Non-celiac subjects (n = 8) were recruited as control group. Two biopsies of the cheek lining were taken from each subject for mRNA analysis and immunohistochemical characterization. We observed a significant decrease in the expression of epithelial junction proteins in all CD patients, indicating that oral mucosa barrier integrity is compromised. FoxP3+ population was greatly increased in CD patients, suggesting that Tregs are recruited to the damaged mucosa, even after avoidance of gluten. Amphiregulin mRNA levels from Peripheral Blood Mononuclear Cells (PBMCs) and epithelial damage in the oral mucosa correlated with Treg infiltration in all the experimental groups, suggesting that recruited Tregs might display a “repair” phenotype. Based on these results, we propose that oral mucosa is altered in CD and, as such, might have diagnostic potential. Furthermore, due to its tolerogenic nature, it could be an important target for oral immunotherapy.Celiac disease (CD) is a chronic autoimmune disease characterized by an immune-triggered enteropathy upon gluten intake. The only current treatment available is lifelong Gluten Free Diet (GFD). Several extraintestinal manifestations have been described in CD, some affecting the oral mucosa. Thus, we hypothesized that oral mucosa could potentially be a target for novel biomarkers and an administration route for CD treatment. Six  Celiac disease (CD) is characterized by an immune-mediated chronic enteropathy of the small intestine, triggered by the ingestion of gluten in genetically predisposed individuals. The prevalence of CD is 1–3% worldwide. Although this prevalence varies from country to country, i.e. 1% in Western countries, due to environmental, autoimmune, and genetic factors. CD is regarded as one of the most common genetic disorders, as virtually all the patients are HLA-DQ2+ and/or HLA-DQ8+ .It is characterized by the atrophy of the villi of the small intestinal mucosa, which entails a syndrome of nutrient malabsorption . CD diagThe typical form of the disease is a malabsorption syndrome with chronic diarrhea, abdominal pain, distention, and weight loss . HoweverCurrently, the only available treatment for CD is the adherence to a lifelong GFD. After gluten removal, an improvement in clinical symptoms and intestinal histological findings is observed. However, some patients find this diet cumbersome, as it is more expensive and socially restrictive than ordinary diets , 10. In The oral mucosa lines the inside of the mouth and consists of primarily non-keratinized stratified squamous epithelia, and an underlying connective tissue, the lamina propria, which is highly vascularized . EpithelAlthough the immune processes taking place in the oral mucosa are still poorly understood, CD pathogenesis in the gut is very well characterized. IL-15, a cytokine structurally related to IL-2 that it is widely distributed, has been found to be upregulated in enterocytes in active CD. It drives epithelial damage by stimulating the production of Th1 cytokines, such as IFNγ, and the cytotoxicity of intestinal intraepithelial lymphocytes (IELs) –31. IL-1Previous studies by our group with allergic patients to grass pollen, olive pollen, or house dust mite (HDM) have demonstrated that they undergo oral epithelial remodeling characterized by reduced expression of claudin-1, occludin, and E-cadherin proteins , 41. In In view of these evidence, we formulated the hypothesis that oral mucosa remodeling could also be present in CD patients and would not only help us understand the extraintestinal manifestations associated to CD, but also provide a rationale for oral IT strategies in CD.In this study, we have found that the oral epithelial barrier of CD patients is compromised, even when they adhere to a GFD. Moreover, increased Treg numbers in the oral mucosa are observed in CD patients. Our data suggest that the characterization of the oral mucosal barrier may be a potential tool for advancing novel oral diagnostic markers and disease modifying and tolerization treatments for CD.de novo diagnosed, seven CD patients under GFD treatment for at least 1 year, and eight non-celiac subjects as a control group. De novo CD patients were included after confirmation of duodenal biopsy histological classification Marsh III (villi atrophy), positive Anti-Transglutaminase Antibodies (ATA), and DQ2+ allele of the Human Leukocyte Antigen (HLA). GFD treated group included patients with positive clinical response to diet and ATA loss. Medical history from all subjects was revised by the gastroenterologist in charge of patient enrolment. All subjects with a recent history of nasopharyngeal disease or severe respiratory allergic disease were excluded from the study. During sample collection, all patients were further investigated to ensure that no significant oral lesions were present. Complying with EU regulation 2016/679 from the European Parliament and the Council and Spanish Royal Decree-Law 5/2018, all subjects provided written informed consent and protocol approval was obtained from the Research and Ethics Committees of San Agustin Hospital of Aviles. Clinical features and detailed information of the studied population are shown in Twenty-one subjects were recruited: six CD patients Duodenal sampling was carried out under local anesthesia, obtaining a total of six biopsies from the second portion of the duodenum and the duodenal bulb. Endoscopy was carried out using a Fujifilm gastroscope series 200 or 500 with a biopsy channel of 2.8 mm. Biopsy specimens were fixed in 4% PFA, and later processed to paraffin blocks, sectioned at 4 µm and stained with H&E following manufacturer´s protocols. Duodenal damage was categorized according to Marsh classification .Serum antitransglutaminase 2 antibodies (IgA class) were measured using Triturus automated ELISA analyzer (Grifols). Total IgA was measured by immunoturbidimetry using a CobasTwo biopsies were taken from the buccal mucosa of each study subject using a 3 mm surgery biopsy punch under local anesthesia. One biopsy was embedded in 4% paraformaldehyde (PFA) and processed to paraffin for immunohistochemical (IHC) studies; and the other was conserved in RNALater™ (ThemoFisher) for qPCR studies.Twenty milliliters of heparinized blood were collected from each study subject. Ficoll-Paque  density gradient centrifugation was performed to obtain both plasma and Peripheral Blood Mononuclear Cells (PBMCs). Plasma samples were frozen at −80°C for cytokine quantification by ELISA. PBMCs were further lysed in RLT buffer (Qiagen) and stored at −20°C for later RNA extraction.Paraffin blocks were cut into 1 µm-thick sections and fixed onto a poly-L-lysine treated glass slide and then used for histological and immunohistochemical analyses. All sections were stained with H&E following manufacturer’s protocol to achieve correct orientation of the biopsy. Immunohistochemical staining was performed using Bond Polymer refine Detection kit on a BOND-MAX Automated IHC/ISH Stainer (Leica Biosystems) following the manufacturer’s protocol. The following antibodies were used: mouse monoclonal anti-human CD19 , rat anti-human CD3 , mouse monoclonal anti-human CD4 , mouse monoclonal anti-human CD8 , mouse monoclonal anti-human Claudin-1 , mouse monoclonal anti-human E-Cadherin (36B5) , mouse monoclonal anti-human FoxP3 , mouse monoclonal anti-human γδ-TCR , mouse monoclonal anti-human Langerin , rabbit recombinant monoclonal [EPR20992] to Occludin , and rabbit polyclonal to Neutrophil Elastase . Positive and negative controls were included for each experiment.Sections were stained with a mixture of hematoxylin and Biebrich scarlet for 5 min. Subsequently, they were rinsed with 1% acid alcohol solution (hydrochloric acid), and water. Finally, samples were counterstained with 0.5% lithium carbonate solution and rinsed in running water for 2 min.Scanning of the samples was performed using pathology scanner Aperio Versa 8 (Leica Biosystems) at 40× magnification. A sufficient number of images covering the whole biopsy were captured at an appropriate magnification for quantification.Epithelial cell junctions image analyses were performed using Image-Pro Plus v4.5.0.29 (Media Cybernetics). Briefly, an adequate threshold that best adjusted to the actual DAB staining was established for a selected area. After applying the same threshold to all the images taken from the same sample, the software calculated the stained area relative to the total selected area. The final value was calculated as the weighted average of all measurements obtained for each image in one sample. These analyses were done by at least two independent observers for each staining.Inflammatory infiltrate analyses were performed by using the “counter tool” integrated in Aperio ImageScope v12.3.2.8013 (Leica Biosystems) covering either epithelium, connective tissue, or the whole sample, when appropriate. Both area and stained cells were determined by three independent observers.Oral mucosa tissue was digested in TRIzol™ (ThermoFisher) manually using a scalpel and later homogenized using TissueLyser (Qiagen). After centrifugation, aqueous phase was purified using RNeasy Mini Kit (Qiagen) columns with DNase treatment, according to manufacturer’s protocol, and later retrotranscribed into cDNA with the High Capacity RNA-to-cDNA kit (Applied Biosystems). RNA from lysed PBMCs was also extracted using RNeasy Mini Kit (Qiagen) columns. SYBR Green master mix (Takara) was used for quantitative RT-PCR in the equipment Real Time HT 7900 (Applied Biosystems).Expression data were normalized to the average median of housekeeping genes β-actin and GAPDH and the results were analyzed using the 2 -ΔΔCT method . OligonuELISA kits for IL-33 (CSB-E13000h-96T), IL-25 (CSB-E11715h-96T), and TSLP (CSB-E03316h-96T) (Cusabio) were used to determined cytokine plasma levels following manufacturer’s recommended protocols for every specific kit. To detect PAF in plasma, LabClinics ELISA kit (EH4331) was used. Optical density of the ELISAs was measured at 450 nm in a Varioskan plate reader. A five-parameter logistic fit curve was generated for each cytokine from the seven standards.GraphPad Prism v8.0.1 software was used for statistical analysis. Non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparison test or One-way ANOVA test followed by Tukey’s multiple comparison test were used when appropriate to compare data among experimental groups. For correlation analysis, Spearman correlation was applied. A p-value <0.05 was considered significant for all the analyses. Descriptive statistics along the text are expressed as “mean (s = standard deviation).” Inferential statistics are expressed as “” when a parametric test is used, and “” when the test is non-parametric.de novo group, 87.5% in the GFD group, and 62.5% in the control group. Average age was 44.69 years: 51 (s = 15.62 years) in the de novo group, 48.57 years (s = 13.16 years) in the GFD group, and 34.5 years (s = 10.81 years) in the control group. All included study subjects were Caucasian.Of the study subjects, 76.2% were female: 83.3% in the de novo diagnosed and 42.21 years (s = 12.12 years) for the GFD group. The most frequent clinical manifestations at diagnosis were iron deficiency and diarrhea, found in 38.46% of the CD patients, being 23.08% diagnosed with anemia. Other frequent manifestations were abdominal pain (30.77%), weigh loss (23.08%), and dyspepsia (23.08%). Comorbidities were described in some patients and included other autoimmune diseases such as vitiligo or rheumatic disease. Of the patients in the GFD group, 25% showed signs of depression  for the pression Table 1.According to the inclusion criteria, all newly diagnosed patients involved in the study tested positive for tissue transglutaminase 2 (tTG2) antibodies, genotype HLA-DQ2+ and reach at least III in Marsh scale for duodenal biopsy. All patients had normal IgA levels, except one that presented IgA deficiency, but clear anti-tTG2 antibodies. In the GFD group, average time under diet was 6.42 years (s = 6.76 years) and ranged between 1.17 and 12.42 years Table 1.de novo diagnosed  and GFD patients  compared with non-celiac controls  , a member of desmosomes. We found increased mRNA expression in GFD patients for both occludin  and PPL  when compared to +, CD4+, or CD8+ T cells among the experimental groups in either epithelium or the lamina propria  and de novo subjects    markers, such CD3, CD4, CD8, FoxP3, Langerin, and CD11c  and GFD  CD patients as compared to the non-celiac (control) group  and GFD  groups. FoxP3+/CD3+ ratio followed the same pattern and was also increased in de novo  and GFD  groups (Fox p 3+ cell numbers in the epithelium were negligible in most cases (data not shown). FoxP3We also stained for the APC markers langerin and CD11c to characterize whether there was a predominant APC population associated to CD. No significant differences were found in these two markers among the experimental groups , i.e. the higher the damage in the epithelium (the lower the E-cadherin expression), the higher the Treg infiltrate in the oral mucosa  for all the experimental groups  and GFD  patients, and IFNγ expression in GFD patients  when compared to the control group  of CD patients  and GFD  CD patients presented significantly higher plasma levels of IL-33 as compared with non-celiac subjects (de novo diagnosed CD patients when compared to GFD (p = 0.09) . In the study by Holtan S et al., high circulating amphiregulin levels reclassified patients into high risk subgroups helping further refine the aGVHD clinical risk score  seems to be absent in the oral mucosa. Furthermore, IL-33, TSLP, or POSTN mRNA levels are not elevated in buccal biopsies. Thus, we hypothesize that the oral mucosa remodeling observed may be a consequence of the systemic inflammation associated to CD. In fact, systemic immune deregulation is reflected in the function of the oral immune system . We, andBased on our results, we conclude that oral mucosal integrity is compromised in CD patients, even after gluten avoidance. A remodeled epithelium may be key for the IT to gain access to both epithelial surfaces  and the local mucosa associated immune system, as previously suggested , 68, 69.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Research and Ethics Committees of San Agustin Hospital of Aviles. The patients/participants provided their written informed consent to participate in this study.LS, CDF, and JSS recruited the study subjects and conducted the biopsy and blood sampling. JSS, CPT, and CGC performed and analyzed the laboratory experiments. JSS, PS, DB, and CGC discussed the results and wrote the manuscript. All the authors contributed to the article and approved the submitted version.This study was financed by Allero Therapeutics BV, Rotterdam. JSS was supported by an FPI‐CEU predoctoral fellowship. CGC was supported by a postdoctoral contract co-funded by the competitive Program “Attracting Talent,” Community of Madrid, Spain.PS is a shareholder of Allero Therapeutics.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Introduction: Bacterial meningitis (BM) is a global public health concern that results in significant morbidity and mortality. Cerebral arterial narrowing contributes to stroke in BM and may be amenable to intervention. However, it is difficult to diagnose in resource-limited settings where the disease is common.Methods: This was a prospective observational study from September 2015 to December 2019 in sub-Saharan Africa. Children 1 month−18 years of age with neutrophilic pleocytosis or a bacterial pathogen identified in the cerebrospinal fluid were enrolled. Transcranial Doppler ultrasound (TCD) of the middle cerebral arteries was performed daily with the aim to identify flow abnormalities consistent with vascular narrowing.Results: Forty-seven patients were analyzed. The majority had Streptococcus pneumoniae (36%) or Neisseria meningitides (36%) meningitis. Admission TCD was normal in 10 (21%). High flow with a normal pulsatility index (PI) was seen in 20 (43%) and high flow with a low PI was identified in 7 (15%). Ten (21%) had low flow. All children with a normal TCD had a good outcome. Patients with a high-risk TCD flow pattern (high flow/low PI or low flow) were more likely to have a poor outcome .Conclusions: Abnormal TCD flow patterns were common in children with BM and identified those at high risk of poor neurological outcome. Bacterial meningitis (BM) results in significant morbidity and mortality worldwide . Over 1 Ischemic stroke is one such event occurring in 20–25% of patients with BM –13. The p < 0.01)  are undertaken. The goal of CPP augmentation in this situation would be to overcome the increased resistance of the cerebral arteries and provide sufficient substrate delivery to meet metabolic demand, thus reducing secondary ischemic injury , 15. Whe < 0.01) . HoweverAdvanced neuroimaging approaches commonly used to diagnose pathophysiological changes to the neurovasculature in developed countries are not widely available in the regions of the world that are most heavily impacted by BM. Transcranial Doppler Ultrasound (TCD) is a portable, non-invasive, inexpensive tool that evaluates cerebral blood flow velocities (CBFVs) –19. It hThere are no reports on TCD findings or associations with outcomes in children with BM in sub-Saharan Africa. Given the high burden of disease, the differing causative pathogens, and the comorbidities in this population, we performed this prospective, observational study. CBFVs become elevated and then profoundly reduced as the degree of arterial narrowing progresses in other disease states , 28. We 3 with >60% neutrophils (culture-negative meningitis).The study was performed from September 2015 to December 2019 at Kalembe Lembe Children's Hospital in the Democratic Republic of the Congo (DRC), L'Hopital General de Reference de Lodja in the DRC, L'Hopital General de Reference de Nyankunde in the DRC, and Queen Elizabeth Central Hospital (QECH) in Blantyre, Malawi. Ethics approval was granted by involved institutions and parents or guardians signed written consent before enrollment. Children 1 month−18 years of age were approached for enrollment when diagnosed with BM according to the following criteria: (1) Gram stain or culture of the cerebrospinal fluid (CSF) positive for a bacterial organism OR (2) CSF with ≥100 leukocytes/mmChildren with sickle cell anemia or cerebral malaria were excluded given the high frequency of abnormal TCD findings in these children. Given the unknown impact of severe malnutrition or advanced HIV disease on CBFVs, children diagnosed with one of these conditions (mid-upper arm circumference <11.5 cm or known HIV positivity with severe wasting) were excluded.Demographic data, vital signs, and Blantyre Coma Score (BCS) at presentation were collected. Finger-prick samples were analyzed to determine blood glucose and lactate concentration . Venous blood was drawn to obtain a complete blood count . CSF was analyzed using the standard methods for cell count, protein, Gram stain, and culture.TCD was performed using a commercially available TCD unit . Daily TCD was performed through hospital day 8, discharge, or death, whichever occurred first. In patients with abnormal TCD findings at day 8, weekly TCD was performed until discharge. Middle cerebral arteries (MCAs) and extracranial internal carotid arteries (EC-ICAs) were insonated at 2-mm intervals using previously described methods . SystoliPulsatility index (PI = (Vs – Vd/Vm)), a marker of downstream cerebrovascular resistance (CVR), was automatically calculated by the TCD unit at each depth. As distal CVR decreases, diastolic flow rises and the PI decreases. As distal CVR increases, diastolic flow falls and the PI increases.To differentiate causes of high measured CBFVs, the Lindegaard ratio (LR = MCA Vm/Ex-ICA Vm) was calculated . A LR <3Autoregulation is the capacity to maintain constant cerebral blood flow over a wide range of blood pressures. The transient hyperemic response ratio (THRR = average Vm of 2 beats after a 10-s carotid compression/average Vm of 5 beats before compression) was used to interrogate cerebral autoregulation . A THRR The definitions used to categorize study patients were:Normal flow—Vs, Vd, and Vm within 2SD of age normative value .(a) Normal PI—PI ≥0.6 and ≤ 1.3.(b) Abnormal PI—PI ≤ 0.5 (low PI) or >1.3 (high PI).High flow—Vs, Vm >2SD above age normative value.(a) Normal PI—PI ≥0.6 and ≤ 1.3.(b) Abnormal PI—PI ≤ 0.5 (low PI) or >1.3 (high PI).Low flow—Vs and Vm <2SD below age normative value.An explanation of the basis for the selected diagnostic criteria is in Pediatric cerebral performance category (PCPC) scoring was performed at hospital discharge , 31. ChiThe study was approved by the ethics committee at the University of Kinshasa, School of Public Health, DRC. The study was also approved by the ethics committee at the University of Malawi College of Medicine Research Ethics Committee (COMREC). All subjects' guardians provided informed consent before enrollment.2 or Fisher's exact test, one-way ANOVA with a Tukey correction for multiple comparisons, or Kruskal–Wallis tests with Dwass–Critchlow–Flinger–Steel corrections for multiple comparisons. Comparisons between patterns were evaluated using χ2 or Fisher's exact tests, two-sample t-tests, or Wilcoxon rank sum tests. As a sensitivity analysis, for variables with some longitudinal collection, mixed-effects models with random subject effects were run to evaluate comparisons using all available data, while accounting for within-patient correlation and differences in number of measurements. Given the sample size, multivariate analysis was not performed. All analyses were conducted using SAS 9.4 .Primary results are restricted to the first observation per patient. Categorical variables are summarized using frequencies and percentages, normally distributed continuous variables are summarized using means with SDs, and non-normally distributed continuous variables are summarized using medians with interquartile range (IQR). Comparisons by organism and pattern were evaluated using χStreptococcus pneumoniae  or Neisseria meningitides  meningitis. Fewer were diagnosed with Salmonella sp.  or culture-negative meningitis . One child was infected with Staphylococcus aureus (2%) and one with Escherichia coli (2%). Children with meningococcal meningitis were more likely to have convulsions than children infected with other organisms (n = 14/17 (82%) for N. meningitides vs. n = 10/17 (59%) for S. pneumoniae vs. n = 1/6 (17%) for Salmonella sp., vs. n = 3/5 (60%) for culture-negative meningitis, p = 0.03). CSF cell count was significantly different depending on infecting organism . Opening pressure was highest in children with meningococcal meningitis . No other variables were different when evaluated by infecting organism. Children with Salmonella sp. meningitis had better PCPC at discharge than children infected with other organisms .Forty-seven patients were included. Demographics, vital signs, and laboratory variables for participants are in p = 0.2).Admission TCD was normal in 10/47 (21%) children. High flow with a normal PI was noted in 20/47 (43%) and high flow with a low PI was noted in 7/47 (15%). Ten of 47 (21%) children met criteria for low flow. Representative images of each flow pattern are in n = 4), the median duration to normalization was 4 days .When excluding children who died before the resolution of abnormal CBFVs, time to normalization varied between groups. All children with an initial normal TCD remained normal on subsequent examinations. Children with high flow/normal PI normalized after a median of 4 days . Children with high flow/low PI normalized at a median of 10 days . One patient with this flow pattern had persistently abnormal flows at discharge on day 40. Evaluation of the duration of abnormality in the low flow group was limited by the high mortality rate, but in survivors (p = 0.001) ]. PCPC was worse for patients with an abnormal CBFV pattern when compared with those with normal CBFVs . If cate= 0.001) . Poor ou= 0.001) .−9/L vs. 17 ·10−9/L], had lower lactate [1.8  mmol/L vs. 4.3  mmol/L], and lower CSF WBC counts [75  vs. 239  WBC/mm3]. When evaluating the LR, PI, and THRR in children with this flow pattern, LR was not significantly different between children with good (2.24 ± 0.75) vs. poor outcome (2.3 ± 0.66) (p = 0.88). PI was lower in children with a good outcome (0.73 ± 0.19) than in those with a poor outcome . Autoregulation was significantly better in children with a good outcome (THRR1.12 ± 0.06) compared with those with a poor outcome (0.98 ± 0.05) (p = 0.03).There was notable heterogeneity in the outcomes of children with high flow/normal PI. There were no differences in the demographics, vital signs, or laboratory parameters of children with a good vs. poor outcome with this TCD pattern. While not significant, children with good outcome were less febrile (mean temperature 37 ± 1.5°C vs. 38.9 ± 0.7°C), had lower peripheral WBC counts [7 ·10BM is an important cause of long-term neurodisability and mortality in developing countries –8. BeyonTCD is a portable, non-invasive tool that evaluates the cerebrovascular hemodynamics , 39–42. p = 0.04) and death (p = 0.03). Our findings are similar to these previously reported works, but in a much larger, distinct patient population.Three previous studies report the use of TCD to evaluate the cerebral vasculature in children with BM outside the neonatal period –24. One For the first time, we also identified clear differences in neurological outcomes associated with different CBFV patterns in BM. Children in our study with normal CBFVs likely represented individuals with no or minimal neurovascular involvement that fully recovered with antibiotics and supportive care. CBFVs are elevated when large vessel narrowing occurs. Children with high CBFVs and a normal PI were found to have significant variability in outcome. While not statistically significant, there were differences in vital signs and laboratory parameters suggesting the children with good outcomes were less severely ill. There was no difference in the LR between those with a good vs. poor outcome, suggesting the degree of vessel obstruction was not different. Rather, TCD measurements revealed that the PI was lower and autoregulation was more intact in children with good outcome. This suggests that perhaps some children in this group had better outcomes because they were able to sufficiently augment flow to meet demand via peripheral vasodilation.p = 0.03 vs. normal flow), suggestive of significant vessel diameter reduction. In this instance, distal vessels maximally vasodilate to reduce resistance to flow. This results in increased flow during diastole and a low measured PI. However, as the obstruction is relatively fixed in BM, flow may not sufficiently increase to meet demand despite peripheral vasodilation, and secondary ischemic injury may occur. This likely explains the significant morbidity in these children. Children with high CBFVs and low PI may be those who would most benefit from adjunctive interventions to augment CPP. Future studies could be designed to answer this question.An extremely low PI in the setting of elevated CBFVs represents clear evidence of impaired distal perfusion beyond a point of flow obstruction. The LR was most significantly elevated in these children (3.8 ± 1.5, We identified a significant proportion of children with low CBFVs. Investigative studies of progressive intracranial vascular occlusion in other diseases outline a “tipping point” of vessel lumen diameter reduction where flow becomes so impaired that measured CBFVs transition from elevated to reduced , 28. AniOverall, while done in a resource-limited setting in Africa, the findings of this study should be applicable to the care of children in any setting. TCD is portable, widely available in centers that care for pediatric patients, and can be performed repeatedly to evaluate the cerebral hemodynamics of children with BM. If a high-risk flow pattern is identified, practitioners could consider further neuroimaging, further investigation with multi-modal neuromonitoring, and potentially CPP augmentation.The relatively small sample size limited the number of children identified with each CBFV pattern. However, the work does represent the largest cohort of children with BM evaluated by TCD to date. Even with small sample sizes, there were significant differences in the PCPC scores between groups. Thus, it is likely that these associations would continue to hold true with a larger sample size. Another limitation is that normative values for TCD measurements do not exist for African children. As such, the results of the TCD studies were compared with previously published pediatric normative values, which were largely Caucasian children. Future studies are needed to ensure appropriate normative values are developed for this patient population given the possible alterations in expected flow velocities based on race and common comorbidities. Lastly, neuroimaging was not available, and thus correlations between clinical findings concerning for neurologic impairment, TCD measurements, and abnormal findings on CT or MRI were not possible.TCD can be used to non-invasively evaluate the neurovasculature of children with BM and identify those at high risk of poor outcome.The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Kinshasa School of Public Health Ethics Committee and the Malawi College of Medicine Ethics Committee. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.All authors participating in study design, interpretation of the data, and manuscript preparation. YF, TT, JP, MU, and NO'B participated in TCD examination performance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


High body mass index- (BMI-) related vascular injury contributes to the pathogenesis of the atherosclerotic cardiovascular disease (ASCVD). Rigorous calorie restriction is one of the major lifestyle interventions to reduce vascular risk in overweight or obese individuals. However, the effects of fasting therapy (FT) on vascular function and the mechanism are still unclear. This study was aimed to investigate the impacts of FT on endothelial function, arterial stiffness, and circulating arterial damage parameters in overweight and obese individuals and possible mechanism.  Overweight and obese individuals participated in FT intervention . Arterial function including brachial arterial flow-mediated dilation (FMD), brachial-ankle pulse wave velocity (baPWV), vascular injury-related markers including trimethylamine N-oxide (TMAO), and leptin and endothelial microparticles (EMPs) were assessed. Endothelial progenitor cells (EPCs) of these participants were isolated and cultured to investigate EPCs function. mRFP-GFP-LC3 confocal microscopy scanning and western blot were carried out to determine autophagy. P < 0.01; 29.93 ± 2.82 vs. 28.47 ± 2.83 kg/m2, P < 0.01). FT remarkably improved FMD  while baPWV kept unchanged. TMAO and leptin levels decreased . EMPs showed a decreased tendency. EPCs function was significantly improved, autophagy fluorescence intensity was enhanced, and the level of Beclin1, Atg5, LC3 II/I also increased after starvation in vitro, and the effects were blocked by autophagy inhibitor.  After FT, body weight and BMI significantly decreased (81.76 ± 12.04 vs. 77.51 ± 12.06 kg, ChiCTR1900024290. Our present study demonstrated for the first time that FT markedly improves endothelial function and reduces the levels of arterial injury markers through improving EPCs function via activating autophagy. These findings provide a novel insight into FT as a lifestyle intervention strategy to promote the maintenance of vascular homeostasis in overweight or obese individuals. The trial was registered with  The prevalence of overweight and obesity is rising rapidly worldwide. High body mass index (BMI) is among the leading causes of elevated morbidity and mortality for atherosclerotic cardiovascular disease (ASCVD). Globally, it is estimated that 4 million deaths are caused by high BMI, more than two-thirds of which are related to ASCVD . ImpaireAccumulating evidence indicates that the abnormal vascular function in overweight and obesity are mainly manifested as impaired endothelial function –6 and enRigorous calorie restriction is an important lifestyle to reduce vascular risk and may help prevent the incidence of ASCVD in high BMI persons , 17. FasEndothelial progenitor cells (EPCs), initially defined as circulating bone marrow-derived CD34-positive/KDR (kinase domain receptor)-positive cells , protectAutophagy is a highly conserved physiological process controlling endothelial homeostasis in vascular beds . StudiesBased on the data mentioned above, we hypothesized that the FT would improve arterial function in overweight and obese individuals and reduce circulating vascular injury-related biomarkers via regulating autophagy to repair EPCs function. To address these assumptions, overweight and obese individuals who were enrolled in this pilot study received FT. FMD and baPWV as well as circulating arterial damage parameters such as TMAO, EMPs, and leptin, were evaluated. EPCs of these participants were isolated and cultured to investigate EPCs function and autophagy level after starvation. The present study may provide novel insight into the beneficial effects of FT as a lifestyle intervention strategy to maintain vascular homeostasis and retard the pathogenesis of high BMI-induced ASCVD.2) aged between 25 and 65 were recruited to participate in this study from The First Affiliated Hospital, Sun Yat-Sen University. Exclusion criteria were persons with abnormal heart, liver, or kidney function, secondary hypertension, essential hypertension above the second grade, insulin use, severe gastric bleeding, cancers, blood diseases, active tuberculosis, and women during pregnancy, lactation or menstruation. All subjects were given written informed consent prior to study participation. Ethics approval was granted by the ethical committee of The First Affiliated Hospital, Sun Yat-Sen University in Guangzhou ([2019] 085). The trial was registered in Chinese Clinical Trial Registry of WHO (ChiCTR1900024290).Thirteen overweight or obese individuals , 5 days of intense calorie restriction , and 1 day with the stepwise reintroduction of a normal diet . Each subject was given 10–20 g of thenardite powder on the first day of the fasting period for bowel-cleansing. During the fasting period, patients were asked to drink at least 2 L of mineral water, have 200 kcal/d diet each day consisting of one serving of the liquid meal replacement and two cups of light vegetable soup, and receive L-carnitine  2 g, bid (9 a.m. and 4 p.m), i.v. infusion . Each serving of the liquid diet provided approximately 150 kcal: 7.9 g protein, 2.8 g fat, and 22 g carbohydrate. The liquid diet was prepared by mixing the powdered product with 250 ml water and was to be consumed at dinner. Two bowls of vegetable soup provided about 50 kcal and were given at breakfast and lunch. All patients were required to engage in low-level physical activity consisting of 2 h of slow walking per day. Furthermore, participants were recommended to avoid alcohol, coffee, or tea during the study. All participants were compulsorily hospitalized during FT intervention , 20.Each subject underwent vascular function examination by trained sonographers, performed between 8 and 11 a.m.; they were recommended to rest for 10 min or longer and were examined in a quiet, temperature-controlled room. FMD was examined through noninvasive ultrasound scan ; it could dynamically record artery diameter, at rest (baseline) and reactive hyperemia period produced by using the cuff inflated to the greater of 50 mmHg above systolic pressure or 200 mmHg for 5 min as previously described . FMD wasArterial stiffness was assessed with baPWV in supine individuals after rest. baPWV was measured by using an automatic device  as reported previously  and was μL of internal standard solution  was added to 100ul plasma; then 300 μl of acetonitrile for protein precipitation was added, vortexed for 1 min, centrifugated at 1000 rpm for 5 minutes, 4°C; at last, we took 200 μL of the supernatant in the vial for HPLC-MS/MS analysis , 1% BSA, 140 mM NaCl, 1 mM CaClor 1.5 h .EPCs were transfected with mRFP-GFP-LC3 adenovirus obtained from Hanbio . Confocal fluorescence microscopy  was used to evaluate autophagy flux by counting the cells with GFP-LC3 (green) puncta, RFP-LC3 (red) puncta, and GFP+/mRFP+ -LC3 (yellow) puncta.Total EPC protein was extracted and quantified by RIPA Lysis Buffer  containing protease inhibitors (Roche) and BCA assay kit  separately. Protein extracts were separated by SDS-PAGE, transferred to PVDF membranes . The following antibodies were used: anti-Beclin1 antibody , anti-Atg5 antibody , rabbit anti-LC3 antibody , and anti-ACTB antibody . Proteins were visualized with HRP-conjugated anti-rabbit IgG or anti-mouse IgG , followed by the use of the ECL chemiluminescence system (Thermo). And the level of protein was analyzed by using Image J.t-test and Wilcoxon test for nonnormal distribution data. Statistical significance of multiple groups was assessed by one-way ANOVA for normal distribution data and followed by Bonferroni test. They were two-tailed. P < 0.05 represented statistically significant. All statistical analyses were performed in Statistical Package for Social Sciences version 23.0 software .All results were expressed as mean ± standard deviation (SD). The normal distribution of continuous variables was determined via the Shapiro–Wilk test. Two groups of normal distribution data were performed using the Students P < 0.01), BMI  after FT; and bodyweight decreased approximately 4.0 kg in average after 7 days, besides fasting plasma glucose  and systolic blood pressure . As for lipid metabolism, triglycerides  decreased, whereas total cholesterol , low density lipoprotein cholesterol  increased, but TC and LDL-C would return to baseline or even lower after getting back to normal diets (unpublished data); it may be due to the steatosis and energy supply during fasting time. The exact mechanism deserves further investigation. All the participants went through FT without hypoglycemia or other severe adverse events. No one showed liver or renal impairment.A total of 13 subjects received FT intervention in this pilot study see . ResultsP=0.01; see Figures P=0.22; see Figures FMD and baPWV are the most reproducible measures to indicate endothelial function and arterial stiffness which are widely used in clinical research. It showed FT ameliorated endothelial function after 7-day intervention. Overall, FMD significantly increased , adhesion (P < 0.01), and tube formation (P < 0.01) of EPCs were all improved compared with the nonstarvation group (see EPCs were incubated in starvation medium for 1.5 h. The migration (roup see . These dP < 0.05), paralleled with the increase of protein levels of Beclin1 (P < 0.01), Atg5 (P < 0.01), and LC3 (P < 0.01). In addition, 3-MA, the autophagy inhibitor, blocks the effects of starvation in improving EPCs function  achieved with diet and lifestyle interventions seems to improve PWV , our stuIn addition to improvements in endothelium-dependent vasodilation, FT also led to a decline in systemic levels of vascular injury-related biomarkers including TMAO and leptin. TMAO has been established as a predictor of cardiovascular events which is involved in the progress of atherogenesis, endothelial dysfunction, and metabolic disorders and significantly elevated in high BMI individuals . And to Large amounts of studies demonstrate that EPCs are pivotal to the maintenance of vascular homeostasis  and defiAutophagy is a highly conserved and complex process to preserve cellular homeostasis involved in many functional proteins. Beclin-1 is a key factor in the formation of autophagy, and Atg5 and LC3 participate in the extension of the autophagic membrane to identify and wrap damaged proteins and organelles, eventually form mature autophagosomes . StudiesThe study still has several limitations. First, this work represents a small, interventional pilot study designed to determine whether FT has favorable effects on arterial function and the levels of vascular injury-related biomarkers in overweight and obese individuals. Surely, a larger, placebo-controlled randomized trial with a long time follow-up is necessary to validate our current findings. Second, whether FT would reduce the morbidity and mortality of ASCVD remains to be investigated in clinical practice. At last, the deeper mechanism of autophagy behind the favorable effects of FT on vascular homeostasis remains unclear and needs further exploration.Our present work demonstrated for the first time that FT improves endothelium-dependent vasodilation and reduces vascular injury-related biomarkers in overweight and obese individuals through improving EPCs function via activating autophagy of EPCs. Overall, these findings showed that FT may be an effective therapeutic method to protect vascular health in overweight and obese individuals and retard the incidence of ASCVD in clinical practice.


Few studies have focused on the reasons for the low cure rate of multidrug-resistant tuberculosis in China and within-host evolution during treatment, which is of great significance for improving clinical treatment regimens. Acquired resistance events were common during the ineffective treatment, among which resistance to amikacin and high-level moxifloxacin were the most common. The cure rate of multidrug-resistant tuberculosis (MDR-TB) is relatively low in China. The reasons for the treatment failure and within-host evolution during treatment have not been sufficiently studied. All MDR-TB patients receiving standard treatment from January 2014 to September 2016 at a designated TB Hospital in Zhejiang Province were retrospectively included and grouped according to their known treatment outcome. Clinical information was collected. Baseline strains of all patients and serial strains of treatment-failure patients were revived. Drug susceptibility tests (DSTs) of 14 drugs and single nucleotide polymorphism (SNP) analysis based on whole-genome sequencing (WGS) were performed. The genetic distance and within-host evolution were investigated based on SNPs. In total, 20 treatment failure patients and 74 patients who succeeded in treatment were included. The number of effective drugs for patients who failed treatment was no more than three. Eighteen (90.0%) treatment-failure patients were characterized by a continuous infection of the primary strain, of which 14 patients (77.8%) developed phenotypic or genotypic acquired drug resistance under ineffective treatment. Acquired resistance to amikacin and moxifloxacin (2.0 mg/ml) was detected most frequently, in 5 and 4 patients, respectively. The insufficient number of effective drugs in the combined treatment regimen was the main reason for MDR-TB treatment failure. The study emphasizes the importance of DST for second-line drugs when implementing the second-line drug regimen in MDR-TB patients. For patients with risk factors for MDR-TB, DST of second-line antituberculosis drugs should be performed at initiation of treatment. Second-line drugs should be selected based on the results of DST to avoid acquired resistance. WGS detects low-frequency resistance mutations and heterogeneous resistance with high sensitivity, which is of great significance for guiding clinical treatment and preventing acquired resistance.IMPORTANCE Few studies have focused on the reasons for the low cure rate of multidrug-resistant tuberculosis in China and within-host evolution during treatment, which is of great significance for improving clinical treatment regimens. Acquired resistance events were common during the ineffective treatment, among which resistance to amikacin and high-level moxifloxacin were the most common. The main reason for the treatment failure of MDR-TB patients was insufficient effective drugs, which may lead to higher levels of drug resistance in MDR-TB strains. Therefore, the study emphasizes the importance of DST in the development of second-line treatment regimen when there is a risk of MDR. By performing whole-genome sequencing of serial strains from patients with treatment failure, we found that WGS can detect low-frequency resistance mutations and heterogeneous resistance with high sensitivity. It is thus recommended to conduct drug susceptibility tests at the beginning of treatment and repeat the DST when the sputum bacteria remain positive. Drug-resistant tuberculosis (DR-TB) constitutes one of the main threats to the End-TB by 2035 strategy of the World Health Organization (WHO) –3. China–4–All MDR-TB patients who failed standardized treatment in accordance with the 2011 version of the WHO guidelines  were ret16–We elucidated the current reality and the factors influencing treatment failure of MDR-TB in China and used WGS to further investigate the molecular evolution of strains during treatment failure, which has been less explored . These fIn total, 123 MDR-TB patients were diagnosed between January 2014 and September 2016. Among these, 94 patients had treatment outcomes by December 2018, of which 20 were treatment failure and 74 cured. We successfully revived 94 baseline strains from the 94 patients and 40 serial strains from the 20 treatment-failure patients .The characteristics of patients, such as history of TB treatment with or without second-line anti-TB drugs and previous treatment duration of over 1 year, were associated with poor treatment outcomes .The higher resistance level of baseline strains  was also associated with poor treatment outcomes .P < 0.001; relative risk [RR], 0.04, 95% confidence interval [CI] 0.01 to 0.18). Further analysis revealed no significant differences in the drug resistance profile or effective use of specific drugs in 27 patients treated with three effective drugs; however, any history of TB treatment with or without second-line anti-TB drugs was associated with treatment failure.Importantly, insufficient administration of effective drugs was associated with poor clinical outcomes. In contrast, treatment regimens using specific effective drugs, such as PZA, levofloxacin (LFX), moxifloxacin (MFX), and prothionamide (PTO), were associated with successful outcomes . We calcAs reinfection by another higher-resistance MDR strain was considered a plausible reason for treatment failure, we used WGS to distinguish between primary infection and reinfection of 20 patients with poor treatment outcome. The number of different SNPs between the serial strains was in the range of 0 to 1,143. Eighteen (90.0%) cases were identified as the same strains, of which the number of SNP differences varied from 0 to 8 in this study. Only two cases (10%) were identified as reinfection by another, more resistant strain (no. 6 and no. 16), which had 1,134 and 313 SNPs in genetic distance, respectively  were observed to develop phenotypic or genotypic acquired drug resistance (ADR) . The ADRmprA  and amount genes . These results provided a new insight into the acquired resistance of TB strains to drugs.As no resistance-related mutation was discovered in the PTO-resistant isolate from patient no. 4 and the CS phenotypic resistance of patient no. 14, we further analyzed all differential SNPs identified between the serial isolates of these two patients. After excluding synonymous mutations, three mutations were identified to distinguish the resistant from the susceptible isolates of patient no. 14. These were Rv0565c Y306C (probable monooxygenase), Rne N649D (RNase E), and Rv2611c I102M (probable acyltransferase). Only two mutations were detected in the intergenic region of the isolate of patient no. 4, located in the promoter of pncA mutation related to PZA (patients no. 2 and no. 13), the gyrA and gyrB mutations related to FQ , the rrs mutation related to SLID , the embB mutation related to EMB (patients no. 8 and no. 20), the alr mutation related to CS (patients no. 8 and no. 14), and the ethA mutation related to PTO   . Moreove no. 17) , Table 3ethA W116C of patient no. 8, rrs a1401g of patient no. 11, and gyrB A504V of patient no. 19 increased from 0% at the initiation of treatment to nearly 100% in the last cultured isolates.In patients no. 8, no. 11, and no. 19, the emergence, selection, and fixation process of a drug resistance-related mutation were entirely recorded by WGS. In detail, the frequency of gyrA D94N mutation, usually characterized by high level of resistance to FQ, gradually replaced the existing A90V mutation, resulting in low level of resistance to FQ. This phenomenon was also observed in isolates from patient no. 10, in which the gyrA D94G mutation was demonstrated to have replaced the previous A90V mutation during treatment containing MFX  and third (23 months later) specimens, only the ethA S57P or ethA Y461* mutations were present  were noted to develop acquired drug resistance, which was higher than that reported previously. A previous study only performed DST of FQ and SLID at the initiation and checkpoint of treatment to determine the resistance pattern, which might have underestimated the prevalence of acquired resistance to other drugs , 28. ThegyrA A90V resistance mutation (low level) was replaced by mutations with higher levels of resistance, gyrA D94N and D94G , clofazimine (CFZ), or ethambutol (EMB) for the period of 6 to 8 months during the intense phase, continued with administration of a combination of 3 to 4 drugs without a SLID for another 18 months.We retrospectively included all MDR-TB patients from January 2014 to September 2016 in the Wenzhou Central Hospital  with a known outcome according to the standard outcome definitions by WHO . DST of Clinical information, including age, gender, comorbid conditions, treatment history, and treatment regimen was extracted from the medical records of patients to analyze the risk factors related to treatment outcomes. Baseline strains, the first sputum culture positive strain, of all enrolled patients were collected and revived from refrigeration. For all treatment failure patients, sputum specimens were collected every month for the first 6 months, and then every 1 to 2 months according to WHO guidelines. In order to observe evolution events and heterogeneity, we selected at least two more serial strains at intervals of more than 6 months. Strains that failed to revive or were contaminated were excluded. All colonies on the plate were scraped off and resuspended in 30% glycerol before freezing.The study was approved by the Ethics Committee of Wenzhou Central Hospital, Zhejiang Province, China. All patients provided written informed consent before the study.−1), RIF (40.0 μg ml−1), EMB (2.0 μg ml−1), LFX (2.0 μg ml−1), MFX (2.0 μg ml−1), AM (30.0 μg ml−1), KM (30.0 μg ml−1), CM (40.0 μg ml−1), streptomycin (SM) (4.0 μg ml−1), PAS (1.0 μg ml−1), CFZ (1.0 μg ml−1), and CS (30.0 μg ml−1) using the proportion method on Löwenstein–Jensen medium . Exceptions were for PZA (100.0 μg ml−1) and PTO (2.5 μg ml−1), the DSTs of which were performed using an automated MGIT 960  according to the manufacturer's guidelines. We conducted the DSTs of EMB, PZA, and PTO at least twice due to their unsatisfactory reproducibility. Where the results were inconsistent, a third test was performed and the interpretation was based on the two most consistent results out of the total three.Drug susceptibility tests (DSTs) were conducted on all included strains to compare the resistance profiles between the two groups. Following the revival of strains on Löwenstein–Jensen medium, we performed DSTs for 14 drugs –50 on th–Whole-genome sequencing (WGS) was performed on all selected strains to provide genotypic resistance, distinguish between a continuous infection with the primary strain or reinfection, and elucidate the microevolution of serial strains. Growth was scraped from Löwenstein–Jensen medium. Genomic DNA was extracted using the Qiagen DNeasy blood and tissue kit (Cat. no. 69506) and sequencing libraries were constructed using the Nextera XT sample prep kit  following the manufacturer's protocol. All strains were sequenced on an Illumina Miseq or X 10 with at least 100-fold coverage.NC_000962.3) reference sequence using Bowtie2 (version 2.3.3.1) with default parameters. Single nucleotide polymorphisms (SNPs) were detected using SAMtools (version 1.6) with a minimum sequencing depth of 10 reads without strand bias and a frequency of no less than 10%. All SNPs with a mutation frequency exceeding 10% were retained in the step of calling SNPs to detect microevolution and minor subpopulations. If the mutation ratio of a certain site was above 80%, it was considered a fixed mutation.WGS data were analyzed  accordinSNPs in highly repetitive regions such as PE and PPE, GC-enriched sequences, and drug resistance-related genes were removed from the analysis. The phylogenetic tree was constructed using the maximum likelihood method using MEGA (version 7.0) software based on the fixed SNPs, with H37Rv as the root. The strains with a genetic distance of no more than 12 SNPs were considered persistent infection with the same strain.The study was approved by the Ethics Committee of Wenzhou Central Hospital, Zhejiang Province, China. All patients provided written informed consent before the study.P < 0.05 were considered significant. We performed all analyses using the SPSS version 21 software.We used the chi-square test or Fisher's exact test to calculate significant differences between characteristics of patients, drug resistance, and treatment regimen and outcome. We performed univariable logistic regression analysis and calculated the relative risk (RR) followed by its 95% confidence intervals. Variables with a PRJNA613359. The SRA accession numbers for these isolates are SAMN14402132 to SAMN14402172. The WGS data for a portion of these strains were previously made available to the public under BioProject number PRJNA522942  values given in boldface indicate statistical significance. Download Table S1, DOCX file, 0.01 MB.Treatment outcomes of MDR-TB patients according to patient characteristics; Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00884-20.2TABLE S2Table S2, DOCX file, 0.02 MB.Diagnostic criteria of genetic sequencing for genotypic resistance. Download Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00884-20.3FIG S1FIG S1, DOCX file, 0.1 MB.Phylogenetic tree of 60 serial strains in 20 patients with treatment failure; branch in red means reinfection by different strains during treatment. Download Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


To investigate long-term outcomes of prediction error after phacotrabeculectomy and to determine risk factors that may cause unstable prediction error after phacotrabeculectomy in glaucoma patients.A total 120 eyes of 120 patients who had underwent uncomplicated phacotrabeculectomy (combined group) or phacoemulsification (phaco-only group) were included. Best-corrected visual acuity (BCVA), intraocular pressure (IOP) were measured before and after surgery, and anterior segment parameters including anterior chamber depth (ACD), lens vault (LV), and anterior vault (AV) measured using anterior-segment optical coherence tomography were compared between the two groups. The mean absolute error (MAE) at 3, 6, 12, and 24 months postoperatively were compared. Risk factors associated with unstable prediction error (MAE ≥ 0.5) were investigated in the combined group.In both groups, BCVA was improved and IOP was decreased significantly. MAE at 3, 6, 12, 24 months postoperatively were not significantly different between two groups. The risk factors for unstable prediction error after 12 months of phacotrabeculectomy were old age and LV. Whereas, the only factor predicting unstable prediction error after 24 months of phacotrabeculectomy was LV. The cut-off value of LV for predicting unstable refractive error analyzed by the ROC curve was 0.855 mm.Phacotrabeculectomy may be an effective treatment with stable long-term outcomes of prediction error similar to phacoemulsification in patients with glaucoma. However, elderly patients or patients with large LV may be predisposed to unstable prediction error after phacotrabeculectomy. When planning the surgery of a patient with cataract and medically uncontrolled glaucoma, it becomes difficult to decide whether to perform trabeculectomy and phacoemulsification at the same time or to perform phacoemulsification after trabeculectomy. It has already been reported that axial length (AL) and anterior chamber depth (ACD) decrease –3 and keIf phacoemulsification is performed with uncontrolled intraocular pressure (IOP), surgery becomes difficult due to high posterior pressure . ComplicAs the calculations for IOL power improve due to the development of preoperative evaluations and surgical techniques, refractive errors are becoming more predictable , 23. TheHowever, most of the previous studies examined short-term outcomes of prediction error less than 6 months, and no studies have investigated risk factors that can cause unstable prediction error after phacotrabeculectomy. Therefore, the present study aims to compare the long-term outcomes of prediction error of phacotrabeculectomy and phacoemulsification, and to determine the factors that predict unstable refractive error after phacotrabeculectomy.We retrospectively reviewed medical records of patients who had underwent uncomplicated phacotrabeculectomy (combined group) or uncomplicated phacoemulsification (phaco-only group) between September 2015 and December 2018. Patients underwent phacotrabeculectomy when IOP was not controlled even with the maximal tolerated medical treatment. Patients who were able to follow-up for more than 1 year were included, and exclusion criteria included prior keratorefractive surgery, ocular disease that may affect refractive errors except glaucoma and cataract, IOL in the ciliary sulcus or sclera fixation of IOL, and bleb needling after phacotrabeculectomy. If both eyes of a patient satisfied these criteria, one eye was chosen randomly. Ethical approval was obtained from the Chonnam National University Hospital Institutional Review Board, and the study protocol followed the guidelines of the Declaration of Helsinki.All patients underwent preoperative evaluations including best-corrected visual acuity (BCVA), manifest refraction using autorefractor keratometer , IOP using Goldmann applanation tonometry, and slit-lamp examination. For comparison, BCVA was converted to logarithm of the minimum angle of resolution (LogMAR) and the refraction value was converted to spherical equivalent (SE) by adding spherical power to 1/2 of cylinder power. And 12 months after the surgery, BCVA and IOP were measured to investigate the effect of the two operations on visual acuity and IOP.K-value using ARK and axial length (AL) using partial coherence interferometry  were measured, and SRK-T formula was used to calculate IOL power and predicted refractive errors. Additionally, anterior segment parameters were measured by using anterior-segment optical coherence tomography (AS-OCT) device . A single examiner (S.W.P) selected the best images with no motion artifacts, good visibility of the scleral spur, and no image artifacts from the eyelids. And then, two independent examiners (Y.S.K and M.S.S) who were blinded to other clinical information analyzed images using custom software . Anterior chamber depth (ACD) was defined as the distance between the center of posterior corneal surface and anterior lens surface, lens vault (LV) was defined as the maximum perpendicular distance between the anterior lens surface and horizontal line connecting the two sclera spurs, and anterior vault (AV) was defined as the sum of ACD and LV .Surgical procedures were performed by a single surgeon (S.W.P) under topical or retrobulbar anesthesia. Standard phacoemulsification was used to remove the cataract through a temporal 2.8 mm clear corneal incision. In all cases, Acrysof SN60WF  IOL was implanted in the capsular bag.For a phacotrabeculectomy, a fornix-based conjunctival flap and rectangular shaped half-thickness scleral flap were created. Phacoemulsification and IOL implantation were performed through clear corneal incision at a different site with trabeculectomy. A sponge soaked in 0.04% mitomycin C was placed under the conjunctiva and Tenon’s capsule on the sclera for 3–5 min according to the discretion of the surgeon and based on the patient characteristics. Sclerotomy and peripheral iridectomy were performed. Finally, the scleral flap was sutured with 2 interrupted sutures using 10–0 nylon and the conjunctiva was closed.Mean absolute error (MAE) was defined as the absolute value of the difference between predicted refractive errors and postoperative refractive errors. MAE at 3, 6, 12, and 24 months postoperatively were compared between two groups. The combined group was further divided into two subgroups based on the prediction errors: stable prediction error (MAE < 0.5 diopters [D]) and unstable prediction error (MAE ≥ 0.5 D). Risk factors associated with unstable prediction error were investigated at 12 and 24 months postoperatively. The number of glaucoma medications used preoperatively and 12 and 24 months postoperatively were also compared to determine the IOP lowering effect of phacotrabeculectomy.t-test or Mann-Whitney U test was used to compare the continuous data of the two groups, and paired t-test was used to compare the values before and after surgery in the same group. Chi-square test was used to compare categorical data, and logistic regression analysis was used to investigate risk factors that may cause unstable prediction error. Variables with a significance level at P < 0.1 in the univariable analysis were included in the multivariable analysis. The receiver operating characteristic (ROC) curve analysis was used to determine the optimal cut-off value of risk factor predicting unstable refractive error. The ability of the cut-off value to predict accurately is represented by the area under the curve (AUC). P values less than 0.05 were considered statistically significant.SPSS version 26.0  was used for all statistical analyses. Independent P < 0.001). ACD was significant smaller in the combined group than that of the phaco-only group (P = 0.018). There were no significant differences in age, sex, preoperative BCVA, AL, LV, and AV.A total 120 eyes of 120 patients were enrolled in this study, and 60 eyes were included in each group. The subject’s demographics and baseline characteristics of the included eyes are summarized in Table P = 0.001 and P < 0.001, respectively) and IOP was significantly decreased  at 12 months postoperatively (Table P = 0.065). Mean postoperative IOP of the combined group was 13.35 ± 3.12 mmHg, which remained stable until 12 months after phacotrabeculectomy. MAE at 3, 6, 12, and 24 months postoperatively were not significantly different between the two groups   and had a larger LV (P = 0.038) and larger AV (P = 0.041) than those in the stable subgroup at 12 months postoperatively. And patients in the unstable subgroup had significantly shallower ACD (P = 0.040) and larger LV (P = 0.010) than those in the stable subgroup at 24 months postoperatively.Table P = 0.031), large LV , and large AV  were associated with unstable prediction error at postoperative 12 months after combined surgery. The multivariable analysis showed that old age  and large LV  were risk factors of unstable prediction error at 12 months postoperatively (Table P = 0.024) was associated with unstable prediction error after combined surgery  and average of 1.27 medications at 24 months (P < 0.001) postoperatively.The patients in the combined group used an average of 2.8 medications preoperatively. The needs for glaucoma medication were significantly reduced to average of 1.12 medications at 12 months  in the combined group, whereas the phaco-only group had twelve patients with ACG. Previous studies reported that the refractive errors of patients with ACG were difficult to predict  eyes in , but ourRecently, the indications of phacotrabeculectomy are as follows: i) medically uncontrolled glaucoma, ii) tolerance of glaucoma medications, iii) postoperative IOP spikes may worsen visual field damage, iv) suspected compliance of glaucoma medications –17, 36. In this study, we initially aimed to investigate prediction error after phacotrabeculectomy at 12 months postoperatively, the medical records of 120 patients in combined group and phaco-only group were analyzed. Among them, there were many patients who had medical records up to 2 years after the surgery, the data of 94 patients were additionally analyzed. And we found that there was no significant difference in prediction error between phacotrabeculectomy and phacoemulsification alone until 24 months after surgery. It means that phacotrabeculectomy was effective not only for IOP control but also for stable prediction error.Previous studies reported that myopic shift occurs after phacotrabeculectomy compared to phacoemulsification alone , 23–25. Law et al.  reportedMost of the studies that focused the prediction errors of phacotrabeculectomy analyzed only short-term outcomes less than 6 months , 22–25, Tzu et al.  reportedOur group previously reported that one risk factor causing unstable prediction errors after cataract surgery in patients with glaucoma was large LV . We specOzaki et al.  reportedThe current study has several limitations. First, we could not analyze which ocular parameters had a significant effect on refractive outcomes because some ocular parameters were not measured after surgery. Second, both patients with ACG and patients with OAG were included in the combined group, so the effect of angle status was not considered. Third, we did not consider digital massage or releasable suture removal that could affect refractive errors after phacotrabeculectomy. Forth, SRK-T formula was used instead of using the widely used Barrett or Haigis formulas, which is known to have high accuracy recently. Finally, we did not analyze the complications that could occur after phacotrabeculectomy because only patients without complication were included. In the future, larger and more long-term studies that consider these factors will be needed.In conclusion, phacotrabeculectomy is an effective treatment that significantly reduces IOP and decreases the use of glaucoma medications in patients with cataract and medically uncontrolled glaucoma. There was no significant difference in prediction errors compared to phacoemulsification alone, and patients who underwent phacotrabeculectomy could also get stable prediction errors. However, the operator should be careful phacotrabeculectomy may increase in elderly patients or patients with large LV.


The prognosis of patients with perihilar cholangiocarcinoma have been unsatisfactory. We established new anatomical resectability classification for patients with localized perihilar cholangiocarcinoma and performed neoadjuvant chemotherapy followed by curative-intent surgery based on its resectability classification and lymph node status to improve prognosis. This study aimed to clarify the long-term outcomes and validation of our strategy.Between September 2010 and August 2018, 72 consecutive patients with perihilar cholangiocarcinoma were classified into three groups: Resectable (R = 29), Borderline resectable (BR = 23), and Locally advanced (LA = 20), based on the two factors of tumor vascular and biliary extension. R with clinically lymph node metastasis, BR, and LA patients received neoadjuvant chemotherapy using gemcitabine plus S-1.Forty-seven patients (65.3%) received neoadjuvant chemotherapy: R in 8, BR in 21, and 18 in LA, respectively. Fifty-nine patients (68.1%) underwent curative-intent surgery: R in 26, BR in 17, and LA in 6. Five-year disease-specific survival was 31.5% : 50.3% (not reached) in R, 30.0% (31.4 months) in BR, and 16.5% (22.5 months) in LA, which were relatively stratified. Among 49 patients with resection, disease-specific survival was 43.8% (57.0 months): 57.6% (not reached) in R, 41.0% (52.4 months) in BR, and 0% (49.4 months) in LA, which were significantly good prognosis compared to 23 patients without resection (17.2 months). Multivariate analysis identified preoperative high carcinoembryonic antigen levels (more than 8.5 ng/ml) and pT4 as independent poor prognostic factor of patients with resection.Neoadjuvant chemotherapy based on resectability classification and lymph node status was feasible, and was considered efficacious in selected patients. In the localized perihilar cholangiocarcinoma, negative tumor margin resection contributes to get an opportunity for long recurrence free survival. In the past two decades, advances in diagnostic and surgical techniques have improved surgical outcomes and survival rates . HoweverThe role of adjuvant chemotherapy (AC) for resected bile duct cancer (BTC) is controversial. Although 3 phase-III randomized trials have been explored in the adjuvant setting for BTC –4, the pIn terms of neoadjuvant chemotherapy (NAC) for localized BTC, there are a few retrospective small reports –7. They In the field of pancreatic ductal adenocarcinoma (PDAC), localized tumors are anatomically classified as resectable (R), borderline resectable (BR), or locally advanced (LA) based on the likelihood of a positive margin resection. Neoadjuvant chemo and/or radiotherapy is introduced PDAC patients based on its classification. In our institution, neoadjuvant gemcitabine based chemoradiotherapy for advanced PDAC based on its resectability has been introduced since 2005 and its prognosis has been improved . TherefoThis study aimed to evaluate the feasibility of anatomical resectability classification and the efficacy of NAC followed by curative-intent surgery based on its classification and LN status.n = 29), BR (n = 23), and LA (n = 20). Of them, 43 were men and 29 were women, with an average age of 71 years (range 44–87 years).Between September 2010 and August 2018, 72 consecutive patients with localized perihilar cholangiocarcinoma, who were all eligible patients identified by us, had been enrolled for our institutional treatment protocol based on our established resectability classification from surgical points of view. The diagnosis of perihilar cholangiocarcinoma was confirmed by means of cytological analysis of bile juice or histological analysis of biopsy specimens obtained using endoscopic retrograde cholangiography (ERC). Patients were excluded when they showed evident distant metastatic lesions at the time of enrollment. On the basis of our resectability classification mentioned below, the 72 patients were classified into the three groups: R . Initial resectability classification was performed based on initial dynamic multidetector-row computed tomography (MDCT) findings before biliary drainage at the visit to our hospital. High resolution CT allows us to make accurate depiction of a thickened bile duct wall and tumor spread into liver parenchyma or hilar vessels [In 2010, we established our own anatomical resectability classification for localized perihilar cholangiocarcinoma which consists of the three categories: R, BR, and LA according to surgical points of view from biliary and vascular factors  vessels . This wa vessels . AdditioBiliary factor is defined whether or not curative resection could be obtained by either side of right or left trisectionectomy or less hepatectomy. Vascular factor is defined whether or not combined vascular resection and reconstruction of PV and/or HA is required. Finally, the three classifications are determined by combination of biliary and vascular factors as follows.R: curative resection can be obtained by either side of right or left trisectionectomy or less hepatectomy (biliary factor), and combined resection with reconstruction of PV and/or HA is not required regardless of vascular invasion (vascular factor).BR: curative resection can be obtained by either side of right or left trisectionectomy or less hepatectomy (biliary factor), and combined vascular resection with safe reconstruction of PV and/or HA can be performed (vascular factor).LA: curative resection cannot be obtained even by either side of right or left trisectionectomy (biliary factor), and/or combined vascular resection with safe reconstruction of PV and/or HA cannot be performed (vascular factor). included 2 cycles of chemotherapy with gemcitabine (800 mg/m2 on days 7 and 21) plus S-1  [As shown in Fig. Fig.  includedeks, GC) , 14. AftIn terms of tumor related factors, serum tumor markers, such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19–9 (CA19–9), were measured before the initial treatment and curative-intent surgery. In terms of host related factors, inflammatory/immunonutritional factors, such as neutrophil-to-lymphocyte ratio (NLR), and prognostic nuritional index (PNI), were evaluated before the initial treatment and curative-intent surgery. Inflammatory/immunonutritional factors have been reported to predict the prognosis of patients with various malignancies .Toxicity of chemotherapy was categorised according to the National Cancer Institute’s Common Toxicity Criteria for Adverse Events, version 3.0. Toxicity was recorded continuously during treatment and serious adverse events were monitored throughout.Based on preoperative imaging studies, we determined which side of hepatectomy with caudate lobectomy could be performed to obtain curative resection according to the biliary and vascular factors. In terms of biliary extension, right hepatectomy was applied to Bismuth type I, II, and IIIa tumors. Left hepatectomy was applied to Bismuth type IIIb tumors. When the tumor obviously extended over the second order biliary radicles, such as Bismuth type IV tumors, trisectionectomy or central bisectionectomy was selected. However, patients with obvious invasion of the right side of the umbilical portion (U portion) and the left side of the origin of the right posterior portal vein (P portion) were contraindication for surgery. In terms of vascular invasion, it was critical point to secure at least 5 mm tumor free hepatic margin of PV and/or HA in the remnant liver side for safe vascular resection and reconstruction. Vascular extension beyond the second branch of PV and/or HA was contraindication because safe vascular resection and reconstruction were usually impossible. When patients had sufficient remnant liver function for proposed operation which achieve R0 resection even if we found tumor progression, curative-intent surgery was underwent because R0 resection contributed to get an opportunity for long recurrence free survival. However, it was difficult to evaluate biliary and vascular extension after biliary drainage. Occasionally, it was up to intraoperative judgment in order not to miss the opportunity of R0 resection.For almost LA patients, they remained unresectable at the time of reevaluation after 2 cycles of chemotherapy, and thus we continued chemotherapy using GS or GC regimen with or without adding external radiotherapy, followed by every three-month interval of reevaluation to seek the timing of curative-intent surgery. When the biliary and vascular factors of unresctability were determined to be overcome, we decided to perform curative-intent surgery.. The future remnant liver function was determined by multiplying the future functional remnant liver ratio (f-rem) by the indocyanine green plasma clearance rate (KICG) (f-rem-KICG). The f-rem was calculated by the fusion image of MDCT and hepatic uptake ratio of 99mTc-GSA scintigraphy at 15 min using 3D simulation software  [Combined with the above mentioned biliary and vascular factors for hepatectomy, the type of hepatectomy was finally determined by the remnant liver function, Japan) . Patient, Japan) . Portal , Japan) .Postoperative complications including morbidity and mortality were graded according to the Clavien-Dindo classification .The resected specimens were fixed in a formalin solution, sectioned approximately 5-mm intervals and embedded in paraffin blocks. A 3-μm section was obtained from each block and stained with hematoxylin and eosin. All specimens underwent routine histopathological work-up according to the American Joint Committee on Cancer staging system, 7th edition. Pathological differentiation, degree of LN metastasis, and assessment of residual tumor and so on was evaluated by an experienced pathologist. R1 status was defined based on microscopic tumor exposure at any biliary, vascular, and hepatic paremchema resection margin of the surgical specimen. R2 status was also defined based on macroscopic tumor exposure or distant metastasis including intrahepatic metastasis.2 biweekly, from February 2005 to May 2013, and S1 orally twice daily at a dose of 60 mg/m2/day on days 1 through 28 of a 42-day cycle from June 2013 to March 2019. Depending on patient tolerability of AC regimen, we changed the regimen from gemcitabine to S1 or vice versa. After operation, all patients were evaluated as follows: physical examination every month; laboratory tests including 12 serum levels and tumor marker levels (CEA and CA19–9) every 2 or 3 months; and 4-phasic contrast-enhanced MDCT every 4 months within 2 years and thereafter every 6 months. If the serum levels of the tumor markers increased, the patients were immediately evaluated by MDCT.From 4 to 6 weeks after resection, we started the AC and continue at least 6 months. Chemotherapy regimen was gemcitabine at a dose of 800 mg/mp < 0.05 in the univariate analysis were entered into the multivariate analysis. Comparisons were performed using the X2 test with Yates correction in the univariate analysis. All statistical analyses were performed using the SPSS version 24  software. A p value less than 0.05 was considered statistically significant.Continuous and categorical variables were expressed as median (range) and were compared using the Mann-Whitney test and chi-square test. In all patients who came for re-assessment, the date of the initial treatment was chosen as the starting point for the measurement of survival time. Patients who were alive or had died of a cause other than perihilar cholangiocarcinoma were censored for analysis of disease-specific survival (DSS) and median survival time (MST: months). Survival was calculated using the Kaplan-Meier method and was compared between the groups using the log-rank test. The day of final follow-up was March 31, 2019. All variables were dichotomized for analyses. A multivariate analysis was performed using Cox proportional hazard model. Variables with a significance of n = 29), BR (n = 23), and LA (n = 20). Figure The enrolled 72 patients with localized perihilar cholangiocarcinoma had been classified into the three groups: R . However, there were no differences between the three groups in clinical LN metastasis based on initial MDCT and PET-CT. The induction ratio of NAC was significantly lower in R  group than in BR  and LA  groups (p < 0.001). there were no differences among the three groups in completion rate of initial GS therapy and adverse reaction rate. The resection ratio was significantly higher in R  and BR  groups than in LA  group (p < 0.001).Characteristics in three groups of R, BR and LA are summarized in Table p < 0.001). In terms of pathological findings, the rate of patients who diagnosed as pT4 was significantly higher in BR  and LA  groups than in R  group (p < 0.001). However, there were no differences among the 3 groups in histological differentiation, LN metastasis, intrahepatic metastasis, and R0 resection rate. However, all four patients with limited resection such as hilar bile duct resection with or without S1 hepatectomy who were classified as Bismuth type I or II were belonged to the R group. Among them, 2 patients could not achieve R0 resection. Additionally, all four patients with pancreatoduodenectomy and hilar bile duct resection who were classified as Bismuth type I were also belonged to the R group. Unfortunately, 3 patients could not achieve R0 resection. Therefore, among 8 patients classified as R group who underwent curative-intent surgery without major hepatectomy, only 3 (37.5%) patients achieve R0 resection resulted in relative low R0 resection rate in the R group. In contrast, among 18 patients classified as R group who underwent curative-intent surgery using major hepatectomy, 13 (72.2%) patients could achieve R0 resection. In terms of intraoperative outcomes, there were no differences among the three groups in operation time and blood loss. The rate of patients who underwent combined PV resection and reconstruction was significantly higher in BR  and LA  groups than in R  group (p < 0.001). In terms of postoperative course, there were no differences among the three groups in postoperative complication, 90-day mortality, and induction rate of AC.The total of 49 patients could undergo curative-intent surgery. Characteristics in three resected patient groups of R, BR and LA are summarized in Tables p < 0.001). Interestingly, the patients’ survival did not differ among the three groups when resected.As shown in Fig. (Table ). Univariate analysis identified the following poor prognostic factors: preoperative CEA levels (more than 8.5 ng/ml), G3 histological differentiation, pT4, intrahepatic metastasis, and non R0 resection. Multivariate analysis identified preoperative high CEA levels (more than 8.5 ng/ml) and pT4 as independent poor prognostic factor. The patients with resection showing preoperative high CEA levels (more than 8.5 ng/ml) and pT4 had very poor prognosis, being comparable to the patients without resection .Prognostic factors in the 49 patients with resection were evaluated by uni- and multi-variable analyses Table . UnivariIn Table In Table p = 0.021, Fig. LA patients with resection had significantly better prognosis compared to LA patients without resection , BR (n = 23), and LA (n = 20), respectively. Based on this classification and LN status, NAC using 2 cycles GS followed by curative-intent surgery was administered for the 47 patients, and its completion rate was 91.4% (43/47), which was feasible and tolerable. The resection rate was similar between R (89.7%) and BR (73.9%), but much lower in LA (30%). The 5-year DSS was stratified according to the resectability classification: 50.3% in R, 30.0% in BR, and 16.5% in LA, respectively. The patients with resection had significantly better prognosis compared to those without resection (5-year DSS: 43.8% vs. 5.9%). Interestingly, the patients’ survival did not differ among the three groups when resected. Among the 49 patients with resection, preoperative high CEA levels (more than 8.5 ng/ml) and pT4 were identified as independent poor prognosis factors, and the patients with resection showing high CEA levels or pT4 had very poor prognosis, being comparable to the patients without resection.In the present study, we proposed anatomical resectability classification for patients with localized perihilar cholangiocarcinoma according to surgical points of view from biliary and vascular factors, and the enrolled 72 patients had been classified into the three groups: R , arterial involvement is incorporated into T4 stage: tumor invades the main PV or its branches bilaterally or the common hepatic artery; or unilateral second order biliary radicles with contralateral PV or HA involvement. Recently, the development of surgical techniques has enabled us to perform technically demanding procedures including trisectionectomy, and combined PV and/or HA resection. Therefore, using these technically demanding procedures, the tumors determined as unresectable according to Blumgard criteria, Bismuth IV, or UICC T4 stage are not always unresectable, and actually some of them can undergo negative margin resection.In terms of survey of distant metastasis, preoperative MRI to evaluate intrahepatic metastases or peritoneal dissemination was not performed routinely. Of 54 laparotomy cases in this study Fig. , 4 casesn = 18) were significantly better than those of patients who underwent other types of resection (n = 13). In that study, most patients did not have invasion of the right hepatic artery, but the distance between the leading edge of the cancer and the outer layer of the hepatic artery was 1 mm in many patients. The authors suggested that the resected margin would have been cancer positive without combined resection of the right hepatic artery. Therefore, they recommend right hepatectomy even when invasion of the right hepatic artery cannot be demonstrated preoperatively by diagnostic imaging. Additionally, 2 small studies also reported that outcomes of limited resection except for those of right hepatectomy with caudate lobectomy were unsatisfactory due to low curative resection rate and survival rate [Regarding the operative procedure for patients of Bismuth type I or II, although limited hilar bile duct resection without major hepatectomy is occasionally underwent, our institution usually employs right hepatectomy with caudate lobectomy, based on previous studies. Ikeyama et al. reported retrospective study using 31 consecutive patients who underwent resection of these types of tumors . R0 reseval rate , 29. FurIn the fields of localized PDAC, the National Comprehensive Cancer Network (NCCN) guidelines for Pancreatic Adenocarcinoma in Version 3. 2019 clearly defines the resectability criteria  based on a likelihood of a positive margin resection. The definition of BR-PDAC is a tumor that is at high risk for positive margin resection when surgery is used as an initial treatment. LA-PDAC is defined a tumor not to achieve R0 resection and not to indicate up-front surgery . In the The role of AC for resected BTC is controversial. Recently, 3 phase-III randomized trials have been explored in the adjuvant setting for BTC –4, but aIt is unclear which patient is most likely to benefit from NAC for perihilar cholangiocarcinoma. Nagino et al.  evaluate(Tables 7), 6 patients with resection had either one of the vascular or biliary factor, while none of the 4 patients with both vascular and biliary factors could not undergo resection. LA patients with both biliary and vascular factors are not for the candidate of conversion surgery after neoadjuvant GS therapy.According to the factor analysis for unresectability in 20 LA patients Tables  and 7, 6There are several limitations in this study. Study cohort was small and data were collected from a single Japanese center. To evaluate the efficacy of resectability classification and NAC for localized perihilar cholangiocarcinoma patients, prospective randomized multicenter trials are needed.NAC based on our resectability classification for patients with localized perihilar cholangiocarcinoma according to surgical points of view from biliary and vascular factors and LN status was feasible and tolerable. NAC enabled the downstaging / downsizing of BR patients and conversion surgery in selected LA patients resulted in improving prognosis.