In addition to researcher-designed sampling biases, population-representative surveys for biomarker measurement of STIs often have substantial missingness due to non-contact, non-consent and other study-implementation issues. STI prevalence estimates may be biased if this missingness is related to STI risk. We investigated how accounting for sampling, interview non-response and non-provision of biological samples affects prevalence estimates in the third National Survey of Sexual Attitudes and Lifestyles .Natsal-3 was a multistage, clustered and stratified probability sample of 16–74 year-olds conducted between 2010 and 2012. Individuals were sampled from all private residential addresses in Britain; respondents aged 16–44 were further sampled to provide a urine specimen based on characteristics including self-reported sexual behaviours. We generated prevalence estimates and confidence intervals for six STIs in five stages: first without accounting for sampling or non-response, then applying inverse-probability weights cumulatively accounting for interview sampling, interview non-response, urine sampling and urine non-response.Interview non-completion occurred for 42.3% of interview-sampled individuals; urine non-completion occurred for 43.5% of urine-sampled individuals. Interview-sampled individuals, interview respondents, those selected for urine samples and those providing urine samples were each in turn slightly more at-risk for most STIs, leading to lower prevalence estimates after incorporating each set of weights. Researcher-controlled sampling had more impact than respondent-controlled response.Accounting for both sampling structures and willingness to interview or provide urine specimens can affect national STI prevalence estimates. Using both types of weights, as was done in Natsal-3, is important in reporting on population-based biomarker surveys. Representative samples of individuals with biomarkers for STIs are vital to understanding both the population burden of STIs and monitoring trends.One common method that can overcome some of these biases is inverse-probability weighting (IPW), which can account for under/oversampling of subpopulations, and for some types of sampled individuals being less/more likely to respond.Non-response IPWs are commonly used in representative health studies using biomarkers, including the National Health and Nutrition Examination Survey (NHANES) and Health and Retirement Survey (HRS) in the USA and the English Longitudinal Study of Ageing (ELSA),Natsal-3 was a multistage, clustered and stratified probability sample survey of the British resident population, using face-to-face and computer-assisted self-interviews.Chlamydia trachomatis, human papillomavirus (HPV), HIV antibody, Mycoplasma genitalium, Neisseria gonorrhoeae and Trichomonas vaginalis. STI testing was anonymised and results not returned.Following the interview, a stratified subsample of participants was invited to provide a urine specimen, with undersampling of lower-risk individuals. Natsal-3 sampled all 16–17 year-olds, all 18–24 year-olds reporting any sexual partner ever, all males aged 25–44 reporting having sex with another man in the past 5 years, and a random 85% of other 25–44 year-olds reporting any sexual partner ever. The survey tested urine specimens for Natsal-3 aimed to generate nationally representative STI prevalence estimates. IPWs were developed to account for unequal probabilities of sampling, and for non-response, at both the interview and urine provision stages. Interview sampling weights corrected for postcode-level differences in multiple-occupancy addresses, multiple-adult households and for oversampling in London. Interview non-response weights corrected for differences in response by sex, age and region. Urine sampling weights adjusted for the non-random urine sampling outlined above, while urine non-response weights were based on models including a range of sociodemographic and sexual behaviour variables reported at interview found to predict response, specifically: for males—ethnicity, education, marital status, past sexual health clinic attendance, past-year condomless sex partner count, partner concurrency and non-prescription injection drug use; for females—residential region, lifetime partner count, anal sex history, HIV testing history, presence of others during the interview; for both—lifetime report of same-sex sexual experiences. A small number of weight values were trimmed to remove extreme values.and non-response are calculated as the product of oversampling and non-response weights.All weights were calculated as the inverse of the probability of participation, that is, IPWs, rescaled to have a mean of one. Weights accounting for more than one type of bias were calculated by multiplying together the weights for each bias, for example, weights for oversampling M. genitalium, trichomoniasis and HPV. HPV results are only provided for females given the low sensitivity of HPV tests in urine in men.We estimated the prevalence of chlamydia, gonorrhoea, HIV, Ethical approval for Natsal-3 was obtained from Oxfordshire Research Ethics Committee A.Natsal-3 sampled 26 274 households. The interview non-response rate, including non-contact and non-consent, was 42.3%. Of the 15 162 interview respondents, 8047 were sampled for urine provision and 4550 (56.5%) provided a sample .8 Preval10.1136/sextrans-2019-054342.supp1Supplementary dataM. genitalium in men where non-providers had higher estimated prevalence). As a result, adjusting for urine sampling and non-response again lowered most prevalence estimates. In all cases, the sampling and non-response effects for urine were in the same direction, and both of similar  magnitude. The overall impact of adjustment for sampling and non-response was to lower prevalence estimates from their crude values to the published values containing all four weights,Similarly, those providing a urine specimen were slightly more likely to have each STI tested for . This reflects participants in Natsal-3 who provided urine having higher-risk characteristics than those who declined.The relative impact of sampling versus non-response on STI prevalence in population-representative surveys depends on both processes. Non-random sampling with respect to the outcome is often used to improve efficiency and power—interviewing higher-risk individuals increases the number of ‘cases’ identified; the more focused the sampling process, the more impact the resulting reweighting will have on prevalence. Non-response bias depends on the degree to which non-response is a function of the outcome of interest—because higher-risk (or lower risk) individuals cannot be found, do not wish to participate in general, or decline to test for STIs because they believe they are at higher or lower risk of testing positive. In Natsal-3, it appears that the sampling process had more impact than non-response, perhaps because many individuals did not know their STI risk or did not decide to provide urine based on such risk.While some non-response predictors can be measured and accounted for via IPW (as we do here), others may be unobserved and continue to bias STI estimates. IPWs rely on the untestable assumption that non-responders differ from responders in their STI risk in a manner that can be predicted from observed covariates . If this untestable assumption does not hold , IPW-adjusted figures will be biased. If MNAR is suspected then, under further assumptions, selection models can potentially be used provide more valid estimates both of STI prevalence and CIs.An important potential extension of the use of IPW would be to look at changes in STI prevalence over time. However, such analysis is complex, requiring taking into account differential sampling if using repeated cross-sectional studies, or of differential loss-to-follow-up and learning effects if repeatedly surveying a cohort. In all cases, there is a risk that changes in willingness to participate across waves are associated with STI risk, and any changes in STI test performance over time.Weighting is a standard and necessary part of good survey methodology. This paper demonstrates the quantified contribution of different elements of the weighting strategy to deriving the best estimates of unbiased population prevalence. This was done for Natsal-2 and Natsal-3. It will be important for Natsal-4 as well as other population-based surveys incorporating biological markers.


This study aimed to determine the change in mental health (depression and anxiety) among HIV-positive gay and bisexual men (GBM) one year after diagnosis and the disparities in trajectories of mental health between them. The potential factors contributing to the disparities were also investigated. This was a one-year follow-up study focusing on the mental health of newly diagnosed HIV-positive individuals. Participants rated their depression, anxiety, stress, and social support levels at baseline and one year later. Information on the utilization of mental healthcare and the initiation of antiretroviral therapy (ART) after diagnosis was collected at one-year follow-up. A total of 171 and 87 HIV-positive gay and bisexual men, respectively, completed two-time points surveys in this study. The depressive and anxiety symptoms experienced by HIV-positive GBM improvement one year after diagnosis. These improvements tended to be smaller in gay participants. Other factors including mental health care utilization and ART status during the one-year follow-up period, changes in social stress scores and objective social support scores were also associated with the changes in depression and anxiety, and all these factors, except for change in objective support, were found to be statistically different between HIV-positive GBM. Special attention should be given to the mental health of HIV-positive gay men. Promoting HIV-positive gay men to assess to mental health services and ART may be important for these populations to improve mental health. Enhancing social support and reducing stress levels may also be necessary for the vulnerable HIV-positive sexual minority groups.  The mental health of HIV-positive individuals is of great concern in public health. There is evidence that HIV-positive individuals experience much higher mental health burden than the general population, with depression and anxiety being the most commonly reported ,2. PreviDue to the growing prevalence of HIV infection among them , the menAccording to the minority stress theory, GBM are vulnerable social groups susceptible to stigma and prejudice, making them more likely to suffer from elevated stress levels . A meta-Some researchers have noticed the heterogeneity in mental health of GBM, which means that gay men and bisexual men may have disparities in mental health ,17,18,19The purposes of this study were: (1) to determine the changes in mental health (depression and anxiety) one year after HIV diagnosis and the disparities in trajectories of mental health between HIV-positive gay and bisexual men; (2) to identify the potential factors contributing to the disparities in mental health between them. According to the findings in previous studies focusing on the mental health of general GBM , we hypoThe data reported in this study derives from a longitudinal study focusing on the mental health among newly diagnosed people living with HIV. A detailed description of study design is available elsewhere . BrieflyThere were 1267 people in Changsha diagnosed with HIV between 1 March, 2013, and 30 August, 2014. Of them, 855 individuals met the criteria and 557 participated in this study. Among the individuals who participated in this study, a total of 354 participants identified their sexual orientation as gay or bisexual, which was the final sample included in the analyses of this study. Socio-demographic information, including age , educational background , monthly income , marital status , employment , living alone or not, and having children or not were collected. Clinical information included HIV-related symptoms, CD4 counts, and ART initiation. HIV-related symptoms included persistent diarrhea, persistent cough, persistent fevers, oral thrush, unintentional weight loss, recurrent herpes simplex, and active tuberculosis. Any other symptoms were recorded. The therapeutic information  was also collected. In this study, sexual orientation was determined by self-identification . The selα coefficient of 0.906 [We used the 17 items Chinese version of the HIV/AIDS Stress Scale (CSS–HIV) to assess HIV/AIDS-related stress. It was originally developed by Pakenham , and theof 0.906 .We use the 10 items Social Support Rating Scale (SSRS) developed by Xiao to evaluate the social support . This scA single yes/no item was used to collect the mental health care utilization among participants: “Have you accessed to mental health care due to the emotional distress after diagnosis?”α coefficient of 0.86 [The 9-item Patient Health Questionnaire Depression Scale (PHQ-9) was used to assess depressive symptoms. It is a 4-point Likert-type scale, with each item ranging from 0  to 3  . Higher  of 0.86 . The 7-items Generalized Anxiety Disorder Scale (GAD-7) was used to measure anxiety symptoms . It is aHIV-diagnosed individuals were enrolled when they attended Changsha CDC to get the HIV-infection diagnosis certification or the first CD4 test after diagnosis. The baseline survey was conducted between 1 March, 2013, and 30 September, 2014. One year later, participants who completed baseline survey were contacted for the follow-up survey and all assessments were completed by 31 October, 2015. Participants, at both baseline and one-year follow-up surveys, were asked to complete a questionnaire, including socio-demographic and psychosocial information, via self-report formats. The socio-demographic information was collected at baseline survey and the psychosocial information was collected at both time surveys. Members of our research team reviewed medical records with participants’ permission from Chinese HIV/AIDS Comprehensive Response Information Management System (CRIMS), to obtain the clinical information, including the CD4 counts and HIV-related symptoms of participants at diagnosis and at one-year follow-up. Information on whether participants started ART during the follow-up period was also reviewed from CRIMS at the one-year follow-up survey. The IEC of the Institute of Clinical Pharmacology at Central South University (CTXY-120033-3) approved this study, and written informed consent was obtained from each participant before participation.Descriptive statistics were presented as frequency with percentages and medians with interquartile range (IQR). The Chi-square and Mann–Whitney U tests were used to compare the baseline sample characteristics between participants who completed two-time surveys and those who dropped out of follow-up survey. The change in scores between baseline and follow-up for depression, anxiety, HIV-related stress, and social support  were calculated by subtracting baseline scores from follow-up scores. The Mann–Whitney tests were used to compare change scores of psychosocial variables between HIV-positive GBM. p value of <0.05 was considered to be statistically significant.Two separate generalized linear models (GzLM) were performed separately to determine whether HIV-positive GBM have different trajectories of mental health (depressive and anxiety symptoms) one year after diagnosis, after controlling age, marital status, education, employment, monthly income, living alone or not, having children or not, CD4 counts, HIV-related symptoms, ART status, mental health care utilization, change in stress scores (3 dimensions), and change in social support scores (3 dimensions). All analyses were performed using SPSS for Windows 25.0 . A Among the 354 participants who identified their sexual orientation as gay or bisexual, 235 were gay and 119 were bisexual, of which 171 and 87, respectively, continued to complete follow-up survey one year after diagnosis. The participant flowchart is shown in There were no significant differences between the 171 HIV-positive gay participants who completed two-time surveys and the 64 who completed only baseline survey, with respect to baseline socio-demographic, clinical, and psychosocial characteristics, except that participants lost to follow-up were more likely to live alone. All baseline sample characteristics were not significantly different between 87 HIV-positive bisexual participants who completed the two-time surveys and 32 of those lost to follow-up .For the overall HIV-related stress and social support, the group differences in changes from baseline to follow-up did not reach statistical significance. However, the change scores of PHQ-9 and GAD-7 as well as the social stress were statistically different between the two groups, with smaller decreases in PHQ-9, GAD-9, and social stress score observed in gay participants. The details of changes in psychosocial status between the two groups are shown in β = 1.61, p = 0.041) and GAD-7  scores, indicating gay participants have a poor recovery from depressive and anxiety symptoms one year after diagnosis.β = −2.14, p = 0.008) and anxiety  symptoms, compared to those who did not. Participants who had access to mental health care after diagnosis were more likely to have better improvement in depressive  and anxiety  symptoms. Increases in social stress scores were associated with increases in PHQ-9  and GAD-7  scores, while increases in objective support were associated with decreases in PHQ-9  and GAD-7  scores. In addition, other factors associated with changes in PHQ-9 and GAD-7 scores were mental health services utilization and ART status during the one-year follow-up period, changes in social stress, and objective support scores. Participants who received ART during the first year after diagnosis had better improvement in depressive . The depressive and anxiety symptoms experienced by HIV-positive GBM at diagnosis alleviated one year later. These improvements, however, contrary to the hypothesis, tended to be smaller in gay participants. After controlling other variables, sexual orientation was found to be independently associated with the changes in depression and anxiety one year after diagnosis, which further demonstrate the disparities in trajectories of mental health between these two groups. Other factors, including mental health care utilization and ART initiation status during one-year follow-up period, changes in social stress scores, and objective support scores were also shown to be associated with the changes in depression and anxiety, which may provide some reasonable explanations for the disparities in trajectories of mental health between gay and bisexual participants.Participants who accessed mental health care after diagnosis had larger improvements in depressive and anxiety symptoms in this study, and gay participants with significant depression or anxiety were found to be less likely to use mental health care than their bisexual counterparts. This may be the possible explanation why gay participants had worse improvement in mental health one year after diagnosis than bisexual participants. Furthermore, a low rate of mental health utilization was also found in the whole sample, with less than one-fourth of participants who reported experiencing significant depression and anxiety at diagnosis accessing mental health care after diagnosis. It is evidenced that the mental health burden related to HIV/AIDS still remains a major challenge for HIV-positive individuals ,38,39. AA previous study reported that the lack of professional mental health care specific to sexual minority men and the fear of discrimination from potentially exposing their sexual minority status to healthcare providers, are important potential barriers that prevent sexual minority groups from seeking mental health care . Some seIn this study, we found that the proportion of ART initiation after diagnosis among gay participants was lower than that among bisexual participants, and ART status was a significant factor associated with the changes in depression and anxiety in the GzLM analysis. Participants who received ART during the first year after diagnosis had better improvements in depressive and anxiety symptoms, compared to those who did not. It is widely accepted that early ART initiation is important to the health of HIV-positive individuals . In prevWe found that change in social stress were associated with changes in depressive and anxiety symptoms. The lesser the decreases in social stress levels one year after diagnosis, the lesser the improvement in the symptoms of depression and anxiety. The majority of individuals have to struggle with multiple social stress after receiving HIV diagnosis, such as disclosure concerns, HIV-related stigma, and discrimination . These hConsistent with previous studies showing that a higher level of social support was linked to a better mental health ,60,61, tWe found that compared to gay participants, bisexual participants are more likely to be married and have children. Although these two factors were not significantly associated with the trajectories of mental health in the GzLM analyses, they may be important factors that indirectly influence the changes in mental health among gay and bisexual men over the one year after diagnosis. Previous studies suggested that compared to single men, married men are more likely to start ART after diagnosis, because they need to consider the potential risk for infecting their family members such as wife, children ,64. PrevThis study has several limitations. First, the sample in this study is recruited consecutively, which may limit the generalizability of results. Second, we have an unbalanced sample size between the HIV-positive gay and bisexual group, and some participants did not participate in the follow-up study a year later. These participants may exhibit different psychosocial status during follow-up, which may bias the final results. Third, our assessments of depression and anxiety were relied solely on self-reported symptoms scales. Multiple assessments of depression and anxiety, especially the diagnosis tools, would provide a more reliable measure of mental health in this sample. Additionally, in the three dimensions of sexual orientation , this study only focused on the identity dimension. This approach used in this study may miss some HIV-positive MSM populations who may have distinct mental health needs.The depressive and anxiety symptoms experienced by HIV-positive GBM at diagnosis alleviated one year later. These improvements tended to be smaller in gay participants. Other factors including mental health care utilization and ART initiation status during one-year follow-up period; changes in social stress scores and objective support scores were also shown to be associated with the changes in depression and anxiety. Moreover, all these factors, except for change in objective social support, were found to be statistically different between HIV-positive GBM. Special attention should be given to the mental health of HIV-positive gay men. Promoting HIV-positive sexual minority men to assess to mental health services and ART may be important for these populations to improve mental health. Enhancing social support and reducing stress levels may also be necessary for the vulnerable HIV-positive sexual minority groups.


Bacteroide in CG and small-bowel inflammation. Combined with differential network analysis, the network-hub microbiota within rewired microbiota networks revealed high-ranked iENA microbiota markers, which were disease specific and had particular pathogenic functions. Our findings illuminate the components of the fecal microbiome and the importance of specific bacteria in CG and small-bowel erosions, and could be employed to develop preventive and non-invasive therapeutic strategies.The development of non-invasive, inexpensive, and effective early diagnosis tests for gastric and small-bowel lesions is an urgent requirement. The introduction of magnetically guided capsule endoscopy (MGCE) has aided examination of the small bowel for diagnoses. However, the distribution of the fecal microbiome in abnormal erosions of the stomach and small bowel remains unclear. Herein, alternations in the fecal microbiome in three groups  were analyzed by metagenomics and our well-developed method [individual-specific edge-network analysis (iENA)]. In addition to the dominant microbiota identified by the conventional differential analysis, iENA could recognize novel network biomarkers of microbiome communities, such as the genus  Choung and colleagues reported that in the United States, 338 out of 31,255 adult residents were diagnosed with diseases of the digestive system . In partCandida albicans, and a decreased abundance of Saccharomyces cerevisiae in inflammatory bowel disease (IBD) (Metagenomics using next-generation sequencing (mNGS) is becoming a promising approach for identifying the microbiome community in human diseases . Some stse (IBD) . Signifise (IBD) . mNGS case (IBD) . Small-bse (IBD)  but alsose (IBD) . HoweverAn accurate clinical diagnosis can enable monitoring, quantification, and progression of a disease , and canHere, we used the method of computational systems biology that we had developed to analyze the dominant microbiota and network on the basis of fecal metagenomics data. We identified specific bacteria that had key roles in the clinical classification of erosive lesions of the small bowel and CG that might offer prevention and non-invasive treatment strategies.The study protocol was approved by the Ethics Committee of Shanghai Sixth People’s Hospital, which is affiliated with Shanghai Jiao Tong University . Written informed consent was obtained from all individuals. Personal data were anonymized and omitted.n = 15) were recruited. The procedures of enrollment, fecal mNGS, and MGCE classification were completed independently by different investigators who were blinded to the results of each other’s examinations. Fisher’s exact test was used for evaluation of statistical difference in comparisons between three groups. p < 0.05 was considered statistically significant.The study ran at Shanghai Sixth People’s Hospital from May 1, 2017 to September 1, 2018. The individuals who agreed to complete examinations of MGCE  and mNGS examination of their stools  were characterized by an absence of lesions in the stomach and small bowel through MGCE. Gastric inflammation (G group) was identified based on the Updated Sydney System . InflammWe recorded (i) mucosal lesions, such as erosions; (ii) capillary lesions ; (iii) mucosal changes ; (iv) changes in villi ; (v) lymphangiectasias/lymphocellular infiltrates.TM ND-1000 spectrophotometer .Samples of total DNA from fecal samples were extracted using a Fast DNA SPIN extraction kit  and stored at −20°C. The quality and quantity of isolated DNA were assessed by agarose gel electrophoresis and a NanoDropTM DNA Sample Preparation kit  and sequenced at Shanghai Personal Biotechnology  on an Illumina HiSeq system with a 150-bp paired-end protocol. Reads from each sample were retained and then merged by megahit . In addition, principal component analysis (PCA) was used to analyze and visualize the sample distribution and discrimination among different disease groups.DNA extracted from fecal samples was used for mNGS. Metagenomic libraries were constructed with a TruSeq megahit  and, if  megahit . The rem megahit  and then megahit , with an megahit  was intr megahit  using KE megahit  and Diam megahit  was applp < 0.05 were selected  to provide a powerful network-analysis tool to quantify disease progression in an individual patient. Recently, we implemented an adjusted iENA using samples from healthy individuals as a network reference due to a limited number of individual samples and applied it in a proof-of-concept study on microbiota dynamics . Here, wAfter selecting reference samples, we constructed a co-expression network for one sample with our single-sample measurement of the Pearson correlation coefficient (sPCC) . BecauseWe selected top-ranked edge-pairs as edge-biomarkers, which have strong high-order compositional correlations and can be viewed as feature candidates represented as a set called “Markers.” In theory, each individual-specific biomarker is related to the clinical phenotype to some extent because the closely contacted candidates are identified in a certain disease state. As a warning signal , we extewhere We could obtain the different individual-specific biomarkers to indicate disease-specific signals. Microbiota features that always present in the same disease group are more robust and representative in terms of disease specificity . Thus, wFrom May 1, 2017 to September 1, 2018, 38 people with symptoms such as chronic abdominal pain, abdominal distention, and diarrhea participated in this study. They were screened across the Department of Gastroenterology of Shanghai Sixth People’s Hospital .Among them, 33 patients completed MGCE and stool collection, and the feces of 15 patients were processed through mNGS . The cohUsing mNGS, we demonstrated the quality of sequencing and obtained the microbe abundance through a standard pipeline. There were 52,820 differential genes between the G group and H group; 22,537 genes between the I group and H group; 17,670 genes between the G group and I group. We obtained two important results .First, the distribution of different species in samples indicated the varying microbiota compositions. Also, the samples in the same group/state tended to have similar microbiota compositions . The difN-Glycan biosynthesis, biosynthesis of secondary bile acids, and metabolism of -Linolenic acid). Recent study has revealed that patients with IBD have a higher abundance of large-size glycans, as well as lower levels of galactosylation and fucosylation . Thus, the variance in microbe abundance was expected to supply more discriminative information, which could be detected by our proposed iENA approaches. Indeed, using one CI score with iENA-identified marker microbes, each sample could be assigned to a suitable group correctly. In accordance with the analysis stated above, CG samples had the highest scores, indicating the greatest variation/alteration in the microbiota community, whereas samples from healthy people had the lowest scores  . More imThree important trends were observed in this part of our study.First, for the key microbiota with differential abundance identified by conventional analysis, some well-known pathogenic microbes were discovered.Roseburia and Enterobacter were related to Helicobacter pylori (HP) in gastritis and Clostridioides difficile infection (CDI) in IBD. The absence of Roseburia has been observed in HP-positive samples  (Blautia and Flavonifractor contribute to discrimination of IBS or CRC from controls (Roseburia and Blautia in feces specimens of patients with ulcerative colitis (UC) indicates a higher risk of pouchitis after ileal–anal pull-through surgery (Blautia than those without CDI (Blautia is enriched after 26 weeks of quadruple treatment with bismuth in patients with asymptomatic HP-related gastritis (Roseburia increases significantly after 1-week administration of vitamin D in Crohn’s disease (CD) cases (Microbes from the genera er (CRC) . The gencontrols . The dec surgery . In parthout CDI . Also, tastritis . Also, tD) cases .E. cloacae is associated significantly with CD patients without antibodies to Saccharomyces cerevisiae  in MGCE-examined subjects according to the data from multiple medical centers . Thus, iAcinetobacter and Bacteroides in patients with IBD , project ID OEP000779.The studies involving human participants were reviewed and approved by the Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this manuscript.J-SZ, JZ, and X-TY supervised the study. X-TY, X-YC, and JZ designed the experiments and analyzed the data. X-YC, H-NF, and H-WQ carried out the experiments. X-TY, X-YC, H-KZ, and LS undertook the data analysis. X-YC and X-TY prepared the manuscript. X-TY and X-YC revised the manuscript.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.


The classical van Benthem theorem characterizes modal logic as the bisimulation-invariant fragment of first-order logic; put differently, modal logic is as expressive as full first-order logic on bisimulation-invariant properties. This result has recently been extended to two flavours of quantitative modal logic, viz. fuzzy modal logic and probabilistic modal logic. In both cases, the quantitative van Benthem theorem states that every formula in the respective quantitative variant of first-order logic that is bisimulation-invariant, in the sense of being nonexpansive w.r.t. behavioural distance, can be approximated by quantitative modal formulae of bounded rank. In the present paper, we unify and generalize these results in three directions: We lift them to full coalgebraic generality, thus covering a wide range of system types including, besides fuzzy and probabilistic transition systems as in the existing examples, e.g. also metric transition systems; and we generalize from real-valued to quantale-valued behavioural distances, e.g. nondeterministic behavioural distances on metric transition systems; and we remove the symmetry assumption on behavioural distances, thus covering also quantitative notions of simulation.


ShinySOM offers a user-friendly interface for reproducible, high-throughput analysis of high-dimensional flow and mass cytometry data guided by self-organizing maps. The software implements a FlowSOM-style workflow, with improvements in performance, visualizations and data dissection possibilities. The outputs of the analysis include precise statistical information about the dissected samples, and R-compatible metadata useful for the batch processing of large sample volumes.ShinySOM is free and open-source, available online at gitlab.com/exaexa/ShinySOM.Bioinformatics online. Flow cytometry enables fast measurement of multiple parameters of individual cells in solution. In recent years, multiple algorithms have emerged for analyzing flow and mass cytometry data. Among them, FlowSOM . The comThe performance of FlowSOM makes it a suitable basis for constructing interactive data analysis platforms. Here, we introduce ShinySOM, a web-based frontend for FlowSOM-style sample dissection and analysis. ShinySOM exposes the FlowSOM functionality in a graphical interface that is useful both for quick interactive and ad-hoc exploration of datasets, and for running the usual FlowSOM-style pipelines in a way convenient for users not familiar with programming interfaces. In addition, ShinySOM provides improved visualization methods and new implementations of unsupervised analysis algorithms, which lead to a substantial speed-up of the operations and further extend the ability to work with large datasets.The implemented analysis workflow  and customized to work with FlowSOM-style clustering.The Mahalanobis-average linkage computation (idendro-inspired (shinyDendro (github.com/exaexa/shinyDendro) was implemented and integrated into ShinySOM, in order to simplify interactive cluster selection and annotation.An The resulting workflow gives results comparable to FlowSOM, while enabling user-controlled cluster selection in the final stage of analysis see . This apAll used algorithm parameters and user-generated metadata can be exported along with the main analysis results. This information describes all expert decisions performed during the analysis and allows for reproducible use of the platform. Moreover, the ‘canned’ expert decisions may be used to automate the analysis, further allowing, e.g. the construction of R-based batch-processing pipelines.ShinySOM provides a new software alternative for analyzing multidimensional flow and mass cytometry data. Importantly, it contains a suite of fast algorithms in a user-friendly environment capable of generating precise results more quickly than the manual gating-based analyses. Accessible use of unsupervised analysis algorithms helps to overcome the common dimensionality overhead and promotes analysis reproducibility.R-based environment provides a link between the expert knowledge gathered using the graphical interfaces and the batch processing required for work with large volumes of samples. A complete cytometry workflow can be constructed with the help of other compatible packages in the R ecosystem  [LM2015047]. D.B. was supported by the Charles University Project Progress Q48. T.S. and K.F. were supported by Czech Health Research Council (AZV) [NV18-08-00385]. Funding for open access publication was provided by the Institute of Organic Chemistry and Biochemistry of the CAS [RVO: 61388963].Conflict of Interest: none declared.btaa091_Supplementary_DataClick here for additional data file.


Routes of drug administration and their corresponding physiochemical characteristics play major roles in drug therapeutic efficiency and biological effects. Each route of delivery has favourable aspects and limitations. The oral route of delivery is the most convenient, widely accepted and safe route. However, the oral route of chemotherapeutics to date have displayed high gastric degradation, low aqueous solubility, poor formulation stability and minimum intestinal absorption. Thus, mainstream anti-cancer drugs in current formulations are not suitable as oral chemotherapeutic formulations. The use of biopolymers such as chitosan, gelatin, hyaluronic acid and polyglutamic acid, for the synthesis of oral delivery platforms, have potential to help overcome problems associated with oral delivery of chemotherapeutics. Biopolymers have favourable stimuli-responsive properties, and thus can be used to improve oral bioavailability of anti-cancer drugs. These biopolymeric formulations can protect gastric-sensitive drugs from pH degradation, target specific binding sites for targeted absorption and consequently control drug release. In this review, the use of various biopolymers as oral drug delivery systems for chemotherapeutics will be discussed. Majority of global deaths are caused by non-communicable diseases, with cancer ranking as the leading cause of death. In 2015, the World Health Organisation estimated that cancer is one of the leading causes of death in 91 of 172 countries -propionate) (SPDP). GEM-SPDP had an the body . In anotThe formulated NPs were pH-responsive and the loading capacity of DOX and BET was 20.5% and 16.25%, respectively. Shape and size of the NPs were analysed, and results showed that the formulated NPs were well rounded, with a uniform size around 162 nm. The pH-response profile was assessed in simulated physiological and tumour environments, and controlled release was shown by adjusting the environmental pH. The formulated NPs were further assessed for cell viability on breast cancer cells (MCF-7), in comparison to free-form DOX and BET. The results obtained showed a decrease in cell viability of the MCF-7 cells. Hence, the group concluded that the developed delivery platform is a promising nanocarrier for cancer treatment . AnotherCollagen is a naturally occurring protein, abundantly present in animals. It has been used in various drug delivery system applications . CollageA study was carried out by Zhong Luo et al., where they formulated mesoporous silica NPs end capped with collagen. The aim of the study was to synthesise redox-responsive NPs for targeted drug delivery of anti-cancer drugs. Targeted delivery and cellular uptake studies were carried out on HepaG2 cells. The results obtained showed a 2.2-fold increase in targeted cellular uptake, as compared to the free drugs in intravenous solution. The group therefore concluded that collagen capped mesoporous silica NPs could successfully serve as redox-responsive nanocarriers. The formulated nanoparticles exhibited excellent biocompatibility, cell specific delivery and excellent cellular uptake properties. The collagen capped mesoporous silica NPs can potentially be used for oral chemotherapeutic targeted delivery .D-glutamic acid/L-glutamic acid units, and or a combination of both. The molecular weight of the polymer controls the release of the entrapped drug. Hence, polymers of varied molecular weights are needed, depending on the desired properties and applications of the polymer [Polyglutamic acid (PGA) is a polyamino acid that can be used to form conjugates with anti-cancer drugs. These polymer–drug conjugates have the potential to increase chemotherapeutic drug efficacy and reduce toxicity towards normal cells. PGA is water soluble and is therefore a good candidate for orally delivering hydrophobic anti-cancer drugs. Additionally, PGA is non-toxic to both the environment and humans . PGA is  polymer ,24.b-(PGA)-b-poly micelles for the dual delivery of paclitaxel (PTX) and an alkylating agent (CDDP). PTX was bound to the polyphenylalanine core, with 9% loading capacity while the CDDP was bound to the PGA layer, with 15% loading capacity. Therapeutic efficacy of the formulated co-delivery platform micelles was tested against A2780 ovarian cancer cells and results obtained showed improved efficacy, as compared to micelles containing PTX or CDDP alone. The group noted that besides multi-drug delivery capability of the PEG-b-(PGA)-b-poly micelles, the micelles also showed improved stability against disassembly in the gastric region and therefore enhanced tumour delivery [Attention has been drawn to γ-PGA because of its diverse properties. Desale et al. performed a study where they synthesised PEG-delivery . In anotdelivery .In 2015, Liadong Deng et al. formulated chitosan-PGA-grafted PEG-DOX NPs. Their aim was to engineer drug gastric survivability, improve intestinal permeability, and enhance hemodynamic stability and intracellular activity of DOX. The formulated NPs demonstrated long-circulating properties, improved therapeutic efficacy and lower systemic toxicity as compared to DOX free in intravenous solution at a concentration of 10 μg/mL. The mucoadhesive properties of chitosan permit the formulated NPs to persist in the stomach and adhere to the mucus layer of the small intestine, and subsequently penetrate through the small intestine into the blood circulation. In vivo studies of the chitosan-PGA-grafted PEG-DOX NPs at a dose of 15 mg/kg, were carried out on Balb/c mice xenografted with Ehrlich ascites tumor, with DOX-HCL (15 mg/kg) intraperitoneal injection (i.p.) solution being used as the control formulation. The group treated with the oral NP formulation showed no deaths up to 4 weeks, while the group treated with i.p. solution had a survival rate of approximately 45.5% over the same period. The chitosan-PGA-grafted PEG-DOX NPs exhibited comparable tumour inhibition effects, as that of i.p. DOX-HCl, with significantly lower toxicity, enhanced cellular uptake and relative stability in physiological circulation. It was thus concluded that the chitosan-PGA-g-PEG/DOX NPs are a promising nano-platforms for oral delivery of chemotherapeutics .Poly(lactic acid-co-glycolic acid) (PLGA) is a polyester, copolymer block made up of polyglycolic acid and poly-lactic acid . It is aBiodegradation of the copolymer occurs by bulk erosion. Its degradation mechanism allows for potential drug survivability in the acidic gastric environment, as the drug will be encapsulated at the core. The polymeric chains of PLGA are cleaved by hydrolysis and the monomeric units, lactic acid and glycolic acid, are metabolically excreted from the body . The degPLGA NPs can be formulated by use of emulsion evaporation, emulsion diffusion, solvent displacement and nanoprecipitation techniques. In 2017, Yan-na Cui et al. formulated paclitaxel (PTX)-loaded magnetic PLGA (50:50) NPs, surface modified with transferrin (TF). The aim of their research was to investigate the feasibility of the drug-loaded magnetic PLGA nanoparticles in enhancing cellular uptake and thus improving the therapeutic efficacy against cancer cells. The drug-loaded PLGA NPs were characterised in terms of encapsulation efficiency, size and morphology, magnetic properties, in vitro PTX release, and lastly, cytotoxicity and cellular uptake were evaluated in vitro against MCF-7 and U-87 cells. The morphology results obtained showed that the NPs were nearly spherical and approximately 150 ± 20 nm. Encapsulation efficiency (EE) was reported to be 82.9% and more than 80% of PTX was released over a period of 10 days, with the release profile following a biphasic profile. The formulated PTX-PLGA NPs showed increased cytotoxicity against the cancer cells and improved cellular uptake, as compared to the free PTX in solution for injection .In a more recent study, Ajinder Kaushik et al. used PLGA 85:15 to formulate cetuximab (CMB) loaded PLGA NPs . Their a3, while the tumours treated with GEM/BA injection solution had a mean volume of 213.5 mm3. The group concluded that the GEM/PLGA-PEG/BA NPs could potentially be used to co-deliver chemotherapeutics to enhance antitumor efficacy [In another study, piperine (PIP)-loaded PEG-PLGA NPs were formulated for the targeted delivery of adjuvant breast cancer therapy. Manendra Pachauri et al. reported that the PIP/PEG-PLGA NPs killed MCF-7 cells by apoptotic mechanism. When used in combination with PTX, the PIP-loaded NPs exhibited significant reduction in the PTX dose. Results obtained from the study led to the conclusion that the PIP/PEG-PLGA NPs were safe to be used as adjuvant cancer therapy against resistant breast tumours . In 2019efficacy .Polysaccharide biopolymers consist of repeating simple sugar units joined by glycoside bonds. They are generally inexpensive and abundantly found in nature. They are stable, non-toxic and are excellent candidates for development of biocompatible anti-cancer drug delivery platforms. Biopolymers such as chitosan, alginates and hyaluronic acid have hydrophilic groups that form bio-adhesions with biological tissues, potentially increasing cellular drug retention which is beneficial in oral delivery of chemotherapeutics . AdditioChitosan is produced from purified and N-deacetylated chitin. Chitosan production can be optimised to control the final product properties . Chitosa2 and –OH groups of the chitosan can be used as sites for molecule modification. Chitosan modification can either be physical or chemical. Chitosan derivatives offer modified properties for controlled drug release and can impact the pharmacokinetic profiles of the loaded anti-cancer drugs [The –NHer drugs . ChitosaDrug release from the chitosan NPs tends to be pH dependent . ChitosamaxC value of DOX after oral administration of DOX loaded NPs was 208.07 ± 20.17 ng/mL, which was nearly 4-folds, being 2.3-folds higher than that of free DOX solution. The results demonstrated improved intestinal absorption of DOX, when loaded onto the chitosan/carboxymethyl chitosan NPs, and hence improved oral bioavailability. Organs were excised from the rats, 24 h post-administration. The rats treated with oral DOX loaded NPs showed accumulation of drug in the liver (5.87 μg/g tissue), spleen (3.65 μg/g tissue) and lungs (2.58 μg/g tissue). While the rats treated with DOX solution , demonstrated DOX concentrated in the kidneys. These results indicated that DOX/carboxymethyl chitosan NPs could prolong systemic circulation and the retention time in the mentioned organs and can therefore be used to target tumours in the liver, spleen and lungs [Chitosan has been used to deliver drugs with low water solubility and acid-labile anti-cancer drugs. For example, tamoxifen (TFN), which is sparingly water soluble and is therefore a good candidate for oral drug delivery. In 2015, S Barbieri et al. developed lecithin-chitosan NPs loaded with TFN. The TFN-loaded NPs showed muco-adhesive properties and increased drug permeation across the intestinal epithelium. Intestinal permeation of the TFN-loaded NPs was assessed in comparison to tamoxifen citrate in ringer solution (160 μg/mL). The amount of TFN absorbed and transported through the intestinal epithelia, from the tamoxifen citrate suspension, after 4 h was 0.76 nmol. On the other hand, the amount of TFN absorbed from the TFN-loaded NPs was reported to be 1.5 times higher than that of the tamoxifen citrate suspension. When the NPs were used, TFN passed through the intestinal tissue without being transformed by the CYP 450 enzyme. Hence, more of the drug was able to be found on the receptor site . In anotnd lungs .In 2017, Asad Khan et al. synthesised and characterised carboplatin (CPN)-loaded chitosan NPs for the treatment of breast cancer. The CPT loaded NPs had an average size of 277.25 ± 11.37 nm and a zeta potential of 31 ± 3.14 mV with low polydispersity index. Maximum drug encapsulation was obtained at 58.43%. The CPT chitosan NPs exhibited significant blood compatibility. When analysed in vitro, the NPs showed improved cytotoxicity effects against MCF-7 cell line. The group concluded that the CPT chitosan NPs could be used as a potential candidate for cancer treatment .Sodium and potassium alginates have emerged as one of the most extensively explored biomaterials. Their unique physical properties permit for sustained release and targeted delivery of drugs, thus making them favourable BPs for use in oral drug delivery for cancer treatment . This isLim Vuanghao et al. published a report on their use of biocompatible disulphide cross-linked sodium alginate derivative NPs, for targeted oral delivery of PTX to treat colon cancer. They formulated self-assembled cysteamine based disulphide cross-linked sodium alginate NPs, aimed at improving the delivery of PTX to the colon cancer cells . The druAnother group developed DOX loaded pH sensitive alginate derivative NPs, targeted to the liver. The DOX loaded glycyrrhetinic acid modified alginate-alginate complex (GA-ALG/DOX-ALG) NPs were formulated by self-assembly method. The formulated NPs showed a pH sensitive release profile of DOX, where at pH 7.4, less than 10% of the doxorubicin had been released, whereas 58.7% of the drug was released at pH 4. Guo et al. carried out an in vivo biodistribution study in Kunming mice. DOX biodistribution from the formulated NPs was assessed in comparison to that of intravenous solution DOX-HCL. Significantly different biodistribution in heart, liver, spleen, lung, and kidney tissues was observed in DOX-HCl and GA-ALG/DOX-ALG NP groups, 1 h after administration. The DOX concentration in the liver of the GA-ALG/DOX-ALG NP group reached 27.6 μg/g, which was significantly higher than that of DOX-HCl (8.1 μg/g). The higher concentration of DOX in the liver was due to glycyrrhetinic acid, having liver-targeting properties . Liver tIn 2014, Ghislain Garrait et al. developed a novel drug delivery system of chitosan NPs entrapped in alginate microparticles. The aim was to develop an ODP that can protect the encapsulated drug from degradation in the gastric tract. In vitro drug release was studied in simulated gastric and intestinal fluids. Regardless of the presence or absence of enzymes, no breakdown of the alginate microparticles was observed. However, in simulated intestinal fluid, with or without pancreatin, the alginate was rapidly disintegrated. The alginate microparticles disappeared within 15 min, therefore releasing the chitosan NPs for loaded drug release. Experimental results obtained showed pH-responsive release of drug, as only 5% of loaded drug was released in the stomach, and at intestinal pH, the drug was completely released. The developed platform proved to be a suitable vehicle to protect anti-cancer drugs from gastric degradation, after oral administration .Hyaluronic acid (HA) is a linear macromolecular mucopolysaccharide, composed of alternating glucuronic acid and N-acetylglucosamine units . The hydThe three functional groups on the main chain of HA can be modified, thus, different anti-cancer drugs can be conjugated to the HA, to formulate HA–drug conjugates. HA–drug conjugates are usually covalently bonded prodrugs and are not easily cracked in the blood but break through enzymolysis or hydrolysis after reaching the target site. HA–drug conjugates can improve drug solubility and are therefore a good candidate for use in oral delivery formulations of anti-cancer drugs with low aqueous solubility. HA also imparts change in drug biodistribution and increase in vivo half-life, as it leads to increased drug accumulation in the tumour sites due to enhanced osmotic retention and exerts better drug efficacy .In 2015, Cai et al. made a novel delivery system by conjugating cisplatin (CIS) to HA. CIS is widely used for the treatment of most solid tumours, but its use is limited due to the serious side effects. The novel delivery system formulated by Cai et al. led to an increase in the concentration of platinum in the lymphatic vessels, reduced systemic toxicity and side effects, all while inhibiting early tumour metastasis of breast cancer . At pH 7In 2019, Nilkamal Pramanik et al. formulated HA-modified graphene oxide (GO) and iron oxide NPs for targeted delivery of PTX and DOX. The resultant NPs exhibited cytotoxicity against breast cancer cell lines, BT-744 and MDA-MB-231. DOX and PTX were successfully loaded and the drug-loaded NPs significantly killed CD44-expressing MDA-MB-231 cells but did not kill the BT-474 cells that did not express CD44. Iron oxide was used in the formulation of the NPs to enable magnetic hyperthermia tumour cell targeting. In conclusion, the functionalized NPs demonstrated improved efficacy in killing tumour cells and thus can be used as a potential platform in cancer therapy . The stuAureobasidium pullulans. Pullulan is non-immunogenic, non-toxic, and non-carcinogenic and it exhibits liver specificity. It is a linear and unbranched polymer, made up of repeating malto-triose units connected by α (1→6) glycosidic bonds. It has essential physiological activity due to the vast number of hydroxyl groups found on the main chain. Its hydrophilic properties make it of great importance in delivering poor water-soluble anti-cancer drugs. [Pullulan is a hydrophilic biopolymer. It is produced by bi-morphic fungi, r drugs. ,57,58,59r drugs. . Althougr drugs. .w/w). The NPs remained stable in physiological conditions. Cell toxicity of P-Dox/S was analysed in comparison to free DOX-HCL, in vitro against 4T1 cells. A stronger DOX signal was demonstrated in the nuclei of cells treated with P-Dox/S, after 0.5 h of incubation. The cell nucleus of the group treated with P-DOX/S began to fade and apoptosis after 6 h of incubation, with nucleus almost disappearing after 17 h. In vivo tumour accumulation and biodistribution of the NPs were tested in 4T1 tumour-bearing nude mice, in comparison to another group treated with free DOX-HCl. Compared with the free DOX-HCl treatment group, the fluorescent signal accumulation of NPs groups was stronger and lasted longer. This may have been due to liver-targeting properties of pullulan, which was the major component of the nanoparticle carrier. These results led to the conclusion that co-delivery of the chemotherapeutics outperformed the conventional delivery of single/free drugs. Combination therapy has vast potential for use in the treatment of tumours exhibiting drug resistance properties [In 2017, Junhui Sui et al. synthesised pH-responsive pullulan-DOX conjugate NPs encapsulating sorafenib (SNB) (P-Dox/S). The formulated NPs were developed as a synergistic delivery system for the treatment of murine breast carcinoma. Characterisation results showed a drug loading capacity of 65.34% , such as, PGP, XLAP, VEGF, BCL2 and MRP-1. siRNA have shown great potential for overcoming drug-resistant genes, as well as improving sensitivity of the resistant tumours to chemotherapeutics. It is noted that the siRNA must be protected from RNAse degradation and there is a need to prevent off-target side effects. Hence, the use of nanotechnology-based delivery . siRNA rIn addition to the numerous engineered NP formulation platforms, nano-enabled systems are also used to develop chemotherapeutic delivery formulations. Liposomes, dendrimers, micelles, hydrogels, and metallic and carbon-based carriers can be used to increase chemotherapeutic efficacy, control drug release and improve cell targeting thus reducing cytotoxicity to normal cells . Each caDendrimers are branched polymeric molecules, which consist of uniform and well-defined shapes and sizes. Dendrimers basic structure consist of three main components—that is, the core, repetitive branching units and terminal groups . The arcThere are three types of dendrimers, namely, poly-amidoamine (PAMAM), poly-propylene imine (PPI) and poly-l-lysine (PLL). They all exhibit good biocompatibility, water solubility, flexibility and biodegradability. Dendrimers can be conjugated with other polymers, for example, PEG. Conjugation with other polymers such as PEG is not only used to reduce the cytotoxic effects of dendrimers, but it can also increase plasma circulation time and tumour accumulation, through enhanced permeability and retention effect . Dendrim2 in the PAMAM cavity. The hydrogen bonds, along with electrostatic interactions with the surface amino groups, enabled physical encapsulation of the drugs. Drug encapsulation led to sustained release of the drugs [The internal cavities of dendrimers, characteristic of their structure, allow for interaction with poorly water-soluble cancer drugs. Sanyamkamdhorn et al. investigated the intramolecular interaction of dendrimers with DOX and TFN ,76. Resuhe drugs .Liposomes are widely used in drug delivery for systemic administration of drugs. They are microscopic unilamellar or multilamellar particles, made up of membrane-like lipid layers . Their aActive site targeting can be achieved by grafting the liposomes with monoclonal antibodies (mAbs), antibody fragments, peptides and glycoproteins . LiposomJoon Myong Song et al. performed an investigation on the liposomal co-delivery-based quantitative evaluation of chemosensitivity enhancement, in breast cancer stem cells . They deA hydrogel is a swellable mesh of hydrophilic polymeric chains dispersed in water. Upon swelling, the hydrogels can release drugs previously loaded onto the mesh. Hydrogels have been and are currently being studied for use in oral delivery due to their polymeric chains which are able to closely interact with saliva glycoproteins . The intMicelles are amphiphilic molecules which self-assemble when in contact with a solvent. If the solvent is hydrophilic, the polar parts of the copolymer will be attracted toward the solvent while the hydrophobic components orient away from the solvent. For this to happen, the amphiphilic molecules in solution must exceed critical micelle concentration ,85. A hyA micellar system entrapped in a pH-responsive hydrogel was used by Wang L et al. to improve oral bioavailability of docetaxel (DTX). The polymeric micelles were formulated using poly-caprolactone (PCL) and PEG, to allow drug release in the intestinal lumen and subsequently absorption from the small intestine. The in vivo studies carried out showed a 10-fold increase in oral bioavailability of DTX and better tumour suppression against the 4T1 breast cancer cells used . In anotNanotechnology delivery systems have gained momentum over the past few years due to their beneficial characteristics such as having a large surface volume ratio, drug protection from degradation, control of drug release and hence allowing for the reduction of administered drug doses. As favourable as the delivery systems are, they also have certain limitations. The chemotherapeutic agents carried by the nano-delivery systems are non-cell specific. The unwanted attack on healthy cells is the main reason for the failure of conventional nano-cancer treatment. Hence, nanotechnology delivery systems have had to further advance to develop novel drug delivery strategies that are able to overcome non-specificity issues, thus the use of targeting moieties as shown in Surface modification of the nano-delivery systems allows for active targeted delivery of the therapeutic agent into the tumours. There are several compounds that can be used for surface modification, for example, antibodies and ligands such as TF, folic acid (FA), lactoferrins, lectins and mannose derivatives.FA the most commonly used compound for drug targeting applications, by conjugating it with anti-cancer drugs or nanocarriers. In one study, PAMAM dendrimers were amine-terminated and thus functionalised with FA and a fluorescein agent (isothiocyanate). This functionalised system acted as both a drug targeted system and a pH-responsive system for delivering DOX into tumour cells. The developed complexes displayed effective therapeutic efficacy and a high affinity for FA receptors, and thus were able to target the cancer cells and inhibit growth .Apart from conjugating targeting compounds onto the delivery system, targeted chemotherapeutic delivery can additionally be accomplished by chemically modifying the biopolymer platform to respond to stimuli. Stimuli responsiveness is advantageous in cancer therapy as it ensures anti-cancer drug delivery at target site, hence sparing the healthy cells. Stimuli can be classified as either internal or external, for example, pH and temperature, activity of enzymes, light, irradiation and magnetic fields . ChemicaTumours show high intra-tumoral heterogeneity, resulting in heterogeneous expression of targets for drug delivery . TargeteAnother complex issue that arises when it comes to targeted drug delivery is the delivery of the entrapped drugs at their site of action. Current targeting systems simply aim to offload the drug at the site of action, working on the theory that, once the delivery system is bound to the target , rapid internalisation occurs. Once internalisation has occurred, the drug is expected to be released and subsequently move to its site of action inside the cell, and not diffuse back into circulation . HoweverEven though targeting the tumour subsequently leads to internalisation at the target site, a wide range of drug delivery systems struggle to offload the drug because of failure to escape from the lysosomal compartments after internalisation ,95. LysoImpaired release of chemotherapeutics may limit treatment efficacy. Therefore, alternative strategies to tackle these issues are needed. Several polymer materials have already been approved for use as drug delivery materials by the Food and Drug Administration (FDA). However, the regulatory body is having a hard time keeping track with the rapidly increasing development of new polymer-based delivery systems. As of 2015, most of the FDA-approved delivery vehicles were either fully liposomal or PEGylated, with none of them addressing higher-level delivery complexities .With regards to the development and approval of more novel strategies, more data are required from the scientific community to prove efficacy of such formulations. The FDA also needs to play its part with a greater sense of urgency in order to keep track of these developments. The burden of proof is assumed to mainly rest on academia as well as private and public sector research facilities, in order to identify the critical factors relating to safety and efficacy of the novel products and strategies .The FDA generally classifies liposomes, dendrimers, micelles, nanoparticles and quantum dots as nanocarriers. From these, combination nanocarrier products have vastly gained interest over the years, leading to the development of co-encapsulation of synergistic cancer agents. The majority of the polymers of the formulations for cancer treatment, in clinical trials or having made their way into the market, are for intravenous use. There are currently several nanomedicines for cancer treatment on the market and in clinical trials. However, to date, a significant number of these formulations are for intravenous use. In addition, despite the FDA’s full inclusion of combination products on their regulatory evaluations, combination products typically have more hurdles to face before being accepted by the FDA.The FDA has established regulatory guidance draft documents to provide recommendations for meeting the regulatory requirements. The FDA recommends that companies developing novel delivery systems carry out in vitro and in vivo studies and include details on the population to be studied, blood sampling times and analytes to be measured in those blood samples .Interest in the application of biopolymers as oral delivery systems for chemotherapeutics is continuously increasing. This is due to polymers offering favourable characteristics, such as being biodegradable, biocompatible, non-toxic, hydrophilic and non-immunogenic. The use of biopolymer-based ODPs in cancer therapy allows for an increase in drug solubility, a reduction of dose, site-specific delivery and protection of the drug from the gastric environment. The drugs can be protected until they reach their site of absorption in the appropriate gastrointestinal segment for systemic circulation. Oral administration provides prolonged drug exposure, compared to intermittent intravenous infusion, which is an important factor for anti-cancer drugs. A drug with a short half-life can achieve greater systemic exposure by either prolonged infusion or by continuous oral dosing. Patients receiving chemotherapy prefer the oral route over the parental due to flexibility, convenience and the potential to reduce in-patient health care costs. Additional research comparing oral chemotherapeutics and parental chemotherapeutics are thus required as clinical trials. Hence, research further necessitates the use of biopolymers as ODPs for chemotherapeutics over a longer duration of evaluation. In addition, further studies are required to gather enough data in relation to toxicity of various polymers. Although many polymers show stability in vitro and ex vivo, these parameters may still need to be validated in vivo, using established diseased models. In the near future, ODPs have the potential to transform chemotherapeutic drug delivery, as they offer solutions to anti-cancer drug solubility issues, drug oral bioavailability and site specificity challenges.


Gastrointestinal stromal tumors are the most prevalent mesenchymal tumors of the gastrointestinal tract. Distant metastases are most often found in the liver or peritoneum with surgery being the preferred treatment option. In our center, fluorescence-guided surgery with indocyanine green is used as standard-of-care for hepatic metastases in colorectal cancer. This case report describes fluorescence-guided metastasectomy for a hepatic gastrointestinal stromal tumor in two patients undergoing open liver resection and radiofrequency ablation.A 69-year old women was seen during follow-up after laparoscopic resection of a GIST in the lesser curvature of the stomach. Contrast-enhanced computed tomography imaging showed two suspicious lesions in liver segment VI and VIII. Intraoperative near-infrared fluorescence imaging of the liver clearly revealed the lesion in segment VIII, and an additional lesion in segment V – which was not seen on preoperative CT-imaging, neither on intraoperative ultrasonography. The lesion in segment VI was not seen with NIRF imaging due to its deeper location in the liver parenchyma. The second case is an 82-year old man who was also diagnosed with liver metastases from a GIST in the stomach and was scheduled for near-infrared fluorescence-guided liver resection and radio frequency ablation.In this case report we demonstrated the feasibility of fluorescence-guided surgery in detection of liver metastases and treatment planning of two patients with hepatic GIST metastases using indocyanine green.NIRF-imaging with ICG is useful for identification of preoperatively discovered lesions, surgical resection planning and margin evaluation, and for detection of additional hepatic GIST metastases. OverallIndocyanine green (ICG) has been used as fluorescent dye for determining liver function, liver anatomy and for guiding liver resections, with increased utilization over the past decade. ICG binds to plasma proteins and is rapidly taken up by hepatocytes before its excretion into the biliary system. Near-infrared fluorescence (NIRF) imaging shows a variability in distribution of ICG between different tumor types . PreviouHere we report on two cases of open liver resection and radiofrequency ablation (RFA) of hepatic GIST metastases. Informed consent for publication was obtained from both patients and this work is reported according to the SCARE criteria ,9.22.1According to the hospital’s standard-of-care protocol for secondary liver tumors, both patients were admitted to the hospital and were intravenously injected with 10 mg (5 mg/mL) ICG  24 h prior to surgery. During the surgical procedure, after access to the abdominal cavity was acquired, a visual inspection of the abdomen was performed. After the liver was fully mobilized, intraoperative open-field NIRF-imaging of the liver was performed, prior to intraoperative ultrasonography (IOUS), using the Quest Spectrum system  to localize the metastases, and identify possible additional lesions. This was followed by resection and/or RFA of the lesions. The surgical procedures were performed by HH and JM, having 12 years of experience with NIRF-guided liver surgery. After surgery the resected specimens were assessed by a pathologist, and closed-field NIRF-imaging was performed with the Pearl Trilogy Imaging System  for further inspection of the tumor bread loaves.2.2A 69-year-old female was seen at the outpatient clinic for follow-up after resection of a GIST of the stomach (mitotic count 10/50 HPF). Other medical history was notable for laparoscopic cholecystectomy, pulmonary embolism, cerebral infarction, and pyramidal tract syndrome. The CT-scan revealed a suspicious superficial hypodense lesion of 34 × 44 mm in segment VIII and a 4 × 5 mm suspicious lesion in segment VI. In segments V and VIII two preexistent calcifications were found, thought to originate from intraoperative spillage of bile stones during previous cholecystectomy. There was no sign of recurrence of the primary tumor, and no lymph nodes or other distant metastases were found. The primary tumor was a ‘wild-type’ GIST and therefore neoadjuvant chemotherapy was not indicated [Intraoperative NIRF-imaging clearly revealed the lesion in segment VIII . The lesAfter completion of the resection, all resected specimens were cut into 5 mm bread loaves by the pathologist, followed by consecutive NIRF bread loaf imaging. The bread loaves were further processed for histopathological analysis and to determine resection margins. Immunohistochemical staining with Discovered on GIST 1 (DOG1), a gene encoding anoctamin 1 – a chloride channel protein  – was peFive months post-surgery a new hypodense lesion was found on CT during regular follow-up. After a multidisciplinary team meeting the patient was scheduled for percutaneous RFA. The first post-RFA scan showed no residual disease after RFA. However, two new liver lesions were found, for which the patient was referred to the oncologist for systemic therapy.2.3The second case represents an 82-year-old male with a solitary suspicious hypodense lesion in segment III of 21 mm, discovered on routine CT-scan during follow-up after previous partial gastrectomy for GIST (mitotic count 2/50 HPF). Previous medical history reported tuberculosis, acute myocardial infarctions, and a GIST in the stomach. After a multidisciplinary team meeting, the patient was planned for percutaneous, CT-guided RFA. However, the metastasis was located in close relation to the intestine and the RFA procedure was aborted. Instead a biopsy of the lesion was taken, confirming a metastasis from a primary GIST. The patient was planned for open resection of the metastasis. Prior to surgery, another CT-scan was performed, revealing a second 4 mm suspicious lesion in segment VIII. Because of its deeper location, the new lesion was considered to be better suitable for RFA.After mobilizing the liver, NIRF-imaging revealed the lesion in segment III a. The seClosed-field NIRF-imaging confirmed the intraoperative findings and showed tumor-free resection margins on the bread loaves b. Immuno3To the best of our knowledge, this is the first case report solely describing the use of NIRF-imaging with indocyanine green in patients with hepatic metastases from GIST. Positive resection margins and missed tumors following surgery are directly related to a decrease in disease free survival and 5-year survival rates in CRLM ,12, we eWe described the use of ICG in hepatic metastases from GIST in two patients in our institution. Intraoperative NIRF-imaging showed to be feasible and effective in both localizing preoperatively found superficial metastases as well as finding additional superficial lesions. ICG is currently used as standard-of-care as intraoperative fluorescent contrast agent in our center for liver metastases.NIRF light penetrates tissue up to 8 mm, allowing the surgeon to identify subcapsular lesions invisible for the naked eye. However, the limited penetration depth remains an important limitation for deeper located lesions. Therefore, IOUS is still required for the identification of deeper (>8 mm) lesions. NIRF-imaging with ICG has proven to be useful for surgical planning and evaluation of resection margins . ImagingNIRF-imaging has shown to be an effective imaging technique in the surgical treatment of CRLM, and now in hepatic GIST metastases as well.4NIRF-imaging with ICG is useful for identification of preoperatively discovered lesions, surgical resection planning and margin evaluation, and for detection of additional hepatic GIST metastases for capsular lesions and subcapsular lesions up to 8 mm below the liver surface. Because of the limited penetration depth of NIRF-imaging should be used as an additional imaging technique next to IOUS. Development of tumor-specific fluorophores for primary GIST and metastases can aid in more accurate detection of these tumors and may lead to lower false-positive rates.The authors report no declarations of interest.No funding was received.According to the local medical ethics committee (METC-LDD) no formal ethical approval was required.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Not applicableOkker D. Bijlstra and Alexander L. Vahrmeijer.Not commissioned, externally peer-reviewed.O.D. Bijlstra: Conceptualization, Methodology, Validation, Formal analysis, Writing - original draft. F.B. Achterberg: Methodology, Writing - original draft. Q.R.J.G. Tummers: Writing - review & editing. J.S.D. Mieog: Writing - review & editing. H.H. Hartgrink: Writing - review & editing. A.L. Vahrmeijer: Writing - review & editing, Supervision.


Background: Antisynthetase syndrome (ASyS) is a rare autoimmune disease characterized by inflammatory myopathy, arthritis, fever, and interstitial lung disease (ILD). Pulmonary involvement in ASyS significantly increases morbidity and mortality and, therefore, requires prompt and effective immunosuppressive treatment. Owing to the rarity of ASyS, limited data exists on progression and prognosis of ILD under immunosuppression.Objectives: The objective of the study was to evaluate the radiological progression and outcome measures of ILD with immunosuppressive therapy in patients with ASyS.Methods: Twelve patients with ASyS-associated ILD (ASyS-ILD) were included. Demographic and clinical data, including organ involvement, pulmonary function tests (PFT), laboratory parameters, imaging studies, and treatment regimens were retrospectively analyzed from routinely collected data. The extent of ground glass opacities, fibrotic changes and honeycombing was analyzed and scored using high-resolution chest computed tomography (HRCT) scans. HRCT findings were compared between baseline and follow-up examinations. In addition, patients were stratified depending on whether they had received rituximab (RTX) or not.Results: Pulmonary function tests revealed stable lung function and follow-up HRCT scans showed an improvement of radiological alterations in the majority of ASyS patients under immunosuppressive therapy. We did not detect significant differences between the RTX- and non-RTX-treated groups, but the RTX-treated patients more frequently had myositis and relapsing disease.Conclusions: Radiographic alterations in ASyS-associated ILD respond to immunosuppressive treatment. RTX is a feasible treatment option with similar clinical and radiographic outcomes in patients with relapsing disease and clinically apparent myositis. Antisynthetase syndrome (ASyS) is a rare autoimmune disease, belonging to the idiopathic inflammatory myopathies (IIM) . Due to In this single-center cohort study, we studied the effect of immunosuppression on high-resolution chest computed tomography (HRCT) findings in the course of ASyS-ILD, focusing particular on RTX.This retrospective observational study used routinely collected clinical data in patients with ASyS. The clinical care of patients with IIMs is organized in an interdisciplinary way among the Departments of Rheumatology, Neurology, Pulmonology, Dermatology, and Neuropathology, and relies on the use of standardized operating procedures. Management decisions are discussed and evaluated in multidisciplinary case conferences held on a monthly basis .All patients fulfilling at least two or more clinical findings consistent with ASyS  were recruited from the University Medical Center Goettingen and their medical records were independently reviewed by three investigators who extracted the data . Additional patients were identified by the analysis of positively detected ARS abs at our DIN:ISO 2001 certified autoimmune laboratory.Tests for myositis-associated (MAA) and myositis-specific (MSA) antibodies including Mi-2 alpha, Mi-2 beta, TIF1 gamma, MDA5, NXP2, SAE1, Ku, PM-Scl100, PM-Scl75, Jo-1, SRP, PL-7, PL-12, EJ, OJ, and Ro-52 were performed using the 16 Ag EUROLINE Blot . The presence of additional antibodies was examined using the Elia™ SymphonyS test assay , which screens for, among others, the presence of anti-SSA antibodies (60 and 52 kDa). Anti-SSA-antibodies have been reported in about 50% of patients with incomplete forms of ASyS . PatientDemographic data and clinical parameters were retrieved from patients' medical records. We evaluated the presence and spectrum of specific abs, organ involvement, laboratory parameters, pulmonary function tests (PFT), and imaging procedures in each patient. Histologic evidence of organ involvement was recorded, if available. As outcome measures for pulmonary involvement, we assessed the alteration of lung parenchyma on HRCT as well as PFTs before and during treatment. To assess the effect of immunosuppression, we recorded all patients' individual therapies used between the first and any subsequent follow-up HRCT scans. Progressive ILD was defined as worsening on imaging studies or worsening of PFT [at least a 10% decline of forced vital capacity (FVC) or at least a 15% decline of diffusion capacity for carbon monoxide (DLCO)].Baseline and follow-up HRCT scans were obtained with 4-, 16-, 64-, and 128-slice scanners from 2011–2019 at the same institute. The scans were interpreted and scored independently and blinded to patient identity and clinical details by a senior registrar-level radiology resident (EMS) and a board-certified thoracic radiologist (JL) with 4 and 22 years of experience, respectively.Scleroderma Lung Study by Goldin et al.  or mixed effects analysis with Tukey test as post-hoc analysis for multiple comparisons (more than two groups). Mixed effects analysis was used because repeated measures analysis of variance (ANOVA) cannot handle missing values. We analyzed the data instead by fitting a mixed model. This mixed model uses a compound symmetry covariance matrix and is fit using Restricted Maximum Likelihood (REML). In the absence of missing values, this method gives the same P-values and multiple comparisons tests as repeated measures ANOVA. In the presence of missing values (missing completely at random), the results can be interpreted like repeated measures ANOVA. Geisser-Greenhouse correction was used.Demographic data of the study population were analyzed by descriptive statistics. The Shapiro-Wilk test was used for testing normal (Gaussian) distribution. Parametric between-group-comparisons were performed with either the Student's P < 0.05 were considered statistically significant. Data analyses were performed with GraphPad Prism  or STATA .Non-parametric between-group-comparisons were performed with Fisher's exact test. The interrater agreement of HRCT scores between the two radiologists was assessed by the weighted kappa statistic. Values below 0.20 were considered as poor, 0.21–0.40 fair, 0.41–0.60 moderate, 0.61–0.80 good, and 0.81–1.0 as very good agreement. We identified 22 patients with positive ARS abs. One additional patient met clinical criteria  for ASyS but tested positive for anti-RO52 abs only. Testing for antinuclear antibodies in this patient revealed a cytoplasmic fine-speckled staining pattern.Of the 22 patients with positive ARS abs, nine were excluded because they did not have ILD; one patient was not eligible due to incomplete data. Therefore, a total number of 12 patients  was included in the final analysis . Of thesMedian follow-up time was 31 (6–156) months. Demographic and clinical characteristics are presented in Anti-Jo-1 abs were present in 10 of 12 patients (83.3%). In addition, 10 of these 12 patients also tested positive for anti-SSA .Arthritis (joint pain and swelling) was clinically evident in 7 patients (58%), mainly involving the hands. Myositis  was present in five patients  and was the second most common manifestation.Creatine kinase levels during follow-up remained stable or improved in all cases. Two of the patients had a remote history of breast cancer, one patient had received a diagnosis of ASyS during pregnancy related to ovarian cancer.Ten individuals received glucocorticoids (GC) while seven patients were treated with RTX during the study period. The reasons for RTX initiation were ILD progression in four patients , ILD at the time of ASyS diagnosis in two patients  and treatment of concomitant anti-CCP antibody positive rheumatoid arthritis in one patient (14.3%). Equally, relapsing disease with ILD flares and clinically apparent myositis in 5/12 (41.6%) patients led to RTX initiation. Six patients  received azathioprine (AZA). Less frequently used immunosuppressants were MTX in three and MMF in two patients. CYC (one patient), leflunomide , hydroxychloroquine , and adalimumab  were used infrequently. The individual therapeutic regimens are presented in PFTs were obtained at baseline and throughout the course of the study. Forced vital capacity (FVC) and diffusion capacity for carbon monoxide  are presented in HRCT of the chest was performed for each of the 12 patients at baseline (CT1). A second CT scan (CT2) was available in 10 patients after a median time span of 14.5 months (6–72). A third CT scan (CT3) was available in six patients, at a median time of 30 months (17–156) after the first scan.The interrater agreement κ between the two radiologists was 0.82 for GGO, 0.54 for FIB and 0.05 for HC, corresponding to very good (GGO), moderate (FIB), and poor (HC) agreement, respectively.Seven patients who received RTX during the study period were compared with five patients without RTX treatment. The total CT scores in the RTX vs. non-RTX groups are presented in Also, the CT scores for the specific findings of GGO, FIB, and HC showed a progressive decrease with treatment over time . This waNo patient died during the study. Radiological findings improved in most patients, exemplified by a decrease of the GGO and FIB scores. We did not observe worsening of the low baseline scores for HC. PFTs were stable or improved in the majority of patients. One patient developed pneumonia and sepsis from a urological source under immunosuppression with the need for hospitalization. No other serious adverse events were documented.We have shown that pulmonary outcomes, as assessed by PFT and HRCT, did not differ between the varying therapeutic regimens. Nevertheless, the use of RTX was employed in patients with more severe disease as demonstrated by a numerically  higher GGO score at baseline, more frequent relapses, and a higher prevalence of myositis.The prevalence of anti-Jo1 abs in our patient cohort was 83.3%. Anti-SS-A abs can be detected in about half of the patients with ASyS ; in our Myositis was histologically proven in 41.7% of our patients. Most case series and registries include ASyS patients when myositis becomes clinically apparent or patients initially diagnosed with IIM are diagnosed as ASyS. For this reason, the reported frequency of 75% for myositis in ASyS may be lower early in the course of the disease . MoreoveGiven the rarity of ASyS, little is known about the long-term effects of immunosuppressive therapy on the course of ILD. Lung involvement represents the most serious and life-threatening complication of ASyS, and, therefore, early commencement of an adequate therapy is crucial. The therapeutic response of the disease to immunosuppression can be assessed by the course of the ILD. Normal PFTs at the initiation of treatment are associated with stable or even an improved course of ASyS-ILD, whereas dyspnea and decreasing FVC correlate with a poor prognosis and progression of ILD in ASyS . The majWith respect to pulmonary function and overall outcome, RTX is a promising agent, especially early in the course of ASyS-ILD , findingIn two of seven cases treated with RTX, the drug was started as a first-line therapy due to an early manifestation of ASyS-ILD. Additionally, our data demonstrate that other immunosuppressive agents also lead to improved HRCT imaging findings over time, but these patients differed in terms of disease severity and extrapulmonary manifestations.The limitations of our study include its retrospective design and the small sample size lacking a control group. The intervals between follow-up HRCT scans varied considerably. Although we only examined patients with the presence of ILD at baseline (the first available HRCT), a clinically apparent lung disease may not have been evident in all patients at first presentation but may have developed over time. Abnormal radiology findings indicate an active underlying pulmonary disease which has not yet become apparent clinically. Consequently, the effect of immunosuppressive treatment must be interpreted with these limitations in mind. Also, we did not assess dyspnea scores, such as the St. George's respiratory questionnaire, in all patients since these are not consistently performed routinely in non-pulmonary clinics. The interrater agreement showed a moderate discrepancy in the assessment of HC. This has also been observed other studies, such as the Scleroderma Lung Study . HoweverIn conclusion, our results indicate a trend toward an improvement of ASyS-ILD outcome under treatment with RTX and other immunosuppressive therapies as well as a stabilization of PFT. RTX seems to be superior in patients with a higher number of clinical manifestations, including ASyS-ILD, myositis, arthritis, and in patients with relapsing disease. Nevertheless, prospective trials with pre-specified endpoints are required to further elucidate the impact of immunosuppression on progression and outcome of ASyS-ILD.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the ethics committee of the University Medical Center Goettingen (Protocol no. 4/8/19). All patients consented to the use of their routinely collected data for research purposes.PK conceived the study, treated patients, abstracted and analyzed data, created the figures, and wrote the manuscript. JG-R abstracted and analyzed data, created the tables, and co-wrote the manuscript. LR abstracted data, reviewed and revised the manuscript critically. EM-S scored imaging data, contributed to the methodology, and revised the manuscript critically. UO analyzed data and revised the manuscript. CS and JS treated the patients, analyzed data, and critically revised the manuscript. JL scored imaging data, contributed figures, and revised the manuscript critically. RV conceived the study, analyzed data, co-wrote, and revised the manuscript critically. All authors contributed to the article and approved the submitted version.PK has received honoraria and travel support by Abbvie, Bristol-Myers-Squibb, Chugai, Gilead, Glaxo Smith Kline, Janssen-Cilag, Pfizer, and Sanofi-Aventis, all unrelated to this study. JG-R has received travel support by Abbvie and Janssen-Cilag, unrelated to this study. JS has received payments for advisory boards, honoraria, travel expenses, and research projects from Alnylam, Bayer, Biogen, BioMarin, Biotest, CSL Behring, Grifols, LFB, Novartis, Octapharma, Pfizer, all unrelated to this study. JL reported honoraria from Toshiba Medical Systems and Bayer Pharma AG as well as travel support from Boston Scientific, all unrelated to this study. RV has received honoraria and travel support by Abbvie, Janssen-Cilag, Pfizer and Sanofi-Aventis, all unrelated to this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Results from several studies show that only obese, unfit subjects, but not obese, fit subjects, are at higher mortality risk than are normal-weight fit subjects. The aim of the study was two-fold: (1) to examine the differences in C-reactive protein levels across different metabolic phenotypes  of weight status and (2) ascertain whether high levels of cardiorespiratory fitness (CRF) attenuate the association of C-reactive protein and metabolic phenotypes of weight status. This was a pooled study, which included data from three cross-sectional projects (1706 youth (921 girls) aged 12–18 years). We used a Shuttle run test to assess CRF. Adolescents were classified into six metabolic phenotypes  of weight status , based on age- and sex-specific cutoff points for triglycerides, systolic blood pressure, HDL-cholesterol, glucose and body mass index. High-sensitivity assays were used to obtain the C-reactive protein as inflammatory biomarker. After adjustment for potential confounders , the analysis of covariance (ANCOVA) shows that C-reactive protein is directly associated with metabolic phenotypes of weight status. Subjects with obesity, regardless of their metabolic profile, had higher levels of C-reactive protein Z-score. In addition,  a two-way ANCOVA showed that high levels of CRF attenuated the associations of C-reactive protein levels in metabolic healthy non-overweight and in adolescents with obesity. In conclusion, higher CRF levels may attenuate the detrimental association between obesity and C-reactive protein independently of metabolic phenotype. Findings from this study are important for prevention, clinical practice on issues associated with adiposity and metabolic disorders. However, accumulated evidence has shown that some obese subjects could have a healthy metabolic profile , and subjects with normal weight could present an abnormal metabolic profile  . Those w factors . There i factors ,2.Chronic inflammation has a key role as a pathogenic mechanism in the origin and development of cardiovascular diseases (CVD) . Of the In the same line, the assessment of fitness during adolescence has called for a better understanding of the interrelationship between body composition and health-related physical fitness in youth . SimilarResults from several studies show that only obese, unfit , but not obese and fit subjects, are at higher mortality risk than normal-weight fit subjects . Based oWe pooled data from three studies, Fuprecol study , the Lab2). Weight and standing height were measured, respectively, with a stadiometer  and an portable electronic weight scale . Body mass index (BMI) was calculated as the ratio of body weight (kg) to body standing height . The adolescents were classified in two groups (low CRF and high CRF), according to the proposed cutoff for this population by Ruiz et al. [Assessment of CRF was done by the 20 m Shuttle Run Test (20 m SRT), as described previously [z et al. . ® equipment  was used to determine concentrations of fasting glucose, TG and HDL-c. For the Azorean Physical Activity and Health Study II, high-sensitive C-reactive protein was measured by enzyme-linked immunosorbent assay . Fasting glucose, TG and HDL-c were measured on a Cobas Integra 400 Plus, by colorimetric methods . The LabMed study used high-sensitive CRP, latex-enhanced immunoturbidimetric assay (Siemens Advia 1600/1800) and measured HDL cholesterol by precipitation of the apolipoprotein B containing lipoproteins with dextran-magnesium-chloride method ; fasting glucose by hexokinase method ; and TG levels by enzyme glycerol phosphate oxidase method . Finally, blood pressure determination was made after the participants had been sitting for at least 5 min, with an automated blood pressure monitor .Blood samples were collected (for Cholesterol linked to high-density lipoproteins (HDL-c), serum triglycerides (TG), fasting glucose and high-sensitive CRP after an overnight fast, according to standardized procedures, as follows: For the Fuprecol study, high-sensitive C-reactive protein was obtained, using the turbidimetric method with QuikRead 101 equipment (Both Orion Diagnostic). CardiocheckAll the adolescents self-assessed their pubertal stage (ranging from stage I to V for boys and girls separately) of secondary sex characteristics based on the Tanner staging system ; for girMetabolically (un)healthy individuals were defined as having 0  or 1  of the following risk factors: TG, glucose, HDL-c and diastolic or systolic blood pressure. For this, we used age- and sex-specific cut-points for the metabolic phenotype classification that has been published for adults  . Descriptive data are presented as means and standard deviation, unless otherwise stated. The assumptions of normality and data distribution were assessed by the Kolmogorov–Smirnov test. Because of a non-normal distribution, high-sensitive CRP values were standardized prior to the analyses /standard deviation). Interaction between our independent variable and databases was tested, and no significant interaction was observed. The one-way ANCOVA  with Bonferroni correction adjusted for age, sex, pubertal stage and country were used to assess the differences between mean values of high-sensitive CRP z-scores across six metabolic phenotypes of weight status. Differences in the high-sensitive CRP according to metabolic phenotypes’ categories and CRF status were analyzed, using a two-way ANCOVA to obtain adjusted means. Additionally, the one-way ANCOVA was used to test the influence of CRF status within each metabolic phenotype ((un)healthy in individuals with normal weight, overweight and obesity) adjusted for age, sex, pubertal stage and country. Bonferroni post hoc multiple comparison tests were used to assess the differences between Fit and Unfit. A polynomial linear test was used to assess linear trends . Descriptive characteristics of the participants, according to weight status and metabolic health status, are presented in p < 0.001), high-sensitive CRP , Diastolic BP , Triglycerides  and total cholesterol , as well as a direct association with HDL-cholesterol , after adjustments for age, sex, pubertal stage and country.The regression analyses  showed ap for trend <0.001), after adjustments for age, sex, pubertal stage and country. p = 0.03). Our study showed that high-sensitive CRP levels elevated linearly across weight status, without significant differences between metabolic phenotypes. Furthermore, these results also suggest that high levels of CRF attenuates the association of CRP levels, particularly with participants within the MHNO, MHOB and MUOB categories. These results add to the findings of the existing literature about the relevance of CRF on inflammation throughout weight status and metabolic phenotypes categories. Moreover, our findings support previous studies which demonstrated that obesity and the metabolic disorders’ risk in children and adolescents are associated with low-grade inflammation, especially on CRP ,33. IndeSome studies have shown that MHOB subjects with low CRP levels had a trend toward lowering CVD risk than those MHOB with high levels of CRP, along with a similar risk to that subjects with healthy profile and non-obese . Our finSeveral epidemiology studies have widely adopted BMI as a marker of adiposity; however, this measure has been questioned by some authors who see it as being inaccurate . NeverthFuture studies are needed in order to confirm the extent that weight status and CRF remains at cardiometabolic disease risk compared with counterparts with no excess weight. For example, exercise interventions in youth have shown to improve CRF on average by 30%, including among children and adolescents with overweight and obesity . Our resThe current study is subject to certain limitations. First, the cross-sectional design of the study made it impossible to draw any causal inferences. Furthermore, blood samples only reflect inflammatory status at one time point. Additional biomarkers, such as fibrinogen, leptin, adiponectin, interleukin-6, interferon alpha and complementary factors could help to address a more complete inflammatory profile. Finally, the current results should be interpreted with caution, since no direct measure of absolute CRF was available in our study.This study has several strengths. The first strength is the novelty of analyzing the data according with the metabolic profile in adolescents. In addition, the relatively large sample size, particularly with data derived from blood samples, is also a strength.In summary, this large analysis suggests that CRF is a potential mechanism underlying the associations between weight status, metabolic profile and inflammation. Additional longitudinal analyses shall be conducted to further investigate the causal relationship between these variables. Finally, the role of other lifestyles determinants—in particular, sleep and dietary intake—shall further be investigated as potential confounders of the associations between CRF, weight status, metabolic profile and inflammation.