Despite evidence that preferential use of balanced/buffered fluids may improve outcomes compared with chloride-rich 0.9% saline, saline remains the most commonly used fluid for children with septic shock. We aim to determine if resuscitation with balanced/buffered fluids as part of usual care will improve outcomes, in part through reduced kidney injury and without an increase in adverse effects, compared to 0.9% saline for children with septic shock.The Pragmatic Pediatric Trial of Balanced versus Normal Saline Fluid in Sepsis (PRoMPT BOLUS) study is an international, open-label pragmatic interventional trial being conducted at > 40 sites in the USA, Canada, and Australia/New Zealand starting on August 25, 2020, and continuing for 5 years. Children > 6 months to < 18 years treated for suspected septic shock with abnormal perfusion in an emergency department will be randomized to receive either balanced/buffered crystalloids (intervention) or 0.9% saline (control) for initial resuscitation and maintenance fluids for up to 48 h. Eligible patients are enrolled and randomized using serially numbered, opaque envelopes concurrent with clinical care. Given the life-threatening nature of septic shock and narrow therapeutic window to start fluid resuscitation, patients may be enrolled under “exception from informed consent” in the USA or “deferred consent” in Canada and Australia/New Zealand. Other than fluid type, all decisions about timing, volume, and rate of fluid administration remain at the discretion of the treating clinicians. For pragmatic reasons, clinicians will not be blinded to study fluid type. Anticipated enrollment is 8800 patients. The primary outcome will be major adverse kidney events within 30 days (MAKE30), a composite of death, renal replacement therapy, and persistent kidney dysfunction. Additional effectiveness, safety, and biologic outcomes will also be analyzed.PRoMPT BOLUS will provide high-quality evidence for the comparative effectiveness of buffered/balanced crystalloids versus 0.9% saline for the initial fluid management of children with suspected septic shock in emergency settings.ClinicalTrials.gov (NCT04102371) on September 25, 2019. Enrollment started on August 25, 2020.PRoMPT BOLUS was first registered at The online version contains supplementary material available at 10.1186/s13063-021-05717-4. Crystalloid fluid is part of the initial resuscitation for 25 million children with septic shock worldwide each year . OptionsThe Isotonic Solutions and Major Adverse Renal Events Trial (SMART) found a small, but significant, reduction in major adverse kidney events within 30 days (MAKE30) with the use of balanced/buffered fluids compared to 0.9% saline in critically ill adults . The larThe primary aim of the Pragmatic Pediatric Trial of Balanced versus Normal Saline Fluid in Sepsis (PRoMPT BOLUS) is to determine if resuscitation with balanced/buffered fluids as part of usual care will improve outcomes compared to 0.9% saline for children with septic shock. Secondary aims are to compare the relative safety and determine the differential effect of each fluid type on kidney injury biomarkers in children with septic shock. We hypothesize that balanced/buffered fluid resuscitation will reduce MAKE30  compared to 0.9% saline, in part through reduced kidney injury, without an increase in adverse effects. Notably, even a small clinical benefit would be important because sepsis is common, life-threatening, and the proposed practice change is inexpensive and readily available.This manuscript was written in accordance with Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines  in the United States (US), Pediatric Emergency Research Canada (PERC), and Pediatric Research in Emergency Departments International Collaborative (PREDICT) in Australia/New Zealand. An international steering committee comprised of the overall study principal investigators  and network-specific principal investigators from each geographic network will oversee all study activities. Enrollment started August 25, 2020, and is anticipated to continue for 5 years. Patients will only be enrolled in an emergency department (ED), where initial resuscitation of suspected septic shock will begin. Ongoing inpatient management of septic shock may occur on any hospital ward or intensive/critical care unit.All patients > 6 months to < 18 years treated for suspected septic shock with parenteral antibiotics and at least one fluid bolus for abnormal perfusion in a study site ED will be eligible for study enrollment. The lower age was selected to adhere to the US Food and Drug Administration (FDA) warning that infants < 6 months may have reduced hepatic capacity to metabolize exogenous lactate in LR. “Suspected septic shock” is operationalized as a) the treating clinician’s diagnosis of septic shock and/or treatment that includes parenteral antibiotic and fluid resuscitation for abnormal perfusion or b) a sepsis alert on a site-specific screening tool with clinician confirmation to proceed with treatment for suspected septic shock. Abnormal perfusion is defined as the treating clinician’s judgment that hypotension or abnormal (either “flash” or “prolonged”) capillary refill is present. To reflect usual clinical practice, we do not define thresholds for hypotension or abnormal capillary refill but rather defer to the treating clinician’s discretion to differentiate abnormal from normal. Recognizing that fluid resuscitation may start prior to ED arrival, only those for whom total volume of crystalloid fluid administration can be confirmed as ≤40 mL/kg will be eligible.Exclusion criteria are (1) clinician judgment that the patient’s condition deems it unsafe to administer either 0.9% saline or balanced/buffered fluids  will provide regulatory approval and oversight for all US study sites. In Canada, the University of Calgary will sponsor regulatory approval through Health Canada, while each site’s Research Ethics Board will oversee local human subjects’ protections . In Australia and New Zealand, a single ethics committee will provide regulatory oversight within each jurisdiction. Protocol modifications approved by the international steering committee will be first reviewed by the CHOP Institutional Review Board and, following approval, will be distributed by the network-specific principal investigators to all other regulatory bodies in the USA , Canada, Australia, and New Zealand, as well as to local investigators.when sufficient time permits. However, because of the life-threatening nature of septic shock and narrow therapeutic window to commence fluid resuscitation, enrollment may proceed in the USA under the federal “Exception From Informed Consent”  for emergency research . RR. RR38].  and sex . SecondaThe rationale for MAKE30 as the primary endpoint is that mediation of kidney injury provides the major mechanistic pathway through which balanced/buffered fluids are likely to improve patient outcomes. In addition, the only prior comparable randomized clinical trials demonstrated a reduction in MAKE30 with the use of balanced/buffered fluids in adult patients with sepsis , 18. FinAdverse events (AEs) will be queried from the medical record at the end of the intervention phase and between study days 5–7. We will limit the time period for the determination of AEs because we are unaware of any biologically plausible reason to anticipate that either fluid type should lead to AEs remote from the immediate intervention period. AEs that are unexpected, serious, and at least possibly related to study fluid type will be reviewed by the international steering committee and reported promptly to regulatory bodies.A baseline incidence of MAKE30 in children with septic shock predominantly resuscitated with 0.9% saline was determined based on a prior pediatric study (9.6% MAKE30) , the proRecruitment of this large sample size requires collaboration across > 40 sites from three research networks. Based on the enrollment of 85% of eligible patients in our pilot study , sites aThe study will be monitored by a Data Safety Monitoring Board (DSMB) comprised of 10 international members. Interim monitoring for the superiority of one treatment approach will be performed after enrollment of 15%, 40%, and 70% of total study participants. The significance levels at each interim analysis for the difference in MAKE30 between groups are 0.000000014, 0.00079, and 0.014, respectively, using the symmetric two-sided O’Brien-Fleming boundaries. The DSMB may also consider early termination of the trial if there is evidence of futility using an informal, conditional power approach .The primary outcome of MAKE30 will be reported using the Mantel-Haenszel (MH) relative risk, stratified by the study site. The primary analysis will be performed using the intention-to-treat (ITT) principle, including all randomized participants for whom the three components of the MAKE30 outcome are not missing, regardless of whether study fluid was administered. To account for the three planned interim analyses, the final analysis will use a significance level of 0.044 for the primary outcome of MAKE30.Secondary outcomes will also be analyzed using the ITT approach incorporating methods of Benjamini and Hochberg to control for multiple analyses , 43. TheA per-protocol analysis will include all participants who receive at least 75% of total crystalloid fluid volume (bolus and maintenance) during the intervention window as the fluid to which the participant was randomized. Because the study intervention (fluid type) is available outside of the study, participants who continue to receive the same fluid to which they were randomized after withdrawal from the study protocol will remain eligible for the per-protocol analysis if they meet the ≥75% threshold. Participants who are found to have been ineligible for enrollment  or who receive no crystalloid fluid after randomization will be excluded from the per-protocol analysis. To account for potential confounding introduced into the per-protocol analysis, we will use generalized mixed effect regression models to adjust for known covariate imbalances. We will also conduct an analysis using instrumental variables to adjust for potential bias created by treatment contamination .We will evaluate treatment differences among subgroups defined by the following stratification variables: age group, sex, cancer comorbidity, AKI at study enrollment, abnormal kidney function using day 0 biomarker measurements Table , total fand all three country-specific percentages missing the primary outcome is ≤7% of country-specific enrollment. If these criteria are not met, we will use multiple imputation for the primary analysis with data assumed to be missing at random unless available data contradict this assumption. If the primary outcome is missing from between 1% and 5% of total participants, we will perform an adjunctive analysis using multiple imputation in addition to the complete case analysis. In addition, we will examine differences between participants with and without missing data and conduct a sensitivity analysis using the inverse-probability-weighting (IPW) method to conduct multiple imputation for the missing data [post hoc and considered hypothesis-generating.The primary ITT analysis will use complete case data if the percentage missing the primary outcome is ≤5% of total enrollment ing data , 47. NewClinicalTrials.gov, including the full study protocol and statistical code. Authorship will adhere to the International Committee of Medical Journal Editors guidelines. To ensure widespread access to study findings, we will seek publication using “open access”. After the publication of main and secondary results, a public use dataset will be submitted to the Eunice Kennedy Shriver National Institute of Child Health and Development Data and Specimen Hub (DASH).Trial results will be communicated to the public through manuscript publication and The flow of participants through the study will be presented as in Fig. PRoMPT BOLUS will provide high-quality evidence for the comparative effectiveness of buffered/balanced crystalloids versus 0.9% saline for the initial fluid management of children with suspected septic shock in emergency settings. Currently, there is insufficient evidence to support one crystalloid fluid type for pediatric shock resuscitation, which was acknowledged as a key knowledge gap by the 2020 Surviving Sepsis Campaign guidelines , 22. ShoWe selected a pragmatic design for several reasons , 49. Firpredominant—rather than exclusive—use of balanced/buffered fluids versus 0.9% saline. Even if exclusive use of a single fluid type could be executed under controlled experimental conditions, it is unlikely that such a practice could be generalized to real-world fluid management.The decision to use patient-level randomization rather than an alternative method based on systematic allocation  or cluster randomization ensured that allocation concealment was maintained until after patient enrollment despite not blinding the study intervention. We were concerned that pre-enrollment awareness of treatment allocation could lead to selection bias. Importantly, there was no allocation strategy we believed could eliminate cross-over between groups. Selection of fluid type is dictated by multiple factors, including a potential lack of personal equipoise across providers, numerous clinical indications for specified fluid and electrolyte titration, ongoing debate about the utility of acid buffers in acutely ill patients , 51, andand the fluid is directed to treat abnormal perfusion rather than dehydration or hypovolemia. This approach balances early enrollment, when it may not yet be clear how much fluid a patient may require, with enrichment of the study population by children for whom fluid type is most likely to have a clinical impact. In our pilot study using the same enrollment criteria, 42% were admitted to the intensive care unit [Several potential threats to the validity of our trial exist. First, enrollment of children with suspected septic shock exclusively in the ED will include a wide range of illness severity and fluid exposure. Prior studies have demonstrated that fluid type has a negligible clinical impact when exposure is limited to low volumes , 18. Theare unit , which rare unit , 52 and are unit , we planclinicalTrials.gov/NCT04102371) under this protocol version 1, dated May 25, 2021.PRoMPT BOLUS is a pragmatic randomized interventional trial of the effectiveness and safety of fluid resuscitation with balanced/buffered fluids compared to 0.9% saline for children with septic shock as part of usual clinical care. Patient enrollment began on August 25, 2020, and is anticipated for 5 years (Additional file 1. SPIRIT 2013 Checklist: Recommended Items to Address in a Clinical Trial Protocol and Related DocumentsAdditional file 2. Suggested Unsafe Conditions for Study EnrollmentAdditional file 3. Data Collection Planned for the PRoMPT BOLUS Pragmatic Clinical TrialAdditional file 4. Analysis Plan for Secondary Outcomes


Scott M. Hammer, on 17 November 2021. Scott is widely acknowledged as having been a brilliant researcher, a gifted clinician and a devoted mentor. He was born in New York City and obtained his undergraduate and medical degrees at Columbia University. This was followed by training in internal medicine and infectious diseases at New York Presbyterian Hospital, Stanford University Hospital and Massachusetts General Hospital, where he trained with many of the titans of the Infectious Disease field. He spent his professional career first at the New England Deaconess Hospital in Boston, and then at Presbyterian Hospital in New York City and the Columbia University Irving Medical Center, taking on the position of Harold C. Neu Professor of Infectious Diseases and Professor of Epidemiology. He expanded the work of the Division of Infectious Diseases in his capacity as Division Chief, focusing on growing its research portfolio.Scott's career began as the HIV epidemic in the United States unfolded and, in many ways, the epidemic shaped his career, which was true for many infectious disease specialists of that era. Few others, however, were able to shape the epidemic in the ways that Scott did. The suffering of so many patients compelled him to seek to identify effective treatments for HIV. He fully engaged with the U.S. NIH‐funded AIDS Clinical Trials Group and led pivotal studies that identified life‐saving antiretroviral regimens at a time when few options existed. His contributions to HIV virology, HIV resistance and the evaluation of virological and immunological markers among persons living with HIV on and off treatment furthered our understanding of the pathogenesis of HIV infection. When the major advances achieved by Scott and others allowed HIV to be transformed into a chronic and manageable condition, he turned his attention to the challenge of HIV prevention. Scott focused in particular on the quest for an HIV vaccine. He was tireless in the pursuit of this goal, and as a leader in the NIH‐funded HIV Vaccine Trials Network, he engaged deeply in the design and implementation of several critical vaccine studies.Some use the expression “a doctor's doctor,” and Scott epitomized this phrase. His clinical acumen was legendary, never resting until he arrived at a diagnosis and a way forward for his patients. He genuinely cared for his patients, and this was palpable to all, particularly to them. His colleagues looked to him for his insights when they exhausted all options, when they were unable to reach a diagnosis or were seeking a way to reassure a patient. His ability to listen carefully and to pull all the strands of evidence together into a coherent whole was not only impressive to his colleagues, but also inspiring to students and trainees.Medicine is in many ways an apprenticeship. While knowledge is critically important for the creation of skilled physicians, beyond that, the art of medicine is learned by observing mentors and emulating them. For generations of students and trainees, Scott was an incomparable teacher and mentor. He demonstrated compassion, wisdom, patience and respect in his every interaction, leaving an indelible mark on all those who were fortunate to be mentored by him over the years.While our professional careers have intersected with Scott's over the past several decades with many wonderful memories of productive discussions and exciting work, we look back to the uniqueness of his contributions. Beyond his many stellar academic achievements, Scott's legacy is his overarching humanity. For all of us who knew Scott, his wisdom, kindness, humility and generosity were fundamentally what defined him. What better legacy can a person hope for!The authors have declared no competing interests.All authors have contributed to the preparation of the manuscript, read and approved the final draft.


The goal of this study is to investigate the pharmacokinetics in plasma and tumour interstitial fluid of two T-cell bispecifics (TCBs) with different binding affinities to the tumour target and to assess the subsequent cytokine release in a tumour-bearing humanised mouse model. Pharmacokinetics (PK) as well as cytokine data were collected in humanised mice after iv injection of cibisatamab and CEACAM5-TCB which are binding with different binding affinities to the tumour antigen carcinoembryonic antigen (CEA). The PK data were modelled and coupled to a previously published physiologically based PK model. Corresponding cytokine release profiles were compared to in vitro data. The PK model provided a good fit to the data and precise estimation of key PK parameters. High tumour interstitial concentrations were observed for both TCBs, influenced by their respective target binding affinities. In conclusion, we developed a tailored experimental method to measure PK and cytokine release in plasma and at the site of drug action, namely in the tumour. Integrating those data into a mathematical model enabled to investigate the impact of target affinity on tumour accumulation and can have implications for the PKPD assessment of the therapeutic antibodies. Immune modulatory drugs are an important pillar for anticancer treatment . TherapeCibisatamab (CEA-TCB) and the analogous CEACAM5-TCB are two 2 + 1 CD3 engaging T-cell bispecific antibodies (TCBs) with a molecular weight of 194 kDa ,17. BothOur goal in this work was to measure and compare the PK of those two TCBs in plasma, total tumour and tumour interstitial fluid in humanised NOD scid gamma (NSG) mice ,20,21. TAll animal studies were approved by the AAALAC International Accredited Animal Care and Use Program at the University of Bergen. The studies were conducted according to, and with approval of, the Norwegian State Commission for Laboratory Animals , which are aligned with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes and Council of Europe (ETS 123).In this work, we performed several experimental studies and a mathematical modelling analysis. Furthermore, some studies were performed as preparation and prerequisite of a main biodistribution study. Therefore, a comprehensive overview across all studies, which are part of this work including their main features and purpose, are summarised in ®Vet, 1.5% isoflurane in 100% O2 . The anaesthesia was monitored, and the body temperature was kept stable using a heating pad. At the end of the study, mice were euthanised under anaesthesia by excision of the heart.All mice were housed under a 12:12 h light/dark cycle and received standard chow and water ad libitum, both before and during experiments. During invasive procedures, the animals were anesthetised with IsoFlo2 in RPMI-1640 culturing medium with 10% FBS  and 1% GlutaMax. 100U/mL penicillin as well as 100mg/mL streptomycin  and plasmocin  were added.Radiotracer studies were performed in 13 male NSG mice  and the biodistribution study was conducted in 45 female humanised NSGTM mice engrafted with CD34+ hematopoietic stem cells . In those humanised NSG mice, human CD4+ and CD8+ T cells are present in circulation and tissues, offering a model in which effects of T-cell dependent immunotherapy can be studied ,20. WhenThe investigators were blinded throughout the in vivo experiments and data acquisition and did not know the identity/characteristics of the compounds. In case of early termination of the mice, terminal measurements were taken and reported at time of termination.125Iodine using the iodogen method, which was previously described in detail [125Iodine. In order to avoid impact of the labelling procedure on the pharmacological activity, the two T-cell bispecific antibodies were labelled by an indirect labelling approach. After labelling, the retention of the biological activity was confirmed and potentially unbound 125Iodine was removed prior to each injection. The labelling procedure and quality control is described in more detail in the Human serum albumin (HSA) was used as plasma volume tracer and was labelled with n detail ,23. Furtn = 4), 17 (n = 4) or 20 days (n = 5) after tumour cell engraftment. Skin was harvested as reference sample. The tumour size and corresponding volume of isolated interstitial fluid was measured. The isolation of interstitial fluid from harvested tissue samples was based on a tissue centrifugation methodology, which has been previously described [The feasibility to isolate a representative interstitial fluid sample from tumour by centrifugation was assessed. About 13 mice were terminated to harvest tumour and a terminal plasma sample at 14  was used as an extracellular tracer in two separate groups of mice, either at day 14 or 20 post-engraftment. Before injection of the tracer, the kidneys of the mice were surgically tied off to limit renal excretion. An equilibration time of 60 min, before terminating the mice, allowed the tracer to equilibrate in all extracellular spaces . Normalising the counts in a harvested tissue sample with the counts in plasma allows deriving plasma and extracellular volume fraction in the respective sample, using the following equations:For the use as plasma volume tracer, ue space ,25. 51CrThe same approach was followed to evaluate the isolated fluid sample by comparing the counts in the isolated fluid to plasma. An undiluted interstitial fluid sample should have an extracellular fluid fraction close to 1.0 and a low plasma fraction, as previously reported in literature .2 × b). Upon reaching 200 mm3, three mice per time point were randomly assigned to each treatment group, untreated control (n = 9), 2.5 mg/kg cibisatamab (n = 18) or 2.5 mg/kg CEACAM5-TCB (n = 18). Unlabelled and labelled antibody was mixed at a given ratio and injected intravenously in the tail vein at a dose of 2.5 mg/kg with 4–6 million cpm per mouse. The mice were terminated at predefined time points, 1, 8, 24, 48, 96 or 240 h or 1, 48 and 240 h after dosing in case of the control group. Due to mortality of some mice, the earliest measured time point for CEACAM5-TCB was 8 h. In case of early termination , all terminal sampling was performed as described below and the time point of termination was reported and used for analysis. A terminal blood sample was collected from each mouse and tumours were excised and centrifuged as described above. The blood sample was centrifuged at 10,621× g in order to prepare a plasma sample. Total tumour samples, plasma and tumour interstitial fluid (TIF) were transferred to separate vials for γ-counting in order to assess the amount of compound in the respective sample. As the isolation of interstitial fluid by centrifugation often yields small sample volumes with a relatively high surface to volume ratio, all sample handling was done in a humidity chamber (100% relative humidity) in order to avoid evaporation from the sample [Tumour size was monitored every other day using calliper measurements  for IL2, IL6, IL10, TNF-α, Granzyme B and IFN-γ according to the manufacturer’s description. In short, samples were prepared by adding calibrator diluent to 60 µL of sample fluid, achieving a two-fold dilution. Total of 50 µL standard, control and samples were each added in duplicates to the designated wells before microparticle cocktail was added to the plate and incubated for 2 h at room temperature on a plate shaker. After three washing steps, biotin-antibody cocktail was added and incubated for one hour before the addition of Streptavidin-PE and additional 30 min of incubation. Finally, Wash Buffer was added to each well and the plate was measured on a MagPix Luminex reader . Duplicate readings were averaged, and the background mean fluorescence intensity (MFI) subtracted. Each sample was then compared to the standard curve of the respective analyte.The in vivo cytokine data were then compared to respective in vitro data, which were collected in a corresponding in vitro system. The measurement of the in vitro cytokine release has been reported in more detail . In shorThe experimental tumour uptake data were used in order to estimate underlying PK and tumour uptake parameters. The tumour uptake model was inspired by previously published work around tumour uptake modelling and performed in Matlab 2018a including the Simbiology toolbox . The plasma PK was described by a previously published PBPK model , which wA graphical illustration of the final model structure is depicted in tumfVpla is the residual plasma fraction in the tumour sample and tumfVint is the interstitial volume fraction in the tumour sample as measured in this work.The total tumour concentration of the antibody changing over time is defined as follows:freeAint is the unbound interstitial amount of antibody and tumVint is the interstitial fluid volume in the tumour sample.The free interstitial concentration of the antibody changing over time is defined as follows:bndAint is the antibody concentration bound to the target in the interstitial space.The bound interstitial concentration of the antibody changing over time is defined as follows:The total interstitial concentration of the antibody changing over time is defined as follows:tum), backflow rate from tumour (Outtum), uptake rate to rest of body (UpRoB), backflow rate from rest of body (OutRoB), plasma volume (Vpla) and tumour target expression (targ). The total tumour volume (TV) was fixed to 250 µL based on the measured tumour volume data under TCB treatment  and data were plotted as mean ± SD unless stated otherwise. When comparing the different treatment groups or readouts, a one way ANOVA was performed followed by a post hoc Bonferroni test. PD effects were compared based on AUCE differences. Differences with n = 4), 17 (n = 4) or 20 days (n = 5) after tumour engraftment). Due to prior experience, the terminal nature of the measurements and the fact that the radiotracer study was mainly a feasibility study to inform and optimise the following biodistribution study, the number of animals per group was kept to a minimum. No formal sample size analysis or statistical analysis between the individual groups was performed for this study. For the biodistribution study with subsequent PD evaluation, mice were each randomly assigned to the two treatment groups (n = 18) and to the vehicle group (n = 9). The vehicle group was smaller in number as less terminal time points were sampled for the PD readouts in the control group to define the baseline. Due to mortality of the mice, the group sizes had to be adjusted to n = 18 for the cibisatamab and n = 17 for the CEACAM5-TCB treatment group. As the measurements were terminal, three mice per treatment group were terminated per time point. In case of early termination of the mice, terminal measurements were taken and reported at time of termination.For the in vivo studies, the group sizes within one experiment were designed to be equal based on the number of available mice and the mice were randomly assigned to the various groups. The group sizes for the radiotracer studies in the NSG mice were defined based on prior experience with the employed methods and expected variability. The mice were randomly assigned to the assessed time points  injection of either cibisatamab or CEACAM5-TCB in order to investigate the distribution of the two TCBs into the tumour interstitial space and their accumulation therein due to target binding. The measured plasma PK showed a higher clearance for cibisatamab (0.043 mL/h) as compared with CEACAM5-TCB (0.015 mL/h). The free interstitial concentration in the tumour was slightly higher than the total tumour concentration for cibisatamab while the opposite was observed for CEACAM5-TCB, which might be due to the higher CEA binding affinity of CEACAM5-TCB a,b. Basep = 0.001) with the post hoc Bonferroni’s test (performed in GraphPad PRISM version 8.0) showing significant differences between the treated and control group, cibisatamab vs. control  difference = 75,038 mm3/h), CEACAM5-TCB vs. control . The difference between CEACAM5-TCB vs. cibisatamab treatment at a dose of 2.5 mg/kg was not significant in that study (p = 0.8) and both showed very strong anti-tumour effect with a lower mean AUCE of 16,042 mm3/h in case of CEACAM5-TCB. Under treatment, the low tumour volume was maintained over the course of the 10 days, despite no additional TCB administration . Despite a pronounced difference in mean AUCE and a consistent trend across all cytokines, the statistical analysis on the other cytokines individually showed no significance in AUCE difference between TIF and plasma , which is probably due to the relatively high inter-individual variability in the measurements. Interestingly, the cytokine release kinetics observed in TIF was similar to the kinetics observed in an in vitro co-culture assay with the same MKN45 tumour target cells and PBMCs  on local drug concentration with additional follow-up studies. As a next step, it is suggested to link the expanded PBPK model to the drug effect on tumour growth. With such a mechanism-based PKPD model, the drug effect can be predicted based on the local drug concentration as a result of target expression as well as binding affinity. It would be suited for compound selection as well as selection and prediction of optimal dose and dosing schedule. Furthermore, the measurement of immune cell dynamics would add mechanistic insights into their role in the cytokine response. Investigating additional compounds in a similar setting would help to elaborate on potential variability and differences in between IgG-based molecules. Together, those aspects would allow mechanistically expanding the mathematical model and moving towards more of a quantitative systems pharmacology approach. Unfortunately, those aspects were out of scope in this work due to the terminal nature of the measurements and thus very high number of humanised mice required to obtain samples at all defined time points. Therefore, we decided here to focus on the comparison of two TCBs with different binding affinities in terms of their biodistribution to the tumour interstitial space, to quantify relevant parameters driving antibody tumour distribution with a mathematical model and to characterise the elicited tumour growth inhibition and cytokine release.In summary, we measured for the first time directly the TCB concentration levels at the target site in isolated interstitial fluid from tumour and show a high tumour uptake of TCBs. We provide a model, which allows simulations of interstitial TCB concentrations in tumour and other tissues considering target-binding properties and discuss total interstitial concentration (TIC) as suitable readout for comparing in vitro to in vivo PD effects. As such, the provided work improves the current understanding and model-based predictions of tumour distribution, which is an important translational aspect for tumour-targeted antibodies. The findings and discussions presented here can have important implications to support the development and preclinical testing of targeted antibody therapeutics.


However, specific primary tumour features might impact accuracy of PSMA-PET for PCa detection. We investigated histopathological parameters and immunohistochemical PSMA expression patterns on radical prostatectomy (RPE) specimens and correlated them to the corresponding 68Ga-PSMA-11-PET between 2016 and 2018 were analysed. WHO/ISUP grade groups, growth pattern (expansive vs. infiltrative), tumour area and diameter as well as immunohistochemical PSMA heterogeneity, intensity and negative tumour area (PSMA%neg) were correlated with spatially corresponding SUVmax on 68Ga-PSMA-11-PET in a multidisciplinary analysis.RPE specimens of 62 patients with preoperative %neg. PSMA%neg, infiltrative growth pattern, smaller tumour area and diameter and WHO/ISUP grade group 2 significantly correlated with lower SUVmax values. A ROC curve analysis revealed 20% PSMA%neg as an optimal cutoff with the highest sensitivity and specificity  for a negative PSMA-PET scan. A multiple logistic regression model revealed tumoural PSMA%neg  and growth pattern  as significant predictors for a negative PSMA-PET scan.All tumours showed medium to strong membranous (2–3 +) and weak to strong cytoplasmic (1–3 +) PSMA expression. Heterogeneously expressed PSMA was found in 38 cases (61%). Twenty-five cases (40%) showed at least 5% and up to 80% PSMA%neg, infiltrative growth pattern, smaller tumour size and WHO/ISUP grade group 2 as parameters associated with a lower 68Ga-PSMA-11 uptake in prostate cancer. These findings can serve as fundament for future biopsy-based biomarker development to enable an individualized, tumour-adapted imaging approach.We describe PSMAThe online version contains supplementary material available at 10.1007/s00259-021-05501-1. PSMA exA levels . AdditioA levels , 10, andA levels , 12. It A levels , 14. In reported , 16. Hetreported , and in A-11-PET . FurtherA-11-PET . Only onA-11-PET . Exact c68Ga-PSMA-11-PET followed by radical prostatectomy to identify tumour characteristics that are associated with low SUVmax values on PSMA-PET scans.The aim of this study was to investigate and colocalize immunohistochemical PSMA expression patterns and histopathological features in patients with a pretherapeutic 68Ga-PSMA-11-PET for newly diagnosed intermediate or high-risk prostate cancer at the University Hospital Zurich from April 2016 to May 2018. All patients with no radical prostatectomy (RPE) specimen available were excluded. The local ethics committee approved the study protocol (BASEC Nr. 2018–01284) and all patients gave a general written informed consent for use of their data. Relevant clinico-pathological characteristics such as patients’ age at the time of operation, tumour stage, (modified) Gleason Score and WHO/ISUP prognostic grade group were collected.This study included consecutive patients who underwent staging with Sixty-two formalin-fixed, paraffin-embedded (FFPE) RPE specimens were evaluated on 2 µm hematoxylin and eosin (H&E)-stained sections. One representative slide from the RPE specimen was chosen for further investigation, harbouring the largest area of tumour and therefore defining the dominant tumour lesion.Staging and grading were done according to the WHO/ISUP/UICC guidelines , 23. Sep2 (radius 1.26 mm).A newly developed type of growth pattern (infiltrative vs. expansive) of each prostate cancer lesion was determined. We defined infiltrative growth as entrapped benign glands within the carcinoma complexes. An expansive growth pattern showed a tumour infiltration of pure carcinoma glands (without intermingled benign glands) within an area of at least 3 circles of 5 mm%neg) as a consent of both pathologists. Heterogeneity was defined by differences in the staining pattern of at least 5% of the representative tumour slide  was performed as described previously [Slides were digitalized  using the Hamamatsu NDP.view 2.8.24 Software.68Ga-PSMA-11-PET/computed tomography (CT) on a Discovery VCT 690 PET/CT  or on a Discovery MI PET/CT  or 68Ga-PSMA-11-PET/MRI  after a single injection of 68Ga-PSMA-11 (mean dose ± standard deviation (SD) 130 ± 18 MBq, range 81–171 MBq). The institutional protocol is in agreement with the EANM and SNMMI procedure guidelines [Patients underwent clinical routine idelines . Detailsmax). There is a wide range of proposed cutoffs to detect significant prostate cancer from SUVmax 3.15 [max 9.1 [max ≥ 5 as definition of PSMA-PET positivity [The acquired PET/CT and PET/MR images were analysed in a dedicated review workstation , which enables the review of the PET and the CT or MR images side by side and in fused mode. Every patient was discussed in a multidisciplinary setup including a pathologist and a nuclear medicine physician and radiologist with the selected pathology slide available alongside with the PET data. The corresponding area on PET images with the dominant tumour lesion was identified and PSMA uptake quantified using the maximum standardized uptake value  analysis. We investigated the association between a combination of histological parameters and immunohistochemical PSMA expression patterns with a negative PSMA-PET scan using a multiple logistic regression analysis.Correlations between histological parameters, immunohistochemical PSMA expression patterns and SUVU test for binary variables and the Kruskal–Wallis test for multiple variables. Correlations were done using bivariate Pearson’s correlation. Discrimination was evaluated using area under the receiver operating characteristic (ROC) curve (AUC). The variables entered in the multiple logistic regression analysis were selected by univariable logistic regression with a p-value cutoff point of 0.05. For the logistic regression analyses, ordinal variables were treated as continuous. Multicollinearity was assessed using variable inflation factors (VIF). To avoid an increase in further parameters, PSMA-PET-derived tumour volume and tumour/background ratios were not included into the multivariant analysis; however, correlation of these parameters with growth patern is given in the supplements. Two-sided p values < 0.05 were considered statistically significant. Correlations and ROC curve analysis were performed using SPSS Version 26 . Logistic regression analyses were performed using R . Graphs were generated using GraphPad Prism v8.Normal distribution was tested using the Kolmogorov–Smirnov test. Comparisons were calculated with the Mann–Whitney 68Ga-PSMA-11-PET scans from 137 patients were available. Patients were excluded because of missing informed consent, treatment before PSMA-PET scan, missing clinical information and/or unavailability of a RPE specimen. A total number of 62 patients were included in this study , and maximum diameter was recorded from 2 to 25.7 mm (mean 13.9 ± 6.0 mm). Four lesions were smaller than 5 mm and were excluded for correlation analysis between histology pattern and PSMA-PET uptake. An infiltrative growth pattern of the dominant tumour was seen in 33 of 62 (53.2%) cases, whereas an expansive pattern occured in 29 of 62 (46.8%) cases . Larger tumour area and higher maximum diameter were significantly correlated with expansive growth pattern . Higher WHO/ISUP grade group showed a significant association with higher pT stage .The dominant tumour lesions showed WHO/ISUP grade groups ranging from 2 to 5 Table . The tum%neg, Fig. PSMA expression was noted in all 62 100%) prostate adenocarcinoma specimen with a range from medium to strong membranous (2 + to 3 +) and weak to strong (1 + to 3 +) cytoplasmic expression . Considering SUVmax ≥ 5 as the definition for PET positivity and excluding lesions smaller than 5 mm on histopathology, 49 of 58 scans (84.5%) were positive, and 9 of 58 (15.5%) negative. Of the four lesions smaller than 5 mm, two had a SUVmax ≥ 5.SUV%neg between 5 and 80%) was significantly associated with lower SUVmax values .The presence of PSMA-negative tumour areas (PSMA%neg ≥ 20% resulting in a sensitivity of 89% and specificity of 86% (area under the curve AUC = 0.923) for a negative PSMA-PET scan  revealed a significant association with lower SUVmax values . On the other hand, 42 of 49 cases with a PSMA%neg < 20% showed a positive PSMA-PET scan (86% specificity). Infiltrative versus expansive growth patterns showed a significant difference in mean SUVmax, with expansive tumours having a higher tracer accumulation   and between groups 3 and 5   was found . A correlation between WHO/ISUP grade groups and PSMA%neg did not reach significance but showed a trend towards lower grade groups associated with higher percentages of PSMA negative areas . Tumour area and maximum tumour diameter showed a significant positive correlation to higher SUVmax values   Fig. . For WHOst) Fig. . No signund Fig. . In tumoon) Fig. . Additioon) Fig. , SUVmax/%neg, growth pattern, WHO/ISUP grade groups, and cytosolic and membranous PSMA expression were selected as variables for the multiple logistic regression analysis . None of the selected variables did show a relevant multicolinearity (VIF < 5). The multiple logistic regression model revealed PSMA%neg  and growth pattern  as independent predictors for a negative PSMA-PET scan. Table PSMA68Ga-PSMA-11-PET results with immunohistochemical PSMA expression patterns as well as histopathological features in prostate carcinomas of 58 RPE specimens using a precise colocalization approach. Significantly lower SUVmax values were found in PSMA-PET of prostate carcinoma with PSMA%neg, infiltrative growth pattern, smaller tumour size and WHO/ISUP grade group 2. No significant differences in SUVmax could be observed regarding cytoplasmic and membranous PSMA IHC expression intensity levels.In the present study, we correlated 68Ga-PSMA-11-PET uptake with immunohistochemical PSMA expression in RPE specimen has been described only by Woythal et al. They demonstrated a significantly lower SUVmax in PCa RPE specimen (n = 31) which showed an immunoreactive score (IRS) smaller than 2 or a PSMA staining in less than 50% of the tumour cells [max values in PCa showing PSMA negative areas (ranging from 5 to 80%) . In terms of PSMA intensity, we scored the cytoplasmic and membranous PSMA expression separately instead of using a score where cytoplasmic and membranous expression is evaluated simutanously (e.g. the IRS). Interestingly, we found only a positive trend of cytoplasmic  and membranous  PSMA expression levels correlating with PSMA-PET positivity, which did not reach statistical significance.Direct correlation of ur cells . In our 0%) Fig. . In a RO%neg and infiltrative growth pattern were found to predict negative PSMA-PET scans in a multiple logistic regression model. This is in concordance with another recently published paper of our group describing PSMA%neg as a parameter able to predict negative PSMA-PET scans in a biochemical recurrence setting [max ≥ 5 for the manuscript, and SUVmax ≥ 4 in the supplements, both yielding PSMA%neg ≥ 20% as the optimal cutoff for negative versus positive scans (Supplements).Although different parameters correlated with PSMA-PET negativity, PSMA setting . The sel2 each (radius 1.26 mm). In an infiltrative growth pattern, the density of tumour complexes is decreased by intermingled benign glands. As Woythal et al. stated, benign glands not only show lower PSMA expression but also have a significant lower SUVmax in the PSMA-PET than prostate carcinoma [p = 0.0497).The investigation of histopathological parameters, such as the growth pattern of the prostate cancer lesion  in exact correspondence to PSMA-PET, has not been published yet. We defined an infiltrative growth as entrapped benign glands within the carcinoma complexes whereas an expansive growth pattern showed pure carcinoma glands within an area of at least 3 circles of 5 mmarcinoma . Multipln = 4) very small prostate carcinomas (diameter < 5 mm) because of a natural resolution limit of PSMA-PET scans due to partial-volume effects [max. Instead, in this study, a significant correlation between tumour size and SUVmax values could be observed. Most likely, this can be explained by different measurement methods. Instead of using the tumour size documented in the pathology report, we measured maximum diameter and area of each dominant tumour lesion on one slide and precisely compared this to PSMA-PET uptake of the corresponding area.We decided to exclude all . This tumour had a relatively small diameter of 8.7 mm  and showed an infiltrative growth pattern . Moreover, two of them showed an expansive growth pattern  revealed a SUVern Fig. . On the ern Fig. . The adjern Fig. .%neg ≥ 20% and 6 cases with PSMA%neg ≥ 50%) due to the known natural low frequency of PSMA-negative tumours, of around 10% of all PCa. The low number of negative PSMA-PET scans (9 cases) limited the multiple logistic regression analysis. Although evaluations were done by two experienced genito-urinary pathologists, semiquantitative PSMA IHC scoring harbours a certain degree of variability.Our study faces some limitations, including its retrospective and single-centre approach. Furthermore, even in our relatively large cohort consisting of 62 RPEs, the number of cases with a high ratio of negative PSMA areas was limited Below is the link to the electronic supplementary material.


Response guided treatment in breast cancer is highly desirable, but the effectiveness is only established based on residual cellularity from histopathological analysis after surgery. Tubule formation, a key component of grading score, is directly associated with cellularity, with significant implications on prognosis. Peri-tumoural lipid composition, a potential marker, can be rapidly mapped across the entire breast using novel method of chemical shift-encoded imaging, enabling the quantification of spatial distribution. We hypothesise that peri-tumoural spatial distribution of lipid composition is sensitive to tumour cellular differentiation and proliferative activity.Twenty whole tumour specimens freshly excised from patients with invasive ductal carcinoma (9 Score 2 and 11 Score 3 in tubule formation) were scanned on a 3 T clinical scanner . Quantitative lipid composition maps were acquired for polyunsaturated, monounsaturated, and saturated fatty acids . The peri-tumoural spatial distribution  of each lipid constituent were then computed. The proliferative activity marker Ki-67 and tumour-infiltrating lymphocytes (TILs) were assessed histologically.p = 0.0119), skewness (p = 0.0116), entropy (p = 0.0223), kurtosis (p = 0.0381), and correlations against Ki-67 in mean (ρ = -0.5414), skewness (ρ = 0.6045) and entropy (ρ = 0.6677), and TILs in mean (ρ = -0.4621). For SFA, there were significant differences between groups in mean (p = 0.0329) and skewness (p = 0.0111), and correlation against Ki-67 in mean (ρ = 0.5910). For PUFA, there was no significant difference in mean, skewness, entropy or kurtosis between the groups.For MUFA, there were significant differences between groups in mean (There was an association between peri-tumoural spatial distribution of lipid composition with tumour cellular differentiation and proliferation. Peri-tumoural lipid composition imaging might have potential in non-invasive quantitative assessment of patients with breast cancer for treatment planning and monitoring.The online version contains supplementary material available at 10.1186/s12885-022-09362-1. Breast cancer is a major and expanding health challenge  with curLipid composition quantification using biochemical method of solvent extraction suffers from complex procedures, invasive nature and single spatial location . Non-invWe therefore hypothesise that the peri-tumoural spatial distribution of lipid composition from chemical shift-encoded imaging is sensitive to tubule formation, and is associated with proliferative activities.To probe this hypothesis, we conducted a two-group cross-sectional study examining the peri-tumoural spatial distribution of lipid composition in whole tumour specimens freshly excised from patients Fig. . The stuTwenty patients  with invasive breast carcinoma participated in the study. Patients undergoing breast conservation surgery, with tumour size larger than 10 mm in diameter on mammography, with no previous malignancies, chemotherapy or radiotherapy prior to surgery were eligible. For accurate delineation of peri-tumoural adipose tissue, a tumour size larger than 10 mm was required to ensure adequate image resolution. The majority of breast tumours that are larger than 10 mm would have tubule formation Score 2 and 3. To avoid highly skewed patient distribution leading to biased results, tubule formation score 1 was hence excluded from the study. The study was completed between September 2016 and February 2018.The specimen, upon excision, was submerged in 10% formalin to prevent tissue degradation and immobilised using a custom-built holding harness in a sealed container. The tissue specimen was immediately transported from the operating theatre to Aberdeen Biomedical Imaging Centre for lipid composition mapping. Routine clinical histopathological examination was subsequently performed to determine histological tumour size, grade and Nottingham Prognostic Index (NPI) . Tumour Images were acquired on a 3 T whole-body clinical MRI scanner , using a 32-channel receiver coil for signal detection and a body coil for uniform transmission. Lipid composition images were acquired using chemical shift-encoded imaging sequence , 18 withImage analysis was conducted in MATLAB . The maps of the number of double bonds in triglycerides were computed from raw data , before t-tests and Mann Whitney U tests depending on the normality of sample distribution) were performed to compare the peri-tumoural spatial distribution of lipid constituents between tubule formation Scores. The Spearman’s rank correlation tests were performed between the spatial distribution in each lipid constituent against proliferative activity marker Ki-67 and TILs. A p-value < 0.05 was considered statistically significant.All statistical analysis was performed in the SPSS software . Shapiro–Wilk test for normality was performed on all the collected data. Descriptive statistics were computed for peri-tumoural lipid composition in Luminal A, Luminal B [human epidermal growth factor receptor 2 (HER2) negative] ), Luminal B-HER2( +) and triple negative (TN) breast cancer. Group comparisons  in mean  stained microscopy slides from two typical invasive breast cancer specimens, one from tubule formation Score 2 and one from Score 3, are shown in Fig. In this work, we found significant differences in all the spatial distribution measures of peri-tumoural MUFA between tubule formation Scores, with strong association with proliferative activity marker Ki-67. There were significant differences in the measures of mean and skewness for SFA, with mean SFA associated with proliferative activity. There were no significant differences in the spatial distribution for PUFA between groups.MUFA, supported by significant differences in all the measures of peri-tumoural spatial distribution, might hold the central role in tubule formation, with strong correlations against proliferative activity marker. For least glandular differentiation, a reduction in mean MUFA indicates lower overall peri-tumoural MUFA, potentially due to accelerated absorption of MUFA into tumour core to support aggressive cancer cell growth . The negSFA, with limited number of significant differences between groups and correlations against proliferative activity marker, might hold secondary role in tubule formation, while PUFA showing no apparent connection. For least glandular differentiation, an elevation of mean SFA indicates higher overall peri-tumoural SFA, reflecting a potential extrusion of SFA from tumour to alleviate SFA-induced apoptosis (lipotoxicity) , 33 arisThe correlation between mean SFA and proliferative activity marker Ki-67 might indicate a dependency of overall tumour growth on the export of SFA . HoweverThis work was the first investigation of the peri-tumoural spatial distribution of lipid composition in breast cancer, showing MUFA might be a central biomarker of glandular differentiation. Since lipid composition in breast is affected by oestrogen in premenopausal patients compared to postmenopausal , the assLipid composition mapping, although limited by overlapping distribution between groups and low to moderate correlations with tumour proliferation, could be a valuable clinical research tool to support image guided treatment. Histopathological analysis to determine tubule formation is the clinical standard, however demands significant expertise. The association between tubule formation score and peri-tumoural lipid composition was based on group difference instead of correlation, and the lipid composition imaging proposed in this research is the first step towards in vivo patient application for treatment planning and monitoring. The imaging marker, although showing significant sensitivity to tubule formation, does not replace traditional histopathology using optical microscope as established clinical routine standard. However, the non-invasive nature of the imaging marker may contribute to improved patient care, particularly in the context of neoadjuvant setting for complementing clinical routine assessments. The association with Ki-67 is of considerable clinical relevance, however concordance with ER, PgR, HER2 positivity and histological grade remains critical for breast cancer treatment. The cohort size was small, limiting the reliability for evaluating the potential association of NPI and histological grade with molecular subtypes . HoweverThere was an association between peri-tumoural spatial distribution of lipid composition with tumour cellular differentiation and proliferation. Peri-tumoural lipid composition imaging might have potential in non-invasive quantitative assessment of patients with breast cancer for treatment planning and monitoring.Additional file 1.


In the past, water homeostasis of the brain was understood as a certain quantitative equilibrium of water content between intravascular, interstitial, and intracellular spaces governed mostly by hydrostatic effects i.e., strictly by physical laws. The recent achievements in molecular bioscience have led to substantial changes in this regard. Some new concepts elaborate the idea that all compartments involved in cerebral fluid homeostasis create a functional continuum with an active and precise regulation of fluid exchange between them rather than only serving as separate fluid receptacles with mere passive diffusion mechanisms, based on hydrostatic pressure. According to these concepts, aquaporin-4 (AQP4) plays the central role in cerebral fluid homeostasis, acting as a water channel protein. The AQP4 not only enables water permeability through the blood-brain barrier but also regulates water exchange between perivascular spaces and the rest of the glymphatic system, described as pan-cerebral fluid pathway interlacing macroscopic cerebrospinal fluid (CSF) spaces with the interstitial fluid of brain tissue. With regards to this, AQP4 makes water shift strongly dependent on active processes including changes in cerebral microcirculation and autoregulation of brain vessels capacity. In this paper, the role of the AQP4 as the gatekeeper, regulating the water exchange between intracellular space, glymphatic system , and intravascular compartment is reviewed. In addition, the new concepts of brain edema as a misbalance in water homeostasis are critically appraised based on the newly described role of AQP4 for fluid permeation. Finally, the relevance of these hypotheses for clinical conditions (including brain trauma and stroke) and for both new and old therapy concepts are analyzed. Apart from the exchange of information, one of the most challenging tasks of the mammalian brain is to maintain the internal water and electrolyte homeostasis independent from the caprices of the external environment, in order to provide the neurons with nourishing substances and guarantee them a constancy of electrolyte concentration and osmolarity, required for their proper function , 2. As tCertainly, this oversimplification is far from even approximating the whole complexity of the structure of brain fluid spaces, not to mention its extremely composed function regarding cerebral water turnover. The multidisciplinary research of recent years has delivered solid evidence that the intracerebral water balance is a highly complex, actively regulated process, involving all types of glia cells as well as the neurons and being highly responsible for the electrolyte and water homeostasis of the latter, thus impacting significantly the proper function of the whole central nervous system as a physiological unit , 4–6.Due to a variable number of (sometimes concurring) theories, it is impossible to outline all neurobiological concepts describing how the brain water homeostasis is maintained in the limited text volume of the journal paper. Thus, the main goal of this narrative review is to provide the Reader with the critical appraisal of some of the latest ideas, which attempt to unify the recent findings in (micro-)anatomy, molecular neurophysiology, and biophysics into the form of a concise model of brain fluid turnover. In particular, the concept of the glymphatic system, conjoining the anatomic spaces filled with cerebrospinal fluid and the ultrastructures of extracellular space needs to be outlined , 7–10. TWith the advent of modern neurosurgery, several concepts of cerebral fluid circulation and water turnover have been developed with the classic model of cerebrospinal fluid (CSF) flow also termed “third circulation” published by Cushing, which has since then become universally accepted , 19. Accin vivo experiments  with the highest levels detected in astrocytes localized near the subarachnoid space, along ventricles and blood vessels. Also in areas engaged with water balance maintaining and responsible for the osmoregulation such as the supraoptic nucleus or subfornical organ, the intense AQP4 expression was recorded . The disSeveral studies underlined the fact, that the regulation of AQP4 activity relies more on the subcellular relocation than on the expression of its gene. Both isoforms of AQP4 can be translated from the same full-length transcript by a “leaky scanning” mechanism , 113. PrIn vitro, the mutagenesis of ar/R motifs of AQP4 consisting in substitution of histidine in position 2 and arginine in position 4 creates glycerol or urea permeable channels. The H201A and H206G substitutions, respectively, allow the glycerol and the urea permeable channels to form, while the R216A substitution creates the channel permeable for both substrates. Some authors hypothesized that the H201A mutation along with F77 composes a hydrophobic corner contacting with the alkyl chain of the glycerol due to van der Waals forces, while the loss of the alanine in the H201G mutation causes a disruption of this corner and accessibility of the V197 backbone carbonyl group for binding with water or solutes such as urea due to hydrogen bounds. Oppositely, analogous mutagenesis of AQP1 (R195A and H180/G) did not lead to the formation of urea or glycerol permeable channels  gene deletion  gained through studies implementing animal lines with the genetic modification of AQP4 function. Accordingly, AQP4 knockout animals demonstrate enlarged interstitial fluid spaces , 133, indeletion  or seen deletion , trauma deletion , or ischdeletion  is relatNotably, based on the results of  and 151151 a comHowever, the recent multicenter research effort, provided by five laboratories implementing independently developed transgenic animal models with impaired AQP function, clearly demonstrated, that transport of the tracers, cleared from extracellular space via perivascular fluid compartment is strongly dependent on the proper function of perivascular aquaporins .In conclusion, the main and widely accepted role of AQP4 is the facilitation of fluid exchange between the extracellular space and the perivascular spaces  as well as in the cleavage of several cerebral metabolites, crucial in pathophysiology of neurodegeneration. Importantly, even under the physiological condition, transportation of cerebral fluid does not represent a steady-state but is a very dynamic process constantly adapting to the current needs, being related to the energetic state of neurons and thus linked to autoregulation of microvasculature. Let us take a closer look at the previous evidence regarding this area.Soon after describing glymphatic system with the continuous fluid flow as its main function, the evidence about its dynamic adaptation to the current physiological status appeared. Of importance, the increased energetic demand of neurons on the one hand clearly increases cerebral blood flow on the level of microcirculation –155 [a p+ exchange related to increased neuronal activity needs to be discussed [albeit some reports deny the importance of Kir4.1/AQP4 complex for the mechanism of astrocytic swelling  physiology of cerebral fluid circulation. According to the canonical concept, forged by Klatzo and his research group, there are two main forms of cerebral edema existing. Vasogenic edema is characterized by extracellular water accumulation due to BBB dysfunction and increased transcytosis of plasma elements, including water . In turnin vivo and in vitro seemed to be inconclusive, since AQP4 and its expression demonstrated both surge and depletion of its activity due to developing brain edema. Thus, Ke et al. reported a reduction of AQP4 expression in areas of the traumatically swollen brain  models exist, in which the dominance of cytotoxic or vasogenic edema type relies not only on the mechanism of primary injury but changes dynamically over time as the influence of AQP4 does. Several studies implementing controlled cortical impact paradigm (CCI) , 247 wi–229 [altn et al.  and Abi-n et al. .Due to the ambiguous properties of AQP4 regarding its impact on water homeostasis in different types of edema, the results of experimental studies in which AQP4 function is blocked or enhanced need to be critically analyzed before being translated into clinical practice. Indeed, recently several compounds have been claimed to execute beneficial impact on the course of secondary brain damage, including brain edema via interference with AQP4 function and expression. Here, neuroprotective and antiedematous action of erythropoietin has been linked with the preservation of AQP4 function in trauma , hydrocein vivo. The possible reason here is the characteristic of AQP4 molecule, with the relatively small diameter of its pore and simple structure of its molecule, that, contrary to regular membrane receptors, lacks any complex intrinsic gating and transport mechanism , competitively binding to AQP4 has proven its efficacy in reducing lesions, at least in preclinical tests (Certainly, analyses of Kitchen et al.  and Sylvechanism . This maechanism , 256. Foal tests –283. It al tests , 285. Fial tests  but alsoal tests , 287–289al tests , 291. Thin vivo studies into clinical practice should await more precise and more critical benefit-risk calculations for an inhomogeneous group of conditions, in which brain edema and/or neurovascular uncoupling play a major role.There is growing interest in the structure and function of cerebral extracellular spaces described recently as the glymphatic system. Certainly, the glymphatic flow as well as water metabolismis dependent on numerous physical laws and molecular factors. However, evidence from recent years, regarding the role of cellular water channels in physiological conditions and diverse brain pathologies clearly point out AQP4 as the key component of cerebral fluid homeostasis, acting not only as a passive channel for water and small molecular substances but playing a key role in the proper functioning of blood-brain barrier and perivascular unit. Hereby adapting the glymphatic flow to the phases of neuronal activity with increased blood flow demand in an alternating manner. The knowledge about the role of AQP4 in cerebral fluid homeostasis is vast and continually growing, however, there is still a lot to discover in this field. For this reason, as well as the ambiguity of the impact of AQP4 on the neurological outcome of cerebral edema, attempts to translate somehow the positive results of JS, MK, AW, and JO contributed the conception and design of the review. JS, MK, and AW wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.JO is a consultant to the Karl Storz Company and receives grants from the Erbe Company. These companies were not involved in the design and workflow during the preparation of the present review. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Infectious complications in cancer patients (pts) who have received T-cell therapies are similar to those in autologous hematopoietic stem cell transplant (HCT) recipients, who - because they lose prior acquired immunity after undergoing conditioning regimens and transplantation- may be at an increased risk for vaccine-preventable infections. We sought to determine seroprotection rates against pneumococcus and tetanus-diphtheria before and after cellular therapies.In this ongoing prospective observational cohort study, we enrolled pts with any type of cancer who received cellular therapy with chimeric antigen receptor modified T cell (CAR-T), natural killer CAR-T, or T-cell receptor- directed immunotherapies at MD Anderson Cancer Center from January 2020 through May 2021. We performed antibody assays for diphtheria, tetanus, and pneumococcus before, at 1 month, and between 3-6 months after T-cell therapy for each pt regardless of vaccination history. Of 38 pts enrolled, 27 (71%) were men and 25 (66%) had non-Hodgkin lymphoma (Table 1); 38 (100%) and 17 (45%) had a history of previous diphtheria-tetanus-acellular pertussis (Tdap) and pneumococcal vaccination, respectively (Table 2). Tetanus serologies were positive for all pts tested before, at 1 month and 3-6 months after T cell therapy . Diphtheria serologies were positive for most pts tested before, at 1 month and 3-6 months after therapy Ansun Biopharma Chimerix Clinigen (Consultant)Genentech Janssen Karius (Grant/Research Support)Merck Molecular Partners Novartis (Grant/Research Support)Oxford Immunotec Partner Therapeutics (Consultant)Pulmotec Shire/Takeda Viracor (Grant/Research Support)Xenex (Grant/Research Support) Fareed Khawaja, MBBS, Eurofins Viracor (Research Grant or Support) Ella Ariza Heredia, MD, Merck (Grant/Research Support)


Bronchogenic cysts that occur within the diaphragm are rare and difficult to diagnose preoperatively. We experienced the case of a patient with an abnormally high serum carbohydrate antigen 19‐9 (CA19‐9) level before surgery. The diagnosis of intradiaphragmatic bronchogenic cyst was made at the time of surgery. The patient was a 50‐year‐old woman with upper abdominal pain with an incidentally elevated serum CA19‐9. Although the tumour location could not be established on images, a tumour within the diaphragm was confirmed during the operation. The diaphragm was incised and the tumour was removed together with the capsule. Bronchial cysts were diagnosed histopathologically, and immunohistochemical examination revealed that the bronchial epithelial cells were positive for CA19‐9. When managing patients with bronchogenic cysts in the diaphragm, it is difficult to make a preoperative diagnosis or determine the location of the tumour; thus, careful planning is required before surgery. Bronchogenic cysts that occur within the diaphragm are rare and difficult to diagnose preoperatively. Here, we report the case of a patient with an abnormally high serum carbohydrate antigen 19‐9 (CA19‐9) level before surgery. The diagnosis of intradiaphragmatic bronchogenic cyst was made during surgery. When managing patients with bronchogenic cysts in the diaphragm, a preoperative diagnosis or location of the tumour can be difficult; thus, careful planning is required before surgery. Bronchogenic cysts that occur within the diaphragm are rare. In such cases, it can be difficult to make a preoperative diagnosis or determine the tumour location.18F‐fluorodeoxyglucose‐positron emission tomography showed only slight uptake in the tumour  value (4330.7 U/ml), and a tumour was incidentally detected during further examinations. A chest x‐ray showed a nodule near the left diaphragm that overlapped the cardiac shadow Figure . Contrasr Figure . On furtr Figure . The tumr Figure . Becauser Figure . The patr Figure . Histolor Figure . UnfortuImaging differentiation between tumours near the diaphragm and within the diaphragm is sometimes difficult. In preoperative examinations, chest CT can only roughly clarify the relationship between a tumour and the surrounding organs, while MRI is often useful if it is used together with CT because the internal intensity values change depending on the properties of the cyst contents.CA19‐9 is mainly elevated in gastrointestinal cancers  and inflammatory diseases . It is also present in normal cells, bronchial glands and bile ducts, and CA19‐9 levels in intrapulmonary bronchogenic cysts have been reported to be high.None declared.Soichi Suzuki, Masaya Aoki and Toshiyuki Nagata participated in the operation of this case. Masami Sato supervised the operation and the editing of the manuscript. Kazuhiro Ueda drafted the manuscript, and all authors read and approved the final manuscript.Appropriate written informed consent was obtained for publication of this case report and accompanying images.


Fluid intake is important to prevent dehydration and reduce recurrent kidney stones. There has been a trend in recent years to develop tools to monitor fluid intake using “smart” products such as smart bottles. Several commercial smart bottles are available, mainly targeting health-conscious adults. To the best of our knowledge, these bottles have not been validated in the literature. This study compares four commercially available smart bottles in terms of both performance and functionality. These bottles are the H2OPal, HidrateSpark Steel, HidrateSpark 3, and Thermos Smart Lid. One hundred intake events for each bottle were recorded and analyzed versus ground truth obtained from a high-resolution weight scale. The H2OPal had the lowest Mean Percent Error (MPE) and was able to balance out errors throughout multiple sips. The HidrateSpark 3 provided the most consistent and reliable results, with the lowest per sip error. The MPE values for HidrateSpark bottles were further improved using linear regression, as they had more consistent individual error values. The Thermos Smart Lid provides the lowest accuracy, as the sensors do not extend through the entire bottle, leading to many missed recordings. There are many reports in the literature of attempts to create systems or devices that can aide in tracking and managing fluid intake3. Unfortunately, most of these studies have not resulted in commercially available products. The bottles that are available in the market mainly target recreational athletes or health-conscious adults hoping to increase their hydration3. In this paper, we aim to determine if common commercially available water bottles are a viable solution for researchers and patients alike. We compare four commercial water bottles in terms of performance and functionality. These bottles are the HidrateSpark 34, HidrateSpark Steel5, H2O Pal6 and the Thermos Smart Lid7, as shown in Fig. Dehydration is a very serious issue as it can lead to adverse complications, including confusion, falls, hospitalization, and death. Fluid intake balance is important especially among the elderly and people with underlying conditions affecting fluid regulation. High fluid intake is recommended for patients at risk of recurring stone formation. Therefore, monitoring fluid intake is a useful way to determine if adequate fluid has been consumed8. This study found that over a 24 h fluid intake period, the HidrateSpark bottle was accurate in measuring total intake within 3% error8. The HidrateSpark has also been used in clinical studies to monitor intake volume in kidney stone patients9. Since then, HidrateSpark has developed new bottles with different sensors. The H2OPal has been used in other research studies to track and promote fluid intake, but there is no specific study to validate its performance10. Plecher et al. compared several commercial bottles in terms of functionality for seniors and available information online, however they did not perform any validation on their accuracy11.Out of the aforementioned bottles, only a previous version of the HidrateSpark has been validated in researchAll four commercial bottles include a free proprietary application to display and store intake events that are transferred via Bluetooth. The HidrateSpark 3 and the Thermos Smart Lid have the sensors down the middle of the bottle, likely using capacitive sensors, whereas the HidrateSpark Steel and H2Opal have sensors in the base, using load or pressure sensors. The sensor locations are shown in Fig. Each bottle was tested in two phases: (1) a controlled sip volume phase and (2) a free-living phase. In both phases, the results recorded by the bottle (obtained from the products’ mobile apps used on Android 11) were compared to the ground truth obtained using a 5 kg weight scale . All bottles were calibrated before data collection using the apps. In Phase 1, sip sizes ranging from 10 to 100 mL in increments of 10 mL were measured in a random order, five times each—for a total of 50 measurements per bottle. These events were not actual drinking events by a human but were poured out so the volume of each sip could be better controlled. In this phase, the bottles were recalibrated if the sip error was larger than 50 mL and were re-paired if the app lost Bluetooth connection with the bottle. In the free-living phase, a single user drank from the bottles freely during the day, taking varying sip sizes of their choice. This phase also consisted of 50 sips over time, but not all in succession. Therefore, each bottle had a dataset of 100 measurements in total.2. If a sip is not recorded or is poorly recorded, it is crucial that the bottle can balance out the volume in the next recordings. Therefore, the error  was adjusted manually. For example, assume that the subject drinks 10 mL and the bottle reports 0 mL, but then subsequently the subject drinks 20 mL and the bottle reports 30 mL total, then the adjusted error is 0 mL.To determine summative liquid intake and ensure proper daily hydration, it is more important to have an accurate volume intake detection throughout the whole day (24 h), rather than of each sip. However, to determine just-in-time intervention prompts, there is a need for each sip to have a low error, as done in the study by Conroy et al.Table mentclass2pt{minimmentclass2pt{minimThe R-square Pearson correlation coefficients for all bottles are also shown in Table The Bland–Altman plots in Fig. The HidrateSpark Steel and H2OPal had similar standard deviations of 20.04 mL and 21.41 mL, respectively. Figure In summary, the H2OPal is the most accurate at estimating sips over time and was the most reliable to measure most of the recordings. The Thermos Smart Lid was the least accurate and missed more sips than the other bottles. The HidrateSpark 3 bottles had more consistent error values, however underestimated the majority of sips leading to poor performance throughout time.The results suggest that the bottles may have a certain offset that could be compensated using a calibration algorithm. This is especially true for the HidrateSpark bottles that have small standard deviations of errors and always underestimate individual sips. The Least-Square (LS) method was used with Phase 1 data while excluding any missed recordings to obtain the offset and gain values. The obtained equation was used on the measured sip intakes of Phase 2 to calculate the actual value and determine the error after calibration. Table In Phase 1 to complete all the measurements, each bottle was refilled multiple times so it is possible that the calculated MAE is impacted by the filled level of the bottle. To determine this, each bottle was divided into three liquid levels as high, medium, and low, based on the total volume of each bottle. For the measurements in Phase 1, a one-way ANOVA test was performed to determine if the liquid level had a significant difference on the absolute error. For the HidrateSpark 3 and Steel, there was no significant differences in the error in each of the three categories. For the H2OPal and the Thermos bottles, there was a borderline significant difference (p < 0.05) using the Welsh test for unequal variance. Subsequently, a multiple comparison Tukey HSD test was performed on these two bottles. In both cases, the significant difference was between the “high” level and the “low” level categories. For the H2OPal, the “high” category had the largest mean and standard deviation, meaning that there is a higher error when the bottle is more filled. However, in the Thermos, the “low” category had the highest error. This is likely because the sensor does not extend to the bottom of the bottle.p > 0.05 meaning that the two populations were not significantly different. However, it was observed that the number of missed recordings was much greater in Phase 2 for both HidrateSpark bottles. For the H2OPal, the number of missed recordings were almost equal (2 vs 3), and for the Thermos SmartLid there were fewer missed recordings in free living scenario (6 vs 10). Since the HidrateSpark bottles were both improved after calibration, a t-test was also conducted after-calibration. For the HidrateSpark 3, the Phase 1 and 2 errors were borderline significantly different (p = 0.046). This is more likely due to the larger number of missed recordings in Phase 2 compared to Phase 1.A two tailed t-test was conducted to compare the Phase 1 and Phase 2 errors for each bottle. For all bottles, we achieved This section provides insight on the usability of the bottles and their apps, as well as additional functionality information. Although the accuracy of the bottles is important, the usability factors are also of interest when choosing a bottle.The HidrateSpark 3 and HidrateSpark Steel are equipped with LEDs that blink to remind the users to drink if they are not on track to meet their goal, or to blink a certain number of times a day (set by the user). They can also be set to blink every time the user drinks. The H2OPal and Thermos Smart Lid do not have any visual feedback to remind the user to drink. However, all purchased bottles have mobile notifications to remind the users to drink via the mobile app. The number of notifications per day can be customized in the HidrateSpark and H2OPal app.The HidrateSpark 3 and Steel use a linear trend to guide the user when to drink, and give an hourly suggested target the user should aim for to reach the goal by the end of the day. The H2OPal and Thermos Smart Lid only provide one total daily target. In all bottles, if the device is not connected to the app by Bluetooth, the data is stored locally and synchs once paired.None of these four bottles focuses on elderly hydration. Additionally, the formulae the bottles use to determine the daily intake goal were not available, so it is difficult to determine if they are appropriate for seniors. The bottles are mostly large, heavy and are not tailored to seniors. The use of the mobile app may also not be ideal for seniors, though it could be useful for researchers to collect data, remotely.All bottles could not determine if the liquid was consumed, discarded or spilled. All bottles also needed to be placed down on a surface after each sip to record the intake, accurately. This means that it is possibile to miss drinks if the bottle is not placed down, especially if it is refilled.Another limitation is that the devices needed to be re-paired regularly with the app to synchronize the data. The Thermos needed to be re-paired every time the app is opened, and the HidrateSpark bottles often had difficulty finding the Bluetooth connection. The H2OPal was the easiest to re-pair with the app if the connection was interrupted. All bottles were calibrated before the testing began, and all had to be recalibrated at least once during the process. The HidrateSpark bottles and H2OPal had to be emptied and filled fully to calibrate.All the bottles did not have the option to download or save data long term. Additionally, none were able to be accessed via an API.The HidrateSpark 3 and H2OPal use replaceable lithium-ion batteries and the HidrateSpark Steel and Thermos SmartLid use rechargeable batteries. The rechargeable batteries should last up to 2 weeks on a full charge, as stated by the manufacturers, however, the Thermos SmartLid had to be charged almost every week when using it frequently. This is a limitation as many people will not remember to charge the bottles regularly.There are various factors that impact the selection of a smart bottle especially when the users are elderly. The heaviness and bulkiness of the bottle is an important factor, as it needs to be easy to use for frail older adults. As mentioned, these bottles are not tailored to older adults. The price and the amount liquid each bottle can contain is also another factor. Table Commercially available smart bottles are very useful to researchers as there is no need to prototype a new device. Though there are many smart water bottles available, the most prevalent issue was that the data or raw signals were not accessible to the users, and only some had the results displayed in a mobile app. The development of a widely available smart bottle with high accuracy and completely accessible data is needed, especially one tailored to older adults. Of the four bottles tested, out of the box the H2OPal had the lowest Sip MPE, Cumulative MPE, and number of missed recordings. The HidrateSpark 3 had the highest linearity and smallest standard deviation and lowest MAE. The HidrateSpark Steel and HidrateSpark 3 can be simply calibrated manually to decrease the Sip Mean Error using LS method. For a more accurate sip recording, the HidrateSpark 3 is the preferred bottle, and for more consistent measurements over a period of time, the H2OPal is preferred. The Thermos SmartLid had the most unreliable performance with the most missed number of sips and a large over estimation of individual sips.This study is not without limitations. In a real-world scenario, many users will drink from other vessels, especially for hot liquids, store bought beverages and alcohol. Future work should evaluate how the form factor of each bottle might affect the error to guide smart water bottle design.