. At the end of the examination, the accelerometer was handed out. After 8 days the accelerometer was collected at school.The participants filled out an electronic health and lifestyle questionnaire including self-reported PA, self-perceived health , and parents’ education as a proxy of socioeconomic status (SES)  , recording accelerations in three axes . Trained technicians instructed the participants to wear the accelerometer on their right hip attached with an elastic band for seven consecutive days, and to remove the ActiGraph only for water-based activities and during sleep. The devices were initialized in ActiLife with sampling frequency 100 Hz and default filter was used to aggregate raw data into epochs of 10 s. Data were collected between 14:00 on the first day and until 23:58 on day eight. The first day of measurements was removed to reduce reactivity . In acco2Non-wear time was identified using a triaxial method described by Hecht et al. 2009 . A minutPA was quantified as counts per minute (CPM) from the vertical axis. The following PA variables were extracted for use in this study: Accumulated minutes per day spent in the different intensity categories; mean number of counts per minute (CPM); percentage of the population fulfilling the WHO minimum recommendations of ≥60 min MVPA per day ; steps pp < 0.05 were considered statistically significant.Differences in PA levels between girls and boys were analysed using Student’s t-test, and differences between weekday and weekend PA levels were analysed using paired-samples t-test. Differences in PA levels by SES, self-perceived health and study program were analysed using Fisher’s one-way ANOVA. In cases of unequal variances, Welch’s ANOVA was used. All analyses were performed using Statistical Package of Social Science (SPSS v. 25) and all values of In total, 611 participants had valid accelerometer measurements . Participants with valid accelerometer data did not differ significantly from those who did not wear an accelerometer with respect to sex, BMI, and self-perceived health (data not shown).In total, mean (SD) accelerometer wear time was 14.1 (1.14) hours per valid day . Study program was associated with the amount of registered MVPA (p < 0.05), with sports-students presenting 79.5% more MVPA than vocational students (Table p < 0.05)  minutes. Boys spent 6.4 min more in MVPA than girls . There was no significant difference in MVPA between BMI groups. Both boys and girls who rated their self-perceived health as excellent accumulated more minutes of MVPA than all the other groups (1.5 minutp < 0.001). The only exception to this was boys studying sports, with 13% more CPM during weekend (data not shown). Variations in CPM were greater during weekends  than during weekdays . Mean CPM increased significantly with better rating of self-perceived health (p < 0.05), and with parents’ education for girls (p < 0.05), but not for boys (p > 0.05). Participants who attended the sports program had considerably higher means of CPM than the other study programs  was 8.3% higher in boys than in girls (p < 0.05). In both boys and girls, PA levels were lower during weekend days compared to weekdays (for girls 12.9% and for boys 15.3% lower on weekends, Mean steps were similar in boys and girls (total steps 7831, 95% CI 7632–8030, Table Our results suggest that approximately 20% of 16–17-year-old boys and girls fulfilled the current WHO recommendations for PA. Boys were more physically active than girls, as they accumulated more minutes in MVPA and higher CPM. However, steps per day were similar between boys and girls. Moreover, both boys and girls had higher mean CPM during weekdays than weekends.Our results are at large in accordance with other studies assessing PA by accelerometry in adolescents , 20–22. The higher activity levels in boys in our study is consistent with previous studies , 22, 38.We expected the PA levels in our sample of adolescents aged 16–18 years to be lower than in younger children but higher than in adults. However, we found that the mean CPM in our sample was similar as that previously observed in Norwegian adults , 41. HerWe found lower PA during weekends compared to weekdays, which is in line with previous studies , 38. AlsThe positive association between self-perceived health and PA is consistent with the findings in several other studies –46. ThisWe have not been able to find other studies comparing levels of PA in different school programs. It might be considered obvious that students in a sports class are more physically active than peers in general studies and vocational studies. This raises the question of whether these students are more active because they are attending a sports study program, or if they attend the sports study program because they lead a more active lifestyle. The two are not mutually exclusive. This study did not differentiate between school time and after school activity. However, several studies imply that increased PA during the school day increases total PA –49. We cWe consider the high participation rate and the objective PA measurements as the main strengths of our study. The ActiGraph wGT3X has high validity compared with self-reported PA  and compPA levels tend to fluctuate during the day, week, and between seasons. A limitation of this study is that the measurements were done during one single week, and do not capture seasonal variability. Previous studies have documented lower PA levels during the winter and during periods with poor weather conditions –60. In NThe majority of 16- to 17-year-old adolescents living in Northern Norway do not fulfil the current WHO recommendations for physical activity. Total PA volumes were similar to those reported in Norwegian adults. PA varied with sex, self-perceived health and study program. Inadequate levels of PA is a significant challenge for public health, and efforts should be made to increase PA to recommended levels. Health officials would profit from a future research focus on identifying the least physically active individuals and specifically target these groups for interventions.Additional file 1. Overview of questions from questionnaire used in this study. Contains the questions and the response alternatives to each question, translated from Norwegian to English.