Retinitis pigmentosa(RP) is a hereditary retinal disease that causes photoreceptor, outer retinal, degeneration. Although the pathogenesis is still unclear, there have been numerous reports regarding inner retinal changes in RP eyes. The aim of this study is to retrospectively evaluate the changes in the thicknesses of different retinal layers of RP eyes, and its association with aqueous flare, which is used for measuring the intensity of intraocular inflammation. A total of 125 eyes of 64 patients with RP and 13 normal eyes were studied. The thicknesses of total neural retina,nerve fiber layer(NFL),ganglion cell layer(GCL),inner plexiform layer(IPL),inner nuclear layer(INL),outer layers and foveal thickness were measured in the optical coherence tomographic images. Aqueous flare was measured with a laser flare-cell meter. The associations between those parameters, visual acuity and visual field were determined in RP eyes using multivariate analysis. The results of this study showed the significant thickening of NFL, GCL and INL, the significant thinning of outer layers and the association of them with increased aqueous flare, whereas NFL and INL thickening associated with outer retinal thinning. These results can suggest the involvement of intraocular inflammation in the pathogenesis of inner retinal thickening as a secondary change following outer retinal degeneration. Retinitis pigmentosa (RP) is a hereditary retinal disease that causes photoreceptor degeneration. The worldwide prevalence of RP is about 1 in 4000, and the disease is a major cause of visual disturbance and blindnessThe treatments, such as artificial retinas, or transplantation of photoreceptor cells or retinal pigmented epithelium (RPE), have been proposed2345There are a number of studies about the inner retinal change of RP. Using Optical coherence tomography (OCT), which can gives high quality imaging of retinal structures, recently some articles have reported that there are inner retinal changes including nerve fiber layer (NFL) thickening in eyes of RP patients78On the other hand, although the pathophysiology of RP has not been established, there have been some reports about intraocular inflammation in RP eyes, which might be related to its pathophysiology101112We hypothesized the association between the inner retinal change and the intraocular inflammation. In order to investigate this relationship, we measured the aqueous flare value, as an intraocular inflammatory parameter, in RP eyes using laser flare-cell meter. We also determined its association with the thicknesses of different retinal layers measured using OCT, best-corrected visual acuity (BCVA) and visual field in this study.P < 0.001, RP eyes with CME vs RP eyes without CME: P < 0.006, RP eyes without CME vs eyes of controls: P < 0.001).The aqueous flare value was 7.9 ± 3.5 pc/ms in RP eyes without cystoid macular edema (CME), 12.0 ± 7.8 pc/ms in RP eyes with CME, and 3.5 ± 1.0 pc/ms in eyes of controls. The differences between any pair of the three groups were significant . The BCVA in RP eyes with and without CME were not significantly different (P = 0.99). The differences in age, IOP, and refractive error between RP eyes without CME, RP eyes with CME, and controls were not significant . The NFL thickness of RP eyes and controls were 41.3 ± 7.1 μm and 27.6 ± 2.1 μm respectively. The GCL thickness of RP eyes and controls were 59.3 ± 9.7 μm and 52.5 ± 2.5 μm respectively. NFL and GCL were significantly thicker in RP eyes than controls (P < 0.001 and P = 0.01 respectively). The IPL thickness of RP eyes and controls were 33.3 ± 4.9 μm and 33.0 ± 2.2 μm respectively, and these were not significantly different (P = 0.84). The INL thickness of RP eyes and controls were 50.3 ± 7.2 μm and 44.4 ± 2.3 μm respectively. INL was significantly thicker in RP eyes than controls (P = 0.004). The thickness of outer layers of RP eyes was 95.9 ± 36.5 μm which was significantly thinner than controls of 172.8 ± 7.9 μm (P < 0.001). The visual field scores of RP eyes without CME were 3.6 ± 3.2.The thicknesses of each retinal layer in RP eyes at different measured points are shown in P = 0.006) and negatively with the thickness of outer layers . GEE showed that the thickness of ganglion cell complex (GCC = NFL + GCL + IPL) was significantly associated with aqueous flare positively  and negatively was aqueous flare  showed that the NFL thickness was significantly and positively associated with aqueous flare  in our study. The strength of this study is that the findings of this study which has pathophysiological implication, is totally based on non-invasively evaluated clinical data.In conclusion, our study first showed the thickening of GCL and INL layer in RP eyes in addition to NFL change previously described. Our study also showed the thinning of outer layers and increased aqueous flare, consistent with previous studies. These results are similar to other studies. However, our study first demonstrated that increased aqueous flare associates with inner retinal thickening, outer retinal thinning and visual field loss in RP eyes, and that their inner retinal thickening associates with outer retinal thinning. These results can suggest the involvement of intraocular inflammation in the pathogenesis of inner retinal thickening as a secondary change following outer retinal degeneration. These findings can provide pathophysiological implication of inner retinal change of RP eyes to this field.The medical records of 64 patients with RP who had been examined at Nagoya University Hospital from April 2012 to June 2014 were studied. There were 26 men and 38 women whose mean ± standard deviation (SD) age was 47.5 ± 15.2 years with a range of 15 to 74 years. Both eyes of one patient with Usher syndrome were also included.Eyes with the vitreomacular traction syndrome (VMTS), macular hole, epiretinal membrane (ERM) which affect the retinal thickness, intraocular lens implanted, glaucoma, intraocular pressure higher than 21 mmHg, diabetic retinopathy, and cataracts that affected the visual acuity were excluded. Eyes after cataract surgery were removed because cataract surgery was reported to cause inflammation and affect the long-term postoperative flare valueThe RP eyes were divided into eyes with and without cystoid macular edema (CME) in the spectral domain (SD) OCT images. There were 104 RP eyes of 53 patients  without CME and 21 RP eyes of 11 patients  with CME .As controls, 13 normal fellow eyes of patients with unilateral idiopathic macular holes or unilateral retinal detachments, and 1 eye of a normal healthy subject were studied. Eyes of controls were age- and refractive error- matched to RP eyes without CME. In controls, there were 6 men and 7 women whose mean ± SD age was 50.4 ± 13.6 years with a range of 27 to 66 years .The protocol for this retrospective study was approved by the Institutional Review board of Nagoya University School of Medicine, and the procedures used conformed to the tenets of the Declaration of Helsinki. Informed consent was not required for this retrospective study.The examinations included SD-OCT , and measurements of BCVA, intraocular pressure (IOP) with a non-contact tonometer , aqueous flare with a laser flare-cell meter , and refractive error  with the KR-8900 auto Kerato-Refractometer . Visual fields were determined with the Goldmann perimeter. For the Spectralis OCT images, image quality less than 15 dB was excluded. Decimal visual acuity was converted to logarithm of the minimal angle of resolution (logMAR) units for statistical analyses.The Spectralis OCT was used to record the cross sectional scans across the retina centered on the fovea , ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), and outer layers were manually measured. The measurements were made at 1 and 2 mm superior, inferior, nasal, and temporal to the fovea in the vertical and horizontal images using the built-in caliper function in the Spectralis OCT . The laser flare-cell meter emits a laser beam (635 nm) into the anterior chamber and measures the backscattered light. The intensity of backscattered light is proportional to the concentration and size of proteins in the anterior chamber282930282930We evaluated the visual field by Goldmann kinetic perimetry using the previously proposed scoring systemThe reproducibility of the thickness measurements was determined by intra- and inter-grader interclass correlation coefficients (ICCs). The thickness of each layer was measured in 15 randomly selected OCT images by a single grader twice for the intra-grader ICCs. For the inter-grader ICCs, each layer was measured by 2 graders independently. For both measurements, the graders were masked to the clinical characteristics of the subjects, e.g., the age, sex, normal or with retinitis pigmentosa.t tests. Age, IOP, refractive error, BCVA, and aqueous flare values in the RP eyes without CME and with CME were compared to those of controls using one-way analysis of variance (ANOVA) with Tukey’s post-hoc test.The thicknesses of each retinal layer in the OCT images of RP eyes without CME were compared to those of the corresponding layers in controls using unpaired P value < 0.05 was considered statistically significant. All statistical analyses were performed using IBM SPSS 22.0 software .In RP eyes without CME, GEE33How to cite this article: Nagasaka, Y. et al. Increased aqueous flare is associated with thickening of inner retinal layers in eyes with retinitis pigmentosa. Sci. Rep.6, 33921; doi: 10.1038/srep33921 (2016).


Logistic analysis found that hyperuricemia  was associated with of cardiovascular diseases clustering among very elderly women. Prevalences of prehypertension, hyperuricemia, visceral obesity and dyslipidaemia are apparent in very elderly women, while diabetes mellitus, renal dysfunction are common in very elderly men. Women are more likely to have ≥3 cardiovascular diseases. Hyperuricemia is associated with cardiovascular diseases clustering among very elderly women.Cardiovascular epidemiological features among very elderly Chinese are still uncertain. This study aimed to describe the distribution of cardiovascular diseases and sex difference, and investigate potential risk factors for diseases clustering among very elderly Chinese. From May 2013 to May 2015, a total of 1056 very elderly were sampled in this cross-sectional study. Demographic characteristics collection, physical examination and biochemical analysis were performed. Totally, 1038 participants (men: 49.8%) with a median age of 83.0 years (age range: 80.0–100.0 years) were included. In this very elderly group, the prevalences of hypertension, diabetes mellitus, renal dysfunction, hyperuricemia, obesity, visceral obesity, and hypercholesterolaemia were 76.9%, 20.0%, 23.3%, 33.0%, 9.4%, 54.5% and 35.8%, respectively. About 17.5% of very elderly (men vs. women: 15.1% vs 19.8%,  When there were no improvement on current trends in smoking, hypertension, diabetes mellitus (DM) and obesity, premature cardiovascular deaths would increase about 32% in 2025, and 60% of these deaths would occur in South Asia, East Asia, and Southeast. Moreover, there would be no detectable change in premature cardiovascular diseases mortality by 2025 in China2.Cardiovascular diseases are predominated causes of death in the world3, but also accounts for about 38% total death in all ages4. Stroke, ischemic heart disease, hypertensive heart disease, and DM are all among the top ten causes of death for Chinese3.In China, cardiovascular disease is not only the first burden of disease7, although in 2010 the proportion of elderly Chinese is already 1.57%8. There is relatively few study on cardiovascular disease risk factors in very elderly Chinese. Hence, the aim of this study was to describe the distribution of cardiovascular diseases and sex difference, and investigate potential risk factors for diseases clustering among very elderly Chinese in Chengdu.As China is becoming a aging society rapidly, there is a growing urgency to tackle cardiovascular and metabolic diseases through effective prevention and treatment strategies, informed by precise estimates of disease prevalence and burden. Most of these current epidemic data are from population younger than 80 yearsp = 0.342). Among all participants, more than 10% were current smokers and 8% of them were current alcohol drinker. Not surprisingly, both proportions in men were much significantly higher than in women (both p < 0.001). Nearly 70% of these men were educated in a primary school or a middle/high school. While, more than 30% of women were illiterate compared with 11% in men (p < 0.001). Moreover, the proportion of women educated in middle/high school and college/university were significantly lower than which in men (both p < 0.001)  were included in final analysis. The median age was 83.0 years , in which men were slightly older than women (p = 0.038), PP (p < 0.001), and heart rate (p = 0.003), while a lower mean DBP level (p = 0.006), respectively.Mean levels of systolic blood pressure (SBP), diastolic blood pressure (DBP), and pulse pressure (PP) in all participants were 146.4 ± 20.6 mmHg, 74.1 ± 11.9 mmHg, and 72.5 ± 17.1 mmHg, separately. In women, there were relatively higher mean levels of SBP (p < 0.001), and heavier . However, there were no significant difference in body mass index (BMI) and waist circumference (WC) between very elderly men and women (both p > 0.05). While, the mean waist–to-height ratio was significant lower in very elderly men .Compared with women, very elderly men were higher . Mean levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in all participants were 4.87 ± 0.99 mmol/L, 1.34 ± 0.57 mmol/L, 2.58 ± 0.74 mmol/L, and 1.60 ± 0.44 mmol/L, respectively. Furthermore, all mean levels of these parameters were significantly higher in very elderly women than in men .The overall mean fast glucose (FG) of all participants was 5.53 ± 1.35 mmol/L  and Creatinine levels  (Table And mean levels of urine acid (UA) and Creatinine in all very elderly participants were 350.1 ± 84.5 μmol/L and 104.1 ± 32.4 μmol/L. Compared with very elderly women, men had a significantly higher UA . The estimated prevalence of DM was about 20.0% among overall very elderly participants. The prevalence of DM among very elderly men did not differ significantly from women . The estimated prevalence of renal dysfunction among the very elderly participants was 23.3%, which was doubled in men than in women . As well, we could not ignore that the prevalence of hyperuricemia among very elderly was as high as 33.0%, which was even higher in women than that in men . We also presented the estimated prevalence of renal dysfunction (eGFR < 60 ml/(min · 1.73 m2)) according to Cockcroft-Gault equation in Table Estimated prevalences of pre-hypertension and hypertension among very elderly participants were 16.6% and 76.9%, respectively. While, both prevalences were not significantly different between sexes, although both of them were a bit higher in very elderly women (both p < 0.001).Compared with obesity, overweight and visceral obesity were more common in very elderly with a prevalence of 30.0% and 54.5%, respectively. Especially in very elderly women, the prevalence of visceral obesity was significantly higher than in men .As among the very elderly, estimated prevalences of persistent atrial fibrillation and three degrees atrial ventricular block were also higher in men than in women, while not significantly . And both prevalences were significantly higher in women than in men (both p < 0.05) (Table 1.6%. Andp = 0.129). Among participants with both hypertension and DM, 54.1% of them had visceral Obesity and 27.3% of them had renal dysfunction. Prevalences of hypertrigleicemiea and hyperuricemia among participants with DM were higher than which among participants with hypertension, while the prevalence of hypercholesterolaemia was lower. Moreover, in participants with both hypertension and DM, prevalences of all other cardiovascular abnormalities were lower than in participants separately with hypertension or DM, except for renal function  were 35.5%, 38.0%, and 17.5%, respectively. Furthermore, the percentage of one participant having ≥3 diseases in very elderly women was significantly higher than in men .Multiple logistic regression analysis found that hyperuricemia  is associated with clustering of ≥3 comorbid diseases  among very elderly women Table .Table 4LReceiver-operating characteristic (ROC) analysis found that the optimal cutoff point for UA predicting cardiovascular diseases clustering was 352.5 μmol/L among very elderly women p < 0.001) To our knowledge, this survey is the first community population-based study providing general epidemiological information across wide spectrum of very elderly Chinese in Chengdu. This survey demonstrates as follows: (1) Cardiovascular risk factors are common among very elderly. (2) Sex differences of risk factors are apparent among very elderly, and women are more likely to cluster ≥3 diseases. (3) Hyperuricemia is associated with cardiovascular diseases clustering among very elderly women.9. The average age of participants in this study is more than 83 years. Although much of the evidence refers to young and middle-aged adults, older people currently account for most cardiovascular disease morbidity and mortality. Our study shows that burdens of cardiovascular diseases in very elderly are even worse than in other age group or in general population7. Hypertension, DM, renal dysfunction, obesity, and lipid abnormalities are currently not rare in very elderly residents. Especially prevalences of some of them are relatively high. It is already reported that among very elderly American the incidence of dementia is higher than that of coronary heart disease, although both diseases are complications of cardiovascular and metabolic diseases10. A recent study in Shanghai community elderly and very elderly residents, which included about 600 very elderly, reported similar cardiovascular and metabolic parameter levels  and prevalences of hypertension and DM compared with results in our study11.According to the newest report by World Health Organization (WHO), the life expectancy at birth for Chinese in 2015 is 76.1 years12. It is already known that total DM prevalence in China rises from 3.2% in population aged 20–39 years to more than 21% in population aged 70 or older13. More than one quarter Chinese adults have two cardiovascular risk factors , and nearly one fifth adults have three risk factors7. In this survey, it is showed that more than 17.5% very elderly residents have ≥3 cardiovascular risk factors. Therefore, there is no doubt that the burden of cardiovascular diseases increases gradually with aging.Based on results of this survey, hypertension is one of the most predominant health problems in very elderly Chinese in Chengdu. The proportion of very elderly with normal blood pressure (BP) is only about 6.5%. In another word, most population would suffer from hypertension when they are older than 80 years. The mean level of SBP is already as high as 146 mmHg, while levels of DBP and PP are almost the same, which is a apparent characteristic in very elderly. Also, the mean levels of glucose, UA, and creatinine are relatively high in very elderly. According to NHANES study, the prevalence of total DM increases sharply with age, from 2.4% in population aged 20–39 years to 21.6% in population aged 65 years and older14. Furthermore, prevalences of overweight , obesity  in very elderly are lower than which in middle aged and elderly population, while the prevalence of visceral obesity  is much higher, which is more obviously in women. Surprisingly, the prevalence of hypertension  was obviously higher in very elderly than in middle aged and elderly population. Both in very elderly men  and women , the prevalence of hypertension is two times than in middle aged and elderly population. Moreover, in very elderly, there is no obvious decrease in prevalence of DM . In contrast, the prevalence of hyperuricemia increases apparently . Besides, prevalences of hypercholesterolemia  and hyper-low density lipoproteinemia  are even higher in the elderly Chinese, while the prevalence of hypertrigleicemiea  is lower. Therefore, cardiovascular risk factors distribute differently among very elderly and middle aged and elderly Chinese.Compared with our previous survey in 2008, current study finds that total smoking rate in very elderly is only half of which in middle aged and elderly population . Moreover, that difference is mainly from men  while not from women 15. Although China remains a developing country with the fastest acceleration in urbanization, industrialization, the burden of disease increases dramatically fast. A meta-analysis included 15 trials found that in people aged 60 years or older, 5 year event rates per 1000 people were reduced by 18 for all deaths, by 19 for cardiovascular deaths, and by 51 for combined cardiovascular morbidity and mortality by diuretics or β blockers16. Another meta-analysis with 14 randomized trials found that benefits from statins based cholesterol-lowering treatment were apparent for patients aged 75 years and older, with risk reduction of major vascular and coronary events17. Since hypertension, DM, dyslipidemia, and obesity are major modifiable risk factors for cardiovascular diseases, benefits of risk factor modification in older people are clear for control of hypertension and hypercholesterolemia.Sex differences in cardiovascular diseases are common in very elderly. Our survey demonstrates that not only prevalences of hypertension, visceral Obesity and obesity, but also prevalences of hypercholesterolaemia, hypertrigleicemiea, and hyper-low density lipoproteinemia in very elderly women are higher than in men. It seems that these abnormalities are more likely to cluster in very elderly women while not in men. Nearly one quarter of the total global disease burden is attributable to diseases in people aged 60 years and older, and half of them is in high income countries and a fifth is in low-income and middle-income countries18 and the increase of incidence of hypertension19. Moreover, hyperuricemia could increase the risk of cardiovascular diseases and DM in patients with hypertension20 and the mortality in patients with coronary heart diseases21. In accordance with results of previous studies above, our study finds that hyperuricemia is associated with cardiovascular diseases clustering among very elderly women.It has been reported that UA could predict mortality in patients with heart failureIn conclusion, prevalences of cardiovascular diseases are high among very elderly Chinese in Chengdu. Cluster of cardiovascular diseases are more apparently among vey elderly women. Hyperuricemia is associated with cardiovascular diseases clustering among very elderly women.There are several limitations in this study. First, this is a cross-sectional study with relatively small sample, which could not describe any causality. Therefore, results of this survey should be applied with caution and longitudinal prospective cohort studies investigating the impact of hyperuricemia on cardiovascular diseases in very elderly are needed. Second, oral glucose tolerance test, hemoglobin A1c, echocardiogram, and pulse wave velocity could not been obtained in this survey because of economic reasons.This cross-sectional survey was conducted from May 2013 to May 2015. Participants were recruited by using a stratified three-stage cluster sampling design. The first stage of sampling involved the random selection of five districts in Chengdu. And the second stage involved the random selection of four neighborhoods from each of the selected districts. From each of the neighborhoods, one residential committees was randomly selected.A total number of 1056 participants from 20 residential committees were sampled according to registration data from Chengdu government. The study participants were defined as permanent residents of the households with a record in the household registration and living in local committees at least 3 years. Ethics approval was obtained from the Ethics Committee of the Second People’s Hospital of Chengdu, China. The methods in the study were in accordance with relevant guidelines and the Declaration of Helsinki. All participants gave informed consent.This study was designed as a cross-sectional survey in general very elderly Chengdu residents. All participants were assessed at a survey site in their committees by well-trained researchers. In oder to avoid uncertain influences of abominable weather, the survey was not conducted when the temperature outside was below 18 °C or over 25 °C. All participants completed a questionnaire-based interview, physical examination, and electrocardiogram and fast venous blood sample taken for subsequent biochemical analysis.The survey questionnaire included questions about basic demographic characteristics, disease history, and family history, and so on. The physical examination involved general estimation, auscultation of lung and heart, measurements of height, weight, WC, and BP . Electrocardiogram was taken for every participant after resting for more than 15 minutes . The biochemical analysis included FG, TC, TG, LDL-C, HDL-C, UA and creatine, which were measured in the central laboratory of the Second People’s Hospital of Chengdu.23. Impaired fasting glucose was diagnosed if 6.1 mmol/L ≤ FG < 7.0 mmol/L. DM was diagnosed if FG ≥ 7.0 mmol/L, or FG < 7.0 mmol/L with a past history of DM24.Pre-hypertension was defined as 120 ≤ SBP < 140 mmHg and/or 80 ≤ DBP < 90 mmHg. Hypertension was defined as SBP ≥ 140 mmHg and/or DBP ≥ 90 mmHg and/or self-reported treatment of hypertension with antihypertensive medication in last 2 weeks according to the Chinese Guidelines on Prevention and Control of Hypertension and the Seventh Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure guidelines26. Hypercholesterolaemia was defined as TC ≥ 5.2 mmol/L according to the guidelines of Chinese adult dyslipidemia prevention and control27. Hypertrigleicemiea was defined as TG ≥ 1.7 mmol/L. Hyper-low density lipoproteinemia defined as LDL-C ≥ 3.4 mmol/L. Hypo-high density lipoproteinemia was defined as HDL-C level of <0.9 mmol/L in men, or HDL-C level <1.0 mmol/L in women. Dyslipidaemia was defined as any or more than one of disorders as follows: hypercholesterolaemia, hypertrigleicemiea, hyper-low density lipoproteinemia and hypo-high density lipoproteinemia. According to WHO guidelines for the Asian Pacific population, overweight was defined as 24.0 ≤ BMI <28.0 kg/m2, and obesity as BMI at least 28.0 kg/m2 28. Visceral obesity was defined as WC at least 85 cm in women and at least 90 cm in men24.Renal dysfunction was defined as creatine ≥115 μmol/L according to the reference ranges recommend by central laboratory. Hyperuremia was defined as serum UA ≥ 416 μmol/L (7 mg/dL) in men and 357 μmol/L (6 mg/dL) in women2 test was applied to compare frequencies between men and women. Prevalences among total elderly participants were standardized for age and sex. Stepwise multiple logistic regression models were used to evaluate the association between potential factors and disease clustering. ROC curves were used to demonstrate the sensitivity and specificity of related parameters. Calculations were performed by SPSS software . A p value < 0.05 was considered statistically significant.Data were inputted by two researchers independently and checked by a third researcher. Continuous variables were expressed as mean ± standard deviation. Frequencies were presented as percentages. Statistical comparison of continuous variables between men and women was conducted by using Student’s t -test (Mann-Whitney U test for skewed data). And x


Impaired ability to make inferences about what another person might think or feel  is recognised as a core feature of schizophrenia and a key determinant of the poor social functioning that characterizes this illness. The development of treatments to target social cognitive impairments as a causal factor of impaired functioning in schizophrenia is of high priority. In this study, we investigated the acceptability, feasibility, and limited efficacy of 2 programs targeted at specific domains of social cognition in schizophrenia: “SoCog” Mental-State Reasoning Training (SoCog-MSRT) and “SoCog” Emotion Recognition Training (SoCog-ERT).n = 19) or SoCog-ERT (n = 12). Treatment comprised 12 twice-weekly sessions for 6 weeks. Participants underwent assessments of social cognition, neurocognition and symptoms at baseline, post-training and 3-months after completing training.Thirty-one participants with schizophrenia or schizoaffective disorder were allocated to either SoCog-MSRT (Attendance at training sessions was high with an average of 89.29 % attendance in the SoCog-MSRT groups and 85.42 % in the SoCog-ERT groups. Participants also reported the 2 programs as enjoyable and beneficial. Both SoCog-MSRT and SoCog-ERT groups showed increased scores on a false belief reasoning task and the Reading the Mind in the Eyes test. The SoCog-MSRT group also showed reduced personalising attributional biases in a small number of participants, while the SoCog-ERT group showed improved emotion recognition.The results are promising and support the feasibility and acceptability of the 2 SoCog programs as well as limited efficacy to improve social cognitive abilities in schizophrenia. There is also some evidence that skills for the recognition of basic facial expressions need specific training.ACTRN12613000978763. Retrospectively registered 3/09/2013.Australian New Zealand Clinical Trials Registry  Impaired social cognition is a core feature of schizophrenia . Social Treatments for social cognitive impairments in schizophrenia tend to be dichotomised into ‘targeted’ treatments focusing on specific impairments , 7 or ‘cKern et al.  concludehttp://www.paulekman.com/micro-expressions/) and found improved emotion recognition in people with schizophrenia that was durable 1 month after testing. The METT uses a series of videos showing facial expressions with verbal commentary to direct attention to relevant facial features of commonly confused emotional expressions . Following from this we piloted a novel 6-week “SoCog-MSRT” program that targets ToM and attribution style, with no specific reference to emotion recognition  dunb = -.24, see Fig. p = .025, CI , dunb = -.85, see Fig. p = .078, CI , dunb = .68, see Fig. p = .025, CI , dunb = .54).Figures p = .18, 95 % CI , dunb = .394, see Fig. p = .001, dunb =1.26, CI , see Fig. p = .349, CI , dunb = .246, see Fig. p = .01, CI , dunb = .63, see Fig. Figure p = .01, dunb = .49, 95 % CI , see Fig. p = .003, CI , dunb = -.38, see Fig. p = .015, CI , dunb = .71, see Fig. p = .137, CI , dunb = -.37, see Fig. Figures p = .14, 95 % CI , dunb = .28, see Fig. p = .94, CI , dunb = -.02, see Fig. p = .078, dunb = .53, see Fig. p = .60, CI , dunb = .28, see Fig. Figures p = .11, dunb = -.83, see results in Fig. p = .01, CI , dunb = -1.9, see Fig. Figures The aim of this study was to explore the feasibility, acceptance, and limited efficacy of 2 novel social-cognitive remediation (SoCog) programs to improve social cognition in schizophrenia. We predicted that participants would accept the program and find it enjoyable and beneficial and they would be motivated to attend training sessions. We further predicted that following training, the SoCog-ERT group would show enhanced recognition of basic facial expressions of emotion whereas the SoCog-MSRT group would show improved mental-state decoding and mental-state reasoning.Attendance rates were better than in similar studies . There was a trend toward improvements immediately after training. Although only 6 participants remained in SoCog-ERT at T3, 5 of these 6 individuals showed moderate increases in accuracy  3 months after training. This is consistent with the results of our previous brief emotion recognition training studies where we also found delayed improvements 1-month post-training and speculated that was due to participants using newly learned skills in their everyday life such that practice over time led to improvements that were not immediately apparent . CIs shoAs predicted, the SoCog-MSRT group did not show improvements in emotion recognition accuracy for basic emotions and indeed this group showed a decrease in accuracy at 3-month follow-up. As this type of training did not provide explicit training about how to recognise basic facial expressions this finding might indicate that it is important to provide specific ERT in order to improve these more basic perceptual aspects of social cognition. However, further investigation in a larger study would be needed to draw any definitive conclusions.Both the ERT and MSRT groups showed an increased ability to recognise complex emotions from static photos of the eyes (RMET) at 3-month follow-up. With the exception of 2 participants in MSRT, all participants showed delayed improvement at 3 months. CIs showed comparable changes for both groups between 1.87 and 5.2 for MSRT and from 1.12 to 5.2 for ERT with large and moderate effect sizes, respectively.Both training groups showed immediate post-training improvements on false-belief reasoning (PSTFB). These improvements were not durable for the SoCog-MSRT group at 3-months but were for SoCog-ERT. Nevertheless, inspection of the paired data and the width of the CIs for this ERT group contrast suggest the result might be imprecise as most participants showed decreased scores, and only 1 showed no change. This imprecision is most likely due to the small sample size but might also have been influenced by task difficulty. Participants were sometimes reluctant, or refused, to do this task a second and third time because they found it too challenging, therefore follow-up results might also be confounded by tolerability and/or missing data .unb = 2.26) however the CI was wide indicating the need for replication in a larger sample before strong conclusions can be drawn. Nevertheless, this outcome is encouraging, because the evidence to date that attributional biases can be successfully remediated is variable with few studies showing success in remediating these complex cognitive processes [The SoCog-MSRT group showed reduced biases in a small subset of participants who showed a personalising bias  at baserocesses , 47.An important issue that we cannot address directly in this study is whether the results have clinical significance. Whilst the limited efficacy found in this study is encouraging, we experienced some significant challenges in conducting the study that prevent us from drawing any firm conclusions about clinical significance. These challenges related to working with some of the treating staff who were reluctant to have their patients randomised to a control group, the rejection of the initial active control treatment by the participants randomised to that arm of the study and drop-out. All of these contributed to the small sample size that restricted analyses and also precludes us from ruling out practice effects on some of our dependent variables. Our assessing staff were not blind to the allocation of the subjects, which may also have led to an overestimation of treatment effects . The stuOur results also highlight the difficulties inherent in conducting randomised controlled trials within real-world clinical settings. We found that both consumers and clinicians were often not responsive to randomisation into a control group. This is because most services encourage active engagement in a range of rehabilitation programs. Thus, clinicians and consumers alike can see 3−12 months (or more) of participation in the control group of a longitudinal RCT as a potential impediment to the rehabilitation process.RCTs are considered the gold standard test for new treatments ; howeverDespite the above limitations, the results of this acceptability and feasibility study are encouraging and are consistent with studies testing similar programs. Importantly, participants reported being motivated to participate, enjoying the program and perceiving it as valuable thus supporting the acceptability and feasibility of SoCog. Limited efficacy was observed despite the significant chronicity of many of the participants. Perhaps most promising was that we were able to work closely over several years with hospital staff to implement SoCog within an existing inpatient rehabilitation program. As such, the difficulties that we encountered arguably resemble more closely clinical practice, thus, our results are likely similar to what a clinical team might expect using SoCog. Moreover, SoCog is now run as part of standard care  in several mental health services across Australia by clinicians who continue to provide feedback for further development and refinement of the program. The results also suggest that basic emotion recognition might not improve without specific training.


In summary, OX14 is a new, highly potent bisphosphonate with lower bone binding affinity than other clinically relevant bisphosphonates. This renders OX14 an interesting potential candidate for further development for its potential skeletal and nonskeletal benefits. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.Bisphosphonates are widely used in the treatment of clinical disorders characterized by increased bone resorption, including osteoporosis, Paget's disease, and the skeletal complications of malignancy. The antiresorptive potency of the nitrogen‐containing bisphosphonates on bone in vivo is now recognized to depend upon two key properties, namely mineral binding affinity and inhibitory activity on farnesyl pyrophosphate synthase (FPPS), and these properties vary independently of each other in individual bisphosphonates. The better understanding of structure activity relationships among the bisphosphonates has enabled us to design a series of novel bisphosphonates with a range of mineral binding properties and antiresorptive potencies. Among these is a highly potent bisphosphonate, 1‐fluoro‐2‐‐ethyl‐bisphosphonate, also known as OX14, which is a strong inhibitor of FPPS, but has lower binding affinity for bone mineral than most of the commonly studied bisphosphonates. The aim of this work was to characterize OX14 pharmacologically in relation to several of the bisphosphonates currently used clinically. When OX14 was compared to zoledronate (ZOL), risedronate (RIS), and minodronate (MIN), it was as potent at inhibiting FPPS in vitro but had significantly lower binding affinity to hydroxyapatite (HAP) columns than ALN, ZOL, RIS, and MIN. When injected i.v. into growing Sprague Dawley rats, OX14 was excreted into the urine to a greater extent than the other bisphosphonates, indicating reduced short‐term skeletal uptake and retention. In studies in both Sprague Dawley rats and C57BL/6J mice, OX14 inhibited bone resorption, with an antiresorptive potency equivalent to or greater than the comparator bisphosphonates. In the JJN3‐NSG murine model of myeloma‐induced bone disease, OX14 significantly prevented the formation of osteolytic lesions ( Bisphosphonates continue to be the major drugs used in the prevention and treatment of diseases or conditions characterized by increased bone resorption, including osteoporosis and Paget's disease. They are also used to prevent the skeletal complications of malignancy in breast, prostate, and other cancers, as well as in multiple myeloma, and may increase the survival of patients with some of these cancers.The first bisphosphonates to be developed for clinical use, etidronate, clodronate, pamidronate, and alendronate (ALN), were already known chemical compounds.The antiresorptive potency of the nitrogen‐containing bisphosphonates on bone in vivo is now recognized to depend upon two key properties, namely mineral binding affinity and inhibitory activity on farnesyl pyrophosphate synthase (FPPS); these properties vary independently of each other in individual bisphosphonates.This better understanding of structure activity relationships among the bisphosphonates has enabled us to design a series of novel bisphosphonates with a range of mineral binding properties and inhibitory potencies on FPPS. During the past decade we have synthesized dozens of new bisphosphonates, and screened them for these properties. Among these is a highly potent bisphosphonate with reduced affinity for bone mineral, namely 1‐fluoro‐2‐‐ethyl‐bisphosphonate, also known as OX14There are several reasons why bisphosphonates with reduced bone binding characteristics, but retaining strong inhibitory activity on FPPS, might have potential value. Such compounds, like RIS, have a more rapidly reversible effect on reducing bone turnover compared with longer‐acting bisphosphonates such as ALN and ZOL. This may have advantages under certain circumstances; eg, in treating women of childbearing years. Furthermore, the risk of some of the side effects that are often attributed to long‐term bisphosphonate treatment, such as osteonecrosis of the jawAll procedures involving mice were conducted at the University of Sheffield, UK, and were approved by the Home Office (PPL 40/3462) and the University of Sheffield's Animal Ethics Committee in accordance with the Animal [Scientific Procedures] Act 1986. All procedures involving rats were conducted at the Procter & Gamble Health Sciences Center, Mason, OH, USA. They were performed in accordance with the guidelines of Procter & Gamble Pharmaceuticals’ Institutional Animal Care and Use Committee, and meet the guidelines established by the Animal Welfare Act.The bisphosphonates were obtained from Procter and Gamble Pharmaceuticals , except for ALN, which was from Sigma , and OX14 ‐ethyl‐bisphosphonate), which was synthesized by AM . The cell line was genetically profiled by American Type Culture Collection  using short tandem repeat analysis to confirm its identity.The JJN3 myeloma cell line  was purchased from DSMZ  and grown in RPMI medium, containing 10% FCS, 1% penicillin/streptomycin (100 U/100 µg/mL), 1% non‐essential amino acids (1×), and 1% sodium pyruvate (1 mM) at 37°C in 5% COn = 8 mice/group using the JJN3‐NSG model to see a 25% difference in tumor burden  and a 40% difference in myeloma‐induced bone disease .Sprague Dawley rats, C57BL/6J and NOD/SCID‐γ  mice were purchased from Charles River Laboratories . All animals were housed in cages under standard conditions (12‐hour light/dark cycle). NSG mice were housed in specific‐pathogen‐free conditions. All animals were healthy and pathogen‐free at the start of each study and once each study had commenced they were monitored daily for any unexpected adverse effects. Animals were housed in groups and the numbers per group were determined using power calculations based on previous studies in which reproducible statistical differences had been shown.The binding affinities of OX14, ALN, ZOL, RIS, and minodronate (MIN) were compared by measuring their differential rates of elution from hydroxyapatite (HAP) columns using a fast protein liquid chromatography (FPLC) system similar to that previously described.50 values are based on preincubation protocols.The inhibitory activity of OX14 was compared to ALN, ZOL, RIS, and MIN in a FPPS inhibition assay as previously described.n = 6) based on body weight. All groups received treatment by subcutaneous (s.c.) injection at the same time daily for 7 days. The first treatment was on day 0, and the last treatment on day 6, with animals euthanized by cervical dislocation on day 7. BMD of the proximal metaphysis of the right tibia was analyzed using dual‐energy X‐ray analysis (DXA) using a Hologic QDR‐4500 densitometer . Efficacy of each bisphosphonate was expressed as percentage change from the vehicle control group. The dose that increased BMD 20% greater than the control was determined via a logistic dose‐response relationship , which estimates an efficacy value using animals in the study, and for comparison of bisphosphonates across studies. Data from compounds run in multiple studies was averaged.The antiresorptive potency of OX14 was measured by its effect on increasing BMD compared to ALN, ZOL, RIS, and MIN in a growing rat model adapted from the Schenk assay.2H4‐RIS was added as an internal standard. Prepared samples were injected onto a 2‐mm × 50‐mm C18 column and separated using a rapid water:acetonitrile gradient and constant modifier composition of 10 mM ammonium formate, 10 mM dimethylhexylamine, and 0.4% formic acid. Chromatographic effluent was passed into the ion source of a Sciex API5000 triple quadrupole mass spectrometer, operated in negative ion mode and employing selected‐reaction‐monitoring, for selective detection of each bisphosphonate. In this, the first quadrupole mass analyzer sequentially transmitted the [M‐H]– ion corresponding to each targeted bisphosphonate into a collision cell, from which a bisphosphonate‐characteristic fragment ion  was selected to pass through the second mass analyzer, for detection. This approach enabled specific detection of each bisphosphonate, even in the presence of closely related compounds and high concentrations of urine matrix components, allowing simultaneous quantification of these co‐administered compounds. With appropriate sample dilution, accurate measurements were achieved over a 0.1‐μg/mL to 10‐μg/mL range, employing analytical calibration curves created from a cocktail containing each bisphosphonate, and the 2H4‐RIS internal standard. Measurement accuracy was assured by also analyzing quality control samples, consisting of naive rat urine, fortified with known concentrations of bisphosphonate reference standards. Analytical recoveries were consistently in the 95% to 105% range. Bisphosphonate concentrations measured in urine were multiplied by the mass of urine collected for each time‐segment, and these results were added together across the 24‐hour period for each rat, yielding the absolute urinary excretion for each bisphosphonate. The percentage excretion data for each bisphosphonate whether given individually or in combination was averaged from all studies performed .It is known that bisphosphonates are rapidly taken up by the skeleton after parenteral administration, and the portion that is not bound to bone is cleared by the kidneys and appears in the urine within a few hours. This provides a convenient way of assessing skeletal uptake as the portion of an administered dose that is not excreted within 24 hours. Male Sprague Dawley rats  were randomized into groups based on weight, and fitted with i.v. femoral catheters. Prior to i.v. dosing, each rat was given a single oral dose of 2.5 mL saline to increase initial urine output. Doses of ALN, ZOL, MIN, RIS, and OX14 (18 to 72 µg P/kg) were given in various combinations and relative concentrations via an i.v. femoral catheter, which was then flushed with 2.5 mL non‐heparinized saline. Rats were then immediately placed in metabolism caging and urine was collected for 0 to 24 hours. At the end of urine collection, the metabolism caging was rinsed with 10 mL of sterile water to collect any urine that had not yet reached the collection container. Collected urine was analyzed for the simultaneously administered bisphosphonates, by employing ion‐pairing, reverse‐phase, gradient HPLC, with tandem mass spectrometry detection. Briefly, urine samples were prepared by dilution in water containing 100 mM dimethylhexylamine (ion pairing reagent) and n = 8/group) based on weight and treated at the same time on each day twice a week s.c. with PBS (100 µL), ZOL (28.4 µg P/kg), RIS (27.1 µg P/kg), or three different doses of OX14  for 3 weeks. All animals were then anesthetized  for cardiac bleeding and euthanized by cervical dislocation. Tibias were then assessed by µCT and histological analyzes as described below (see Assessment of bone integrity).Female C57BL6 mice  were randomized into six groups (n = 8/group). Group 1 was a non–tumor‐bearing control group (naive). Groups 2 to 4 were injected via the tail vein with 1 × 106 JJN3 cells and then treated twice a week s.c. from the time of tumor cell injection for the duration of the study with vehicle control , ZOL , or OX14 . At the first signs of morbidity (after 3 weeks) all animals were anesthetized  for cardiac bleeding and euthanized by cervical dislocation. Bone disease and tumor burden were assessed as described below .Female NSG mice  were randomized based on weight into four groups (2) were assessed by taking the µCT datasets, removing the trabecular bone and then volume‐rendering the datasets to create 3D models using Drishti , followed by analysis of three different sides of the bone using ImageJ software . After decalcification, wax embedding, and sectioning of the tibias, the numbers of osteoclasts (following tartrate‐resistant acid phosphatase [TRAP] staining) were assessed using OsteoMeasure Advanced Bone Histomorphometry Video System  and standard histomorphometric methods.At euthanasia, the right tibias were dissected free of soft tissue and fixed in 10% formalin before µCT analysis was used to measure the percentage of BV/TV. Tibias were scanned on a Skyscan 1272  at 4.3 μm with a 0.7‐degree rotation step, reconstructed using NRecon  and analyzed using CTAn . The number and area of cortical bone lesions . All data are expressed with error bars representing standard error of the mean (SE) unless otherwise stated.A). The retention time for OX14 (6.17 ± 0.08 min) was significantly lower (p < 0.0001) than for ALN, ZOL, RIS or MIN.To assess the mineral binding characteristics of OX14 compared to clinically relevant bisphosphonates, each compound was run separately on a HAP column  after i.v. dosing. After i.v. injection, the percentage of the dose of OX14 excreted over the ensuing 24 hours was greater than for all of the comparator bisphosphonates. The rank order for increasing excretion was ALN (22.5% ± 3.6%), ZOL (27.95% ± 3.18%), RIS (34.91% ± 9.3%), MIN (44.80% ± 1.6%), and OX14 (52.85% ± 3.82%) . The numbers of animals dosed with each compound ranged from three to 42. The results obtained from these and other studies have shown that the skeletal retention of individual bisphosphonates appear to be similar, regardless of whether the drugs are given individually, in pairs, or given all together, even at different relative doses, implying that there is no significant binding competition among the bisphosphonates under the conditions used. For example, when RIS was given over a 10‐fold dose range 0.1, 0.5, and 1 mg, the % excretion was 38.1 ± 5.6, 45.1 ± 1.0, and 43.0 ± 1.8, respectively. Similar values were obtained when RIS was co‐dosed with equal or greater amounts of ALN or ZOL (43.2 ± 2.6), or with MIN or OX14 (32.5 ± 0.1).The skeletal uptake of OX14 in vivo compared to other bisphosphonates was assessed by measuring its excretion into the urine of rats  to ZOL, RIS, and MIN, and to be significantly more potent than ALN (p < 0.0001) . The antiresorptive potency of OX14 was then tested in vivo and compared to ALN, ZOL, RIS, and MIN by assessing  the BMD increase in a growing rat model. OX14 was shown to have a similar potency to ZOL, RIS, and MIN, and to be significantly more potent than ALN (p < 0.0001) . These results are summarized in Table The potential cellular potency of OX14 was initially assessed in an FPPS inhibition assay in vitro, where it was shown to have similar potency  and was as effective as ZOL and RIS when given at the same dose. However, lower doses of OX14 (2.3 and 0.23 µg P/kg) were not significantly different from the vehicle control group. Despite this, osteoclast numbers on the trabecular bone surface were significantly reduced with all compounds except for the lowest dose OX14 group (0.23 µg P/kg) when compared to the vehicle control group . A similar trend was observed when the osteoclast perimeter on the trabecular bone perimeter was assessed ; however, neither of the lower doses of OX14 produced significant reductions compared to the vehicle control. The variability in assessment of osteoclasts, and the variability in animals, is greater than the variability in assessment of BMD. The trends are consistent with decreases in osteoclasts correlating with the increases in bone mass. In addition, we analyzed the number of osteoblasts on the trabecular bone surface . A significant reduction in osteoblasts was only observed in the ZOL‐treated group. Therefore, taking all the data into account, the significant differences in BV/TV between the vehicle and bisphosphonate groups is likely to be due to the reduction of bone turnover caused by the inhibition of osteoclastic bone resorption in the bisphosphonate‐treated groups with no inhibition caused in the vehicle‐treated group.In non–tumor‐bearing mice OX14 increased BV/TV as assessed  by µCT Fig. A, B andAi–iv, B) and flow cytometric analysis . Histological analysis showed a significant reduction in tumor burden in the ZOL‐treated group (p < 0.05) compared to the tumor control group (JJN3). Interestingly, flow cytometric analysis showed no significant reduction in tumor burden with either OX14 or ZOL compared to the tumor control group (JJN3). However, a significant reduction in osteoclast number was observed when tumor mice were treated with OX14 (p < 0.05) or ZOL (p < 0.05) . OX14 inhibited osteoclastic bone resorption to the same extent as ZOL, with osteoclast numbers similar to those seen in non–tumor‐bearing mice (vehicle).To assess the effects of OX14 in a disease setting we treated JJN3‐bearing NSG mice with OX14 and compared its effects to ZOL  and ZOL (p < 0.001) prevented the osteolytic disease which was observed in the tumor control group  and the BV/TV was significantly higher  in tumor‐bearing mice treated with OX14 (p < 0.001) or ZOL (p < 0.0001) compared to the tumor control group.μCT analysis revealed that both OX14  were observed, indicating that OX14 has a similar safety profile to other bisphosphonates.In this series of studies we have evaluated the properties of OX14, a new bisphosphonate compound designed to have lower bone binding dynamics and a high antiresorptive potency.50) on their main enzyme target, FPPS. The pharmacological differences among bisphosphonates may be of practical clinical importance,Over the years many hundreds of different bisphosphonates have been made and more than a dozen have been registered for clinical use for various indications in various countries. One of the gratifying advances in recent years has been the elucidation of the molecular mechanisms of action through which bisphosphonates act. This has led to the recognition that the pharmacological effects of bisphosphonates as inhibitors of bone resorption appear to depend upon two key properties: their affinity for bone mineral, and their inhibitory effects on osteoclasts. Thus, although different bisphosphonates share many pharmacological properties, every bisphosphonate has a specific and often unique profile, based on its mineral binding affinity and biochemical actions. In the case of nitrogen‐containing bisphosphonates such as those studied herein, their biochemical potency is highly dependent on their inhibitory activity (ICOne concept to emerge from these considerations is whether there might be value in designing compounds with somewhat reduced affinity for bone to enhance their potential direct anti‐tumor effects, while reducing their long‐term retention in bone, which has been thought to be possibly associated with adverse events. OX14 is an example of applying this concept.Here we have shown that the binding affinity of OX14 to HAP was lower than that of ALN, ZOL, RIS, and MIN. Furthermore, OX14 given to rats was excreted into the urine to a greater extent than ALN, ZOL, RIS, and MIN, indicating lower skeletal retention over the 24‐hour study period. After a single i.v. dose, there is rapid skeletal uptake within the first few hours, and this determines how much stays in the skeleton over the ensuing days and weeks.To assess the antiresorptive potency of OX14 we used a growing rat model where it was as effective as ZOL, RIS, and MIN at increasing BMD. Similarly, in non–tumor‐bearing mice, OX14 was as effective as ZOL and RIS at increasing BV/TV. In a murine model of myeloma‐induced bone disease, OX14 was shown to prevent the development of osteolytic lesions but not to reduce tumor burden. Interestingly ZOL was shown to significantly inhibit tumor burden by histological analysis of tibial sections. However, flow cytometric analysis of tumor burden in the ZOL‐treated JJN3 mice did not show a significant reduction compared to the tumor control. Because the JJN3‐NSG model is an aggressive short‐term model, it is perhaps not that surprising that no anti‐tumor effects were observed for OX14, and perhaps a less aggressive longer‐term model would be more suitable to use to assess any nonskeletal effects of the bisphosphonates in future studies.50 on FPPS. MIN has emerged as probably the most potent bisphosphonate to be approved for clinical use by this FPPS measure, but is only used in Japan.Interestingly, OX14 is closely related to MIN, with fluorine substituted for a hydroxyl group at the R1 position. This profoundly alters its properties in terms of reducing its mineral binding with a modest reduction in its ICThere is an increasing realization that bisphosphonates have an extensive range of biological actions in addition to their well‐established effects on bone resorption. Examples of observed clinical benefits outside the field of bone diseases include a reduction in mortality observed with ZOL in the Horizon trial.Bisphosphonates have an overall excellent safety profile. The development of new and even more potent compounds with lower bone‐binding dynamics, such as OX14, offer promising opportunities for use in these emerging and novel applications. A key question will be whether the lower bone‐binding properties will offer real pharmacological advantages.One recent example of the potential utility of nonskeletal effects of OX14 is its effectiveness in preventing experimentally induced inflammatory colitis.In summary, OX14 is a new, highly potent bisphosphonate with reduced bone binding properties compared with other clinically relevant bisphosphonates. This renders OX14 an attractive candidate for clinical development for its potential skeletal or nonskeletal benefits. A full evaluation of its potential will require further studies of its possible uses and safety profile.MWL and FHE are consultants for Biovinc. RGGR has acted as legal expert for Novartis, Merck, and Lilly.Supporting Information.Click here for additional data file.


The incidence of type 2 diabetes is increasing, creating a huge burden for China’s social healthcare system. This study aimed to evaluate hospital length of stay (LOS) based on admission characteristics and direct costs correlated with various types of complications for type 2 diabetic inpatients in Hubei Province, China.A total of 1528 inpatients diagnosed with type 2 diabetes discharged between April 1, 2013, and March 31, 2014, were included in this study. Information regarding patients’ admission and hospitalization were obtained from the hospital information system. The relationship between admission characteristics and LOS, distribution of total costs, and types of complications were described and analysed.p < 0.001). (3) Mean total costs per patient were US$225.40 ± 115.32 (median: US$200.34), US$221.25 ± 177.64 (median: US$170.05), and US$275.18 ± 193.14 (median: US$217.91) for inpatients with microvascular complications, macrovascular complications, and microvascular + macrovascular complications, respectively. Total costs were significantly higher for patients with microvascular + macrovascular complications than for those with other types of chronic complications (p < 0.001). (4) Drugs were the greatest expense for patients, and the least expensive treatment was nursing care.(1) The mean LOS was 11.65 days (median: 10 days). Multiple linear regression analysis demonstrated that inpatients with New Cooperative Medical Scheme (NCMS), aged 80 and above, had longer LOS than the reference group, and inpatients with chronic or acute + chronic complications had shorter LOS than those without. (2) Mean total costs per patient were US$159.72 ± 130.83 (median: US$135.33), US$240.60 ± 166.58 (median: US$192.09), and US$247.98 ± 166.22 (median: US$200.99) for inpatients with no complications, chronic complications, and acute + chronic complications, respectively. Total and individual costs were significantly less for patients without complications than for those with the two types of complications  contains supplementary material, which is available to authorized users. With global industrialization, urbanization, and modernization, the number of people afflicted with type 2 diabetes is growing year by year, with a projected increase from current estimates of 240 million, 6% of adults, affected to some 380 million, or 7% of adults, by 2025 . In deveIn recent years, huge changes have been taking place in Chinese society in terms of lifestyle and diet , 4. IncrIn China, the treatment of patients with diabetes has created significant problems for the social healthcare system. A systematic review of the direct economic burden of type 2 diabetes in China showed that the direct medical cost increased from US$250 million in 1993 to US$8.65 billion in 2008. The annual total inpatient cost increased from US$30 million in 1993 to US$3.13 billion in 2007, with an average annual growth rate of 19.90%, which was more than the average annual growth rate of the gross domestic product (GDP (12.77%) and national health expenditures (12.88%) from 1993 to 2003 [Inpatient costs were a substantial portion of health expenditures and were comprised of both direct and indirect costs; direct costs included cost of diagnosis and treatment of diabetes and its complications, and indirect costs represented productive losses resulting from prolonged length of stay (LOS), temporary or permanent disability, and premature death. This has been the subject of much research globally, but few studies have been carried out in Hubei Province, China. Therefore, assessing diabetic inpatients’ LOS and hospitalization expenses to reflect the patients’ burden was both necessary and urgent. This study aimed to estimate the LOS and direct costs of inpatients by analysing data on admission characteristics and a variety of complications and provide some suggestions for reducing the financial burden associated with type 2 diabetes.A number of previous studies of LOS and direct costs of type 2 diabetes and its complications have been made , 10. HowThe study was carried out in Zhongnan Hospital, a large tertiary general hospital located in the capital of Hubei Province, China. Data on inpatients between April 1, 2013, and March 31, 2014, whose principal discharge diagnosis was type 2 diabetes were obtained from the hospital information system (HIS), with diagnosis of type 2 diabetes determined in accordance with World Health Organization guidelines [http://apps.who.int/classifications/icd10/browse/2010/en#/E10–E14), or (2) chronic, including macrovascular complications  and microvascular complications .Numeric variables were reported as Mean ± standard deviation (SD), Median/25th–75th percentile, and categorical variables were described as both numbers and percentages.U test (with 2 subgroups) and Kruskal-Wallis test (with multiple subgroups) were used for a preliminary exploration of factors associated with LOS in univariate analysis and to evaluate the distributions of hospitalization costs between subgroups. Multiple linear regression was used to select variables which significantly predicted LOS. To avoid overfitting the model, variables found to be significant  in the univariate analysis were included.Because LOS and hospitalization costs did not conform to the normal distribution, the Mann–Whitney P < 0.05 was considered to be statistically significant.Data filtering was performed using Excel and analyses were carried out using IBM SPSS Statistics 20. n = 685) of patients, and 96.20% (n = 1470) patients were admitted as outpatients. Most of the patients were retired (47.19%). Data from Table A total of 1528 patients were included in the study. Of them, 944 were men (61.78%), with a mean age of 61.78 years (median: 60 years), and mean LOS of 11.65 days (median: 10 days). Urban Employee Basic Medical Insurance, Urban Resident Basic Medical Insurance, and the NCMS covered 44.82% (U test (with 2 subgroups) and Kruskal-Wallis test (with multiple subgroups) showed that no significant difference was found between LOS and gender (p =0.056), mode of admission (p =0.057), or year of admission (p =0.998). Age group, type of medical insurance, occupation, and type of complications (p < 0.001) were significantly associated with LOS. Inpatients aged 90 and over, receiving public financial assistance, retired, and without complications had a longer LOS than other members within the group, their mean LOS were 19.94 ± 5.96 days, 14.19 ± 6.11 days, 12.54 ± 6.29 days, 15.43 ± 7.17 days, respectively . The distribution of costs for therapeutics and sanitary materials were significantly different between the three different types of complications (p < 0.001). We can conclude inpatients without complications spent less than those with other types of complications on total costs and on the various individual expenses. Drugs and laboratory analysis were found to be the greatest expenses, and the least was nursing care . The distribution of costs for therapeutics and nursing care were significantly different not only between inpatients with microvascular complications and with microvascular + macrovascular complications but also between inpatients with macrovascular complications and with microvascular + macrovascular complications (p < 0.001). The distribution of costs for drugs, imaging, laboratory analysis, sanitary materials, and diagnostics showed significant differences between inpatients with macrovascular complications and with microvascular complications as well as between inpatients with macrovascular complications and with microvascular + macrovascular complications (p < 0.001). From the perspective of the total costs, inpatients with microvascular + macrovascular complications spent the most. Drugs and laboratory analysis were the highest costs and nursing care was the lowest  were slightly shorter than those reported in Shantou, China   but sligThe findings of the multiple linear regression analysis were unlike much previous research , 19 in tIn addition, the multiple linear regression analysis showed another result different from many studies which indicated that people without complications had a longer LOS than people with chronic complications and acute + chronic complications –23. The http://data.worldbank.org.cn/indicator/SH.XPD.PCAP/countries). This means diabetes is a huge burden both for residents and for the health care system. Other studies have come to the same conclusion [The result of the study showed that the average per patient cost of diabetes was US$232.47 and the median cost was US$185.00. This is different from that found in some other parts of China and abroad , 25. Fornclusion .This study found that costs for inpatients with complications were higher than for patients without complications, and numerous studies have drawn similar conclusions , 30. ThiRegardless of the kind of complication, drugs were the highest type of cost and nursing care the least. Therefore, in order to reduce the burden of the disease in patients with type 2 diabetes, measures should be taken to reduce drug costs in conjunction with the implementation of drug price reform policies in Hubei Province. Moreover, because diabetes is a metabolic disease, in addition to access to affordable medication, health education and lifestyle interventions are also very important, as many studies have shown , 32. In Because this study was conducted with data from a single hospital, findings might have been different in other parts of the country or in other types of hospitals. In addition, the data was limited to the period between April 1, 2013, and March 31, 2014, a relatively short time span; thus, a continuous trend comparison could not be made. Also, for patients undergoing two or more hospitalizations, each hospitalization was studied as a separate case, which may have led to an underestimate of hospital costs. Moreover, some cardiovascular diseases are not limited to patients with diabetes but may occur before diabetes, so some of the costs might not have been caused by diabetes, leading to an overestimation of hospital costs. Finally, the data for demographic and clinic characteristics were limited , if more data for demographic and clinical characteristics had been available, this research would be more substantial.The LOS for patients with diabetes in China were found to be longer than those in foreign countries. It should be appropriate to take measures, such as integrating the medical insurance system of urban and rural areas as well as monitoring and educating patients at home. Overall, the costs for patients with complications were higher than for patients without complications, so preventing and controlling the complications of diabetes is imperative, especially microvascular complications.


Within this study, silver (Ag) and four carbon-supported Ag catalysts were synthesized by the γ-radiation method and their morphology and structure examined using transmission electron microscopy, X–ray diffraction, and UV-Vis analysis. The nanocatalysts were tested for Br− sensing in aqueous media using cyclic voltammetry. All five Ag materials exhibited electroactivity for sensing of Br− ions, with pure Ag catalyst giving the best response to Br− ions presence in terms of the lowest limit of detection. Sensing of bromides was also explored in tap water after addition of bromides suggesting that herein prepared catalysts could be used for bromides detection in real samples. Furthermore, sensing of other halogen ions, namely, chlorides and iodides, was examined, and response due to chloride presence was recorded.Monitoring bromides (Br Concentrations of these bromoorganic compounds are typically far below the drinking water standards. However, the main by-product of concern is bromate (BrO3−), generated by the oxidation of bromide by both O3 and OH∙, including several pathways and up to six oxidation states of bromine, According to the UN-Water Global Analysis and Assessment of Sanitation and Drinking Water (GLAAS) 2017 report, nearly two billion people use drinking water from sources polluted with faeces with over 500,000 diarrhoeal deaths per year. Ozone is a powerful disinfectant which can disarm even those resilient pathogenic microorganisms that common disinfectants such as chlorine and chlorine dioxide cannot. Still, high ozone exposure is necessary to neutralize these microorganisms, resulting in generation of undesirable disinfection by-products [Bromate, unlike many other organic by-products, is not degraded in biological filters applied upon the ozonation process. Bromate is known to be the genotoxic carcinogen inducing, for instance, renal cell tumor in rats. It is believed that bromate toxic effects arise from the elevated levels of lipid peroxides (LPO) or from oxygen radicals formed from LPO and to induce DNA damage.Thereafter, monitoring the level of halogen ions in water is nowadays of great importance for the protection of human health. Methods so far used or suggested for the detection of bromide and other halogens in water include neutron activation analysis, inductively coupled plasma–mass spectrometry, ion and liquid chromatography, and fluorescence spectroscopy –7. These+ ions in aqueous solution using different reducing agents that can be toxic or non-environmentally friendly and the procedure comprises several time-consuming steps, including addition of reducing agent in the solution, filtering, washing, and drying.On the other hand, electrochemical methods for detection of halogen ions offer benefits of low cost, simplicity, and rapidity –11. Silv+ ions were reduced by radiolytic products of water, where polymers, such as the poly and the chitosan, act as stabilizers for controlling the size of Ag NPs [γ–radiation method and investigated them as electrocatalysts for the oxygen reduction and borohydride oxidation reactions [Radiation methods of synthesis have several advantages over the other methods: rapidity, low operational costs, no reducing agent required, easier control of the synthesis parameters, absence of unnecessary synthesis products, convenience for large scale use, and environmental friendliness. A major advantage of the radiation synthesis of metal NPs is a homogenous reduction of metal ions throughout the solution. Radiation methods have already been used for preparation of Ag NPs for biomedical applications. In this method, Agf Ag NPs . We haveeactions . With reγ-radiation method, as electrode materials for detection of the halogen ions . As mentioned above, the radiation method, used in this work, enables direct preparation of the sensing electrode in a simple and a much faster way than a conventional approach, avoiding filtering, washing, and drying steps. The polymer used for controlling the Ag NPs size during the radiation procedure (synthesis of the Ag NPs) can later be used as the active material binder. Thus, in our approach, the Ag NP ink was prepared from the precursor in one-step radiation process. Prepared Ag NPs electrocatalysts were tested for detection of bromides, chlorides, and iodides using cyclic voltammetry.The goal of this research is to investigate the potential application of Ag nanocatalysts, synthesized by the Mw of 72 kDa (degree of hydrolysis of 99%), silver nitrate (AgNO3), glutaraldehyde , and 2-propanol ((CH3)2CHOH) were purchased from Merck, while argon gas was obtained from Messer Tehnogas, Serbia. The medium molecular weight chitosan  and potassium sulfate (K2SO4), bromide (KBr), chloride (KCl), and iodide (KI) were supplied by Sigma-Aldrich. Carbon black, Vulcan XC 72R, was produced by Cabot Co. The chemicals were used as received, without additional purification. Water from Millipore Milli-Q system was used in all experiments.Poly (PVA) with mean molecular weight γ–radiation reduction method using PVA and PVA/chitosan as a stabilizer 2CHOH were prepared. All solutions had constant concentration of AgNO3 (0.4 M) and (CH3)2CHOH (0.2 M), while concentration of polymer varied . Different amounts of Vulcan XC 72R were added to the four out of five solutions (γ–rays (60Co source) at the temperature of 22°C. The absorbed dose and the dose rate were 700 kGy and 12 kGy h−1, respectively.Both types of Ag NPs, nonsupported and supported on the carbon substrate (Ag/C), were synthesized by the abilizer , as descolutions  and the α1 radiation, λ = 0.1541 nm). Optical characterization of the as-prepared Ag NPs was carried out using Thermo Scientific Evolution 600 UV-Vis spectrophotometer in the 300–800 nm wavelength range.Transmission electron microscopy (TEM) analysis was done using the JEOL JEM–1400Plus device, while the X–ray diffraction (XRD) analysis was done using Bruker D8 Advance Diffractometer  resulting in Ag loading on the electrode of 20 μg cm−2. Crosslinking of the polymer with GA was achieved by HCl during the drying under the nitrogen atmosphere, and the gel that was formed acted as a binder. In order to wash out the Cl− ions from the electrodes, they were immersed for 10 minutes in deionized water.The working electrode was prepared as follows: 0.5 wt.% GA solution was added to each of the obtained catalyst dispersions (molar ratio of GA to polymer was 8.2 : 1), after which the dispersions were diluted and the appropriate volume of 1 M HCl was added, in order to obtain HCl concentration of 0.1 M in the final dispersion. 10 2SO4 solution as supporting electrolyte. Br− ions detection was done by CV measurements in bromide solution in 0.1 M K2SO4 in the potential range from −0.9 V to 0.5 V at scan rate of 100 mVs−1. Cl− and I− ions detection was assessed under the same experimental conditions.Electrochemical characterization of the five Ag catalysts was done by cyclic voltammetry (CV) using a Gamry PCI4/750 potentiostat/galvanostat. For all electrochemical measurements within this study, catalyst-coated GCE served as working electrode; Pt foil was employed as the counter electrode and a saturated calomel electrode  as reference. All potentials are reported relative to the SCE. CVs were recorded in 0.1 M K+ starts with the radiolysis of water and formation of hydrated electrons, hydrogen (H∙) and hydroxyl radicals (OH∙). Hydrated electrons and H∙ radicals reduce Ag+ to Ag0, which further dimerize when they encounter or associate with Ag+ and progressively grow yielding the formation of metal clusters and particles. The detailed mechanism of Ag NP formation by radiation methods has been explained by Belloni [∙ radicals can oxidize the metal atoms into a higher oxidation state and thus counterbalance the reduction, (CH3)2CHOH is used as a scavenger to convert them to 2–propanol radicals (∙(CH3)2CHOH), which further act as strong reducing species. It should be mentioned that the Ag NP dispersions are stable at room temperature for several days. However, at lower temperatures (around 7°C), the pure Ag dispersion (101) can withstand more than a year without precipitation of Ag.The radiation-induced reduction of Ag Belloni . Since tγ–radiation method was reported in our previous publication [214/C, Ag114/C, and Ag116/C, and mixture of metallic Ag and Ag oxide NPs in case of Ag112/C [Ctxt), interplanar spacing between atoms (d), and lattice parameter (a) of Ag, Ag214/C, Ag114/C, and Ag116/C were determined from the XRD pattern data. Thus, crystallite size of the samples determined by XRD was found to be from 14.6 nm to 20.0 nm, which is in good agreement with the crystallite size evaluated by TEM. Obtained values of Ctxt for (111), (200), (220), and (311) planes were evaluated to be in the range from 0.55 to 1.05, with these values indicating planes with no preferential growth orientation. Δd/d0(111) ranging from −0.0001 to −0.0018 and Δa/a0sr values ranging from −0.0008 to −0.0022 confirmed formation of bulk FCC Ag structure. The lattice parameters were found to be lower than the bulk lattice constant indicating presence of strain and stress that could further lead to the change in the bond length and to the change of energy levels of the bonding electrons [The detailed XRD and TEM analysis of the Ag NPs obtained by the lication , in whiclication . XRD ana Ag112/C . It was lectrons , 17.101) colloid is shown in rexp is the particle radius, Vf is the Fermi velocity of the metal, and Δω1/2 is the FWHM for the SPR band in units of angular frequency [UV-Vis spectrum of Ag/polymer  and D is the diameter of Ag NPs.The calculated values of average Ag as well , while t2SO4 at scan rate of 100 mV s−1. 2O is observed as peak at ca. 0.15 V. On the cathodic scan, pronounced oxide reduction peak could be seen at ca. −0.05 V in agreement with the literature data [214). Charge under the reduction peaks was used to evaluate the electrochemical surface area (ECSA), assuming that charge associated with one Ag oxide monolayer is 2.10 C m−2 [Electrochemical characterization was done in 0.1 M Kure data . Qualita2SO4 in the potential range from −1 V to 0.5 V at scan rate of 100 mVs−1 (j) value (1.19 mA cm−2), followed by Ag214/C (0.59 mA cm−2), Ag114/C (0.36 mA cm−2), Ag116/C (0.23 mA cm−2), and Ag112/C (0.07 mA cm−2). It is worth noting that these peak current/current density values are one order of magnitude higher than the current values obtained by different Ag NPs reported in the literature [− sensing can be attributed to the differences in their particle size, surface structure, and C and polymer percentage. The highest peak current density observed for Ag and then Ag214/C can be correlated with the smaller particle size of these two electrocatalysts and consequently to their higher specific surface area. Furthermore, it can be observed that the lower peak current densities are observed for electrocatalysts containing higher percentage of C , suggesting that C hinders access of Br− ions to Ag surface. Surface structure also plays significant role in the catalytic activity of Ag-based materials as it was shown that different Ag surface planes catalyze electrochemical reactions at different rate [Subsequently, possibility of using prepared Ag and Ag/C catalysts for electroanalytical sensing of bromides was examined. Therefore, CVs of all five Ag materials were recorded in 0.25 mM KBr + 0.1 M K00 mVs−1 . All fivterature . Althougent rate .2SO4 solution increasing the KBr concentration with 50 μM additions of KBr to 0.1 M K2SO4, except in the case of Ag214/C catalyst where 100 µM additions were made (ip) could be observed with increase of KBr concentration (c), with ip versus c dependence resulting in straight lines with correlation coefficients in the 0.96008–0.99648 range, Subsequently, CVs of all five Ag catalysts were recorded in 0.1 M Kσ is the standard deviation of the y coordinates from the line of best fit and b the slope of the same line. The lowest limit of bromides detection of 18 µM was evaluated with pure Ag catalyst. Ag114/C and Ag116/C gave similar LODs of 36 and 40 μM, respectively, followed by Ag112/C that gave LOD of 53 μM, while the highest LOD of 102 μM was obtained using Ag214/C, μM range) [μM at AgNPs-GC-epoxy composite electrode and of 22 μM at GCM-Ag/MWCNT/GCE  [μg L−1 using ion chromatography [The limit of detection (LOD) of bromide at Ag and Ag/C catalysts was evaluated using the 3-sigma methodM range) –29. For ectrode) . FurtherμM. Dependence of the oxidation peak current on KCl concentration resulted in a straight line with correlation factor of 0.98678, and data from that line were used for evaluation of the limit of chloride detection using Ag catalyst. Thus, LOD of chloride was determined to be 51.3 μM with standard deviation (10 measurements) of 6.9%. Sánchez-Polo et al. reported LOD for bromide and chloride ions of 3 and 1 μg L−1, respectively, by ion chromatography [As pure Ag catalyst gave the lowest LOD for bromides, it was further tested for sensing of other halogen ions, namely, chlorides and iodides. For that purpose, CV of pure Ag catalyst was recorded in 0.25 mM KCl solution under the same experimental conditions as in the case of bromides detection, tography .μM range did not result in the noteworthy increase of the oxidation peak current. Thus, due to the poor signal, limit of iodide detection using Ag catalyst could not be determined.Contrary to CVs of Ag catalyst recorded in 0.25 mM KBr and 0.25 mM KCl solutions, where well defined oxidation peaks were observed, only a vague peak at ca. −0.25 V could be seen at the CV recorded in 0.25 mM KI solution. Increase of KI concentration in the 50 to 500 Finally, possibility of using Ag catalyst for sensing of bromides in real sample was explored by recording CV of Ag catalyst in tap water after addition of 0.25 mM KBr + 0.25 mM KCl + 0.25 mM KI without any pretreatment of the sample. No peak was observed at CV recorded in tap water prior to halides addition. Conversely, two peaks could be observed, a peak at ca. 0.18 V corresponding to bromide presence and a peak at ca. −0.6 V corresponding to chloride presence, − and Cl− ions. Among studied catalysts, pure Ag NPs showed the best performance for halogen ions sensing.The results indicate that Ag and Ag/C catalysts prepared within this study could be used as sensing electrode materials for detection of different halogen ions, namely, Brγ-radiation method and evaluated as sensing electrodes for bromide detection. Limit of bromides detection was evaluated using the 3-sigma method. Pure Ag electrocatalyst gave the best results as evidenced by the lowest LOD of 18 μM. Furthermore, this catalyst was shown to be active for chloride detection as well. Finally, bromide sensing was assessed in real sample and the catalyst gave a clear response to bromides presence suggesting catalysts' potential application for sensing of bromides in real samples.Ag NPs and carbon-supported Ag NPs of ca. 14–18 nm diameter were prepared using


Noninvasive EIS data analysis showed differences in both impedance magnitude ratio  and phase angle slope  in EBIG compared with control animals. Moreover, in EBIG‐treated animals, the infarct size was 48% smaller , less inflammation was found by means of CD25+ lymphocytes , and a lower collagen I/III ratio was detected . An EBIG composed of acellular pericardium refilled with pATPCs significantly reduced infarct size and improved cardiac function in a preclinical model of MI. Noninvasive EIS monitoring was useful for tracking differential scar healing in EBIG‐treated animals, which was confirmed by less inflammation and altered collagen deposit. Stem Cells Translational Medicine2017;6:647–655Cardiac tissue engineering, which combines cells and biomaterials, is promising for limiting the sequelae of myocardial infarction (MI). We assessed myocardial function and scar evolution after implanting an engineered bioactive impedance graft (EBIG) in a swine MI model. The EBIG comprises a scaffold of decellularized human pericardium, green fluorescent protein‐labeled porcine adipose tissue‐derived progenitor cells (pATPCs), and a customized‐design electrical impedance spectroscopy (EIS) monitoring system. Cardiac function was evaluated noninvasively by using magnetic resonance imaging (MRI). Scar healing was evaluated by using the EIS system within the implanted graft. Additionally, infarct size, fibrosis, and inflammation were explored by histopathology. Upon sacrifice 1 month after the intervention, MRI detected a significant improvement in left ventricular ejection fraction (7.5% ± 4.9% vs. 1.4% ± 3.7%;  This article describes the development and testing of a new custom‐designed engineered bioimpedance graft (EBIG) to noninvasively monitor myocardial scar healing. The EBIG was made of a scaffold of decellularized human pericardium, green fluorescent protein‐labeled porcine adipose tissue‐derived progenitor cells, and an electrical impedance spectroscopy (EIS) monitoring system. In a noninvasive manner, cardiac function was evaluated by magnetic resonance imaging (MRI) and scar healing by a customized‐design EIS system incorporated within the implanted graft. A significant improvement in MRI left ventricular ejection fraction was detected in EBIG‐treated animals, and histopathology confirmed less inflammation and altered collagen deposit.Cardiac tissue engineering, which combines cells and biomaterials, is a promising therapy after myocardial infarction (MI) Prat‐Vidal et al. previously reported preliminary data on a novel engineered bioactive impedance graft (EBIG) comprising a scaffold of decellularized human pericardium, green fluorescent protein (GFP)‐labeled porcine adipose tissue‐derived progenitor cells (pATPCs), and an electrical impedance spectroscopy (EIS) monitoring system http://www.ethicon.com) http://www.cardiolink.es). Finally, the animals were allowed to recover and housed for 1 month before sacrifice. In order to ensure that all animals had a similar MI, we analyzed circulating troponin I (cTnI) and creatine kinase in serum samples collected by jugular venipuncture at baseline and 2 hours after MI induction. Cardiac biomarkers were measured in a fluorometric immunoassay analyzer .After a left lateral thoracotomy, an MI was induced via double‐ligation of the first marginal branch of the circumflex artery, 1.5 cm distally from the atrioventricular groove , MI induction treated with apposition of a cell‐free pericardial scaffold connected to the EIS system; (b) EBIG‐treated arm (n = 12), MI induction treated with the EBIG; and (c) sham arm (n = 4), no MI, but the EBIG was implanted on top of healthy myocardium.Twenty‐six cross‐bred Landrace × large white swine (30.2 ± 3.6 kg) were randomly distributed into three groups : (a) conAll animal studies were approved by the local Animal Experimentation Unit Ethical Committee (no. ES 100370001499) and complied with all guidelines concerning the use of animals in research and teaching as defined by the Guide for the Care and Use of Laboratory Animals . Human pericardial samples were obtained after written informed consent from patients undergoing cardiac surgery. The local ethics committee approved this study, and the protocol conformed to the principles outlined in the Declaration of Helsinki.http://www.bdbiosciences.com), and in treated animals, the EBIG included 2 × 106 GFP‐pATPCs embedded in 175 μl of 10% sucrose. Finally, to ensure jellification, 350 μl of α‐minimal essential medium was applied  in all animals by using a four‐channel phased array surface coil . Breath‐held, ECG‐gated cine steady‐state precession MRI was acquired . Delayed enhancement images were acquired after intravenous gadolinium (0.2 ml/kg) using a phase‐sensitive inversion recovery sequence . Left ventricular ejection fraction (LVEF), cardiac output (CO), stroke volume (SV), left ventricular end‐systolic volume (LVESV), left ventricular end‐diastolic volume (LVEDV), and left ventricular end‐diastolic wall mass (LVEDWM) were measured at baseline, 48 hours after MI, and before sacrifice. Independent blinded investigators carried out MRI data acquisition and analysis.Cardiac MRI was performed at 1.5 T . The impedance measurements were processed with a median filter  to exclude sudden artifacts. Subsequently, a moving average filter  was used to smooth the time series by using the zero‐shift double pass filter filtfilt . In order to correct for long artifacts, the magnitude at the highest frequency (200 kHz) was subtracted from the magnitude at the other frequencies. Two estimators were chosen to display the integrity of the tissue in the monitored area: the slope of the time course of the impedance magnitude ratio between low frequency  and high frequency , and the slope of the time course of the phase angle difference between the LF and HF. The rationale for these estimators is presented in Discussion.Impedance (magnitude and phase angle) was acquired every 5 minutes at 14 frequencies logarithmically spaced between 100 and 200 kHz. System temperature and battery voltage were also acquired. The contents of the EIS systems were downloaded after explantation through the Zigbee radio link controlled by a custom application .Sacrifices were performed an average of 30.6 ± 3 days after MI with an overdose of anesthesia. After lateral thoracotomy, the hearts were excised. Left ventricle (LV) infarct size was measured by examination of sections obtained 1.5 cm distally to the artery ligation by using the following equation: infarct size (%) = [(LV infarct area)/] × 100. Quantitative morphometric and histological measurements were completed with Image‐Pro Plus software  microscope with a polarized filter.On 4‐μm paraffin slices, modified Gallego's and Masson's trichrome and Picrosirius Red staining were performed to analyze both pathological and histological changes and collagen deposition (type I red/yellow and type III green) under a computer‐associated Leica DMI 6000B , smooth muscle actin , and elastin  antibodies to quantify vessel area and detect blood vessels within the scaffold. For the inflammatory state study, CD3 (1:100) and CD25 (1:10)  antibodies were applied to determine the presence of lymphocytes and activated lymphocytes, respectively, in the infarct zone. Finally, to study the endothelial and cardiac differentiation of GFP‐pATPCs, and proliferation of cardiomyocytes, anti‐GFP (1:1000: Abcam), c‐Kit , MEF‐2 , cardiac troponin I , NKX2.5 , cardiac troponin T , SMA , von Willebrand factor (vWF) , CD31 , and Ki67  antibodies were used. Alexa Fluor 488‐conjugated streptavidin, Alexa Fluor 488 and 568 , and Cy2, Cy3, and Cy5  secondary antibodies were also used. All sections were counterstained with 4′,6‐diamidino‐2‐phenylindole  and analyzed by confocal microscopy .Frozen sections of 10 μm were stained by using biotinylated GSLI B4 isolectin  with the Tukey's procedure for multiple comparisons, and the Mann‐Whitney test for nonparametric data, using SPSS 19.0.1 . MRI data were analyzed as repeated measures by using ANOVA with the Greenhouse‐Geisser correction. Bioimpedance data analyses were performed with the paired samples t test using SigmaStat software . Values of p < .05 were considered significant.Data are represented as the mean ± SEM. Statistical analyses were performed by using the Student n = 8), EBIG‐treated (n = 10), and sham (n = 3) groups (p = .5 between groups). In all animals, the graft was seen covering the infarct area; absence of deleterious graft‐driven effects was detected in sham animals . Baseline infarct size between EBIG‐treated and control animals was not significantly different as assessed by the necrosis biomarkers cTnI and creatine kinase‐MB .Two animals died during MI induction due to ventricular fibrillation, and three were excluded from the study after postoperative infections. Therefore, 21 animals were included in the experimental protocol in the control  groups . Swine wp = .85), CO , SV , LVESV , LVEDV , and LVEDWM  .Baseline cardiac function did not differ between the EBIG‐treated and control groups in LVEF  and SV  between EBIG‐treated and control animals, respectively (p = .13), LVESV (p = .91), LVEDV (p = .07), or LVEDWM (p = .56).The ANOVA test with Greenhouse‐Geisser post hoc correction showed differences over time in LVEF   –3F. Prop+‐pATPCs de novo expressed the endothelial markers IsoB4, SMA, vWF, and CD31+ within the EBIG and at the infarct zone, suggesting graft‐driven neovascularization . Moreover, GFPrization . Relativnd cTnT. .Out of the 18 systems implanted , 11 provided useful data . Four of the remaining EIS systems were in deceased or excluded animals, and the remaining three stopped measuring after 1–3 days for unknown reasons.p = .002). There was also a significant difference between the control and sham groups , whereas no differences were found between the EBIG‐treated and sham groups (p = .39).Within 8 days, significant differences were observed between EBIG‐treated and control animals. p = .004). There was also a significant difference between the control and sham groups . The slopes were calculated as the first‐order coefficient of the linear regression for every magnitude ratio and phase difference time courses without any normalization. Regarding the phase angle difference slope estimator, the results were −3.52° ± 0.84° per day for controls and −0.18° ± 0.24° per day for EBIG‐treated (p = .1), collagen III , collagen volume fraction , and the collagen I/III ratio  were analyzed in the infarct cores of EBIG‐treated compared with control animals, respectively (p = .79) and EBIG‐treated  animals , infarct , and border  zones were found (data not shown). After 30 days, in all implanted bioprostheses some SMA‐positive vessels were detected . Analysis of inflammation at the infarct core showed no differences in CD3+ content (p = .49) between groups; however, CD25+content  and the CD25+/CD3+ ratio  were lower in EBIG‐treated animals  in the EBIG‐treated group was more than fivefold higher than that of controls; second, the EBIG positively impacted the healing process, providing impedance values similar to those of sham‐operated animals, and altered scar collagen content.+ cells within both the EBIG and in underlying myocardium de novo expressing cTnI and NKX2.5. cTnI plays a key role in mature sarcomere organization, and NKX2.5 is central in activating the cascade of cardiomyogenic genes. Thus, it is likely that pATPCs are partly responsible for the beneficial effects of the EBIG because many studies have reported that ATPCs secrete factors such as adipokines, growth factors, and proteins from the extracellular matrix that exert an important role in cardiac regeneration + pAPTCs within the scaffold also expressed SMA. Regarding cardiomyocyte proliferation, and in agreement with previous studies Indeed, the remarkable cardiac function benefits with the EBIG in terms of LVEF and SV improvement by MRI analysis are in agreement with the 48% reduction in infarct size in EBIG‐treated animals. Similar, but lower‐magnitude, benefits were previously described when adipose‐derived progenitor cells were implanted using different routes of administration  is observed in patients under immunosuppressive treatment Because inflammation is also a key step in myocardial healing after MI injury, we explored the impact of EBIG as an inflammatory modulator. Indeed, fewer activated lymphocytes accumulated in EBIG‐treated animals, a phenomenon most likely caused by the adipose progenitors contained within the graft. Perea‐Gil et al. have reported the immunomodulatory potential of cardiac adipose tissue progenitors abrogating T‐cell alloproliferation in vitro An advanced EBIG composed of acellular human pericardium refilled with pATPCs significantly reduced infarct size and improved cardiac function in a preclinical model of MI. In this context, the integration of a sophisticated noninvasive EIS monitoring system was useful to analyze the evolution of myocardial healing in EBIG‐treated animals. Collectively, from an EIS perspective, myocardial tissue was preserved in EBIG‐treated animals despite MI induction, which was confirmed by histopathological measurements of less inflammation and altered collagen I/III ratio.C.G.‐M.: conception and design, animal experimentation, collection and/or assembly of data, data analysis and interpretation, manuscript writing; R.B.: collection and/or assembly of data, data analysis and interpretation, bioimpedance device creation, final approval of manuscript; C.S.‐B. and I.D.‐G.: collection and/or assembly of data, data analysis and interpretation, animal experimentation; C.P.‐V., V.C., and A.L.‐V.: collection and/or assembly of data, data analysis and interpretation; F.M.S.‐M.: provision of study material, final approval of manuscript; P.B.‐F.: collection and/or assembly of data, data analysis and interpretation, bioimpedance device creation; I.P.‐G.: collection and/or assembly of data; S.R.: manuscript writing, final approval of manuscript; A.B.‐G.: conception and design, manuscript writing, final approval of manuscript.The authors indicated no potential conflicts of interest.Supporting InformationClick here for additional data file.