Exposure to famine during early life was associated with increased risks of abdominal obesity in adulthood, which was partially alleviated by healthy lifestyle factors .Undernutrition during early life may lead to obesity in adulthood. This study was conducted to examine the relationship between famine exposure during early life and the risk of abdominal obesity in adulthood. A total of 18,984 and 16,594 adults were surveyed in 2002 and 2010–2012 in two nationally representative cross-sectional surveys, namely China Nutrition and Health Survey, respectively. The risk of abdominal obesity was evaluated for participants born during 1956–1961 and compared with that of participants born during 1962–1964. The overall prevalence of abdominal obesity in adulthood showed a positive association with famine exposure during early life. The odds ratios of famine exposure were 1.31 (1.19–1.44) and 1.28 (1.17–1.40) in 2002 during fetal life and infancy and 1.09 (1.00–1.19) in 2012 during fetal life, respectively. The relationships between famine exposure and abdominal obesity across the famine exposure groups were distinct among females and those who lived in urban areas and were physical inactive ( Overweight and obesity, as well as abdominal obesity, in middle age are strongly related to all-cause mortality and morbidity of chronic diseases such as diabetes and hypertension and other metabolic diseases ,2. SeverPrevious studies have primarily focused on the relationship between famine and body mass index (BMI) and/or systemic obesity ,10,11,12The Great Chinese Famine that occurred during 1959–1961 is one of the most disastrous catastrophes in human history, resulting in 20–30 million deaths ,16,17 thTwo nationally representative cross-sectional surveys were conducted 40 and 50 years after the Great Chinese Famine in 2002 and 2010–2012 in China, respectively. These surveys could be good sources for analyzing the relationship between famine and abdominal obesity in participants of middle age and pre-elder age. Data were extracted from the China Nutrition and Health Survey (CNHS), a nationally representative cross-sectional study on nutrition and chronic diseases. A stratified, multistage probability cluster sampling design was used in this survey, which has been described in detail previously . In the Participants were categorized into the following three predefined groups according to their DOB: (1) nonexposed, with DOB between October 1, 1962, and September 30, 1964, (2) fetal-exposed, with DOB between October 1, 1959, and September 30, 1961, and (3) infant-exposed, with DOB between October 1, 1956, and September 30, 1958. Mean ages of the participants in these three exposed groups were respectively 38.6, 41.6, and 44.6 years in 2002 and 48.2, 51.2, and 54.1 years in 2010–2012.Although the Great Chinese Famine affected the entire mainland of China, its severity varied across provinces due to different weather conditions, population density, and local policies pertaining to food shortage . The sev2). Systemic obesity and abdominal obesity were defined using the Chinese criteria of weight for adults [2. Overweight was defined as BMI ≥24 and <28 kg/m2. Abdominal obesity was defined as waist circumference >90 cm in men and ≥85 cm in women.Anthropometric measurements included body weight, height, and waist circumference. Height was measured using a stadiometer  after removing shoes, and body weight was measured with light clothes using a beam scale . Waist circumference was measured using a waist circumference tape  only after breathing out. The accuracy of the height, waist circumference, and weight measurements was 0.1 cm, 0.1 cm, and 0.1 kg, respectively. The anthropometric measurements were made according to standard anthropometric measurement methods in health surveillance . All antr adults . SystemiResidential areas were classified into urban and rural. Physical activity was categorized into active and inactive level, wherein regular exercise for >20 min each time, including various activities such as running, swimming, and performing Tai Chi, was defined as active level. Current drinking status and current smoking status were considered as dichotomous variables based on the answer in the questionnaires (yes or no). Current drinking was defined as participants drinking alcohol in the past year, irrespective of the amount drunk. Current smoking was defined as smoking regularly every day or irregularly. Education level was categorized into dichotomous variable, where high school or above was considered as high educational level, otherwise as low educational level.p value <0.05 was considered to be statistically significant. Odds of abdominal obesity for the fetal-exposed group and the infant-exposed group, compared with the nonexposed group, were examined by the maximum likelihood method using the logistic regression model. Analyses were adjusted for sex, residential areas, education level, marital status, household income, current drinking status, current smoking status, and physical activity. To investigate whether the associations between fetal and infant exposure to famine and abdominal obesity were affected by social environment in later life, we subsequently stratified the analyses according to sex, residential areas, physical activity, and education level. The odds ratios ) of abdominal obesity in the fetal- and infant-exposed groups compared with the nonexposed group were calculated within each category of the stratified variables. The stratified variables were not adjusted in the corresponding models. The odds ratios (95% CI) were plotted in a graph using Stata 13.0. Sensitivity analysis was also performed in this study. First, we selected the 75th percentile of EDR as the cutoff point, and an EDR of 150.0% was used to define the severity of famine for the purpose of distinguishing severely and moderately severely affected famine areas more significantly. Second, participants with BMI ≥28.0 kg/m2 who were categorized as having systemic obesity according to the Chinese criteria were excluded to prevent the interaction between systemic obesity and abdominal obesity.The SAS version 9.4  was used for all statistical analyses, and a two-sided p < 0.05 after Bonferroni correction), whereas the infant-exposed group showed a greater waist circumference only in 2002. The prevalence rates of abdominal obesity in nonexposed, fetal-exposed, and infant-exposed groups were 15.5%, 19.4%, and 18.9% in 2002 and 31.6%, 33.3%, and 32.6% in 2010–2012, respectively.2 were excluded in sensitivity analyses B. The results of the fetal-exposed group were similar to those in sensitivity analyses A. After excluding the effect of systemic obesity, the rate of abdominal obesity was higher in the severely affected famine areas than that in the moderately severely affected famine areas in 2010–2012.In addition, participants with BMI ≥28 kg/mIn CNHS 2002 and CNHS 2010–2012, the risk of famine exposure associated with abdominal obesity was detected among female, inactive participants, those who lived in urban areas, and those with high level of education . The resBased on two nationally representative studies, the present investigation found that exposure to the Great Chinese Famine during early life increased the risk of abdominal obesity in later adulthood, especially among female, inactive participants, those who lived in urban areas, and those with high education level. The risk of abdominal obesity had a longer lasting effect on those who experienced the Great Chinese Famine in their fetal period. Current unhealthy lifestyle factors would exacerbate the effect of early-life exposure to famine on abdominal obesity.Low birth weight has been considered to be a risk factor for obesity in adults, which indicated that exposure to famine during early life might increase the risk of obesity in adulthood ,9. Some The fetal origin of adult disease hypothesis, also referred to as the Barker hypothesis, proposed that alterations in fetal nutrition and endocrine status lead to developmental adaptations that permanently change the body structure, physiology, and metabolism, thereby predisposing individuals to cardiovascular, metabolic, and endocrine diseases in adult life ,5,6. SevThe adaptive sex ratio adjustment hypothesis suggested that mothers who experience nutritional stress would be more likely to give birth to female babies. This is because nutrition cost was less expensive for female babies than for male babies, and female babies had a better opportunity to survive in the harsh environment . Male suA mismatch between early life and adult life environment may explain the association between famine exposure and risk of abdominal obesity ,34,35. NThe association between famine exposure and abdominal obesity disappeared in the 2010–2012 survey, such as in rural participants and those with low education level. The significant association existed for the female participants, but the odds ratio became smaller. Overall, the association was attenuated among participants, except for physically inactive participants in 2010–2012. This might be related to the trajectory of the human body shape over the course of the lives. Zhai et al [This study has some limitations. First, the lack of birth weight data might be a concern. However, fetal programming could also occur without any marked effects on birth size , and henEarly-life exposure to the Great Chinese Famine exacerbated the risk of abdominal obesity, especially in females or those who lived in urban areas or were physically inactive. A healthy lifestyle might partially alleviate the adverse effects. The results suggest that promoting a healthy lifestyle should be considered as a critical strategy for the prevention of chronic diseases in adult life.


Spondias mombin L. (yellow mombin) is a tree with a nutritional fruit that is commonly consumed in the North and Northeast of Brazil, as the juice of its pulp is rich in antioxidant compounds. This study aimed to investigate the gastroprotective and ulcer healing activities of yellow mombin juice (YMJ) in Wistar rats, and to elucidate the possible involved mechanisms. Phytochemical characterization of the lyophilized fruit juice was performed by high-performance liquid chromatography (HPLC). The gastroprotective activity of YMJ was investigated in ethanol  and indomethacin (100% YMJ) models of acute gastric ulcer in rats. Then, the effect of YMJ on mucus production and gastric secretions, and the involvement of non-protein sulfhydryl groups and prostaglandins in the gastroprotective process were examined. Moreover, the ulcer healing effect of YMJ was investigated in a model of acetic acid-induced chronic ulcer through histological and immunohistochemical analyses. HPLC results identified the presence of epicatechin (7.1 ± 1.6 μg/mL) and quercetin (17.3 ± 2.5 μg/mL) in YMJ. Ethanol-induced gastric lesions were inhibited by YMJ  by 42.42, 45.09, and 98.21% respectively, and indomethacin-induced lesions were inhibited by YMJ (100%) by 58.96%, compared to control group. Moreover, YMJ reduced gastric content and total acidy by 57.35 and 71.97%, respectively, compared to the control group. Treatment with YMJ also promoted healing of chronic ulcer, regeneration of the gastric mucosa, and restoration of mucus levels in glandular cells, as confirmed by histological analysis. It also increased cellular proliferation, as demonstrated by high reactivity to Ki-67 and bromodeoxyuridine. In conclusion, YMJ was found to possess gastroprotective and ulcer healing activities that are correlated to its antisecretory action. These results support the commercial exploration of YMJ as a functional food. Helicobacter pylori infection /g/4 h) were determined.This experiment was carried out according to the pylorus ligation method described by Shay et al.  with sli2).Chronic ulcer induction was carried out based on the method described by Takagi et al.  with somIn order to reduce the number of animals, our experimental procedure of gastric cicatrization using acetic acid followed the Russell-Burch Principles of reduction, substitution, and refinement in animal use (known as the 3R’s Principle). The experiments for this study and a related study were carried out jointly so that for the control and ranitidine groups, data from the same animals is used for both this study and a previously published study , allowinStomach tissue specimens with chronic ulcers (from acetic acid-induced rats) were sectioned and fixed in 10% buffered formalin. Then, tissue specimens were washed with water, immersed in 70% ethyl alcohol for 3–4 days, and embedded in paraffin. Finally, 5-μm thick paraffin sections were cut and stained with hematoxylin and eosin (H&E) and Periodic Acid–Schiff (PAS). Stained gastric tissue sections were examined under a light microscope and photographed using Leica camera, model EC3, attached to the microscope and connected to the Leica Application Suite software EZ .The immunohistochemical staining of Ki-67 and BrdU was carried out in 4-μm thick sections cut from paraffin blocks of samples containing ulcerated areas. Initially, samples were deparaffinized in xylene and hydrated .The expression of Ki-67 and BrdU proteins was evaluated using the free-biotin method in conjugation with horseradish peroxidase (HRP). Antigen retrieval was performed using a pressure cooker for 2 min, after which the slides were cooled to room temperature, and endogenous peroxidase was blocked using bovine serum albumin for 1 h. After cooling, slides were incubated overnight with primary mouse monoclonal anti-Ki-67 antibody  and anti-BrdU antibody . Visualization was carried out using HRP. Slides were washed, incubated with diaminobenzidine (DAB) chromogen solution , washed in water, counterstained with hematoxylin, dehydrated, and mounted. Cells positive for Ki-67 and BrdU were detected by the presence of a dark reddish-brown chromogen in the nucleus or nucleus/cytoplasm, respectively, of epithelial cells in the lesion area. Reactivity was scored using the following system: positive, mild , moderate , strong ; or negative .®. The level of significance for rejection of the null hypothesis was set at 5% (p < 0.05).Results were expressed as mean ± standard error of the mean (S.E.M). The differences between means were analyzed by analysis of variance (ANOVA) followed by Tukey’s test for one-way analysis. Statistical analysis was performed using GraphPad Prism 6.0The manual extraction of juice from 5000 g of yellow mombin was able to yield approximately 400 mL of YMJ. A 2-mL aliquot was frozen, lyophilized, and the lyophile (0.48 g) was used for the phytochemical analysis. From the chromatographic analysis of YMJ, it was possible to identify and quantify two components by the similarity of their retention times and ultraviolet absorption spectrum (λ = 340 nm) to those of the analytical standards used: epicatechin at 20.2 min (7.1 ± 1.6 μg/mL), and quercetin at 14.5 min (17.3 ± 2.5 μg/mL).Ethanol administration resulted in extensive damage to the gastric mucosa with hemorrhagic erosions in the control group (pretreated with 0.9% NaCl). However, pretreatment with YMJ  and lansoprazole significantly inhibited the gastric lesions by 42.42, 45.09, 98.21, and 89.22%, respectively, compared to the control group  and ranitidine (60 mg/kg) significantly inhibited the gastric lesions by 58.96% and 86.43%, respectively, compared to the control group  resulted in injury to the gastric mucosa with an area of 16.36 ± 3.44 mm0 mg/kg rThe gastric mucus level in control group animals submitted to pylorus ligation was found to be 8.79 ± 0.67 μg of Alcian Blue/g of tissue. Pretreatment with carbenoxolone (200 mg/kg) significantly increased the production of mucus by 76.8%, while pretreatment with YMJ did not significantly alter the mucus production, compared to the injured control group .Administration of NEM exacerbated ethanol-induced gastric lesions by 45.38% compared to the groups administered NaCl solution. However, pretreatment with YMJ (100%) and carbenoxolone (100 mg/kg) significantly inhibited the ulcerative lesions induced by absolute ethanol in both NaCl and NEM-administered groups, i.e., YMJ continued to exert its gastroprotective effect even after NEM administration .Administration of indomethacin exacerbated ethanol-induced gastric lesions in groups pretreated with NaCl and misoprostol by 65.54% and 553.08%, respectively, compared to the NaCl-administered groups pretreated with NaCl and misoprostol, respectively. However, pretreatment with YMJ (100%) significantly inhibited the ulcerative lesions induced by absolute ethanol in both NaCl and indomethacin-administered groups, with no significant difference between NaCl-administered/YMJ-pretreated and indomethacin-administered/YMJ-pretreated groups .Four hours after pylorus ligation and intraduodenal administration of NaCl, ranitidine, and YMJ, animals were euthanized and gastric secretions were collected, It was found that the intraduodenal administration of YMJ (100%) and ranitidine (60 mg/kg) significantly reduced the total acidity by 71.97 and 51.50%, respectively, and the content of gastric secretions by 57.35 and 66.17%, respectively .2 (Results of this experiment showed that treatment with YMJ (100%) and ranitidine (60 mg/kg) for 14 consecutive days decreased the area of the chronic ulcer by 57.34 and 70.65%, respectively, compared to the control group, which demonstrated an ulcer area of 58.19 ± 3.29 mm2 . These r2 . Further2 .Histological analysis of H&E-stained stomach specimens revealed well-defined ulcers with complete destruction of the mucosal and submucosal layers caused by acetic acid administration in control group rats. However, stomach specimens from rats orally treated with YMJ (10 mL/kg) and ranitidine (60 mg/kg) demonstrated regeneration of the gastric mucosa, as revealed by the reappearance of the epithelial and stromal layers, compared to the control group. Moreover, PAS staining showed increased mucus production, as demonstrated by the areas intensely stained by magenta color in the mucosal epithelial layer .Immunohistochemical investigation using monoclonal antibodies against Ki-67 and BrdU showed strong reactivity to BrdU, marked by the great quantity of BrdU-positive nuclei, and moderate reactivity to Ki-67, as demonstrated by the dark reddish-brown color in the gastric mucosa of animals treated with YMJ and ranitidine for 14 days. On the other hand, tissues from the control group demonstrated no reactivity for any of the two markers due to the destruction of the epithelial layer, as shown in S. mombin fruit, in acute gastric lesions induced by different necrotizing agents, and its healing effect in chronic gastric lesions induced by acetic acid, as well as the mechanisms involved in its action.This study was carried out to investigate the gastroprotective activity of YMJ, the juice of It was found that induction of gastric lesions by ethanol results in depletion of gastric defensive mechanisms, starting from the formation of reactive oxygen species (ROS) such as superoxide anions, hydroxyl radicals, and lipid peroxides  that resAdministration of indomethacin was found to suppress prostaglandin synthesis and thus, increase susceptibility to gastric mucosal lesions, as shown in the control group. However, pretreatment with YMJ significantly reduced mucosal damage compared to the respective control group pretreated with NaCl.Quercetin, a compound found in YMJ, was previously reported to inhibit the production of ROS and act as an anti-apoptotic during gastric injury . MoreoveFrom our results, YMJ was found to possess gastroprotective properties in ethanol and indomethacin models of acute gastric injury, which suggests the potential involvement of prostaglandins, mucus production, and/or antioxidant capacity in the antiulcer activity of YMJ .S. mombin. Tiburski et al. [in vitro antioxidant capacity of the frozen pulp of yellow mombin. The antioxidant activity was determined by the Trolox equivalent antioxidant capacity assay using the radical cation ABTS+ ., and was found to be 17.47 ± 3.27 mmol Trolox equivalent/g. Data from the literature suggest that agents that increase the antioxidant defenses of the gastric mucosa are considered to be beneficial in the treatment of gastric lesions [As far as we know, this is the first study to investigate the gastroprotective effect of the juice from the fruit pulp of i et al.  reported lesions , 28.After confirming the gastroprotective action of the juice, we aimed to elucidate the possible mechanisms of this action through a number of experiments. First, the effect of YMJ on mucus production was evaluated. As expected, carbenoxolone increased mucus production; however, YMJ did not affect it. This result negates the enhancing effect of YMJ on mucus production. Increased mucus secretion is among the important mechanisms of gastric mucosal defense against necrotizing agents , 30. In Then, the roles of–SH groups and prostaglandins in the mechanism of YMJ action were investigated. Our results showed that there was no significant difference between the gastroprotective effect of YMJ in the absence and presence of NEM, as well as in the absence and presence of indomethacin. These data indicate that the gastroprotective effect of YMJ is not associated with the production and/or presence of sulfhydryl compounds and does not depend on the production of prostaglandins. The–SH groups are responsible for increasing the production and maintaining the stability of gastric mucus through the formation of disulfide bridges, and are involved in maintaining gastric integrity by limiting the production of free radicals involved in tissue damage . The relProstaglandins may be involved in the gastroprotection process by inhibiting gastric acid secretion or enhancing gastric mucosal protective factors such as stimulation of mucus secretion, bicarbonate, and increased membrane phospholipids .+ ions, thereby decreasing the gastric acidity, and also decreasing the content of gastric secretions. These results indicate that the antisecretory activity of YMJ could contribute to its gastroprotective activity. In addition, data from the literature [+/K+-ATPase, which supports the antisecretory activity of YMJ demonstrated in this study.After pylorus ligation, the intraduodenal administration of YMJ was effective at reducing the concentration of Hterature , 34 showMoreover, YMJ assisted the healing of acetic acid-induced gastric injury in rats. The acetic acid model highly resembles human peptic ulcer regarding pathological features, the healing process, and the ulcer recurrence cycle . Thus, iAdministration of YMJ once daily for 14 days after the induction of gastric injury with acetic acid accelerated the healing process through mucosal and submucosal re-epithelialization, as demonstrated by H&E staining. Moreover, the YMJ-treated group was found to restore mucus production in glandular mucus producing cells, as demonstrated by PAS staining. The mucus layer acts as the first line of defense against luminal pepsin by forming a pre-epithelial mucus-buffer barrier along with phospholipids .Immunohistochemical analysis was used to verify the regenerative capacity of YMJ on the gastric mucosa through the detection of proliferating cells at the site of the chronic ulcer. Ki-67 is a nuclear matrix protein expressed by proliferating cells during the G1, S, G2, and M phases of the cell cycle, but not in quiescent cells , while BYellow mombin juice (YMJ) was found to exhibit a significant gastroprotective activity, the mechanism of which may be associated with its antisecretory action. The juice was found to be effective in accelerating the process of re-epithelialization in rats. Findings of this study open the perspective for the exploitation of YMJ as a functional food.


Recent studies have confirmed the efficacy of sorafenib for patients with advanced renal cell carcinoma; however, its efficacy and safety as an adjuvant therapy in patients with non-metastatic and loco-regional renal cell carcinoma after surgery remains controversial. Thus, the aim of the present retrospective study was to evaluate the efficacy of adjuvant sorafenib therapy in such patients from 8 centers in northwestern China that were treated from August 2009 to December 2016.After surgery, the patients (n = 48) received oral sorafenib for 3 months. The control group (n = 48) comprised patients that underwent the same surgery from December 2009 to June 2016 but without adjuvant therapy who were matched 1:1 with the sorafenib group with respect to sex, age, pathological findings, disease stage and grade, operation time, and surgical procedure. The primary outcome compared between the groups was disease-free survival. Adverse events were also recorded to evaluate the safety of sorafenib. The influence of patients’ characteristics and laboratory tests on recurrence was analyzed using unconditional logistic regression.P = .66) or median disease-free survival between the 2 groups . In multiple logistic regression analysis, increased blood urea nitrogen (BUN) emerged as an independent predictor of recurrence risk (P = .02).Overall, the demographic characteristics of the 2 groups were similar. There was no significant difference in the rate of recurrence (8.3% for sorafenib patients and 6.2% for the matched patients, These results indicate that postoperative sorafenib adjuvant therapy did not achieve the expected beneficial effect, pointing to the need for further studies to evaluate its utility in such cases. Approximately 134,000 people die of RCC annually, although the proportion of long-term survivors may be higher in developed countries.,3 Some studies indicated that 20–30% of patients had already developed metastases at diagnosis, whereas those with localized RCC  still have an approximately 40% recurrence rate after surgery.,4,5 The recurrence risk for primary resected RCC can be estimated based on clinical and histological features in conjunction with TNM staging.–8Renal cell carcinoma (RCC) is a cancer derived from the renal tubular epithelial cells, and is among the 10 most common cancers worldwide,–12 These factors are all beneficial for tumor angiogenesis, and further lead to activation of the Raf/MEK/ERK pathway that promotes tumor cell survival and proliferation. Moreover, RCC pathogenesis has been related to immune system dysfunction. For example, loss of mature dendritic cells and the reduction of central memory T cells in lymphoid tissues surrounding the tumor can significantly suppress the anti-tumor function of T cells.,15 In addition, certain gene mutations have been linked to RCC. For example, de Martino et al found that patients with JAK3 mutations have a higher possibility of metastasis.The major pathological type of RCC is clear cell renal cell carcinoma (ccRCC), which is characterized by an inactive Von Hippel-Lidau (VHL) gene due to mutations and aberrant methylation, leading to abnormal overexpression of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and hypoxia-inducible factor (HIF)-1α and HIF2-α. targeted tyrosine kinase inhibitor (TKI) are the first-line drugs for treatment of RCC. In particular, the TKI sorafenib is an oral multi-kinase inhibitor that mainly targets the VEGF and PDGF pathways, thereby suppressing tumor proliferation and angiogenesis, and shows potent anti-tumor activity in patients with metastatic RCC.,20 Furthermore, sorafenib also showed a significantly greater therapeutic effect for RCC patients compared with interferon treatment.Based on these characteristics and its high rate of resistance to conventional chemotherapy,Although the efficacy of sorafenib has been extensively studied in patients with advanced-stage RCC, there are relatively few studies on its effectiveness as adjuvant therapy for early-stage RCC. Previous prospective studies, ASSURE and S-TRAC, explored these effects, but found different results. Based on these conflicting findings, we conducted the present retrospective analysis including patients from 8 centers in northwestern China that received sorafenib treatment and matched controls without adjuvant treatment post-surgery.22.1This multicenter retrospective study was conducted using a matched-pairs design with a 1:1 ratio between sorafenib and control patients. The sorafenib patients received the drug postoperatively via oral administration. The matching criteria were based on pathological examination, TNM stage, Fuhrman grade, sex, age, operation time, and surgical procedure. If the patients could not be completely matched, we appropriately broadened the matching criteria and chose the most similar patient as the paired control.2.2From August 2009 to December 2016, we collected the data of 96 patients that underwent tumor resection for localized RCC from 8 centers in northwestern China, with 48 patients each in the sorafenib and matched non-sorafenib group. All patients were pathologically diagnosed with RCC, and were >18 years of age. Other inclusion criteria included: no significant liver and kidney function damage , no second tumor within 5 years, no major cardiovascular events within 6 months prior to treatment, no severe uncontrolled blood pressure (>150/100 mmHg). None of the patients received any systemic anti-tumor therapy. All of the patients were supported by ethics committee of Xijing Hospital.The patients in the sorafenib group received 400 mg of sorafenib twice daily for 3 months continuously after the operation. Adverse events were monitored every month during the treatment. Within 3 months of the start of treatment, the patients were followed up once a month, which was subsequently changed to once every 6 months. Tumor recurrence, metastasis, or the presence of new tumors was evaluated by imaging examinations (computed tomography or magnetic resonance).2.3The safety evaluation of sorafenib included adverse events, laboratory tests, score on the Eastern Cooperative Oncology Group (ECOG) scale , and 12-lead echocardiogram. The assessment of adverse events included the type, duration, and grade, according to the Common Terminology Criteria for Adverse Events version 3.0 (CTCAE v3.0).2.4t tests, and the count or categorized data were compared using the chi-squared test. The influence of patients’ characteristics and laboratory variables on recurrence was analyzed by unconditional logistic regression. Statistical significance (P < .05) was analyzed using SPSS 21.0 .Disease-free survival (DFS) was the main outcome measure used for comparison between the groups, which was defined as the duration from surgery until tumor recurrence, and was visually assessed with a Kaplan–Meier plot. Continuous data are presented as means ± standard deviations, and count data are represented by the number of cases and their percentages. Continuous data were compared using independent 33.1The basic clinical features of the patients are listed in Table In the sorafenib group, there were 38 male and 10 female patients. According to the UISS, 25 and 23 patients were classified as low and intermediate risk, respectively. In the matched group, there were 37 male and 11 female patients, with 23 and 25 classified as low and intermediate risk, respectively.The matched patients were followed up with no further treatment, whereas the patients in the sorafenib group received 400 mg of sorafenib twice daily within 4 to 12 weeks after surgery, and continued the therapy for 3 months. No patients had to discontinue treatment owing to adverse events.3.2P = .557) , with liver, lymph node, lung, and bone metastasis in 1 patient each. The overall rate of recurrence in the matched patients was 6.2% (3/48), with liver, bone, and lung metastasis in 1 patient each  and serum creatinine (Scr)  (Table P = .01), hypertension (P = .02), UISS risk group (P = .03), Fuhrman grade (P = .02), decreased alkaline phosphatase (ALP) (P = .03), increased total bilirubin (TBI) (P = .04), increased BUN (P = .01), and increased Scr (P = .01) may be potential recurrence risk factors (Table P = .02) (Table The patients that experienced recurrence had increased levels of blood urea nitrogen (BUN) . However, the opposite result was obtained in the S-TRAC trial, showing a median DFS of 6.8 years and 5.6 years in the sunitinib and placebo group, respectively. Following assessment of an independent blinded central review board, sunitinib adjuvant therapy was concluded to be superior to placebo.Previous studies have shown that TKIs have significant therapeutic efficacy in patients with advanced RCCThis is the first study to investigate the efficacy and safety of a TKI as an adjuvant treatment for early and local RCC in a northwestern Chinese population. The main pathological type in this cohort was ccRCC. Since sorafenib targets VEGF and PDGF, this group was appropriate for evaluation of the effect of TKIs. In addition, the study population was distributed across the major provinces of northwestern China, making it a geographically representative sample. Similar to the ASSURE trial, there were no significant differences in the survival time between the sorafenib-treated patients and matched controls, and the median DFS was not reached in either group. which is largely attributed to advances in surgical techniques. In northwestern China, the administration of sorafenib adjuvant therapy in patients with RCC was mainly only started in 2011. Therefore, in this retrospective study, we did not reach the median DFS. However, analysis of the overall survival time still showed no difference between the groups. Hence if we get the media DFS, the results may be different.In general, 40% of patients with RCC can now survive for 10 years after surgery, reported that in the S-TRAC trial, the higher-risk patients  were those that received a benefit from sorafenib. Haas et al reanalyzed the results of the ASSURE trial by selecting only patients with ccRCCs who were at least at middle risk, and found no significant difference in the 5-year PFS between the sorafenib and placebo groups, further contributing to the controversy of the benefit of postoperative adjuvant therapy. In our study, 95% of the patients were at AJCC stage 1–2 and there were far fewer patients at stage T3 compared with those in the ASSURE trial. Hence, this may have contributed to the difference in the results between trials. In contrast to the ASSURE and S-TRAC trial, the patients received sorafenib for 1 year, whereas our patients only received sorafenib for 3 months; thus, it is possible that sustained therapy will have some benefits down the road. However, we do not recommend a longer course owing to the financial burden for the patients and adverse effects of the drug. Moreover, we hypothesize that immune system dysfunction plays a dominant role in the development of RCC; however, the targeted drug in this case cannot affect the patient's immune system. Hence, adjuvant therapy may have a limited benefit for patients with early-stage RCC.Thus, our results provide further support for no benefit of sorafenib as adjuvant treatment after surgery in early-stage RCC patients; thus, the controversy remains, and is worthy of further study. The different results obtained among studies may be related to variations in the distribution of pathological type and tumor risk stage in the patients in each trial. In the ASSURE trial, 79% of the patients had ccRCC, and 33% were classified in AJCC stage 1–2. In the S-TRAC trial, 99% of the patients also had ccRCC; however, they were all at AJCC stage 3 or higher. Similarly, in the ASSURE trial, the mid-high risk was based on T1, Fuhrman grade 3–4; T2, T3, Fuhrman grade 1; T3, Fuhrman grade ≥ 1; and the ECOG score was 0, whereas in the S-TRAC trial, all patients were at T3 or higher. Staehler et al The UISS risk group and Fuhrman grade are not only used to evaluate the RCC grade but also serve as prognostic predictors. TBI, Scr, and BUN, can reflect renal function, and thus their abnormal levels often raises alarm, leading to closer monitoring. However, our multiple logistic regression analysis demonstrated that only increased BUN can be used as a predictor of recurrence risk. Nevertheless, this result may have been influenced by the relatively small sample size; hence, further data with larger populations will be needed to verify this association.According to the results of the chi-squared test, the frequencies of increased BUN and increased Scr were higher in the patients that experienced recurrence. However, this does not necessarily mean that these factors are the cause of the recurrence. Univariate non-conditional logistic regression analysis showed many factors that could affect recurrence, including older age, hypertension, UISS risk group, Fuhrman grade, decreased ALP, increased TBI, increased BUN, and increased Scr. It is generally believed that the human organ function gradually declines with age, leading to an increased risk of tumorigenesis in older individuals. Indeed, a previous study showed that older female RCC patients had a worse prognosis.,20 Nevertheless, the dose of sorafenib did not need to be reduced and the drug did not need to be discontinued in any patient due to unacceptable adverse events. This may be related to the fact that our patients only took sorafenib for 3 months after surgery, which is a relatively short time.The most common adverse events were hand-foot syndrome, alopecia, rash, diarrhea, hypertension, and cutaneous pruritus, which is consistent with previous studies on sorafenib.5In this first study of the effects of sorafenib as postoperative adjuvant therapy for RCC in a northwestern Chinese population, we did not observe a beneficial therapeutic effect, contributing to the ongoing controversy. Thus, continued follow-up and inclusion of new patients in this study will help to supplement these results and hopefully provide resolution to the controversy or explanation as to why these effects differ from those for patients at more advanced disease stages.The authors are grateful to the clinicians and staffs at the 8 clinical centers in northwestern China who contributed to the samples and data collection for this study.Conceptualization: Di Wei, Guojun Wu, Yu Zheng, Jianlin Yuan.Data curation: Di Wei, Fubao Chen, Jingyi Lu, Yangmin Wang, Dalin He, He Wang, Zhiping Wang, Peng Chen, Yujie Wang, Zhiyong Wang, Yongli Ye, Zheng Zhu.Formal analysis: Di Wei, Yu Zheng.Supervision: Guojun Wu, Jianlin Yuan.Visualization: Guojun Wu.Writing – original draft: Di Wei.Writing – review & editing: Jianlin Yuan.


Exhaled breath condensate (EBC) was introduced more than two decades ago as a novel, non-invasive tool to assess airway inflammation. This review summarizes the latest literature on the various markers in EBC to predict asthma in children. Despite many recommendations and two comprehensive Task Force reports, there is still large heterogeneity in published data. The biggest issue remains a lack of standardization regarding EBC collection, preservation, processing, and analysis. As a result, published studies show mixed or conflicting results, questioning the reproducibility of findings. A joint, multicenter research study is urgently needed to address the necessary methodological standardization. EBC is obtained by cooling exhaled breath, and mainly contains water vapor and a small fraction of volatile and non-volatile macromolecules , 2. Thesytokines , 2. In cytokines . Despiteytokines . This isytokines . Therefoytokines , 2, therIn this review, we aim to provide a concise update on markers in EBC to predict asthma in children. For this purpose, the PubMed database was searched for relevant publications, with at least an English abstract, in the last 5 years (January 2013–December 2018). For review of EBC literature older than 5 years, we refer to two other excellent reviews , 7. All n = 10), acidity (n = 9), and eicosanoids . For measurement of the various biomarkers, 14 different analytical methods were used .A total of 30 studies with original data in the past 5 years were identified, of which 20 had a cross-sectional design  and 10 aThese numbers illustrate quite well that the methodological issues, as described in both Task Force reports , 2, are As previously stated, the different cross-sectional studies published in the past 5 years are difficult to compare .Cytokines were the most studied biomarkers. Earlier studies have shown increased cytokines in EBC from children with asthma and preschool wheeze compared to healthy controls . These f1) after exercise (Two other studies assessed cytokines in EBC of asthmatic children with exercise-induced bronchoconstriction (EIB) , 23. In exercise . A majorn = 8), IL-2, MCP-1, and two markers of airway remodeling  showed a good correlation with fractional exhaled nitric oxide (FeNO)  . Again, Interestingly, all the above described studies found neither detectable nor increased levels of IL-5 or IL-13, two major T helper 2 (Th2) cytokines that are considered to play a major role in allergic asthma . Only onAcidity is another frequently investigated biomarker. Earlier studies have shown a decreased pH in children with severe compared to mild asthma . With reRegarding eicosanoids in EBC, earlier literature has shown mixed results. 8-isoprostane (8-IP) is increased in childhood asthma, and then particularly in children with severe asthma or with an asthma exacerbation . This fi10), Cys-LTs were higher in asthmatics compared to healthy children  in EBC showed a tendency to increased levels in the moderate persistent asthma group, but not a significant difference . Both thchildren . Furtherchildren .4 (LTB4) in children with asthma compared to healthy controls  had significantly higher LTB4 than children without obstructive lung function  of children with an asthma exacerbation . As shown above, these recent studies showed mixed results, which was also the case in reviews regarding older literature , 7. Morex and H2O2, markers of oxidative stress that got a lot of attention in the early days of EBC research, were not incorporated in recent research. This may reflect persistent methodological problems, as illustrated by the only study that assessed EBC NOx, which reported a detection rate of only 48% . The optimal method for measuring pH in EBC has been part of debate for more than a decade  children, and despite the publication of many recommendations by both the ERS and ATS, the development of EBC into maturity seems to have stagnated. The main concern remains the various methodological issues before, during, and after breath condensation, as clearly shown by all methodological recommendations from the latest ERS Task Force . NaturalWe urge to recognize that the current use of EBC resembles a popular hit-and-run game, almost without clear rules or a single solution within reach. In our opinion, the necessary procedural rules or methodological standardization can only be achieved through a joint multicenter research initiative and a study design that will clearly address each step of EBC collection, preservation, processing, and analysis for each type of biomarker. Meanwhile peer-reviewed recommendations for publication of new data on EBC should be based on a thorough and obligatory presentation and description of each procedural step as mentioned.MB and PR included the studies, extracted the data, and drafted the manuscript. QJ and ED helped to draft the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bone graft material should possess sufficient porosity and permeability to allow integration with native tissue and vascular invasion, and must satisfy oxygen and nutrient transport demands. In this study, we have examined the use of three-dimensional (3D)-printed polycaprolactone/tricalcium phosphate (PCL/TCP) composite material in bone grafting, to estimate the scope of its potential application in bone surgery. Adipose-derived stem cells (ADSCs) and bone marrow stem cells (BMSCs) are known to enhance osteointegration. We hypothesized that a patient-specific 3D-printed solid scaffold could help preserve seeded ADSCs and BMSCs and enhance osteointegration. Diffuse osteogenic tissue formation was observed by micro-computed tomography with both stem cell types, and the ADSC group displayed similar osteogenesis compared to the BMSC group. In histological assessment, the scaffold pores showed abundant ossification in both groups. Reverse transcription polymerase chain reaction (RT-PCR) showed that the BMSC group had higher expression of genes associated with ossification, and this was confirmed by Western blot analysis. The ADSC- and BMSC-seeded 3D-printed PCL/TCP scaffolds displayed promising enhancement of osteogenesis in a dog model of maxillary bone defects. Charles W. Hull, who founded 3D Systems in the United States in 1986, invented the world’s first three-dimensional (3D) printer. Since then, 3D printing has revolutionized a variety of manufacturing technologies, as well as tissue engineering and regenerative medicine .Tissue engineering is an important medical technology that combines biological and engineering techniques for the restoration of damaged or missing tissues or organs. Materials used to construct scaffolds for tissue engineering should have biocompatibility and biodegradability, and mechanical properties that can be supported in the body. In addition, an artificial scaffold prepared according to the characteristics of the transplantation site should allow differentiation and proliferation of cells attached to the pore ,4,5,6. TBased on computed tomography (CT) or magnetic resonance imaging (MRI) data, it is possible to make patient-customized scaffolds using 3D printing technology. An advantage of this approach is that the mechanical properties, pore size, and porosity of the scaffold can be controlled. In addition, the excellent internal connective structure formed in the 3D shape improves the penetration and differentiation of the cells into the scaffolding, resulting in excellent biocompatibility. This technology is widely used for the reconstruction of ears and craniofacial defects in plastic surgery ,9,10,11.We have previously published literature introducing blended polycaprolactone (PCL) and tricalciumphosphate (TCP) as a 3D printable material for guided bone regeneration . The bleStem cells are cells that can infinitely regenerate over a lifetime, and can differentiate into various cell types in response to specific biological signals and external stimuli. Techniques for differentiating mesenchymal stem cells (MSCs) isolated from bone marrow  and adipose tissue , into chondrocytes, osteoblasts, and muscle cells are well established, and these cells can be used in tissue engineering to regenerate various tissues such as cartilage, bone, and muscle ,15,16,17MSCs are the main constituents of bone marrow stromal cells, and are the precursors of bone, cartilage, muscle, and connective tissue . ImmunolWe hypothesized that ossification between normal bone and a 3D-printed scaffold is an important factor in the reconstruction of maxillary bone defects, and that substances secreted from ADSCs and BMSCs could enhance osteointegration and thus increase both the degree of contact between the normal bone and the 3D-printed scaffold and bone formation inside the scaffold. This study was conducted to investigate the use of polycaprolactone/tricalcium phosphate (PCL/TCP) scaffolds, which are 3D-printed with a mixture of PCL, a polymeric material, and TCP, a bioceramic material, with ADSCs and BMSCs in bone regeneration in a dog model of maxillary bone defects.Biodegradable PCL  and TCP  were used as materials for the scaffolds. PCL was dried at 105 °C for 1 day before use. TCP had a particle size of 100 nm or less. The 3D defect model was created by commercial 3D medical image editing software , and customized to maxilla defect of animals used in this study. A steel syringe containing the PCL/TCP mixture was equipped to a 3D printer and maintained at 120 °C. The molten PCL/TCP mixture was precisely dispensed through stainless steel nozzle with diameter of 300 µm. The inner microstructure and surface composition were characterized by field-emission scanning electron microscope  and energy dispersive spectrometer (EDS) analyses .g. Contaminating erythrocytes were removed by the addition of erythrocyte lysis buffer  at pH 7.3. The stromal cells were rinsed twice with PBS, and the ADSCs were collected.Adipose tissues obtained from the abdominal cavity of the beagles were washed with an equal volume of phosphate buffered saline (PBS). Then, enzymatic degradation was carried out at 37 °C for 30 min using 0.075% collagenase type I . To remove debris from the connective tissues, the degraded tissue was filtered and the supernatant of the adipocyte layer was separated. The cell suspension was centrifuged for 10 min at a force of 200× Under general anesthesia, the humerus of each beagle was punctured with an 18-gauge injection needle and 1 mL of bone marrow tissue was collected with a 1 mL syringe. The collected bone marrow aspirates were combined with Histopaque  at a ratio of 1:1 in a 15-mL tube and centrifuged at 2000 rpm for 30 min. After centrifugation, the white layer was collected in a 15-mL tube, diluted 10-fold in PBS, and then centrifuged at 2000 rpm for 10 min. After aspiration of the supernatant, the cells were again diluted 10-fold in PBS and centrifuged as above to collect BMSCs.n = 3 in each group) for application of ADSCs and BMSCs.Six healthy beagles, each 12 months old and weighing 9 kg, were included in the study. Prior to the 3D printing model experiment, spiramycin and metronidazole were injected under general anesthesia, the teeth were scaled, and the left molar and premolar were removed. Prior to implanting 3D-printed PCL/TCP scaffolds, the dogs were divided into two groups  was injected intravenously and general anesthesia was performed with halothane . During the operation, Lactated Ringer’s Solution and 1 g of cephalosporin were administered. After exposure to anesthesia, the fur on the left side of the beagle’s face was removed and the skin was disinfected with Betadine and alcohol. The maxillary bone was exposed through a skin incision from the zygomatic arch to the infraorbital foramen, and the maxillary bone defect was created from the infraorbital rim to the zygomatic arch in accordance with the 3D-printed scaffold prepared before surgery. The infraorbital nerve was preserved.First, the bottom pores of the 3D-printed PCL/TCP scaffolds were blocked with fibrin glue. ADSCs and BMSCs were injected into the pores according to the experimental group, and then fibrin glue was applied to the exposed outer pores to prevent cell leakage. The prepared 3D-printed scaffold was inserted into the defect, fixed with a plate and screw, and the skin was sutured . Both grTo evaluate the ossification of the implanted 3D-printed scaffold, CT images of coronal, axial, and sagittal views were taken immediately after surgery, and 4, 8, and 12 weeks postoperatively. The ossification activity was determined by calcification of the marginal area, increased bone density, and decreased internal pore size. The level of tissue density depending on the time was analyzed with the Hounsfield unit (HU), which is a quantity commonly used in CT imaging. The HU value of the tissue in the pore was randomly measured at 5 sites for each individual, and the average values were obtained . The measured HU values were normalized and calculated. To measure the extent of ossification, 3D-CT reconstruction was used.For histological evaluation, all beagles were euthanized 12 weeks after 3D-printed scaffold implantation and the grafts were harvested, including 1 cm of marginal bone tissue. Tissues were taken from three normal bone/scaffold junctions and the center of one scaffold, and each was divided into three pieces. One third was fixed in 10% neutral formalin, and paraffin embedded tissue sections were stained with Hematoxylin and Eosin (H&E) and Goldner’s trichrome (GT). New ossification of the entire graft, the thickness and extent of periosteum produced, the degree of bone ingrowth into the pores, the infiltration of inflammatory cells into the graft, and the formation of collagen fibers were observed with an optical microscope . Each slide was measured for optical density using an image analysis program . In each group, the optical density of normal bone, tissue in the pore, and soft tissue around the scaffold including the periosteum were measured randomly at 5 sites for each tissue sample, and average values were obtained.One third of each collected tissue was frozen in liquid nitrogen, and after grinding, total RNA was isolated using TRIzol . RNA was reverse transcribed to cDNA using a RevertAid First Strand cDNA Synthesis Kit  and polymerase chain reaction (PCR) was performed using an AccuPower RT-PCR kit . Type 1 collagen (COL1), osteocalcin (OCN), runt-related transcription factor 2 (RUNX2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression were measured by RT-PCR .Proteins were extracted from obtained skin tissue by PRO-PREP kit  and protein concentration was determined with BCA method. Proteins and 5X Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer were mixed and boiled at 95 °C for 5 min before incubation on ice. This protein was separated with 10% SDS-polyaclamide gel at 80~100 V for 2 h and transferred to nitrocellulose membrane at 100 V for 70 min on ice. Membranes were blocked with 5% skim milk at room temperature for 1 h and probed with primary antibody such as OCN , COL1 , RUNX2 , β-actin . β-actin was used as a housekeeping protein. After incubating the membranes with antibody solution overnight at 4 °C, the membranes were washed with Tris buffered saline with Tween-20 (pH 7.4) and probed with secondary antibody for 1 hour at room temperature. Membrane was then exposed to ECL. The blots were followed immediately by exposure to X-ray film and the bands were quantified using ImageJ software .t-test. Statistical significance was set at p < 0.05.Statistical analysis was performed with SPSS Ver. 22.0. . A subgroup analysis was performed using the paired For fabrication of the customized scaffold, 3D defect was reconstructed using Mimics software a. The 3DIn both groups, ossification was not observed until four weeks after surgery. After eight weeks, the bone density along the scaffold margins was similar to that of normal bone. This can be regarded as progression of ossification along the margin, and showed a marked increase in density after 12 weeks. This suggests that ossification of the margin continuously increased for up to 12 weeks after surgery.In both groups, the size and number of pores inside the scaffold also decreased after eight weeks. However, the bone density of the scaffold was only partially increased, and did not reach the density of the surrounding normal bone. This suggests that most of the pores are formed by the increase in connective tissue and the inflammation reaction with the surrounding tissues and the ossification is partially occurring. After 12 weeks, pore size decreased further, and bone density was increased compared to eight weeks. However, at both 8 and 12 weeks, the BMSC group showed more pronounced marginal ossification and greater pore size reduction than the ADSC group .When the density of the normal bone was set to 100, the normalized density value of the new tissue (including the new bone) in the pores was 36.5 and 35.7 in the ADSC and BMSC groups, respectively, which was higher than the relative density of the periosteum around the scaffold  around the scaffold were obtained for the two groups. In GT and H&E staining, the optical density inside the pores was higher than that of the periosteum and other soft tissues around the scaffold, and was higher with GT than with H&E, however, there was no significant difference .Histological evaluation 12 weeks after surgery displayed ossification in the pore of the scaffold and an increase of fibrinogen in the periphery. The increase of fibrinogen was confirmed by an increase of COL1 gene expression, and ossification was confirmed by increases in OCN and RUNX2 gene expression. By RT-PCR, the band intensity of COL1, OCN, and RUNX2 was normalized to that of GAPDH. The degree of ossification-related gene expression was slightly increased in the BMSC group compared to the ADSC group, however, there was no significant difference .Twelve weeks after surgery, expression of the ossification-related proteins COL1, OCN, and RUNX2 was increased in both groups. The expression of these proteins was higher in the BMSC group than in the ADSC group . In partDue to its ease of use and adaptability, 3D printing technology is being rapidly developed and applied in various medical fields. For example, 3D printing technology can be used to visualize the complex deformities of congenital cardiovascular anomalies, to map intricate blood vessels during renal cancer surgery, and allow the development of approaches to intracranial tumor neurosurgery. These applications help surgeons analyze and evaluate the individual condition of the patient before surgery, aiding in the development of a surgical plan ,24. HoweWith tissue engineering, a new era in bioengineering has begun. In general, tissue engineering involves three components: cells, scaffolds, and growth factors. Recently, 3D technologies have been used to develop cell and tissue printing technologies, allowing tissue engineering to go beyond conventional cell culture technology. Scaffold development and 3D cell culture both have remaining limitations, but it is expected that in the near future, 3D cell-printing technology will enable the simultaneous formation of 3D biological structures and scaffolds ,28,29,30Currently, 3D printing technology is most often used in craniomaxillofacial surgery, and several challenges must be overcome for its widespread application. First, more specialized computer software is required. Pre-operative 3D printing is not easy to plan because it takes too much time to perform a computer simulation of a differentiated process. This requires more automated functionality and simulation via a variety of software.Second, there must be a sufficient relationship between the pre-operative simulation and the actual operating environment, because the pre-operative simulation serves as a guide for the operation. For example, to apply 3D-printed titanium implants in surgery, bone must be resected precisely according to the preoperative plan, as 3D-printed implants are generally quite hard, and cannot easily be cut or bent.Third, accuracy during measurement is very important. Due to technological advances, it is possible to obtain very thin CT images, but the combination of these images to form 3D images introduces error, particularly for very thin bones such as the orbital bones.Finally, there is a possibility of artifacts due to metal materials. The use of CT scans in 3D printing for prosthetic dentistry, which mainly uses metal models, may be less accurate due to these artificial shadows. Tooth occlusion requires very fine 3D rendering, and CT may not provide images of adequate resolution because of these artifacts ,32.Despite these problems, 3D printing technology is attracting attention as a new medical technology. Using 3D printing, the medical industry could visualize various characteristics of individual patients and apply them to personalized medical care.Scaffolds used in tissue engineering are generally made of natural and synthetic polymeric materials. Polymers derived from natural materials, animals, and human bodies have superior biocompatibility and are nontoxic. These include gelatin, collagen, fibrin, elastin, and alginate. Synthetic polymeric materials are relatively inexpensive, have excellent mechanical properties, and are either hydrolyzed in vivo or degraded by enzymes, making them ideal polymers for scaffolds. PCL, polylactide-co-glycolide, and polylactic acid (PLA) are typical synthetic polymer materials. In particular, various scaffolds for bone tissue regeneration are made using PLA and TCP, a bioceramic material. Since TCP is made of powder, it confers decreased mechanical strength to the scaffold in comparison with a solid polymer material that can be produced using heat, and is more difficult to form into 3D scaffold with excellent internal pore structure. To overcome this problem, many studies have fabricated scaffolds by mixing ceramic and polymeric materials ,35,36,37The 3D printing system enables precise control and rapid production of desired 3D shape structures at the micron scale. It also has the advantage of determining the line width and line height of laminated materials according to pneumatic pressure, temperature, and moving speed. Generally, when a scaffold is produced by 3D printing with a mix of polymer and a bioceramic material, the ceramic material content is limited. Because of the difficulty of nozzle discharge with increased amounts of bioceramic powder, controlling the mixing method of the bioceramic powder is a very important aspect of scaffold development. In this study, a 3D printing system was used to fabricate 3D scaffolds from PCL mixed with β-TCP, and evaluated their effects on bone formation.MSCs grow adherently in the form of fibroblasts in vitro, form cell clusters from single cells, and differentiate into osteocytes, adipocytes, and chondrocytes. Friedenstein first reported the presence of MSCs in the bone marrow in 1970 . SubsequIn order to improve osteointegration in bone grafting and the regeneration of bone defects, ADSCs and BMSCs have recently been used in tissue engineering. However, when harvesting BMSCs, complications such as pain and infection are likely to occur, and there is a limit to the amount of BMSCs taken. In contrast, ADSCs are readily available in large quantities under local anesthesia ,14,15,16The use of scaffolds for the application of ADSCs and BMSCs to bone defects has many advantages. The scaffold serves as a bridge, allowing adequate time for stem cell survival and subsequent bone formation. Generally, the scaffold should be strong enough to withstand external impact, and the pores should disassemble after the bone has adequately regenerated. In this study, an artificial scaffold was prepared using biodegradable PCL/β-TCP, which is currently in clinical use as an osteoinductive material. β-TCP is highly compatible with bone morphogenetic protein 2 (BMP-2), a bone growth-promoting factor, and thus increases osteoinduction ,45,46,47To evaluate the bone regeneration ability of PCL/β-TCP scaffolds injected with ADSCs and BMSCs, 3D CT and histological analysis were performed after implantation of the scaffolds into maxillary bone defects in beagles. Diffuse osteogenic tissue formation was observed by micro-computed tomography with both stem cell types, and the ADSC group displayed similar osteogenesis compared to the BMSC group. RT-PCR and Western blot analysis confirmed increased expression of proteins associated with osteogenesis in both groups, with higher expression of COL1 of the BMSC group observed in Western blot. As a result, new bone formation inside the scaffold was effective in the reconstruction of maxillary bone defects in a dog model using PCL/TCP biomaterials manufactured using a 3D printing system, with ADSCs and BMSCs. However, unlike other previous studies, gross and histological results suggested that ADSC treatment was superior to BMSC treatment.These results suggest that ADSCs induce osteogenesis similar to BMSCs when 3D-printed PCL/TCP scaffolds are implanted in bone defects. Clinically, ADSCs have many advantages over BMSCs because ADSCs can be harvested easily, quickly, safely, and in relative abundance by lipoaspiration in the office-based setting. On the other hand, BMSCs harvest is an invasive procedure with low yield, which may impart donor site morbidity, such as pain, bleeding, hematoma, and deep infection. Therefore, if used with a 3D-printed PCL/TCP scaffold, ADSC can be a good alternative to BMSC to improve osteointegration in bone grafting and the regeneration of bone defects.


Supplemental Digital Content is available in the text Benralizumab, a humanized, anti-interleukin-5 (anti-IL-5) receptor α monoclonal antibody that directly and rapidly depletes eosinophils, has shown significant efficacy in reducing asthma exacerbations and improving lung function in moderate to severe eosinophilic asthma patients. However, there is some controversy regarding the adverse events (AEs) of benralizumab and a comprehensive analysis of these AEs has not been performed. This study aimed to assess the incidence of these AEs in published randomized controlled trials (RCTs).We searched for RCTs in the Embase, PubMed and Cochrane databases that compared benralizumab with placebo in moderate to severe eosinophilic asthma patients. The outcome was the incidence of AEs during the observation period.Eight RCTs were analyzed in this study. Patients treated with benralizumab had a lower risk of overall AEs (risk ratio (RR) 0.94; 95% confidence interval (CI) 0.90–0.98), serious adverse events (SAEs) , asthma exacerbation , bronchitis  and sinusitis , but had a higher risk of headache  and pyrexia  than patients treated with placebo. No increased incidence of death, hypersensitivity, injection-site reactions, nasopharyngitis, rhinitis, upper respiratory tract infection, influenza, cough, nausea, back pain or arthralgia was observed with benralizumab compared with placebo.Benralizumab reduced the risk of SAEs, asthma exacerbation, bronchitis and sinusitis, and aggravated the risk of headache and pyrexia. Other AEs were comparable between the benralizumab group and placebo group. Therefore, benralizumab is a relatively safe drug, but vigilance regarding AEs is imperative during long-term treatment. However, the potential long-term side-effects of ICS and LABA cause suffering in many patients. Therefore, we urgently need more effective and safe treatments to improve the quality of life in these patients. For patients with uncontrolled severe eosinophilic asthma, the recently updated GINA guidelines recommend add-on treatment with anti- interleukin (IL)-5 receptor therapy for patients ≥12 years of age.Asthma is a common, chronic inflammatory, respiratory disease that affects approximately 1% to 18% of individuals in different countries around the world. IL-5 is the major cytokine in activating eosinophils and is pivotal for eosinophil development, maturation, and survival in tissues. Eosinophils release the contents of granules, causing tissue damage, promoting the progression of inflammation and are a classic feature of asthma.,6 Benralizumab is an anti-IL-5 receptor α monoclonal antibody that can efficiently block the effects of IL-5.,8 The published data indicate that benralizumab can completely deplete eosinophils and their (late) bone marrow progenitors by inducing antibody-dependent cell-mediated cytotoxicity executed by natural killer cells and/or macrophages both in vitro and in non-human primates in vivo. Numerous studies in humans have shown an adequate safety profile with a significant reduction in blood eosinophil counts following the administration of benralizumab.IL-5 and eosinophils play key roles in the inflammatory response in T-helper 2-high asthma.–18 Benralizumb has also been approved by the FDA as add-on maintenance treatment for severe asthma in patients ≥12 years old with an eosinophilic phenotype. Some AEs of benralizumab during treatment in eosinophilic asthma patients are still controversial, and almost none of the available articles classify and analyze these AEs in detail.–18 This meta-analysis is the first article to assess the risk of common AEs induced by benralizumab, and includes eight clinical RCTs involving patients with moderate to severe eosinophilic asthma who received benralizumab by subcutaneous (SC) injection,12,14–18 or intravenous (IV) injection.–13 The purpose of this meta-analysis was to estimate whether there are differences between the benralizumab and placebo groups in relation to common AEs to further demonstrate the safety of benralizumab.Recently, several clinical trials have shown that benralizumab has a significant role in reducing the rate of exacerbation and improving lung function in patients with moderate to severe, uncontrolled, persistent asthma.2This study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.2.1Two researchers (WSL and XSM), expert in EndNoteX8, performed systematic and independent searches of the MEDLINE, EMBASE, and the Cochrane Library databases using MeSH and free text search terms from inception to September 2018, and evaluated the title and abstract for eligibility. A combination of MESH terms such as “asthma”, “MEDI-563” and “anti-interleukin-5” and terms such as “eosinophilic asthma” and “benralizumab” were used to search. There were no limitations on study design or language, but only human and clinical studies were included in the evaluation.2.2Studies that met the following criteria were included:1.the studies must be placebo-controlled RCTs;2.patients recruited into these studies were diagnosed with moderate to severe eosinophilic asthma;3.recruited patients were ≥12 years old;4.the intervention in these studies must include benralizumab regardless of the different drug doses; and5.study outcomes must include AEs.Studies that met the following criteria were excluded:1.review articles, case reports and conference abstracts; and2.articles where the full text was unavailable.According to the above criteria, both reviewers independently analyzed the literature data and resolved any differences by discussion.2.3Both reviewers read the full text, supplementary appendix and extracted the data independently and meticulously. The following descriptive data were obtained from all included studies: first author, publication year, study type, patient characteristics, methods, duration, interventions, and follow-up time. The reviewers checked the accuracy of data extraction and resolved any differences by discussion. E-mails were sent to the authors if key data were missing.2.4The reviewers completed an assessment of the risk of bias independently. This risk of bias assessment included: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting and other bias methods. Bias judgment was also carried out  according to the Cochrane Collaboration's tool for assessing the risk of bias.2.52) test and I2 statistics. A χ2 value < 0.1 and an I2 value of 50% or more was considered to represent significant heterogeneity. The heterogeneity between studies was assessed with the I2 statistic by calculating the percentage of total variation in the trials. When I2 was < 0.5 the results of a fixed effect analysis are presented; otherwise, the results of a random effect analysis are presented. A subgroup analysis of the dose of benralizumab or placebo was performed. A post hoc sensitivity analysis was performed by changing the effect model from a random effect model to a fixed effect model. The overall effects were calculated by combining the Z value with P < .05 to indicate statistical significance. When there were at least 10 trials for an outcome, a funnel plot was constructed to evaluate publication bias. All data were analyzed with the Review Manager (RevMan) software version 5.3. . The descriptive statistics were calculated using Microsoft Excel 2010 .We calculated risk ratios (RRs) and 95% confidence interval (CI) for all dichotomous outcomes. We assessed heterogeneity between studies using the Chi-square , Cochrane (n = 98), EMBASE (n = 754), and manual searches (n = 10). After identification of duplicates and screening of abstracts and titles, 43 full text studies were evaluated. Thirty-five articles were excluded as the full text was unavailable, or were conference abstracts, reviews and clinical trials lacking outcome data. Eight articles were finally selected, all of which were RCTs  at a stable dosage during the study. All AEs were assessed up to day 1 after the last day of the study. Table All the included trials were double-blind, placebo-controlled RCTs involving a total of 3788 patients with moderate to severe eosinophilic asthma. Patient age ranged from 12 to 75 years, and included 1573 males and 2215 females. Of these 3788 patients, 2277 received benralizumab treatment and the remaining 1511 received placebo, and a comparative analysis of different dose groups was included. All patients enrolled in the trials were from various experimental centers in a number of countries. All patients had a history of physician-diagnosed asthma requiring treatment with medium-to-high dose ICS or ICS/LABA for at least 1 year or 2 years prior to screening, and post-bronchodilator reversibility of airflow obstruction ≥12%. All female patients required adequate contraception from screening until trial completion. The patients in these trials experienced an exacerbation of asthma for at least 7 days or 12 months. The benralizumab group and placebo group were randomized to receive IV or SC injections as add-on to their standard background treatment. In addition to the patients in the study by Ferguson 2017 ,3.3,16,17; thus, these trials were classified as having an unclear risk of bias. Allocation concealment was clear in the remaining trials. Blinding of participants was considered adequate in all studies; thus, blinding of participants and personnel, and blinding of outcome assessment in all studies included in this analysis were classified as having a low risk of selection bias. Blinding of outcome assessor was not mentioned in two trials,16; thus, we classified these two trials as unclear risk. In all included trials, the loss to follow-up was less than 10%; therefore, all trials were classified as having a low risk of attrition bias. On account of all the outcomes specified in the methods reported, all trials were classified as having a low risk of reporting bias. The results of each risk-of-bias item for the included studies are summarized in Figure All the included trials were randomized trials with an interactive voice response system; thus, they were classified as having a low risk of selection bias. Only 3 trials did not describe the allocation concealment strategy3.3.1–17 reported overall AEs in this analysis: 7.61% (1448/2008) of patients in the benralizumab group developed bronchitis as compared to 10.04% (799/1063) of patients in the placebo group. Fewer patients treated with benralizumab vs placebo experienced overall AEs  of patients treated with benralizumab and in 12.38% (159/1284) of patients treated with placebo. In the pooled analysis, patients receiving benralizumab had a lower chance of suffering SAEs than patients in the placebo group , and the incidence in the placebo group was 17.53% (193/1101). A statistically significant difference in worsening asthma between the patients receiving benralizumab and patients receiving placebo was observed  of patients in the benralizumab group developed bronchitis as compared to 10.04% (99/986) of patients in the placebo group. The benralizumab group had a lower probability of bronchitis than the placebo group  of patients receiving benralizumab experienced sinusitis as compared to 7.91% (78/986) of patients receiving placebo in 5 trials.3.3.6,13–18 reported the incidence of headache: 8.32% (173/2080) of patients receiving benralizumab developed headache compared to 5.63% (62/1101) of patients receiving placebo. The benralizumab group was more likely to suffer headache than the placebo group  of patients receiving benralizumab developed pyrexia as compared to 1.76% (16/911) of patients receiving placebo. In these 4 trials, we found a higher odds of pyrexia in the benralizumab group as compared to the placebo group  in the subgroup analysis. We pooled the data on injection-site reactions and performed a subgroup analysis according to the dose of benralizumab received by the patients. The first subgroup included the patients in the study by Castro and Park based on mixed doses of benralizumab. Patients treated with 2 mg, 20 mg, or 100 mg benralizumab were included in the mixed dose group. The first subgroup displayed a higher probability of injection-site reactions in the mixed dose benralizumab group than in the placebo group (17.53% (81/462) and 3.38% (8/237), respectively; RR 4.46, 95% CI 2.22–8.93, P < .0001; Fig.  FitzGerald, Ferguson, and Nair were included in the second subgroup as all patients were treated with benralizumab at the dose of 30 mg. No significant difference in the probability of injection-site reactions was observed between the 30 mg benralizumab group and the placebo group (2.60% (47/1814) and 2.04% (21/1027), respectively; RR 1.25, 95% CI 0.69–2.27, P = .46; Fig. P = .15; Fig. Six trialsant I2 = 9% in theant I2 = 9% in theant I2 = 9% in the4 and more effective therapies without severe AEs are required. The latest GINA guidelines advise the addition of anti-IL-5 treatment or IL-5 receptor treatment  for severe eosinophilic asthma in the absence of a curative effect with ICS or ICS/LABA. Due to favorable efficacy and fewer AEs, benralizumab has been approved by the FDA as add-on maintenance therapy to treat severe asthma patients (≥12 years) with an eosinophilic phenotype. Although the efficacy and safety of benralizumab have been demonstrated, to date, there is some controversy regarding its AEs. There are few meta-analyses on the safety of benralizumab and these provided only a basic statistical analysis of the overall incidence of AEs and SAEs, and the types of AEs were not classified and analyzed. The present meta-analysis is the first to classify the most important and frequently observed AEs  in multiple trials and analyze the differences in these AEs between the benralizumab and placebo groups. It was found that some of these results were contrary to those observed in previous research.It is difficult to control persistent, moderate to severe eosinophilic asthma and reach a steady state with traditional ICS or ICS/LABA, Nowak, Bleecker, Nair, Ferguson, Tian, Liu, and Wang showed a comparable overall incidence of AEs in patients in the benralizumab and placebo groups during the study period. In addition, Castro and Park demonstrated that slightly more patients treated with benralizumab versus placebo experienced AEs. However, our results agree with those of FitzGerald that patients treated with benralizumab had a slightly lower risk of overall AEs than those treated with placebo . It is worth mentioning that SAEs either did not occur or only a few occurred in previous trials of benralizumab, but these SAEs were still important during treatment. Henriksen analyzed nine studies involving 3193 patients and found a reduced risk of SAEs in the mepolizumab (SC) and reslizumab group (IV) groups compared to the placebo group. Farne found that significantly fewer SAEs occurred in the mepolizumab group (SC and IV), but there was no significant difference in the number of SAEs between that group and the benralizumab (SC) group. However, our meta-analysis found that patients treated with benralizumab had a lower chance of suffering SAEs than those treated with placebo . The difference between the analysis by Farne and our analysis is that Farne only included patients treated with benralizumab (SC), and the subjects included in our study were treated with benralizumab (SC and IV). The patients receiving benralizumab IV had more asthma-related SAEs such as exacerbations requiring admission. Therefore, we believe that benralizumab can directly and rapidly deplete eosinophils and effectively reduce airway inflammation, thus alleviating the symptoms and signs of SAEs. Castro, Nowak, FitzGerald, and Bleecker showed that the most common SAE was worsening asthma–17 and some SAEs . In summary, benralizumab, mepolizumab and reslizumab are all registered anti-IL-5 treatments for severe eosinophilic asthma. From the current study, these agents are safe, and can significantly alleviate symptoms in patients with moderate to severe asthma, and reduce the incidence of SAEs due to increased asthma.Laviolette,P = .0001, I2 = 30%),,13–18 bronchitis  –17 and sinusitis  –17 compared with the placebo group. This was consistent with our expectation. Eosinophils, stimulated by IL-5, can cause inflammation and the release of histamine. Platelet-activating factor secreted by eosinophils increases mucosal secretion of leukotriene C4, oxygen free radicals, degranulation of eosinophils, and aggravates asthma exacerbations, inflammation and local edema of nasal mucosa, and causes a decline in lung function. Benralizumab can directly, rapidly, and almost completely remove eosinophils via enhanced antibody-dependent cell-mediated cytotoxicity. Therefore, benralizumab improves the typical symptoms and signs in the eosinophilic asthma phenotype, reduces the rate of deterioration, increases FEV1, and alleviates the inflammation and symptoms of bronchitis and sinusitis. The above results indicate that benralizumab has a better additive effect on bronchitis in asthmatic patients. It is worth noting that there is no biological therapy for sinusitis approved by the FDA at present,[31] and the data from this meta-analysis may provide vital information for the treatment of sinusitis. However, further investigation is required.We also observed that benralizumab reduced the frequency of the moderate to severe eosinophilic asthma phenotype ,,13–18 while the incidence of pharyngitis was comparable between the 2 groups.–18 . This is a new discovery and deserves further research. Patients treated with benralizumab were more likely to experience pyrexia than patients receiving placebo. We suspect that this may be related to the pharmacokinetics, and the specific mechanism is not detailed here.The center for drug evaluation and research of the FDA consider that headache and pharyngitis are the most common AEs in patients receiving benralizumab.,12,14–18 However, heterogeneity (I2 = 59%) was observed in the entire group analysis of injection-site reactions, but in the subgroup analysis, in patients receiving different doses  of benralizumab vs placebo and 30 mg benralizumab vs placebo, there was 0% and 11% heterogeneity (I2), respectively. The subgroup analysis showed that there was an increased risk of injection-site reactions in the different dose groups  compared with the placebo group when the 2 effects models were changed. However, the data on patients who received 2 mg, 20 mg, and 100 mg of benralizumab could not be separately extracted, and these results require further investigation to support these findings. There was no significant difference in injection-site reactions between the 30 mg benralizumab group and placebo group when the 2 effects models were changed. Therefore, we consider that the heterogeneity observed in the entire group analysis of injection-site reactions was closely related to the dose of benralizumab received.The incidence of injection-site reactions did not differ significantly between the benralizumab group and the placebo group.The incidence of other AEs, such as death, hypersensitivity, rhinitis, upper respiratory tract infection, influenza, nausea, cough, back pain, and arthralgia was comparable in patients treated with benralizumab and placebo .Meta-analysis plays an important role in assessing relevant disputes. As far as we know, this article is the first meta-analysis to classify and analyze the AEs of benralizumab. This meta-analysis compared the AEs of benralizumab, mepolizumab and reslizumab, and found that all three are safe and can reduce SAEs during the treatment of asthma patients. This study provides doctors with an essential reference on the efficacy and safety of benralizumab in clinical treatment. The advantage of this meta-analysis is that all the studies were placebo-controlled RCTs. Another strength was the low heterogeneity in the majority of outcomes among the studies.Our study had several limitations. Firstly, the data on specific doses  in patients treated with benralizumab were not extractable in the subgroup analysis of injection-site reactions. Secondly, this meta-analysis included only eight clinical trials that were all funded by the pharmaceutical industry. Furthermore, the possibility of publication bias is an ever-present limitation of the statistical method in the meta-analysis. Nevertheless, it was difficult to assess publication bias due to the small number of studies included in the meta-analysis.5This meta-analysis is the first study to classify and comprehensively assess the risk of AEs caused by benralizumab. Like mepolizumab and reslizumab, benralizumab reduces the risk of SAEs and significantly alleviates the symptoms of moderate to severe asthma. In addition, benralizumab can reduce asthma exacerbations, and the incidence of bronchitis, and sinusitis. Furthermore, we found that headache, and pyrexia were more likely to occur in patients treated with benralizumab than with placebo. There was no difference in the likelihood of certain AEs occurring in patients receiving benralizumab compared with those receiving placebo, including the most common AEs reported in a number of trials nasopharyngitis, death, hypersensitivity, injection-site reactions, rhinitis, pharyngitis, upper respiratory tract infection, influenza, nausea, cough, back pain, and arthralgia. In conclusion, benralizumab as an add-on treatment has commendable efficacy and safety in patients with moderate to severe eosinophilic asthma with little benefit on ICS or ICS/LABA treatment. However, vigilance to assess the occurrence of AEs in any patient receiving immunomodulatory therapy must be maintained. The long-term safety and effectiveness of benralizumab require further research.Wanshu Liu and Xuesu Ma contributed equally to the work as co-first authors. Wanshu Liu and Xuesu designed the study, and wrote the manuscript; Wanshu Liu, Xingru Wu and Zuzhen Ou analyzed the data. Weikang Zhou edited the manuscript. In addition, all authors approved the final draft.Formal analysis: Wanshu Liu.Methodology: Weikang Zhou.Writing – original draft: Wanshu Liu, Xuesu Ma.Writing – review & editing: Weikang Zhou.Wanshu Liu orcid: 0000-0003-0848-1147.