Although after adjustment for sex, GA, birth weight, and growth restriction, GA  and writhing GMs  were not significant, a strong trend still persisted. The HNNE scores significantly correlated with both the HINE evaluation  and fidgety GMs . Linear regression confirmed the HNNE was highly predictive of the HINE . Writhing GMs did not significantly correlate with either fidgety GMs  or the HINE . Conclusions: Abnormal writhing GMs in the neonatal period were non-significantly associated with early CP/high-risk diagnoses in extremely preterm infants in a multivariate analysis. Additionally, the HNNE significantly correlated with both fidgety GMs and the HINE.Background: Early diagnosis of cerebral palsy (CP) in high-risk infants is possible at 3–4 months’ corrected age (CA) using standardised assessments. Aim: To assess the utility of neonatal screening assessments—writhing general movements (GMs) and the Hammersmith Neonatal Neurological Examination (HNNE)—to predict CP/high-risk status at 3–4 months’ CA in extremely preterm infants. Methods: Retrospective cohort study of high-risk preterm infants (born < 29 weeks’ gestation and/or birth weight < 1000 g) attending an Early Neurodevelopment Clinic. Data from neonatal assessments were compared with CP/high-risk diagnosis at 3–4 months’ CA, fidgety GMs, and Hammersmith Infant Neurological Examinations (HINE) using logistic regression, linear regression, and Spearman rank correlation. Results: Two hundred and two preterm infants (median gestation age at birth 27.3 (IQR 25.4–28.3) weeks, mean birth weight 870.3 (SD 248.4) grams) were included. A total of 26 (12.8%) infants received early CP/high-risk diagnoses at 3–4 months’ CA. A lower gestational age (GA) (OR = 0.78;  Cerebral palsy (CP) is the most common physical disability diagnosed in children, with an overall prevalence of approximately 2.0–2.11/1000 live births internationally and 1.4/1000 live births in Australia . The early diagnosis of CP is now possible in high-risk infants at 3–4 months’ corrected age (CA). Early intervention may promote positive cognitive and motor developmental outcomes amongst high-risk infants, and thus the early diagnosis or early identification of infants at high risk of CP is a critical step towards enabling this .There is a range of assessment tools currently utilised to detect movement difficulties and predict CP in infants. Prechtl’s general movement assessments (GMA), Hammersmith Infant Neurological Examinations (HINE), and MRI brain scans have the highest predictive validity before 5 months’ corrected age and are recommended to be used to predict CP with high sensitivity before 5 months’ corrected age ,7,8. NotThe Prechtl GMA is an observational tool used to assess the spontaneous movements of an infant, known as general movements (GMs). GMs arise during early foetal life and persist until 4–5 months’ CA. GMs can be recorded after a preterm birth until 6 weeks’ CA. Known as “writhing movements”, these are characterised by variable movements through the whole body, including arm, leg, neck, and trunk movements. Normal writhing movements appear smooth and unpredictable and show variability in the intensity, range, direction, and complexity of movement ,10. AbnoThe Hammersmith Infant Neurological Examination (HINE) is a validated scored assessment of 26 items, each scored from 0 to 3, used to evaluate infants between 2 and 24 months’ CA to inform a diagnosis of CP. It functions as both a quantitative and qualitative assessment, evaluating movement, cranial nerves, tone, reflexes, and posture . A globaCurrently, a combination of GMA, the HINE, and neonatal MRI is the gold standard for the diagnosis of CP in high-risk infants and can be used to accurately predict CP before 5 months’ CA ,7,8,14.The Hammersmith Neonatal Neurological Examination (HNNE) is a scored neurological assessment presently limited to use as a screening tool in term and preterm infants, though it has not been validated for use in infants <34 weeks’ GA. It consists of 34 items across six domains: tone (10 items), tone patterns (5 items), reflexes (6 items), spontaneous movements (3 items), abnormal signs (3 items), and behaviour (7 items), with a maximum global optimality score of 34 ,20. In aSeveral studies have identified a relationship between GMs and CP diagnosis amongst very preterm infants assessed at preterm or term-equivalent age ,23,24. HPresently, no studies have analysed the utility of early neonatal assessments in the preterm period for predicting the later diagnoses made based on fidgety age assessments in very preterm (<29 weeks) or extremely low birth weight (1000 g) infants at 3–4 months of corrected age. There are also no studies tracking the correlation between the HNNE and HINE scores.We conducted an exploratory study to assess how early neonatal screening assessments correlate with 3–4-months’ CA assessments of high-risk infants in the setting of a clinical surveillance clinic (Early Neurodevelopment Clinic (ENC)), in order to answer the question: can early neonatal screening assessments accurately predict early diagnoses of CP/high-risk status in high-risk infants at 3–4 months’ corrected age in a real-world setting? The answer to this question may be useful in guiding the approach to high-risk infants and maximising early intervention at term age to improve outcomes amongst the high-risk preterm cohort, or in guiding further research that needs to be undertaken. This retrospective cohort study analysed the data of high-risk preterm infants (born < 29 weeks’ gestation and/or birth weight < 1000 g) attending the Early Neurodevelopment Clinic at Monash Children’s Hospital, Melbourne, Australia. This clinic was established in 2018 to facilitate the early diagnosis of CP in line with current recommendations .Data were collected from electronic records of clinic notes between May 2019 and November 2021. The records of 393 patients attending ENC were reviewed; 190 did not fit the inclusion criteria (<29 weeks/<1000 g) and were excluded. One further patient was excluded due to non-attendance at their ENC appointment. Early neonatal assessment results and demographic information were obtained from the electronic patient records of the remaining 202 infants who attended the clinic. Assessments were performed and videos were scored in practice by experienced clinicians at Monash Newborn and ENC, and videos were not re-reviewed during the study. Where practicable, infants had multiple writhing GM videos captured and assessed during their neonatal admission between 32 and 46 weeks’ GA. Thirty infants had only one writhing GM video recorded during their admission. Factors preventing multiple recordings included short admission duration, inter-hospital transfer, and patient behaviour unsuitable for assessment. The HNNE was performed between 35 and 45 weeks’ GA. Participants were divided into two cohorts based on their diagnosis at 3–4 months’ CA:CP or high-risk for developing CP, and normal development or developmental delay other than CP. Diagnoses were made based on fidgety GMA and the HINE in combination with neuroimaging findings, such as cranial ultrasounds or MRI brain scans, where available. CP and high-risk status were distinguished as some infants meeting criteria for CP  did not receive a definitive diagnosis at 3–4 months’ CA but were still referred for close follow-up to monitor progression. Neonatal neuroimaging findings were included in the 3–4-month assessment for CP, and thus cannot be included in the comparison between these two time points. Therefore, this paper focuses on clinical assessments.Normal = 0;PR = 1;PR/CS = 2;CS on one occasion = 3;CS on two or more occasions = 4.All available writhing GM scores from the neonatal period were reviewed, and patients were categorised according to the worst recorded score, as follows: The group for which CS > 2 was distinguished from the CS group to illustrate the difference between a one-off CS GM and a trajectory of persistent CS GMs. Of the 27 infants who received a score of 3, only 1 did not have multiple writhing GM assessments performed. Present = 0;Sporadic = 1;Abnormal = 2;Absent = 3.Fidgety GMs were categorised as follows:Global cut-off scores of 27 and 57 were used for the HNNE and the HINE, respectively.This study was exempt from review by Monash Health Human Research Ethics Committee and received ethics approval by Monash Health as a quality assurance project (ERM Reference No. 81908). It followed NHMRC Ethical Considerations in Quality Assurance and Evaluation Activities (2014) guidelines.Statistical analyses were performed using Stata Version 16 . Additional analyses were performed in GraphPad Prism Version 9.0 . The Wilk–Shapiro test was performed to determine the normality of numerical data. The mean and standard deviation were calculated for normally distributed data, while the median and inter-quartile range were calculated for non-normally distributed data. Data from neonatal assessments (writhing GMs and HNNE scores) were correlated against results of 3–4-months’ CA assessments (fidgety GMs and HINE scores) using Spearman’s Rho correlation. Linear regression was performed to directly compare the HNNE with subsequent HINE scores. Neonatal assessment scores (writhing GMs and HNNE) were studied for association with the 3–4-months’ CA diagnosis using a parsimonious model univariate logistic regression and then adjusted for demographic characteristics  with the results expressed as odds ratios, with 95% confidence intervals and significance set at <0.05. Two hundred and two preterm infants were included in the study. The median gestational age of participants was 27 + 3 weeks (IQR 25 + 4–28 + 3), and the mean birth weight was 870.3 g (SD 248.4 g). Twenty-six infants (12.8%) received a CP or high-risk clinic diagnosis—eight (4.0%) infants were diagnosed with CP, and eighteen (8.9%) were considered at high-risk. Eighty-two (40.6%) infants were deemed developmentally normal at ENC, and ninety-four (46.5%) were deemed delayed in specific parts of development but not high-risk for CP. Additional demographic characteristics of the participants are outlined in One infant did not receive a diagnosis at ENC. Eight did not receive HINE scores, and three did not receive fidgety GM scores. Thirty-three infants did not have available HNNE scores, and three did not have available writhing GMs. All infants attending the clinic were within the fidgety period (between 10 and 20 weeks’ CA) .p = 0.029, 95% CI ) and all abnormal writhing GMs  were significantly predictive of CP/high-risk diagnosis at 3–4 months’ CA. The relationship between writhing GMs and 3–4-month diagnosis is represented in Parsimonious model univariate logistic regression  determinp = 0.068, 95% CI ) or writhing GMs .This trend persisted in multivariate analyses , though s) of 0.43 for the HINE  and −0.18 for fidgety GMs , indicating a significant correlation between the HNNE and both the HINE and fidgety GM scores. Linear regression .Correlation analysis  showed tgression  confirmep = 0.723, 95% CI ) and −0.10 for the HINE . The writhing GMs had a non-significant Spearman’s Rho coefficient of 0.03 for fidgety GMs . However, once the data were adjusted for birth weight, sex, GA, and SGA/IUGR, this correlation was not statistically significant . Abnormal writhing GMs were also found to be weakly correlated with abnormal fidgety GMs   and abnormal HINE scores . Both writhing and fidgety GMs were scored with normal being 0 and increasing scores indicating worsening performance, which is reflected in the negative coefficients with both the HINE and HNNE. A review of the literature found fidgety GMs had sensitivity between 95 and 100% with a specificity of 89–98% for predicting CP diagnosis at 2 years of age ,13,14,26p = 0.05, 95% CI ). This is consistent with current evidence, which suggests that the HNNE performed at two weeks post-term in term infants, or at term-corrected age in preterm infants, is highly predictive of a neurodevelopmental deficit [Our study also found the HNNE scores to be somewhat predictive of early CP diagnosis at 3–4 months (OR= 0.88). Because the HNNE scores increase with improving performance, an odds ratio <1 indicates that an increasing HNNE score is associated with a decreased likelihood of early CP diagnosis. However, this association was not statistically significant  for early CP diagnosis at ENC. As with the HNNE, the risk of CP decreases with increasing gestational age. It is known that reduced gestational age at birth is a risk factor for CP [Our study also identified gestational age at birth as a significant predictive factor in the development of CP. Reduced gestational age had an odds ratio of 0.78  hospitals. Three patients (1.5%) did not have scores for writhing GMs; thirty-three (16.3%) did not have scores for the HNNE. A small number of infants attending the ENC had incomplete 3–4-month assessments; three (1.5%) did not have recorded results for fidgety GMs, and eight (4.0%) did not have HINE scores. The reasons cited included telehealth appointments limiting the ability to perform assessments, infant irritability impeding the assessment, and parental reluctance. Additionally, whilst all participants attending ENC appointments were within the 10–20-week fidgety period, there was some variation in the age at the time of appointment, which may have impacted the data. This is due in part to the COVID-19 pandemic, which resulted in some patient clinic appointments needing to be rescheduled due to lockdown requirements or moved to telehealth, which also impacted the examination accuracy. However, it is reflective of the real-world setting in which these assessments are conducted, and thus still provides valuable insight into their utility.The findings of this study suggest that early neonatal assessments, in particular writhing GMs, are non-significantly associated with the early diagnosis of CP at 3–4 months’ CA. Writhing GMs and the HNNE may have combined value as supportive assessments for infants at risk of poorer outcomes at 3–4 months. This information may be utilised to identify infants who are likely to be diagnosed with CP or high-risk status from a high-risk population and ensure that long-term follow-up is planned from inpatient discharge. Additionally, it may be beneficial in organising access to early intervention and support services for high-risk infants.


Neisseria gonorrhoeae susceptibility to therapeutically relevant antimicrobials across the European Union/European Economic Area (EU/EEA). We present the Euro-GASP results from 2019 (26 countries), linked to patient epidemiological data, and compared with data from previous years.The European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) performs annual sentinel surveillance of . gonorrhoeae isolates from 26 countries across the EU/EEA. Significance of differences compared with Euro-GASP results in previous years was analysed using Z-test and the Pearson's χ2 test was used to assess significance of odds ratios for associations between patient epidemiological data and antimicrobial resistance.Agar dilution and minimum inhibitory concentration (MIC) gradient strip methodologies were used to determine the antimicrobial susceptibility  of 3239 NN. gonorrhoeae isolates collected between 2016 and 2019 displayed shifting MIC distributions for; ceftriaxone, with highly susceptible isolates increasing over time and occasional resistant isolates each year; cefixime, with highly-susceptible isolates becoming increasingly common; azithromycin, with a shift away from lower MICs towards higher MICs above the EUCAST epidemiological cut-off (ECOFF); and ciprofloxacin which is displaying a similar shift in MICs as observed for azithromycin. In 2019, two isolates displayed ceftriaxone resistance, but both isolates had MICs below the azithromycin ECOFF. Cefixime resistance (0.8%) was associated with patient sex, with resistance higher in females compared with male heterosexuals and men-who-have-sex-with-men (MSM). The number of countries reporting isolates with azithromycin MICs above the ECOFF increased from 76.9% (20/26) in 2016 to 92.3% (24/26) in 2019. Isolates with azithromycin MICs above the ECOFF (9.0%) were associated with pharyngeal infection sites. Following multivariable analysis, ciprofloxacin resistance remained associated with isolates from MSM and heterosexual males compared with females, the absence of a concurrent chlamydial infection, pharyngeal infection sites and patients ≥ 25 years of age.European Resistance to ceftriaxone and cefixime remained uncommon in EU/EEA countries in 2019 with a significant decrease in cefixime resistance observed between 2016 and 2019. The significant increase in azithromycin “resistance” (azithromycin MICs above the ECOFF) threatens the effectiveness of the dual therapy (ceftriaxone + azithromycin), i.e., for ceftriaxone-resistant cases, currently recommended in many countries internationally and requires close monitoring. The European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) is a sentinel surveillance system that since 2009 has been coordinated by the European Centre for Disease Prevention and Control (ECDC) and supported by a European network of microbiologists and epidemiologists. The programme aims to provide quality-assured antimicrobial susceptibility data to inform European and other regional and national gonorrhoea treatment guidelines as well as to detect emerging antimicrobial resistance and monitor trends in antimicrobial resistance.N. gonorrhoeae infections, 1 g ceftriaxone  [In addition, cefixime (400 mg single dose) is recommended in the European guideline only as a substitute for ceftriaxone in dual therapy in cases where injections are refused or contraindicated. Cefixime is not recommended for use in monotherapy. Ciprofloxacin (500 mg single dose) is suggested as an alternative treatment regime for those with history of severe hypersensitivity to any β-lactam antimicrobial or when injections are contraindicated or refused, however, only where susceptibility has been confirmed phenotypically or molecularly (testing) .As azithromycin is only recommended for use in dual therapy with another effective agent, the correlates between azithromycin in vitro susceptibility and treatment outcome are limited, and appropriate clinical treatment data to inform clinical breakpoints are lacking, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) replaced their clinical resistance breakpoint with an epidemiological cut-off (ECOFF) value of 1 mg/L in January 2019 . This ECIn the present study, we describe the results from the 2019 Euro-GASP sentinel surveillance, in conjunction with patients’ epidemiological data, and compare them to the Euro-GASP results obtained from previous years (main focus on the published 2016 Euro-GASP data ).N. gonorrhoeae isolates collected in their country as described previously [Annually, laboratories participating in Euro-GASP report antimicrobial susceptibility profiles  and pateviously , 15. All. gonorrhoeae isolates (one isolate per patient per gonorrhoea episode) to The European Surveillance System (TESSy) at ECDC. Some countries were over-represented in the complete 2019 TESSy data set with Austria submitting data for 434 isolates, France 243 isolates, the Netherlands 364 isolates and Norway 641 isolates whereas other countries with lower gonorrhoea incidence were under-represented with Cyprus submitting data for two isolates, Latvia seven isolates, Estonia eight isolates and Croatia nine isolates. The same imbalance was also present in previous years’ TESSy datasets. In order to reduce biases caused by high isolate numbers from some countries, only data for the first 200 isolates with complete antimicrobial susceptibility testing results submitted to TESSy in each year were included in the analysis of the present paper including data from 2016 which was used as the main comparator for this publication unless otherwise stated. For comparison of azithromycin MIC data, data collected prior to the introduction of the ECOFF was converted to equal to or below the ECOFF (referred to as susceptible hereafter) or above the ECOFF (referred to as resistant hereafter). Azithromycin MICs ≥ 256 mg/L will be discussed as “high-level azithromycin resistant”.In 2019, 26 countries participated in Euro-GASP, submitting total data for 4166 N2 test was used to measure if these odds ratios differed significantly from 1. For small cell numbers (n < 5), Fisher’s exact test was performed. Statistical significance for all tests was assumed when p < 0.05. Statistical analysis was performed using Stata v15 .The Mann–Whitney test was used to analyse differences in age distribution and the Z-test was applied to compare proportions when analysing antimicrobial susceptibility and epidemiological data. Associations between epidemiological characteristics and antimicrobial susceptibility were assessed using odds ratios (OR) and 95% confidence intervals (CI); the Pearson’s χ. gonorrhoeae isolates collected in 2019 from 3239 episodes of gonorrhoea were compared to the Euro-GASP data from 2016 (n = 2556)  . The proportion of heterosexual males decreased from 33.6% in 2016 to 27.9% in 2019  and pharyngeal  sites which is likely a consequence of the significant increase in specimens from men-who-have-sex-with-men (MSM) in 2019  and one in Portugal (MIC = 0.25 mg/L). Both isolates were also resistant to cefixime  and ciprofloxacin  but were susceptible to azithromycin . For comparison, there were three urogenital isolates with ceftriaxone resistance identified in 2018 , zero in 2017 and 2016. Despite the detection of ceftriaxone-resistant isolates in both 2018 and 2019, the proportion of gonococcal isolates that were most susceptible to ceftriaxone (MIC ≤ 0.016 mg/L) increased from 82.9% (2117/2555) in 2016 to 89.7% (2903/3238) in 2019  increased from 74.9% 1805/2411) in 2016 to 80.9% (2621/3239) in 2019  increased from 3.8% 97/2532) in 2016 to 9.0% (284/3159) in 2019 , pharyngeal sites  and patient age ≥ 25 years .There was an increase in resistance to ciprofloxacin from 47.2% 1003/2124) in 2016 to 57.4% (1665/2884) in 2019 , which has become increasingly common in the most recent years in the EU/EEA countries. The same shift towards cefixime susceptibility was observed in the United States (US) between 2014 and 2018 with the proportion of isolates with decreased susceptibility to cefixime (MIC 0.032–0.125 mg/L) decreasing from 37.8 to 29.9% and the proportion of cefixime-resistant isolates decreasing from 0.7 to 0.3% . In theN. gonorrhoeae but for also for other bacterial STIs. The same increase in higher MICs (> 1 mg/L) was observed in the US between 2014 and 2018 although less pronounced than in Europe with an increase from 2.4 to 4.6% , and/or replacement of some major azithromycin-resistant gonococcal clones with more azithromycin-susceptible clones.Azithromycin MICs are, in contrast to those observed for ceftriaxone and cefixime, shifting towards MICs above the azithromycin ECOFF, and this has been recently reported in many countries internationally , 9, 25. ented in ). By conented in . In the ented in . This maNeisseria species in the pharynx often with previous exposure to antibiotics allows for horizontal transfer of resistance genes to N. gonorrhoeae [In the Euro-GASP 2009–2016 data, ciprofloxacin resistance was significantly associated with urogenital sites , in 2019orrhoeae –33. Pharorrhoeae , 35.N. gonorrhoeae isolates  from many diverse countries are examined, many gonorrhoea cases in the EU/EEA are diagnosed with molecular diagnostics and no N. gonorrhoeae isolates are available from many of these cases, the majority of examined N. gonorrhoeae isolates are obtained from urogenital sites and the number of isolates from rectal and pharyngeal sites are more limited, and in many countries the completeness of reported epidemiological data  is suboptimal. However, despite these limitations a previous representativeness analysis showed that Euro-GASP appropriately reflects the antimicrobial resistance situation for N. gonorrhoeae in the EU/EEA [The limitations of Euro-GASP and, accordingly, the present study have been previously described in detail , 36, 37.N. gonorrhoeae to the third-generation cephalosporins ceftriaxone and cefixime remained uncommon in 2019 in EU/EEA countries with a significant decrease in cefixime resistance observed between 2016 and 2019. The significant increase in the proportion of isolates with azithromycin resistance is concerning for the future effectiveness of any ceftriaxone plus azithromycin dual therapy, particularly if ceftriaxone resistance starts to spread more widely. It is imperative that the MICs for N. gonorrhoeae isolates continue to be monitored very closely over the next years, particularly of azithromycin and ceftriaxone.Resistance in


This retrospective analysis from the registry of the Acute Leukemia Working Party (ALWP) of the European Society of Bone Marrow Transplantation (EBMT) database included 1427 adult patients (median age 58.2 years) receiving either Flu/Mel (n = 1005) or Flu/Treo (n = 422). Both groups showed similar 3-year overall survival (OS)  for patients conditioned with FluMel and FluTreo, respectively. However, patients treated with FluMel showed a reduced 3-year relapse incidence  and slightly increased non-relapse mortality (NRM)  compared to patients treated with FluTreo. Our data may serve as a basis for further studies examining the role of additional agents/ intensifications in conditioning prior to allo-HCT.In recent years considerable variations in conditioning protocols for allogeneic hematopoietic cell transplantation  protocols have been introduced for higher efficacy, lower toxicity, and better outcomes. To overcome the limitations of the classical definition of reduced intensity and myeloablative conditioning, a transplantation conditioning intensity (TCI) score had been developed. In this study, we compared outcome after two frequently used single alkylator-based conditioning protocols from the intermediate TCI score category, fludarabine/melphalan 140 mg/m Allogeneic hematopoietic cell transplantation  protocols are in continuous evolution and are constantly being evaluated for higher efficacy, lower toxicity, and better outcomes . The opt2) showed lower relapse incidence and slightly higher non-relapse mortality (NRM) compared to patients treated with lower-intensity fludarabine/busulfan , resulting in similar leukemia-free survival (LFS) and overall survival (OS) [2) have been shown to be safe and effective and are good alternatives to busulfan or total body irradiation (TBI)-based regimens in the conditioning for acute myeloid leukemia (AML) treatment [2, showing the lowest relapse rate with similar toxicity [n = 138) FluMel  and FluTreo  conditioning protocols were compared. Patients conditioned with FluMel suffered more frequently with grade III–IV oral mucositis and diarrhea but had similar outcomes to patients conditioned with FluTreo [Conditioning with fludarabine/melphalan (FluMel) has been suggested to have significant anti-leukemia activity while showing moderate toxicity , 7. Patitoxicity , 11. In  FluTreo . NeverthWe therefore used the registry of the EBMT to retrospectively analyze data from patients treated with two of the most used conditioning protocols with an intermediate TCI score, based on the single alkylating agent combinations of FluMel and FluTreo. Outcome variables included OS, LFS, relapse incidence, NRM, graft-versus-host disease (GvHD), and GvHD-free, relapse-free survival (GRFS). Characteristics of patients, donor type, cause of death, complications including incidence of GvHD, as well as immunosuppression and GvHD prophylaxis were also analyzed.2, melphalan 140 mg/m2) or with FluTreo , (2) first allo-HCT from a matched sibling donor (MSD) or matched unrelated donor  for patients with AML including secondary AML in complete remission (CR), (3) transplantation date between January 1st, 2008 and December 31st, 2018, (4) with an unmanipulated peripheral blood stem cell graft (no in vitro T-cell depletion (TCD)). Patients undergoing haplo-identical or cord blood allo-HCT were excluded. CR was defined as less than 5% blasts in bone marrow at the time of allo-HCT. GvHD prophylaxis was conducted according to local institutional guidelines. Doses and sources were not always documented in the ALWP registry. Post-transplant events such as hematological relapse, GvHD among others were defined based on standard clinical and laboratory criteria. All patients gave signed informed consent for data submission and scientific analysis within the registry. The study was approved by the general assembly of the ALWP of the EBMT.In this retrospective multicenter analysis, data were provided by the ALWP of the EBMT in which >600 transplant centers report annually all consecutive allo-HCTs after patient authorization via informed consent, and approval of the study from the ALWP EBMT general assembly. We focused on (1) adult (aged > 18 years) patients who received conditioning with FluMel  with the 95% confidence interval (95% CI).https://www.R-project.org/).All tests were two sided. The Type I error was fixed at 0.05 for factors associated with time-to-event outcomes. Statistics were performed with SPSS 25.0  and R 4.0.3R Core Team (2020). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. The patient and transplant characteristics of the 1427 AML patients are shown in Table p < 0.0001) and adverse risk cytogenetics . However, more patients in the FluTreo group were transplanted in CR1 compared with FluMel group . Hence, the donor distribution was different. MSD was used more frequently in FluMel (46.8% vs. 29.9%) and MUD 9/10 in FluTreo . Further imbalances between the two groups are depicted in Table Some clinical features were remarkably different between both treatment groups. AML from patients treated with FluTreo were associated with high-risk characteristics for relapse as secondary AML  and ATG (9.9% vs. 68.7%) was more often used in FluTreo conditioning. The frequency of use of GvHD prophylaxis treatments is shown in Table Graft failure was observed in 9 (0.6%) patients in the entire cohort Table . Overallp = 0.001). There was a corresponding lower NRM in the FluTreo group  but this was of borderline significance. Although the LFS was higher in FluMel than in FluTreo treated patients . The OS was similar in the two treatment groups   Table , Fig. 1.p = 0.002) and lower risk of NRM  as compared to FluMel. However, there were no significant differences in LFS , GFRS  or OS  between treatment groups  and with ATG  comparing both conditioning protocols. As only 3 patients of the FluTreo group received alemtuzumab, it was not possible to adjust the comparison on this variable. Univariate analysis stratified on in vivo TCD with ATG or no in vivo TCD were consistent with the entire population  and 900 patients aged ≥55 years (626 patients with FluMel and 274 patients with FluTreo) were included. Similar to the previously described results in the entire cohort, no differences were found by conditioning in both patient age subgroups regarding outcome and GvHD incidence, suggesting that both protocols are also suitable for older patients (Suppl. Tables n = 144) or two (n = 219) controls who received FluMel using exact and propensity-score matching. We included in this analysis a total of 582 patients in FluMel and 363 patients in FluTreo groups. Exact matching was performed for status at allo-HCT (CR1/CR2), cytogenetics (adverse vs other) type of AML (de novo or secondary), donor type . The propensity score was based on patient age and in vivo TCD. The patients were well matched, with standardized mean difference estimates of less than 5% for all matched parameters except age. To take into account for similarity within each pair, a cluster was used and comparison was adjusted on patient age. The results are consistent with those found in the entire population, but GRFS was significantly lower in the FluTreo group  score receiving MAC, outcomes improved compared to a similar patient population receiving RIC , 15. Hen2) was compared with FluBu2 in a prospective randomized clinical trial for patients with AML/MDS and improved outcome was observed in patients treated with FluTreo [2, busulfan 16 mg/kg orally or 12.8 mg/kg intravenously, 4 days), Busulfan/Cyclophosphamide , total body irradiation/Cyclophosphamide  or RIC including FluBu2  and FluMel . Patients receiving MAC showed significantly better outcomes than patients receiving RIC, although these data may have been confounded by the heterogenous conditioning regimes used in each group and the smaller proportion (20%) of patients conditioned with FluMel in the RIC group [2 × 4 days, AMSA 100 mg/m2 × 4 days, Busulfan -total dose 11.2 mg/kg) did not improved outcome of patients conditioned with FluBu2 neither in MRD positive nor negative patients with AML [2 and melphalan at 140 mg/m2 (FluMel), and fludarabine at 150 mg/m2 and treosulfan at 42 g/m2 (FluTreo).Conditioning intensity is a crucial factor, influencing the prognosis of patients in the allo-HCT setting. Recently, FluTreo , adverse cytogenetic risk, unrelated donor, female donor to male recipient, in vivo TCD and pre-transplant MRD. In vivo TCD  was more often used in the FluMel group than in the FluTreo group. In the former, in vivo TCD was given not only to patients receiving a transplant from an unrelated donor but also from a related donor. Previously, unrelated, and related donor recipients receiving ATG have shown better outcomes in terms of GRFS compared to standard GvHD prophylaxis –24.This study has several other limitations. This is an analysis of a retrospective registry-based study. Although the gold standard method of comparing conditioning protocols on outcomes after allo-HCT is the randomized controlled trial, the performance of such a clinical trial with enough patients to elucidate the appropriate conditioning protocol for a patient populations in an allo-HCT setting is no trivial matter. The value of a registry database analysis is the large number of patients. The conditioning protocols are country/center dependent. FluMel, mainly in combination with alemtuzumab for in vivo TCD as GvHD prophylaxis, and is most commonly used in United Kingdom, Belgium, and Switzerland. However, FluTreo in combination with ATG for in vivo TCD as GvHD prophylaxis, is more frequently used in Germany, Finland, Sweden, and Italy. As mentioned above, distribution of donor type was also imbalanced. Allo-HCT from an MSD was more frequently performed with FluMel (46.8%) conditioning than with FluTreo (29.9%). GvHD prophylaxis based on in vivo TCD  was more often used together with FluMel (90.5%) than with FluTreo (69.4%), which might also reflect center/country differences in the combination of conditioning with a specific GvHD prophylaxis regimen. The source of ATG  was not always documented in the ALWP registry, which might also have affected the GvHD incidence, and therefore, outcome. We included in vivo TCD in the multivariate analysis, which was associated with decreased NRM and GFRS. However, we did not differentiated between patients receiving alemtuzumab (more often used in FluMel patients with 80.7%) und ATG (more often used in FluTreo patients with 68.7%) among patients receiving in vivo TCD in the entire cohort. However, in a subgroup analysis, univariate subgroup analysis stratified on in vivo TCD with ATG or no in vivo TCD were consistent with the entire population , in patients with AML in CR, using registry data from the ALWP/EBMT. Both protocols showed similar outcomes regarding OS.Supplementary tablesSupplementary Figure 1


To validate the comprehensive complication index (CCI) for mini-percutaneous nephrolithotomy (mPCNL).Data from 287 patients who underwent mPCNL were analyzed. Complications after mPCNL were classified using both the CCI and the Clavien–Dindo classification (CDC). Descriptive statistics and linear/logistic regression analyses detailed the association between clinical predictors and mPCNL outcomes.r = 0.32; p < 0.01 vs. CDC: r = 0.26; p = 0.01). Patients with multiple complications had higher stone volume (p = 0.02), longer operative time and LOS . A higher rate of post-operative hospital readmission  and lower rate of stone free  were found in patients with multiple complications than in those with single complication. Linear regression analysis revealed that multiple complications were associated with longer LOS (p < 0.001) after accounting for BMI and stone volume. Similarly, having multiple complications was associated with fivefold higher risk of readmission (p = 0.02).After mPCNL, 83 (28.9%) patients had complications, of which 12 (4.2%) patients with multiple complications had a higher CCI score compared to the traditional CDC system accounting only for the highest grade. The CCI enabled a more accurate prediction of length of stay (LOS) than CDC (CCI: The CCI is a valuable metric for assessing post-operative complications after mPCNL. The cumulative CCI is a better predictor of LOS than the CDC for mPCNL. Minor complications not captured by the highest CDC score are relevant since patients with multiple complications have longer LOS and higher rate of readmission than those with single ones.The online version contains supplementary material available at 10.1007/s00345-022-04045-9. Percutaneous nephrolithotomy (PCNL) is the gold standard surgical technique for large renal stones in adult patients . VariousIn clinical practice, the Clavien–Dindo classification (CDC)  is the mTo this aim, the Comprehensive Complication Index (CCI) has been recently introduced in clinical practice to score the burden of post-operative adverse events . The CCIIn particular, we wanted to introduce the CCI in mPCNL, which is the most widely employed PCNL option by the urological community . TherefoWe performed a retrospective analysis of all consecutive patients who underwent mPCNL for renal stones at a single tertiary-referral academic center between January 2017 and December 2021. Mini-PCNL represented the technique of choice when PCNL was planned, except in case of complete or almost complete staghorn stones, for which standard PCNL (24 Ch) was indicated.Patient’s demographics were collected. Health significant comorbidities were scored with the Charlson comorbidity index (CHI), further segregated as 0 vs. ≥ 1 . Weight All procedures were performed by two experienced (> 150 PCNL performed) endourologists  in a standardized fashion as previously described , 25.Briefly, with the patient placed in the supine Valdivia position, under general anesthesia, a ureteral catheter was placed in the renal pelvis to inject contrast medium. Subsequently, the renal puncture was performed with combined fluoroscopic/ultrasonographic control and tract dilation was executed one-shot with a 16 Ch metallic dilator. A 16 Ch metallic sheath was placed and the 12 Ch Karl Storz “minimally invasive PCNL” (MIP) nephroscope was introduced . A 550 μNumber of the percutaneous tracts and operative time (OT) were recorded. The evaluated post-operative data included hemoglobin drop and length of hospital stay (LOS). According to internal protocol of our institution, uncomplicated procedures were managed as follows: on post-operative day one the bladder catheter was removed and the nephrostomy tube was closed; on post-operative day two a percutaneous pyelography was performed to assess ureteral canalization and the presence of residual stones. If ureteral canalization was confirmed, the nephrostomy tube was removed. Patients were discharged on post-operative day three.www.assesssurgery.com [Only events that occurred during the hospital stay or within 30 days of the initial procedure were considered. Post-operative complications were graded according to the PCNL-adjusted Clavien Score (CDC) . For thegery.com . Of notePatient counseling and follow-up were standardized among the cohort. All patients were instructed to return to the emergency department of the same hospital if they developed post-PCNL complications. Phone calls for the evaluation of possible complications were scheduled 15 and 30 days after surgery as per standard clinical protocol. Patients were evaluated within 3 months after surgery with CT scan to identify residual stones. The stone-free (SF) rate was defined as the absence of residual fragments. Patients with residual fragments were offered, according to stone dimension, observation or auxiliary procedures including second-look PCNL, extracorporeal shockwave lithotripsy, or retrograde intrarenal surgery.N = 15); scheduled staged procedures for large stone burden (N = 27); concomitant additional procedures other than PCNL (N = 15); endoscopic combined intrarenal surgery procedures (N = 2). A final cohort of 287 patients who underwent mPCNL for kidney stones was considered for statistical analysis.We excluded patients with congenital renal anomalies .Distribution of data was tested with the Shapiro–Wilk test. Data are presented as medians  or frequencies (proportions). Descriptive statistics were used to describe the whole cohort. Clinical parameters, intraoperative and post-operative characteristics were compared among patients with single vs. multiple post-operative complications with the Mann–Whitney test and Fisher Exact Test, as indicated.p < 0.05.Univariable and multivariable linear regression models were used to investigate potential predictors of hospitalization time. Similarly, logistic regression models tested the association between clinical variables and re-hospitalization event in the whole cohort. Statistical analyses were performed using SPSS v.26 . All tests were two sided, and statistical significance level was determined at 2, respectively. A CHI ≥ 1 was found in 105 (36.6%) participants. Above all, 191 (66.6%) patients had multiple stones, with a median stone volume of 2.2 (1.0–4.6) cm3. Multiple access tracts were used in 49 (17.1%) cases. Median operative time and LOS were 107 (80–140) min and 4 (3–6) days, respectively.Table Of 287, 83 (28.9%) patients had post-operative complications, of which 24 (8.4%), 45 15.7%), 10 (3.5%) and 4 (1.4%) were of highest CDC of I, II, IIIa and IIIb, respectively. Supplementary Table 1 reports the type and the severity of post-operative complications. Median CCI score was 20.9 (8.6–20.9) .Of 83 participants with post-operative adverse events, 12 (4.2%) patients had multiple complications resulting in a higher total CCI score compared to that calculated from the traditional CDC system accounting only for the highest grade [total CCI mean 24.8 (range 12.2–34.8) vs. non-cumulative CDC mean 19.8 (range 8.7–33.7), p = 0.02), longer operative time and LOS  than those with single adverse event. A higher rate of post-operative hospital readmission  and lower rate of stone free  were found in patients with multiple complications (Table Table p < 0.01) (Table p = 0.02), stone volume  and one  or multiple complications  were independent predictors of longer LOS, after accounting for CHI. Of clinical relevance, the average LOS for mPCNL with multiple complications was expected to be 5 days longer than that of surgeries with single adverse event (p < 0.001).Table 1) Table . Multivap = 0.02).Table This is the first study which introduces and validates the CCI in mPCNL cases for kidney stones. We found that the CCI was more accurate than the CDC in depicting the overall burden of post-operative complications and that the CCI enabled for a more precise prediction of LOS than the Clavien classification. Approximately 4.2% of patients showed a higher CCI value than that corresponding to their highest CDC grade, thus suggesting that the CCI is relevant in capturing minor complications which are underestimated by the Clavien–Dindo score. Of note, having multiple complications after mPCNL was associated with longer LOS and higher rates of hospital readmission.Quality control in urological surgery is crucial to ensure patient safety and improve patient care. Therefore, it is of utmost clinical importance that post-operative complications are reported systematically, objectively and reproducibly . To thisThe CCI has been first validated in major uro-oncological series. Vetterlein et al. analyzed a cohort of 506 patients treated with radical cystectomy for bladder cancer and showed that the proportion of cases with severe complications was higher using the CCI than the CDC (31% vs. 11%) . Of noter = 0.32; p < 0.01 vs. CDC: r = 0.26; p = 0.01). We showed that, along with patient’s BMI and stone volume, having multiple complications was independently associated with longer LOS. Of clinical importance, the average LOS for mPCNL with multiple complications was 5 days longer than that of surgeries with single adverse event. Therefore, in research setting and in clinical practice, the use of CCI, which takes into account multiple adverse events and not only the most severe, gain even more importance in terms of patient’s management and health-related costs [Nowadays, the miniaturization of the instruments has improved PCNL outcomes in terms of SFR and post-operative complications, widening the indications of PCNL to a greater range of stone volumes . In facted costs . We alsoThis study is innovative because it specifically validates the CCI in a series of mPCNL, which is the most preferred surgical technique for stone diseases by endourologists . TherefoOur study is not devoid of limitations. The retrospective nature of our investigation might lead to data loss in terms of complications; however, as for our internal protocol, patients were checked by phone calls 15 and 30 days post-operatively. Therefore, we can assume that our scoring of complication is rigorous. Also, this was a single center-based study, which raises the possibility of selection biases; thereof, larger studies across different centers and cohorts are needed to externally validate our findings. Lastly, the relatively low rate of complications in our cohort precluded the investigation of association between the CCI and more severe outcomes  in a multivariable fashion. For that reason, multicenter studies should further investigate the role of CCI in PCNL.In this study, we validated the CCI as a score for reporting post-operative complications after mPCNL. We found that the CCI was more accurate than the CDC in depicting the overall burden of post-operative complications and that the CCI enabled for a more precise prediction of LOS than the Clavien classification. Only 4.2% of patients had multiple complications leading to a higher CCI value than that corresponding to their highest CDC grade. Of note, having multiple complications after mPCNL was associated with longer LOS and higher rates of hospital readmission. Therefore, the CCI is relevant in clinical practice to capture the overall burden of complications, in terms of major and minor adverse events, which have a strong impact in patient’s management and health costs.Supplementary file1 (DOCX 13 KB)Below is the link to the electronic supplementary material.


Protein C is an anticoagulation agent, and protein C deficiency results in vascular thrombosis disease. Hereditary protein C deficiency is a risk factor for pulmonary embolism in adults. Pathogenic variants of the Protein C, Inactivator Of Coagulation Factors Va And VIIIa (PROC) gene which encodes protein C have been identified as a cause of protein C deficiency.We describe a patient with a novel mutation in the PROC gene who was diagnosed with pulmonary embolism in a Chinese family.According to the results of the pulmonary computed tomography angiography (CTA) and the level of blood protein C, the patient was diagnosed with pulmonary embolism caused by protein C deficiency.Whole-exome sequencing (WES) was performed for the molecular analysis.The results of patient’s deoxyribonucleic acid revealed a heterozygous mutation (c.237 + 5G > A) in intron 3 of the PROC gene. His father also harbored the same mutation in the PROC gene. We also reviewed the protein C deficiencies caused by PROC gene mutations in cases.A novel mutation in intron 3 of PROC gene has not been previously reported in patients with pulmonary embolism caused by protein C deficiency. After anticoagulation therapy, the patient recovered, and CT showed resolution of the thrombosis. Pulmonary embolism may be caused by protein C deficiency and the rare compound heterozygous mutation in intron 3 of the PROC gene could cause protein C deficiency via impairment of the secretory activity of protein C. It has been estimated that only 7% of the patients with PE who died were correctly diagnosed before death. Genetic abnormalities in proteins involved in the coagulation pathway leading to hypercoagulability have been found to be the cause of thrombophilia disease. Factor V Leiden (FV Leiden) and prothrombin G20210A gene mutations are highly prevalent in the Caucasian population. In contrast, in the Asian population, protein C (PC) and protein S (PS) deficiencies have a higher prevalence. PC is a vitamin K-dependent plasma zymogen that is synthesized mainly in the liver and plays an important roles in anticoagulation and fibrinolysis by inactivating the blood coagulation factors Va and VIIIa. Protein C is encoded by the Protein C, Inactivator Of Coagulation Factors Va And VIIIa (PROC) gene on chromosomes 2q13–q14 and is composed of 9 exons and 8 introns. Several studies have reported the spectrum of PROC mutation, and at least 300 mutations have been reported to date.Pulmonary embolism (PE) is responsible for more than 100,000 cardiovascular disease-related deaths annually in the United States and is the third leading cause of cardiovascular mortality.Here, we reported a patient diagnosed with pulmonary thromboembolism associated with protein C deficiency caused by a novel PROC gene mutation that has not been previously reported before.The PROC gene mutation analysis was performed in accordance with the ethical committee of the PLA General Hospital. Peripheral blood samples were also obtained from his parents after obtaining consent for deoxyribonucleic acid analysis.We use whole-exome sequencing (WES) in panel to determine the possible mutations. If possible pathogenic mutations were found, his parents mutations wound be identified.–31We searched PubMed for PROC gene mutations and protein C deficiency in English. Articles that lacked genetic analysis and basic patient information were excluded, and 26 studies were included in our study.A 23-year-old man was admitted to Hainan Hospital with headache for 24 days and hemoptysis for 4 days in 2018-3-12. The patient had headaches since February 2018 and hemoptysis since 2018-3-8. The patient was previously healthy and had a negative medical history. Magnetic resonance imaging of the brain was normal, and ultrasonography of both cephalic veins showed thrombosis.Physical examination revealed a temperature of 37.1 °C, pulse of 88 beats/minute, respiratory rate of 20 breaths/minute, and blood pressure of 130/80 mm Hg. The patient’s state of consciousness was poor. Crackles were heard in both lungs. His heart rate was normal, and no murmur was heard in the auscultation area of each valve. The abdomen was soft with no tenderness or rebound pain, and the liver and spleen ribs were not observed. Muscle strength was normal, and meningeal stimulation was negative.Laboratory tests showed the following results: white blood cell (WBC), 9.48 × 109/L , neutrophils: 0.747 , platelets: 507 × 109/L , C-reactive protein 13.15 mg/dL , interleukin-6 34.82 pg/mL (0.373–0.463 pg/mL), procalcitonin 0.040 ng/mL , D-dimer: 3654 ng/mL , international normalized ratio (INR):1.46 , prothrombin time, 15.7 s (11–13 s), fibrinogen level: 7.16 g/L . We further performed computed tomography angiography (CTA) of the pulmonary artery, which revealed an embolism in the middle segment of the left pulmonary artery  activity of 112.4%  and PC activity of 44.6%  were measured using the StaClot  PS and PC activity assay, which indicated a diagnosis of PC deficiency.Molecular analysis was performed by targeting inherited diseases presenting as vasculitis or coagulopathy using next-generation sequencing. A heterozygous pathogenic variant located on intron 3 of the PROC gene (c.237 + 5G > A(2q14|NM-000312)) were identified  anticoagulation at 14,000 IU per day (100 IU/kg) based on a weight of 70 kg. One month after treatment with LMWH, hemoptysis and chest pain were relieved. Subsequently, rivaroxaban (20 mg per day) was administered. The patient was discharged after taking rivaroxaban for 15 days without any adverse effects and continued taking rivaroxaban out of hospital. The symptoms of headache and hemoptysis disappeared, and reexamination of CTA of the pulmonary artery showed that the embolism had disappeared . There have been only been a few case reports of pulmonary embolism caused by PROC gene mutation.,33 In this case, we reported the case of an adult male with pulmonary embolism with a novel mutation in the PROC gene that had not been previously reported before. We further tested the genetic condition of the patients. His father had the same mutation; however, he did not show any evidence of deep venous thrombosis or pulmonary embolism. Therefore, we deduced that the intron 3 mutation (c.237 + 5G > A) in the PROC gene may be an autosomal recessive mutation, and patients with this mutation may or may not present with protein C deficiency.PC plays an important roles in anticoagulation and fibrinolysis and is often associated with pulmonary embolism. The incidence of heterozygous protein C deficiency is approximately 0.14% to 0.50% of the general population,,27,28,31 These PROC mutations are located in exons 4 to 9 and intron 8. Exons 7 and 9 were most frequent involved, most of the mutations were point mutations, 5 cases showed a del mutation of the PROC gene,,15,17,21 one case showed a frameshift mutation of duplicated of c246_247. The mutation in 20 cases were mutation in exons, which can directly change the expression of proteins. For example, changes in c.1152C > G leads to p.N384K (replacement of asparagine by lysine) and c.1207G > T leads to p.G403W (glycine by tryptophan). Changes in the homozygous missense mutation, c.1198G > A leads to the replacement of Gly with Ser in the 400 amino acid. However, not all cases had exon mutations; 1 case reported that a patient suffered from deep venous thrombosis at the age of 26 years, and genetic analysis showed a transition at nucleotide 7054 in intron 7 (7054G to A). The 7054 mutation G to A caused the amplified exon 8 fragment to cause the R87H mutation and led to protein C deficiency and deep venous thrombosis. Interestingly, our case also had a mutation of introns in the PROC gene, resulting in protein C deficiency. This suggests that introns may not be nonsense gene sequences; they may affect the expression of upstream and downstream exons. However, the exact mechanism of intron 3 mutation (c.237 + 5G > A) of the PROC gene requires further study to reveal its function.More than 300 mutations that disrupt protein C levels have been identified. Our article reviewed 26 cases showing that protein C deficiency with a PROC mutation is rare Table . These cIn conclusion, we identified a novel heterozygous mutation in the PROC gene in a patient with protein C deficiency, which has not been previously reported. The patient was treated with LMWH and rivaroxaban for anticoagulation, with good progress. This article provide a more complete gene mapping with PROC gene in Chinese population. We hope that our report will further assist other clinicians in diagnosing and treating this rare disease.The authors gratefully acknowledge the patient and his family for allowing us to share their information.Zhaorui Zhang participated in the writing of the paper. Yuzhu Li participated in the study design of the work. Zhen Yang participated in the primary data acquisition and clinical analysis. Mei Chen participated in the genetic analysis of patients.Conceptualization: Zhaorui Zhang, Zhen Yang, Yuzhu Li.Data curation: Zhaorui Zhang, Zhen Yang, Mei Chen, Yuzhu Li.Formal analysis: Zhen Yang, Yuzhu Li.Investigation: Zhen Yang, Yuzhu Li.Methodology: Zhen Yang, Yuzhu Li.Writing – original draft: Zhaorui Zhang.Writing – review & editing: Zhaorui Zhang.


Olea europaea methanolic extract (MEOE) and aqueous extract (AEOE) were orally administered to normoglycemic and alloxan-treated diabetic rats so as to determine their hypoglycemic effect. High-performance liquid chromatography (HPLC) analysis showed gallic acid, ferulic acid, quercetin, and vanillic acid in MEOE. It was found that the methanolic and aqueous extracts exhibited the maximum DPPH and alpha-amylase inhibition activities, respectively. MEOE and AEOE exerted a significant decline in the fasting blood sugar in diabetic animals (p < 0.05); however, they did not cause hypoglycemia in nondiabetic animals. Treatment with MEOE and AEOE reduced the aggravated liver and kidney function biomarkers. Aggravated levels of oxidative stress biomarkers including superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and malondialdehyde (MDA) were restored by treatment with MEOE. Moreover, MEOE improved the count of islets of Langerhans in the pancreas, fatty changes, and enlarged sinusoidal spaces in the liver and necrosis of the glomerulus and tubular cells of the kidney in diabetic rats. This study showed that the African olive seed extract effectively managed experimental diabetes and restored the normal functions and histology of the liver and kidney in diabetic rats through the reduction of oxidative stress.The aim of the present study was to evaluate the antioxidant and antidiabetic potential of Indian olive seed extracts. Plant seeds were sequentially extracted with n-hexane, chloroform, methanol, and water. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging and alpha-amylase inhibitory activities of extracts were carried out.  Diabetes mellitus (DM) is associated with an altered metabolism of lipids, proteins, and carbohydrates. It is the most widespread metabolic disorder in the world that affects about 2.8% population globally . AntidiaOlea europaea leaves showed in vitro antidiabetic and antioxidant activities [Several plants and their individual constituents have been investigated for antidiabetic potential in animal and human studies. More than five hundred plants belonging to 140 different genera have exhibited antidiabetic activity when investigated on the basis of ethnomedicinal significance . Extracttivities . A previtivities .Olea europaea subsp. Cuspidata  Cif. belongs to the family Oleaceae and is commonly called as wild olive, African olive, Kao, and Kahu [Olea europaea have exhibited antioxidant activity [Olea europaea [and Kahu . It is tand Kahu . Masliniactivity . Variouseuropaea .in vitro, and in vivo antidiabetic potential of different extracts of Olea europaea subsp. Cuspidata seeds so as to demonstrate their effectiveness in combating DM.This study was carried out to evaluate the antioxidant, Chloroform, n-hexane, dimethyl sulfoxide (DMSO), and sodium hydroxide were acquired from RDH®, Germany. Vitamin C, 3,5-dinitro-salicylic (DNS) acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), alpha-amylase, and starch were obtained from Unichem®, UK. Sodium potassium tartrate and alloxan monohydrate , glimepiride , and Glucometer (Roche Inc.) were also used in the study.Olea europaea subsp. Cuspidata were collected from the subtropical region of Pakistan . The plant was identified by the taxonomist at GC University, Lahore, who assigned voucher no LAH#322017B. Seeds were washed, dried, and ground to coarse form. For extraction, 200 gm of ground material was extracted with 1 L of n-hexane. The coarse powder was then extracted with equal volumes of chloroform, methanol, and distilled water sequentially by the Soxhlet apparatus. The extracts were collected and dried at 40°C with a rotary evaporator. Semisolid extracts were stored in a refrigerator until further evaluation [Seeds of aluation .Qualitative phytochemical screening of different plant extracts was carried out according to the previously described standard procedures .Olea europaea (MEOE) was carried out to quantify the phenolic acids and flavonoids by following the previous method. The plant extract (25 mg) was dissolved in 8 ml methanol and 12 ml water to prepare a sample for analysis. The mobile phase is comprised of two solvents with gradient elution. Solvent A consisted of methanol and acetonitrile at a ratio of 30 : 70, while solvent B comprised of water and acetic acid at a ratio of 6 : 94. A 20 μl sample was injected to reverse phase HPLC  equipped with a C18 column. The analytes were identified by matching with retention times of standard phytochemicals with a UV spectrum obtained at 275 nm, and the quantities were estimated from the area of standard phytochemicals [The chemical analysis of the methanolic extract of hemicals .The free radical scavenging activity of all extracts was evaluated by the DPPH inhibition method . A 40 mgin vitro antidiabetic activity of different extracts, alpha-amylase inhibitory activity was carried out by following the procedure described earlier [For an appraisal of  earlier . A 2.36 A 1 ml alpha-amylase solution was added to an equal volume of the extract solution and incubated for 10 min at 25°C. Then, 1 ml starch solution was added to it. The reaction mixture was incubated again for 30 min at 37°C followed by the addition of 1 ml DNS reagent as a stop solution. The reaction mixture was finally incubated for 5 min in a boiling water bath, cooled to 25°C, and the final volume was made up to 10 ml by adding distilled water. Absorbance was determined at 540 nm with a UV-visible spectrophotometer and percentage inhibition was calculated. Control incubation, showing 100% enzymatic activity, was prepared in the same way. However, the extract was replaced with the solvent in the control solution. Acarbose was used as a standard alpha-amylase inhibitor.Wistar rats of both sex  acquired from the University of Lahore were housed in stainless steel cages at 25 ± 02°C with a 12-hour day and night cycle with free access to standard pellet diet and water. The study was approved and conducted according to the guidelines of the Animal Ethics committee, the University of Lahore.Normoglycemic Wistar rats were orally given 250, 500, and 750 mg/kg MEOE and aqueous extract (AEOE) for one week. Fasting blood glucose (FBG) in rats was tabulated daily with a glucometer .A single intraperitoneal dose of alloxan monohydrate (150 mg/Kg) was administered to Wistar rats for the induction of DM . The serth day, the rats were anesthetized. Blood was collected by heart puncture for the determination of biochemical markers. Anesthetized rats were killed by cervical dislocation to remove the kidney and liver for histopathological evaluation. The complete blood count (CBC) and liver function tests (LFTs) and renal function tests (RFTs) were carried out on blood serum.Group 1 was comprised of nondiabetic rats which were orally administered with normal saline. Group 2 served as a standard control in which diabetic rats were treated orally with a standard antidiabetic agent, Glimepiride (0.2 mg/kg). Group 3 was comprised of diabetic rats (negative control) which received normal saline only. Other groups comprised of diabetic rats receiving an oral daily dose of 250, 500, or 750 mg/Kg of aqueous or methanolic extract. Treatment was carried out for 14 consecutive days. Blood samples were collected from tail veins to determine FBG with the glucometer. On the 15μm thickness) were prepared with a rotary microtome. The tissues embedded in paraffin were stained with eosin and hematoxylin [Isolated rat organs (liver and kidney) were kept in 10% formaldehyde. Small parts of the kidney and liver were embedded in paraffin wax, and tissue sections  pH 7.4 followed by centrifugation at 3000 rpm to collect the supernatant. The protein content in the tissues was estimated by the Lowry method. The oxidative stress biomarkers were determined by following earlier procedures .To determine the SOD activity in liver and kidney tissue homogenates, equal volumes of tissue homogenate and pyrogallol solution (0.1 ml) were mixed with 2.8 ml of PBS. Changes in absorbance were determined 5 min apart with a UV-Vis spectrophotometer at 325 nm. The activity of SOD was expressed as U/mg of protein.μl supernatant, 1.95 ml phosphate buffer of pH 7, and 1 ml 30 mM hydrogen peroxide were admixed, and absorbance was determined at 240 nm 0.5 min apart with UV-Vis spectrophotometer. The activity of CAT was expressed as U/mg of protein.To determine the CAT activity, 50 μg/mg of protein.To determine the GSH level in the liver and kidney tissue homogenates, 1 ml tissue supernatant was precipitated with an equal volume of 10% trichloroacetic acid solution followed by the addition of 4 ml PBS. The absorbance of the resultant solution was determined after adding 0.5 ml 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) reagent. The GSH amount was expressed as The level of MDA in the liver and kidney tissue homogenates was determined by mixing 1 ml tissue homogenate with 3 ml thiobarbituric acid reagent. The solution was thoroughly mixed and heated over a water bath. The absorbance of the cooled solution was determined at 532 nm which was obtained after centrifugation at 3000 rpm for 10 min. The amount of MDA was expressed as nM/ml.The data were presented as mean ± standard deviation. Statistical analysis of in vitro antioxidant and alpha-amylase inhibitory activities were performed by two-way analysis of variance (ANOVA) by GraphPad Prism®. The in vivo parameters were analyzed by one-way ANOVA. In case of statistically significant results, Tukey's post hoc evaluation was carried out.Qualitative phytochemical analysis revealed that the MEOE was comprised of carbohydrates, glycosides, saponin, proteins, fixed oils, tannins, and phenols. AEOE consisted of alkaloids, saponin, and proteins. Only saponins and fixed oils were present in chloroform and n-hexane extracts.HPLC analysis of the plant methanolic extract revealed the presence of gallic acid, ferulic, and quercetin. Vanillic acid (37.755 ppm) was present in the highest amount in the extract followed by ferulic acid, quercetin, and gallic acid. The phytochemicals detected in the plant extract along with the retention time, area, and concentration are presented in μg/ml concentration followed by chloroform (90.73%), aqueous (82.21%), and n-hexane (81.63%) at the same concentration.In vitro free radical scavenging activity of seed extracts showed that the methanolic extract exhibited the maximum percentage inhibition (91.2%) at 500 μg/ml, respectively. Detailed results of the free radical scavenging activity of all seed extracts are shown in The decreasing order of DPPH scavenging activity was methanolic > chloroform < aqueous >  n-hexane. IC50 of methanolic extract, aqueous extract, chloroform extract, n-Hexane extract, and ascorbic acid were 311.9, 118.1, 153.4, 112.5, and 136.3 It was also demonstrated that the aqueous extract exhibited the maximum percentage inhibition of alpha-amylase (82.10%) at 1.6 mg/ml concentration followed by chloroform (65.60%), methanol (64.90%), and n-hexane extract (58.25%). IC50 was of methanolic extract, aqueous extract, chloroform extract, n-Hexane extract, and acarbose of 1.331, 0.3194, 0.3730, 0.9374, and 0.1105 mg/ml, respectively. A concentration-dependent effect was observed as shown in It was found that the statistically considerable difference was not observed in the FBG of normal rats and extract-treated rats at 250, 500, and 750 mg/kg/day dosage of the aqueous and methanolic extracts. The results of the effect of plant seed extracts on normoglycemic rats are shown in MEOE and AEOE  in diabetic rats showed a remarkable hypoglycemic effect. It was revealed that all extract-treated groups showed a significant reduction FBG when compared to untreated diabetic rats. The FBG levels of untreated and treated diabetic animals are shown in It was seen that the administration of seed extracts had reversed weight loss in diabetic rats. A statistically significant difference in treated and untreated diabetic rats was found at day 14 .p < 0.05) in the level of renal and liver function markers of the extract-treated animals opposite to diseased animals  the body weight of rats than untreated diabetic rats. It also showed that Olea europaea subsp. Cuspidata had tried to restore the body weight possibly through normalizing metabolic processes of the body. However, there was no significant difference in the hematological parameters of the experimental animals which showed that the administration of aqueous and methanolic extracts were devoid of any toxicity [Citrullus colocynthis, had also shown hypoglycemic potential and body weight gain in diabetic rats [A statically nonsignificant hypoglycemic effect was seen in normoglycemic animals which showed that the olive seed extracts might be devoid of any hypoglycemic effect in normoglycemic individuals in contrast to the hypoglycemic effect in diabetic rats . It is atoxicity . The livtic rats . It can tic rats .It is previously established that alloxan administration had resulted in the generation of reactive oxygen species (ROS) through cyclic redox reactions of dialuric acid through metabolism . Thus, dThis study shows that African olive seed extracts slightly restored body weight possibly by normalizing the metabolic processes of the body. Evaluation of complete blood count among untreated and treated diabetic rats showed that the treatment with MEOE was devoid of any pathological changes in blood cell count .It is evident from the current study that MEMO reduced the alloxan-induced hepatic damage in diabetic rats. Previously, it is known that olive oil had reduced the hepatotoxicity induced by ethanol or acetaminophen by the reduction of oxidative stress due to the presence of phytochemicals such as gallic acid . MoreoveOlea europaea subsp. Cuspidata seeds were helpful in managing DM due to their antioxidant and alpha-amylase inhibitory activities. Antioxidant and antidiabetic activities of the plant seed extract may be due to the presence of specific phytoconstituents such as gallic acid, quercetin, ferulic acid, and vanillic acid which resulted in a reduction of oxidative stress. The seeds of African olive may serve to relieve the long-term complications associated with DM. There is a need to elucidate the molecular mechanism involved to treat diabetes.In the light of the above findings, it can be concluded that


In some patients with congenital biliary dilation (CBD), biliary cirrhosis has been reported to rapidly progress from the neonatal period to the infantile period. We herein report an infantile case of CBD that showed severe biliary cirrhosis at the diagnosis, which was treated by laparoscopic surgery.3, 123 μg/dl). We initially performed laparoscopic exploration and bile drainage via the gallbladder, noting severe hepatic fibrosis resembling end-stage liver cirrhosis. After placing a drainage tube in the gallbladder, cholangiography was performed. Cholangiography showed Todani type IVa CBD with pancreaticobiliary maljunction. Contrast agent flowing into the duodenum could not be confirmed. The patient received liver-supporting therapy and nutritional support for 7 weeks before definitive surgery. Following the improvement of the hepatic synthetic capacity , we performed extrahepatic bile duct excision and hepaticojejunostomy laparoscopically. Laparoscopic surgery was successfully performed along with liver biopsy. Histopathologically, the liver specimen showed chronic hepatitis and fibrosis (F3A2). Biliary scintigraphy showed good bile excretion at postoperative day 15. The postoperative course uneventful, and the patient was discharged on the 23rd day after surgery. A needle liver biopsy six months later showed mild improvement of chronic hepatitis and fibrosis (F2-3A1). The patient was regularly followed at the outpatient clinic.A 16-month-old girl underwent conservative therapy for liver dysfunction and cholangitis on a remote island of our prefecture. She was transferred to our hospital after the detection of a huge dilated common bile duct on imaging at the previous hospital. Contrast-enhanced computed tomography showed a dilated common bile duct (maximum diameter: 5 cm), thus suggesting CBD. However, her laboratory data on admission showed a poor nutritional status and severe liver dysfunction (Alb, 2.5 mg/dl; AST, 79 IU/L; ALT, 43 IU/L; γ-GTP, 491 mg/dl; D-bil, 0.3 mg/dl; CHE, 90 IU/L; NHSevere liver fibrosis was suspected to be continuous cholestasis of CBD after birth. CBD with severe liver fibrosis may avoid liver transplantation by two-stage surgery with hepatoprotection therapy and immunonutritional support. Congenital biliary dilatation (CBD) is more in some Asian region as than in the Western countries, including United States, and some Asian populations have an incidence as high as 1 in 1,000 births . In contThe patient was a 16-month-old girl. No prenatal abnormalities were noted. She visited a nearby doctor on a remote island for fever and was treated with antibiotic therapy as an outpatient. She gradually developed clouding of consciousness and dyspnea. She was transferred to our hospital on the 13th day after the onset of symptoms due to a huge dilated common bile duct that was detected on imaging at the previous hospital.3, 123 μg/dl)  , suggest3 μg/dl) . The Chiirrhosis . A 5-mm irrhosis . Cholang3, 75 μg/dl)  were inserted into the right upper abdomen  and at the right side of the umbilicus , the left lateral abdomen , and the left upper abdomen . The dilated CBD was dissected and then taping was performed. After imaging the lower bile duct and confirming that the bile duct on the side of the pancreas was sufficiently detached, the lower bile duct was clipped and transected. Subsequently, after dissection and transection of the cystic duct, the hepatic duct just above the dilated common hepatic duct was transected. The jejunum was then extracted from the umbilical wound, and Roux-en Y jejunojejunostomy was performed. The mucosa and serosa of the opened hole was approximated using 6–0 absorbable sutures to secure hepaticojejunostomy. The jejunum was pulled up through the retro-colic route. Both the posterior and anterior walls were approximated using interrupted intracorporeal knot-tying with 5–0 absorbable sutures. Laparoscopic surgery was successfully performed along with liver biopsy. Histopathologically, the liver specimen showed chronic hepatitis and fibrosis (F3A2) based on the new Inuyama classification  , we perf5 μg/dl) . Laparosfication  , type Ic (n = 2) and type IVa (n = 4), and their ages ranged from 0.7 to 37.3 years. The grade of liver fibrosis ranges from F2 to F4. The causes leading to liver transplantation were postoperative portal vein thrombosis, postoperative anastomotic stenosis, repeated shunt surgery, recurrent cholangitis and pancreatitis, and slowly progressive postoperative cirrhosis. Many of these causes underwent liver transplantation due to the worsening clinical course after extrahepatic bile duct resection and hepaticoenterostomy although these CC patients are not indicated for liver transplantation at the time of initial surgery. We evaluate that our case was not indication for liver transplantation at the time of diagnosis. Liver transplantation may be avoided with appropriate therapeutic intervention in many of the liver fibrosis patients with CC. However, there are various reports of liver cirrhosis in the neonatal period and reports of the progression of postoperative liver fibrosis (Our search of the PubMed database using the keywords , yielded 46 reports. Most of the reports describing cases in which CBD led to liver transplantation involved patients with Caroli disease, which is a rare inherited disorder characterized by cystic dilatation of the bile ducts within the liver. On the other hand, type I–IVa CBD was reported in only 6 patients by Hori et al.  and 1 pafibrosis ; thus, cfibrosis . Some CBfibrosis . Oral nufibrosis , but it fibrosis . A hepat


P < 0.001). Comparison between NSSDE and healthy group demonstrated that DC density, dendrites and field were significantly higher in NSSDE. However, there was no significant difference in DC size (P = 0.076). DC density and morphological parameters showed significant associations with the systemic severity  and ocular surface damage. The corneal epithelium DC density and morphological alterations were obvious in SSDE, which reflected higher level of immune activation and inflammatory response in SS. Marked correlations were found between DC density/morphology and systemic/ocular severity. Dynamic assessment of corneal DC may facilitate to clarify pathogenesis, stratify patient, and tailor treatment in SS patients.We aimed to investigate the density and morphology of corneal dendritic cells (DCs) in dry eye (DE) patients with or without Sjogren’s syndrome (SS). This study included 28 patients with Sjogren’s syndrome dry eye (SSDE), 33 patients with non-Sjogren’s syndrome dry eye (NSSDE), and 30 age and sex matched healthy volunteers. In vivo confocal microscopy (IVCM) was used to investigate density and morphology  of DC. Compared with NSSDE and healthy group, SSDE showed significantly higher DC density, larger DC size, more DC dendrites with larger DC field (all  Additionally, the most common form, aqueous tear deficient DE, can be subdivided into Sjogren’s syndrome dry eye (SSDE), associated with a progressive autoimmune disease characterized by chronic lymphocyte infiltration of the salivary and lacrimal glands, and non-Sjogren’s syndrome dry eye (NSSDE), tear deficiency by other causes2. The SSDE patients typically have been suffering terrible xerophthalmia and ocular surface injury for a long time, with multiple organ dysfunction and even under the threat of blindness and death (because of multiple organ failure and non-Hodgkin’s lymphoma)4. Therefore, early diagnosis and appropriate treatment play important roles in SSDE management.Dry eye disease (DED) is one of the most frequent ophthalmic multifactorial disorders, which is assigned to aqueous tear deficient dry eye (DE) and evaporative DE by mechanism5. Recent researches have shown that immune and inflammation play key roles in the pathogenesis of DED6, especially in SSDE and lead to severe xerophthalmia and ocular surface damage7. There is an increasing evidence that antigen-presenting cells (APCs), especially corneal dendritic cells (DCs) which are equipped to induce T-cell activation and inflammatory cascade, are crucial for the DED pathogenesis11. With the help of in vivo confocal microscopy (IVCM), the density and morphology of DCs in DED have been identified and provide a better insight to the pathogenesis of clinical manifestations7.DED is characterized by tear film instability, visual disturbance, inflammation and damage of the ocular surface12. Some studies also demonstrated increased DCs in the central corneal epithelium of SSDE7. Besides quantity, DCs morphology changes  are other biomarkers in response to inflammation and immunity15. Nevertheless, these studies have limitations such as small sample sizes and non-detailed DC parameters. Moreover, the correlations between the DC parameters and systematic/ocular characteristics may aid in the pathogenesis elucidation and severity assessment of SS. But there was no study has clarified the associations between the DC and serum antibodies/salivary gland biopsy in SS. We aimed to evaluate the DC density and morphology  of the central corneal epithelium in normal subjects and DE patients with or without SS. Such a determination was made to clarify whether there was an association among these parameters and the pathogenesis and severity of SS.In patients with Sjogren’s syndrome (SS), DCs have been found to infiltrate into the lacrimal and salivary glands in an ex vivo studySixty-one consecutive patients with aqueous tear deficient DE, including 28 with SSDE and 33 with NSSDE, and 30 healthy volunteers were enrolled. Only the right eye was evaluated. The study was performed according to the principles of the Declaration of Helsinki and was approved by the Human Research and Ethics Committee, Peking University Third Hospital (No. M2019236). Written informed consent was obtained from each study participant.16. The diagnosis of SS was made according to the American College of Rheumatology-European League Against Rheumatism (ACR-EULAR) criteria3. The DED patients without SS were categorized to NSSDE group16. The healthy controls were asymptomatic participants without any ocular injury and medication, with normal tear functions and negative ocular surface staining (OSS)16. The exclusion criteria included the systemic/ocular disease except for DE and SS, especially ocular inflammation , rheumatoid arthritis, systemic lupus erythematosus and other diseases which might affect DC density and morphology, history of contact lenses wear and ocular surgery, diabetes mellitus, and pregnancy. Since the anti-inflammatory medications are likely affecting the ocular surface, the patients enrolled in the study have not been treated with these medications.DED was defined according to the following international diagnostic criteria by the Dry Eye WorkShop II (DEWS II) of the Tear Film and Ocular Surface Society (TFOS): ocular surface disease index (OSDI) ≥ 13 and tear break-up time (TBUT) < 10 s or ocular surface staining 16. The TMH was recorded with a Keratograph 5 M  and repeated three times, then the average value was analyzed. The OSS score (0–12) for each eye was the summation of the fluorescein score for the cornea and the lissamine green score for the nasal and temporal bulbar conjunctiva16. Serum levels of anti-SSA52, anti-SSA, anti-SSB antibodies and salivary gland biopsy were assessed in DE patients according to the diagnostic criteria3. All salivary gland biopsies were performed by an experienced dentist and evaluated by the same experienced pathologist. Salivary gland biopsies were collected as follows17: (1) evert the lower lip to visualize the minor salivary glands; (2) inject 0.5–1.0 mL 1:100,000 1% lidocaine:epinephrine solution into the submucosa; (3) make a 1.0–1.5 cm linear incision, parallel to the vermilion border; (4) Blunt dissect on a plane perpendicular to the mucosal incision and parallel to the direction of the sensory nerve fibers; (5) recover 4–7 glands from each patient and store them in 10% formaldehyde; (6) close the incision with simple interrupted 4–0 chromic sutures. The salivary gland biopsies were stained with hematoxylin–eosin (HE) and analyzed under microscope. The result was considered positive if the focus score (defined as the number of mononuclear infiltrates containing ≥ 50 lymphocytes/4 mm2 of glandular tissue) was ≥ 117. All examinations were carried out in the same examination room under the same conditions. The eye and systemic measurements were performed by the same masked observers.A careful case history and the OSDI were recorded for each participant. Schirmer-I test (ST), tear meniscus height (TMH), TBUT and OSS score were performed to evaluate aqueous production, tear film stability and ocular damage using standard protocols18. The corneal sub-basal nerves shift centripetally and form a corneal vortex just inferior to the nose of the corneal apex20. The corneal vortex is a land mark of central cornea and easy to identify with IVCM20. At least 100 images of corneal sub-basal layer (approximately at a depth of 50–70 μm) were obtained in each eye by the same masked professional technician. According to previous literature, three non-overlapping images with optimal quality in the central cornea were chosen for corneal sub-basal nerve23 or DCs analysis24. Therefore, three images with the best focused and contrast, without motion or folds were chosen for analysis. DC density and morphology were determined for each image, the morphology parameters including DC size (represented the DC activation and scored based on the cell body area with processes number), number of dendrites per DC and DC field (area bounded within cell span and the length of dendritic processes).All participants had undergone imaging with a digital corneal confocal laser-scanning microscope of the central cornea using Heidelberg Retina Tomograph 3 with the sequence mode . After topical anesthesia with 0.4% oxybuprocaine hydrochloride . A drop of carbomer  was served as a lubricant gel both in ocular surface and the disposable sterile polymethylmethacrylate cap . Then advanced the cap manually until the gel contacted the central corneal surface as previously dsecribed6. The DC size was further scored based on a 3-point grading scale (1–3): 1 (globular cell: hyperreflective cell body without dendritic process), 2 , 3 (large cell body with more than two processes)27. The number of dendrites per cell was calculated manually. The DC field was measured using a polygon joining the dendrite tips around each cell according to Kheirkhah et al. study6. Two masked observers assessed all parameters and the averaged values were calculated for further analysis. A third observer is needed if more than 10% difference between the two observers and the average of three values was recorded for analysis.A semiautomatic Java-based image processing software  dedicated to the tracing and quantification of elongated image structures, such as dendrites and neurons, were used to evaluate the density and morphologic parameters of DC in IVCM images. The Cell Count tool was used to count DC density and the average for three images was recorded. The 10 most representative DCs (the largest cell body with more dendrites) in three images were selected for morphologic analysis according to Kheirkhah et al. studyP value < 0.05 was considered significant for all comparisons. The sample size was calculated by Stata/SE 15.0 . According to previous literature7, the DC density in the central corneal epithelium of SSDE, NSSDE and healthy controls were 127.9 ± 23.7 cells/mm2, 89.8 ± 10.8 cells/mm2, and 34.9 ± 5.7 cells/mm2, respectively. The sample size of each group was determined to be 12 eyes (α = 0.05 and β = 0.10). However, small sample size may be difficult to further clarify the associations among these parameters and pathogenesis and severity of SS. Therefore, we enrolled 28 eyes in SSDE group, 30 eyes in NSSDE group and 30 eyes in healthy group.Statistical analysis was performed using SPSS version 26.0 . The normality of the data distribution was verified by the Kolmogorov–Smirnov test. Continuous variables were described as the mean ± standard deviation (SD) or median with interquartile range. Categorical variables were expressed as frequencies and percentages. Continuous variables were compared with ANOVA or Kruskal–Wallis tests among three groups. Continuous data between two groups were compared using Least Significant Difference (LSD) tests or Mann–Whitney nonparametric U-tests. Categorical variables were compared with the Chi Square test. Spearman’s rank order correlation was performed to identify the potential factors related to the density and morphologic parameters of DC from the variables of age, sex, systemic characteristics, as well as dry eye symptoms and signs. Logistic regression analysis and multiple linear regression analysis were used to evaluate the validity of DC density and morphology  compared with salivary gland biopsy/serum antibodies and ocular parameters  in SSDE patients, respectively. Since the OSS did not show a continuous distribution, normality transition (a logarithmic transformation) was performed before regression analysis. A P = 0.052 and P = 0.088, respectively). Compared with the NSSDE patients and the healthy controls, the participants in the SSDE group showed higher OSDI (67.88 ± 12.25) and OSS [4 ] scores, and lower ST (1.43 ± 0.92), TMH (0.09 ± 0.02) and TBUT (1.94 ± 0.72) . The NSSDE patients also showed more serious OSDI score (35.12 ± 11.23 vs. 8.61 ± 1.71) and signs  than the healthy controls .This study included 28 eyes of 28 patients with SSDE , 33 eyes of 33 patients with NSSDE  and 30 eyes of 30 healthy controls . The mean age was 56.61 ± 15.01 years  in the SSDE group, 50.41 ± 13.53 years  in the NSSDE group, and 57.73 ± 8.42 years  in the control group. The demographic data and systematic/ocular characteristics of the three groups have been presented in Table 2), DC size [3 ], number of dendrites (3.84 ± 0.44), and DC field (393.21 ± 57.13 μm2) in the SSDE group . Compared with the healthy control group, the patients with NSSDE had a higher DC density , higher number of dendrites per cell , and larger DC field . However, the difference in DC size between the healthy controls and NSSDE group was not statistically significant .Table P values were shown in Table P < 0.001). Positive correlations were also noted among anti-SSA52, anti-SSA, anti-SSB and DC dendrites ; anti-SSA52, anti-SSA and DC size ; as well as anti-SSA and DC field (r = 0.302) . OSDI scores correlated positively with DC density , DC dendrite  and DC field . Meanwhile, ST, TMH, and TBUT showed inverse correlations with the DC density  and DC field . Moreover, TBUT also demonstrated an inverse correlation with DC dendrites . The OSS score showed marked positively correlations with DC density , DC dendrites , and DC field , however, a weak positive correlation with DC size .To determine whether there is a correlation between DC density/morphology and systematic/ocular manifestations of SS, we performed Spearman’s rank order correlation analysis of clinical data and IVCM parameters. The correlation coefficients (r) and P = 0.046] for positive salivary gland biopsy. DC size had an OR 1.319  for positive anti-SSA. DC dendrites had an OR 1.980  for positive salivary gland biopsy and OR 1.473  for positive anti-SSA. DC field presented an OR 2.073  for positive salivary gland biopsy. No significant association of DC parameters with anti-SSA52 and anti-SSB was observed .The associations between DC density/morphology and systematic manifestations  in SSDE are shown in Fig. P = 0.049, per 1 cells/mm2 increase], increased DC dendrites , and increased DC field . Significant associations were shown between increased OSS and higher DC density , DC dendrites , and DC field . Higher DC density was also associated with decreased ST  and TBUT . Higher DC field was associated with decreased ST  and TMH .The effects of DC parameters on ocular features in SSDE patients are shown in Table 29. Development of IVCM made it feasible to investigate and quantify DCs alterations in various ocular surface diseases, such as DED33, keratitis18, contact lens wear35, and corneal graft rejection36, both in DC density and morphology. This study showed significant increases in DC density and morphologic parameters  in aqueous tear-deficient DE patients with or without SS. Furthermore, as compared with NSSDE patients, increased DC density with higher activation state (higher DC size scores) and larger DC fields with more dendrites were noted in patients with SSDE. Additional, DC density and morphologic parameters showed marked correlations with systemic severity  and ocular surface discomfort (OSDI) as well as damage . Those results may contribute to stratify patient and evaluate treatment effect.Corneal epithelial APCs, particularly DCs, play an essential role in corneal immune surveillance and homeostasis, including the initiation and induction of ocular surface immune reactivity and tolerance4. However, it should be bear in mind that SS is a chronic inflammatory disease with more severe clinical manifestations than NSS4. Furthermore, comparison of SSDE and NSSDE revealed that DC density was higher in the SSDE subtype (P < 0.001). Therefore, corneal immune and inflammatory responses were seen in both subtypes of aqueous tear-deficient DE and were more severe in SSDE. This was in accordance with previous reports, increased DC density was found in eyes with SS compared with non-SS31. Furthermore, it was reported by Lin et al. that the density and size of DC increased in the central cornea of DED with and without SS, whereas higher DC density and larger DC size in peripheral corneas were only found in SS patients7. In addition, Wakamatsu and coworkers demonstrated that increased DC number was found both in cornea and conjunctiva of patients with SSDE compared with NSSDE37.In our study, the SSDE and NSSDE patients demonstrated differential degrees of increased DC parameters Table  and 2. C38. Notably, the upregulation of MHC-II DC has been verified in patients with SS and implicated in this complex autoimmune disorder40. Interestingly, human leukocyte antigen (HLA)-DR, encoded specific MHC II DC, is upregulated in SSDE and NSSDE patients42. However, the levels of HLA-DR are significantly higher in SSDE42. These researches suggested that HLA-DR upregulation and MHC II DC overexpression played essential roles in the pathophysiology of aqueous tear-deficient DE, especially in SSDE. Clinically, upregulated HLA-DR has been acted as an active marker in SSDE, and reduced HLA-DR expression could be observed in experimental testing of the treatment efficacy44. Since the visibility of DC density and morphology with IVCM, which reflected the corneal immune and inflammatory changes. It may be possible to evaluate the activity of the immune system and the severity of the inflammatory response using IVCM, and help to stratify patient and tailor treatment.Corneal DCs will increase and mature throughout the cornea on inflammatory stimulation, with the increased expression of major histocompatibility complex (MHC) class II and co-stimulatory molecules45. It is well known that mature DCs are larger with longer processes, whereas immature DCs are smaller ones and lack dendrites46. In our study, we found that small DCs with lacked dendritic extensions often seen in healthy controls , whereas inverse correlations with hyposecretion conditions (ST and TMH) and tear film instability (TBUT). In other words, there were positive correlations between the density and morphology of the DC and eye severity in SS patients. The presence of DCs as the immune sentinel and biomarker of the corneal immune response has been identified both in animal and human studies47, and simultaneously MHC class II molecules were also upregulated in salivary gland in SS patients49. With continuous injury to the system and ocular surface in SS patients, the cycle of antigen recognition, immune response, and inflammatory damage are perpetuating and exacerbating39. Thus, the density and the morphologic features of DCs may provide an index for evaluating immunoinflammatory status in SS patients. Since the salivary gland biopsy and serum antibody tests are not feasible during repeated follow-ups in SS patients, the observation of DC density and morphologic parameters with IVCM may be used as an approach to assess the activity and severity of immune and inflammation in SS and may also facilitate to early diagnose as well as monitor the therapeutic effect.In addition, it is worth noting that the associations between salivary gland biopsy and DC density (OR = 2.292), DC dendrites (OR = 1.980) as well as DC field (OR = 2.073), between anti-SSA antibody and DC size (OR = 1.319) as well as DC dendrites (OR = 1.473). These results demonstrated that increased DC density and morphology changes could result in higher risks of positive salivary gland biopsy and anti-SSA antibody in SS. Previous studies have identified that DCs were significantly increased in the central corneal epithelium47. With immune and inflammatory stimuli, corneal DCs, which at least in part recruited from the blood, are increased and activated50. The blood-derived DCs initially reach the limbus and later migrate to peripheral and central cornea47. Both in humans and rodents, the DCs have been reported to reside very close to the corneal nerves51. According to the neuro-immune interaction, corneal nerves play an important role in DCs morphological changes and kinetic alterations47. In this present study, there was a clear difference on corneal nerve density and morphology among SSDE, NSSDE and healthy controls (shown in Fig. Resident corneal immune cells are considered long-lived and can maintain corneal homeostasis6. Moreover, the meibomian gland may also affect the DC density and morphology and should be demonstrate in further research. Second, only the central cornea was investigated in our study since previous research has found that DC changes were more obvious in the central than the peripheral cornea in SSDE7. The DC parameters in the peripheral cornea/conjunctiva cannot be extrapolated. Currently a study with larger patients is underway to assess the DC changes both in central and peripheral cornea. Third, further larger sample investigations are needed to determine the efficacy of using DC density and morphology to evaluate the therapeutic effects of DED.Inevitably, our study has several limitations. First, the evaporative DE was not included in this study, which also showed DC alterations in previous studyNevertheless, this present study provides a quantitative description of DC density and morphology in DE patients with or without SS. Moreover, DC parameters showed significant correlations with activity and severity of SS and DED. Additionally, the demonstration of differential responses in DC parameters may further elucidate the pathogenesis of this complex disease.In summary, the SSDE are characterized by increased DC density, higher DC activation, more dendrites and larger fields. Such DC alterations could contribute to activity and severity of SS and can be detected and quantified by IVCM objectively, allowing its use in evaluating pathogenesis and guiding clinical treatment.