The diagnosis of periprosthetic joint infection (PJI) remains a challenge in clinical practice. Many novel serum and joint fluid biomarkers have important implications for the diagnosis of PJI. The presented study evaluated the value of joint fluid interleukin-6 (IL-6) combined with the neutral polymorphonuclear leukocyte (PMN%) ratio for chronic PJI diagnosis after arthroplasty.Sixty patients with chronic PJI or aseptic failure who underwent hip or knee revision from January 2018 to January 2020 in our department were included in this retrospective study. According to the 2013 MSIS diagnostic criteria, the 60 patients were divided into a PJI group and a non-PJI group (30 patients per group). We collected the joint fluid before surgery and determined the level of IL-6 and the PMN% by ELISA, and the differences between the two groups were compared. The diagnostic efficacy of joint fluid IL-6 combined with PMN% in chronic PJI was analyzed using a receiver operating characteristic curve (ROC curve).The diagnosis of PJI using joint fluid IL-6 combined with PMN% presented an area under the curve of 0.983, which was more accurate than the areas under the curve for diagnosis using IL-6 and PMN% individually . The optimal threshold values for IL-6 and PMN% were 662.50 pg/ml and 51.09%, respectively. Their sensitivity and specificity were 96.67% and 93.33%, respectively. The accuracy of the diagnosis of PJI was 95.00%.Joint fluid IL-6 combined with PMN% can be used as an auxiliary method to detect chronic infection around the prosthesis after hip/knee arthroplasty.Patients who underwent hip/knee revision at the First Hospital of Chongqing Medical University for periprosthetic infection or aseptic failure of the prosthesis after hip/knee arthroplasty from January 2018 to January 2020 were included.Trial registration This study was approved by the ethics committee of the First Hospital of Chongqing Medical University on September 26, 2018  and registered with the China Clinical Trials Registry (registration number: ChiCTR1800020440) with an approval date of December 29, 2018. Total joint arthroplasty (TJA) is an effective treatment modality for end-stage bone and joint disease , 2. AlthMany novel serum and joint fluid biomarkers, such as CD14, TREM-1, and TLR2, have shown promising potential in the diagnosis of PJI –12. Qin Considering that the level of inflammatory markers in the joint fluid better reflects local inflammation , in thisThis study was approved by the ethics committee of the First Hospital of Chongqing Medical University on September 26, 2018  and registered with the China Clinical Trials Registry (registration number: ChiCTR1800020440) with an approval date of December 29, 2018. Patients who underwent hip/knee revision at the First Hospital of Chongqing Medical University for periprosthetic infection or aseptic failure of the prosthesis after hip/knee arthroplasty from January 2018 to January 2020 were included. Inclusion criteria: age > 18 years; periprosthetic infection or aseptic failure of the prosthesis after initial replacement, with a recommendation for second-stage revision surgery; no other infection during the 2-year follow-up after hip/knee replacement. Exclusion criteria: (1) inflammatory arthritis, such as rheumatoid arthritis; (2) patients who had used antibiotics within the previous 2 weeks; (3) a history of petechiae, prosthetic heart valves, or hypercoagulation; (4) heavy smokers or patients with malignancy; (5) acute infections , 17.The diagnostic criteria for chronic PJI were the isolation of pathogens from two or more separate tissue or fluid samples from around the involved prosthetic joint and the presence of at least four of the following six criteria: (1) elevated erythrocyte sedimentation rate (ESR) and CRP; (2) elevated joint fluid white blood cell count; (3) elevated joint fluid percent neutrophils (PMN%); (4) pus from the involved joint; (5) the isolation of microorganisms from periprosthetic joint tissue or fluid cultures; (6) periprosthetic histopathological specimens with a mean neutrophil count of 5 in five high-powered fields under high-powered microscopy. Patients were divided into a periprosthetic infection group  and a non-infected (non-PJI) control group .Clinical data relevant to the study included medical histories and laboratory examinations from patients undergoing revision surgery. The medical history included the patient’s age, gender, BMI, previous surgery, functional status of the affected limb at the current admission, presence of nocturnal or resting pain, presence of fever or a definite history of infection and comorbidities, and physical examination . Laboratory examinations included serum CRP, ESR, PMN%, and joint fluid IL-6 testing on admission. In addition, all patients discontinued antibiotics for 2 weeks before arthrocentesis, and joint fluid was obtained by arthrocentesis at admission or preoperatively, with cell sorting and pathogenic microorganism culture. Histopathology and pathogenic microorganism culture of at least three periprosthetic tissues were performed intraoperatively. Microbial cultures of all specimens included aerobic, anaerobic, fungal, and prolonged cultures.Immediately after admission, 1–2 ml of joint fluid were collected. 2 h after collection, all cellular and particulate components were removed by centrifugation at 1000 rpm for 10 min. The levels of IL-6 and PMN% in the joint fluid were measured using an IMMULITE 1000 immunoassay system . Serum ESR and CRP were measured by blood collection at admission and 1 day before surgery, respectively.t-test was used for measures that conformed to a normal distribution, and the Mann–Whitney U test was used to compare the differences between the two groups for measures that did not conform to a normal distribution. Correlations between variables were analyzed using Pearson’s correlation coefficient. MedCalc 15.2.2 software  was used to analyze the receiver operating characteristic (ROC) curves and the area under the curve (AUC) values. Youden’s j statistic was used to determine the optimal cut-off value for the diagnosis of chronic PJI. The sensitivity, specificity, accuracy, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio (DOR) were calculated for two serum markers  and joint fluid IL-6. p < 0.05 was considered statistically significant.Categorical variables were analyzed using SPSS version 25 software . Continuous variables were expressed as mean ± standard deviation, and categorical variables were expressed as counts and percentages. The independent samples P > 0.05).Table P < 0.001). The PMN% of joint fluid in the PJI group, 69.71% (25.85%), was significantly higher than that in the non-PJI group: 40.06% (22.49%) (P < 0.001). Serum ESR and CRP concentrations were not statistically different in the PJI (ESR: 35.00 (33.50) mm/h; CRP: 19.10 (18.40) mg/L) and non-PJI (ESR: 30.00 (16.00) mm/h; CRP: 16.40 (19.79) mg/L) groups (P > 0.05).The levels of IL-6 and PMN% in joint fluid and of ESR and CRP in serum in both groups are shown in Table To assess the predictive value of joint fluid IL-6 and PMN% for chronic PJI, we developed a ROC diagnostic analysis model Fig. , determiTable The clinical symptoms and laboratory markers that patients with chronic PJI may show are not obvious, which greatly increases the difficulty of diagnosing chronic PJI , 18–20. Previous studies have demonstrated that IL-6 in joint fluid and blood is useful in the diagnosis of chronic PJI but cannot be used as the sole diagnostic criterion . A prospThis study has some limitations. First, there is currently a lack of a real gold standard for diagnosing PJI. We used the 2013 MSIS diagnostic criteria as the basis for grouping in this study, and some patients in the non-PJI group may have been false-negative patients, but this is a situation faced by all studies evaluating the diagnosis of PJI infection. Second, the sample size of our study was small. However, as an initial test, the current study has shown promising results, so a larger multicenter study could be conducted to further validate the findings. Finally, patients on antibiotics for two weeks before surgery were excluded from this study to eliminate confounding factors. However, this may have created testing prerequisites that differ from those used in clinical practice, limiting the generalizability of the results of this study.Our study found significantly elevated IL-6 and PMN% values in the synovial fluid of PJI patients. Based on the MSIS criteria, IL-6 combined with PMN% had higher sensitivity and specificity for the diagnosis of chronic PJI. From the above analysis, we believe that IL-6 combined with PMN% in synovial fluid is a promising diagnostic index for PJI and may be included in the diagnostic criteria for PJI in the future. This combination of joint synovial fluid biomarkers can improve the accuracy of PJI diagnosis, providing clinicians with a more timely and accurate diagnosis of PJI, and bringing benefits to patients. Therefore, more research that focuses on finding biomarkers with high accuracy, cost-effectiveness, and feasibility is needed.In this study, it was shown that the combination of joint fluid IL-6 and PMN% can be used as a specific molecular marker for the diagnosis of chronic PJI, and the most appropriate thresholds for IL-6 and PMN% were found to be > 662.50 pg/ml) and > 51.09%, respectively. This combination is more sensitive and specific in distinguishing aseptic failure after arthroplasty from chronic PJI than IL-6 or PMN% in joint fluid alone. However, there are few clinical examples of the combined use of joint fluid IL-6 and PMN% for the diagnosis of PJI, so more evidence is needed to support the use of joint fluid IL-6 combined with PMN% as a valid reference indicator for diagnosing PJI and determining the timing of prosthetic reimplantation.


While not traditionally included in the conceptual understanding of circulation, the interstitium plays a critical role in maintaining fluid homeostasis. Fluid balance regulation is a critical aspect of septic shock, with a well-known association between fluid balance and outcome. The regulation of transcapillary flow is the first key to understand fluid homeostasis during sepsis.Capillary permeability is increased during sepsis, and was classically considered to be necessary and sufficient to explain the increase of capillary filtration during inflammation. However, on the other side of the endothelial wall, the interstitium may play an even greater role to drive capillary leak. Indeed, the interstitial extracellular matrix forms a complex gel-like structure embedded in a collagen skeleton, and has the ability to directly attract intravascular fluid by decreasing its hydrostatic pressure. Thus, interstitium is not a mere passive reservoir, as was long thought, but is probably major determinant of fluid balance regulation during sepsis. Up to this date though, the role of the interstitium during sepsis and septic shock has been largely overlooked. A comprehensive vision of the interstitium may enlight our understanding of septic shock pathophysiology. Overall, we have identified five potential intersections between septic shock pathophysiology and the interstitium: 1. increase of oedema formation, interacting with organ function and metabolites diffusion; 2. interstitial pressure regulation, increasing transcapillary flow; 3. alteration of the extracellular matrix; 4. interstitial secretion of inflammatory mediators; 5. decrease of lymphatic outflow.We aimed at reviewing the literature and summarizing the current knowledge along these specific axes, as well as methodological aspects related to interstitium exploration. Interstitium can be defined as the interstitial, often virtual space existing between cells of any given tissue, but it is also a structured tissue in its own right. Advances in in vivo microscopy allowed the description of large anatomical spaces with thick collagen bundles delimiting fluid-filled polygons Fig. , especiaAs septic shock patients can gain up to one-third of their weight in a few days due to interstitium expansion, this “third space” deserves closer attention and should be understood as a whole by the intensivist. The interstitium has long been neglected and considered as a passive reservoir, whereas the vascular compartment was the only focus. Major physiology discoveries were made in the past decades, revealing how the interstitium plays a major role during acute inflammation, especially in regulating capillary filtration .Overall, we have identified five potential intersections between septic shock pathophysiology and interstitium Fig. : 1. incrTo shed light on the potential role of the interstitium in the pathophysiology of septic shock, we aimed at reviewing the literature and summarizing the current knowledge along these specific axes, as well as methodological aspects related to interstitium exploration.The interstitium is traditionally defined as a fluid compartment, separating blood vessels and cells. It was recently shown that the interstitial space was continuous across tissue and organ boundaries, allowing movement of particles between layers of the colonic wall or through subcutis and fascia . An inteWhen inflammation is present in a circumscribed area, vascular flow and permeability are regionally increased to enable rapid recruitment of humoral  and cellular elements , required to control the pathogens. During sepsis, global hyperactivation of innate immunity results in diffuse endothelial alterations leading to macro- and micro-circulatory dysfunction. Endothelial barrier alteration is probably the most relevant consequence at the capillary level, with impairment of each strata of the endothelial wall, enabling capillary leak . VE-cadhThe pathophysiology of capillary filtration, and thereby of oedema formation was first described by Ernest Henry Starling in 1896 . Transcawhere:Jv is the trans-endothelial filtration volume per second; Kf is the coefficient of filtration of the membrane, Pc is the capillary hydrostatic pressure, Pi is the interstitial hydrostatic pressure, πc is capillary colloid osmotic pressure (COP), πi is the interstitial colloid osmotic pressure, σ is the reflection coefficient for plasma proteins, and ΔPf is the net filtration pressure.Pc and πi at this point, and compensate in part the filtrated volume .Fig.Fig10].FThe role of ß1 integrin subunit in the formation of oedema has been demonstrated in vitro by the assessment of fibroblast-mediated contraction of floating collagen type I gels . In thisFurthermore, ECM’s GAGs are also involved in interstitial volume regulation. They are negatively charged and interact in particular with sodium, the most abundant cation in the extracellular fluid. Depending on their spatial conformation, GAGs accumulate a varying quantity of sodium, which can contribute to variations in interstitial volume, oedema formation and resolution , 67. DurThe ECM forms the physical structure of the interstitial space. We described the functional changes induced by inflammation, leading to ECM decompaction. Moreover, inflammation also leads to the direct alteration of ECM structural compounds due to the release by infiltrating neutrophils of potent catalytic enzymes, such as MMPs , heparanases and hyaluronidases , activatA small descriptive study from Koskela and collaborators , reporteOther ECM macromolecules, such as proteoglycans and small leucine-rich proteoglycans (SLRP), can be synthesized de novo or released from collagen association during ECM remodelling. Recently, Maiti G and collaborators reported that during sepsis, lumican (a proteoglycan from the SLRP family) is endocytosed by immune cells and is able to control receptor ligand trafficking . Lumican. [The consequences of ECM alteration during sepsis are not known yet. It could participate in the increased capillary filtration, as ECM physical structure is a key to the regulation of interstitial pressure: collagen and proteoglycans alteration could enhance the “decompaction” of the ECM. This is supported by experimental data: enzymatic digestion potentiates the ex vivo swelling of loose connective tissue immersed in isotonic saline, which is normally restrained by the collagen fibers network . Further. . Indeed,. . AlthougIncreased interstitial flow has biophysical effects on ECM and residing cells. Mechanical stress, as well as inflammation, can induce the differentiation of fibroblast into myofibroblasts, and may ultimately lead to fibrogenesis . It was It is yet unknown if interstitial fibrosis can develop in critically ill patients with persistent inflammation, but it might then contribute to inadequate healing with a persistent state of fluid overload, as seen in the more severe patients.In the view of the emerging role of the interstitium, it appears that interstitial fluid is a promising medium for the discovery of new biomarkers. However, access to this fluid is difficult. In the 1900s, metal tubes were introduced subcutaneously for the evacuation of oedema in patients with heart failure (“Southey’s tubes”) . This inThe interstitial compartment is much more difficult to access than the blood for the analysis of biomarkers. However, numerous markers of inflammation are of local origin. Olszewski et al. performed lower leg lymphatic cannulation in patients with rheumatoid arthritis and in control subjects and collected prenodal lymph continuously for 72 h . A lymphUsing a microperfusion catheter inserted into the abdominal cutaneous adipose tissue of nine patients with severe sepsis, Ikeoka et al. found that the concentrations of IL-1β, IL-6 and IL-8 were higher in subcutaneous adipose tissue than in serum, indicating a subcutaneous interstitial production of these pro-inflammatory mediators, remotely from the infection site . In addiThe lymphatic system plays a critical role in the circulation. At steady state, pre-nodal lymph flow equals capillary filtration, with an estimated 8–12 L reabsorbed daily through the lymphatic system , beginniMoreover, lymphatic alterations are also critical in the development of organ damage, and may prevent healing mechanisms. In the lungs, the role of lymphatic circulation is evidenced in lung transplantation, during which lymphatic vessels are interrupted. Lung transplant recipients exhibit persistent oedema, and the role of lymphatic vessels and lymphangiogenesis is suspected in primary graft dysfunction . ResorptThe interstitium is not merely a passive bystander but finally emerges as a key player in the regulation of transcapillary flow, in particular with its ability to aspirate fluid from the blood vessels. This important role in the regulation of capillary filtration is increasingly well-documented in the setting of local inflammation and in tumours, but evidence of interstitium involvement during sepsis is still scarce. A lot of questions remain open as to its exact role during sepsis: what is the magnitude of pressure changes? Are there organ-specific responses? What is the respective role of subcutaneous fibroblasts and adipocytes in cytokine secretion? What is the role of ECM damage on oedema formation and resolution? In the current era of personalized medicine, the study of this little-known compartment could yield valuable insights, enhance our understanding of the pathophysiology and open unique perspectives for the investigation of new biomarkers and therapies.


Percutaneous hepatic melphalan perfusion (chemosaturation) is a treatment option in patients with inoperable liver metastases which is associated with considerable procedural challenges, especially hemodynamic depression, due to a reduced preload and impaired coagulation caused by the use of heparin. Studies on factors that contribute to bleeding complications and a prolonged intensive care unit length of stay are not available. In this retrospective analysis, we found that high perioperative infusion volumes and the omission of heparin reversal with protamine were associated with postoperative bleeding complications, while high infusion volumes also contributed to a length of stay in the intensive care unit of more than one day, which usually is not required. Furthermore, protamine use was not significantly associated with anaphylactic or thromboembolic complications. Our findings suggest a restrictive perioperative infusion regime and support the use of postoperative protamine for heparin reversal in chemosaturation procedures.p = 0.003) and protamine  to be associated with bleeding complications. Furthermore, the amount of perioperative fluid volume was associated with an ICU LOS > 1 d . Heparin dosage, melphalan dosage, extracorporeal circulation time, and noradrenaline dosage had no significant effects on outcomes. Protamine use was not associated with anaphylactic or thromboembolic complications. Despite the limited sample size, these results suggest a restrictive perioperative fluid regime to be beneficial, and support the use of protamine for heparin reversal after chemosaturation procedures. Further prospective randomized trials are needed to confirm these findings.Percutaneous hepatic melphalan perfusion (chemosaturation) in patients with liver metastases is known to be associated with procedure-related hemodynamic depression and coagulation impairment, which may cause bleeding complications and/or a prolonged intensive care unit length of stay (ICU LOS). We retrospectively analyzed possible predictive factors for bleeding complications and an ICU LOS > 1 d in a cohort of 31 patients undergoing 90 chemosaturation procedures. Using a multivariable mixed-model approach, we identified the amount of perioperative fluid volume (OR 12.0, 95% CI 2.3–60.0,  Percutaneous hepatic melphalan perfusion (chemosaturation) is a treatment option for patients with inoperable liver metastases. It may be a promising option particularly for liver metastases of ocular melanomas, which are known for their invasive growth pattern, but also for hepatocellular carcinoma, intrahepatic cholangiocarcinoma, and other carcinomas ,2,3,4. TExtracorporeal chemofiltration using blood pump circulation requires anticoagulation with an estimated activated clotting time (ACT) of >400 s to maintain functionality and prevent clotting of the filter system. After a bolus administration of heparin, frequent ACT measurements are performed throughout the procedure to maintain the estimated ACT. After the discontinuation of the bypass, reversal of the heparin effect may be provided by protamine. Additionally, the chemosaturation procedure itself is associated with further coagulation disorders , and there is a risk for postoperative bleeding complications ,6,7,8,9.The purpose of this analysis was to identify factors that contribute to bleeding complications and a prolonged intensive care unit length of stay (ICU LOS).−1 min−1), crystalloid fluid volume (mL kg−1), heparin dosage (U kg−1), and protamine use (yes/no). Variables including surrogate parameters of selected predictors, e.g., mean arterial blood pressure and coagulation values ), were not considered. Data are reported as medians and interquartile ranges (IQRs) or frequencies and relative percentages.After receiving approval from the ethical commission, we analyzed our cohort of patients who underwent hepatic chemosaturation procedures from 2016 to 2022; postoperative bleeding complications were defined according to Clavien Dindo classes III-IV, and a prolonged ICU LOS was defined as more than one day after chemosaturation. Investigated predictors were extracorporeal circulation pump (ECCP) time (min), melphalan dosage (mg), noradrenaline (norepinephrine) dosage . Mixed-effects logistic regression was performed in the framework of generalized-linear mixed models using scaled numeric predictors and applying the R package lme4 1.1.31, while regression diagnostics were supported by the DHARMa 0.4.6 package. Thereby, confidence intervals were calculated by applying the Wald z-distribution approximation, which were implemented in package parameters 0.20.2. We considered a p < 0.2, which was followed by the sequential removal of nonsignificant predictors from this model at the 0.05 significance level. Thereby, in each step, the nonsignificant predictor with the largest p-value was removed unless only predictors with p-values larger than 0.05 remained in the model. We assessed model fit and model assumptions, including residual plots, using the R package DHARMa 0.4.6.We used backward elimination to select the best multivariate mixed-effects logistic regression model, including a random intercept term to account for subjects contributing to our study with more than one observation. This procedure involved first including all predictors in the initial model that were significant at the level of Possible trends in the management of investigated predictors over time were analyzed using the Spearman correlation coefficient rho (ρ) based on the first melphalan perfusion of each patient. All data and calculation codes of this study are publicly available without restrictions.n = 17), intrahepatic cholangiocarcinoma (n = 8), hepatocellular carcinoma (n = 2), and other carcinomas (n = 4).The study cohort consisted of 31 patients who underwent 90 chemosaturation procedures in total . Twenty-The median total crystalloid infusion volume was 5000 mL , whereas 13 cases (14.4%) had volumes of ≥8000 mL, and 4 cases (4.4%) exceeded volumes of 10,000 mL. Three patients received an additional albumin infusion, two of which had crystalloid infusion volumes ≥8000 mL. The perioperative transfusion of red cells, fresh frozen plasma, and platelets was not performed.An initial heparin bolus administration of 300–400 U/kg reached the ACT target of >400 s in 67 cases (74.4%), whereas in 23 cases (25.6%), additional heparin was required before chemofilter bypass activation was achieved. Postoperative heparin reversal with protamine was performed in 68 procedures (in 16 cases (24%), full reversal with a heparin-to-protamine ratio of 1:1, and in 52 cases (76%), reduced protamine dosages and heparin-to-protamine ratios of 4:3 (31 cases), 3:2 (10 cases), 2:1 (4 cases), and 3:1 (7 cases)), whereas in 22 procedures protamine was omitted. The characteristics of the procedures are presented in TM ) was required in twelve cases, including five cases with femoral puncture site suturing. Postoperative blood transfusion due to bleeding was required in ten cases, which included red blood cells in five cases (nine units), fresh frozen plasma in six cases (sixteen units), and platelet concentrates in five cases (five units). Fibrinogen and tranexamic acid were each administered in four cases. All patients, including the one requiring cardiopulmonary resuscitation, recovered well and had no long-term sequelae. Mixed-effects logistic regression analysis revealed univariable associations of extracorporeal circulation pump time, the amount of crystalloid fluid volume, and protamine use with postoperative bleeding complications (Postoperative bleeding complications matching class III or greater were observed in 13 cases (14.4%) . Of thesications . After mications . The hepAccording to the model, on average protamine lowered the probability of postoperative bleeding complications from approximately 15% to 1% at the median applied crystalloid fluid volume of 5000 mL. Considering the upper 95% prediction interval, the risk of bleeding complications exceeded 50% when the crystalloid fluid volume was more than 4000 mL in cases without protamine, and more than 7000 mL in cases with protamine.A prolonged ICU LOS of more than one day was observed in nine cases (10%). Our analysis revealed that postoperative bleeding complications were associated with a prolonged intensive care unit length of stay, but not vice versa. Univariable associations were seen with crystalloid fluid volume, protamine use, and extracorporeal circulation pump time. After the multivariable generalized linear mixed-effects model analysis, crystalloid fluid volume was the only significant predictor .p = 0.003) and mL/kg ), while noradrenaline dosages increased . The extracorporeal circulation pump time decreased significantly . Heparin and melphalan dosages did not differ significantly across the observation period   .Our main findings suggest that high perioperative crystalloid fluid infusion volumes and the omission of heparin reversal with protamine may be associated with postoperative bleeding complications after hepatic chemosaturation procedures. Furthermore, high infusion volumes may contribute to a prolonged ICU LOS. Adverse events regarding protamine use, particularly anaphylaxis or thromboembolic complications, were not observed. However, this should be considered with caution, since the number of analyzed procedures was limited to 90 cases. A recent dual-center study involving 256 hepatic chemosaturation procedures reported 10 thromboembolic complications, including 6 major events in 192 cases, in which full heparin reversal with protamine was performed. In 21 cases with reduced protamine dosages and 43 cases without reversal, no thromboembolic events were observed [Although protamine was previously applied only at the discretion of the interventional team, we found it to have an independent protective effect on postoperative bleeding complications. As a result of this analysis, we have changed our strategy back to a mandatory reversal while we continue to discuss the correct protamine dose for each case individually after termination of the extracorporeal circulation. Recent studies of other endovascular procedures, particularly percutaneous coronary intervention ,12,13, tIn the literature, the hemodynamic management of common chemosaturation procedure-related hypotension using infusions and vasopressors is regarded as mandatorily needing to be performed by those with expertise ,5,6,7. HOur analysis indicated that there were no significant effects of extracorporeal circulation pump time and dosages of melphalan, noradrenaline, and heparin on the risk for both bleeding complications and a prolonged ICU LOS. Notably, pump time showed a non-significant trend which might be related to the low sample size, but it should be considered in further studies. In other populations, particularly in those who undergo cardiopulmonary bypass surgery, prolonged pump time may be a relevant prognostic factor for postoperative morbidity and bleeding complications, as it may be possibly related to complex inflammatory triggers due to the shearing forces of non-pulsatile blood flow and exposure to the artificial surfaces of the bypass circuit ,36,37.The present results reveal the learning process of a single center, showing a significant decrease in pump time and crystalloid fluid infusion volumes and an increase in noradrenaline dosage, while melphalan and heparin administration remained constant within the ranges of the recommended dosages. This demonstrates that performing hepatic chemosaturation procedures requires special centers with appropriate infrastructure and attending teams with professional interdisciplinary expertise. Although there are hepatic chemosaturation procedures that occur without any complications, suggesting a postoperative transfer to the recovery room or post-anesthesia care unit, we recommend an enhanced observation of the patients in the ICU at least for one night until the coagulation parameters have normalized and all large-bore vascular catheters have been safely removed. In this context, we acknowledge that angiography units are usually not based in central parts of hospitals and there may be the need for postoperative mechanical ventilation. Furthermore, our case series demonstrates that delayed complications, particularly respiratory distress due to neck swelling, may occur hours after the procedure, which warrants the ability and preparedness for emergency airway management and resuscitation measures.The main limitations of this analysis are the retrospective design and the moderate number of observations. Although we used a mixed-model approach for repeated measures, including a random intercept, confounding effects cannot be excluded due to small sample sizes. Furthermore, the use of protamine for heparin reversal as well as the treatment with crystalloid fluid infusion was performed under the discretion of the attending team and was not based on a study protocol. However, we present relevant real-world data on bleeding complications and prolonged intensive care after percutaneous hepatic chemosaturation procedures, which have to be confirmed in future studies.In hepatic chemosaturation procedures, high perioperative crystalloid fluid infusion volumes and the omission of heparin reversal with protamine may be associated with postoperative bleeding complications. Furthermore, high fluid infusion volumes may contribute to a prolonged ICU LOS.


Background: Hyponatremia is common among hospital inpatients. It is generally due to excess free body water resulting from increased water intake and decreased water elimination due to underlying pathology and hormonal influence. However, supporting evidence is lacking for treating mild hyponatremia with fluid restriction. Our study examines the association between hyponatremia and fluid intake in acutely ill inpatients. We hypothesize that fluid intake is not closely associated with serum sodium (SNa).Methods: We conducted a retrospective study of hyponatremia using the Multiparameter Intelligent Monitoring in Intensive Care (MIMIC) III dataset, a public ICU registry. We analyzed fluid, sodium, and potassium intake with a mixed model linear regression with SNa as the outcome for hyponatremic and non-hyponatremic patients and cumulative total input from one to seven days. In addition, we compared a group of patients receiving less than one liter of fluid per day to a group receiving more than one liter.Results: The association of SNa with fluid intake was negative and statistically significant for most cumulative days of intake from one to seven for the total population and those with sporadic hyponatremia. For those with uniform hyponatremia, the negative association was significant for three and four days of cumulative input. The change in SNa was almost always less than 1 mmol/L of additional fluid intake across all groups. SNa for hyponatremic patients who received less than one liter of fluid per day were within one mmol/L of those who received more .Conclusions: SNa is associated with a change of less than 1 mmol/L across a wide range of fluid and sodium intake in adult ICU patients. Patients who received less than one liter per day had SNa almost identical to those who received more. This suggests that SNa is not tightly coupled with fluid intake in the acutely ill population and that hormonal control of water elimination is the predominant mechanism. This might explain why the correction of hyponatremia by fluid restriction is often difficult. Hyponatremia is the most common electrolyte disorder among hospital inpatients, with an estimated prevalence of 15-40% . DespiteWe conducted a single-center, retrospective, observational study of hyponatremia using data from the Multiparameter Intelligent Monitoring in Intensive Care (MIMIC) III database. MIMIC is a publicly available registry of deidentified ICU data from multiple admissions over 11 years at Beth Israel Deaconess Medical Center. The population is a mixture of medical and surgical ICU patients. Although surgical patients may require more fluid than medical, we chose to include both to make the results more broadly applicable. This database is distinctive for its granularity, including details down to specific lab values and detailed recording of drug and fluid administration . We seleWe totaled electrolyte and fluid intake for specified days prior to each SNa measurement. We included only sequential days because data for missing days could affect results. We analyzed the association between SNa and fluid intake over the previous two, three, four, and seven days. Hyponatremia is defined as SNa<135 mmol/L [A total of 37,754 patients were analyzed. The mean age was 64.1 years. Male patients made up 56.5% of the cohort. A majority of the patients (54.5%) were on the medicine service, and hypertension was the most prevalent ailment (46.7%)  is the most common electrolyte disorder treated in clinical practice. It is a chronic and acute illness present in 15-20% of emergency admissions . Our stuFurther subcategories are hypotonic versus non-hypotonic hyponatremia. Non-hypotonic is due to the presence of other solutes, such as glucose. It is rare, occurring mainly in severe hyperglycemia and renal failure, and is not the focus of our analysis. Euvolemic hypotonic hypovolemia is the most common subtype and is most commonly due to the syndrome of inappropriate antidiuretic hormone secretion (SIADH) . SIADH rGiven the underlying endocrine cause of most hyponatremia, we sought to examine how solute and fluid intake interact with the hormonal state in ICU patients and what are the implications for treatment. Normally functioning kidneys can excrete free water or conversely concentrate urine sodium to excrete solute, thus conserving free water. This allows the body to maintain normal SNa values despite widely varying solute and volume intake. However, variations in fluid and sodium intake may exceed the ability of the kidney to adapt . Some stThe fluid does not have to be hypotonic to contribute to hyponatremia. A long-recognized phenomenon known as desalination occurs when the kidney excretes solute in urine at higher concentrations than the fluid that is infused. In this circumstance, even isotonic fluid infusions can lead to hyponatremia . For thiOur study also showed an association between hyponatremia and intake of volume and sodium, which was significant but very small. These results are displayed in Table American and European guidelines recommend hypertonic saline  for acute or symptomatic hyponatremia . AccordiIn a 2016 review, Cuesta et al. stated that there have been no randomized controlled trials (RCTs) proving the efficacy or safety of fluid restriction in the clinical setting . Since tFluid restriction alone failed to achieve prospectively defined Na improvement goals in >50% of cases, and 75% were still hyponatremic at discharge. Correction of hyponatremia was not associated with survival, suggesting that it is a marker of disease severity rather than an independent cause of mortality . In an aOur findings are subject to some limitations. Strict diagnosis of SIADH requires measurement of urine and plasma osmolality, and urine sodium. We rely on a clinical diagnosis without extensive laboratory confirmatory tests. Fluid restriction must limit intake below a certain threshold to be effective. We addressed this by comparing a group that received liberal fluid intake with another that received less than one liter per day of fluid, consistent with a typical fluid restriction regimen. SNa following the lower intake was almost identical to the liberal fluid group, however, it is possible that more severe fluid restriction could be effective. Another possibility is that low SNa prompted clinicians to treat some of their hyponatremic patients with fluid restriction, thus low SNa could indirectly be a cause of low fluid intake. We address this possibility by analyzing a subgroup of patients who were uniformly hyponatremic throughout the period of study. The fluid restriction would likely be prescribed for all these patients; thus, the effects of selective fluid restriction should be minimized. Record keeping, especially intake and output, may not be accurate, which is an inherent limitation of retrospective data.SNa is almost unchanged across a wide range of fluid and sodium intake in acutely ill hospital inpatients. This includes volumes typically associated with a regimen of fluid restriction used to treat hyponatremia. We believe that this supports hormonally mediated water elimination over water intake as the primary determinant of SNa in the critically ill adult population. Fluid restriction is based on a sound physiologic mechanism, but the practical application has been challenging and strong evidence of efficacy has been lacking. Volume intake within the variation documented for this cohort of ICU patients showed minimal association with SNa. We consider it unlikely that deliberate restriction within these same parameters would have a greater effect. The resistance of SNa to variations in fluid volume suggests that treatment of hyponatremia with fluid restriction alone will be difficult in the acutely ill population. The practice is based on a model of renal function rather than empirical data. Withholding free water below the minimum that the kidney requires to excrete a solute load will eventually concentrate SNa, but our study helps explain why this is difficult to implement as a practical treatment protocol.


During septic shock, fluid therapy is aimed at increasing cardiac output and improving tissue oxygenation, but it poses two problems: it has inconsistent and transient efficacy, and it has many well-documented deleterious effects. We suggest that there is a place for its personalization according to the patient characteristics and the clinical situation, at all stages of circulatory failure. Regarding the choice of fluid for volume expansion, isotonic saline induces hyperchloremic acidosis, but only for very large volumes administered. We suggest that balanced solutions should be reserved for patients who have already received large volumes and in whom the chloremia is rising. The initial volume expansion, intended to compensate for the constant hypovolaemia in the initial phase of septic shock, cannot be adapted to the patient’s weight only, as suggested by the Surviving Sepsis Campaign, but should also consider potential absolute hypovolemia induced by fluid losses. After the initial fluid infusion, preload responsiveness may rapidly disappear, and it should be assessed. The choice between tests used for this purpose depends on the presence or absence of mechanical ventilation, the monitoring in place and the risk of fluid accumulation. In non-intubated patients, the passive leg raising test and the mini-fluid challenge are suitable. In patients without cardiac output monitoring, tests like the tidal volume challenge, the passive leg raising test and the mini-fluid challenge can be used as they can be performed by measuring changes in pulse pressure variation, assessed through an arterial line. The mini-fluid challenge should not be repeated in patients who already received large volumes of fluids. The variables to assess fluid accumulation depend on the clinical condition. In acute respiratory distress syndrome, pulmonary arterial occlusion pressure, extravascular lung water and pulmonary vascular permeability index assess the risk of worsening alveolar oedema better than arterial oxygenation. In case of abdominal problems, the intra-abdominal pressure should be taken into account. Finally, fluid depletion in the de-escalation phase is considered in patients with significant fluid accumulation. Fluid removal can be guided by preload responsiveness testing, since haemodynamic deterioration is likely to occur in patients with a preload dependent state. The first therapeutic measure in septic shock, volume expansion, is seemingly simple: a few hundred millilitres of fluid injected intravenously over a few minutes. However, nothing is simple in the strategy of using fluids in septic patients. Several different types of fluids are available today . The effTo these questions, one can provide a standard and unequivocal answer. This is what the current guidelines do , withoutIn this review, we will describe what a personalized fluid strategy in septic shock can be, based not only on the existing publications, but also on the pathophysiological concepts underlying the effects of fluids. From the choice of fluid type to the decision to remove the fluid administered in excess, we will try to show that, at each stage, the choices can reasonably be individualized.In septic shock, crystalloids are indicated in first intention by the Surviving Sepsis Campaign (SSC) . HoweverThe current debate is mainly between the use of balanced or unbalanced crystalloids. Using saline as the main fluid source can induce hyperchloremic acidosis, which may decrease kidney cortical perfusion and could induce renal failure. Several studies have suggested that compared to chloride-rich solutions, balanced solutions reduce the incidence of renal failure , 11 and Accordingly, the 2021 SSC guidelines suggested to prefer balanced crystalloids in all septic shock patients . This weShould we favour these balanced solutions as first intention in all septic shock patients? Like other authors , we thinvs. normal saline in critically ill patients (not only septic) . Th. Th36]. Reviews detailing indices and tests to detect preload responsiveness can be found elsewhere . Our goa2O, without intra-abdominal hypertension, with sinus cardiac rhythm and with a ratio of heart rate to respiratory rate ≥3.6 [The passive leg raising (PLR) test and the mini-fluid challenge do not require mechanical ventilation. This makes them usable, for example, in the emergency department, while patients are not yet intubated . Of courate ≥3.6 , i.e. inate ≥3.6 . The chaate ≥3.6 .The end-expiratory occlusion (EEXPO) test  in the event of preload responsiveness ), can beIn ventilated patients with a Vt of 6 mL/kg, the Vt challenge  is valiThe great advantage of the Vt challenge , the PLRThe EEXPO test requires a precise measurement of CO, because the changes in stroke volume induced are small . If echoThe mini-fluid challenge requires the injection of 100-150 mL of fluid, which cannot be withdrawn if ineffective . It is tNorepinephrine is usually started once fluid boluses no longer exert any benefit, to increase arterial pressure . HoweverThese venous effects of norepinephrine are synergistic with the administration of fluid, which may reduce the quantity of fluid administered for resuscitation Fig. . ObservaThe CLOVERS study randomized patients between a restrictive strategy, prioritizing lower intravenous fluid volumes and vasopressors, and a liberal strategy, prioritizing higher fluid volumes before starting vasopressors. Although less fluid was administered within the first 24 hours, mortality at day-90 did not differ between groups . HoweverMoreover, in this study, patients in whom norepinephrine was administered earlier were not selected . This eaIn addition, the effectiveness of a fluid bolus on CO is ephemeral. In the perioperative context, the effects on CO of a 250-mL crystalloid bolus were shown to disappear after 10 minutes . This duGood fluid management should include safety criteria, indicating that the risk of fluid infusion outweighs the expected benefit . These c2) to fractional inspiration of oxygen (FiO2). However, it can also be lowered by pulmonary consolidations, shunt related to microthrombi, etc., which are not influenced by additional fluid infusion.In these patients, to alert to the risk of additional fluid infusion, one often uses the ratio of arterial partial pressure of oxygen  should be considered to estimate the risk of fluid accumulation which, ases IAP . Intra-aases IAP . This vaases IAP .Regardless of the patient, even if CVP does not predict preload responsiveness, its measurement provides a great deal of information that is often overlooked . CVP is Fluid boluses are aimed at increasing mean systemic pressure, cardiac preload, consequently stroke volume and CO, and ultimately at improving tissue oxygenation. However, the therapeutic targets must be adapted to the available haemodynamic monitoring and to the context.When fluid therapy is conducted in the wards, in the pre-hospital or in the emergency department, the clinical examination must look for clinical signs of improvement in tissue perfusion: disappearance of skin mottling, shortening of capillary refill time, which disappears quite faster and might change even after a single fluid bolus , 81. ImpThe primary effect of a bolus of fluid is to increase CO, and arterial pressure increases only incidentally . Of noteHowever, even changes in pulse pressure imperfectly reflect CO , 87, 88,More variables are available to estimate fluid efficacy. CO is easily measured, either by transpulmonary thermodilution, pulmonary arterial catheter, recommended in the most severe patients , or by eAlso, the effects on tissue oxygenation must be assessed , as an iOne of the ways to reduce the total fluid balance is to undertake active fluid depletion, at the “evacuation” phase of the resuscitation–optimization–stabilization–evacuation (ROSE) concept Fig. . Indeed,Fluid depletion should be the most beneficial in patients with the largest fluid accumulation, as indicated by the variables mentioned above: high left ventricular filling pressure, high CVP, very lung water, signs of pulmonary congestion on CT scan and elevated IAP. The presence of soft tissue oedema does not necessarily have to be required, as they may on the contrary be accompanied by a depleted intravascular sector.Conversely, if fluid accumulation is low, for example in easily resolving septic shock, the benefit of fluid depletion is less obvious. The risk of undertaking it must therefore be weighed against the possible benefits.Diuretics are used first. However, in case of oliguria or anuria, ultrafiltration during renal replacement therapy is the alternative solution . In caseThe risk of too much fluid withdrawal is to alter the haemodynamic state. To ensure the safety of fluid depletion, physiological conditions must be considered, which is rarely done in practice . First, unresponsiveness. In a study in critically ill patients in a stabilized phase of shock, fluid removal undertaken by ultrafiltration did not induce intra-dialytic hypotension in patients who did not have preload responsiveness, as well as proved by a negative PLR test before depletion [Then, decreasing cardiac preload with fluid removal can only alter the haemodynamic condition in case of preload responsiveness. Conversely, if ventricles are preload unresponsive, fluid removal must be well tolerated. One should condition the decision to withdraw fluid to the existence of a preload epletion  Fig. 1)unresponsepletion , other fDespite its apparent simplicity, fluid therapy is complex. Fluids are drugs, inconsistently effective and endowed with notable deleterious effects. Like any medicine, they must be administered at the right dose and only to patients who need them. To guide fluid therapy, rather than relying on an identical strategy for all patients, it is possible to personalize decisions. Consider the characteristics of the patient , his/her clinical history , his/her physiological conditions (in particular preload responsiveness), should logically ensure effective and safe fluid delivery and removal. If these individual criteria are not considered, clinical studies investigating the various fluid therapy strategies are and will remain negative.


Enterococcus faecalis , a gram-positive facultative anaerobic bacterium, is likely to survive root canal treatment because of its extremely high alkaline tolerance, which may contribute to the refractory nature of apical periodontitis (AP). In this study, protamine was combined with calcium hydroxide to evaluate its efficacy in killing E. faecalis. First, the antibacterial activity of protamine against E. faecalis was investigated. Protamine reduced the E. faecalis growth rate at concentrations above the MIC (250 μg/mL), but was not bactericidal at any of the concentrations tested. Next, we investigated the calcium hydroxide tolerance of E. faecalis, using a 10% 310 medium, adjusted for pH by adding a calcium hydroxide solution. The results showed that E. faecalis could survive and proliferate in alkaline environments up to pH 10. However, the complete killing of E. faecalis was observed when protamine (250 μg/mL) was added. In addition, compared with treatment with protamine and calcium hydroxide alone, membrane damage and internalization of protamine into the cytoplasm of E. faecalis were enhanced. Therefore, the synergistic increase in antibacterial activity may be related to the action of both antimicrobial agents on the cell membrane. In conclusion, co-treatment with protamine and calcium hydroxide seems to be very effective in sterilizing E. faecalis, and has the potential to provide a novel control method against E. faecalis for root canal treatment. Enterococcus faecalis  is frequently detected in root canals with failed treatment [E. faecalis, and the presence of these bacteria may be one of the factors that make AP difficult to treat. Therefore, it would be indispensable to investigate more effective methods for controlling E. faecalis in root canals, to improve the success rate of AP treatments.Apical periodontitis (AP) is an inflammatory dental disease caused by microbial infection of the root canal , which oreatment ,7,8. ThiE. faecalis has an extremely high alkaline tolerance, due to its unique membrane durability, impermeability, and proton pump mechanism [E. faecalis disinfection are not met deep in the dentin tubules because of the buffering effect of these dentin tubules. [E. faecalis survival may be promoted in real root canal environments. To improve the success rate of root canal treatment, it is essential to consider more effective treatment methods of calcium hydroxide against E. faecalis.The purpose of root canal treatment is to eradicate pathological flora from the root canal system and achieve complete tooth asepsis . Calciumechanism ,11. Therechanism . Probabltubules. . Thus, EE. faecalis, and evaluated the efficacy of co-treatment with protamine and calcium hydroxide in killing E. faecalis.In this study, we focused on antimicrobial peptides (AMP), which have recently attracted attention as novel antimicrobial agents ,15,16. PE. faecalis, a type of bacteria involved in AP, and proposes a novel method for root canal treatment.Our study provides new insights into the bacterial control, especially A 310 medium (NBRC) was used to culture the microorganisms. In this study, assuming a poor nutritional environment after endodontic cleaning, a 310 medium, diluted with ultrapure water to a final concentration of 10%, was used for various evaluation tests.E. faecalis purchased from NBRC (NBRC No. 100481) was used in this study. E. faecalis was restored by 310 medium and cultured anaerobically at 37 °C using an anaerobic jar  and AnaeroPack® Kenki . The culture was stored at −80 °C in 15% glycerol.The strain of Protamine was supplied by Maruha Nichiro Corporation . Protamine (0.5 g) was dissolved in 50 mL of ultrapure water . The solution was sterilized in an autoclave , diluted appropriately, and used for the various evaluation tests.To prepare the calcium hydroxide solution, supersaturated amounts of calcium hydroxide powder  were dissolved in ultrapure water. The solution was centrifuged at 8000 rpm for 10 min. The supernatant was used for evaluation tests.E. faecalis to protamine was evaluated by alamarBlue® assay and colony count assays. Protamine (20 μL), bacterial suspension (5 × 105 CFU/mL) diluted by 10% 310 medium, and 20 μL of alamarBlue® solution  were added to 96 well plates. Testing was performed using appropriate controls. After incubation for 24 h at 37 °C, the color of the solution changed. The minimum inhibitory concentration (MIC) was determined as the lowest concentration at which no color change occurred (red indicates growth and blue indicates no growth). For concentrations above the MIC, 100 μL of the suspension from each well was plated onto a 310 agar plate, incubated for 24 h at 37 °C in an anaerobic environment, and then observed for the presence of colonies. Minimum bactericidal concentration (MBC) was determined as the lowest concentration that resulted in no growth on agar plates.The susceptibility of E. faecalis, we treated planktonic cells with protamine, and determined changes in viable bacterial counts over time using a colony count assay. E. faecalis was incubated anaerobically overnight in 310 mL of medium, and the cell density was adjusted to 5 × 106 CFU/mL. Antimicrobial treatment was performed by mixing 100 μL of the bacterial suspension, 100 μL of Protamine , and 800 μL of sterile water. After incubation of these samples at 37 °C for 1, 2, 4, 8, and 24 h, 100 μL of suspension from each sample was plated to a 310 agar plate, and the viable bacterial count was determined by colony count.To evaluate the growth-inhibitory ability of protamine against E. faecalis, a calcium hydroxide solution was added to 10% 310 medium, and the planktonic cells were incubated for 24 h in an arbitrary alkaline environment. Viable bacterial counts were determined using the colony count assay. E. faecalis was incubated anaerobically overnight in 310 medium. The suspension was replaced with 10% 310 medium, and the cell density was adjusted to 5 × 107 CFU/mL. Antimicrobial treatment was conducted by mixing 10 μL of bacterial suspension and 990 μL of 10% 310 medium, with the pH adjusted to 8, 10, or 12 by adding a calcium hydroxide solution. After incubation of these samples at 37 °C for 24 h, 100 μL of suspension from each sample was plated to a 310 agar plate, and the viable bacterial count was determined by colony count.To evaluate the calcium hydroxide tolerance of E. faecalis, planktonic cells were incubated in an arbitrary alkaline environment, and changes in viable bacterial count over time were determined by a colony count assay. E. faecalis was incubated anaerobically overnight in a 310 medium. The suspension was replaced with 10% 310 medium, and the cell density was adjusted to 5 × 107 CFU/mL. Antimicrobial treatment was conducted by mixing 10 μL of the bacterial suspension and 990 μL of a 10% 310 medium, with the pH adjusted to 8 and 10 by adding calcium hydroxide solution. After incubation of these samples at 37 °C for 2, 4, 8, and 24 h, 100 μL of suspension from each sample was plated to a 310 agar plate, and the viable bacterial count was determined by colony count.To evaluate the effect of calcium hydroxide treatment on the metabolic activity of E. faecalis, planktonic cells were co-treated with protamine and calcium hydroxide, and the viable bacterial count was determined using a colony count assay. E. faecalis was incubated anaerobically overnight in a 310 medium. The suspension was replaced with a 10% 310 medium, and the cell density was adjusted to 5 × 107 CFU/mL. Antimicrobial treatment was conducted by mixing 10 μL of the bacterial suspension and 990 μL of a 10% 310 medium, with the pH adjusted to 8 and 10 by adding calcium hydroxide solution. If necessary, protamine was added to 990 μL of a 10% 310 medium, to a final concentration of 250 μg/mL. After incubation of these samples at 37 °C for 1 or 24 h, 100 μL of suspension from each sample was plated in a 310 agar plate, and the viable bacterial count was determined by colony count.To evaluate the effectiveness of co-treatment with protamine and calcium hydroxide in killing E. faecalis, we observed the bacteria after treatment using scanning electron microscopy (SEM) after treatment, as described in ® Plastic Coverslips  and air-dried overnight. They were then coated with gold and observed by SEM .To evaluate the effect of co-treatment with protamine and calcium hydroxide on the surface structure of E. faecalis, we examined the presence of cell membrane damage by Live/Dead®Baclight™ Bacterial Viability Kit . E. faecalis was incubated anaerobically overnight in 310 medium. The suspension was replaced with 10% 310 medium, and the cell density was adjusted to 2 × 109 CFU/mL. We mixed 50 μL of the bacterial suspension and 950 μL of a 10% 310 medium, with pH adjusted to 8 and 10 by adding a calcium hydroxide solution . Antimicrobial treatment was conducted by mixing 324.97 μL of Suspension A, 8.33 μL of protamine , 0.5 μL of SYTO9, and 0.5 μL of Propidium Iodide (PI). After incubation of these samples at 37 °C for 1 h, they were washed three times with PBS, and observed by an all-in-one fluorescence microscope .To evaluate the effect of co-treatment with Protamine and calcium hydroxide on the cell membrane of E. faecalis was incubated anaerobically overnight in a 310 medium. The suspension was replaced with a 10% 310 medium, and the cell density was adjusted to 2 × 109 CFU/mL. We mixed 50 μL of bacterial suspension and 950 μL of 10% 310 medium, with pH adjusted to 8 and 10 by adding a calcium hydroxide solution . The antimicrobial treatment was performed by mixing 324.97 μL of Suspension B, 8.33 μL of FAM-PP , and 0.5 μLof P. After incubation of these samples at 37 °C for 1 h, they were washed three times with PBS, and observed by an all-in-one fluorescence microscope .To investigate the relationship between cell membrane damage and localization site of protamine, and the relationship between the localization site of protamine and antimicrobial activity, fluorescence microscopy was conducted using N-terminal Carboxyfluorescein hydrate (FAM), labeled protamine peptide (FAM-PP), synthesized by SCRUM Corporation . In this study, various ratios were calculated using the following formulae:E. faecalis to protamine, MIC and MBC were determined using alamarBlue® assay and colony count assays, respectively. E. faecalis (MIC: 250 μg/mL), although it was not bactericidal at any of the concentrations tested (MBC: >1000 μg/mL).To examine the antimicrobial susceptibility of E. faecalis, cells were treated with protamine at concentrations above the MIC, and changes in viable bacterial count over time were determined by a colony count assay. E. faecalis treated with protamine was significantly lower than that of the negative control (sterile water). However, an increase in the viable bacterial count was observed with prolonged incubation. The bactericidal activity of the positive control (2.5% NaClO aq.) was not detectable. These results suggest that a single treatment of protamine is difficult to control E. faecalis. No concentration dependence was observed in the growth inhibitory ability of protamine against E. faecalis. Therefore, protamine at a concentration of 250 μg/mL (MIC) was used for subsequent tests.To evaluate the growth inhibitory ability of protamine against E. faecalis, a calcium hydroxide solution was added to 10% 310 medium, and the planktonic cells were incubated for 24 h in an arbitrary alkaline environment. The viable bacterial counts were determined using a colony-count assay , E. faecalis survived. Cell growth was also observed and compared with the initial number of bacteria. After incubation, the pH of the culture medium decreased to the same level as that of the control ). E. faecalis, a lactic acid bacterium, produces lactic acid via homofermentation [E. faecalis in alkaline environments at pH 8 and 10. These results show that E. faecalis maintains its metabolic activity, even in a strongly alkaline environment, at pH 10. We further examined the effect of calcium hydroxide treatment on the metabolic activity of E. faecalis by measuring the viable bacterial count and the pH of the culture medium over time. The evaluation was limited to the alkaline range (up to pH 10), where metabolism was possible. E. faecalis growth was significantly enhanced in the alkaline environment at pH 8  significantly reduced the growth rate of E. faecalis. However, no bactericidal activity was observed at any of the concentrations used in this study, and no concentration-dependent antibacterial activity was observed. Previous studies investigating the MIC and MBC of protamine against various oral bacteria have shown that the MBC of E. faecalis is extremely high compared to that of other oral bacteria [E. faecalis with protamine alone.The use of calcium hydroxide as an intracanal medication has been reported to be beneficial for endodontic treatment because of its excellent antibacterial activity, as well as its ability to inhibit exudate, induce hard tissue, and dissolve organic material ,4. Neverortality ,11. Calcortality . Howeverortality . E. faecctory AP ,7. Therectory AP ,19. In actory AP ,23. Protbacteria . TherefoE. faecalis. E. faecalis was suspended in a 10% 310 medium, adjusted to pH 8, 10, and 12 by adding a calcium hydroxide solution, and incubated for 24 h. The results showed that an alkaline environment at pH 12 completely killed E. faecalis; however, its survival and growth were observed in alkaline environments up to pH 10. Previous studies have reported that a strongly alkaline environment with a pH of 11.5 or higher is necessary for the complete mortality of E. faecalis [Next, we investigated the calcium hydroxide tolerance of faecalis , and theE. faecalis, based on the observation of one point after 24 h in a sample with a pH of up to 10. Therefore, we limited the conditions up to pH 10, where biological activity could be maintained, and observed changes in viable counts over time. Growth was promoted in an alkaline environment at pH 8, whereas that was suppressed in an alkaline environment at pH 10. In relation to this result, previous studies have reported that E. faecalis can maintain an internal pH of 7.5–8.5 within the pH range of 8–10, while other bacteria have equal values of external and internal pH [E. faecalis and affect its metabolism.It is not clear whether calcium hydroxide has any effect on E. faecalis. The results showed that 24 h of co-treatment with protamine and calcium hydroxide completely killed E. faecalis under alkaline conditions at pH 8 and 10. In addition, the 1 h co-treatment also showed complete mortality at pH 10, and over 99.9% mortality even at pH 8. These results suggested that co-treatment with protamine and calcium hydroxide was effective in killing E. faecalis. In clinical application, calcium hydroxide is considered the first choice of root canal treatment. However, it is difficult to control microorganisms, especially E. faecalis, using only calcium hydroxide. Additionally, a highly alkaline environment by calcium hydroxide damages the bacteria, but it also has adverse effects on humans. In contrast, our approach can provide a safe and less toxic environment, and has potential clinical applications.Finally, we limited the conditions to an alkaline range of up to pH 10, where biological activity could be maintained, and evaluated the efficacy of co-treatment with protamine and calcium hydroxide in the disinfection of E. faecalis. It has been reported that the mechanisms of action of calcium hydroxide are as follows: (i) disruption of cell membranes by hydroxide ions, (ii) increase in cytosolic pH due to hydroxide ion influx, (iii) metabolic inhibition by enzyme denaturation, and (iv) destruction of DNA [E. faecalis in an alkaline environment up to pH 10 is likely to depend mainly on the durability of its cell membrane. A previous study expressed the same view [+, ATP, and other intracellular enzymes [E. faecalis cell membranes; (ii) membrane disruption is promoted by hydroxide ions; (iii) this damage promotes protamine internalization into the cytoplasm; and (iv) protamine and hydroxide ions act synergistically in the cytoplasm. However, further studies are needed to determine the antimicrobial mechanisms of protamine after its entry into the cells.Based on these results, we investigated the background mechanisms of the effect of co-treatment with protamine and calcium hydroxide on the synergistic increase in antimicrobial activity. Previous studies have reported the importance of cell membrane permeabilization in the antimicrobial activity of protamine . In thisn of DNA ,11. Ther enzymes , inhibit enzymes , increas enzymes . TherefoIn both experiments, more bacteria were observed at pH 10 than at other pH values. It is clear, from the results shown in E. faecalis disinfection. Our results showed that complete sterilization is now possible, even in an alkaline environment below pH 11.5, which is considered necessary for E. faecalis sterilization. Although more detailed studies are needed for clinical applications, such as the evaluation of biofilm formation and verification of deep reachability in the dentinal tubules, we believe that this approach has the potential to provide a novel control method against E. faecalis for root canal treatment.In this study, we proposed a new method of cotreatment with protamine and calcium hydroxide, an existing intracanal medication, and evaluated its efficacy against


Social media has emerged as a prominent approach for health education and promotion. However, it is challenging to understand how to best promote health-related information on social media platforms such as Twitter. Despite commercial tools and prior studies attempting to analyze influence, there is a gap to fill in developing a publicly accessible and consolidated framework to measure influence and analyze dissemination strategies.We aimed to develop a theoretical framework to measure topic-specific user influence on Twitter and to examine its usability by analyzing dietary sodium tweets to support public health agencies in improving their dissemination strategies.We designed a consolidated framework for measuring influence that can capture topic-specific tweeting behaviors. The core of the framework is a summary indicator of influence decomposable into 4 dimensions: activity, priority, originality, and popularity. These measures can be easily visualized and efficiently computed for any Twitter account without the need for private access. We demonstrated the proposed methods by using a case study on dietary sodium tweets with sampled stakeholders and then compared the framework with a traditional measure of influence.More than half a million dietary sodium tweets from 2006 to 2022 were retrieved for 16 US domestic and international stakeholders in 4 categories, that is, public agencies, academic institutions, professional associations, and experts. We discovered that World Health Organization, American Heart Association, Food and Agriculture Organization of the United Nations (UN-FAO), and World Action on Salt (WASH) were the top 4 sodium influencers in the sample. Each had different strengths and weaknesses in their dissemination strategies, and 2 stakeholders with similar overall influence, that is, UN-FAO and WASH, could have significantly different tweeting patterns. In addition, we identified exemplars in each dimension of influence. Regarding tweeting activity, a dedicated expert published more sodium tweets than any organization in the sample in the past 16 years. In terms of priority, WASH had more than half of its tweets dedicated to sodium. UN-FAO had both the highest proportion of original sodium tweets and posted the most popular sodium tweets among all sampled stakeholders. Regardless of excellence in 1 dimension, the 4 most influential stakeholders excelled in at least 2 out of 4 dimensions of influence.Our findings demonstrate that our method not only aligned with a traditional measure of influence but also advanced influence analysis by analyzing the 4 dimensions that contribute to topic-specific influence. This consolidated framework provides quantifiable measures for public health entities to understand their bottleneck of influence and refine their social media campaign strategies. Our framework can be applied to improve the dissemination of other health topics as well as assist policy makers and public campaign experts to maximize population impact. Health promotion is important for updating knowledge, attitudes, and behaviors regarding essential health topics among individuals, households, and communities. However, it is challenging to understand which way of promotion works and which does not and how to promote health-related information outside of traditional approaches such as radio broadcasting, television, newspapers, and magazines. The percentage of US adults who use at least one social media tool has grown from 5% in 2005 to 72% in 2020 . Social Twitter has become one of the largest social networking services in the world since its launch in 2006. Twitter has been extensively used by government agencies, nongovernment organizations, academic organizations, and experts for health information dissemination , promoteTo optimize public influence on Twitter, it is important to quantitatively evaluate different dissemination strategies. However, there is no consensus yet on how to quantify a user’s influence on Twitter. An influencer is not necessarily the one creating the most popular tweets or amassing the highest number of followers. There is a broad categorization of influencers based on different behavior patterns. Influencers include but are not limited to authoritative policy makers, domain experts, and opinion leaders . OpinionCommercial tools have been developed to analyze the social media engagement of Twitter accounts. Currently active commercial tools include Socioviz, Tweet Binder, Social Bearing, Hootsuite Insights, Followerwonk, Twitonomy, Audiense, Keyhole, and Meltwater  in 2015 , accessiMany measures have been proposed to quantify influence on social media. Note that the definition of influence on social media is slightly different from the notion of influence in other interactions. Although one could assess changes in people’s awareness, knowledge, attitudes, or intentions, it is more common in social media research and practice to assess influence in terms of engagement with social media content. We followed the categorization in Riquelme et al  to synthInfluence, sometimes called authority, measures the degree of reach to other users in the Twitter network. One approach of social network analysis is to construct a graphical representation of Twitter data and then measure the accessibility of each node (user) with respect to other nodes (users) in the network. Existing methods in this category differ in how edges between nodes are defined  and the centrality measure employed, such as closeness, betweenness, H-index, and eigenvector centrality . SeveralActivity refers to a user’s participation in a social network. Examples of activity measures include TweetRank , Tweet count score , and general activity (sum of visible actions of a user) . To measPopularity refers to the attention a user receives from the social network. A convenient proxy for popularity is the number of followers in Twitter and its variants, such as the follower-following ratio (relative number of followers versus following) and follower rank (proportion of followers out of the sum of followers and following) . HoweverThis research aims to develop a consolidated framework to measure topic-specific user influence on Twitter. We first specified our working definitions for influence, activity, priority, originality, and popularity. Then, we proposed a framework that decomposes a summary indicator of influence into 4 measures—activity, priority, originality, and popularity—to characterize the different tweeting dissemination strategies and emphasize topic-specific influence to tailor dissemination strategies on topics of interest. We demonstrated this framework by using a case study of dietary sodium tweets. The aim of this case study was to analyze the influence of 16 organizations or individuals on the topic of dietary sodium, quantify their tweeting patterns, and provide personalized recommendations for the promotion of sodium-related content. Unlike existing commercial tools that are copyright protected and targeted toward private access–granted Twitter accounts, our framework can efficiently measure public influence for any Twitter account based on publicly accessible information provided by the Twitter API. We aim to develop a generic framework intended for public use and for providing support to public health agencies to improve their dissemination strategies. More importantly, our framework can be easily applied to various applications with social purposes.In this section, we first describe the development of a 4D framework to measure topic-specific influence and then perform a usability study of dietary sodium tweets. Diseases related to high dietary sodium consumption are cardiovascular diseases, as high sodium intake is a risk factor for cardiovascular diseases. The sample population in this study consisted of 33 Twitter accounts from June 30, 2006, to May 31, 2022, with data accessible through Twitter API v2.Our working definition of topic-specific influence refers to the capacity of an individual or entity to engage the public on Twitter within a specific subject area. The degree of engagement between the influencer and their audience is assessed by the cumulative number of topic-related interactions generated through content creation over a given period. In Twitter, public engagement is captured by 4 public metrics: number of likes, quotes, replies, and retweets. Thus, we compute total tweet-level public engagement as the sum of the number of likes, quotes, replies, and retweets. At the user level, the summary indicator of topic-specific influence is the total public engagement received from all tweets that are relevant to the topic of interest. We emphasize that this summary indicator can be computed with public metrics from Twitter API without the need for private access. Considering N as the number of tweets, R as the number of retweets, P as the number of public engagements, and superscript t as topic-specific, the summary indicator of influence can be calculated as the product of 4 measures:In the next section, we discuss these 4 dimensions of influence in detail. An overview of the framework is shown in An overview of the 4 dimensions of influence is shown in In our study, activity is defined as a user’s level of effort on Twitter. Taking into consideration commonly used activity measures such as TweetRank  ,23, Tweet) out of the user’s total number of tweets (N): Existing studies of Twitter either measured general influence or topic-specific influence. We establish a relationship between general tweets and topic-specific tweets called as priority. Priority is defined as the percentage of topic-specific tweets , quotes (Pquote), replies (Preply), and retweets (Pretweet) as the total public engagement of each tweet. At the user level, we measured popularity as the average number of public engagements per content-creation tweet. The topic-specific popularity is computed as:Popularity is measured by the amount of public attention that an organization or individual receives on Twitter. Twitter defines engagement as the “total number of times a user interacted with a tweet. Clicks anywhere on the tweet, including retweets, replies, follows, likes, links, cards, hashtags, embedded media, username, profile photo, or tweet expansion” . Four ofNote that retweets were excluded from this computation because public metrics are not collected for retweets.We selected 8 pairs of domestic and international stakeholders with scopes of work relevant to dietary sodium. This selection included public agencies, academic institutions, professional associations, and domain experts. A total of 33 Twitter accounts from 16 organizations and individuals were analyzed . Note thData were retrospectively collected from the 33 Twitter accounts from June 30, 2006, to May 31, 2022, via Twitter API v2. The Academic Research license enabled us to carry out a full-archive retrieval of every tweet since Twitter’s conception. We retrieved tweet-level data, including creation date, author username, full tweet text, language, public metrics , mentions, and hashtags. The account-level data retrieved included username, display name, creation date, location, number of total tweets, number of followers, and number of followings . The tweets from multiple accounts of the same stakeholder were concatenated into 1 data set. The definitions of Twitter-specific terminology used in this study can be found in A keyword-based algorithm was used to determine whether a tweet was dietary sodium–related. The algorithm first removed content involving English slangs that contained the keyword “salt.” See Since this is a second-hand data analysis with publicly available data, it is exempted in terms of institutional review board review. Data used in this study received explicit consent for research purposes from Twitter. Twitter users have the right to make changes to the availability of their public content, such as tweet addition, deletion, and other user-driven changes. Users of Twitter data respect these changes and only access data that has current public disposition. It is also worth noting that this study only used summary statistics such as total counts of tweets and public measures without analyzing tweet content in detail.A total of 585,600 tweets were retrieved from the 16 stakeholder accounts, of which 12,393 were considered sodium-related. The 4D analysis of influence for each stakeholder is shown in As hypothesized, the top influencers in this sample exhibited different tweeting patterns. WHO’s high influence was due to its high performance in 3 out of 4 dimensions: large tweeting volume, high portion of original content, and high popularity. AHA, Harvard, Center for Science in the Public Interest, CDC, and United States Department of Agriculture had a similar pattern for activity and originality but low level of priority and popularity. In contrast, Tom Frieden and Alexey Kulikov were low-volume content creators but still influential due to their high popularity. While having a similar overall influence as UN-FAO, WASH had a noticeably different influence profile with the highest priority in creating sodium content but the lowest popularity in this sample. At the lower end of this spectrum, Oxford, Stanford, and World Heart Federation had low influence in dietary sodium due to relatively low performance in 3 out of 4 dimensions, that is, low activity, priority, and popularity. Simon Capewell, Kirsten Bibbins-Domingo, and University College London also had low influence measures with regard to sodium.In terms of priority, most sampled stakeholders had very low priority for dietary sodium in their communications on Twitter (<0.5%), showing that sodium was not a popular dissemination topic in these large health organizations. WASH, an international group, focused on reducing salt and sugar intake and had, by far, the highest priority for sodium , followed by Center for Science in the Public Interest  and AHA .In terms of originality, UN-FAO, Tom Frieden, and Stanford were the most dedicated content creators. Although content creation is traditionally seen as a priority in information dissemination and the design of Twitter’s metrics also favors content creation, Simon Capewell showed as an exception. Despite having the lowest originality level in this sample and less followers, Simon Capewell still had a higher influence measure than Stanford. This case shows that retweeting can be a relatively time-efficient approach to increase influence and boost public engagement of the content-creation tweets.In terms of popularity, UN-FAO, WHO, and Tom Frieden had the highest popularity, with more than 100 public engagements per sodium-related content-creation tweet on average. However, WASH had the lowest popularity in this sample despite having the highest priority for dietary sodium. Two of the top 3 stakeholders in popularity were not the ones with the highest number of followers, which reflects that the number of followers does not necessarily indicate topic-specific popularity. Note that Tom Frieden and Alexey Kulikov were among the stakeholders with the highest popularity for sodium-related tweets despite joining Twitter relatively late, that is, in 2017 and 2016, respectively.P<.001). These results suggest that the proposed summary indicator is notably aligned with this traditional measure of influence. Note that the main driver of this association is the popularity measure, as users with more followers have more public exposure and thus tend to receive more public engagement per tweet. Our summary indicator of influence is a more granular measure than the number of followers, as it captures tweet-level public engagement and topic-specific influence.A common measure of influence used by commercial tools as well as in literature is the number of followers, which is a publicly available measure that can be conveniently obtained without computations. We conducted a statistical comparison of our summary indicator of influence with this traditional measure of influence. Since the number of followers is clearly non-Gaussian in our sample , we used a nonparametric correlation test, Kendall rank correlation test, which does not require distribution assumptions. The Kendall rank correlation coefficient between the summary indicator of influence and number of followers for this stakeholder sample was 0.742  was outside the scope of this work. Lastly, our case study only focused on tweets in English, while international organizations such as WHO publish a small portion of tweets in other languages.This study lays out efficient and accessible methods to measure user influence for any Twitter account and given topic of interest. The proposed framework can be used to identify influencers and characterize their distinctive dissemination strategies on Twitter, accompanied by a quantifiable visualization of influence with its 4 dimensions: activity, priority, originality, and popularity. We illustrated this framework by using a case study on dietary sodium by evaluating 16 health domain stakeholders’ influence. The case study showed that each stakeholder had different strengths and weaknesses in their dissemination strategies, and 2 stakeholders with similar overall influence could adopt significantly different tweeting patterns. Our framework can be applied to improve the dissemination of other health topics by enhancing influence visibility and forming tailored recommendations to optimize user’s time and financial investments on health education and promotion on Twitter. These capabilities can assist public health entities and policymakers in refining their social media campaign strategies on Twitter to maximize their population impact.


Stem Cells Translational Medicine, Volume 10, Issue 6, June 2021, Pages 922–938, https://doi.org/10.1002/sctm.20-0469This is a correction to: Huipu Yuan, Hui Hu, Rui Chen, Wenbo Mu, Liangliang Wang, Ying Li, Yuelei Chen, Xiaoyan Ding, Yongmei Xi, ShanShan Mao, Mizu Jiang, Jie Chen, Yong He, Lang Wang, Yi Dong, Jinfa Tou, Wei Chen, Premigratory neural crest stem cells generate enteric neurons populating the mouse colon and regulating peristalsis in tissue-engineered intestine, In the originally published version of this article, there was an error in the funding information that was presented in the second sentence of the Acknowledgments section. The sentence should have read as follows: “This research was financially supported by Xiamen’s two hundred talent Program; National Key Research and Development Program of Zhejiang, Grant/Award Number: 2020C03113; Zhejiang Provincial Natural Science Foundation of China, Grant/Award Number: LZ18C090001; National Natural Science Foundation of China, Grant/Award Number: 31171423; National Key Research and Development Program of China, Grant/Award Numbers: 2014CB541705, 2012CB967903.”These details have been corrected only in this correction notice to preserve the published version of record.


Understanding the transmissibility of respiratory viruses by symptoms is important for public health.Persons who tested positive for SARS-CoV-2 and their household contacts (HHC) were recruited from 7 US sentinel sites or by remote invitation nationwide during Sep. 2021—Mar. 2023. The household primary case was the person with the earliest symptom onset or positive test. Starting ≤6 days after primary case onset, primary cases and HHC completed symptom logs  and collected nasal or saliva specimens (daily for 10 days) that were tested by RT-PCR. Infected individuals were counted as having developed fever, lower respiratory symptoms , other symptoms , or as being asymptomatic based on all logs. Risk of secondary infection (any PCR positivity) among eligible, tested HHC (Methods 1) by symptoms of primary cases was estimated using Poisson regression with generalized estimating equations. We estimated days from onset to last PCR positive in a survival model.Methods upload 1. Enrolled and analytically included household members in case-ascertained studies of household transmission of SARS-CoV-2, United States, Sept 2021 - Mar 2023.This analysis included 842 households . Most primary cases (99%) and infected HHC (81%) were symptomatic (Results 1). Primary cases had higher frequencies of fever or LRS than infected HHC (Results 2). HHC exposed to primary cases who developed fever or LRS were more likely to become infected than HHC exposed to primary cases who did not have fever or LRS (Results 3). Post-hoc comparisons by individual symptoms supported this for fever and all LRS but chest pain . Primary cases with fever or LRS were PCR positive for a median of 14 days (95% CI: 14 – 15) post-onset, compared to 10 days (95% CI: 9 – 11) for cases who did not have fever or LRS.Results upload 1. Characteristics of included household members in case-ascertained studies of household transmission of SARS-CoV-2, United States, Sep 2021 - Mar 2023.Results upload 2. Proportion of primary cases and infected household contacts who experienced individual symptoms.Results upload 3. Unadjusted and adjusted risk of household contacts becoming infected with SARS-CoV-2, by symptoms in the primary case.Contacts of primary cases with fever or lower respiratory symptoms may have been more likely to become infected than contacts of primary cases without, suggesting higher transmissibility.Joshua Petrie, PhD, CSL Seqirus: Grant/Research Support Yvonne A. Maldonado, MD, Pfizer: Grant/Research Support|Pfizer: Site Investigator, DSMB member Suchitra Rao, MBBS, MSCS, Sequiris: Advisor/Consultant Edward Belongia, MD, Seqirus: Grant/Research Support Huong McLean, PhD, MPH, Seqirus: Grant/Research Support Edwin J. Asturias, MD, Hillevax: Advisor/Consultant|Moderna: Advisor/Consultant|Pfizer: Grant/Research Support Carlos G. Grijalva, MD, MPH, AHRQ: Grant/Research Support|CDC: Grant/Research Support|FDA: Grant/Research Support|Merck: Advisor/Consultant|NIH: Grant/Research Support|Syneos Health: Grant/Research Support