Traditional Chinese medicine (TCM) prescriptions have multiple bioactive properties. “Gui Zhi–Shao Yao” herb pair is widely used to treat chronic pain (CP), as well as anxiety and depression. However, its related targets and underlying mechanisms have not been deciphered.In this study, the network pharmacology method was used to explore the bioactive components and targets of “Gui Zhi–Shao Yao” herb pair and further elucidate its potential biological mechanisms of action in the treatment of CP with comorbid anxiety disorder (AD) and mental depression (MD).Following a series of analyses, we identified 15 active compounds, hitting 130 potential targets. After the intersections the targets of this herb pair and CP, AD and MD – sorted by the value of degree – nine targets were identified as the vital ones: Akt1, IL6, TNF, PTGS2, JUN, CASP3, MAPK8, PPARγ and NOS3. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results demonstrated 11 pathways, such as AGE-RAGE signalling pathway, IL-17 signalling pathway, TNF signalling pathway, which primarily participate in the pathological processes.In vivo and in vitro experiments will be required to further investigate the mechanisms.A network pharmacology approach was applied to identify key targets and molecular mechanisms.Nine targets were regarded as the vital targets for chronic pain with comorbid anxiety and depression.Predicted 11 pathways were the potential therapy targets and pharmacological mechanism of “Gui Zhi–Shao Yao” herb pair.This study preliminarily predicted and verified the pharmacological and molecular mechanisms of “Gui Zhi–Shao Yao” herb pair for treating CP with comorbid AD and MD from a holistic perspective.  Recently, the International Association for the Study of Pain (IASP) has revised the definition of pain to “an unpleasant sensory and emotional experience associated with, or resembling that associated with actual or potential tissue damage” . Among vRamulus Cinnamomi (Gui Zhi in Chinese)–Paeonia lactiflora (Shao Yao in Chinese) is a classic traditional Chinese herb pair, which can reconcile the camp and guard, clear heat and relieve pain, and warm meridians and dredge collaterals [Traditional Chinese medicine (TCM) is a treasure of China and has formed a unique and complete theoretical system different from Western medicine . Chineselaterals . AccordiWith the development of system biology, bioinformatics and high-throughput histology, the network pharmacology technology, which integrates pharmacology and information network, has attracted much more attentions. The major features of TCM  and the characteristics of Chinese herb pair  are consistent with the main view of the emerging concept of network pharmacology ,11,12. Thttps://tcmspw.com/tcmsp.php, version 2.3) with screening conditions of oral bioavailability (OB) ≥30%, drug-likeness (DL) ≥0.18 and half-life (HL) ≥4 h, as described before [http://www.megabionet.org/tcmid/) through SwissADME [http://www.swissadme.ch/). After deleting the duplicate data, there were 26 herbal compounds of Gui Zhi and 22 herbal compounds of Shao Yao (Table S1).The chemical compounds of each herb in this herb pair were obtained from (1) Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform   [http://www.swisstargetprediction.ch/) [Homo sapiens”. We took the intersections of the above two difference analyses, yielding a total of 130 targets genes. Then, the National Centre for Biotechnology Information’s (NCBI) Gene database (www.ncbi.nlm.nih.gov/gene) [Homo sapiens” (Table S2).We input all the active ingredients into PubChem (ih.gov/)  – the woion.ch/)  and TCMSov/gene)  was usedhttps://www.disgenet.org/, version 7.0) [https://www.genecards.org/, version 5.0) [The gene targets associated with diseases were collected from DisGeNET (ion 7.0) , a discoion 5.0) , a searchttp://jvenn.toulouse.inra.fr/app/example.html) [https://string-db.org/, version 11.0) [Homo sapiens” and a medium confidence score with correlation degree ≥0.400 as the cut-off value. PowerPoint  was utilized to render the results.We utilized an online tool, jvenn (le.html) , to takeon 11.0)  – a datahttps://metascape.org/) [p value cut-off, .01). The bar chart of GO enrichment analysis and bubble chart of KEGG pathway chart were visualized by http://www.bioinformatics.com.cn, an online platform for data analysis and visualization.The Metascape (pe.org/)  was usedNetwork construction was performed using the network visualization software Cytoscape (version 3.7.2) as follows: (1) compound–compound target network, which connects the chemical compounds of this herb pair and the related targets; (2) “Gui Zhi–Shao Yao” herb pair–diseases PPI network obtained from the STRING; (3) targets–disease–KEGG pathway network.A total of 130 predicted targets were identified from 15 candidate compounds of the “Gui Zhi–Shao Yao” herb pair ; the othTable S3.The Venn diagram of the common targets between “Gui Zhi–Shao Yao” herb pair and disease and the PPI network of these common targets are shown in Table S4). In terms of AD, the BP results suggested that these targets were related to neurotransmitter  and synaptic transmission (GABAergic), molecule biosynthetic and metabolic process, and macrophage differentiation; the CC results suggested that these targets mainly localized at neuronal membrane; the MF results suggested that these targets were also mostly involved in regulation of receptor activity, enzyme activity, ion channel activity (Table S5). In terms of MD, the BP results suggested that these targets were related to synaptic transmission (GABAergic and dopaminergic), chemokine biosynthetic process, lipid storage, etc. the CC results suggested that these targets mainly localized at neuronal membrane; the MF results suggested that these targets were similar mostly involved in regulation of receptor activity, enzyme activity and ion channel activity (Table S6).The GO and KEGG enrichment analysis was conducted to systematically discern the multiple therapeutic mechanisms of “Gui Zhi–Shao Yao” herb pair for CP with comorbid AD and MD. Sorted based on the value of “Enrichment”, we selected the top 10 of biological processes (BP), cellular components (CC) and molecular functions (MF), separately, as the pivotal results of GO enrichment  pathway, is involved in nociceptive information processing, anxiety and depression-like behaviours. For example, intrathecal injection of Akt inhibitor MK-2206 or PI3K inhibitor LY294002 significantly attenuated mechanical allodynia and thermal hyperalgesia induced by paclitaxel . AKT1 afTables S7–S9). Among these, 11 pathways were considered the vital mechanisms involved in all of these three diseases. Neuronal apoptosis is a significant contributor to the development of hyperalgesia and sensitization particularly in neuropathic pain [The KEGG database has been developed to understand the conservation and variation of genes and genomes at the level of cellular organisms . In thishic pain , and it hic pain . CP is ahic pain . Persisthic pain . As an ehic pain  in the phic pain . Advancehic pain . AGE–RAGhic pain . RAGE sihic pain . The norhic pain . IL-17 ihic pain . It conthic pain . IL-7 mahic pain . The anxhic pain . IL-7 ishic pain ,93. TNF hic pain . TNF relhic pain ,96. In lhic pain ,98. Delehic pain . The paihic pain . Nicotinhic pain ,102. Forhic pain ; howeverhic pain . Macrophhic pain . Mincle hic pain . Adminishic pain . Howeverhic pain ,109. CP hic pain . By conthic pain . Insulinhic pain . In othehic pain ,114. Meahic pain , which phic pain . Relatedhic pain ,118. Numhic pain . Moreovehic pain . Adipocyhic pain . By conthic pain . Adipocyhic pain , which shic pain ,127.There have been some reports about the effects of Gui Zhi or Shao Yao on these targets. For example, Gui Zhi has the anti-inflammatory effect of down-regulating the expression of various genes related to inflammatory responses in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells, including IL6, TNF and COX-2 . PaeonifOur study has several limitations. First, during the decoction process, various chemical components of different herbs may interact, causing certain changes in the composition. It is very likely that the chemical compositions are differences between the herbal compounds of Gui Zhi, Shao Yao and the “Gui Zhi–Shao Yao” herb pair. Second, the further experimental validation is essential to reveal these targets and pathways in anti-CP effect of the “Gui Zhi–Shao Yao” herb pair. Third, our study was based on the currently available scientific evidence. Some druggability of the compounds of “Gui Zhi–Shao Yao” are not well elucidated at present. We cannot completely exclude this possible information bias.In this study, using the network pharmacology, our study has predicted the targets of the ingredients of the “Gui Zhi–Shao Yao” herb pair and explored the underlying mechanism of the potential anti-CP effects. The extensive analysis results showed that “Gui Zhi–Shao Yao” herb pair elicits its pharmacological effects in CP by modulating the multiple pathways and multiple targets.Click here for additional data file.


Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases; however, there has been little research into its impact on gestational diabetes mellitus (GDM).This study included 308,095 women registered in the Korean National Health Insurance Service database, who delivered between 2011 and 2015 and received a health examination within 52 weeks before pregnancy. Insulin-requiring GDM was defined as no insurance claims for diabetes mellitus and a fasting blood glucose level of < 126 mg/dL before pregnancy, and initiation of insulin treatment during pregnancy. A fatty liver index (FLI) was calculated using body mass index, waist circumference, and blood triglyceride and γ-glutamyl transferase levels. FLI scores < 30 ruled out hepatic steatosis, while FLI scores ≥ 60 indicated NAFLD.The prevalence of NAFLD was 0.8%  and 1984 (0.6%) subjects developed insulin-requiring GDM. FLIs of 30–59 and ≥ 60 were significantly associated with increased risk of insulin-requiring GDM , respectively. Further exploration of the association of FLI with GDM across FLI decile categories revealed a steady increase in OR across the categories. The association was more prominent among those without metabolic syndrome.NAFLD in women is an independent risk factor for insulin-requiring GDM.The online version contains supplementary material available at 10.1186/s13098-021-00710-y. Nonalcoholic fatty liver disease (NAFLD) is defined as an increase in liver fat content, in the absence of any secondary cause of steatosis , 2. The GDM is a common international health problem in pregnant women, which can lead to adverse pregnancy outcomes . As the th revision (ICD-10) codes, medical treatments, and drug prescriptions is routinely collected and undergoes quality control before being released for research purposes [Using the Korean National Health Insurance Service (NHIS) database, we retrospectively recruited pregnant women from the population for the current study. The NHIS is managed by the government and is the sole insurer for health-care services with a coverage rate of approximately 97% of the population in the Republic of Korea. The NHIS database is available for population-based cohort studies. Information on demographics, national health screening data, diagnosis statements defined by the International Classification of Disease 10purposes –14. EnroWe calculated the fatty liver index (FLI) according to the formula below, which incorporated levels of triglycerides and γ-glutamyl transferase (GGT), and body mass index (BMI) and waist circumference (WC) , 16:\docWe classified the study population into three groups according to the FLI as follows , 14: low2, according to the World Health Organization Western Pacific Region guideline [We defined obesity as a BMI ≥ 25.0 kg/muideline . Abdominuideline . InformaInsulin-requiring GDM was defined as having no history of previous diabetes and receiving a prescription for insulin during the pregnancy. Participants with non-GDM or GDM without insulin treatment were treated as the control group.P value < 0.05 was considered to indicate statistical significance.Continuous variables are presented as mean ± standard deviation (SD) and median (25–75%), while the categorical variables are presented as n (%) for each group. The participants were classified into three groups according to cut-off scores of FLI (30 and 60). We performed one-way analyses of variance (ANOVA) or Chi-square test, as appropriate to compare each group. Multiple logistic regression analysis was performed to obtain odds ratios (ORs) and 95% confidence intervals (CIs) for GDM. The multivariable-adjusted models used in the analysis were as follows: model 1 was adjusted for age; model 2 was adjusted further for socioeconomic status , fasting blood glucose, and dyslipidemia; and model 3 was adjusted further for family history of diabetes. The potential modification effect caused by age, smoking, hypertension, dyslipidemia, and MetS was identified through a stratified analysis and interaction testing using the likelihood-ratio test. We also performed the same analyses according to the FLI components . SAS software  was used for the analyses and a The prepregnancy characteristics of the study population according to their baseline FLI categories are shown in Table There were 1,984 (0.6%) women with GDM who received insulin therapy. Compared to subjects with FLI scores < 30, the age-adjusted ORs for subjects with FLI 30–59 and FLI ≥ 60 were 5.70 (95% CI 4.98–6.53) and 9.96 (95% CI 8.35–11.90) for insulin-requiring GDM, respectively Table . These aFurther exploration of the association of FLI with GDM across FLI decile categories revealed a steady increase in OR across the categories Fig. . Women wIndividual components of the FLI were also associated with the risk of insulin-requiring GDM Table , Fig. 1.P values for interaction were 0.03 and < 0.001, respectively). Higher adjusted ORs for insulin-requiring GDM were observed in the younger-aged (age < 35 years) and non-MetS groups. When compared with subjects having FLI scores < 30 and without MetS, having a FLI score ≥ 60 without MetS was associated with a 3.5-fold increased risk of insulin-requiring GDM . Compared with subjects with a FLI score < 30 and MetS, having a FLI ≥ 60 and MetS was associated with a 2.1-fold increased risk of insulin-requiring GDM . These findings suggest that the utility of the FLI as a risk factor for GDM may be more valid in these subpopulations.We performed subgroup analyses according to age, smoking, absence or presence of hypertension, dyslipidemia, and MetS Table . In all In additional sensitivity analyses, with the combination of FLI category and MetS status as a composite exposure variable, the risk of GDM was compared to subjects having FLI scores < 30 and without MetS (reference group). Women having a FLI score ≥ 60 and MetS were at the greatest risk of insulin-requiring GDM  of hepatic steatosis . Second,In the current study, the OR (95% CI) of NAFLD for GDM was 4.83 , implying that NAFLD should be considered a major risk factor for the development of GDM. We found a stronger association among women without MetS before pregnancy, which supports the hypothesis that NAFLD is an independent risk factor for GDM, regardless of MetS status. Early identification of women with NAFLD is important and more intensive screening and preventive strategies are needed for this subpopulation.Additional file 1: Figure S1. Flowchart of the study population. Figure S2. Timeline for the Study data collection. Figure S3. Adjusted odd ratios  of insulin-requiring gestational diabetes according to the presence of metabolic syndrome (MetS) and fatty liver index (FLI) category. Subjects with a FLI <30 and no MetS were analyzed as a reference group. Adjusted for age, smoking, alcohol drinking, regular exercise, income status, fasting blood glucose, and dyslipidemia.


Fatal hemorrhagic pneumonia is one of the most severe manifestations of Stenotrophomonas maltophilia (SM) infections. Here, we aimed to investigate the clinical characteristics of SM bacteremia and to identify the risk factors of hemorrhagic pneumonia caused by SM in patients with hematologic diseases.The clinical records of 55 patients diagnosed with hematologic diseases and SM bacteremia were retrospectively reviewed. We compared patients’ clinical characteristics and outcomes between the hemorrhagic pneumonia group and non-hemorrhagic pneumonia group.P = 0.020) was an independent risk factor of 30-day mortality in hematological patients with SM bacteremia. Compared with the non-hemorrhagic pneumonia group, patients in the hemorrhagic pneumonia group were older and showed clinical manifestations as higher proportions of isolated SM in sputum culture, neutropenia and elevated procalcitonin (PCT). Multivariate analysis showed that neutropenia, high levels of PCT, prior tigecycline therapy within 1 month were independent risk factors associated with hemorrhagic pneumonia.Twenty-seven (49.1%) patients developed hemorrhagic pneumonia. The overall mortality rate of SM bacteremia was 67.3%. Hemorrhagic pneumonia (adjusted HR 2.316, 95% CI 1.140–4.705; Neutropenia, high level of PCT and prior tigecycline therapy within 1 month were significant independent predictors of hemorrhagic pneumonia in hematologic patients with SM bacteremia. Due to no effective antibiotics to prevent hemorrhagic pneumonia, prophylaxis of SM infection and its progression to hemorrhagic pneumonia is particularly important. Stenotrophomonas maltophilia (SM) is a non-fermentative gram-negative bacterium without highly virulent widely found in nature, and rarely causes infection in the normal immune population . HoweverDue to the intrinsic resistance of SM to many broad-spectrum antimicrobial agents, it is difficult to determine the appropriate antibiotic treatment before the SM is detected , 7. TherIn this study, we identified cases of SM bacteremia in patients with hematologic disorders and performed a retrospective review of these patients’ clinical characteristics and outcomes. Further, we attempted to confirm the risk factors of SM bacteremia with hemorrhagic pneumonia.This study was conducted at a 318-bed hematology department, the first affiliated hospital, College of Medicine, Zhejiang University, in China. From January 2015 to July 2019, all patients who were diagnosed with hematologic diseases and had positive blood cultures for SM were included in the study, with each patient being included only once at the time of initial blood culture. All patients were categorized into 2 groups: hemorrhagic pneumonia group and non-hemorrhagic pneumonia group. Clinical and microbiological characteristics were compared between the two groups to identify the risk factors for hemorrhagic pneumonia in SM bacteremia patients.We retrospectively reviewed the electronic medical records for all enrolled patients and extracted the clinical data. The data included the following: age, gender, underlying hematologic disease, therapeutic treatment for hematologic diseases, sputum culture, polymicrobial bacteremia, laboratory tests, antibiotics susceptibility testing results, prior antibiotic therapy within 1 month, indwelling central venous catheter, inadequate initial empirical antimicrobial treatment and mortality.SM bacteremia was defined as one or more positive blood cultures with clinical symptoms of infection. SM bacteremia with hemorrhagic pneumonia was diagnosed as follows:1) SM isolated from a blood culture, with or without SM in sputum culture at the same time, 2) newly detected imaging findings by chest X-ray or computed tomography (CT) scan when the blood culture was positive, 3) with pulmonary hemorrhage symptoms, such as continuous blood in sputum or hemoptysis. Polymicrobial bacteremia was defined as the isolation of another pathogen within 24 h of index SM isolate, satisfying the Centers for Disease Control and Prevention (CDC) criteria for blood stream infection (BSI) . NeutropEnterobacteriaceae was usedhttps://www.fda.gov/drugs/development-resources/tigecycline-injection-products), and the relevant criteria for . For Enterobacteriaceae isolates, ≤2 / ≥8 μg/ml were the sensitivity/resistance MIC breakpoints against tigecycline based on FDA criteria. Additionally, Cefoperazone/sulbactam susceptibility was based on the breakpoints for cefoperazone alone against Enterobacteriaceae  according to the criteria for susceptibility testing by CLSI [The BacT/Alert 3D  automated blood culture system was used. Species identification was performed by the automated Vitek 2 system with the matched gram-negative (GN) test card . Antimicrobial susceptibility was tested with the agar dilution method. The antibiotics tested included trimethoprim-sulfamethoxazole (TMP-SMX), cefoperazone/sulbactam, minocycline, levofloxacin and tigecycline. The MICs of antimicrobial agents were interpreted according to the 2018 Clinical and Laboratory Standards Institute (CLSI) criteria except for tigecycline . The intP-value of less than 0.10 in univariate analyses were enrolled in further multivariate analysis. The survival curves were performed by the Kaplan-Meier method. Univariate and multivariable logistic regression was used to evaluate the risk factors of hemorrhagic pneumonia in hematologic patients with SM bacteremia. Variables with P-value of less than 0.10 in univariate analyses were included in a multivariable logistic regression analysis. In addition, thrombocytopenia and prolonged activated partial thromboplastin time (APTT) which are considered as the potential variables associated with bleeding were also enrolled in the multivariate model. Results from the univariate and multivariate analysis are expressed as a hazard ratio (HR) or an odds ratio (OR) and 95% confidence interval (CI). Statistical tests were two-sided, and significance was defined as P-value < 0.05.All statistical analyses were performed using SPSS version 22.0 for Windows. The independent sample t-test was used for continuous variables and the Chi-square test or Fisher’s exact test was used for categorical variables. Cox’s proportional hazard model with forward selection was used to find out which independent risk factors were predicting 30-day mortality. All variables with Escherichia coli in 3 cases, Staphylococcus haemolyticus in 2 cases, Burkholderia cepacia in 2 cases, Staphylococcus epidermidis in 1 case, and Enterococcus faecium in 1 case. Fifty-two (94.5%) patients with SM bacteremia had thrombocytopenia and 50 (90%) patients’ platelet counts were less than 50,000/uL. Bleeding was one of the most common clinical manifestations in this study. There were 30 (54.5%) patients with hemoptysis, 11 (20.0%) patients with gastrointestinal bleeding and 5 (9.1%) patients with intracranial hemorrhage. Hemorrhagic pneumonia was confirmed in 27(49.1%) patients. However, SM was only identified in 14 (25.5%) patients from sputum culture simultaneously.Of the 288 patients with SM positive culture in the hematology department during the study period, 55 19.1%) with SM bacteremia were included. The median age was 51 (range 12–72) years old and 56.4% were male in our study population. The clinical characteristics of this cohort were shown in Table 9.1% withP = 0.049). There were more patients (40.7%) isolated SM from sputum in the hemorrhagic pneumonia group. The proportions of neutropenia and elevated procalcitonin (PCT) were significantly higher in the hemorrhagic pneumonia group than the non-hemorrhagic pneumonia group . There were no significant differences for patients’ gender, polymicrobial bacteremia, anemia, platelet count, coagulation function, underlying hematologic diseases, C-reactive protein level, inadequate initial antimicrobial treatment and prior courses of chemotherapy between the two groups.The cohort was categorized into hemorrhagic pneumonia group and non-hemorrhagic pneumonia group . The comparisons of clinical characteristics between the two groups were listed in Table The overall mortality was 67.3% (37/55), and all patients died within 30 days after SM bacteremia onset in non-survivor group. It was noteworthy that 20 (36.4%) patients died before obtained the positive blood culture reports, and all these patients had hemorrhagic pneumonia. The 30-day mortality rate was significantly higher in patients with hemorrhagic pneumonia than those without hemorrhagic pneumonia . In this study, 35 (63.6%) patients received inadequate initial antimicrobial treatment. The 30-day mortality rate was 77.1% in patients who received inadequate initial antimicrobial treatment, which was significantly higher than that in patients treated with adequate initial antimicrobial (50.0%).P = 0.002), globulin< 20 g/L , inadequate initial antimicrobial treatment  and hemorrhagic pneumonia  were the independent high-risk factors associated with 30-day mortality. Kaplan-Meier survival curves stratified by these four independent risk factors were shown in Fig. The results of Cox’s proportional hazard analysis of risk factors associated with 30-day mortality in hematological patients with SM bacteremia was shown in Table P = 0.010).All patients in this study were administrated with antibiotics before SM bacteremia onset, either due to a previous other infection or as an empirical therapy for unknown origin infection Table . The majThe antimicrobial susceptibilities of SM isolate in vitro are shown in Fig. P = 0.045), high levels of PCT , prior tigecycline therapy within 1 month .For patients with SM bacteremia, once hemorrhagic pneumonia develops, the patients’ conditions always rapidly deteriorate and nearly all these patients die in a very short time. Univariate analysis showed that neutropenia, high levels of PCT (> 0.5μg/L) and prior tigecycline therapy within 1 month were significant risk factors for hemorrhagic pneumonia in SM bacteremia patients. Variables finally included in the multivariate analysis were neutropenia, platelet< 50,000/uL, prothrombin time (PT) > 13.5 s, APTT> 33.5 s, high levels of PCT (> 0.5μg/L) and prior tigecycline therapy within 1 month. In multivariate analysis showed in Table In the present study, we found two main results. First, the mortality rate of SM bacteremia with hemorrhagic pneumonia was significantly higher than those without hemorrhagic pneumonia. Hemorrhagic pneumonia was an independent risk factor of 30-day mortality in hematologic patients with SM bacteremia. Second, neutropenia, high level of PCT and prior tigecycline therapy were found to be independent risk factors associated with hemorrhagic pneumonia in hematologic patients with SM bacteremia.Hemorrhagic pneumonia is one of the most severe manifestations of SM infections. Almost all patients die within a few days after the onset of hemoptysis and before the isolate is detected from blood or sputum culture. Previous literature has proven that inadequate initial antimicrobial treatment is an independent adverse prognostic factor for SM bacteremia , which wA previous review summarized the clinical information of 30 cases of hemorrhagic pneumonia caused by SM . The autPCT is a prohormone of calcitonin, which has a short half-life and is highly upregulated during the acute phase of sepsis. PCT is valuable and sensitive in predicting bacteremia and distinguishing Gram-negative sepsis in hematologic patients with febrile neutropenia –27. Its . This process might lead to tissue invasion, destruction of the alveolar micro vessels and hemorrhage [P = 0.015, χ2 = 5.908). Furthermore, the majority of enrolled patients occurred thrombocytopenia concurrently in our study. If destruction of alveolar micro vessels by protease secreted by SM, coagulative dysfunction induced by tigecycline, and thrombocytopenia happen at the same time, the probability of hemorrhagic pneumonia occurring might increase. On the other hand, it was identified that the resistance to tigecycline was easily selected during exposure to this antimicrobial and cross-resistance to other antimicrobials, such as aztreonam and quinolones, was also presented [P = 0.223). However, there may be some bias based on the nature of retrospective study. Tigecycline is a broad-spectrum antibiotic used to treat infections that do not respond to first-line treatments. It might be indicated that these patients with prior tigecycline use had suffered from severe infections in a short time before SM bacteremia onset. It was unknown whether this previous infection was associated with hemorrhagic pneumonia. We need to verify the potential association between hemorrhagic pneumonia and prior tigecycline use by expanding the case samples or in a SM-infected mouse model.Tigecycline is a potential alternative antimicrobial for SM infection, and the susceptibility to tigecycline is ranged from 70.6 to 90.6% according to previous studies , 31, 32.morrhage . It was morrhage . The mosresented . UnfortuThe study still has several limitations. First of all, 59.3% of cases did not obtain a positive result of SM in sputum culture in the hemorrhagic pneumonia group. It might lead to the argument about whether SM was the true pathogen of hemorrhagic pneumonia. For this special group of patients, sputum culture was difficult to carry out under certain conditions, such as the sudden appearance of massive hemoptysis. Besides, the majority of these patients had thrombocytopenia, so it was unable to perform bronchoalveolar lavage fluid culture to improve the positive rate of culture. According to our definition, the blood cultures of all patients were positive for SM. Then combined with clinical and imaging findings, we reasonably identified that hemorrhagic pneumonia was caused by SM. On the other hand, the antimicrobial susceptibilities of SM isolates were insufficient. There were only a few cases completed the susceptibility test to tigecycline. Consequently, it is difficult to confirm the relationship between the susceptibility to tigecycline and the development of hemorrhagic pneumonia.Our study suggested that SM bacteremia with hemorrhagic pneumonia developed in patients with hematologic diseases was a condition with rapid progression and high mortality. In addition, neutropenia, high level of PCT and prior tigecycline therapy within 1 month were independent predictors of hemorrhagic pneumonia in hematologic patients with SM bacteremia, and thus patients with these conditions should be treated carefully. Futher studies are needed to explore effective approaches for prevention of the life-threatening hemorrhagic pneumonia in these patients.


Babies born early and/or small for gestational age in Low and Middle-income countries (LMICs) contribute substantially to global neonatal and infant mortality. Tracking this metric is critical at a population level for informed policy, advocacy, resources allocation and program evaluation and at an individual level for targeted care. Early prenatal ultrasound examination is not available in these settings, gestational age (GA) is estimated using new-born assessment, last menstrual period (LMP) recalls and birth weight, which are unreliable. Algorithms in developed settings, using metabolic screen data, provided GA estimates within 1–2 weeks of ultrasonography-based GA. We sought to leverage machine learning algorithms to improve accuracy and applicability of this approach to LMICs settings.This study uses data from AMANHI-ACT, a prospective pregnancy cohorts in Asia and Africa where early pregnancy ultrasonography estimated GA and birth weight are available and metabolite screening data in a subset of 1318 new-borns were also available. We utilized this opportunity to develop machine learning (ML) algorithms. Random Forest Regressor was used where data was randomly split into model-building and model-testing dataset. Mean absolute error (MAE) and root mean square error (RMSE) were used to evaluate performance. Bootstrap procedures were used to estimate confidence intervals (CI) for RMSE and MAE. For pre-term birth identification ROC analysis with bootstrap and exact estimation of CI for area under curve (AUC) were performed.p < 0.001). This model performed better than Iowa regression, AUC Difference 14.4% .Overall model estimated GA had MAE of 5.2 days (95% CI 4.6–6.8), which was similar to performance in SGA, MAE 5.3 days (95% CI 4.6–6.2). GA was correctly estimated to within 1 week for 85.21% (95% CI 72.31–94.65). For preterm birth classification, AUC in ROC analysis was 98.1% (95% CI 96.0–99.0; Machine learning algorithms and models applied to metabolomic gestational age dating offer a ladder of opportunity for providing accurate population-level gestational age estimates in LMICs settings. These findings also point to an opportunity for investigation of region-specific models, more focused feasible analyte models, and broad untargeted metabolome investigation.The online version contains supplementary material available at 10.1186/s12884-021-04067-y. Of 15 million preterm births annually, 90% happen in Low and Middle Income Countries (LMICs)  contribuThe estimation of accurate gestational age at birth is essential for identifying both preterm and SGA births. Early ultrasonography examination, considered as gold standard for gestational age (GA) assessment is unavailable due to high equipment cost and lack of trained manpower in most LMICs settings. Recall of last menstrual period (LMP)  used in In global health a need for novel tools is required that could help monitor these metrics in LMIC countries on a population scale . AlgoritWe hypothesized that application of ML to metabolite profile datasets of Alliance for Maternal and New-born Health Improvement (AMANHI) All children thrive (ACT) cohorts (representing both South Asia and Sub-Saharan Africa) would potentially improve the prediction of GA as compared to conventional approaches previously reported. The rationale for these cohorts and associated bio-bank procedures, and cohort characteristics have been described in previous publications , 21. OneThis study was undertaken using data from the Alliance for Maternal and New-born Health Improvement (AMANHI) All children thrive (ACT), community based, prospective pregnancy and New-born cohorts from Pemba (Tanzania), Sylhet (Bangladesh) and Karachi (Pakistan). These studies received ethical approval from the local and institutional ethics committees of all the three sites: ICDDR, B and John Hopkins University for Bangladesh, Aga Khan University for Pakistan and ZAMREC for Tanzania. The protocols were also approved by the Ethical Review Committee (ERC) at World Health Organization (WHO). Briefly, women were enrolled in early pregnancy and followed through delivery and the postpartum period , within 24–72 h of birth from new-born’s as per standard procedures. All 903 cards were labelled with barcoded unique identifier (ID’s), were air-dried and stored in air tight Ziplock bags with desiccant at − 80 °C. The 903 cards were shipped in dry ice to the University of Iowa where they were examined for quality. Then samples were sent to State Hygienic Laboratory, Ankeny, Iowa, USA at regular intervals (ensuring processing before potency window). Sixty-six metabolites  bootstrap sampling -with replacement and 2) Random feature –m = 10 in RF regressor. In this generation procedure was repeated until 10 decision trees were created to form a randomly generated “Forest”. We denoted the hyper-parameters for RF regressor as “mtry” (a variable imputed in the model) at each split node for performing regression; the default value of “mtry” is p/3 where p is the number of predictors . Random Sklearn.ensemble   from the profile of the blood metabolites along with birthweight and gender.Model 2: was designed to replicate model published by Ryckman et al.  and Model 2 [2.5% ]  of 5.2 days (95% CI 4.5–6.8), compared to gold standard ultrasound dating. Accuracy was slightly lower in Africa MAE 5.3 days (95% CI 54.8–6.2) than Asia MAE 5 days (95% CI 4.3–6.2) , we repeated the analysis with machine learning being trained by African sample for Africa estimations and Asian sample for Asia estimations. The model performance in spite of reduced samples for training improved both for Africa and Asia. The precision of MAE improved rather being reduced for Africa to 5 days (95% CI 4.1–6.0) and Asia to 4.8 days (95% CI 4.1–6.1) Table . The samp < 0.001). Criterion of ≥37 providing a sensitivity of 100% and specificity of 92.61%  over predicting GA by regression models in the same dataset, AUC 84% (95 CI 78.6–88.0),  in AUC between Africa and Asia  , model in ROC analysis showed an area under curve (AUC) of 92.6%  a sampling frame which utilized samples from both South Asia and East Africa, home to most of the global mortality associated with preterm and SGA births, 2) the study design was nested in a well-described population-based cohort of pregnancy with WHO coordinated and harmonized protocols and SOP, 3) Active surveillance for early pregnancy identification with added measures , culminating in harmonized ultrasonography based gestation assessment between 8 and 19 weeks of gestation and 4) Sample collection, storage and shipment SOP based on pilot QC, resulting in high quality of samples. The primary limitation of this study is the participation bias against early preterm and early deaths before sample collection window. Relatively small proportion of actual births in this sub-sample limits our ability to comment on model performance in these sub-groups. Our finding of lower accuracy in pre-term ≤34 weeks may either reflect lack of association of the metabolites in that sub-group, a function of lack of sample in that group and/or bias introduced by selective exclusion of early deaths. Additionally, we were working with the limitation of a smaller sample size as compared to the usual sample sizes in machine learning universe. We did try to use methods appropriate to accommodating smaller sample sizes, however would not have been protected against extreme chance affecting the sample. The ability to train the model and precision of estimates is somewhat reassuring but would need confirmation.Preterm births and SGA account for a substantial burden of mortality in first 5 years , 4. TracTowards implementing preterm birth surveillance initiatives  ML algorAdditional file 1: Fig. S1. Pregnancy cohort and Study design. Table S1. Performance metrics of the different machine learning algorithms. Table S2. RMSE and Mean Abs Error in weeks obtained in three comparative models. Fig. S2. ROC analysis comparing discriminatory ability of four models evaluated. Table S3. Newborn screening metabolomic analytes estimated. Table S4A. Description of the model. Table S4B. Description of the model.


Checkpoint inhibitor therapy with monoclonal antibodies against programmed cell death protein 1 (PD1) has been implemented in the treatment of numerous malignancies. Pembrolizumab is one such medication. While severe toxicities are very rare, mild immune-mediated reactions with a variety of end organ injuries are among the most commonly encountered adverse events attributed to these medications. Acute liver injury manifesting as biochemical abnormalities with or without synthetic dysfunction is one such potential adverse reaction. Rarely, a relatively severe hepatitis can occur. While such severe adverse events are often successfully managed with systemic corticosteroids and drug discontinuation, our patient was refractory to standard management. We present a case of pembrolizumab-induced hepatitis in a patient with squamous cell carcinoma and prior orthotopic liver transplantation. Through a combination of serial plasmapheresis and intravenous immunoglobulin (IVIG) infusion, the patient's hepatitis resolved as evidenced by virtual normalization of his liver indices. This illustrates the effectiveness of a relatively novel treatment strategy for this rare side effect of checkpoint inhibitor antineoplastic therapy. A 76-year-old Caucasian man with end-stage liver disease secondary to alcoholic cirrhosis underwent orthotopic liver transplantation and was maintained on low-dose immunosuppression of tacrolimus monotherapy with good graft function. His course was complicated by stage III metastatic laryngeal squamous cell carcinoma  with a positive lymph node on the left side of the neck with negative laryngoscopy.He underwent three cycles of chemotherapy consisting of weekly cisplatin followed by radiotherapy with reduction in the dose of tacrolimus to minimize side effects. Following radiation therapy, he developed severe dysphasia and aspiration requiring feeding tube placement.Nine months after the completion of radiotherapy, he had a recurrence of squamous cell carcinoma with lung metastasis. The metastasis was diagnosed in a painful left supraclavicular lymph node that required a left-sided neck dissection. As a result of this metastasis, he received pembrolizumab (Keytruda). His tacrolimus dose was decreased, and prednisone was added at 5 mg daily.Pretreatment liver function values (LFT's) included serum alanine aminotransferase (ALT) 24 U/L, aspartate amino transferase (AST) 16 U/L, alkaline phosphatase (ALP) 108 U/L, gamma glutamyl transferase (GGT) 17 U/L, and bilirubin of 0.2 mg/dl.Repeat lab test results after 2 weeks of the first dose of pembrolizumab were within normal limits. However, repeat lab tests after 3 weeks were abnormal with ALT 783 U/L, AST 413 U/L, ALP 444 U/L, and bilirubin 2.1 mg/dl.At this time, his tacrolimus trough was <2, and the only clinical symptom was jaundice. An abdominal ultrasound ruled out biliary obstruction and portal vein thrombosis.Viral serologic testing for cytomegalovirus (CMV), Epstein–Barr virus (EBV), BK virus (BKV), hepatitis viruses, herpes simplex virus (HSV), varicella zoster virus (VZV), human herpes virus (HHV), and respiratory syncytial virus/severe acute respiratory syndrome coronavirus 2 (RSV/SARS-CoV-2) were negative.Prednisone was increased to 60 mg PO daily and then to 100 mg daily without improvement in his liver function.He was admitted to the hospital and treated with 80 mg IV methylprednisolone with minimal improvement. Due to minimal steroid response, mycophenolate 500 mg BID, thymoglobulin 1.5 mg/kg, and ursodiol 300 mg BID were given. A liver biopsy revealed portal venous and capillary endothelialitis and complete duct dropout without a classic portal mixed inflammatory infiltrate indicative of cellular rejection Figures .Severe immune-mediated hepatitis with vanishing bile duct syndrome and cholangiopathy was diagnosed. Given the clinical history of dramatic elevation in alkaline phosphatase since starting treatment with pembrolizumab instead of a gradually progressive elevation of alkaline phosphatase, drug-induced immune mediated injury was favored over ductopenic rejection. In other words, given the strong temporal relation to pembrolizumab therapy and laboratory trends, a drug-induced immune mediated injury was felt to be the most likely etiology in our patient. A RUCAM score of 9, which indicates a high probability that the liver injury was due to pembrolizumab, was calculated.Liver function tests were still not improving. Thus, IV dexamethasone 50 mg and antithymocyte immunoglobulin 1.5 mg/kg per day were added. Tacrolimus was increased to 2 mg BID with a goal trough of 6–8. Mycophenolate was increased to 1000 mg BID. A magnetic resonance colangiopancreatography (MRCP) revealed mild anastomotic narrowing of the common bile duct (CBD). Given the histologic diagnosis of immune-mediated hepatitis, this mild narrowing was not considered to be the primary cause of the histologic changes.His LFTs started to improve. Thymocyte globulin was withheld but was restarted again after declining function was observed again. In sum, despite treatment with a variety of immunosuppressive agents including high-dose IV corticosteroids, antithymocyte globulin, mycophenolate, and increased doses of tacrolimus in addition to ursodiol, his liver failed to show significant improvement. Thus, his liver injury appeared to be refractory to medical immunosuppression.Given the IgG nature of pembrolizumab, we started plasmapheresis for drug removal and IVIG for immunomodulation. Each centrifugal plasmapheresis was a 1-volume exchange that used 5% albumin as the replacement fluid. Daily plasmapheresis was initially performed on four consecutive days. Of note, the third plasmapheresis lasted only 15 minutes and was terminated due to shoulder pain and hypotension. An infusion of 100 m/kg IVIG was added after each of these four initial plasmapheresis procedures.Before these four initial plasmapheresis procedures, LFT's were AST 87 U/L, ALT 222 U/L, ALP 469 U/L, and bilirubin 15.3 mg/dl. Following these treatments, his liver function improved with LFT's, AST 60 U/L, ALT 82 U/L, ALP 143 U/L, and bilirubin 10.3 mg/dl.After receiving additional two plasmapheresis procedures with IVIG, he was discharged from the hospital. Total number of inpatient plasmapheresis with IVIG was 6 (this includes 1 incomplete). As an outpatient, he received both plasmapheresis and IVIG twice a week for 4 more weeks for a total of 8 outpatient procedures.After a total of 14 inpatient and outpatient treatments, his procedures were discontinued, and liver function values were monitored continuously while he was on tacrolimus, mycophenolate, and a prednisone taper. Currently, he is receiving non-immune-mediated chemotherapy with carboplatin and docetaxel for his lung metastasis. His most recent LFT's were AST 19 U/L, ALT 16 U/L, ALP 147 U/L, and bilirubin of 0.5 mg/dl.The programmed cell death protein 1 (PD1) is a checkpoint that regulates the immune response. Ligation of PD1 with its ligands PDL1 and PDL2 results in transduction of negative signals to T-cells. The expression of PD1 on effector T-cells and PDL1 on neoplastic cells allows tumor to evade the immune response. Blockade of PD1 is an important immunotherapeutic strategy for cancers. Pembrolizumab (Keytruda) is a humanized monoclonal anti-PD1 antibody that has been extensively investigated in numerous malignancies [Though adverse events occurred in up to 60% of patients, grade 3/4 toxicities were observed in <10% of cases. Besides infusion reactions, most of the AEs are thought to be immune-related AEs, occurring in any organ. Since the optimal duration of treatment is undefined, pembrolizumab may be given for up to 2 years. Consequently, immune-related AEs can occur late or even after cessation of treatment [One of the known side effects of checkpoint inhibitor immunotherapy is the onset of mild liver biochemical abnormalities at 3–6 months after starting treatment is . It geneMultiple patterns of histological injury have been documented in the literature . In a stThough corticosteroids are the mainstay for most high-grade immune-related adverse events such as pembrolizumab-induced hepatitis, the dose and duration of corticosteroid therapy are not well-established. Literature search revealed a case of successful management of grade 3 pembrolizumab-induced hepatitis with a combination of low-dose corticosteroids and bicyclol .In addition, plasmapheresis was performed for another patient with pembrolizumab-induced myocarditis because of deterioration in his hemodynamic status refractory to conventional immunosuppression. The plasmapheresis seemed to cause a rapid reduction of serum pembrolizumab levels and marked clinical, radiological, and biochemical improvement. To the authors' knowledge, that was the first reported case on the successful use of plasma exchange for pembrolizumab-associated fulminant myocarditis .Some degree of our patient's short-term improvement in biochemical parameters was likely due to direct removal via plasmapheresis. There was clear downtrend in alkaline phosphatase, AST, ALT, and total bilirubin each day after plasmapheresis followed by an uptrend or stagnant value the following day when patient did not receive plasmapheresis. However, his long-term improvement cannot be solely due to direct removal, as his recovery of liver injury was durable.Also, some of these same laboratory markers were on a downward trend prior to plasmapheresis. Thus, it is plausible that plasmapheresis did not hasten his recovery. The total bilirubin was an exception, as it plateaued after all of our other immunosuppression therapies prior to the initiation of plasmapheresis. While alkaline phosphatase and aminotransferases were on a downward trend prior to initiation of plasmapheresis, the improvement trend increased on the day of initiating plasmapheresis and remained increased until normalization. It is plausible that the patient may have recovered on his own but at a slower rate. In other words, plasmapheresis may have increased the rate of recovery at worst and may have been an essential factor in his recovery at best. The half-life of IgG is about 23 days, so there was most likely significant amounts of pembrolizumab in the patient about 4–6 weeks after administration. Moreover, IgG is about 50% intravascular, so the rationale for some degree of drug removal via plasmapheresis seemed sound.


The 1-year overall survival (OS) was significantly lower in the SOS group compared to patients without SOS . In conclusion, we found a low incidence of SOS in patients receiving low-dose heparin and UDCA prophylactically, but among SOS patients, a high mortality. Low-dose heparin and UDCA might be a prophylactic approach for SOS.Hepatic sinusoidal obstruction syndrome (SOS)/veno-occlusive disease (VOD) is a complication after allogenic hematopoietic stem-cell transplantation  with high mortality. The purpose of this study was to assess the incidence and outcome of SOS in patients after allo-HSCT with the impact of ursodeoxycholic acid (UDCA) and low-dose heparin as SOS prophylaxis. Out of 1016 patients, 23 developed SOS, with a cumulative incidence of 2.3% (95% CI 1.3–3.3) 6 months after HSCT. Approximately one quarter of these patients (26.1%) had late-onset SOS. A high proportion were very severe SOS cases (74%), and 83% of the patients were treated with defibrotide (DF). In multivariate analysis, advanced disease ( Hepatic sinusoidal obstruction syndrome (SOS) (formerly veno-occlusive disease (VOD)) is a potentially life-threatening complication after allogenic hematopoietic stem cell transplantation  . An injuRisk factors to develop SOS may be related to the patient’s condition before transplantation  less than 90%) , 8, to tSOS of 8.9% after allo-HSCT (children and adults) [n = 275/8,341) [Varying incidence of SOS has been reported in literature. An older, but one of the only available prospective studies about SOS, indicated an SOS incidence of  adults) . In a sy adults) . A more 5/8,341) .n = 102) with historical controls (n = 32) found a significantly higher day+100 survival rate (38.2% versus 25.5%), and a significantly higher SOS resolution rate on day+100 after HSCT (25.5% versus 12.5%) in patients treated with DF [n = 41), the other without (n = 55), and found a trend towards better survival on day + 100 with DF treatment, although not significantly [Defibrotide (DF), a polydisperse oligonucleotide with antithrombotic, anti-ischemic and anti-inflammatory activity on microvasculature, is the only approved agent for the treatment of SOS . Whereas with DF  comparedficantly . In an eficantly , indicatficantly . In a poficantly . These oficantly . DF treaficantly , 16. Conficantly .p = 0. 0488; log-rank test p = 0.0507) [n = 32, treated between 1995 and 2007) and the results were adapted to the Spanish health system (2019) [The role of DF as prophylaxis of SOS is not yet fully evaluated. In a pediatric randomized controlled open-label trial, the prophylactic use of DF was associated with a lower incidence of SOS by day + 30 after HSCT , compared to the control arm  . In the  0.0507) . Results 0.0507) . Expert  0.0507) . The hig 0.0507) . They fom (2019) . In an eAnticoagulation with low-dose heparin or low-molecular-weight heparin (LMWH) has previously been used for SOS prophylaxis. It is currently not recommended by expert groups given the absence of conclusive results and potential side effects , 15. OneIn contrast, the prophylactic administration of UDCA in patients undergoing HSCT is recommended . In a syn = 82) with patients receiving heparin only (n = 83) as SOS prophylaxis in a prospective RCT [p = 0.348). In the heparin only subgroup there were more severe SOS cases than in the subgroup with ursodiol and heparin (5 vs 2), although the difference was not statistically significant (p = 0.321) [There is no literature available about a synergistic effect of UDCA and low-dose heparin. However, nineteen years ago, Park et al compared patients receiving heparin and UDCA (tive RCT . They fo= 0.321) .Heparin may interact in the pathomechanism of SOS development by reducing the procoagulant effect of injured endothelial cells, leading to less SOS occurrence and less severe SOS cases. This may have a synergistic effect with UDCA, which reduces hepatotoxic hydrophobic bile acids, leading to a liver protective effect .The purpose of the present study was firstly to assess the incidence of SOS 6 months after HSCT in a retrospective cohort study from 2006 to 2020 of patients receiving UDCA and low-dose heparin as SOS prophylaxis. Secondly, to compare patients with and without SOS, with regard to patient, disease and transplant characteristics, and the clinical course . Further, the incidence of SOS depending on the provision of DF as treatment or prophylaxis was examined.n = 41) or died before transplantation (n = 1). If patients received more than one allo-HSCT in the period of observation (n = 92), the last one was counted. In total, n = 1,016 patients were included , clinical course and outcome . In all patients diagnosed with SOS with the modified Seattle criteria, additionally the EBMT diagnosis criteria and severity criteria for SOS were retrospectively applied .With access to data from an accounting program, a sub-analysis from 01.01.2016 to 22.09.2020 could be performed. All patients who received DF (for treatment or prophylaxis) in this period could be extracted and the EBMT diagnosis criteria for SOS were applied retrospectively.The primary endpoint of the study was the incidence of SOS in the period examined, calculated as cumulative incidence 6 months after HSCT. Secondary, uni- and multivariate analysis were performed to detect potential risk factors for SOS development. The overall survival (OS), progression-free survival (PFS), incidence of non-relapse-mortality (NRM) and relapse one year after transplantation were calculated for patients with and without SOS. The day+100 survival was calculated for SOS patients. In the sub-analysis for 2016–2020, the occurrence of SOS was analysed depending on the provision of DF. The study was approved by the Ethics Committee of Northwestern and Central Switzerland (EKNZ study number: 2020-02784).MAC protocols consisted of cyclophosphamide combined with busulfan, cyclophosphamide and TBI ≥8 Gy, cytarabine, carmustine, etoposide, melphalan, and fludarabine (BEAM- fludarabine) and further protocols . RIC proWithin MAC conditioning regimens, GvHD prophylaxis consisted of cyclosporine A (CsA) and methotrexate (MTX) or other protocols. In patients with RIC containing fludarabine and busulfan the GvHD prophylaxis included CsA and MTX, if RIC was based on fludarabine and low-dose TBI it contained CsA and mycophenolate mofetil (MMF) . In caseRed blood cell transfusion was given to patients with hemoglobin concentrations <70 g/l, platelet transfusion if the level was <10 G/l, or <20 G/L, in case of bleeding, mucositis, or fever. Generally, patients received antiviral prophylaxis with valaciclovir  until day+30, prophylaxis against Pneumocystis jirovecii and Toxoplasmosis with trimethoprim/sulfamethoxazole  at least for 6 months after HSCT, and fluconazole  prophylactically against yeast infections until day + 30. Most patients did not receive mold-active prophylaxis but were treated empirically or pre-emptively, following a diagnostic-driven approach, based on chest CT scans and serum galactomannan that were performed regularly .SOS diagnosis was made by the treating physician after HSCT by clinical criteria, using modified Seattle  and fromDate of diagnosis of SOS was defined by the day when the treating clinician made the diagnosis. Date of SOS onset was defined by the first day with bilirubin level ≥2 mg/dl (34 µ/ml), or first documentation of ascites or weight gain >5% without another explanation, retrospectively.MOF was defined by dysfunction of two or more organs: renal failure (creatinine >2x baseline or GFR < 50% of baseline), pulmonary failure  <90% room air or/and need for positive pressure or ventilation dependence not attributable to any other cause) or CNS failure  ., and low-dose heparin was stopped. DF was administered until resolution of symptoms or until engraftment, if given prophylactically.Oral UDCA (3×250 mg daily) and continuous infusion of low-dose heparin (5000 IE/day) was used as SOS prophylaxis and was usually started simultaneously as the conditioning regimen and stopped after engraftment and when liver values were near the normal range. Low-dose Heparin was administered without monitoring, and only stopped in case of major bleeding or treatment initiation with DF. In selected cases with increased risk for SOS , DF was administered as SOS prophylaxis according to the treating physician’s decision. When DF was administered for prophylaxis or treatment, dose of 6.25 mg/kg every 6 h was given intravenously.p values are two-sided and p < 0.05 was considered statistically significant.Continuous variables are reported as median (minimum to maximum) and categorical variables are presented as absolute values and percentages. We assessed univariable differences between patients with and without SOS with regard to patient, disease or transplant characteristics, testing statistical significance by Chi-squared or Fisher’s exact test for categorical variables and by the Mann–Whitney U test for continuous variables. A multivariable analysis was then performed using logistic regression analysis. Variables were included in the model using a backward selection by likelihood ratio test. Results were expressed as odds ratio (OR) with 95% confidence interval (CI). OS and PFS were calculated using the Kaplan-Meier method. Comparisons of OS and PFS were performed using the log-rank test. OS was calculated from allo-HSCT until death from any cause or last follow up, with censoring of survivors. PFS was calculated from allo-HSCT to relapse, death or last follow up, with censoring of relapse free survivors. NRM was defined as death without prior relapse. Cumulative incidence curves for relapse and NRM were constructed considering NRM or relapse as a competing event, respectively, using the Fine and Gray method to test for difference. All Statistical analysis was performed using the SPSS  and Stata  software.n = 1,016 patients were included in the final analysis  had late-onset SOS. Almost all patients had a weight gain >5% , in most of them, ascites was detected , and most suffered from liver pain . Nineteen patients (82.6%) were treated with DF for a median of eight days (2–32). Thereof, two patients (8.7%) received DF already before, as a prophylactic treatment.Out of 1,016 patients, 23 developed SOS, with a cumulative incidence of 2.3% (95% CI 1.3–3.3) six months after HSCT. The median time to diagnosis was 14 days (3–151) and the median time to onset nine days (1–150). Approximately one quarter of the patients  compared to severe , moderate  and mild . In 69.6% (n = 16) of the cases MOF occurred. The overall survival on day + 100 of the SOS patients was 39.1% (95% CI 18.7–59.5). A more detailed presentation of the clinical course of the patients who developed SOS is displayed in Supplementary figs. Twenty-two patients (95.7%) fulfilled the EBMT-criteria retrospectively, only one patient did not, due to lower bilirubin levels than 2 mg/dl. However, in this case the modified Seattle criteria were met. In nine patients (39.1%) the diagnosis was confirmed by biopsy or autopsy. The EBMT severity grading very severe was clearly the most frequent  received DF as a treatment for suspected SOS. Of these, 12 patients (60%) were finally diagnosed with SOS (fulfilling the EBMT-criteria retrospectively), corresponding to a cumulative incidence of 2.8% (95% CI 1.2–4.4) six months after HSCT. DF as a SOS prophylaxis had been administered to 17 patients (4.0%), thereof two patients developed SOS . Of notice: in total, only 35 patients received DF, the two patients developing SOS under prophylaxis were counted in both groups (treatment and prophylaxis).p = 0.003) and previous HSCT  (p = 0.025) were significantly associated with the development of SOS. An association was also found between the use of PTCy and the development of SOS, although it was not statistically significant (p = 0.055).Both in univariable analysis Table  and confp = 0.0001)    Fig. . PFS was01) Fig. , whereas01) Fig. .Table 3An = 2,572 [n = 1,016 (the present work)).Firstly described forty years ago, as a complication after allo-HSCT, SOS remains a complication with high mortality until today , 34. The = 2,572  and n = In recent studies, the SOS incidence after allo-HSCT varied from 7.5% (patients 15 years and older) , to 5.2%A possible explanation for the low survival at day+100 of the SOS patients in our study is the high proportion of severe and very severe SOS cases (83%). Our finding is similar to the survival rates at day + 100 of patients with severe or very severe SOS described in literature , 12, 41.In the current study, 26% were late onset SOS cases, which is consistent with existing literature , 38, as As presented in Supplementary fig. post-HSCT GvHD prophylaxis with PTCy as a risk factor for SOS. Further research is needed to evaluate this association.The risk factors significantly associated with SOS development in our multivariable analysis are already well described in literature  in patients considered at high risk for SOS receiving prophylactic DF [n = 2/17, 11.8%), compared to the patients with standard SOS prophylaxis  . It is noteworthy, that the patients receiving DF prophylactically were considered as high risk patients for SOS. Given the lack of data for DF as SOS prophylaxis in adults, more research is needed, especially when taking into account the high costs of the agent. UDCA and low-dose heparin are, in contrary, a low-price SOS prophylaxis.A systematic review analyzing studies using DF as SOS prophylaxis reported a mean SOS incidence of 5% (only abstract available) . A smallactic DF . In our Our study has several draw backs. Most importantly, a control group is lacking. Due to the practically non-existent side effects of UDCA and low-dose heparin, these were administered to nearly all patients, assuming that the benefits are outweighing potential side effects. Another main limitation of the study is the observational and retrospective setting, which may be related to the markedly high proportion of severe and very severe SOS cases. We cannot exclude a potential underdiagnosis of mild SOS cases. Furthermore, given the low number of identified SOS cases, the power for assessing risk factors was limited.In conclusion low-dose heparin and UDCA might be a promising low-cost prophylactic approach to prevent SOS, although the mortality of SOS cases remains very high, even when they are treated with DF. Further research is warranted on SOS prophylaxis and treatment, especially prospective and randomized trials.Supplementary material


The study included 20 IBD patients receiving anti-TNF-α therapy, 20 IBD patients receiving anti-integrin-α4β7 therapy and 20 IBD patients without immunomodulatory therapy. Participants completed questionnaires on medical information, oral lesions and symptoms. For each patient, clinical examination and a salivary flow rate test were performed, followed by a swab of the oral mucosa. The swab samples were cultured to identify Candida spp. and oral bacteria. No bacterial opportunistic infections were detected. Candidiasis was detected in four participants, with no significant difference between groups (p = 0.765). Hyposalivation was most common in the anti-TNF-α group, with a significant difference between groups (p = 0.036). There were no significant differences between groups in self-reported oral mucosal lesions and symptoms (p > 0.05), or in the distribution of oral mucosal lesions (p > 0.05). This study suggests that IBD patients receiving biological therapy are at no greater risk of developing oral opportunistic infections than IBD patients not receiving immunomodulatory therapy.Biological therapy of inflammatory bowel disease (IBD) carries an increased risk for the development of opportunistic infections due to immunomodulation. The aim of this study was to determine the prevalence and types of oral infections in IBD patients treated with biological (anti-TNF-α and anti-integrin-α Over the past decade, the treatment of inflammatory bowel disease (IBD) has become increasingly dependent on immunomodulation, including biological therapy ,2. Given4β7 is considered to be gut-selective, meaning that its action is primarily limited to the site of inflammation in the gastrointestinal tract [The goal of treating IBD with biological therapy is not only to control symptoms but also to modify the progression of the disease and preserve the intestinal function . Anti-tual tract .Although it has been suggested that the risk of opportunistic infections is increased with anti-TNF-α therapy, recent studies have shown that the risks have varied, while vedolizumab, for example, has not been associated with major infectious complications because of its primary gut selectivity .In addition to immunomodulatory therapy, IBD patients are at increased risk for opportunistic infections because of age, comorbidity, reduced nutritional status, parenteral nutrition and bowel surgery , of whicDespite the numerous research papers on infections in IBD patients receiving biological therapy ,4,10, no4β7) and conventional therapy. Our hypothesis is that patients receiving anti-TNF-α therapy have more infections than patients receiving anti-integrin-α4β7 or conventional therapy.The present study aimed to determine the prevalence and types of oral infections in patients with inflammatory bowel disease treated with biological therapy (anti-TNF-α and anti-integrin-αn = 60) from the Department of Internal Medicine, Clinical Hospital Center Rijeka, were asked to participate in this study. Patients were diagnosed with Crohn’s disease (CD) or ulcerative colitis (UC) according to clinical, radiological, endoscopic and histopathological criteria described in the European Crohn’s and Colitis Organization (ECCO) guidelines [4β7 therapy for more than three months and UC and CD patients treated with non-immunosuppressive therapy. The exclusion criteria were UC and CD patients treated with other forms of biological therapy and immunosuppressive therapy , intermittent smokers, smoking fewer than five cigarettes per day, systemic or topical antifungal therapy, antibiotics used in the previous month and/or oral mouth rinses used in the previous month.A group of IBD patients , patients receiving anti-integrin-α4β7 biological therapy (n = 20) and patients receiving conventional treatment (n = 20) without any immunomodulatory therapy.Participants were divided into three groups: patients receiving anti-TNF-α biological therapy (4β7 groups): dry mouth, burning sensation, taste changes, halitosis, presence of lesions in the mouth and labial herpes.Participants were interviewed using a questionnaire that included information on age, sex, type of IBD, other diseases, prescribed medications, smoking status (more than five cigarettes per day), denture wearing and the following oral complaints and symptoms . Oral infections were diagnosed using the clinical features listed in the World Health Organization guidelines  and the Samples were collected with sterile swabs and cultured on Sabouraud dextrose agar at 37 °C for 48 h. All isolates were identified by germ tube production, grown on a selective nutrient medium containing chromogenic enzyme substrates  and the Vitek 2 YST  automated identification system .Conventional in vitro culture techniques were used for bacterial-specific identification. Swab material was cultured aerobically on agar containing 5% defibrinated sheep blood  at 37 °C for 48 h. Simple biochemical tests and the automated identification system Vitek 2  were used to distinguish pathogenic bacteria from the microflora of the healthy oral cavity.The medical data on patient history and medication were taken from the hospital’s information system. Laboratory tests were performed in all patients, including blood glucose and iron level, C-reactive protein (CRP) level and fecal calprotectin level. Clinical activity of the disease was assessed using the Harvey–Bradshaw index (HBI) for CD and the Mayo score for UC.p-value < 0.05 was considered statistically significant.Statistical analysis of the data was performed using JASP for Windows, version 0.14.1.0 . The Shapiro–Wilk test was used to test the normality of the distribution. The chi-square test was used to test the differences between the groups in terms of sex, type of disease, prevalence of candidiasis and hyposalivation, self-reported oral lesions and symptoms and distribution of oral mucosal lesions. ANOVA was used to determine the differences between the groups in terms of age and duration of biological therapy, and the Kruskal–Wallis test in terms of duration of disease, calprotectin and CRP values. A The study protocol was approved by the Ethical Committee of the Clinical Hospital Center Rijeka . The ethical guidelines of the Declaration of Helsinki were followed. All participants provided their informed consent before being enrolled in the study.p > 0.05). A statistically significant difference was found between the groups in terms of duration of disease (p = 0.005). Of the participants with CD, 11 had clinically active disease (HBI score > 5), while 20 were in clinical remission (HBI score < 5). Five participants with UC had clinically active disease (Mayo score > 2), while 24 were in clinical remission (Mayo score < 2).The demographic and disease data of the participants are shown in p = 0.036).The evaluated predisposing factors for the development of candidiasis were elevated glucose levels and iron deficiency (systemic factors), and hyposalivation, denture wearing and smoking . The results of the predisposing factors of each group are shown in C. albicans infection followed by C. glabrata, C. krusei and C. dubliniensis, as shown in C. albicans, with others showing infection by C. albicans, C. krusei and C. dubliniensis. Clinically, candidiasis presented as denture-associated erythematous stomatitis in three participants and as angular cheilitis in one participant. According to the Newton classification scale, candidiasis was observed as type I in two participants and type II in one participant. The duration of biological therapy in participants with candidiasis in the anti-TNF-α group was five years in one participant and six months in the other participant, whereas the duration of treatment in the anti-integrin-α4β7 group was four years. All participants with oral candidiasis were in clinical remission. The ages of participants with oral candidiasis were 64 in the conventional group, 70 and 82 in the anti-TNF-α group and 35 in the anti-integrin-α4β7 group. Predisposing factors for candidiasis in the four participants with candidiasis were hyposalivation (n = 4), dentures (n = 3), smoking (n = 1), elevated glucose level (n = 1) and iron deficiency (n = 1) , predominantly 4β7 group. There were no significant differences in self-reported oral lesions and symptoms between the groups (p > 0.05). None of the participants reported changes in taste sensation.The self-reported oral lesions and symptoms are shown in p > 0.05).The distribution of the oral mucosal lesions detected during the clinical examination is summarized in Treatment of IBD with biologics has led to a significant reduction in symptoms and progression of inflammatory processes, resulting in a marked improvement in quality of life. Despite the benefits, biological therapies have increased the risk of opportunistic infections. This is not only dependent on the immunomodulatory therapy but can also be increased by known risk factors . A large4β7 biological therapy. The study was conducted only with participants who received anti-TNF-α and anti-integrin-α4β7, as these types of biologics are used more frequently than others. Participants receiving conventional therapy were included as a control group to rule out other known infection predisposing factors as well as disease- and treatment-associated risk factors in IBD patients.To our knowledge, our study is the first to investigate the occurrence of oral opportunistic infections in IBD patients receiving anti-TNF-α and anti-integrin-αCandida spp., and azathioprine is associated with viral infections. These medications are often prescribed concomitantly with a higher possibility of infection development because of cumulative immunosuppression [Immunomodulatory therapy in IBD poses a safety concern given the elevated risk of opportunistic infections . An assopression ,11,20.Higher disease severity has been identified as a risk factor for the development of opportunistic infections , indepenIt has been suggested that the duration of therapy is related to the risk of developing opportunistic infections, with the first year of anti-TNF-α therapy (and particularly the first few months) being associated with a higher risk ,23. As rAs age is a risk factor for infection, 17% of patients over 60 years of age receiving vedolizumab have been found to develop an infection in the first year of treatment and 20% of patients receiving anti-TNF-α for IBD . In our Although immunomodulators have been reported to carry an increased risk of infection, the extent of this risk is not clear . The oddThere are conflicting data on whether some biologics, such as anti-TNF-α agents, carry a higher risk of developing opportunistic infections. To our knowledge, there have been no studies investigating oral opportunistic infections in IBD patients receiving biological therapy; however, several studies have investigated opportunistic infections in IBD patients with no reference to oral infections. In the meta-analysis by Ford et al. , anti-TNCandida spp. are considered part of the normal flora, but under certain conditions, such as immunosuppression, they can cause disease [Candida spp. associated with invasive infections, including C. albicans, C. glabrata, C. parapsilosis and C. tropicalis. Our finding of C. albicans and C. glabrata in swab samples concurs with the reported findings, while C. parapsilosis and C. tropicalis were not found in our samples. Corazza et al. [C. albicans, C. glabrata and C. parapsilosis, whereas in patients with psoriasis without immunosuppressive therapy, candidiasis was caused by C. albicans, C. dubliniensis and C. krusei. In our study, only the finding of C. albicans aligns with Corazza et al. [Candida spp., except for C. parapsilosis, which we did not find at all. In the meta-analysis by Ford et al. [In the gastrointestinal tract,  disease . The spe disease . Ahmad a disease  reporteda et al.  found tha et al. . We found et al. , only siThe limitations of our study are the relatively small sample size, the fact that not all types of biologics for the treatment of IBD were included and the absence of a control group in the form of IBD patients without therapy. A larger sample size and inclusion of all types of biological therapy should be considered in future studies.4β7 therapy. Future studies are needed to investigate oral opportunistic infections in patients receiving other types of biologics.In conclusion, our study suggests that IBD patients receiving biological therapy are at no greater risk of developing oral opportunistic infections than IBD patients not receiving immunomodulatory therapy. Anti-TNF-α therapy was also not shown to be associated with a higher risk of developing oral opportunistic infections than anti-integrin-α


We designed an experimental model of sepsis in rats to investigate the effects of agomelatine (AGO) on lung tissues using molecular and histopathological methods.In our experimental model, the 32 rats were divided into 4 groups: group 1: control group (HEALTHY); group 2: lipopolysaccharide group (LPS); group 3: LPS plus 50 mg/kg AGO group (LPS + AGO50); and group 4: LPS plus 100 mg/kg AGO group (LPS + AGO100). An LPS-induced sepsis model was performed to replicate the pathology of sepsis. Rats from all 4 groups were killed after 12 hours, and their lungs were quickly collected. To investigate the therapeutic strategy, we evaluated tumor necrosis factor-alpha (TNF-α) and nuclear factor-kappa B (NF-κB) messenger RNA expressions by real-time polymerase chain reaction using molecular methods and lung tissue damage indicators using histopathological methods.The expressions of TNF-α and NF-κB were reduced in the groups treated with AGO. The histopathology results supported the molecular results.In this experimental study, we demonstrated for the first time the positive effects of AGO on LPS-induced sepsis in lung tissue using molecular and histopathological methods, indicating that it contributes to the prevention of lung damage. Sepsis is a life-threatening condition that can cause death in hospitalized patients.4To create polymicrobial sepsis to better understand the mechanism of clinical sepsis and develop more effective treatments, a wide variety of models are used, including cecal ligation and puncture, mesenteric ischemia, and reperfusion models, and high-dose lipopolysaccharide (LPS) administration.Melatonin is a hormone synthesized from a substance called tryptophan. It is secreted from the pineal gland in the brain in the dark at night. Melatonin has positive effects on asthma bronchiale, chronic obstructive pulmonary disease, infectious respiratory diseases, lung cancer, pleural diseases, and vascular diseases of the lung.Agomelatine , henceforth referred to as AGO, is an approved drug for the treatment of major depression in adults and a nonselective agonist for MT1 and MT2 and has anti-inflammatory properties.Thirty-two female albino Sprague Dawley rats (10–12 weeks old and 240–260 g) were taken from the Atatürk University Medical Experimental Research Center. The experiments were carried out under normal temperature conditions (22 °C), regular ventilation, and controlled light conditions (12-hour light/dark cycle). The use and care of laboratory animals was approved by the Atatürk University Institutional Animal Care and Use Committee, and the experiments were carried out according to international guidelines . Rats were housed in plastic cages with a sawdust bottom, and standard rat food and tap water were provided ad libitum.LPS (E. coli O55: B5), which is derived from the cell wall of most Gram-negative bacteria, was purchased from Sigma-Aldrich . AGO  from Pfizer, Turkey; and xylazine (Basilazin 2%) from BioTek, Turkey. Metamizole sodium  was obtained from Sanofi-Aventis, and all other chemicals for the laboratory experiments were purchased from Sigma and Merck (Germany). Oral drug administration was given by calculating the drug per gram for each rat with the help of a gavage according to the size of the rats. Similarly, in intraperitoneal applications, the amount of drug to be applied per gram was calculated, and drug applications were completed with the help of an injector.In our experimental model, we divided 32 rats into 4 groups to carry out our research;Group 1 (HEALTHY): Control groupGroup 2 (LPS): Rats were administered only LPSGroup 3 (LPS +AGO50): Rats were administered LPS + 50 mg/kg AGOGroup 4 (LPS +AGO100): Rats were administered LPS + 100 mg/kg AGOAGO at doses of 50 and 100 mg/kg were administered orally 1 hour before intraperitoneal LPS administration.21LPS obtained via phenol extraction from serotypes were used.Lung tissues were collected from all the rats, fixed in 10% neutral formalin for 48 to 72 hours, then routinely processed and embedded in paraffin wax. Serial sections of 3-μm thickness were cut and stained with hematoxylin and eosin (HE) for histopathology before being examined under a light microscope . To evaluate septic lung damage, edema, alveolar wall thickness, and inflammatory cell infiltration were evaluated and scored histopathologically. At least 5 areas were evaluated for each lung slide at ×40 magnification, and the severity of the changes was determined using the following grading: grade 0: − (0% negative), grade 1: + (0%–33% positive), grade 2: ++ (33%–66% moderately positive), and grade 3: +++ (66%–100% severely positive).4We investigated molecularly the expression of inflammatory mediators such as TNF-α and NF-κB by real-time polymerase chain reaction (RT-PCR). For this, we made the following order process: homogenization of lung tissues, RNA isolation, complementary DNA (cDNA) synthesis, and quantitative determination of mRNA expressions.Tissues (20 mg) were homogenized with nitrogen with Tissue Lyser II . RNA extraction was performed on the QIAcube. The total amount of mRNA was measured at 260 nm using a nanodrop spectrophotometer . The obtained RNA was stored at −80 °C under the required conditions.2O were used.Production of cDNA from RNA was achieved with the High Capacity cDNA Reverse Transcription Kit  according. For cDNA synthesis, 10-μL RNA was used in each reaction with Thermal Cycler  according to temperature values. The amount of cDNA was determined by the nanodrop spectrophotometer (EPOCH Take 3 Plate), and the obtained cDNA was stored at −20 ° C. For the cDNA synthesis reaction, 10 μL of total RNA, 2 μL of 10 × Reverse Transcription Buffer, 0.8 μL of a mixture of 25 × Deoxynucleotides, 2 μL of 10 × Reverse TranscriptionRandom Primers, 1 MultiScribe Reverse Transcriptase, and 4.2 μL of diethylpyrocarbonate H2O, for 40 cycles. The cycle threshold (CT) is the cycle number at which the amount of fluorescent signal in RT-PCR experiments exceeds the minimum value  required to be observed. CT values were automatically converted into delta Ct (2–ΔΔCt), and the results were statistically evaluated in the SPSS 25.00 program .4TNF-α and NF-κB mRNA expression was achieved using the TaqMan Gene Expression Master Mix with B-actin  as the reference gene. Amplification and quantification were performed using the StepOnePlus Real-Time PCR System . The following TaqMan Gene Expression Assays for 200 ng cDNA were continued by pipetting, using X μL cDNA (200 ng), 10 μL TaqMan Master Mix, and 1 μL Assay and completed to 20 μL with RNase free HP < .05 was accepted as statistically significant.Data are expressed as meansstandard deviation (SD). To test for any difference among the groups, a one-way analysis of variance test and Duncan’s tests were performed. HEALTHY: The structure of the terminal bronchioles (TB) and alveoli (A), the thickness of the alveolar septa, and the appearance of the arteriolar areas were healthy .LPS: General appearance is compatible with sepsis findings. An increase in alveolar wall thickness and vasodilation (triangles) in alveolar capillaries were observed. Leukocyte infiltration was detected in the whole lung parenchyma, although more intense around the pulmonary artery and terminal bronchioles (stars). Edematous areas were observed in the periphery of the pulmonary artery (points). It was observed that alveolar wall integrity was lost, and lung tissue was destroyed in some parts of the section. LPS+ AGO 50/100 mg/kg: Compared with the sepsis group, there were partial non–dose-related signs of improvement in the treatment groups. Edematous areas around the arterioles, leukocyte infiltration spreading to the lung parenchyma, increase in alveolar wall thickness, and vasodilation findings continue .When histopathological findings were examined, it was concluded that the causative agent had a positive effect, albeit limited, in the elimination of sepsis-induced damage in the lung tissue. To better understand the histopathological data, intracomparative scoring was done according to edema and inflammation assets: none (−), mild (+), medium (++), and intense (+++) .4,2525P < .05) (P < .05).Using RT-PCR, we investigated TNF-α and NF-κB mRNA expressions in the rats’ lung tissue. TNF-α and NF-κB mRNA levels were significantly higher in the LPS group than in the HEALTHY group (P < .05)  and 3. IWe investigated the effects of AGO on septic lung tissues of rats with an experimental sepsis model. We analyzed lungs using molecular and histopathological methods. We molecularly evaluated TNF-α and NF-kB m RNA expressions by RT-PCR and histopathologically evaluated edema, alveolar wall thickness, and inflammatory cell infiltration of lung tissues.Sepsis causes an increase in inflammatory mediators such as TNF-α and NF-κB in lung tissue.It was observed that increased alveolar wall thickness and cellular infiltration in the lungs in the LPS group decreased in the Ago treatment groups.We found in histopathological examination that the occurrence of lung damage was less in the groups that received AGO treatment. The positive effects of AGO, a peripheral melatonin agonist, on the lung may be due to melatonin’s anti-inflammatory, vascular regulatory, and antioxidant properties.28In this study, we demonstrated the molecular and histopathological effects of AGO on LPS-induced sepsis for the first time and revealed its protective efficacy. It remains necessary to conduct detailed research on the effects of AGO on sepsis.In conclusion, this is the first report to demonstrate that AGO prevents lung damage in sepsis. Thus, the use of AGO may be preferred as antidepressant therapy to protect lung tissue in patients at risk of developing sepsis. However, further research is required.In an experimental sepsis model, we investigated molecularly and histopathologically, the effects of agomelatine (AGO), a peripheral melatonin agonist that was found to be suitable for long-term use without central side effects on the lung tissues of rats.To investigate the therapeutic strategy, we evaluated tumor necrosis factor α (TNF-α) mRNA expressions and nuclear factor kappa B (NF-κB), by real-time polymerase chain reaction molecularly, and edema, alveolar wall thickness, and inflammatory cell infiltration histopathologically.The expressions of TNF-α and NF-κB were reduced in the groups treated with AGO.The histopathology results supported the molecular results. We showed for the first time the effects of AGO on experimentally induced sepsis—molecularly and histopathologically—and showed that it contributes to the prevention of lung damage.AGO may be preferred as antidepressant therapy to protect lung tissue in patients with the possibility of developing sepsis. However, further research is required.


Total hip arthroplasty (THA) causes acute blood loss. It may lead to a deficiency in coagulation factors, which, in turn, may lead to increased bleeding during the postoperative period.Thirty patients (18 women) with a mean age of 67 years (range: 63–72 years) participated in this prospective diagnostic study. THA was performed without tranexamic acid administration in the perioperative period. Activities of clotting factors II, VIII, X, and fibrinogen concentration were evaluated before surgery, 6 hours after the procedure, 2, 4, and 6 days after the operation. All laboratory tests were performed using ACL TOP 500 CTS analyzer.No thromboembolic complications were noted during hospitalization. Mean fibrinogen concentration was 366 mg/dL before surgery, which decreased to 311 mg/dL 6 hours after the operation and peaked at 827 mg/dL on the 4th day after the procedure. Activities of factors II and X decreased on the second and fourth days after surgery. Although the activity of factor VIII decreased after the procedure, it remained within the normal range. Increased baseline fibrinogen concentrations were observed in 6 out of 30 (20%) patients. Mean blood loss was 1332 mL  and did not correlate with changes in clotting factor activities.In patients undergoing THA, fibrinogen acts as an acute-phase protein. Activities of clotting factors II and X normalize within 6 days, and although the activity of factor VIII decreases, it remains within the normal range.ClinicalTrials.govThe study was pre-registered May 1st, 2020 on  Total hip arthroplasty (THA) leads to increased coagulation through ischemia, reduced perfusion, and inflammatory reaction . FactorsVarious concepts have been proposed to reduce both blood loss in the surgical field and the volume of blood products transfused after the surgery. One such method involved prophylactic fibrinogen concentrate transfusion in the perioperative period ; howeverStrategies for preventing blood loss also include the blood salvage systems, postoperative flexion of the knee joint, hypotensive anesthesia, and tourniquet; however, the most widespread method for preventing blood loss is the use of anti-fibrinolytic agents, such as tranexamic acid (TXA), and ε-aminocaproic acid , 23. TraA deeper understanding of hemostasis after surgically induced trauma might help to devise better ways of identifying patients requiring optimization of the coagulation and fibrinolysis processes and managing blood loss after those procedures. Therefore, this study aimed to analyze changes in clotting factors II, VIII, and X and fibrinogen concentration in patients undergoing THA for idiopathic osteoarthritis during the first 6 days after surgery. We hypothesized that (1) blood loss correlates with diminished activities of clotting factors, and (2) fibrinogen concentrations will decrease immediately after THA and return to normal levels after that.NCT04372173) on May 1st, 2020. Participation in the study was offered to all patients hospitalized in the Orthopedic Ward, who were eligible for hip arthroplasty for idiopathic osteoarthritis in the study period (from February to April 2018). Exclusion criteria were (1) hip osteoarthritis of non-idiopathic etiology, (2) history of venous thromboembolism (VTE) or arterial embolism, (3) anticoagulation treatment, (4) coagulopathy, (5) infection, and (6) renal failure defined as a creatinine clearance below 50 ml/min. Ultrasound examination was performed only in patients who had a clinical suspicion of VTE, and this decision was left to the discretion of the attending surgeon. All study participants gave written informed consent to participate in the study.The local Bioethics Commission (Military Institute of Medicine) approved the study (84/WIM/2017) on December 20th, 2017). The study was performed accordingly with the tenets of the Declaration of Helsinki. The study was registered in the clinical trials registry (Blood samples were collected as part of routine hemostatic parameter monitoring to determine clotting system parameters (before surgery); blood samples were also collected to determine blood cell counts (6 h post-surgery and fourth-day post-surgery) and, for the study alone, on the 2nd and sixth days post-surgery. Venous blood samples were collected from peripheral veins using 2.7 Ml BD Vacutainer tubes with 3.2% sodium citrate . Other than the six-hour post-surgery data point, samples were collected at fixed hours (8 a.m.).All clotting parameters  were determined using an ACL TOP 500 CTS automated coagulation analyzer .Fibrinogen concentrations were determined by the Clauss coagulation method using HemosIL Q.F.A. Thrombin reagent (Bovine) . The Clauss method measures the coagulation time of a patient’s diluted plasma after the addition of high concentrations of thrombin. Coagulation time is inversely proportional to fibrinogen concentration in the tested sample.Clotting activities of factors II and X were measured by the coagulation method using HemosIL RecombiPlasTin 2G reagent  and lyophilized substrate plasma: HemosIL Factor II deficient plasma  and HemosIL Factor X deficient plasma  to determine prothrombin time values. The patient’s plasma is diluted and added to plasma without a given coagulation factor (substrate plasma). Substrate plasma clotting time changes proportionally to the clotting factor activity in the patient sample.Factor VIII activity was measured by the coagulation method using HemosIL SynthASil reagent  and HemosIL Factor VIII deficient plasma lyophilized substrate plasma  to determine the activated partial thromboplastin time. The measurement consists of adding diluted patient plasma to the substrate plasma and determining the activated partial thromboplastin time. Substrate plasma clotting time changes proportionally to the clotting factor activity in the patient sample.We calculated blood loss using Mercuriali’s formula . This foAll patients were operated on by a single surgical team using a posterolateral approach, with cementless hip replacements implanted, and without tranexamic acid administered in the perioperative period. All patients underwent VTE prophylaxis (enoxaparin 40 mg 1 × 1 subcutaneously from the surgery day), per local guidelines [The following data were entered into the database: age, body mass index (BMI), gender, perioperative risk assessment scale according to the American Society of Anesthesiologists score (ASA score), blood transfusions, hemoglobin levels, and hematocrit before surgery and 2 days post-surgery, and instances of thromboembolic complications. This study was observational and did not require any changes in the treatment of patients.p < 0.05 were considered statistically significant.Statistical analysis was performed using MedCalc 18.5 software , and normal distribution was verified using the Kolmogorov-Smirnov test. Continuous variables of normal distribution were presented as an average with a 95% confidence interval, minimum, and maximum values. In the absence of normal distribution, variables were transformed logarithmically, and their normality was retested. Student’s t-test for related variables was used to compare averages after logarithmic transformation. In the absence of a normal distribution of post logarithmic transformation, variables were compared using the Wilcoxon test. Medians were compared using the Mann-Whitney test. We used stepwise multiple regression to examine the correlation between blood loss and demographic parameters, concentrations, and changes in clotting factors. For hypothesis testing, values of Forty-four patients scheduled for hip arthroplasty were hospitalized, and all of them qualified for hip replacement; however, only 32 met the inclusion criteria, and two refused to participate in the study. Finally, data from 30 patients were analyzed. Fig..Fig. 1S2 (21–29 kg/m2), and an average ASA score was II . Preoperative hemoglobin level was 13.1 g/dL (12.9–15.4 g/dL) and postoperative hemoglobin level on the 2nd day was 10.5 g/dL (9.9–11.1 g/dL). Transfusions were not needed, and VTE symptoms were not observed during patients’ hospital stay and up to 6 weeks postoperatively. Mean blood loss was 1332 mL .In the study group, 18 patients were women, and the average age was 67 years (63–72.2 years). Average BMI was 26 kg/mp < 0.0001). For all patients, fibrinogen concentrations exceeded the reference level on the 4th and sixth days post-THA.Changes in fibrinogen concentration are shown in Fig. p < 0.0001) on the 2nd and fourth days post-surgery. Average factor VIII activity was significantly (p < 0.0001) lower between 2nd and sixth-day.Factor II activity decreased significantly (p = 0.009) until the 4th day after the procedure (p < 0.0001). Average clotting factor activity values with 95% confidence intervals and value ranges are presented in Figs. post-surgery. Significantly reduced factor X activity was recorded from 6 h  before THA, which is likely a manifestation of hip osteoarthritis inflammatory component or the influence of comorbidities . Althoug0% before. Perhaps low molecular weight heparin may have caused a drop in the said factor activity because heparin, apart from its anticoagulant activity, displays an anti-inflammatory effect [.Surgical trauma is associated with increased systemic activities of procoagulant and anticoagulant pathways of coagulation . In thisy effect . A slighy effect .Our study had some limitations. This study consisted of a relatively small group of patients and did not evaluate factors potentially influencing the concentration levels of coagulation factors. However, all patients referred for THA in this study were free from infection foci, their comorbidities were well managed, and the perioperative risk levels were limited (ASA I - III). In this study, blood samples were collected preoperatively and after 2, 4, and 6 days, which might have caused us to miss the actual peak and nadir times. There is some variability in methodologies used in this aspect—another research group studied clotting factor activities alone pre-and postoperative,  and otheAfter THA, marked blood loss may lead to higher transfusion rates, which may negatively affect surgical outcomes and lead to higher complication rates . It woulFibrinogen behavior is similar to that of an acute-phase protein—its concentration drops temporarily on the day of surgery (6 h after surgery) and increases after that. Elevated fibrinogen concentration persisted from days 2 to 6 post THA, with a maximum concentration recorded 4 days after the procedure. These changes in concentrations are probably associated with the inflammation that occurs during the healing process. No significant decrease in blood clotting factors was observed in patients undergoing THA. The use of low-molecular-weight heparin may be responsible for a slight decrease in activities of factors VIII and X. Blood loss was not correlated with changes in clotting factor concentrations and changes in clotting factors’ activities.