The following parameters were evaluated: the number and type of recovered WBCs, RBCs contamination, PLTs contamination, vitality of neutrophils, and chemotactic properties of neutrophils. Clinical comparison was performed between 80 patients  injected with 99mTc-HMPAO-WBCs, using HES as the sedimentation agent, and 92 patients  injected with 99mTc-HMPAO-WBCs using Gelofusine as the sedimentation agent. Patients were affected by prosthetic joint infections, peripheral bone osteomyelitis, or vascular graft infection. We compared radiolabelling efficiency (LE), final recovery yield (RY), and diagnostic outcome based on microbiology or 2-year follow-up. Results showed that HES provides the lowest RBCs and PLTs contamination, but Gelofusine provides the highest WBC recovery. Both agents did not influence the chemotactic properties of WBCs, and no differences were found in terms of LE and RY. Sensitivity, specificity, and accuracy were also not significantly different for WBCs labelled with both agents . In conclusion, Gelofusine can be considered a suitable alternative of HES for WBCs separation and labelling.The scintigraphy with radiolabelled autologous leukocytes (WBCs) is considered the gold-standard technique for imaging infections. Leukokit® is a commercially available, disposable, sterile kit for labelling WBCs ex vivo. In this kit, WBCs isolation from red blood cells (RBCs) was performed using poly(O-2-hydroxyethyl)starch (HES) as the RBCs sedimentation agent. Due to its poor availability, HES has been recently replaced by Gelofusine as the RBC sedimentation agent. The aim of this study was to compare the labelling efficiency and the diagnostic accuracy of WBCs labelled with Leukokit® with HES vs Leukokit® with Gelofusine. WBCs were isolated using HES or Gelofusine for 45 minutes and then purified from platelets (PLTs) and labelled with 1.1 ± 0.3 GBq of freshly prepared  WBCsidelines –12. Howein vitro the suitability of Gelofusine as an alternative to HES. This was achieved through the assessment of several parameters after erythrocyte separation: the number and type of recovered WBCs; RBCs contamination; platelets (PLTs) contamination; viability of neutrophils; chemotactic properties of neutrophils.The aim of the study is to test 99mTc-HMPAO, as requested by the Italian legislation.After the evaluation of safety and efficacy of the new sedimentation agent (Gelofusine) performed by the producers of Leukokit® , we aimed at evaluating the performance of this “new Leukokit®” , as compared to the previous kit containing HES , for WBCs purification and labelling with The second goal of the study consists in the complete validation of the “new Leukokit®”, containing Gelofusine, as compared to the previously commercialized kit, containing HES, applying our standard operating procedure (SOP) for the validation and annual revalidation of the WBCs purification and labelling procedure.Secondly, the “new” and the “old” Leukokit® were compared in terms of WBCs labelling efficiency, recovery yield, and diagnostic accuracy in patients with suspected infections.in vitro study, WBCs were isolated from the blood of 5 healthy volunteers. In brief, 30 ml of blood was withdrawn from each subject with a syringe containing 6 ml of anticoagulant citrate dextrose (ACD). The blood was then divided in 3 Falcon-type tubes (12 ml each) containing 3 ml of HES, Gelofusine, or 0.9% NaCl solution, respectively. Gelofusine was provided at 4% concentration of a clear, transparent, and slightly yellowish sterile solution . After approximately 40 minutes of sedimentation, cell-rich plasma (CRP) was collected from each vial and an aliquot was used for FACS analysis to evaluate the number of WBCs, RBCs, and PLTs contaminations. Another aliquot was used for the viability testing by the trypan blue exclusion test. The remaining CRP was then centrifuged on Lymphoprep® for 10 minutes at 1000 rpm to isolate granulocytes. After the centrifugation, the supernatant was discarded and the pellet was resuspended in 5 ml of phosphate buffered saline (PBS). FACS analysis and viability test were then repeated, and the rest was used to evaluate the retention of the migrating capabilities of granulocytes.For the μm pores (Corning®) placed in a 24 multiwell plate. In the upper chamber of each well were placed 105 granulocytes in 100 μl of RPMI, whereas the lower chamber of each well contained 650 μl of RPMI supplemented with 10% FBS. The plate was then incubated overnight in an incubator at 37°C and 5% CO2. The day after, the upper portion of the membrane contained in each well was cleaned to remove residual granulocytes and then the membranes were rinsed in Coomassie blue followed by distilled water. Each membrane was cut from the support and placed on a microscopy slide for counting.Granulocytes migration was evaluated using a 24-well permeable support with 5 99mTc-HMPAOQC1 for the evaluation of hydrophobicity of QC2 for the evaluation of clumps after WBCs purification and labelling by visual inspectionQC3 to calculate the labelling efficiency (LE) and labelling yield (LY) of WBCsQC4 to evaluate the sterility of the final productQC5 to evaluate the apyrogenicity of the final productQC6 to evaluate the vitality of radiolabelled cells by trypan blue exclusion test99mTc-HMPAO from labelled WBCs at different time pointsQC7 to evaluate the percentage of spontaneous release of The SOP for the validation includes the following quality control tests (QC):Leukokit® validation was performed in 6 patients who donated 60 ml of blood each (age 30–60), once given the written informed consent. For each patient, 60 ml of blood was withdrawn in two syringes with 6 ml of ACD each (30 ml and 30 ml of blood).The first 36 ml was used for WBCs labelling with the “old Leukokit®” containing HES as the sedimentation agent; the other 36 ml was used for WBCs labelling with the “new Leukokit®” containing Gelofusine as the sedimentation agent.The whole procedure requires between 2 h 45 min and 3 h 30 min depending on the erythrocyte-sedimentation rate (ESR) of the patient. Additional 4 h were necessary to complete all quality controls.99mTc-HMPAO-WBCs, labelled using HES as the sedimentation agent, and 92 patients  injected with 99mTc-HMPAO-WBCs, labelled using Gelofusine as the sedimentation agent. Patients were affected by prosthetic joint infections, peripheral bone osteomyelitis, or vascular graft infection, as reported in Clinical comparison was performed between 80 patients  injected with Several parameters were considered: the radiolabelling efficiency (LE), final recovery yield (RY), and diagnostic outcome based on microbiology or 2-year follow-up.For each group of patients, diagnostic accuracy, sensitivity, specificity, negative-predictive value (NPV), positive-predictive value (PPV), and their confidence intervals (CI) were calculated considering the number of patients as true positive (TP), true negative (TN), false positive (FP), and false negative (FN) based on the correspondence between the WBC scan and microbiology or follow-up.t-test for continuous variables after confirmation of normal distribution by the Kolmogorov–Smirnov test. Results in patients were statistically compared performing the Student's t-test, if normally distributed, otherwise performing the Mann–Whitney test.Comparisons of WBCs concentration, RBCs and PLTs contamination, and migration results were performed using Student's p values were <0.05. All calculations were performed using Prism 7 .All results were given as mean values ± SD or SE, unless otherwise indicated. Differences were considered significant when p=0.02 and p=0.03 for WBCs concentration (before), respectively, for HES and Gelofusine vs control; p=0.04 and p=0.07 for GRs concentration (before), respectively, for HES and Gelofusine vs control).Gelofusine showed the best results in terms of number of recovered WBCs and granulocytes isolated from the blood of five healthy volunteers compared to HES and control . Differep=0.04).On the contrary, the use of HES gave slightly lower RBCs and PLTs contamination . DiffereHigh viability of isolated granulocytes was observed before and after purification, as revealed by the trypan blue exclusion test .No statistical differences were observed between samples analyzed immediately after sedimentation  or after centrifugation over the Lymphoprep® gradient (granulocytes). The same applies for samples sedimented with HES or Gelofusine or control.p=0.001, p=0.007, and p=0.0006 for HES, Gelofusine, or control groups, respectively).Isolated granulocytes retained their ability to migrate in response to attracting stimuli, as revealed by migration assays performed in medium with or without 10% FBS Figures  and 5. TIn addition, there was no significant difference between cells prepared with HES or Gelofusine or without any sedimentation agent as control.in vitro release of 99mTc-HMPAO from labelled WBCs at different time points incubated at 37°C .The main QCs that are reported here (and of interest for the comparison of Gelofusine vs HES) are the labelling efficiency (LE), the labelling yield (LY), the vitality of labelled cells using the trypan blue exclusion test, and the spontaneous The CRP volume was different for each patient, depending on ESR of each one (range 20–30 ml).The average labelling efficiency (LE) was similar between the two sedimentation agents: 72.3 ± 4.8% for HES and 72.5 ± 8.9% for Gelofusine; the labelling yield (LY) was slightly better for HES (54.5 ± 4.1%) than Gelofusine (52.7 ± 5.8%). Differences were not statistically significant .99mTc-HMPAO from labelled WBCs was evaluated. Results showed a less release from cells at 10 minutes for Gelofusine (4.9 ± 1.7%) in comparison to HES (5.4 ± 1.5%), showing similar results at 1 h and 4 h   . All dift-test p=0.06 and p=0.57, respectively, for LE and RY).For the “new Leukokit®,” the LE and RY, calculated on 92 samples, were 71.4 ± 11.4% and 55.6 ± 9.4%, respectively, whereas for the “old Leukokit®”, the LE and RY, calculated on 80 samples, were 74.5 ± 9.6% and 54.8 ± 10.4%, respectively. Both differences were not statistically significant .A possible criticism to our work can be raised by the consideration that we used blood of normal subjects for the Both HES and Gelofusine sedimentation agents allowed reproducible separation of granulocytes from whole blood with a high percentage of purity and vitality as required by EANM guidelines.In particular, Gelofusine can be considered a suitable alternative of HES for WBCs separation and labelling, yielding to high labelling efficiency, without cell damage and high diagnostic accuracy.


P < 0.05) between RT, FT, and FT + ISBS groups. The hemoglobin level in Group FT + ISBS was significantly higher than in Group RT and Group FT. Comparing the duration of hospital stay of 3 groups, Group RT stayed for a significantly longer time than Group FT and Group FT + ISBS, Group FT + ISBSmuch shorter than Group FT. Comparing the hospitalization expense, GroupFT + ISBS significantly expended less than Group RT and Group FT. Our study shows that laparoscopic splenectomy with fast-track care is feasible, effective, and safe for patients who require splenectomy. Fast-tracking with intraoperative blood salvage improved the fast-track laparoscopic splenectomy procedure.Fast-track surgery is claimed to reduce medical morbidity, eliminate the hospitalization needs, and shorten the convalescence period. Intraoperative bleeding as the main complication is also the primary cause of conversion from laparoscopic to open splenectomy. Intraoperative blood salvage can reduce transfusion requirements, decrease the conversion rate to open, and promote fast-tracking in laparoscopic splenectomy (LS). From November 2007 through December 2016 we collected medical data of 115 LS patients. There were three groups: 54 patients receiving routine care (we marks them as Group RT), 33 patients with fast-track care (Group FT), and 28 receiving fast-track care receiving intraoperative splenic blood salvage and autotransfusion (Group FT + ISBS). These medical data are comprised of included three phases . There were significant differences ( However, open splenectomy is associated with a high risk of intraoperative hemorrhage and postoperative pain, and it is contraindicated in patients who have poor liver function2. Laparoscopic surgery is considered as a minimally invasive technique to reduce the systemic immunologic and inflammatory responses resulting from the surgical trauma of open surgery5. Laparoscopic splenectomy (LS) was first introduced by Delaitre and Maignien6 and has been well studied in patients with splenomegaly due to its promising clinical benefits7. In the last 20 years, studies have shown that LS is safe and feasible. There are no significant differences between LS and open splenectomy regarding complications and mortality10.Splenectomy has generally been the treatment of choice for splenomegaly and hypersplenism since the 1950s11. FT surgery is used to enhance the recovery from “single-modality” evidence-based surgical care procedures by applying “a multimodal effort” pattern. FT surgery combines various practices and care regimens in the perioperative period, which include epidural or regional anesthesia, minimally invasive techniques, optimal pain control, preoperative oral nutrition, and postoperative early ambulation12. Applying these procedures, fast-tracking can reduce stress responses and organ dysfunction to shorten the recovery time, avoid complications, and decrease cost13.Fast-track (FT) surgery was first introduced in 1991 for colorectal surgeryet al.14 showed that while LS in patients with massive splenomegaly is feasible, it is associated to a longer median operating time, higher conversion rate, higher postoperative morbidity, and longer median postoperative stay. Intraoperative bleeding is the main complication and cause of conversion during LS. Combining LS with intraoperative blood salvage with autotransfusion retains the advantages of autotransfusion and eliminates the risks associated with homologous transfusion.Patel The evidence for the use of FT with or without intraoperative blood salvage in LS remains incomplete. We performed this study to investigate the application of FT surgery with or without intraoperative blood salvage in LS.This study was conducted in the General Surgery Department, Zhejiang University School of Medicine Second Affiliated Hospital, China. From November 2007 through December 2016, a retrospective review of medical charts was performed of patients who underwent LS by the same surgeons’ treatment team. Totally 115 patients involved in this study, 54 patients received routine care (group RT), 33 received FT care (group FT), and the rest 28 received both FT care and intraoperative splenic blood salvage (group FT + ISBS). The data included 3 perioperative  phases  or shears. The 12-mm trocar was positioned in the left midaxillary line halfway between the left costal margin and the iliac crest or below the border of the spleen for the use of the endoscopic linear vascular stapling device or other supplementary device.This study team has 20 years experience in LS performing, the surgery procedures: After general anesthesia administration and endotracheal intubation, patients were placed in the right lateral decubitus and 60° semilateral position. Four trocars, one with a diameter of 12 mm, two 10 mm, and one 5 mm were placed with COUsing LigaSure instruments, mobilization of the spleen was performed inferiorly. After that, the splenocolic attachments and the gastrocolic ligament were divided and opened to expose the lesser sac. The gastrosplenic ligament (including the short gastric vein) and the splenorenal ligament were dissected and coagulated carefully and then cut. The splenic hilum was raised, and the splenectomy was completed while cutting the splenic artery and vein using an endoscopic linear vascular stapler . The spleen was then freed and placed into a hard plastic bag to prevent spillage of blood or splenic pulp into the abdominal cavity.16. In this study, these specific techniques were applied to FT and FT + ISBS groups. The control group underwent conventional laparoscopy.FT surgery includes various techniques that are also used in the care of patients undergoing LS Table 16. In thPatients in the FT + ISBS group underwent intraoperative cell salvage. Blood collected from the operative field or the freed spleen in a hard bag were transferred to an autotransfusion device  using a double-tube suction device Fig. . The RBCP value < 0.05 was considered statistically significant. IBM SPSS Statistics for Windows, Version 19.0  was used for statistical analysis.Other analysis variables included the patient characteristics, intraoperative parameters, and postoperative course. Numerical data were expressed as mean ± standard deviation (SD). Group means were compared using Student’s t-test  or the Mann–Whitney U-test , and the Chi-square test was used to compare percentages. A As shown in Table Comparing these three groups, there were no significant differences in operative time, blood loss, volume of blood autotransfused, and intraoperative complications Table .Table 4OP < 0.05). Furthermore, the increase in Hb in the FT + ISBS group was (2.3 ± 1.0) g/L at postoperative day one, which was significantly higher than (0.5 ± 0.2) g/Lin the RT and (0.6 ± 0.4) g/Lin the FT groups. At postoperative day three, the Hb increase in the FT + ISBS group was (2.5 ± 0.8) g/L, which was significantly higher than (0.6 ± 0.3) g/L in the RT and (0.5 ± 0.2) g/L in the FT groups. There were significant differences (P < 0.05) between the groups without splenic blood autotransfusion and the FT + ISBS group. Meanwhile, the other blood test data  were not statistically different between the groups. The postoperative blood test data are shown in Table The Hb level in the FT + ISBS group was (11.2 ± 1.1) g/Lafter surgery, which was significantly higher than (9.3 ± 0.7) g/Lin the RT group and (9.4 ± 0.9) g/L in the FT group, showing a significant difference P < 0.0. FurtherFever was the most common complication after LS. Nine patients of RT group had fever (temperature > 38.5 °C), while four in the FT group, and five in the FT + ISBS group had fever after operation. There are other complications including pulmonary infection, pancreatic leakage, subphrenic abscess, and injuryto the colon. Four patients in the RT group, two patients in the FT group, and one patient in the FT + ISBS group experienced one of these complications. All complications were managed by conservative treatment, no patients died in the perioperative period. There were no significant differences in postoperative data among the three groups.P < 0.05). The median length of the hospital stay of patients in FT + ISBS group was (5.4 ± 1.3) days after surgery, which was significantly shorter than (6.7 ± 2.8) days in FT group, a significant difference (P < 0.05). The hospitalization expense of FT + ISBS group was (41.2 ± 13.6) thousand RMB, which was significantly less than that of RT (66.5 ± 21.5) and FTgroups (53.5 ± 16.8).Our discharge criteria included the following parameters:postoperative pain adequately controlled with oral analgesics, no need for intravenous nutrition or fluids, mobilization (out of bed ≥ 6 h daily), recovery of bowel function (stool or repeated flatus), and no complications requiring hospital treatment. The hospital stay of RT group was (8.4 ± 2.2) days after surgery, which was significantly longer than (6.7 ± 2.8) days of FT group and (5.4 ± 1.3) days of FT + ISBS group, showing a significant difference  after surgery, and the expense was (66.5 ± 21.5) thousand yuan. The hospital stays and expenses of the FT and FT + ISBS groups  were significantly less than those of the RT group. Fast-tracking was very important in reducing the stress responses and organ dysfunction, which led to a decreased recovery time, fewer complications, and less cost. Information about FT LS was provided not just to patients but to all concerned. The patients who underwent FT surgery were well prepared and acknowledged with all procedures25, improve patient well-being  preoperatively26, and lower the risk of postoperative fluid overloading and conditions delaying discharge27.Other preoperative elements of FT surgery include fluid optimization, alcohol and smoking abstinence, oral glucose fluid intake 2 hour before surgery, and no bowel preparation, all of which could reduce postoperative insulin resistance28, epidural analgesia by anesthetists can effectively relieve the patients’ pain. There are other ways to control postoperative pains including self-control venous analgesic pumps, intravenous analgesics, and oral analgesics, depending on the patient’s needs. Good pain control can reduce the length of the hospital stay after surgery and therefore decrease hospitalization expenses.In the intraoperative period, using less intraoperative grainageWith good pain control, patients in the FT and FT + ISBS groups were encouraged to move on the day of surgery and get up from bed on postoperative day 1. These procedures can reduce the risk of complications such as deep venous thrombosis, because the platelet level in patients who have undergone splenectomy is much higher than the preoperative level, even more than 1000e9/L in some hypersplenism patients with splenomegaly, increasing the risk of thrombosis.FT applied to LS promotes patient rehabilitation. In our study, we found that the hemoglobin level in the FT + ISBS group was significantly higher than in the RT and FT groups. This result held true for the postoperative hemoglobin determinations on days 1 and 3 after surgery. In other words, there were significant differences between the patients who did not receive autotransfused blood intraoperatively and the FT + ISBS group of patients who did. Autotransfusion in patients in the FT + ISBS group played an important role in increasing the hemoglobin level, which is important for patients of variceal bleeding due to cirrhosis, and thus reducing symptoms such as the dizziness and fatigue, all of which can lower the patients’ willingness to be discharged.28. The conversion to open splenectomy in this situation is easier with intraoperative blood autotransfusion. Furthermore, there are additional potential postoperative complications that come into play if the surgery is converted to open splenectomy28. Examples of these complications are delays in pain control, mobilization, and resumption of oral nutrition, which are not achieved easily.Bleeding is the main intraoperative complication of LS and the main reason to convert to open surgery, especially for the splenomegaly patients, although LS is known to be safe. Conversion to open splenectomy should be considered when intraoperative bleeding cannot be managed promptly29. This comes from conditions such as low platelet level, impaired coagulation factors, splenomegaly, and the development of collateral hepatic vessels. In our study, blood from the operative field and resected spleen were recovered by an autologous blood recovery system. In LS, intraoperative splenic blood autotransfusion provides the advantages of autotransfusion and eliminates the risks associated with homologous transfusion. Intraoperative blood autotransfusion is relatively easy on a technical level and has the advantage of significantly increased postoperative hemoglobin levels, as described earlier.The risk of intraoperative bleeding in splenomegaly with or without portal hypertension from liver cirrhosis is highTherefore, we believe that FTLS with intraoperative autotransfusion is worthy of consideration and clinical application.FT LS is feasible, providing effective and safe surgical care for patients who require splenectomy. FT LS with intraoperative blood autotransfusion has the advantage of further LS improvement, especially for patients with potentially massive intraoperative bleeding or rare blood types (such as Rh negative), or in patients with religious concerns related to blood transfusion. Our study showed that additional improvements in FT surgery can be developed and applied optimizing both the procedure and patient outcomes.


Vitamin D levels may differ between migrant and non-migrant populations, especially among non-western immigrants living in a country with limited sun exposure such as Germany. This study examined serum vitamin D concentration and associated factors among Berliners with and without Turkish background.Two samples (with and without Turkish roots) were recruited in the inner city of Berlin for a cross-sectional study assessing serum vitamin D concentration. Linear regression analyses were used to examine sociodemographic, lifestyle and medical factors associated with serum vitamin D levels.p < 0.001. In the Turkish female subgroup, veiled women had considerably lower levels than unveiled women: 14.4 nmol/L  vs 24.9 nmol/L , p < 0.001. Multivariable regression analysis revealed that among the Berliners of Turkish descent, being active less than 150 min per day, and being overweight/obese were independently associated with a lower vitamin D concentration. In the non-migrant sample besides being overweight and obese, female sex was associated with lower vitamin D concentrations.In the analyses, we included 537 subjects  years) with and 112 without Turkish background . The Turkish sample had lower mean  vitamin D levels than the non-Turkish sample: 22.7 nmol/L  vs 34.7 nmol/L , Serum vitamin D levels were considerably low in Berliners of Turkish descent, and especially in veiled women. Potentially modifiable factors of low vitamin D levels were high BMI and low physical activity. These findings should be considered in the development of future public health strategies for subpopulations with Turkish migration background.The online version of this article (10.1186/s12889-019-6446-5) contains supplementary material, which is available to authorized users. Levels > 75 nmol/L have been defined as vitamin D sufficiency, while 50–75 nmol/L have been considered as vitamin D insufficiency and < 50 nmol/L as vitamin D deficiency [Vitamin D is a steroid hormone with regulatory effects on the calcium and phosphate homeostasis, parathormone (PTH) and thyroxine secretion . Vitaminficiency , 3.The main source of vitamin D is sunlight exposure inducing vitamin D production in the skin through ultraviolet B radiation (UVBR). Only a few foods such as fatty fish or cod liver oil contain vitamin D in a relevant amount. Vitamin D can be ingested as supplement, and in some countries foods are fortified with vitamin D . The maiVitamin D has become a worldwide research issue and vitamin D deficiency has been described as pandemic by several authors; the lowest population-based vitamin D levels have been assessed in Asian countries such as China, Mongolia, India and the Middle East –9. In GeIt is known that non-western immigrants migrating to a country with limited sun exposure are at higher risk for vitamin D deficiency, however there are still considerable knowledge gaps regarding factors other than darker skin or veiling behavior , 19–23. The present cross-sectional analysis combined two samples from the pre-test phase of the German National Cohort Study (GNC) . The firDetails of the recruitment strategy of the “P1 project” were published by Reiss et al. . BrieflyFor the first recruitment strategy, randomly chosen persons received an invitation letter including study information and a response form. Up to two reminder letters were sent out in case of non-response. If a phone number was available, potential participants were contacted by phone.To identify persons with Turkish origin, an onomastic procedure  was applied for the “P1 project” sample by the Robert Koch-Institute, the national Public Health institute in Germany. Potential participants received all study material in both the German and Turkish language. Bilingual study assistants, mostly medical students, conducted up to three home visits on different days and times. If a potential participant was not at home, an information card with contact details was left at the home address.For the community-based recruitment approach, key persons in Turkish networks were invited to provide information about the study and to participate in focus groups. Bilingual study staff distributed study materials in migrant settings  and invited people to the study.Inclusion criteria were 20–69 years of age, the ability to understand the aims of the study, and a signed informed consent (in German or Turkish). All participants received a 15€ expense allowance to cover travelling costs and a letter with results from the study center visit by post.The study was approved by the ethical review committee of the Charité-Universitätsmedizin Berlin, Germany.The modified health questionnaire from the German Health Interview and Examination Survey was used to assess socioeconomic status, medical history, health behavior, physical and mental diseases and migration background . A partiIn the “P1 project”, weight, height, circumference of hip and waist, blood pressure and heart rate were measured by trained physicians and nurses according to SOPs; in the control sample, weight and height were assessed by self-report.A blood sample was taken by trained physicians or nurses according to SOPs. The blood samples were analyzed for 25(OH) D, full blood count, liver enzymes, kidney function, cholesterol levels, and blood glucose. Vitamin D was measured as 25(OH) D in serum via chemiluminescence immunoassay by the “hospital-laborverbund Brandenburg-Berlin”. Participants received their results as postal letter two weeks after their visit in our study center.2, overweight as BMI from 25 to less than 30 kg/m2 and obesity as a BMI of 30 kg/m2 and higher. Alcohol consumption was categorized into no consumption, moderate consumption (up to 12 grams/day (g/d) for women and up to 24 g/d for men) and hazardous consumption (> 12 g/d for women and > 24 g/d for men). Smoking was dichotomized in never or ex-smoker versus smoker. A person was defined as a smoker, if he or she indicated smoking daily or occasionally, while ex-smokers were defined as former smokers who quit smoking at least one year ago.Physical activity was dichotomized into categories of 150 min or more vs less than 150 min physical activity per week according to WHO recommendations . Body maWe computed descriptive statistics for sociodemographic, lifestyle and medical characteristics using mean with standard deviation (SD) and median for continuous variables and absolute and relative frequencies for categorical variables.p < 0.2. In the final model for the sample with Turkish background the variables BMI, physical activity and sex were included. In the final model for the sample without Turkish background, BMI and sex were included. Resulting Beta-estimates are presented with 95%-CI and p-value.Potential influences on the serum vitamin D concentration as the primary outcome were evaluated using univariable and multivariable linear regression models that were stratified for study sample. Means with 95% confidence intervals (95% CI) are presented from univariable regression in the subgroup of women with Turkish background. We used a stepwise backwards approach for the selection of the co-variables in the multivariable linear regression model with a limit for inclusion of p < 0.2. In the final model for the sample with Turkish background the variables age, physical activity and chronic disease were included. All odds ratios are presented with 95%-CIs. For the sample without Turkish background the variables sex, BMI, physical activity, smoking status and chronic disease were included.As a sensitivity analysis we conducted for each study sample stratified univariable and multivariable logistic regression analyses with the binary outcome variable “vitamin D deficiency” based on the recommendations by the Institute of Medicine . Crude oAll analyses were explorative rather than strictly hypotheses testing. Missing data were not imputed. All analyses were performed using The SAS System®, Version 9.4 under Windows operating system.The study included 597 persons of Turkish descent and a random sample of 129 Berliners without Turkish roots. After excluding participants without vitamin D measurement, with regular vitamin D intake, and those with a high percentage of missing data the final analysis sample consisted of 537 persons with Turkish background and 112 unselected Berliners without such a background as the control group , and 22.9 ± 13.0 nmol/L vs 38.5 ± 26.7 nmol/L among men (p = 0.002). In the Turkish sample 94.8% of the women and 95.2% of the men had vitamin D levels below 50 nmol/L (suggesting “vitamin D deficiency” [p < 0.001). Vitamin D concentrations of under 25 nmol/L  D level was lower in the Turkish sample: 22.5 ± 13.8 nmol/L vs 31.5 ± 13.8 nmol/L among women (iciency” ) vs 86.9iciency” ) could bp < 0.05   revealed that among the sample with Turkish background a low physical activity level, being overweight and obese were independently associated with lower vitamin D levels Table . Among t5) Table . A firstAdditionally, we conducted a logistic regression analysis as sensitivity analysis with vitamin D deficiency as the outcome stratified for both study samples  by potentially associated factors. Table S3. Vitamin D status of Berliner sample without Turkish roots (n = 112) by potentially associated factors. Table S4. Vitamin D concentration as the outcome based on univariable and multivariable linear regression analyses (beta-estimates with 95% confidence levels) – unselected sample without Turkish background (including one participant with outlier: Vitamin D of 164 nmol/L). Table S5. Vitamin D deficiency as outcome based on univariable and multivariable logistic regression analyses (odds ratios with 95% confidence levels) – sample with Turkish background. Table S6. Vitamin D deficiency as the outcome based on univariable and multivariable logistic regression analyses (odds ratios with 95% confidence levels) – unselected sample without Turkish background. (DOCX 28 kb)


Patients with type 2 diabetes have a significantly increased risk of cardiovascular disease (CVD) compared to the general population—with CVD accounting for two out of every three deaths in patients with diabetes. In 2008, the FDA suggested that CVD risk should be evaluated for any new antidiabetic therapy, leading to a multitude of large CVD outcome trials to assess CVD risk from these medications. Interestingly, several of these outcome trials with new novel antidiabetic therapies have demonstrated a clear and definite CVD advantage at mid‐term follow up in high‐risk patients with T2DM. In this review, we discuss two relatively new classes of diabetic drugs, sodium‐glucose cotransporter 2 inhibitors and glucagon‐like peptide 1 agonists, and their efficacy in improving cardiovascular outcomes. The secondary outcome, cardiovascular death, occurred in 12.8% of patients in the empagliflozin group compared to 14.3% of the placebo group, although this difference was not statistically significant (P = 0.08).P = 0.002).In 2008, the US FDA suggested that new antidiabetic therapies be evaluated for associated alterations of cardiovascular risk, given concerns that specific glucose‐lowering drugs might worsen cardiovascular outcomes (CVOs).P = 0.02).P = 0.24).P = 0.04).P < 0.001); infection of male genitalia ; mycotic genital infection in females ; bone fractures ; and volume depletion .Subsequently, the effects of treatment with canagliflozin, another SGLT2 inhibitor on cardiovascular events were investigated in the CANVAS and CANVAS‐R studies, which were published in combined form as the CANVAS Program Table .27 The cP = 0.17) but resulted in a lower rate of cardiovascular death or hospitalization for heart failure  when compared with placebo.More recently, the effects of treatment with dapagliflozin, another SGLT2 inhibitor upon cardiovascular events were evaluated in the DECLARE—TIMI trial, which randomized 17 160 patients with type 2 diabetes and either established cardiovascular disease or multiple cardiovascular risk factors to 10 mg dapagliflozin or placebo once daily.P < 0.0001) in favor of those treated with SGLT‐2 inhibitors and the RR of MACE was 0.84 .P = 0.049). Notably, over 50% of the participants included in this meta‐analysis were from the EMPA‐REG OUTCOME study.In addition, Wu et al performed a meta‐analysis of six regulatory submissions (37 525 participants) and 57 published trials (33 385 participants), which included seven different SGLT‐2 inhibitors.The mechanism through which SGLT2 inhibitors affect changes in cardiovascular outcomes is not entirely known. It is suspected that, in addition to the decrease in glucose levels, small but favorable effects on other CV risk factors contribute. These include decrease in body weight, favorable modifications to lipid levels, and lower blood pressure.1.2In 1964, Elrick et al showed that oral glucose administration stimulated greater insulin secretion than intravenous glucose administration.P = 0.01, HR 0.87).P = 0.046), no significant trends were observed in rates of fatal MI, stroke, or hospitalization for heart failure.P = 0.06)..The LEADER trial, one of the first GLP‐1 agonist trials evaluated the effects of the addition of liraglutide, a GLP‐1 agonist, on MACE, defined as CV death, nonfatal MI, or nonfatal stroke, in 9340 patients with diabetes and elevated cardiovascular risk when compared with placebo...P = 0.81 for superiority); 4.0% of patients in the lixisenatide group and 4.2% in the placebo group were hospitalized for heart failure (P = 0.75)..Another independent GLP‐1 agonist study, the ELIXA study, evaluated the effects of lixisenatide when compared to placebo in 6068 diabetes patients who had a history of myocardial infarction or hospitalization for unstable angina in the previous 180 daysP = 0.02).P = 0.04).P < 0.001 for both)..Furthermore, the SUSTAIN‐6 trial evaluated the effects on incidence of composite cardiovascular death, nonfatal stroke, and nonfatal MI of 0.5 mg or 1.0 mg of another GLP‐1‐agonist, semaglutide, given once weekly against placebo in 3297 patients.P < 0.001), though this difference did narrow over time.P = 0.06)...Finally, the EXSCEL study tested the effects of 2 mg exenatide, another GLP‐1 agonist, administered once weekly against placebo on the incidence of cardiovascular death, nonfatal MI, or nonfatal stroke in 14 752 patients followed for a median period of 3.2 years.This year, the HARMONY trial investigated almost 9500 patients treated with either albiglutide (which was removed from the market in 2017) or placebo for a median 1.6‐year follow‐up period. Albiglutide was shown to significantly reduce the risk of MACE by 22%; however, the study did not show a significant reduction in death from cardiovascular causes, as was demonstrated with liraglutide, another GLP‐1 agonist in the LEADER trial.The ongoing REWIND trial is a randomized placebo‐controlled trial which will test the cardiovascular effects of once‐weekly dulaglutide, another GLP‐1 agonist in patients with type 2 diabetes. The highlight of the trial is its high proportion of women and a predominantly primary prevention population.Collectively, the overall signal that seems to emerge from the literature about the effects of these novel drugs is a positive effect on CV outcomes among patients at risk for structural heart disease, a lack of effects on heart failure outcomes among those with early cardiac remodeling, and possible detrimental effects on heart failure outcomes in patients with advanced symptomatic heart failure.2In summary, SGLT‐2 inhibitors (empagliflozing and canagliflozin) and GLP‐1 agonists  have been shown to improve CVOs in patients with T2DM. The American Diabetes Association 2018 standard of care guidelines recommended for the first time the addition of a second antihyperglycemic agent that can reduce MACE in select patients with T2DM and pre‐existing atherosclerotic cardiovascular disease.The authors declare no potential conflicts of interests.


CYP1A1 rs1048943, EGF rs444903, HOTAIR rs920778, MMP2 rs243865, and PLCE1 rs2274223, 10 variants were rated as moderate, and 18 variants were rated as weak. Additionally, 17 SNPs were verified noteworthy in six genomewide association studies (GWAS) using FPRP methods. Collectively, this review offered a comprehensively referenced information with cumulative evidence of associations between genetic polymorphisms and EC and ESCC risk.An increasing number of publications had reported the association between single‐nucleotide polymorphisms (SNPs) and esophageal cancer (EC) risk in the past decades. Results from these publications were controversial. We used PubMed, Medline, and Web of Science to identify meta‐analysis articles published before 30 July 2018, that summarize a comprehensive investigation for cumulative evidence of genetic polymorphisms of EC and its subtype risk. Two methods, Venice criteria and false‐positive report probability (FPRP) tests, were used to assess cumulative evidence of significant associations. At last, 107 meta‐analyses were considered to be in conformity with the inclusion criteria, yielding 51 variants associated with EC or esophageal squamous cell carcinoma (ESCC). Thirty‐eight variants were considered to be nominally significant associated with risk of EC or ESCC, whereas the rest showed non‐association. In additional, five variants on five genes were rated as strong cumulative epidemiological evidence for a nominally significant association with EC and ESCC risk, including  Ioannidis et al indicated that mechanisms for summarizing and assessing genetic epidemiological evidence require periodic updates of all appropriate association studies based on widely accepted assessment criteria.Therefore, we attempt to collect cumulative evidence of associations between genetic variants and EC risk from published meta‐analyses and GWAS, and evaluate these associations, which may offer referenced information for further investigation of genetic risk factors for EC and its subtype.22.1The following items were used in search process:  and (“cancer” or “adenocarcinoma” or “carcinoma” or “tumor” or “squamous cell carcinoma”) and  and (“genetic association” or “Genetic” or “SNP” or “polymorphism” or “single nucleotide polymorphism” or “genotype” or “variant” or “variation” or “mutation” or “susceptibility”). Additionally, we also checked all the relevant references to find other potential meta‐analyses that could offer relevant data.‐8 of P value was considered statistically significant; (f) they were studied of EC incidence/susceptibility .Meta‐analysis articles met to the following criteria: (a) The publications were in English; (b) cancer type was EC or including subtypes; (c) the patients with EC were diagnosed by pathological or histological examination; (d) sample size was not fewer than 1000; (e) they were studied of EC incidence/susceptibility . The following criteria should be met for screening the SNPs in GWAS on PubMed: (a) The publications were in English; (b) cancer type was EC, which includes all the subtype of EC; (c) the patients with EC were diagnosed by pathological or histological examination; (d) the studies included two phases (discovery and replication); (e) OR and 95% CI were provided and the less than cutoff of 1 × 102.2P value. In meta‐analysis, the following data were collected: first author, publishing year, gene name, genetic variant, OR and 95% CI,the number of studies, the number of subjects (cases and controls), ethnicity, I‐square, test for heterogeneity (Q test) between studies,Q testP value used for between‐study test of heterogeneity (Q‐test) was 0.10. P value >0.10 represents little heterogeneity while P value <0.10 indicates the presence of heterogeneity. We attempted to extract information of EC, including ESCC and EADC; however, the results showed that almost all of the articles were studied for EC and ESCC, not for EADC. Therefore, we were unable to conduct assessment of EADC, and then, we just evaluated cumulative epidemiological evidence for EC and ESCC. In addition, the eligible studies reported two major ethnicities, Asian and Caucasian. Majority of the meta‐analyses included a combination of two or more ethnicities, which were defined as “diverse populations.” Therefore, we extracted the information of diverse population, when applicable, Asian and Caucasian population were also extracted. Multiple studies concerning the same SNP reported conflicting results due to varied sample sizes and the selection of different association models. Therefore, given study quality and result credibility, we selected the most recently published study with the greatest number of and most integrated participants and the standardized report of the genetic association study based on guidelines of the Human Genome Epidemiology Network for systematic review of genetic association studiesP value was <0.05.Data were extracted by J.T and checked by two authors (C.L and GL). Information extracted from each eligible publication in GWAS included PMID of article, first author, publishing year, gene name, genetic variant, ethnicity of participants, the amount of subjects (cases and controls), minor allele frequency (MAF), OR, 95% CI, 2.3I2 < 25%), grade B (25% < I2 < 50%), or grade C (I2 > 50%). Protection from bias was mainly determined by sensitivity analysis and a series of bias tests including publication bias, small‐study bias, as well as an excess of significant findings  assay with a prior probability of 0.001 and an FPRP cutoff value of 0.2 to uncover potential false‐positive results among significant associations and evaluate whether these associations should be omitted, and we used the statistical power to detect an odds ratio of 1.5 for alleles with an elevated risk in FPRP calculations, as suggested by Wacholder et al.33.1As presented in Figure 3.2For 51 SNPs identified by meta‐analysis, 38 SNPs had statistically significant association with EC or ESCC susceptibility.CYP1A1 rs1048943, PLCE1 rs2274223, MMP2 rs243865 in EC and HOTAIR rs920778 in ESCC, from weak to moderate for ADH1B rs1229984 and COX‐2 rs20417 in EC, based on FPRP <0.05. Cumulative evidence were downgraded from strong to moderate for IL‐18 −607C>A, MMP1 rs1799750, SLC52A3 rs13042395 and MDM2 rs2279744 in EC, and TNF‐α rs1800629, C20orf54 rs13042395, microRNA124 rs531564 in ESCC, from moderate to weak for GSTT1 null/present, XRCC1 rs1799782, Hsa‐mir rs3746444, hOGG1 rs1052133, STK15 rs2273535 in EC, and microRNA‐34b/c rs4938723, NAT2 rapid/slow in ESCC, based on FPRP >0.2. Finally, five SNPs on five genes were rated as strong for cumulative epidemiological evidence of association by combining Venice criteria and FPRP results, including CYP1A1 rs1048943, EGF rs444903, MMP2 rs243865, PLCE1 rs2274223 for EC and HOTAIR rs920778 for ESCC. Seven SNPs with EC and three with EC were rated as moderate. Sixteen with EC and two with ESCC were rated as weak. Great discrepancy between the calculated amount and the true amount makes the grade arduous to determine. Therefore, calculated amounts of less than 3000 were not included for assessment in MAFs obtained from the dbSNP.As presented in Table C20orf54 and rs2274223 on PLCE1) were performed both in meta‐analysis and in GWAS.The associations including 17 SNPs identified in six GWAS studies were presented in Table 3.3XRCC1) with sample sizes >10 000 were also not significantly associated with EC; further investigations for this variant may not be fruitful. Certain variants presented with relatively small sample sizes; as such, the evidence for nonsignificant , Fas 1800682, XPA rs1800975, microRNA146 rs2910164, microRNA196 rs11614913, XRCC1 rs25487. The results of two variants, EPHX1 (Try113His and His139Arg)XRCC1 (Arg399Gln and Arg194Trp),Twenty variants presented inconsistent results in our review  which may combine with DNA to form adducts via a series of biochemical reactions. The ultimate carcinogens converted from the DNA adducts were considered to be associated with the development of EC.EGF, located on chromosome 4q25‐q27,MMP‐2 is a sort of zinc‐dependent endopeptidases, which can regulate various cell behaviors such as tumor initiation and growth by modulating cell proliferation, apoptosis and angiogenesis.PLCE1, located on chromosome 10q23, participate in cell growth, differentiation, gene expression and oncogenesis.HOTAIR, located on chromosome 12q13.13, might transform normal cells to malignant.IL‐8 −607C>A, MMP‐1 rs1799750, TNF‐α rs1800629, MicroRNA124 rs531564, SLC52A3 rs13042395 and MDM2 rs2279744 based on a high FPRP (> 0.2). The FPRP method considers the P value, prior probability, and statistical power of the test; as we calculated FPRP at prior probability of 0.001 and used the statistical power to detect an odds ratio of 1.5 for alleles with an elevated risk in FPRP calculations, certain otherwise significant associations may have been excluded. Previous studies using different prior probabilities have classified their results as more noteworthy. Further investigations on these six variants may be necessary to analyze their associations in greater depth. Additionally, SNP (rs13042395) on C20orf54 also showing moderate evidence of association with ESCC susceptibility, which downgraded from strong to moderate. This SNP was both verified in GWAS and assessed in meta‐analysis. The association in GWAS, however, may be more statistically significant and convincing when related meta‐analysis results are inconsistent or the association evidences are not strong by combination Venice criteria and FPRP method. The Venice criteria could assess multiple reasons of potential bias such as genotype error or misclassification and ethnicity stratification, which were difficult to perform in meta‐analysis. Therefore, results might be more convincing if different weights in the Venice Criteria were reset.There are six variants showing moderate evidence of association in our review, all of which downgraded from strong to moderate: ADH1B rs1229984 and COX‐2 rs20417) with risk of EC was upgraded from weak to moderate based on FPRP <0.05. Additional assessment of two variants were necessary, particularly the variant (COX‐2 rs20417) since sample size of study for this variant are relatively small .Cumulative evidence of two variants (Thirteen variants were found not to be significantly associated with EC risk, to include nine variants on seven genes and two mRNAs in a sample of approximately 4000 patients, at approximately 85% power to detect an OR of 1.15 under different model for a variant with MAF of 20%. The MAFs of the aforementioned seven variants were almost all over 0.3 despite sample sizes >4000. We can safely conclude, therefore, that these seven variants are unlikely to be associated with EC (see Table Certain limitations do apply to this report. Although we did a comprehensive literature search, some articles may have been missed. A variability in sample size presented among different studies; the smaller sizes may have impacted the credibility of the data. The evaluated data extracted from only one source which may be main cause of bias. Finally, only the susceptibility/incidence between genetic variants and EC risk were evaluated; however, other roles of genetic polymorphisms such as tumor progression, metastasis, drug resistance for EC were not be assessed due to lack of data or information. Despite the limitations of our method, we believe that our study, as an updated summary and evaluation of the existing literature reporting genetic predisposition of EC, is of value for further genetic studies.This review evaluated the cumulative epidemiological evidence of significant associations by combining the Venice criteria and FPRP results which identified five SNPs having strong evidence of true association. Collectively, our review provides referenced information for further investigation into genetic susceptibility of EC and ESCC.None declared. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Physicians are faced with a growing number of patients after renal transplantation undergoing graft-unrelated surgery. So far, little is known about the postoperative restitution of graft function and the risk factors for a poor outcome.One hundred one kidney transplant recipients undergoing graft-unrelated surgery between 2005 and 2015 were reviewed retrospectively. A risk analysis was performed and differences in creatinine, GFR and immunosuppressive treatment were evaluated. Additional, a comparison with a case-matched non-transplanted control group were performed.Preoperative creatinine averaged 1.88 mg / dl [0.62–5.22 mg / dl] and increased to 2.49 mg / dl [0.69–8.30 mg / dl] postoperatively. Acute kidney failure occurred in 18 patients and 14 patients had a permanent renal failure. Significant risk factors for the development of postoperative renal dysfunction were female gender, a preoperative creatinine above 2.0 mg / dl as well as a GFR below 40 ml / min and emergency surgery. Patients with tacrolimus and mycophenolate mofetil treatment showed a significant lower risk of renal dysfunction than patients with other immunosuppressants postoperatively. Contrary to that, the risk of patients with cyclosporine treatment was significantly increased. Transplanted patients showed a significantly increased rate of postoperative renal dysfunction.The choice of immunosuppressant might have an impact on graft function and survival of kidney transplant recipients after graft-unrelated surgery. Further investigations are needed.The online version of this article (10.1186/s12882-019-1358-2) contains supplementary material, which is available to authorized users. During the past decades, kidney transplantation has become a safe therapy for end-stage renal disease . The groThis was a retrospective monocentric and controlled cohort study of kidney transplant recipients and healthy controls, reported in line with the STROCSS criteria . Data waGFR was calculated via CKD-EPI equation. The occurrence of renal dysfunction was noted on the basis of the discharge documents. Acute kidney failure was defined as an increase in creatinine level (reference range 0.51–0.95 mg / dl) by more than 0.3 mg / dl in 48 h or as an oliguria (< 0.5 ml / kg / h) for more than six hours.p-value < 0.05 were considered as statistically significant. See Additional file IBM SPSS® (version 23.0) was used for exploratory data analysis. Results were checked for normal distribution. Evaluation of group differences was carried out by a T-test as an analysis of two independent groups. A chi-squared test was utilized to test for trends and significance and compare groups of categorical data. Differences with a Study was approved by the ethical committee of the Medical Faculty of the University of Freiburg (EK: 203/17) and was performed according to the principles of the declaration of Helsinki. It was registered in an approved primary register of the WHO (DRKS00015440).From January 2005 to December 2015 a total of 1535 kidney transplant recipients were admitted to the Department of General and Visceral Surgery at the Medical Center of the University of Freiburg. Out of these, 101 patients underwent abdominal or abdominal wall surgery and were included in our study .n = 38; 38%), followed by cystic kidney disease . The mean age of included patients was 59 years [range 41–77 years], two thirds of the patients  underwent elective surgery.The meantime since renal transplantation was 15.8 years . The vast majority of patients were transplanted once . The main diagnosis that led to transplantation was chronic glomerulonephritis , intractable hemorrhagic shock , cardiovascular complications (n = 2) and progress of neoplastic disease (n = 1).Overall, 20 patients out of 101 (20%) died postoperatively. The leading causes of death were sepsis (n = 11) had demand on acute dialysis. Overall, 14 patients (14%) developed a permanent renal failure with need of long-time dialysis.Acute kidney failure occurred in 18 of the 101 patients (18%). More than 60% of the patients with acute kidney failure (n = 20), 65 % had acute renal failure, and half of them also required dialysis.In the subgroup of patients who died during the postoperative hospital stay (n = 81) were discharged with an averaged creatinine of 1.70 mg / dl [0.24–6.0 mg / dl] and a GFR of 53.85 ml / min [9.14–146.38 ml / min].Preoperative creatinine averaged 1.88 mg / dl [0.62–5.22 mg / dl] and increased to 2.49 mg / dl [0.69–8.30 mg / dl] on the first postoperative day. The calculated preoperative GFR averaged 43 ml per minute [8.74–110.30 ml / min] (vs. postoperative GFR 32 ml /min [6.50–101.74 ml / min]). The surviving patients (p = 0.026). Similar to that, patients with a GFR lower than 40 ml / min showed significant higher rates of acute renal failure postoperatively (p = 0.040). Female patients suffered more frequently from acute and permanent kidney failure (see Table p = 0.009) and had a higher need for dialysis (p = 0.005). We found no influence of the extent of surgery (major vs. minor), localization of operation , number of transplantations and time since transplantation on the development of renal dysfunction  received steroids preoperatively. Slightly more than half of the patients  were treated with tacrolimus (45 combined with Mycophenolate mofetil (MMF)). 32% (n = 32) of the patients were treated with cyclosporine (16 combined with MMF). Further two patients had combination of cyclosporine and azathioprine. Five patients were treated with sirolimus. One patient each had immunosuppression with combination of tacrolimus and cyclosporine, of tacrolimus and azathioprine or a single treatment with everolimus or basiliximab. Due to the low frequency of patients with other immunosuppressants, we focused on the three most common immunosuppressant regimens for further subgroup analysis of the influence of immunosuppressive therapy on postoperative renal function and mortality (see Table n = 45) was associated with the lowest rates of renal dysfunction and mortality. The highest rates were found in tacrolimus only treated patients (n = 10) and in patients with cyclosporine and MMF (n = 16).Nearly all patients (The preoperative creatinine of patients with tacrolimus was on average 1.8 mg / dl (±0.7 mg / dl) and the early postoperative creatinine was 2.5 mg / dl (±1.3 mg / dl). Those patients were discharged with a creatinine of 1.67 mg / dl (±0.8 mg / dl). The preoperative creatinine of patients with cyclosporine was on average 1.93 mg / dl (±0.9 mg / dl), and the early postoperative creatinine was 2.6 mg / dl (±1.3 mg / dl). Patients under cyclosporine were discharged with a creatinine of 1.88 mg / dl (±1.0 mg / dl). The differences of creatinine and GFR between the patients with tacrolimus and with cyclosporine were not significant.We were able to find a case-matched non-transplanted control for 84 of above mentioned 101 transplanted patients. Due to the performed surgical treatment it was not possible to find a suitable case-matched control for 17 patients  and permanent  renal failure postoperatively, compared to non-transplanted patients. Additionally, the rate of postoperative dialysis was significantly increased in transplanted patients .Descriptive statistics, course of creatinine and GFR as well as the incidence of renal dysfunction of 84 renal transplant recipients and 84 control patients are shown in Table Our study evaluated the graft outcome of 101 renal transplant recipients, providing new insights into the outcome of transplanted patients undergoing graft-unrelated surgery. It is not surprising that kidney-transplanted patients have an increased creatinine in comparison with non-transplanted patients. However, the increased rate of postoperative kidney failure and mortality in transplanted patients is striking. So far, recent literature shows only results of low numbers of transplant recipients or single case studies. Sharma et al. investigated the outcome of 36 renal or liver transplant recipients undergoing cardiac surgery and compared the results with non-transplanted patients. Three patients in the transplant group had demand on dialysis compared to one patient in the control group . Reshef However, regarding our results we assume that kidney transplant recipients are at a clearly increased risk for postoperative renal dysfunction and death. We could demonstrate that patients with preoperatively worse renal function, conditions requiring emergency surgery and, possibly, female gender were risk factors for impaired renal function postoperatively. A kidney-friendly perioperative treatment, which focuses at the preservation of graft function, is essential for a good graft outcome. Due to the results of our risk analysis special attention should be paid to patients with a limited graft function preoperatively and with a longer time since transplantation. Interestingly, female patients in our study showed a higher risk for postoperative renal dysfunction. Even if gender differences in postoperative complication rates are often discussed, we found no plausible explanation for this observation.To our knowledge, the study is the first to investigate the postoperative graft function of kidney transplant recipients after graft-unrelated surgery in relation to the chosen immunosuppression. The results suggest that the individual immunosuppressant may influence the postoperative graft outcome. Several publications of the recent years reported advantages of tacrolimus over cyclosporine regarding nephrotoxicity, graft rejection and side effects. Krämer et al. performed a six-month randomized controlled trial and evaluated data of 286 patients with tacrolimus treatment vs. 271 patients with cyclosporine treatment –18. TherThe choice of immunosuppressant might have an impact on graft function and survival of kidney transplant recipients after graft-unrelated surgery, but further investigations are needed.Additional file 1:Whole strategy of creation of control group, searched encryptions and definitions. (DOCX 42 kb)


S. pneumoniae and S. aureus. Importantly, the tolerance of the biofilms to multiple antibiotics and proteolytic degradation is significantly affected. Additionally, our results show that black carbon impacts bacterial colonisation in vivo. In a mouse nasopharyngeal colonisation model, black carbon caused S. pneumoniae to spread from the nasopharynx to the lungs, which is essential for subsequent infection. Therefore our study highlights that air pollution has a significant effect on bacteria that has been largely overlooked. Consequently these findings have important implications concerning the impact of air pollution on human health and bacterial ecosystems worldwide.Air pollution is the world's largest single environmental health risk (WHO). Particulate matter such as black carbon is one of the main components of air pollution. The effects of particulate matter on human health are well established however the effects on bacteria, organisms central to ecosystems in humans and in the natural environment, are poorly understood. We report here for the first time that black carbon drastically changes the development of bacterial biofilms, key aspects of bacterial colonisation and survival. Our data show that exposure to black carbon induces structural, compositional and functional changes in the biofilms of both  Stricter limits are set for PM2.5 as this size fraction is associated with more damaging health effects, and should be kept below an annual mean of 10 µg/m3, and 25 µg/m3 over a 24 hour period. However despite WHO and EU legislation to reduce air pollution, PM levels exceed these recommended guidelines , a major component of air pollution , a by‐product of fossil fuel combustion. In developed countries diesel exhaust fumes are the major source of BC, whereas in the developing world BC mostly arises from indoor burning of biomass for heat and fuel. BC is a chemically and biologically active pollutant that can generate oxidative stress, induce inflammation, and be mutagenic . TEM showed that the pneumococcal cells within the biofilm were closely associated with the BC particles , a feature also observed by SEM . This shows that the effect of BC on biofilm formation is not merely due to the physical presence of exogenous particles acting as a scaffold for the biofilm rather it demonstrates that BC induces a biological effect on the bacteria.The BC‐induced complex architecture and increased thickness of the biofilm did not affect biofilm integrity because there was no change in the proportion of loosely associated and planktonic iae Fig. h and i. S. aureus biofilm formation, but different effects were observed. Similar to S. pneumoniae, BC significantly increased the average thickness of S. aureus biofilms . Additionally, the biofilm structure was altered in that BC induced development of bulky masses that protruded from the normally smooth surface of the biofilms, although the structure observed was not as complex as that of S. pneumoniae BC‐biofilms , and a significant increase in the loosely associated cells , showing that biofilm structural integrity had been altered.BC also altered S. pneumoniae, BC was found to be detrimental to S. aureus strains SH1000 and Newman, because exposure to BC significantly decreased (p ≤ 0.05) the total number of viable S. aureus cells compared with the control  strain USA300 , demonstrating that the effect of BC differs between S. aureus strains. TEM analysis did not reveal any significant effect of BC on the structure of the S. aureus cells . Together our data show that BC changes bacterial biofilm formation and structure by two model bacteria, but importantly, there is intra‐ and inter‐species variation in the impact of BC.Unexpectedly, unlike et al., S. aureus Newman and S. aureus USA300 were significantly disrupted by treatment with proteinase K, showing that these biofilms are mainly proteinaceous in composition . In contrast, S. aureus SH1000 and the S. pneumoniae PR201 control biofilms were not significantly disrupted by proteinase K treatment, suggesting that these biofilms contained less protein  needed for planktonic bacteria . BC‐biofilms of S. aureus Newman also showed significant decreases in tolerance to daptomycin and tetracycline ; S. aureus Newman and USA300 also still demonstrated the observed significant decrease in tolerance to oxacillin using this method .Importantly, biofilms of S. pneumoniae formed in the presence of BC showed a significantly increased resistance to penicillin G, a front line antibiotic . S. pneumoniae is the major cause of pneumonia worldwide therefore this has major implications for the treatment of this disease.Our data show that BC has a differential effect on bacterial biofilm tolerance to antibiotics irrespective of the mode of action. S. aureus Newman biofilms after treatment with daptomycin is 0.043%  S. pneumoniae with BC or (iv) saline as a control. Blood was taken and nasopharyngeal and bronchoalveolar lavages were performed to determine bacterial carriage at predetermined time‐points. A concentration of 7mg/ml BC was used because this does not produce any adverse host response and therefore any observed effect is due to the bacteria and not due to a significant effect on the host. The lungs, spleen, cervical lymph nodes and nasopharyngeal tissue were taken from separate mice for histological analysis.Mice were intranasally inoculated with (i) BC alone, (ii) non‐BC treated S. pneumoniae from the nasopharynx to the lungs . No S. pneumoniae were found in the blood in any condition. Histological analysis of the nares, lungs, cervical lymph nodes and spleen of control and BC‐exposed mice did not reveal any overt signs of an inflammatory response nor were any BC particles detected at these sites . The mice exposed to BC and S. pneumoniae did not display any overt clinical signs of disease despite the observation of ∼3.68 Log10 CFU/ml in the bronchoalveolar lavages. The fact that S. pneumoniae is only detected in the lungs of mice exposed to BC shows that inoculation in the presence of BC promotes the dissemination of S. pneumoniae to the lower respiratory tract, increasing the opportunity for bacteria to cause invasive disease such as pneumonia.Significantly, the data show that BC promotes the spread of ngs Fig. . At 7 daone Fig. a. In conIn this study we show that a major component of air pollution, BC, significantly affects bacterial colonisation and biofilm formation, thereby demonstrating that a biological effect of air pollution has been overlooked. Consequently this work establishes a new paradigm; that the detrimental impact of particulate pollutants on human health is not only just due to direct effects on the host, but also involves the effect on bacterial behaviour in the host.S. pneumoniae biofilms were considerably thicker and more complex than their control counterparts, as well as substantially more sensitive to proteolytic cleavage and importantly, more resistant to antibiotics. The biofilm structure induced by BC in this study is characteristic of the honey‐comb structure of pneumococcal biofilms only previously observed in vivo or on biotic surfaces in vitro  (SH1000 and Newman), methicillin resistant Staphylococcus aureus (MRSA) USA300, and encapsulated (D39) and non‐encapsulated (R6 strain PR201) S. pneumoniae isolates were investigated. S. pneumoniae strains were cultivated on 5% (v/v) horse blood agar, and S. aureus strains cultured on Luria agar (LA). Prior to each assay, overnight cultures were centrifuged, and pellets were resuspended to a matching CFU in the chosen medium.Methicillin sensitive 2O which had been sterilised by autoclaving at 120˚C at 15 pSI for 15 min. It is noteworthy that BC aggregates in liquid therefore size of particles can change. Quartz reference particles (BCR66 ‐ Distrilab) with a size distribution of 3500–350 nm were also employed in this work. Quartz is chemically stable and therefore does not react with most substances even at high temperatures.Black Carbon was purchased from Sigma‐Aldrich (UK) under product number 699632. This was provided as a powder with a size distribution of <500 nm, with <500 ppm trace metals, and a weight of 12.01 g/mol. For use in assays, a stock solution of BC was made by dispersing the powder in dHet al., S. pneumoniae D39 biofilms were formed in Todd‐Hewitt Broth + 0.5% (w/v) yeast extract (THY), and PR201 in Brain Heart Infusion (BHI). S. aureus SH1000 biofilms were formed in BHI + 4% (w/v) NaCl, and S. aureus Newman and USA300 in BHI + 1% (w/v) glucose. 12 well plates were then incubated at 37°C in a 5% (v/v) CO2 atmosphere for 24 h. After incubation, planktonic bacteria were gently removed without disrupting the biofilm, and retained. The remaining biofilms were carefully washed with 3 ml phosphate‐buffered saline (PBS) and this wash was also kept. Biofilm bacteria were then removed with CellScrapers (VWR) into 1ml PBS. Each fraction was vortexed for 30 s and serial dilutions were plated to determine colony forming units (CFU).A novel method to elucidate biofilm structure was developed from previous work  NaCl  was used in BHI.To assess antibiotic sensitivity, the biofilm protocol detailed in the previous section was used. Biofilms were formed in the presence of 100 µg/ml black carbon, and then exposed to antibiotics. For this, the protocol was adjusted so that after the removal of planktonic bacteria and the wash step, either 3 ml medium alone or medium with antibiotics were added to each well and incubated at 37°C 5% (v/v) COet al., To investigate the role of protein in the extracellular biofilm matrix, the standard biofilm protocol previously detailed was again employed. Biofilms were formed in the presence of 100 µg/ml black carbon, and then 3 ml of 100 µg/ml Proteinase K in 10 mM Tris‐ HCl, adjusted to pH 7.5, was added to each well after supernatant removal and washing (Nicholson 2 atmosphere for 24 h. For scanning electron microscopy (SEM), coverslips were then washed with PBS, fixed in 2.5% (v/v) glutaraldehyde, washed in 0.1 M Sörensens buffer, gold coated, and viewed with a Hitachi S‐3000H SEM  COad libitum. For the colonisation experiment, mice were intranasally inoculated with 15 µl of either (i) PBS as a control, (ii) 6.45 × 105 CFU S. pneumoniae D39, (iii) 7 mg/ml BC alone, or (iv) a mixture of 6.45x105 CFU S. pneumoniae D39 with 7 mg/ml BC, whilst held in a horizontal position. For inoculation, mice were anaesthetised with 2.5% (v/v) isoflurane and held in a horizontal position during inoculation. Seven mice were used per group, 28 in total. Allocation of mice to each group, the order in which groups were inoculated, and the order in which each cage was culled for sample recovery, was randomised. Mice were regularly monitored (at least once daily) for signs of disease for the duration of the experiment. At 7 days post‐infection, five mice were culled from each group to assess bacterial load. Mice were deeply anaesthetised with 2.5% (v/v) isoflorane and blood was collected by cardiac puncture in which the animal was exsanguinated under terminal anaesthesia. Death was confirmed by cervical dislocation. Bronchoalveolar and nasopharyngeal lavages were performed to determine bacterial carriage at each time‐point. Bronchoalveolar lavages were performed by instilling 500 µl tripticase soy broth supplemented with 10% (v/v) glycerol (TSBG) into the lungs via the trachea, recovering the lavage fluid, and repeating this twice. Nasopharyngeal lavages were performed by washing through the nasopharynx with 500 µl TSBG three times. Lavages were then serially diluted and plated to determine bacterial viability. For histological analysis, an additional two mice per group were deeply anaesthetised with 2.5% (v/v) isoflurane and culled by cervical dislocation. Death was confirmed by exsanguination. The lungs, spleen, cervical lymph nodes and nasopharyngeal tissue were then removed and fixed in 10% (v/v) formalin for 24–48 h. Samples were embedded in paraffin for histological sectioning, and were stained with haematoxylin and eosin and analysed by light microscopy.All experiments were carried out in accordance with the Home Office Project Licence and adhered to the UK Animals (Scientific Procedures) Act (1986). Naïve 8 week old female outbred MF1 mice were obtained from Charles River, UK. Upon arrival, mice were housed in groups of 2–5, depending on study requirements, and allowed to acclimatise for one week in the Division of Biomedical Services, University of Leicester, on a 12 h light‐dark cycle and access to food and water In vivo experiments were carried out with n = 7 in each group. Quantification of biofilm thickness was carried out on n = 18 distinct biofilm sections per condition. ANOVA or t‐tests were used to determine significance as appropriate.Data are summarised as standard error of the mean (SEM) and were analysed using GraphPad Prism version 6.04 . Biofilm quantification results were validated by at least three biological replicate experiments. Additional Supporting Information may be found in the online version of this article at the publisher's website:Fig. S1. The effect of black carbon on internal biofilm structure. Biofilms of S. pneumoniae , S. aureus SH1000  and Newman  were cultured in the presence or absence of 100 μg/ml BC and imaged by transmission electron microscopy (TEM). Images are representative of the entire biofilm structure.Fig. S2. Quartz has no effect on biofilm architecture. Biofilms of S. aureus SH1000  and S. pneumoniae PR201  were cultured with and without 30 μg/ml Quartz and imaged by scanning electron microscopy (SEM). Images are representative of the entire biofilm structure.Fig. S3. The effect of black carbon on S. aureus Newman biofilms. Biofilms of S. aureus Newman were cultured in the presence or absence of 30 ‐ 100 μg/ml BC. Biofilms were imaged by scanning electron microscopy (SEM) at increasing resolution (a‐d) and light microscopy was used to quantify biofilm thickness . Viable bacterial cells were measured by sequential removal and quantification of planktonic, loosely‐ adhered, and biofilm bacteria . Error bars represent ± 1 SEM. Significance was determined by t‐tests (g) or ANOVA (h) * p≤0.05, *** p≤0.001, **** p≤0.0001.Fig. S4. The effect of black carbon on S. aureus USA300 biofilms. Biofilms of S. aureus USA300 were cultured in the presence or absence of 30 ‐ 100 μg/ml BC. Biofilms were imaged by scanning electron microscopy at increasing resolution (a‐d). Images are representative of the entire biofilm structure. Viable bacterial cells were measured by sequential removal and quantification of planktonic, loosely‐ adhered, and biofilm bacteria . Error bars represent ± 1 SEM. Significance was determined by ANOVA.Fig. S5. BC alters biofilm antibiotic tolerance. Biofilms of S. aureus SH1000 (a), Newman (b), and USA300 (c) were cultured in the presence or absence of 100 μg/ml BC for 24 h. Planktonic and loosely‐adhered bacteria were then removed and biofilms were either incubated with 3 ml of 50 mg/ml oxacillin in PBS supplemented with 2% (w/v) NaCl, or with buffer alone as a control, for 3 h at 37°C in 5% (v/v) CO2. After incubation, planktonic and loosely adherent bacteria were removed and discarded, and the remaining biofilm bacteria were quantified. For S. aureus, oxacillin (50 mg/ml in BHI + 2% (w/v) NaCl), daptomycin (256 µg/ml in BHI + 50 µg/ml CaCl2) and tetracycline (512 µg/ml in BHI) were used. For S. pneumoniae PR201, penicillin G (1 µg/ml) was used in BHI. Planktonic and loosely‐adhered cells were then removed and biofilm bacteria quantified. n≥3. Error bars represent ± 1 SEM. Significance was determined by unpaired t‐tests. * p≤0.05, ** p≤0.01.Fig. S6. Histological analysis of lungs and nares at 7 days post‐inoculation. Female MF1 mice were mice intranasally inoclated with 15 µl of PBS, Black Carbon (BC), S. pneumoniae D39 (D39), or D39 and BC together (D39+BC). Mice were culled 7 days post inoculation. Formalin fixed, paraffin embedded tissue sections of lungs (A‐D) and nares (E‐H) were stained with haematoxylin and eosin (H+E). Sections were imaged using light microscopy using 5x or 10x magnification. Physical features of notes are labelled for identification.Fig. S7. Histological analysis of cervical lymph nodes and spleen at 7 days post‐inoculation. Female MF1 mice were mice intranasally inoculated with 15 µl of PBS, Black Carbon (BC), S. pneumoniae D39 (D39), or D39 and BC together (D39+BC). Mice were culled 7 days post inoculation. Formalin fixed, paraffin embedded tissue sections of cervical lymph nodes (A‐D) and spleen (E‐H) were stained with haematoxylin and eosin (H+E). Sections were imaged using light microscopy using 5x or 10x magnification. Physical features of notes are labelled for identification. Lymph nodes: HEV ‐ High Endothelial Venules, the entry point for lymphocytes into lymph nodes. Spleen: PALS ‐ Peripheral Arteriolar Lymphoid Sheath. Follicles comprised of a germinal centre (GC), a mantle (Mn) zone, and a marginal (Mg) zone. White pulp comprises PALS, follicles, and lymphoid cells, surrounded by Red Pulp (RP).Click here for additional data file.


According to cardiovascular outcome trials, some sodium‐glucose contransporter‐2 inhibitors (SGLT2i) and glucagon‐like peptide‐1 receptor agonists (GLP‐1RA) are recommended for secondary cardiovascular prevention in type 2 diabetes (T2D). In this real‐world study, we compared the simultaneous reductions in HbA1c, body weight and systolic blood pressure after initiation of dapagliflozin or GLP‐1RA as second or a more advanced line of therapy.DARWIN‐T2D was a retrospective multi‐centre study conducted at diabetes specialist clinics in Italy that compared T2D patients who initiated dapagliflozin or GLP‐1RA (exenatide once weekly or liraglutide). Data were collected at baseline and at the first follow‐up visit after 3 to 12 months. The primary endpoint was the proportion of patients achieving a simultaneous reduction in HbA1c, body weight and systolic blood pressure. To reduce confounding, we used multivariable adjustment (MVA) or propensity score matching (PSM).Totals of 473 patients initiating dapagliflozin and 336 patients initiating GLP‐1RA were included. The two groups differed in age, diabetes duration, HbA1c, weight and concomitant medications. The median follow‐up was 6 months in both groups. Using MVA or PSM, the primary endpoint was observed in 30% to 32% of patients, with no difference between groups. Simultaneous reduction of HbA1c, BP and SBP by specific threshold, as well as achievement of final goals, did not differ between groups. GLP‐1RA reduced HbA1c by 0.3% more than the reduction achieved with dapagliflozin.In routine specialist care, initiation of dapagliflozin can be as effective as initiation of a GLP‐1RA for attainment of combined risk factor goals. According to the most recent consensus on the glycaemic management of T2D, the choice of GLM should primarily consider the presence of atherosclerotic cardiovascular disease (CVD) or chronic kidney disease (CKD).Certain combined endpoints have become popular in diabetes phase III RCTs, typically the achievement of HbA1c targets without hypoglycaemia and weight gain.Real‐world evidence from retrospective studies should not substitute for RCTs, but can inform physicians and payers concerning therapeutic effectiveness in clinical practice. Evaluation of surrogate endpoints, such as glycaemia, blood pressure and BW, although correlated with cardiovascular risk, cannot substitute for CVOTs. Nonetheless, several clinically‐relevant questions may remain unanswered, simply because no RCT on that topic is available or planned. For instance, no CVOT is planned to compare SGLT2is and GLP‐1RAs. Thus, in the absence of dedicated RCTs, real‐world data can provide medium‐level evidence to fill such gaps and generate new hypotheses. To address such a gap, we conducted a retrospective real‐world study to compare the effectiveness of the SGLT2i dapagliflozin and GLP‐1RA concerning composite outcomes in clinical practice.22.1The DARWIN ‐T2D trial was a retrospective multi‐centre study conducted at 46 diabetes specialist outpatient clinics in Italy. The protocol was approved by all local ethical committees. The study design and primary results have been published previously.2.2Dedicated software automatically extracted relevant information from the same electronic chart system  at all centres. The following data were collected at baseline (date of first prescription of the above‐mentioned GLMs): age, sex, BW and height to calculate BMI, diabetes duration, systolic and diastolic blood pressure, smoking status, fasting glucose, HbA1c, complete lipid profile, serum creatinine, estimated glomerular filtration rate , urinary albumin excretion rate , prior and concomitant GLM and other concomitant medications. Based on ICD‐9 codes recorded in the chart, microangiopathy was defined as the presence of one or more of the following: retinopathy (any stage), neuropathy (somatic or autonomic), nephropathy . Macroangiopathy was defined as ischemic heart disease or stroke/transient ischemic attack or peripheral arterial disease or revascularization of coronary, carotid or peripheral arteries.Updated information was collected concerning HbA1c, BW, BP and medications at the end of follow‐up, which was set as the date of the first visit between 3 and 12 months after baseline. Detailed information concerning drug dosages was not available, but a previous analysis of the DARWIN‐T2D study estimated the final dose of liraglutide to be closer to 1.2 mg than to 1.8 mg.2.3Three variables were considered in the composite endpoints: change from baseline to end of follow‐up in HbA1c, BW and SBP. The primary endpoint was the proportion of patients achieving a simultaneous reduction in HbA1c, BW and SBP, without thresholds. Thus, to meet the primary endpoint, patients must have had values of HbA1c, BW and SBP at the end of follow‐up that were all lower than the respective values recorded at baseline. Secondary endpoints were (a) the proportion of patients with simultaneous reduction of HbA1c >0.5%, of BW >2 kg and of SBP >2 mm Hg; (b) the proportion of patients simultaneously achieving specific targets at follow‐up: HbA1c ≤7%, BW loss ≥3% and SBP <140 mm Hg; (c) change in the individual components of the composite endpoints.2.4Based on preliminary data,2.5t test for continuous variables and with the chi‐squared test for categorical variables. Intragroup comparison of the change from baseline to the end of follow‐up in continuous variables was performed using the paired two‐tailed Student's t test. Multiple imputation (MI) of missing data was performed using the Multiple Imputation by Chained Equation (MICE) algorithm,Continuous variables are described as mean ± standard deviation, unless otherwise specified, whereas categorical variables are presented as percentages. Comparison between two groups was performed with the unpaired two‐tailed Student's P value less than 0.05 was considered statistically significant.For the MVA approach, logistic regression models were implemented on composite outcomes and linear regression models were built for the change from baseline of HbA1c, BW and SBP. Covariates included clinical characteristics that differed at baseline between the two groups. Non‐normal covariates  according to the Kolmogorov–Smirnov test were log‐transformed. PS were computed in each imputed dataset and the baseline covariates included in the PS models were the following: age, gender, duration of diabetes, BW, BMI, FPG, HbA1c, systolic and diastolic blood pressure, total and HDL cholesterol, triglycerides, eGFR, insulin and metformin therapy, microangiopathy and macroangiopathy.33.1From a background population of 281 217 patients with T2D, detailed information was retrieved for 17 285 patients who initiated new GLMs. Of these, 2484 initiated dapagliflozin and 2247 initiated a long‐acting GLP‐1RA (EOW or liraglutide). As the main objective of the DARWIN‐T2D trial was to describe baseline patient characteristics, patients did not require a follow‐up visit to be included in the study database. A follow‐up examination was available for 830 patients who initiated dapagliflozin and 811 patients who initiated GLP‐1RA. Reasons for the absence of a follow‐up visit were (i) patients had not yet returned to follow‐up after initiation of new drugs (78%), and (ii) patients discontinued drug use before follow‐up (22%). Information concerning change in HbA1c, BW and SBP for computing the combined endpoints was available at both baseline and follow‐up visits for 473 patients in the dapagliflozin group and for 336 patients in the GLP‐1RA group . These two groups differed significantly in terms of fasting glucose, total and LDL cholesterol, eGFR, and concomitant use of insulin and ACE inhibitors or angiotensin receptor blockers.3.2As expected in routine clinical practice, some patients underwent changes in concomitant medication at the time they initiated dapagliflozin or a GLP‐1RA . Patients initiating dapagliflozin had a slightly more frequent initiation of insulin and a prescription for sulphonylureas/repaglinide and pioglitazone than patients initiating GLP‐1RAs. On the other hand, patients initiating GLP‐1RAs were more frequently switching from a DPP‐4i‐based regimen. Minor changes were noted for BP‐ and BW‐lowering drugs.3.3P = 0.642). After adjusting for confounders, the proportion of patients attaining the primary endpoint also did not differ between groups, and the odds ratio (OR) was 0.91   , this difference disappeared after MVA  duration of follow‐up was 5.9 (4.0–6.5) months in the dapagliflozin group and 6.0 (4.4–6.6) months in the GLP‐1RA group. Table P < 0.001)     , each composed of an average of 229 patients , allowing direct comparison of outcomes . There was no change in the overall study results, with no significant difference between groups in any of the three combined endpoints (Table S2). In the second sensitivity analysis, IPW was performed with or without incorporation of the prior number of GLM classes in the PS and there was no difference in any of the outcomes between the two groups (Table S2). Finally, when analyses were further adjusted for follow‐up duration, which, overall, did not differ between the two groups, results did not change substantially (Table S3).4This observational study shows that, during treatment of T2D within routine specialist care, a similar proportion of patients initiating the SGLT2i dapagliflozin or a GLP‐1RA after a median of two GLM classes reached composite endpoints of simultaneous reductions in HbA1c, BW and SBP. This finding has implications for the care of T2D patients because certain specific SGLT2is and GLP‐1RAs are now recommended for secondary cardiovascular prevention.Both SGLT2is and GLP‐1RAs exert favourable extra‐glycaemic effects on risk factors, but these have not yet been formally analysed in RCTs as combined endpoints. In our study, the proportion of patients experiencing a simultaneous reduction in HbA1c, BW and SBP was identical in the dapagliflozin and GLP‐1RA groups. GLP‐1RA was significantly more effective than dapagliflozin in reducing HbA1c by 0.3% in both the MVA  and PSM  analyses ; Giovanni Grossi ; Achiropita Pucci ; Giorgio Sesti and Francesco Andreozzi ; Giuseppe Capobianco ; Adriano Gatti ; Riccardo Bonadonna, Ivana Zavaroni and Alessandra Dei Cas ; Giuseppe Felace ; Patrizia Li Volsi ; Raffaella Buzzetti and Gaetano Leto ; Gian Pio Sorice ; Paola D'Angelo ; Susanna Morano ; Antonio Carlo Bossi ; Edoardo Duratorre ; Ivano Franzetti ; Paola Silvia Morpurgo ; Emanuela Orsi ; Fabrizio Querci ; Massimo Boemi† and Federica D'Angelo ; Massimiliano Petrelli ; Gianluca Aimaretti and Ioannis Karamouzis ; Franco Cavalot ; Giuseppe Saglietti† ; Giuliana Cazzetta ; Silvestre Cervone ; Eleonora Devangelio ; Olga Lamacchia ; Salvatore Arena ; Antonino Di Benedetto ; Lucia Frittitta ; Carla Giordano ; Salvatore Piro ; Manfredi Rizzo, Roberta Chianetta and Carlo Mannina ; Roberto Anichini ; Giuseppe Penno ; Anna Solini ; Bruno Fattor (Comprensorio Sanitario di Bolzano ‐ Azienda Sanitaria della Provincia Autonoma di Bolzano); Enzo Bonora and Massimo Cigolini ; Annunziata Lapolla and Nino Cristiano Chilelli (Complesso Socio Sanitario Ai Colli ‐ Azienda ULSS n.6 Euganea); Maurizio Poli ; Natalino Simioni and Vera Frison ; Carmela Vinci .Table S1. Comparison between patients included in the composite outcome analysis and patients excluded from the analysis for missing outcome information.Table S2. Sensitivity analyses.Table S3. Adjustment for follow‐up duration.Figure S1. Concomitant change in medication prescription.Figure S2. Rebalancing of patient characteristics after propensity score matching.Figure S3. Common support between the two groups of patients.Click here for additional data file.