Methodology. Forty-eight male New Zealand white (NZW) rabbits were grouped into normal diet (C), normal diet with 0.6 g/kg/day of Trihoney (C + H), 1% cholesterol diet (HCD), 1% cholesterol diet with 0.3 g/kg/day of Trihoney (HCD + H1), 1% cholesterol diet with 0.6 g/kg/day of Trihoney (HCD + H2), and 1% cholesterol diet with 2 mg/kg/day of atorvastatin (HCD + At.). Animals were sacrificed after 12 weeks of treatment. Serum lipids and liver function test (LFT) were measured prior to and at the endpoint of the experiment for total cholesterol (TC), low-density lipoprotein (LDL-c), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), and total bilirubin (T. Bil.). Liver was processed for histopathology study. Liver homogenate was analysed for oxidative stress parameters: superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA). Results. Lipid analysis approved the induction of hypercholesterolemia. A significant elevation (p < 0.01) of serum AST and ALT levels showed by the HCD group was compared to C and C + H groups. Trihoney exhibited a significant reduction (p < 0.001) of AST and ALT compared to the HCD group. Likewise, AST and ALT reduced significantly in the HCD + At. group (p < 0.001). Trihoney supplementation induced significant (p < 0.05) enhancement of SOD and GPx activities. Atorvastatin treatment was associated with significant (p < 0.05) reduction of SOD and GPx activities in the liver. Trihoney and atorvastatin showed marked (p < 0.001) reduction of hepatic lipid peroxidation. Trihoney showed histological protection against progression of NAFLD to nonalcoholic steatohepatitis (NASH). Atorvastatin exhibited no beneficial impact on hepatic architecture. Conclusion. Trihoney was able to maintain normal liver function and showed hepatoprotection against progression of NAFLD to NASH probably through hypocholesterolaemic and antioxidant functions.Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disorder worldwide with no curative therapy. The aim of this study was to investigate the hepatoprotective effects of a novel Trihoney against biochemical and histological manifestations of NAFLD in hypercholesterolemic rabbits.  Nonalcoanalysis . The curioxidant  and antiioxidant .Pure cholesterol powder is obtained from Nacalai-Tesque . Cholesterol-free extra virgin coconut oil is bought from Philippines. Masson's Trichrome (MT) stain kit is from Clin-Tech (UK). Other solvents, chemicals, and haematoxylin and eosin (H&E) stain were supplied by Sigma-Aldrich (USA) and Leica Biosystems (Germany).m factor = animal equivalent dose × animal Km factor), with the adult human Km factor being 37 and the rabbit Km factor being 12. Km factor is a constant calculated based on normalisation of body surface area for accurate translation of drug doses between mammalian species [Trihoney is a product made in Department of Nutrition Sciences laboratories of Kulliyyah of Allied Health Sciences, International Islamic University Malaysia (IIUM). Trihoney is a combination of three types of natural honey: Trigona, mellifera, and dorsata, at a ratio of 45%, 15%, and 10%, respectively . Trihone species . Atorvas species –25, at a species , 26.Preparation of 1% cholesterol diet was performed according to Alfarisi et al.  as folloForty-eight NZW rabbits of male gender were purchased from certified experimental animal supplier . Animal weight ranged from 2 to 2.5 kg, and animals' age was 20 weeks. Animals were randomly housed in stainless-steel cages designed for rabbits as a single rabbit per cage with free access to water and standard rabbits' pellet, in addition to the standard animal care housing condition of 12 hours dark/light cycle, temperature 15–21, and humidity 45–65%. The procedure of animal handling was conducted in accordance with the guidelines of Malaysian Code of Practice for the Care and Use of Animals for Scientific Purposes (AEPC) , and the1), 1% cholesterol diet with 0.6 g/kg/day of Trihoney (HCD + H2), and 1% cholesterol diet with 2 mg/kg/day of atorvastatin (HCD + At.). At the end of 12 weeks, animals were sacrificed at the animal surgical laboratory of central research and animal facility, International Islamic University Malaysia (CREAM)-IIUM. Induction of general anaesthesia was done by intramuscular injection of a combination of ketamine and xylazine [Forty-eight male NZW rabbits were grouped into the following 6 groups: normal diet (C), normal diet with Trihoney dose of 0.6 g/kg/day (C + H), 1% cholesterol diet (HCD), 1% cholesterol diet with 0.3 g/kg/day of Trihoney  [Liver tissue was retrieved from −80°C freezer and defrosted. Using bullet blender homogeniser, liver tissue was homogenised to a ratio of 10% (w/v)  as folloch, USA)  supplemech, USA) . Homogench, USA) , 43. Thech, USA) . Protein-c = TC－HDL-c－(TG/5)mmol/L.Serum samples for TC, ALT, AST, ALP, GGT, and T. Bil. were immediately analysed by automated analysis machine . Serum LDL-c was calculated according to Friedewald equation : LDL-c =Concentration of MDA in liver homogenate was determined quantitively using OxiSelect MDA Adduct competitive enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol .Superoxide dismutase enzyme activity was assayed using OxiSelect™ Superoxide Dismutase Activity Assay Kit . Enzyme activity as function of optical density (OD) was expressed as units/µL and calculated as percentage (%). Activity of GPx was assayed using GPx assay kit . The principle of GPx assay was based on the decrease in NADPH (measured at 340 nm) which is proportionate to GPx activity. Activity of GPx in the homogenate was expressed in mU/mg tissue protein.μm thick ribbon were performed using a semiauto rotator microtome  and mounted onto frosted glass microscope slides. Haematoxylin and eosin [Liver tissue was fixed in 10% NBF for at least 72 hours and then was processed in automated tissue processor , clarified in xylene, and embedded by embedding Centre . Trimming and sectioning of 3 nd eosin  and MT snd eosin  were empnd eosin  grading M) and standard deviation (SD) and analysed by one-way analysis of variance (ANOVA). One-way ANOVA followed by the post hoc test was used for determination of any significant differences between means of two or more independent groups. Statistical significance was considered at p < 0.05. Correlations were investigated using Pearson's correlation coefficients (r).Statistical Package for Social Sciences  software was used for data processing. Data were expressed as mean (p > 0.05) difference between all experimental groups prior to induction phase (week 0) and after treatment phase (week 12). However, comparison by repeated measurements of animal's food intake pre- and posttreatment (p < 0.05) in animals' food intake at the end of the treatment duration when compared to pretreatment starting point.Results of animal's food intake are displayed in reatment  showed ap > 0.05) in liver function parameters between all experimental groups before phase of induction (p < 0.001) of serum AST when compared to C and C + H groups and a significant elevation (p < 0.01) of serum ALT in comparison to the C group. More significant elevation (p < 0.001) of serum ALT level was seen between the HCD group and C + H group. Trihoney-treated groups showed significant reduction (p < 0.001) of both liver enzymes AST and ALT when compared to the HCD group. Likewise, the atorvastatin-treated group exhibited significant (p < 0.001) lower serum AST and ALT compared to the HCD group. Serum transaminases in the atherogenic-treated groups were comparable to the controls. Serum T. Bil. and GGT were lower in Trihoney-treated groups in comparison to HCD and HCD + At. groups despite no statistical significance.Definitely, there was no difference  positive correlation was found between serum AST, TC, and LDL-c levels. Moreover, T. Bil. had significant (p < 0.01) positive correlation with serum TC level and significant (p < 0.05) correlation with LDL-c (A significant (th LDL-c .p < 0.001) reduction of SOD and GPx (p < 0.05) activities in the HCD group compared to the control group (1 exhibited the highest (p < 0.001) SOD activity among all atherogenic groups; moreover, this group showed the highest GPx activity despite no statistical significance difference compared to HCD and HCD + H2 groups. Activity of GPx was significantly (p < 0.05) enhanced in the HCD + H1 group compared to the HCD + At. group. Group HCD + H2 had SOD and GPx activities higher than the HCD and HCD + At. groups though no statistical significance difference. On the contrary, the atherogenic group received atorvastatin HCD + At. showed low SOD and GPx activities in liver homogenate when compared to all untreated and treated groups.There were a very significant (ol group . Contrarp < 0.001) elevation of MDA concentration in liver compared to the control groups (p < 0.001) reduction of MDA concentration in the liver compared to the HCD group. Both HCD + H2 and HCD + At. had liver MDA concentration comparable to that of the control groups.High cholesterol diet induced significant  in comparison to the control group that was maintained only on commercial diet. This may indicate the medical beneficial role of Trihoney even in normocholesterolemic status. These findings are supported by other animal studies investigating the hepatoprotective role of honey , 59, 60.Statins are recommended treatment option for NAFLD as the dyslipidemia controlling agent because 70% of patients with NAFLD was found to have dyslipidemia . Use of In this study, we reported evidence of oxidative stress by investigating MDA concentration in homogenate of the liver. In line with our study, elevated MDA in NAFLD/NASH has been reported by various experimental as well as by clinical studies –74. PathIn this investigation, atherogenic group treated with atorvastatin expressed suppressed SOD and GPx activities in the liver coincidently with reduced lipid peroxidation manifested by low MDA concentration in hepatic tissue. Regarding hepatoprotective function of atorvastatin in this investigation, we found the following: reduced lipid peroxidation and normal biochemical liver function; these two effects may be consistent to indicate the role of atorvastatin in management of NAFLD, but simultaneously, we reported marked suppression of SOD and GPx activities in the liver in addition to the nonprotective histological effect; collectively, these results make the effects of atorvastatin in NAFLD inconsistent and nonconclusive and necessitate future research studies to further explain and resolve this issue. In line with this conclusion, Sigler et al.  showed eAs far as histopathology of NAFLD is concerned, histopathological examination remained the cornerstone diagnostic tool for this disease . In thisN-ethylmaleimide. Hepatoprotective histological effect of natural honey was reported also by Yaman et al. [On the contrary, macroscopic features of the liver in Trihoney-supplemented groups were in pale yellowish colour and had smooth surface, sharp edge, brown cut surface, and smooth consistency. These gross findings were in line with the microscopic histological features of these tissues. These groups had (mild) grade-1 NASH and fibrotic stage-1 according to Brunt et al.  grading n et al.  who inven et al. . This isn et al. , and thin et al. . Likewisn et al. , and we Since inflammation and oxidative stress secondary to hepatic lipid accumulation are implicated in the pathogenesis of NAFLD/NASH , and becIn a status of sustained hypercholesterolemia, Trihoney was an effective hepatoprotective supplement protecting against functional and structural changes in NAFLD. Trihoney was able to maintain liver biochemical parameters within the normal range and showed ameliorative potential against hepatic oxidative stress. Trihoney exhibited protective function against histological progression of NAFLD to NASH and restrains hepatic fibrosis. Trihoney may be introduced as an adjuvant therapy with atorvastatin for hypercholesterolemic NAFLD patients. Although this study showed the hepatoprotective functions of Trihoney against NAFLD, some underlying mechanisms at molecular and at immunohistopathological levels have not been addressed, which represents a limitation in this study. Future studies covering alpha-smooth muscle staining in addition to therapeutic experimental model assessment for Trihoney are highly recommended.


Childhood-onset nephrotic syndrome has a variable clinical course. Improved predictive markers of long-term outcomes in children with nephrotic syndrome are needed. This study tests the association between baseline urinary epidermal growth factor (uEGF) excretion and longitudinal kidney function in children with nephrotic syndrome.The study evaluated 191 participants younger than 18 years enrolled in the Nephrotic Syndrome Study Network, including 118 with their first clinically indicated kidney biopsy  and 73 with incident nephrotic syndrome without a biopsy. uEGF was measured at baseline for all participants and normalized by the urine creatinine (Cr) concentration. Renal epidermal growth factor (EGF) mRNA was measured in the tubular compartment microdissected from kidney biopsy cores from a subset of patients. Linear mixed models were used to test if baseline uEGF/Cr and EGF mRNA expression were associated with change in estimated glomerular filtration rate (eGFR) over time.2 per year (P < 0.001) adjusted for age, race, diagnosis, baseline eGFR and proteinuria, and APOL1 genotype. In the biopsied subgroup, uEGF/Cr was correlated with EGF mRNA expression , but uEGF/Cr was retained over mRNA expression as the stronger predictor of eGFR slope after multivariable adjustment .Higher uEGF/Cr at baseline was associated with slower eGFR decline during follow-up (median follow-up = 30 months). Halving of uEGF/Cr was associated with a decrease in eGFR slope of 2.0 ml/min per 1.73 muEGF/Cr may be a useful noninvasive biomarker that can assist in predicting the long-term course of kidney function in children with incident nephrotic syndrome.  See Commentary on Page 383,,,Most cases of primary nephrotic syndrome in children are caused by 1 of 2 disorders: minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS).Standard assessment of kidney biopsies can yield more than the histological diagnosis of MCD or FSGS. The severity of interstitial fibrosis and tubular atrophy (IFTA) has been associated with kidney disease progression.With or without a kidney biopsy, there is no clinical method to accurately distinguish at presentation the patients who are likely to have progressive loss of kidney function from those who will maintain kidney function long-term. The availability of a noninvasive test to improve prognostic evaluation for children with nephrotic syndrome could help guide decision making about further diagnostic assessment, therapy, and patient monitoring.,EGF is the prototypical peptide growth factor of the EGF/EGF receptor signaling pathway, which plays important roles in proliferation, differentiation, and migration of a variety of cell types, especially epithelial cells.Therefore, we conducted the following study among a cohort of pediatric nephrotic syndrome patients to test the hypotheses that (i) urinary EGF excretion is associated with tubular compartment renal gene expression profiles of children with biopsied MCD and FSGS, and (ii) urinary EGF excretion is associated with cross-sectional and longitudinal kidney function in children with nephrotic syndrome, independent of demographic and clinical markers of disease.This longitudinal study used existing prospectively collected data and specimens from the National Institutes of Health–sponsored Nephrotic Syndrome Study Network (NEPTUNE).NEPTUNE, launched in 2010, is an ongoing prospective observational cohort study of children and adults with primary proteinuric kidney diseases.2. To reduce the effect of outlier measurements during periods of hyperfiltration wherein reduction in eGFR from hyperfiltration to normal range is considered improvement, all longitudinal eGFR measurements were Winsorized to 120 ml/min per 1.73 m2.Serum creatinine was measured in the central laboratory from specimens collected during study visits and enriched with data collected from local health records. GFR was estimated using the bedside serum creatinine–based pediatric estimating equation.Urine protein and creatinine were measured in the central laboratory from 24-hour or spot urine samples as available. Urine protein:creatinine ratios (UP:C) were expressed as mg/mg. These central measures were enriched by results collected from local medical records.APOL1 risk alleles were genotyped directly via Sanger sequencing of the last 250 bases of exon 7.The local clinical kidney biopsy report was redacted of participant identifiers and submitted via the NEPTUNE electronic data capture system for review. Glass slides from the local clinical diagnostic kidney biopsy were submitted to the NEPTUNE Digital Pathology Repository for scanning and subsequent scoring by the NEPTUNE Core Pathology Committee.,http://brainarray.mbni.med.umich.edu).2 transformed and batch corrected using ComBat.A single core of kidney biopsy tissue collected for research purposes during the initial local clinical kidney biopsy was preserved in RNAlater and submitted to NEPTUNE laboratories at the University of Michigan. Biopsy tissue was microdissected into tubular and glomerular compartments and renal gene expression data were generated using Affymetrix GeneChip Human Genome ST2.1.uEGF was assessed in duplicate from urine collected at study visits using enzyme-linked immunosorbent assay  and optimized in the laboratory as previously reported.2 tests for categorical variables and Kruskal-Wallis tests for continuous variables. To reduce skewness, a log2 transformation was applied to the mean of the uEGF/Cr duplicate values. Pearson correlations were calculated to examine potential associations between log2(uEGF/Cr) and log2 when both samples were collected within a 7-day interval. All eGFR values were Winsorized (capped) at 120 ml/min per 1.73 m2.Descriptive analyses were conducted on all participants who had a baseline uEGF measurement using frequencies and percentages for categorical variables and medians and interquartile ranges for continuous variables. Comparisons across disease categories were made using χP value. Then main effects underwent backwards selection, but any main effects terms part of a statistically significant interaction with time were retained in the final models. In addition, age was included in all models (regardless of significance) to account for the known inverse relationship between EGF and age.Longitudinal linear mixed effects models with random intercepts and slopes were used to predict the outcome of eGFR. Analyses began at the earliest time of study data collection: time of biopsy for the MCD and FSGS cohorts; time of nephrotic syndrome onset for the nonbiopsied CO-NS cohort. Covariates of primary interest were time from biopsy, baseline uEGF/Cr, and an interaction term between time and uEGF/Cr to test whether trajectories of eGFR differ by uEGF/Cr at baseline. For example, a significant main effect for uEGF/Cr would yield parallel eGFR slopes over follow-up, differing by a linear function of uEGF/Cr at baseline; a significant interaction would indicate nonparallel (fan-shaped) slopes in eGFR over time, reflecting both a shift up or down and a change in slope as a linear function of baseline uEGF/Cr. Other variables included in the models were baseline values of age, prior disease duration, prior therapy, UP:C, and eGFR, as well as sex, race, ethnicity, kidney disease diagnosis, and APOL1 genotype. Interaction terms between time and each predictor of interest were tested as potential predictors of eGFR slope, using Wald tests to assess significance. All variables were included in a backwards multivariable model selection. The backwards selection process ensured the model was hierarchically well-formulated. First, nonsignificant interactions with time were removed in descending order of EGF RNA expression were predictors of eGFR slope using the same longitudinal mixed models and backwards selection approach. Sensitivity analyses were conducted refitting final models using age-normalized values of uEGF/Cr. Normalization was done using median-interquartile range age-normalization of uEGF/Cr based on data from healthy children reported by Meybosch et al.,A separate analysis was conducted in the biopsied participants alone to further examine whether or not renal biopsy IFTA, global sclerosis, or P < 0.001), as did median baseline eGFR  and median uEGF/Cr . At baseline uEGF/Cr collection, 37% of participants had nephrotic range proteinuria (UP:C ≥3.0 g/g), 27% sub-nephrotic, but active, proteinuria (UP:C <3.0 g/g and UP:C ≥0.3 g/g), and 35% were in a complete remission of proteinuria (UP:C <0.3 g/g). However, there was no correlation between baseline uEGF/Cr and UP:C . There was also no association between uEGF receptor/Cr and steroid response pattern, both initially and after 1 year  in the 16 participants with matching samples within 1 week of biopsy . In addition, uEGF/Cr was moderately correlated with baseline eGFR .Urinary EGF/Cr was strongly correlated with renal 2/year increase in eGFR slope . Conversely, halving of EGF/Cr was associated with a 2.0 ml/min per 1.73 m2 decrease in eGFR slope. In addition, a 1-loge increase in UP:C was associated with a steeper decline in eGFR by −1.1 ml/min per 1.73 m2/year . Native American participants also had more rapid eGFR decline compared with white participants , as did FSGS compared with CO-NS participants . Significant main effects, in the absence of a significant interaction with time, indicate that a variable was associated with higher or lower eGFR, but that it was not associated with change in slope of eGFR over time. For example, high-risk APOL1 genotype was associated with lower eGFR , but not a significant interaction between genotype and eGFR slope. Model 1 was refit after removing uEGF/Cr (P < 0.001).The adjusted relationship between uEGF/Cr and eGFR over time was assessed using multivariable linear mixed effects models developed with backward selection , model 1 uEGF/Cr , model 22/year (95% CI: −3.0 vs. −0.6), whereas a participant with a baseline uEGF/Cr of 16 ng/mg had an estimated slope of −3.8 ml/min per 1.73 m2/year (95% CI: −5.5 to −2.1). Model derived estimates of eGFR over time with 95% CIs are also shown in 2/year improvement in eGFR slope . This model was also adjusted for biopsy characteristics , which were also independently associated with eGFR. As seen previously in P < 0.001).2(uEGF/Cr). These analyses refit final models both from all participants . SE has research funding through the University of Michigan with AstraZeneca PLC, Eli Lilly and Company, Novo Nordisk A/S, Gilead Sciences, Inc., and Moderna, Inc., and owns shares of Gilead Sciences, Inc., Johnson & Johnson, ThermoFisher Scientific, Inc, and AbbVie, Inc. TS has received research support from Bristol-Myers Squibb, Mallinckrodt Pharmaceuticals, Goldfinch, and Retrophin, Inc. HT is a consultant to Otsuka  and Chemocentryx (DMC) and a consultant to Goldfinch and Retrophin (in contract through New York University). KJR is a site principal investigator for a Complexa study and Advicienne study unrelated to these data. MK receives research support through the University of Michigan with AstraZeneca PLC, Eli Lilly and Company, Novo Nordisk A/S, Gilead Sciences, Inc., Jansen, Angion, Certa, and Moderna, Inc. WJ and MK have a patent pending on biomarkers for chronic kidney disease (CKD) progression . All other disclosures are unrelated to these data. All the other authors declared no competing interests.


Facebook’s advertising platform reaches most US households and has been used for health-related research recruitment. The platform allows for advertising segmentation by age, gender, and location; however, it does not explicitly allow for targeting by race or ethnicity to facilitate a diverse participant pool.This study looked at the efficacy of zip code targeting in Facebook advertising to reach blacks/African Americans and Hispanics/Latinos who smoke daily for a quit-smoking web-based social media study.We ran a general market campaign for 61 weeks using all continental US zip codes as a baseline. Concurrently, we ran 2 campaigns to reach black/African American and Hispanic-/Latino-identified adults, targeting zip codes ranked first by the percentage of households of the racial or ethnic group of interest and then by cigarette expenditure per household. We also ran a Spanish language campaign for 13 weeks, targeting all continental US zip codes but utilizing Facebook’s Spanish language targeting. The advertising images and language were common across campaigns. Costs were compared for advertisement clicks, queries, applications, and participants, and yields were compared for the final three outcomes. We examined outcomes before and after the Cambridge Analytica scandal that broke in March 2018. Finally, we examined 2 promoted Facebook features: lookalike audiences and audience network placement.P<.001) and applicants . The Hispanic-/Latino-focused versus general market campaign increased Hispanic/Latino weekly queries  and applicants . Cost metrics did not differ between campaigns at generating participants . Costs increased post- versus prescandal for the black-/African American–focused campaign for queries  and applicants  and for the Hispanic-/Latino-focused campaign for queries  and applicants .Zip code targeting campaigns were effective for yielding the racial or ethnic groups of interest. The black-/African American–focused versus general market campaign increased black/African American weekly queries (mean 9.48, SD 5.69 vs general market mean 2.83, SD 2.05; Zip code targeting in Facebook advertising is an effective way to recruit diverse populations for health-based interventions. Audience network placement should be avoided. The Facebook lookalike audience may not be necessary for recruitment, with drawbacks including an unknown algorithm and unclear use of Facebook user data, and so public concerns around data privacy should be considered.ClinicalTrial.gov NCT02823028; https://clinicaltrials.gov/ct2/show/NCT02823028 The distrust of health care and health-related studies among blacks/African Americans and Hispanics/Latinos has been documented extensively -3. To coFacebook is a leading web-based social media platform for adults in the United States, with approximately 221 million monthly active users who represent about 69% of the adult population ,16. FaceThis paper discusses recruitment of racially and ethnically diverse participants by means of Facebook advertising for a quit-smoking web-based social media study. Our research team developed a web-based Twitter peer-support group intervention for quitting smoking called Tweet2Quit ,25. We rThis study evaluated 4 Facebook advertising campaigns that we used to recruit individuals for our quit-smoking web-based social media study. Our general market campaign reached all continental US zip codes, targeting individuals who expressed an interest in smoking or quitting smoking on Facebook, and it served as our baseline. Two additional Facebook campaigns targeted primarily black/African American or Hispanic/Latino zip codes with high cigarette expenditures. Our final Facebook campaign targeted Spanish language speakers living in continental US zip codes who expressed an interest in smoking or quitting smoking on Facebook. For each campaign, we examined yields and costs for 4 standard advertising campaign outcomes: advertisement clicks, queries, applications, and participants. We hypothesized that zip code campaigns would generate higher yields of diverse ethnic groups without higher costs.In addition, we compared costs before and after major news broke in March 2018 concerning the Facebook privacy scandal involving Cambridge Analytica’s alleged breach of privacy of 50 million Facebook users, which raised serious concerns about the protection of private user information. Finally, we examined the costs associated with 2 features Facebook recommends to its advertisers, use of lookalike audiences and audience network placement, and compared these to our general market campaign.Demographics, Interests, and Behaviors. The Demographics and Interests sections do not include race or ethnicity. The Behaviors section includes Multicultural Affinity, which allows advertisers to choose African American, Asian American, or Hispanic affinity. Facebook does not provide information on how individuals are classified into these behavioral affinity categories or what data are used. In addition, categories within the Interests and Behaviors sections change often, which then requires advertisers to recreate existing advertising campaigns using new options. Furthermore, advertising using Multicultural Affinity is restricted by Facebook and can lead to extended advertising review times and automated advertisement disapproval [Facebook does not allow for direct demographic targeting of advertising by users’ ethnicity or race. The main demographics that Facebook advertisers can choose are age and gender. These demographics are offered because when Facebook users create their accounts, they are required to specify their age and gender. Advertisers can further narrow their audience by using the 3 sections labeled approval . Due to As a baseline, we created a general market campaign, selecting all continental US zip codes. The advertising images and language were common in all 4 advertising campaigns we studied. In addition, in all 4 campaigns, we restricted our advertisements to be seen by individuals aged between 21-59 years. We required individuals to be ≥21 years because many states restrict the sale of tobacco to these individuals. We set the maximum age at 59 to focus on younger and middle-aged adults rather than retirees. Moreover, in all 4 of our advertising campaigns, we targeted appropriate study participants based on their Facebook interests. We showed our advertisements only to individuals who expressed an interest in smoking-related topics such as quitting smoking, nicotine, and tobacco or cigarettes, while logged into Facebook.For our black-/African American–focused and Hispanic-/Latino-focused campaigns, we also used more specific zip code targeting. We obtained data on all continental US zip codes and sorted the zip codes from highest to lowest based on the percentage of households in the target racial or ethnic group to ensure that those racial or ethnic groups were reached. After this, we sorted the zip codes from highest to lowest based on the mean annual household expenditure on cigarettes to try to ensure that we reached smokers. We removed any zip codes with fewer than 100 households. We then selected the top 1000 zip codes because Facebook only allowed us to upload 1000 customized zip codes out of the 41,702 total zip codes in the United States -29. All Behaviors or Interests sections. Spanish language targeting was chosen to increase the number of Hispanic/Latino applicants to our program. Advertisements across all campaigns were identical, as noted above. Advertising imagery and wording were chosen using Facebook A/B testing campaigns placed in all US zip codes. A/B testing allows advertisers to run nearly identical, simultaneous campaigns to test a singular variable, such as an advertising image.Finally, for our Spanish language campaign, we used all continental US zip codes; however, we selected Spanish as the spoken language, using Facebook’s designated Language Targeting feature. This feature allows advertisers to show advertisements to users who speak a specific language and is not related to targeting based on the Data on the ethnic makeup of US zip codes came from the US Census Bureau’s American Community Survey and are publicly available . Data onOur study compared 4 different Facebook advertising campaigns: A general market campaign, a black-/African American–focused zip code campaign, a Hispanic-/Latino-focused zip code campaign, and a Spanish language campaign. We compared these campaigns on 4 standard outcome measures: advertisement clicks, queries, applicants, and participants. Our advertisement click measure showed whether an individual tapped or clicked on our web-based advertisement, which automatically directed the individual to our study website. Hence, our advertisement click measure assessed if a campaign possibly brought interested individuals to our website. On our website, individuals could read about the study and fill out a short query form that asked for their contact information (name and email) and their race or ethnicity. Our query measure assessed whether the campaign resulted in individuals providing contact information. Individuals who provided contact information received our full screening survey, and if they finished the survey, they were considered applicants. Hence, our applicant measure assessed whether the campaign resulted in individuals completing the study screener, regardless of whether they passed or failed. Finally, our participant measure assessed whether the campaign resulted in individuals getting enrolled into the study.Our initial Facebook advertising testing began in October 2016 and included testing of the Facebook-recommended audience network placement. We began recruitment for our study in January 2017 but did not commence our sophisticated Pixel-based measurement of our Facebook campaigns (explained below) until mid-June 2017 and that continued until September 2018 for a total of 61 weeks. The Facebook scandal occurred in March 2018 .Across our 4 Facebook campaigns, we received a total of 92,677 advertisement clicks between June 20, 2017, and September 9, 2018. The general market campaign received 66,681 advertisement clicks, the black-/African American–focused campaign received 12,544 advertisement clicks, and the Hispanic-/Latino-focused campaign received 10,969 advertisement clicks. Our shorter Spanish language campaign received 2483 advertisement clicks between June 20, 2017, and September 30, 2017. On average, about 13.92%  of individuals who clicked our advertisement filled out the interest form, constituting a query. After receiving an email with a link to our web-based screening survey, about 19.31%  of individuals completed the survey and about 20.32% (506/2490) of these applicants were enrolled in our study. We could not capture ethnicity at the advertisement click stage because we had no mechanism for doing this. However, we began collecting self-reported race or ethnicity at the query stage and continued this through the applicant and participant stages.2Χ1=7.8; P=.005). Ethnicity related to study exclusion because of refusing mobile phone verification, in that whites  were more likely to be excluded for this reason than blacks/African Americans . Ethnicity related to study exclusion because of smoking too few cigarettes per day to permit use of the study-provided nicotine replacement therapy, in that blacks/African Americans  were more likely to be excluded for this reason than whites . No other effects were significant , in that blacks/African Americans  were more likely to be excluded for health reasons than non-Hispanic whites  using the Sidak correction for multiple comparisons.Facebook’s basic advertisement reporting system measures advertisement clicks by default. To obtain the query, applicant, and participant metrics, we utilized a Facebook Pixel, which is a web code our website developer installed to measure and track actions Facebook users performed on our study website. The Facebook Pixel also tracked which of our Facebook campaigns drew them to our website . We instWe could not directly compare campaign yields because expenditures differed by campaign . To compare yields across campaigns, we standardized expenditures to US $140 per week (US $20 per day), reflecting our average budget for the 2 main ethnic campaigns. For example, on a given week, we may have spent US $700 on the general market campaign versus US $140 on the black-/African American–focused campaign. If we received 100 queries from the general market campaign, we divided this number by 5 and estimated the yield as 20 based on a standardized expenditure of US $140 per week (US $700/5=US $140). This standardized yield of 20 for the general market campaign would be compared with the yield of the black-/African American–focused campaign that ran at US $140.F3,194=19.79; P<.001), with the general market–focused campaign at 19.28 people (SD 6.11), black-/African American–focused at 10.82 people (SD 9.20), Hispanic-/Latino-focused at 12.10 people (SD 7.25), and Spanish language–focused at 7.02 people (SD 2.24). The query yield of whites was higher for the general market campaign than for the black-/African American–focused , Hispanic-/Latino-focused , or Spanish language–focused campaigns . The query yield of whites was similar for the black-/African American–focused campaign versus Hispanic-/Latino-focused  or Spanish language campaigns . The weekly mean count of whites was similar for the Hispanic-/Latino-focused versus Spanish language campaign , with the general market–focused campaign at 2.83 people (SD 2.05), black-/African American–focused at 9.48 people (SD 5.69), Hispanic-/Latino-focused at 2.89 people (SD 2.12), and Spanish language–focused at 0.60 people (SD 0.62). The query yield of blacks/African Americans was higher for the black-/African American–focused campaign than for the general market–focused , Hispanic-/Latino-focused , or Spanish language–focused campaigns . The query yield of blacks/African Americans was similar for the general market–focused versus Hispanic-/Latino-focused  or Spanish language–focused campaigns . The query yield of blacks/African Americans was similar for the Hispanic-/Latino-focused versus Spanish language–focused campaign , with the general market–focused campaign at 0.71 people (SD 0.48), black-/African American–focused at 0.56 people (SD 0.82), Hispanic-/Latino-focused at 3.10 people (SD 2.16), and Spanish language–focused at 3.70 people (SD 1.77). The query yield of Hispanics/Latinos was higher for the Hispanic-/Latino-focused campaign than for the general market–focused  or black-/African American–focused campaigns  but was similar to the Spanish language–focused campaign . The query yield of Hispanics/Latinos was similar for the general market–focused campaign as compared with the black-/African American–focused campaign  but was lower than that of the Spanish language–focused campaign . The query yield of Hispanics/Latinos was higher for the Spanish language–focused versus African American–focused campaign , with the general market–focused campaign at 4.12 (SD 2.08), black-/African American–focused at 2.59 (SD 2.86), Hispanic-/Latino-focused at 2.39 (SD 1.55), and Spanish language–focused at 1.78 (SD 0.95). The applicant yield of whites was higher for the general market–focused campaign than for the black-/African American–focused , Hispanic-/Latino-focused , or Spanish language–focused campaigns . The applicant yield of whites was similar for the black-/African American–focused versus Hispanic-/Latino-focused  or Spanish language–focused campaigns . The applicant yield of whites was similar for the Hispanic-/Latino-focused versus Spanish language–focused campaign , with the general market–focused campaign at 0.54 (SD 0.58), black-/African American–focused at 1.11 (SD 1.21), Hispanic-/Latino-focused at 0.35 (SD 0.55), and Spanish language–focused at 0.15 (SD 0.24). The applicant yield of blacks/African Americans was higher for the black-/African American–focused campaign compared with the general market–focused , Hispanic-/Latino-focused , or Spanish language–focused campaigns . The applicant yield of blacks/African Americans was similar for the general market–focused versus Hispanic-/Latino-focused  or Spanish language–focused campaigns . The applicant yield of blacks/African Americans was similar for the Hispanic-/Latino-focused versus Spanish language–focused campaign , with the general market–focused campaign at 0.10 (SD 0.14), black-/African American–focused at 0.11 (SD 0.32), Hispanic-/Latino-focused at 0.36 (SD 0.55), and Spanish language–focused at 0.52 (SD 0.50). The applicant yield of Hispanics/Latinos was higher for the Hispanic-/Latino-focused campaign compared with general market–focused  or black-/African American–focused campaigns  but similar to that of Spanish language–focused campaign . The applicant yield of Hispanics/Latinos was similar for the general market–focused campaign compared with the black-/African American–focused campaign  but lower than that of the Spanish language–focused campaign . The applicant yield of Hispanics/Latinos was higher for the Spanish language–focused campaign versus the black-/African American-focused campaign , with the general market–focused campaign at 0.80 (SD 0.52), black-/African American–focused at 0.27 (SD 0.43), Hispanic-/Latino-focused at 0.21 (SD 0.28), and Spanish language–focused at 0.28 (SD 0.28). The participant yield of whites was higher for the general market–focused campaign than for the black-/African American–focused , Hispanic-/Latino-focused , or Spanish language–focused campaigns . The participant yield of whites otherwise did not differ across campaigns , with the general market–focused campaign at 0.09 (SD 0.13), black/African American–focused at 0.11 (SD 0.28), Hispanic-/Latino-focused at 0.04 (SD 0.12), and Spanish language–focused at .001 (SD 0.001). However, none of the pairwise comparisons between specific campaigns were significant , with the general market–focused campaign at 0.03 (SD 0.07), black-/African American–focused at 0.01 (SD 0.06), Hispanic-/Latino-focused at 0.01 (SD 0.05), and Spanish language–focused at 0.04 (SD 0.11). However, none of the pairwise comparisons between specific campaigns were significant  but not at the individual level  because at the individual level, internal auctions are used to determine if an advertisement is shown, and Facebook does not share these individual-level costs with its advertisers . The FacF3,194=97.18; P<.001), with the general market–focused campaign at US $0.67 (SD 0.19), black-/African American–focused at US $0.78 (SD 0.13), Hispanic-/Latino-focused at US $0.91 (SD 0.17), and Spanish language-focused at US $1.56 (SD 0.34). The cost per advertisement click was lower for the general market–focused campaign, compared with black-/African American–focused , Hispanic-/Latino-focused , or Spanish language–focused . The cost per advertisement click was lower for the black-/African American–focused campaign than for the Hispanic-/Latino-focused  or Spanish language-focused  campaigns, and lower for the Hispanic-/Latino-focused than for the Spanish language–focused campaign , with the general market–focused campaign at US $4.04 (SD 1.99), black-/African American–focused at US $7.00 (SD 2.77), Hispanic-/Latino-focused at US $7.98 (SD 4.13), and Spanish language–focused at US $11.06 (SD 3.23). The cost per query was lower for the general market–focused campaign than for the black-/African American–focused , Hispanic-/Latino-focused , or Spanish language–focused campaigns . The cost per query was lower for the black-/African American–focused campaign than for the Hispanic-/Latino-focused  or Spanish language–focused campaigns , and lower for the Hispanic-/Latino-focused than for the Spanish language–focused campaign , with the general market–focused at US $22.61 (SD 12.21), black-/African American–focused at US $47.82 (SD 33.00), Hispanic-/Latino-focused at US $47.63 (SD 32.16), and Spanish language–focused at US $75.65 (SD 67.60). The cost per applicant was lower for the general market–focused campaign, compared with the black-/African American–focused , Hispanic-/Latino-focused , or Spanish language–focused campaigns . The cost per applicant was comparable for the black-/African American–focused campaign than for the Hispanic-/Latino-focused campaign  but lower than that of the Spanish language–focused campaign . The cost per applicant was also lower for the Hispanic-/Latino-focused than for the Spanish language–focused campaign , with the general market–focused at US $133.90 (SD 103.92), black-/African American–focused at US $121.05 (SD 60.02), Hispanic-/Latino-focused at US $128.90 (SD 67.86), and Spanish language–focused at US $171.66 (SD 97.37). Likewise, comparing the specific campaigns pairwise, there were no differences in cost per participant . Additionally, there were no significant differences in cost per advertisement click when we conducted specific pairwise comparisons of the campaigns  and a scandal by campaign type 2-way interaction , meaning the scandal effect varied by campaign. For the general market–focused campaign, there was no scandal effect on cost per query , with prescandal at US $4.32 (SD 2.49) and postscandal at US $3.66 (SD 0.94). For the black-/African American–focused campaign, there was a scandal effect on cost per query , with prescandal at US $5.87 (SD 1.89) and postscandal higher at US $8.51 (SD 3.08). Likewise, for the Hispanic-/Latino-focused campaign, there was a scandal effect on cost per query , with prescandal at US $7.04 (SD 3.39) and postscandal higher at US $9.24  and a scandal by campaign type 2-way interaction , meaning the scandal effect varied by campaign. For the general market–focused campaign, there was no scandal effect on cost per applicant , with prescandal at US $22.48 (SD 14.68) and postscandal at US $22.77 (SD 8.19). For the black-/African American–focused campaign, there was a scandal effect on cost per applicant , with prescandal at US $38.96 (SD 28.31) and postscandal higher at US $59.64 (SD 35.63). Similarly, for the Hispanic-/Latino-focused campaign, there was a scandal effect on cost per applicant , with prescandal at US $38.19 (SD 21.20) and postscandal higher at US $61.19 . Additionally, there were no significant differences in cost per participant when we conducted specific pairwise comparisons of the campaigns , with the lookalike audience at US $0.73 (SD 0.30) and general market–focused at US $1.07 (SD 0.14). However, the cost per query for the lookalike audience campaign did not differ from the general market campaign , with lookalike audience at US $3.98 (SD 2.24) and general market at US $4.27 (SD 2.19). The cost per applicant for the lookalike audience campaign also did not differ from the general market campaign , with lookalike audience at US $15.58 (SD 6.07) and general market at US $20.13 (SD 10.13).The cost per advertisement click for the lookalike audience campaign was lower than the general market–focused campaign , with audience network at US $0.11 (SD 0.05) and Facebook alone at US $0.69 (SD 0.13). However, the cost per query was substantially higher when audience network placement was used compared with placement on Facebook alone , with the audience network at US $13.40 (SD 14.83) and Facebook alone at US $5.62 (SD 2.76). After 2 test weeks, based on noticeably poor query results, we stopped using the audience network placement.Here, we compare data from our general market campaign that used audience network placement  to standard advertisement placement on Facebook alone . The analysis shows that the cost per advertisement click was lower when the audience network placement was used compared with placement on Facebook alone , we successfully increased our outreach to and yield of black/African American– and Hispanic/Latino applicants. This study also found that Facebook’s recommended audience network placement, although cheaper at producing advertisement clicks, was far more expensive at producing website queries. This means that many individuals who clicked on our advertisements were not interested. Many individuals may have clicked on our advertisements to use a free app , not because they were interested in our study. Thus, we do not recommend using the audience network placement. We also learned that the Facebook privacy scandal involving Cambridge Analytica had a negative impact on recruitment for our campaigns that were black/African American–focused and Hispanic-/Latino-focused. This scandal increased our cost per query and cost per applicant. Hence, researchers should keep Facebook scandals in mind, as they may raise recruitment costs.source audience can be identified based on website behavior as tracked by a Facebook Pixel (we used queries on our website). Potential drawbacks to this feature include privacy and institutional review board (IRB) concerns. Facebook’s audience lookalike feature seems to involve using data collected whenever people are logged into Facebook, including data that are both public and relatively more private, including likes, posts, visits to other websites, friend networks, and friends’ web-based behavior. This data use could raise potential privacy and IRB approval issues because it is not possible in IRB consent forms or information sheets to describe to potential participants what Facebook data of theirs will be used for the audience lookalike feature (this is proprietary) and it is not possible to obtain consent from their Facebook friend networks.Facebook’s recommended audience lookalike feature produced advertisement clicks at a lower price point than our baseline general market campaign; however, query and applicant price points were similar to our general market campaign. Researchers may want to consider using Facebook’s audience lookalike feature because it may cost less at the advertisement click stage, if this is a high priority, and if the required Facebook-based recruitment for health-related research has been the focus of previous studies, allowing for cost and yield comparisons with our study. Looking at cost per advertisement click, Whitaker et al  reportedOur findings on recruitment yields standardize for spending at US $140 per week, so they are relatively independent of costs. However, our findings on recruitment costs are limited by the fact that recruitment costs are strongly affected by study inclusion/exclusion criteria, participation time demands, and study benefits and incentives. Recruitment costs can also fluctuate daily and seasonally. In addition, we recruited throughout the continental United States for our web-based study, and studies that are more local may yield different outcomes. Moreover, our costs per yield for a Spanish language campaign may be overestimated because English fluency was a requirement for inclusion in our study. Studies with translated materials and no English-speaking requirements may find higher enrollments and lower costs.A strength of our study is that it was a national study with a large sample size; 980 people were recruited overall. Furthermore, we examined Facebook advertising campaigns considering both yields and costs and considered 4 separate outcomes: advertisement clicks, queries, applicants, and participants. In addition, our study lasted for 61 weeks, allowing us to compare costs of Facebook recruitment both before and after a major Facebook privacy scandal. Our main goal was to innovatively test the efficacy of targeting specific zip codes on Facebook to reach ethnically or racially diverse populations. Advertiser intent can have ethical ramifications when utilizing zip code targeting, especially when protected characteristics are targeted such as race or ethnicity. As of August 2019, Facebook restricts audience targeting options, including zip code targeting in the case of housing, credit, or employment to help prevent discrimination . In our Our main conclusion is that Facebook advertising campaigns that employ suitable zip code targeting can help to find and recruit blacks/African Americans and Hispanics/Latinos for web-based health-related studies.


Age-associated lung tissue degeneration is a risk factor for lung injury and exacerbated lung disease. It is also the main risk factor for chronic lung diseases . So, it is particularly important to find new anti-aging treatments.We systematically screened and evaluated elderly senile multiple organ dysfunction macaque models to determine whether BMMSCs inhibited lung tissue degeneration.p < 0.05), while the capillary density around the alveoli was significantly low than in young macaque models (p < 0.05). Intravenous infusion of BMMSCs reduced the degree of pulmonary fibrosis, increased the density of capillaries around the alveoli (p < 0.05), and the number of type II alveolar epithelium in elderly macaques (p < 0.05). In addition, the infusion reduced lung tissue ROS levels, systemic and lung tissue inflammatory levels, and Treg cell ratio in elderly macaque models (p < 0.05). Indirect co-cultivation revealed that BMMSCs suppressed the expression of senescence-associated genes, ROS levels, apoptosis rate of aging type II alveolar epithelial cells (A549 cells), and enhanced their proliferation (p < 0.05).The average alveolar area, mean linear intercept (MLI), and fibrosis area in the elderly macaque models were significantly larger than in young rhesus monkeys (BMMSC treatment inhibited age-associated lung tissue degeneration.The online version contains supplementary material available at 10.1186/s13287-021-02201-3. The rising aging population in China is a risk for various diseases. It is estimated that by 2050, those aged 65 years and above will represent about 20% of the Chinese population . Age enhMesenchymal stem cells are multipotent stem cells. They are characterized by low immunogenicity, self-renewal ability, and multi-directional differentiation potential. Due to immune-associated regulation of anti-apoptosis, angiogenesis, migration and differentiation of stem cells in target organs, enhancing the growth and differentiation of local stem cells, progenitor cells and anti-scarring have been used in the treatment of various diseases .Studies have documented that mesenchymal stem cells can repair lung injuries and effectively treat acute and/or chronic lung diseases –11. HoweMacaques are popular non-human primates whose biological characteristics are identical to those of humans. As model organisms, macaques are physiologically identical to humans. Their genomes have a 93% average sequence identity with humans. They are, therefore, ideal models for studies of human health and disease.In addition, as model animals for basic and applied research in biomedicine, macaques have numerous advantages including environmental factor control and ease of scale , 13.Therefore, the aim of this study was to determine the in vivo and in vitro effects of bone marrow mesenchymal stem cells (BMMSCs) on age-associated lung tissue degeneration.Thirty macaques aged between 2 and 26 years and weighing 2.2–12 kg were obtained from the Kunming Institute of Zoology of the Chinese Academy of Sciences [SCXK (Yunnan) K2017-0003]. Among them, 25 were female macaques aged between 6 and 26 years while 5 were male macaques aged between 2 and 3 years. The animals were housed in the Experimental Animal Center of the 920th Hospital of the Chinese People’s Liberation Army Joint Logistics Support Force, experimental animal license number: SYXK (Military) 2017-0051. Ethical approval for the use of animal models was obtained from the experimental animal council of the 920th Hospital of Joint Logistics Support Force.Bone marrow A549 cell lines were purchased form the Wuhan Sevier company, sub-cultured in a culture flask with DMEM medium supplemented with 10% fetal bovine serum. They were then incubated at 37 °C in an atmosphere of 5% carbon dioxide.18F-FDG was provided by the First Affiliated Hospital of Kunming Medical University.FBS and penicillin-streptomycin solution were purchased from Servicebio; DMEM/F12 media was purchased from Hyclone; 0.25% pancreatin-0.04% EDTA was purchased from Invitrogene; 30% hydrogen peroxide solution was purchased from Solarbio; Cell senescence β-galactosidase staining kit, apoptosis detection kit, active oxygen detection kit, and cell cycle and apoptosis detection kit were purchased from Beyotime Biotechnology; Anti-proSP-C antibody (AB3786) was purchased from Sigma; Mouse anti-human CD45 (555555), mouse anti-human CD73 (344007), Cell staining buffer, True-Nuclear Transcription Factor Buffer Set, Alexa Fluor® 647 anti-human FoxP3 (320113), FITC anti-human CD4 (317408), PE anti-human CD25 antibody (356103), PE Mouse IgG1 κ Isotype Ctrl and Alexa Fluor® 647 Mouse IgG1 κ Isotype Ctrl (400135) were purchased from Biolegend; monkey interleukin 1β (IL-1β), monkey interleukin-17A (IL-17A), and human tumor necrosis factor alpha (TNF-α) ELISA kits were purchased from MeiMian; fluorescent secondary antibody HRP, fluorescent secondary antibody CY3, and primers were purchased from Servicebio; GoScriptTMReverse Transcription System and GoTaq® qPCR Master Mix were purchased from Promega Corporation; TNF alpha (7B8A11), IL-10 (20850-1-AP), and CEBPB (2B6E10) antibodies were purchased from Proteintech group; adipogenic differentiation medium, osteogenic differentiation medium, and chondrogenic differentiation medium were purchased from Guangzhou Saiye Biological Technology Co., Ltd.; Female macaques aged between 22 and 26 years were used as the elderly model group, while young female macaques aged between 6 and 8 years old were used as the control group. A total of 5 animals were randomly distributed in each group. Five milliliters of peripheral blood was obtained from cynomolgus monkeys and centrifuged to obtain serum. Lung length and width were determined using a ruler. Serum samples were used to quantify TNF-α and IL-1β levels through the ELISA technique. Macaques were anesthetized with 3% pentobarbital sodium (5 ml/kg body weight) and sacrificed to obtain lung tissues. Tissue samples were divided for size, morphology, and textural analysis as well as hematoxylin-eosin staining. The Masson’s Trichrome stain was used to determine the degree of pulmonary fibrosis in the two groups. Capillary density was examined by immunohistochemistry.Bone marrow mesenchymal stem cells of 2–3-year-old young rhesus monkeys were isolated and cultured by the adherence method. The morphology and growth characteristics of P4 BMMSC generation cells were observed while CCK8 was used to determine their proliferative capacities. A flow cytometer was used to analyze the positive rate of their surface antigens. A special induction medium was used to induce the differentiation of P4 BMMSCs into bone, cartilage, and adipocytes. Their differentiation abilities were analyzed by staining  levels in the peripheral blood were detected by ELISA.After BMMSC treatment, ROS staining was done on the left lung tissue. Sections were then subjected to the same laser intensity at equal exposure times to obtain images. To obtain the H-scores, the Densito Quant in the Quant Center was used to set dark red, brown red, light red, and blue nuclei as strong positive, moderate positive, weak positive, and negative respectively. The protein levels of proinflammatory factors  and anti-inflammatory factor (IL-10) in the lung tissues were detected by Western blot. VEGF expression levels in the lung tissues after BMMSC treatment was determined by Western blot. ImageJ was used to analyze the gray values of all western blot images, and to compare the gray values of the internal control band to the gray values of the target protein band. The effects of BMMSCs on peripheral blood Treg cell and FOXP3 ratios in lung tissues of elderly macaques were also determined.Lymphocytes were isolated from blood samples obtained from the animals at 0, 30, 60, and 90 days after BMMSC treatment. Changes in Treg cell ratios in peripheral blood were detected by flow cytometry. Treg cells were labeled with FOXP3 and changes in FOXP3 content assayed by immunohistochemistry.Statistical analyses were performed using the SPSS 21.0 statistical software. Data is expressed as mean ± standard deviation. Statistical differences in the means of three or more than three groups were analyzed by one-way ANOVA (One-Way ANOVA).Elderly macaques were found to have a dull coat that turned white, especially around the head and face. Furthermore, their skin was loose and dry while their faces appeared red Fig. a.Fig. 1p < 0.0001). Masson’s Trichrome stain colored the collagen fibers blue. Compared to the young control group, the collagen area in the lungs of the elderly model group was significantly increased . Immunohistochemical labeling of the vascular endothelial cells with CD31 revealed that the nucleus of lung tissue cells were stained blue while the surface markers of vascular endothelial cells were stained brown. In the elderly group, CD31 expression in the lungs was significantly low when compared to the young control group .Lung tissues of the young control group and the elderly model group were soft, butterfly-shaped, flexible, and pale red. However, lung sizes of the elderly macaque models were larger visually compared to those of the young control models . In addition, HRCT exhibited an irregular thickening of the leaflet intervals before treatment. After treatment, the small blood vessels in the leaflet were pronounced. This was attributed to the thickening of the wall in the treatment group. PET showed that the 18F-FDG uptake that was quantified as the glucose uptake in the lungs decreased after treatment. At 90 and 180 days post-treatment, lung textures were clear with normal hilar. The Hounsfield unit was higher than before treatment. The average Hounsfield units were − 685 ± 12.53 and − 705 ± 18.53, respectively . However, the differences in the average alveolar area and alveolar lining interval between the model group and the treatment group were not significantly different . Masson’s Trichrome stain showed blue collagen. The collagen area of the treatment group was significantly reduced (p < 0.05) when compared to the model group .After 180 days of treatment, lung tissues appeared dark white and red without embolism. However, there were no significant changes between the treatment and the model groups  in the treatment group compared to the model group. However, the expression levels of TCAB1 increased significantly  . To verify the in vitro effects of BMMSCs on aging A549 cell model, proSPC was used as an in vivo marker for type II alveolar epithelial cells. It was found that type II alveolar epithelial cells were either round or oval and scattered in the alveolar wall. The number of type II alveolar epithelial cells in the model group was significantly low when compared to the control group (p < 0.001). However, in the treatment group, type II alveolar epithelial cells were significantly elevated when compared to the model group .Type II alveolar epithelium plays a significant role in lung tissue aging. Among the elderly, the quantity and quality of type II alveolar epithelial cells are significantly reduced . In thisly) Fig. b, c. In p < 0.05). After BMMSC treatment, VEGF levels in the treatment group were significantly elevated when compared to the model group . Since the collagen area in the lung tissue was changed after the cells had been transferred, TGF-β1 levels in the lung tissue were determined. The expression levels of TGF-β1 in the lung tissues were found to significantly increase with age. However, there were no significant changes after BMMSC treatment . To elucidate on the regulatory effects of BMMSCs on aging-associated inflammation, IL-1β, IL-17A, and TNF-α levels were detected by ELISA. Compared to the model group, IL-1β levels were found to be significantly low in blood serum (p < 0.05) at 30 and 60 days after BMMSC treatment. After 90 days of treatment, these levels had reverted to normal. Moreover, TNF-α levels were found to be significantly low after 30 days (p < 0.05). However, they reverted to their normal levels after 60 days and remained unchanged. There were no significant variations in IL-17A levels , but were significantly elevated after BMMSC treatment .Studies have revealed that the nine aging hallmarks are stem cell failure, intercellular communication changes, genomic instability and telomere wear, epigenetic changes, loss of protein homeostasis, nutrition changes, mitochondrial dysfunctions, and cellular senescence . The maip < 0.01), and reached their minimum levels at 60 days after treatment (p < 0.0001). Treg cell ratio changes at 60 and 90 days were not significant . To determine the consistency of Treg cell changes in the periphery and lung tissues, a Treg cell surface marker (FOXP3) was used for immunohistochemical detection. FOXP3 levels of the model group lung tissue were found to be significantly elevated when compared to the control group (p < 0.01). In addition, FOXP3 levels in the treatment group were significantly low when compared to the model and control groups  and some RNA-rich microvesicles. This paracrine effect delays cell senescence and apoptosis and lung tissue repair and inhibits fibrosis. Mitochondrial transfer and communication between MSCs and neighboring cells are involved in tissue repair –22.In this study, we report on the effects of BMMSCs on lung tissue degeneration. Lung tissue changes were detected by PET-CT. There was a significant increase in x-ray transmittance (Hounsfield unit) in lung tissues after BMMSC treatment. A significant decrease in SUV max values and fibrosis were observed. Compared to the model group, collagen deposition in the treatment group was significantly inhibited while the alveolar vascular density was significantly elevated. However, the reduction in alveolar size was not significant. These results were consistent with those reported by previous studies on mesenchymal stem cells in the treatment of COPD –25. BreaSA-β-gal is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides in senescent cells. P53 and its downstream genes (P21 and TCAB1) control cellular aging , 26.We confirmed that BMMSCs reversed age-associated type II alveolar epithelial cell (A549 cells) characteristics. These results were consistent with those reported in a previous study of the effects of bone marrow mesenchymal stem cells on 293T cell senescence models .High inflammation and oxidative stress enhance tissue and organ degeneration during aging , 15, 27.Treg cells regulate immune responses. Specifically, they play a central role in immune homeostasis and in preventing autoimmunity. They are secreted by the thymus and lymph and transported through the entire body. They inhibit the activation and proliferation of potential self-reactive T cells, thereby regulating the body’s immunity and inflammation. Studies have documented that the number and functions of Treg cells change significantly in the aging body. Zhao et al. reported that the ratio of Treg cells in the CD4+ cells was significantly elevated and their function was significantly inhibited in the peripheral blood of elderly mice . IL-10 iInterleukin 10 levels obtained in this study were consistent with those reported in a previous mesenchymal stem cell treatment model of lung injury . DecreasIn summary, BMMSCs inhibit aging-associated lung degeneration. The limitations of this study were that the impact of BMMSCs on lung functions and the mechanisms by which BMMSCs improved lung degeneration were not determined.i.The elderly pulmonary degenerative macaque models revealed that the alveolar cavity was enlarged, lung structure was disordered, there was increased pigmentation, and the degree of fibrosis was elevated while the capillary density was decreased.ii.BMMSCs inhibited the degree of pulmonary fibrosis in elderly macaques, suppressed lung and peripheral blood inflammatory levels, enhanced VEGF expression in lung tissues, increased capillary density around the alveoli, and suppressed Treg cell levels in peripheral blood and lung tissues.iii.BMMSCs suppressed the expression of type II alveolar epithelial aging-related genes, inhibited its apoptosis and oxidative stress levels, and enhanced its proliferation. BMMSCs also elevated the number of type II alveolar epithelium in lung tissues.Additional file 1. Preparation and identification of bone marrow mesenchymal stem cells of macaqueAdditional file 2. Comparison of lung size between the young control group and the elderly model groupAdditional file 3. No significant changes between the treatment and the model groupsAdditional file 4. Hydrogen peroxide-induced senescence of A549 cells and β-galactosidase staining (100 ×)


Hypothyroidism is a common complication of therapeutic irradiation in the neck area. Several dose-response models have been proposed to predict its’ occurrence based on clinical and radiomic features. We aimed to externally validate the results of five such models in a prospectively recruited cohort of 108 patients with oropharyngeal cancer. Two of the evaluated models, published by Rønjom et al. and by Boomsma et al., had satisfactory performance. Both models are based on mean thyroid dose and thyroid volume. Three remaining models, by Cella et al., Bakhshandeh et al. and Vogelius et al., performed significantly worse. Short-term change in the level of thyroid-stimulating hormone (TSH) after radiation therapy was not indicative of hypothyroidism development in long term. We conclude that the models by Rønjom et al. and by Boomsma et al. are feasible for long-term prediction of hypothyroidism in oropharyngeal cancer survivors treated with intensity-modulated radiation therapy.p = 0.825). In conclusion, the models by Rønjom et al. and by Boomsma et al. demonstrated external validity and feasibility for long-term prediction of RIHT in survivors of OPC treated with Intensity-Modulated Radiation Therapy (IMRT).We aimed to externally validate five normal tissue complication probability (NTCP) models for radiation-induced hypothyroidism (RIHT) in a prospectively recruited cohort of 108 patients with oropharyngeal cancer (OPC). NTCP scores were calculated using original published formulas. Plasma thyrotropin (TSH) level was additionally assessed in the short-term after RT. After a median of 28 months of follow-up, thirty one (28.7%) patients developed RIHT. Thyroid mean dose and thyroid volume were significant predictors of RIHT: odds ratio equal to 1.11 (95% CI 1.03–1.19) for mean thyroid dose and 0.87 (95%CI 0.81–0.93) for thyroid volume in univariate analyses. Two of the evaluated NTCP models, published by Rønjom et al. and by Boomsma et al., had satisfactory performance with accuracies of 0.87 (95%CI 0.79–0.93) and 0.84 (95%CI: 0.76–0.91), respectively. Three remaining models, by Cella et al., Bakhshandeh et al. and Vogelius et al., performed significantly worse, overestimating the risk of RIHT in this patient cohort. A short-term TSH level change relative to baseline was not indicative of RIHT development in the follow-up (OR 0.96, 95%CI: 0.65–1.42,  Radiation-induced hypothyroidism (RIHT) commonly develops in cancer survivors that receive radiation therapy (RT) for head and neck cancer (HNC). The median interval between RT and hypothyroidism is approximately 1.5 years, however later toxicities are also observed ,3,4.Despite clinical benefits, the use of Intensity-Modulated Radiation Therapy (IMRT) was one of the factors that were reported to increase the incidence of RIHT ,6 in conAs the symptoms of hypothyroidism may be non-specific, appropriate thyroid hormone levels monitoring in patients who have undergone RT in the head and neck region is of great importance. Inadequate levels of thyroid hormones negatively impact the patients’ quality of life , their mA recent systematic review of normal tissue complication probability (NTCP) models relevant to RT of the HNC  criticalWe aimed to externally validate the five available NTCP models for RIHT in an independent cohort of patients with oropharyngeal cancer (OPC) to verify which, if any, should be used to inform clinical decision making. We tested also which of the variables included in the original NTCP models were predictive of RIHT in the validation cohort and verified if recalibration would improve the model performance. Furthermore, we addressed the shortcomings of the previous studies by prospectively collecting data from three radiation oncology centers in Poland and by including an additional timepoint (shortly after RT) in the study design, to verify the hypothesis that monitoring TSH level shortly after RT may be useful to predict RIHT.In total, 195 patients met the inclusion criteria and were recruited in the study. Among these, 83 were excluded from the final analysis due to a lack of follow-up thyroid function assessment before recurrence/death or loss of contact, 3 due to baseline plasma TSH > 4 mIU/L and 1 due to known thyroid disease . A summap = 0.869 and p = 0.299, respectively).In the whole group, the median follow-up was 28 months (IQR 21–38 months). Thirty-one patients (28.7%) developed RIHT that required thyroid replacement therapy, meeting our primary endpoint; median time to RIHT development was 16 months (IQR 14–22 months). Neither RIHT frequency nor median time to RIHT development differed significantly between the three centers  for the whole cohort and for the development cohort from each study is presented in A summary of models’ performance in the whole validation cohort and in each center is presented in Model by Rønjom et al. had better specificity for RIHT prediction (90% vs. 81%), and the model by Boomsma et al. had better sensitivity (94% vs. 81%). While NTCP scores for these two models were also highly correlated—as expected given that both models included the same parameters—the model by Rønjom et al. predicted slightly lower RIHT development probabilities in patients with low risk of developing RIHT  and slightly higher probabilities in the high-risk patients  from center B  0.91–0.94) in patient cohorts from centers A and C and moderate in the smallest patient cohort  that had baseline TSH levels <0.3 mIU/L. Noteworthy, although the baseline TSH level is not included in any of the validated NTCP models, the model by Rønjom et al. correctly predicted the RIHT development in all these 14 patients  and the one by Boomsma et al. misclassified only a single patient , while classifying the remaining 13 patients correctly.p = 0.004 and p < 0.001, respectively); in the multivariable analysis, both these variables remained significant , the short-term change in TSH was not different between patients with and without RIHT in follow-up .Finally, we aimed to assess whether short-term changes in the TSH or fT4 level are indicative of RIHT development in the long term. Although TSH levels decreased significantly in the short term after RT completion  and by the differences between the original and presented patient groups. While Boomsma et al. and Rønjom et al. NTCP models were developed in homogenous populations of HNC patients treated with chemo-RT, the study cohort for Cella et al. consisted of patients with Hodgkin’s lymphoma and the study by Vogelius et al. was a meta-analysis of four studies, two of which included patients with Hodgkin’s lymphoma. Given the differences in treatment for HNC and Hodgkin’s lymphoma, i.e., lower total target dose, a dose–response model developed using data for one of these conditions is unlikely to perform well for the other. This disparity might also explain why the model by Cella et al., which includes thyroid V30 as a predictor in the logistic regression formula, predicted RIHT in 98.1% of the patients in our cohort, where the median thyroid V30 was 100 (IQR 99.8–100).All other three evaluated models, by Cella et al. , BakhshaA recent retrospective study of RIHT after IMRT in 360 OPC patients  reached Noteworthy, the NTCP models published by Rønjom et al.  and by Bn = 13), which prevents an appropriate comparison of the models’ performance between all participating centers. The definition of the endpoint , differs from the one defined in most studies that reported the validated NTCP models . We chose an endpoint based on the CTCAE classification because it is a robust and validated tool used both in state-of-the-art clinical trials and everyday practice. Another difference regarding the original studies is the inclusion of patients with hyperthyroidism . We chose to include these patients to allow the evaluation of models’ performance in a pragmatic, real-life clinical setting. Notably, NTCP models by Boomsma et al. and Rønjom et al. performed well also in this group, correctly predicting RIHT for all  patients  or almoa et al. ).Considering the potential long-term sequelae of hypothyroidism and the relatively high incidence of RIHT, it is important to include the thyroid gland as an organ at risk in routine treatment planning of RT. Recommendations for the constraint doses for the thyroid gland have been proposed by Rønjom et al.  with resThe incidence of HPV-positive OPC patients is rapidly increasing  and trea2, liver failure—C or D score in Child–Pugh classification). The study cohort involved 108 patients prospectively recruited at three oncology centers in Poland: Copernicus Regional Specialist Hospital in Łódź , Radom Oncology Centre and Maria Sklodowska-Curie National Research Institute of Oncology, Gliwice Branch  between 01.05.2016 and 31.12.2018 and followed up until 02.2020; details regarding patient inclusion are presented in 2), every three weeks platinum (cisplatin 100 mg/m2) and induction chemotherapy according to the PF protocol  or the TPF protocol . Detailed description of the RT protocol either or not combined with systemic treatment for all patients is presented in Staging was performed according to the American Joint Committee on Cancer (AJCC) 7th edition staging system . All patINK4a histology kit  with a 70% nuclear and cytoplasmic staining cutoff [For p16 immunohistochemistry, we used a CINtec p16g cutoff . Both poTreatment planning was carried out using Eclipse software  version 13.6 or 15.1. Dose was calculated by means of an anisotropic analytical algorithm (AAA) with 2.5 mm grid size. The aim of the planning optimization was to cover at least 95% of the planning target volume (PTV) with 100% of the prescription dose. The main OARs considered were brain stem, spinal cord, larynx, mandible, parotid glands and esophagus. Sparing the thyroid gland was not part of the optimization objectives. For this study, thyroid glands were therefore retrospectively contoured by two experienced radiation oncologists according to the recent guidelines for OARs in the head and neck region .Laboratory assessments were performed at baseline (during the 7 days before the beginning of RT), shortly after RT (during the 7 days since the last fraction) and at follow-up (median time to follow-up 28 months). The specimens were collected during standard assessments associated with the RT treatment and follow-up visits. The normal range for TSH was defined as 0.3–4 mIU/L and the normal range for fT4 was defined as 7–22 pg/mL. To avoid bias, laboratory assessment, contouring and statistical analysis were performed by independent researchers and the person responsible for the outcome assessments was blinded to the results of the NTCP models being validated.We defined the primary endpoint as grade ≥2 RIHT per the Common Terminology Criteria for Adverse Events grading system, version 4.03 .Since no generally accepted approaches exist to calculate the sample size for validation studies , we did The models by Rønjom et al. , Boomsmap values were reported. The following variables were considered for multivariate analysis: age, sex, surgery, tobacco use, thyroid volume, minimum dose to thyroid, mean dose to thyroid, maximum dose to thyroid, percentage of thyroid gland volume receiving no more than 30 Gy, TSH level at baseline and short-term change in TSH level. Stepwise regression with backward feature elimination was implemented, with threshold p = 0.1 for feature elimination. Laboratory parameters from before and after RT were compared using Wilcoxon rank sum test. Differences between RIHT and no-RIHT groups were evaluated using Mann–Whitney U test.Univariate analysis of RIHT association with an outcome was conducted by creating logistic regression models with a single predictor; odds ratios (ORs) with 95% confidence intervals (95%CI) and p = 0.5. The calibration performance of the NTCP models was assessed by calibration plots, depicting grouped observed outcome frequencies with 95% confidence intervals (95%CI) vs. mean predicted probabilities [NTCP predictions were calculated using the original formulas reported for each model ,16,17,18bilities . SmoothiPlatt calibration was used to recalibrate the model predictions . In thisp values <0.05 were considered statistically significant. Statistical analysis was conducted using STATISTICA 13.1  and R version 3.6.3 (2020-02-29), packages: pROC  [For all analyses, two-sided tests were used and Country) ,41 and CCountry) ,41.The analyses and reporting were done in accordance with the TRIPOD  statement  File S1File S1. The study was approved by the Bioethics Committee of the Medical University of Lodz .Our results from a prospectively evaluated multicenter cohort of patients with OPC confirmed that low thyroid volume and high mean dose to the thyroid gland were both significant risk factors for RIHT. We showed that the models by Rønjom et al.  and by B


Tumor budding (TB) is used as an indicator of poor prognosis in various cancers. However, studies on TB in perihilar cholangiocarcinoma are still limited. We examined the significance of TB in resected perihilar cholangiocarcinoma with or without neoadjuvant therapy.Seventy-eight patients who underwent surgical resection at our institution for perihilar cholangiocarcinoma from 2004 to 2017, (36 with neoadjuvant therapy), were enrolled in this study. TB was defined as an isolated cancer cell or clusters (< 5 cells) at the invasive front and the number of TB was counted using a 20 times objective lens. Patients were classified into two groups according to TB counts: low TB (TB < 5) and high TB (TB ≥5).p = 0.007, pN1/2: 70.8% vs 39.6%, p = 0.011, M1: 20.8% vs 1.9%) and higher histological grade . Disease specific survival (DSS) in high TB was significantly inferior compared to that in low TB group . Interestingly, DSS in high TB showed similar to survival in unresected patients. In addition, high TB was also associated with advanced tumor status and poor prognosis in patients with neoadjuvant therapy. Multivariate analysis identified high TB as an independent poor prognostic factors for DSS .In this 78 patient cohort, high TB was significantly associated with advanced tumor status (pT4: 50.0% vs 22.2%, This study demonstrated that high TB was strongly associated with advanced tumor status and poor prognosis in resected perihilar cholangiocarcinoma patients. High TB can be a novel poor prognostic factor in resected perihilar cholangiocarcinoma regardless of neoadjuvant therapy. Perihilar cholangiocarcinoma, which is an epithelial cell malignancy localized to the area between the second degree bile ducts and the insertion of the cystic duct into the common bile duct, represents the most common form of cholangiocarcinoma , 2. Althn = 47 (16%), extrahepatic: n = 144 (48%), gallbladder: n = 50 (17%), ampulla: n = 58 (19%)). In addition, Tanaka et al. [Tumor budding (TB) is a histological phenomenon encountered in various cancers typically described as the presence of single cells or clusters in the tumor stroma, and was first described by Imai in Japanese literature . TB is wa et al.  demonstrIn recent years, neoadjuvant therapy has become a critical treatment for improving the outcomes of various cancers. Additionally, neoadjuvant therapy is becoming the standard of treatment of locally advanced pancreatic adenocarcinoma. However, there are few reports on the impact of neoadjuvant therapy in the patients with cholangiocarcinoma, although the efficacy of neoadjuvant therapy followed by surgery for “unresectable” locally advanced cholangiocarcinoma has been reported , 15.Studies on the relationship of TB and neoadjuvant therapy remain limited. In the field of rectal and esophageal carcinoma, where the use of neoadjuvant therapy is common, there are several reports showing the prognostic significance of high TB in patients who underwent neoajuvant therapy , 16, 17.Between January 2004 and December 2017, 81 patients with perihilar cholangiocarcinoma underwent surgical resection at our institution. Three patients with hospital death were excluded, and the remaining 78 patients were included in this study. In addition, 28 patients with locally advanced perihilar cholangiocarcinoma who did not undergo surgical resection in the same period were included in the survival analysis with comparison to resected patients.Multidetector-row computed tomography (MDCT), magnetic resonance cholangiopancreatography (MRCP), endoscopic retrograde cholangiography (ERC), and intraductal ultrasonography (IDUS) were used for preoperative diagnosis and tumor staging. Tumor and negative biopsies by ERC were used for confirming diagnosis and definition of biliary cancer invasion. Endoscopic retrograde biliary drainage (ERBD) tubes were inserted into the future remnant liver in patients with obstructive jaundice.The use of neoadjuvant therapy was depended on clinical practice from 2004 to 2009. Chemotherapy or chemoradiotherapy (CRT) was used before and/or after resection. As for chemotherapy, gemcitabine-based regimen in combination with S-1, cisplatin, and 5-FU were used. Radiation therapy (RT) was used as local therapy with a total dose of 36–45 Gy. From 2010, we had introduced a new protocol of preoperative chemotherapy. After evaluation of tumor extension to the hepatic artery (HA), portal vein (PV), and bile duct by preoperative imaging studies, two cycles of chemotherapy with gemcitabine (600 mg/m2 on days 7 and 21) plus S-1 (60 mg/m2 daily on days 1–21 every 4 weeks), followed by surgery, was administrated in patients with locally advanced perihilar cholangiocarcinoma with (1) main, bilateral, or contralateral PV and/or HA invasion with or without possible vascular reconstruction; or (2) invasion of the right side of the umbilical portion and the left side of the origin of the right posterior PV; or (3) regional lymph node metastasis –20.The type of hepatectomy was determined using the following factors: the indocyanine green retention rate at 15 min (ICGR15), the hepatic uptake ratio of 99mTc-GSA scintigraphy at 15 min (LHL15), and the future remnant liver volume using CT volumetry . Right h2 using a 20 times objective lens by microscopy. The independent two pathologists were blinded to the clinical characteristics of the patients and selected a single field for evaluation, so-called ‘hot-spot’ that would include the maximal budding area for determining the TB count. To find a hot-spot, whole invasive front of tumors were evaluated. The maximal value of tumor budding was defined as TB counts for each tumor. Based on previous studies [All resected specimens were fixed in 10% buffered formalin and paraffin-embedded (FFPE). FFPE blocks were then sliced into 4-μm-thick sections and stained with haematoxylin and eosin (H&E) for microscopic examination. Primary tumor staging (pT) and regional lymph node metastasis (pN) were defined according to Union for International Cancer Control (UICC) 8th edition. TB was defined as an isolated cancer cell or clusters composed of fewer than 5 cancer cells at the site of tumor invasive front according to previous studies . The inv studies , 13, TB p < 0.05, and the confidence interval was determined at 95%. All analyses were performed using IBM® SPSS® Statistics version 25 .Continuous data are expressed as median and range. Continuous and categorical variables were compared using the Mann-Whitney U test and Chi-square test, respectively. Disease-specific survival time (DSS) was calculated from the date of initial treatment to the date of cancer related death or the date of last follow-up if death did not occur. Recurrence free survival time (RFS) was calculated from the date of initial treatment to the date of first recurrence of cancer or the date of last follow-up without recurrence if recurrence did not occur in patients without R2 resection. Patients with R2 resection were excluded from RFS analysis. Survival curves were generated by the Kaplan–Meier method, and differences in survival rates were analyzed using the Log rank test. To identify predictors for disease specific survival, COX hazard regression model with significant variables in univariate analysis was used for multivariate analysis. As prognostic factors, age, gender, preoperative tumor marker , TMN stage, status of tumor lymphovascular (LV) and perinueral invasion, residual tumor status (non-curative resection), tumor budding status (High TB) were analyzed. All tests were two-sided, and the significance level was n = 27, 35%), followed by left hepatectomy with caudate lobectomy . Thirty-eight patients (49%) had tumor with regional lymph node metastasis and 6 patients (8%) had distant metastasis: intrahepatic metastasis (n = 3) and extrahepatic metastasis (n = 3). Fifty-three patients (68%) achieved R0 resection. Postoperative complication with grade III or more (Clavien-Dindo classification) was occurred in 32 patients (41%) and median (range) postoperative hospital days were 45.5 (13–325) days. Median follow-up time was 2.4 years.Patient demographics of 78 patients are shown in Table n = 54) and high TB . Then we compared patient characteristics and outcomes.Fig. p = 0.050). In terms of histologically factors, high TB patients had higher rates of tumor with grade G3 , pT4 , lymph node metastasis , and distant metastasis . There were no significant differences in postoperative factors such as length of postoperative hospital stay, complication and adjuvant therapy.As shown in Table p < 0.001 in DSS, p = 0.001 in RFS, Fig. As for survival, high TB patients had significantly poor as compared to low TB patients on both of DSS and RFS (n = 25) and high TB (n = 11), and classified the 42 patients without neoadjuvant therapy into low TB (n = 29) and high TB (n = 13). Among the patients with neoadjuvant therapy, high TB patients had a significantly higher rate of combined vascular resection  compared to low TB patients. In the patients without neoadjuvant therapy, there were no significant differences in pre- and intra-operative factors. In the patients with neoadjuvant therapy, high TB patients, as compared to low TB patients, had significantly higher rates of G3 , pT4 , lymph node metastasis , and distant metastasis . As for postoperative factors, there were no differences between the two groups. Figure p < 0.001 in DSS, p = 0.001 in RFS). In the patients without neoadjuvant therapy, high TB patients had significantly poor DSS, as compared to Low TB patients, but RFS had no significantly difference between two groups.To confirm the significance of TB in the patients who received neoadjuvant therapy Table , we clas TB n = 1. Among tTo identify predictors for DSS and to confirm the significance of TB, multivariate COX regression analysis was performed. As shown in Table In order to improve prognosis and implementation of therapeutic strategies for patients with perihilar cholangiocarinoma, it is crucial to identify new significant prognostic factors. In the present study, we first elucidated the prognostic significance of high TB (TB counts ≥5) at the tumor invasive front by analyzing our patient database, including approximately half of patients having received neoadjuvant therapy. In all patients, high TB was significantly associated with advanced tumor status including rates of pT4, pN1/2, M1, and histological grade 3. Survival in patients with high TB was significantly inferior than that in patients with low TB. By multivariate analysis, high TB was identified as one of independent poor prognostic factors for DSS among 4 factors including regional lymph node metastasis, LV invasion, and non-curative resection. Interestingly, DSS in high TB group did not show statistical difference compared to that in unresected patients. In addition, the impact of high TB in patients with neoadjuvant therapy showed similar results, withhigh TB significantly associated with advanced tumor status and poor prognosis.Many studies have reported several prognostic factors, such as presence of higher histological grade G3), higher T stage, lymph node metastasis, and positive surgical resection margin, associated with poor survival in resected patients with cholangiocarcinoma , higher –24. In t2 using a 20 times objective lens by microscopy. The pathologist then decided on a “hot-spot” location and calculated the TB counts, that were classified into two groups by using 5 as a cut-off value. As for cut-off value, Okubo et al. classified patients according to ≥5 or < 5, whereas Ogino et al. [Evaluation of TB can easily provide useful feedback on the patient’s clinical situation, which can then be easily disseminated from pathologist to clinical physician, because it can be examined in the H&E-stained specimens as a part of conventional pathologic diagnosis. In the present study, the number of TB was counted in a field measuring 0.785 mmo et al.  classifio et al. . Severalo et al. . Okubo eo et al.  demonstro et al. . TherefoMany previous studies on various malignant tumors have reported a correlation between high TB and advanced tumor. In cholangiocarcinoma, Ogino, et al.  reportedA noteworthy point of the present study, is the demonstration of the prognostic significance of TB in patients with neoadjuvant therapy. There are several reports showing the significance of high TB in patients who underwent neoajuvant therapy , 16, 17 Interestingly, DSS in resected patients with high TB did not show a significant difference compared to that in unresected patients, suggesting that they may not have better prognosis irrespective of whether they can achieve R0 resection. For these patients, we should consider the necessity of additional peri-operative therapy. The TB evaluation method employed in the present study, a pathologist determined “hotspot,” is limited in that it cannot evaluate TB before surgery. Therefore, there is an urgent need to identify preoperative predictors of high TB and establish new therapeutic strategies. This should include improving surgical technique, as well as, developing effective new preoperative and postoperative adjunctive therapy.Our present study included several limitations. This study was retrospective study with a small number of patients. In addition, the indications and types of neoadjuvant therapy in each patient were not uniform. However, it is noteworthy that TB was strongly associated with poor prognosis even in small number cohort. Additional prospective studies are warranted.Our present study demonstrated that high TB at the invasive front of tumors in resected perihilar cholangiocarcinoma patients with or without neoadjuvant therapy, is strongly associated with advanced tumor status and poor prognosis, including DSS/RFS. High TB could be a novel prognostic factor in resected perihilar cholangiocarcinoma even if patients received neoadjuvant therapy.Additional file 1: Figure S. Disease specific survival (DSS) and recurrence free survival (RFS) according to TB counts. Both of DSS (A) and RFS (B) did not show differences between patients with TB 5–9 and those with TB 10 or more.