We found that TNF-α induced the production of IL-1, IL-6, IL-12, reactive oxygen species (ROS), nitric oxide (NO), Caspase-3, and Caspase-9 in chondrocytes. We also provided evidence that TNF-α inhibited autophagy markers  and prevented LC3 I translate to LC3 II. Furthermore, TNF-α induced matrix metalloproteinase (MMP)3 and MMP9 expression. The negative effects of TNF-α on chondrocytes can be partially blocked by treating with icariin or ammonium pyrrolidinedithiocarbamate . The present study data also suggested that icariin suppressed both TNF-α-stimulated p65 nuclear translocation and IκBα protein degradation. These results indicated that icariin protected against OA by suppressing inflammatory cytokines and apoptosis, through activation of autophagy via NF-κB inhibition. In conclusion, icariin appears to favorably modulate autophagy and apoptosis in chondrocytes making it a promising compound for cartilage tissue engineering in the treatment of OA.Osteoarthritis (OA) is a common chronic and degenerative joint condition that is mainly characterized by cartilage degradation, osteophyte formation, and joint stiffness. The NF-κB signaling pathway in inflammation, autophagy, and apoptosis plays a prominent role in the progression of OA. Icariin, a prenylated flavonol glycoside extracted from Epimedium, have been proven to exert anti-osteoporotic and anti-inflammatory effects in OA. However, the action mechanisms of its effect on chondrocytes have yet to be elucidated. In the present study, we demonstrated that the  Osteoarthritis (OA) is a chronic degenerative joint disease that occurs mostly in the elderly . As the During the progression of OA, the increased expression of inflammatory cytokines (such as TNF-α and IL-1β) in articular cartilage and synovium contribute to the degradation and erosion of cartilage . At the 2O2-induced human umbilical vein endothelial cell apoptosis, icariin suppresses NF-κB signaling in macrophages . Cell Counting Kit-8 (CCK8) was purchased from MedChemExpress . Antibodies against ATG5, ATG7, LC3, p65, phosphorylated p65 (p-p65), IκBα, MMP3, MMP9 were purchased from Abcam . Antibody against GAPDH was purchased from Cell Signaling Technology, Inc. . The NF-κB inhibitor pyrrolidinedithiocarbamate (PDTC) was purchased from Abcam . Enzyme-linked immunosorbent assay (ELISA) kits were purchased from Bio-Swamp Life Science . The Sprague-Dawley (SD) rat was purchased from The Center of Experimental Animal, Tongji Medical College, Huazhong University of Science and Technology. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology.2. When the cells were treated with 50 ng/ml TNF-α, 10 μM icariin, 10 μM PDTC, icariin or PDTC were added to the medium 2 h prior to TNF-α addition. In the autophagic flux assay, 0.1 μm bafilomycin A1 was added to the medium 1 h prior to icariin or PDTC addition. In the experiments, chondrocytes were treated with PBS (control), TNF-α, icariin with TNF-α, or PDTC with TNF-α for 24 h. All experiments were conducted in triplicate.Chondrocytes were isolated from the articular cartilage of 7–10-day-old male SD rats. Briefly, pieces of articular cartilage were digested with 0.25 mg/mL trypsin for 30 min and 2 mg/mL collagenase type II for 8 h at 37°C. After digestion, isolated chondrocytes were passed through a 180-μm filter and the cells were centrifuged and washed with PBS several times. The cells were then isolated and stained with trypan blue to evaluate cell viability. Chondrocytes with viability greater than 85% were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 IU/ml penicillin, 100 μg/ml streptomycin at 37°C, 5% CO3 cells/well) overnight, followed by treatment with various concentrations of icariin for 24 h, 48 h, and 72 h at 37°C in an atmosphere containing 5% CO2. Following this, 10 μl CCK8 solution was added to each well and the cells were cultured at 37°C for 2 h. The OD value was then measured with a microplate reader  at 450 nm.Rat chondrocytes were seeded in 96-well plates . Proteins were quantified using the bicinchoninic acid protein assay kit (BCA kit) according to the manufacturer’s instructions. Equal amount of proteins (10 μg) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes . Membranes were blocked in 5% bull serum albumin  in TBST for 2 h, then incubated with primary antibody at 4°C overnight. The primary antibodies were as follows: p65 (1:2000), p-p65 (1:2000), IκBα (1:5000), ATG5 (1:5000), ATG7 (1:10000), LC3 (1:3000), MMP3 (1:10000), MMP9 (1:10000), and GAPDH (1:1000). After washing with TBST  three times, the membrane was incubated with HRP-conjugated corresponding secondary antibodies  for 1 h at room temperature. Following washing, the immunoreactive proteins were visualized with the ECL western detection kit . The band density was quantified using TANON GIS software .-ΔΔCt method using GAPDH as the internal control. The primer sequences were as follows: ATG5, forward (5′-AA CGAGAAGCAGAGCCA-3′) and reverse (5′-ATGCCAT TTCAGGGGTG-3′); ATG7, forward (5′-GAAGAACCAGAAA GGAGG-3′) and reverse (5′-CAGGCACTTGACAGACAC-3′); Bax, forward (5′-TGGTTGCCCTCTTCTA-3′) and reverse (5′CACCCTGGTCTTGGAT-3′); Bcl-2 forward (5′-CACAG AGGGGCTACGAGT-3′) and reverse (5′-CAGGCTGGAAGG AGAAGA-3′); GAPDH, forward (5′-CAAGTTCAACGGCA CAG-3′) and reverse (5′-CCAGTAGACTC CACGAC AT-3′).Total RNA from chondrocytes was extracted using Trizol reagent . Total RNA was reverse-transcribed to cDNA with RT Master Mix (Takara Japan). The RT-PCR was performed with SYBR Master Mix using StepOne-Plus system  under the following conditions: denature at 95°C for 30 s, anneal at 60°C for 1 min and extend at 95°C for 5 s. The gene expression was analyzed by 2Collected cells were fixed with 2.5% glutaraldehyde for 24 h, which followed by fixed with 1% osmium tetroxide for 1 h at 4°C. After dehydrated with a series of ethanol concentrations  for 10-min intervals, the samples were incubated in a mixture of acetone and epoxy resin (v:v = 1:1) for 6 h, followed by incubation with pure epoxy resin for 4 h. After semi-thin sectioning, cells were stained with 0.5% toluidine blue and observed under the microscope. Finally, the ultra-section sections were observed using a TEM .Collected cells were washed and suspended in 0.3 mL PBS containing 10% FBS and 0.7 mL ethyl alcohol for 24 h at -20°C. The cells were then suspended with 0.1 mL RNase A (1 mg/mL) and 0.4 mL propidium iodide (PI) (50 μg/mL) for 10 min. The percentage of cells in the different stages was measured using Flow Cytometry.Collected cells were stained with a mixture of calcein-AM and PI solution for 20 min. Fluorescence images of cells were then recorded using an inverted fluorescent microscope. The percentages of cell death were evaluated by calculating the number of PI-stained  and calcein-AM-stained  cells. The number of cells were counted in five random fields by three independent authors. The mean value of each measurement was used for analysis.The ROS level was measured using Reactive oxygen species assay kit  according to the manufactures’ instructions. Briefly, treated cells were incubated with 10 μM dichlorodihydrofluorescein diacetate (DCFH-DA) in the dark for 30 min. Then, the cells were rinsed with DMEM and analyzed on a flow cytometer with excitation wavelength of 500 nm and emission wavelength of 525 nm. ROS level in the experimental group was normalized to the control group.NO production was detected with nitrate/nitrite colorimetric assay kit of Griess reaction  according to the manufacturer’s protocol. In brief, cultured medium was collected from treated cells and mixed with equal volume of Griess reagent. Following a 10-min incubation, absorbance was measured at 550 nm on a microplate reader .Caspase-3 and caspase-9 was detected with Caspase colorimetric assay kit . Briefly, collected cells were lysed in the provided lysis buffer. The absorbance was measured at 405 nm on a microplate reader .Cells were collected and washed with PBS, and then fixed with 4% paraformaldehyde for 15 min. After washing with PBS, the cells were blocked with 5% BSA for 2 h at 37°C. Then, p-p65 antibody (1:150) was added and incubated overnight at 4°C, followed by adding Max Vision TMHRP-polymer for 1 h, and followed by incubation with DAB for 5 min. The number of p65 in the nucleus was counted in five random fields by three independent authors. The mean value of each measurement was taken for analysis.SD). Statistical analysis was performed in GraphPad Prism 6.0  using Student’s t-test and one-way analysis variance (ANOVA). Statistical significance was considered when P < 0.05.Data were presented as means ± standard deviation  The dose of 10 μM was selected for the subsequent experiments.To determine whether icariin was toxic to chondrocytes, CCK8 assay was used after the cells had been treated with increasing concentration of icariin  for 24, 48, and 72 h. As shown in Figure 2), suggesting that icariin had an anti-inflammatory effect on chondrocytes.There are various inflammatory cytokines involved in the pathologic process of OA. These cytokines, such as IL-1β, induce the production of the other inflammatory cytokines , leadingFigures C). In addition to Atg 5 and Atg 7, western blotting result of LC3 (a classic marker of autophagy) was also used to establish the effect of icariin on autophagy. Icariin significantly enhanced LC3-II level when adding to the TNF-α-treated cells . During the late phase of autophagy, the fusion of lysosomes with autophagosomes may lead to a lower accumulation of LC3-II in the cytoplasm. To demonstrate this effect, bafilomycin A1 was added 24 h prior to cells co-treated with TNF-α and icariin to inhibit the fusion of lysosomes with autophagosomes. The level of LC3-II increased when bafilomycin A1 was added to the TNF-α-icariin-treated cells , suggesting that autophagy may fail to detect when autophagosomes fusion with lysosomes. Furthermore, TEM images illustrated that the number of autophagosomes in cells co-treated with icariin and TNF-α was higher than that cells treated solely with TNF-α . These results indicated that icariin activated autophagy in TNF-α-treated chondrocytes.To investigate the effect of icariin on the autophagy of chondrocytes, the autophagy markers Atg 5 and Atg 7 were detected. The western blot and PCR results showed that autophagy markers were significantly reduced in TNF-α-treated cells when compared with those cells co-treated with icariin and TNF-α, indicating that icariin protected against TNF-α-induced inhibition of autophagy . These results suggested that cell cycle progression might be delayed by TNF-α, a delay that is partially reversed by icariin treatment.It is well known that inhibition of the proliferation rate correlates with decreased cell cycle progression in chondrocytes. We analyzed the cell cycle division of chondrocytes treated with PBS, TNF-α and icariin with TNF-α. According to the PI staining of flow cytometry analysis, TNF-α arrested chondrocytes in the G1 phase. However, the percentage of the cell population in the S phase increased from 22.27% to 26.11% when addition of icariin to the TNF-α-treated chondrocytes , suggesting that icariin protected against TNF-α-induced cell apoptosis. To further determine whether icariin affects the mitochondrial apoptosis pathway of chondrocytes, the anti-apoptotic protein Bcl-2 level and the pro-apoptotic Bax level were detected using western blot and PCR. TNF-α significantly inhibited Bcl-2 expression but induced Bax expression. These effects were partially blocked by icariin treatment . Furthermore, the effect of that TNF-α-induced Caspase-3/9 activation in chondrocytes was blocked by addition of icariin. .We also analyzed the effect of TNF-α on chondrocyte apoptosis by calcein-AM/PI double-staining and found that TNF-α significantly induced chondrocyte apoptosis. However, when icariin was added to TNF-α-treated cells, the percentage of cell death decreased from 46.33% to 24.33% .The level of NO and ROS is known to increase when chondrocytes are exposed to inflammatory cytokines. In the present study, we found that TNF-α significantly induced NO and ROS production, an effect which could be blocked by addition of icariin . These results indicated that icariin inhibited NO and ROS production and reconstituted homeostasis of metabolism in chondrocytes.Disruption of homeostasis in cartilage metabolism impairs the extracellular matrix and chondrocytes, resulting in cartilage degradation. The excessive matrix catabolism was usually caused by excessive mechanical joint loading or inflammatory cytokines, which was reflected by the increased levels of matrix-degrading enzymes matrix metalloproteinase (MMP) family. In the present study, TNF-α induced the production of MMP 3 and MMP 9, an effect which could be blocked by addition of icariin . This finding also proven by the result of immunocytochemical staining that the number of p65 translocate to nucleus in TNF-α-treated cells is higher than the control group . In contrast, icariin caused the TNF-α-induced p-p65 increase and IκBα degradation reversal. Furthermore, the number of p65 translated to the nucleus also decreased by addition of icariin to TNF-α-treated cells. .In the present study, western blot results showed that TNF-α significantly increased the level of p-p65 and decreased the level of IκBα in chondrocytes when compared with the control group, suggesting that TNF-α stimulation of the NF-κB pathway .The NF-κB pathway plays a crucial role in various pathogenesis process of OA. To investigate whether this pathway was involved in the TNF-α-induced negative effect on chondrocytes, we used an NF-κB inhibitor, PDTC. In the present study, we found that PDTC partially blocked the TNF-α-induced inflammatory cytokines production, proliferation and autophagy inhibition, apoptosis activation, and hypercatabolism. (Inflammatory cytokines including TNF-α and IL-6 contribute to cartilage catabolism and degeneration in OA . ExcessiThe cytotoxic effects of icariin on other cell types have been investigated . In the Previous studies reported that activation of the NF-κB signaling pathway induced pro-inflammatory cytokines release . ExposurTissue engineering is an evolving interdisciplinary field integrating medicine, material science, biochemistry, and biomedical engineering, which centered on development of biological alternates to restore and/or to improve tissue and organ function . Along win vivo evaluation using macro-hydrogels to explore icariin’s therapeutic potential in OA treatment is currently under way in our laboratory.In conclusion, we demonstrated that icariin had no cytotoxic effects on chondrocytes up to the dose of 20 μM. TNF-α induced inhibition of autophagy and activation of apoptosis, and increased inflammatory cytokines, NO and ROS, as well as stimulating catabolism. These negative effects could be partially reversed by adding icariin to TNF-α-treated chondrocytes via inhibition of NF-κB signaling. Thus, the present study highlights that icariin induces autophagy activation and apoptosis inhibition of chondrocytes via suppression of the NF-κB signaling pathway. Future BM, WZ, and GL conceived and designed the experiments. JL, YL, and LH performed the experiments. BM and JW wrote the manuscript and made the same contribution to the manuscript. AP revised the language of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewers and handling Editor declared their shared affiliation.


Better stability of red color was observed in liqueurs with sugar, stored at 15 °C. The content of the dominant aroma compound, benzaldehyde, increased during storage. Long-term storage and sugar addition decreases color attributes but increases organoleptic value without of great influence on antioxidant activity. Studies on a half-year period of liqueur storage showed that their characteristic features are almost unchanged if stored at 15 °C and without sugar added, but organoleptic properties were better in samples stored at 30 °C.The aim of the study was to evaluate changes of phenolic and anthocyanin contents, antioxidant activity, aroma compounds and color of sour cherry liqueurs with and without sugar during 6 months of storage at temperatures of 15 °C and 30 °C. Contents of phenolic compounds  and antiradical activity (ABTS) changes were measured. Color changes were measured by an objective method (ColorQuest XE). During storage fluctuations of phenolic compounds and antioxidant activity content were observed. The content of substances which react with Folin-Ciocalteu reagent was comparable before and after 24 weeks. During the 24 weeks of storage, the highest average antioxidant activity against ABTS radicals was shown by sour cherry liqueurs without sugar, stored at 15 °C. Quicker degradation of anthocyanins was observed in liqueurs without sugar, stored at 30 °C (t Fruit liqueurs are traditional alcoholic beverages made by maceration of fruits in an aqueous solution of ethanol. The way they are prepared is important to achieve the proper flavor and aroma of liqueurs. Strength and the proportions of alcohol to the components, the method of preparation as well as time of maceration may vary  ,2,3,4. Liqueurs and tinctures have been used in folk medicine for many years. Today, it is known that the therapeutic effects of liqueurs are associated with the presence of phenolic compounds, which are valuable components of the diet. Recent dietary recommendations permit small amounts of alcoholic beverages. According to the recommendations of the Dietary Guidelines for Americans for 2010–2015 and 2015–2020 , moderatp-coumaric, caffeic. They are one of the few fruits to contain melatonin [Cherry fruit is characterized by a red color, juicy flesh, a sour taste and a pleasant aroma. Therefore, in many countries sour cherry liqueurs are especially popular. Cherries contain more than 200 mg of polyphenols/100 g of fruit. They are rich in flavonoids represented by anthocyanins (mainly glycosides of cyanidin), flavan-3-ols , flavonols  and phenolic acids: chlorogenic, elatonin ,9,10,11.Cherry stones contain cyanogenic glycosides, including amygdalin. These compounds are characterized by potential toxicity after hydrolysis into hydrogen cyanide. During the process of maceration of fruit, they can be extracted into the liqueur. The concentration of these toxic ingredients in the finished product depends on the way and time of fruit maceration, but also on the time the liqueurs are stored. Due to the amygdalin content, especially in stones, sour cherry liqueurs should be prepared from partially pitted cherries or in a short period of extraction time to avoid the presence of excessive amount of cyanogenic glycosides in the liqueur. According to Sokół-Łętowska  the contThere are very few research papers on the characteristics of fruit liqueurs. Nikicevic et al. investigated the flavors of cherry spirits made from five Serbian varieties of cherries after fruit fermentation. Ethyl octanoate and ethyl decanoate were determined in the highest concentrations in distillates . RodtjerIn the production of fruit liqueurs, different technologies are used . It is important to maintain the high quality of the product. These issues have not been tested so far, therefore it was decided to determine whether the addition of sugar during the production of liqueurs and storage temperature will affect the final quality of the product. The purpose of the work was to determine whether and how the sugar added to the fruit extract influences the quality of the obtained liqueur. The second aim of this study was to identify phenolic compounds and quantify the main anthocyanins, flavonoids, and phenolic acids that were found in sour cherry liqueurs stored for six months at 15 and 30 °C.Compounds identified before and after storage of cherry liqueurs are presented in p-coumaroylquinic (about 53.3%), neochlorogenic (25.1%) and chlorogenic (21.6%) acids. Hydroxycinnamic acids were quite stable during storage. After 6 months of storage a concentration of 165.1–177.0 µg/mL was determined. In samples without and with sugar added the phenolic acid content was 176.0–177.0 µg/mL and 165.0–170.7 µg/mL, respectively, that is about 4–7% and 15–17% less than the initial content, respectively. Losses of coumaroylquinic acid—the main phenolic acid in cherry liqueur—were slightly greater than those of other acids (7.4–20.7%). Similar results were obtained in a previous study by Kallithraka et al. on changes in phenolic composition of white wines during storage [Directly after preparation of liqueurs 188.4–203.4 µg/mL of phenolic acids were determined . The mai storage .(−)-Epicatechin, procyanidins B1, B2, C1 and other unidentified dimers, trimers and tetramers were present in liqueurs . The conIn samples stored at 30 °C the content of flavanols increased or decreased in the first 6–12 weeks and then consequently decreased due to the disappearance of monomers and dimers, faster in liqueurs with added sugar. The content of trimers and tetramers decreased, but slower than that of the monomers and dimers, probably due to polymerization of monomers and dimers, because near to 6–12 weeks of storage the content of trimers and tetramers increased and later decreased to 25–30% of the initial values. The total flavanol content in stored liqueurs was 4.7–5.9% of the initial value (at 30 °C) and 78.5% and 42.3% of initial values in samples stored at 15 °C without and with sugar respectively. Fluctuations of flavanol contents were observed during storage, as is seen for example for the sum of epicatechin and an unknown dimer  and the An amount of 139.7 to 158.6 µg/mL of flavonols was determined in liqueurs before storage and from 105.0 to 130.0 µg/mL after 24 weeks of storage . FlavonoThe content of anthocyanins immediately after preparation of sour cherry liqueurs was 216.8–230.8 µg/mL . As expeO-glucoside-rutinoside and cyanidin 3-O-rutinoside—were 62.2% and 25.6% of total anthocyanin content respectively before storage and 66.0% and 21.4% after storage at 30 °C. It means that cyanidin 3-glucoside-rutinoside (three sugar moieties) was more stable than cyanidin 3-rutinoside (two sugars). It is in agreement with literature data, indicating that the sugar moiety stabilizes anthocyanin stability [The proportions of the main anthocyanins—cyanidin 3-tability ,20,21,22During the first 12 weeks of storage the anthocyanin content was higher in samples with sugar added, but later the content of anthocyanins decreased faster in these liqueurs, and at the end of the experiment a slightly lower content of them was determined. This can be explained by the fact that presence of anthocyanin degradation products results in more rapid degradation ,24. The Samples without added sugar degraded a little slower than samples with added sugar, the half time being about 12% shorter in samples with sugar added. More important was the temperature of storage. At 30 °C anthocyanins degraded about 2.5 times faster than at 15 °C. The half-lives (t½) of the anthocyanins were in agreement with the Hellstrom et al.  researchCherry liqueurs were characterized by a nice red, stable color, which was due to the quite good stability of anthocyanin pigments, especially at 15 °C. The differences among samples after 26 weeks of storage were very small , so we dAfter 24 weeks of storage, the differences in lightness ΔL* were in the range −4.4 to +0.4 units, and after one year of storage the ΔL* was between −7.6 and +8.7, and only the liqueur with sugar, stored at 30 °C, was found to brighten. The red parameter (a*) decreased after 24 weeks by 2.2–4.2 units at 15 °C and 5.8–7.1 units at 30 °C, and after 52 weeks by 4.0–8.5 and 14.2–21.6 units respectively. Larger changes in this parameter were observed for liqueurs without sugar added. The color parameter b* (yellowness) increased during storage of liqueurs at 30 °C (Δa = 2.6 ÷ 3.0 units), and decreased at 15 °C (Δa = 2.8 ÷ 5.3 units). After one year only in the sample with added sugar stored at 30 °C was an increase of parameter b* observed (Δb* = 17.4). Also, the dominant wavelength decreased, but at 15 °C values were irrelevant (ΔDomWL < 1), and at 30 °C the difference compared to initial values was approximately 10 units in the samples stored at 15 °C and 18–24 units in the samples stored at 30 °C. An important parameter is ΔE, describing the ability of the human eye to discriminate between the colors of products. Pérez-Magariño and González-Sanjosé  stated tColor changes are mainly the result of anthocyanin degradation. Therefore, the anthocyanin polymer percentage was determined in liqueurs Anthocyanin polymer content increased during storage from about 9% to 22–26% and 65–76% at 15 °C and 30 °C respectively , increasAll tested cherry liqueurs had a strong and distinctive aroma. The main component was benzaldehyde, one of the 20 identified substances, representing more than 95% . The cheIn organoleptic evaluation all sour cherry liqueur samples were characterized by the typical flavor of sour cherries. Samples stored at 15 °C were significantly less intense in smell and taste. In samples without sugar, stored at 15 °C, and liqueurs with sugar, stored at 30 °C, there was an acidic “compote” note—not desired in such products. The sample without sugar stored at 30 °C was evaluated as the worst in terms of both taste and smell. The liqueur made with sugar and stored at 15 °C had the best flavor. Unfortunately, there was a characteristic bitter almonds note only during the first weeks of storage, maybe because of the short time of alcohol-pits contact during liqueur preparation, which is in agreement with Loch et al. .Antioxidant activity of liqueurs was measured by two methods: ABTS and by determination of substances which react with Folin-Ciocalteu reagent (FC) . During 24 weeks of storage only small changes in antioxidant activity were observed. Total phenolic content before storage was at the level of 1.15 mg GAE/mL and 1.10–1.14 or 1.21–1.25 µg GAE/mL after storage in samples without and with sugar respectively. Our results are in agreement with other authors. Heinonen et al.  showed tPiljac-Žegarac et al.  studied 2) of ABTS and phenolic compounds were 0.80 (flavan-3-ols), 0.78 (phenolic acids), 0.91 (flavonols) and 0.85 (anthocyanins).The antioxidant activity of cherry liqueurs measured by the ABTS test changed during storage. Average antioxidant activity before storage was 11.6–12.6 µM TE/1 mL (ABTS) and after storage it decreased by 6.4–9.2% in samples stored at 15 °C and by 19.4–37.8% in samples stored at 30 °C, decreasing more in liqueurs with added sugar. A similar effect was observed by Montoro et al.  in myrtlPrincipal component analysis (PCA) was conducted to confirm some relationships among analyzed variables. PCA was performed using Statistica 13.1 software on mean values of 20 samples  and 19 variables  for detecting the most important factors of variability and to describe the relationship between variables and observations. The first principal component (PC1) accounted for 63,98% of the variability in the data set. The second principal component accounted for 17.67% of the variance in the data set. The sum of 81.65% is high enough for a good presentation of multidimensional space in the two-dimensional projection. The loading plot PC2 versus PC1  shows a Liqueurs samples prepared with and without sugar and at different temperatures can be distinguished in the scores plot. Storage longer than 12 weeks negatively correlated with antioxidant activity and phenolic compound contents. In addition, the early, and late stages of the storage can be distinguished as indicated by the green lines. Liqueurs with sugar are well separated, by being on positive region of PC2, from liqueurs without sugar laying on negative region of PC2.The sour cherries used to produce liqueurs were purchased from local market retailers and stored at −20 °C before preparation of liqueurs. The fruit (2 kg) was soaked in glass jars with the same mass of ethanol (65% vol.). Process was carried out at room temperature for three weeks without light. The contents of the jars were stirred gently every day. After 3 weeks of maceration, the fruit extract was separated from the fruit and divided into two parts. To one of them sugar was added in the amount of ¼ of the extract mass, and to the other one the same mass of water. After the dissolution of sugar each liqueur was divided into two parts and stored at temperatures of 15 °C and 30 °C for 6 months. The liqueurs prepared with sugar were labeled 15 s and 30 s, and those without sugar—15 ns and 30 ns—according to the storage temperature of 15 °C and 30 °C. The experiment was carried out in two technological duplicates. 3/min and the injection volume was 5 μL. The column temperature was 30 °C. The major the Q-TOF MS operating parameters were: capillary voltage, 3.0 kV; cone voltage, 40 V; cone gas flow of 11 L/h; collision energy, 50 eV; source temperature, 100 °C; desolvation temperature, 250 °C; collision gas, argon; desolvation gas (nitrogen) flow rate, 600 L/h; data acquisition range, m/z 100–1500 Da; ionization mode, negative. The data were collected by Mass-Lynx v. 4.1. software  [Identification of compounds was performed on an ultra-performance liquid chromatography (Acquity UPLC System) connected to a quadrupole time of flight (Q-TOF) MS instrument  equipped with an electrospray ionization (ESI) source. Separation was achieved on the Acquity BEH C18 column . 4.5% formic acid in water was used as the eluent A and acetonitrile as eluent B. The gradient program was as follows: 0 min—99% (A), 12 min—75% (A), 12.5 min—100% (B), 13.5 min—99% (A). The flow rate was 0.45 cmMA, USA) .O-glucoside, flavonols into quercetin 3-rutinoside, flavanols into (+) catechin and hydroxycinnamic acids into chlorogenic acid. Analyses were performed in three repetitions. The results were expressed as µg/mL. The contents of anthocyanins (TA), phenolic acids (PA), procyanidins and flavan-3-ols (TPC), flavonols (TF), and their derivatives were calculated on the basis of HPLC-PDA assays as described previously by Sokół-Łętowska et al. . PhenoliO glucoside—Extrasynthese, Lyon Nord, France, quercetin 3-O rutinoside, (+) catechin and chlorogenic acid—Sigma Aldrich, Steinheim, Germany), with three injections per level  with ABTS cation radical and was carried out according to Re et al. , TEAC rePolymerized anthocyanin determination was performed with the sodium bisulfite method according to Wrolstad . The res−1 from m/z 39 to 400 in the electron impact (EI) mode at 70 eV. helium was used as the carrier gas at a flow rate of 1 mL min−1 in a split ratio of 1:20 and the following temperature program: rate of 5 °C min−1 from 80 to 200 °C; rate of 25 °C min−1 from 200 to 280 °C; final hold at 280 °C for 5 min. The injector and detector were kept at 200 and 300 °C respectively. Most compounds were identified by means of three methods: (1) Kováts indices; (2) GC/MS retention times (authentic standards); (3) mass spectra with similarity indices >90% .The chemical composition of the volatile compounds was performed according to the procedure described by Tešević . BrieflyThe color of the liqueurs was determined with an on-camera instrumental method on the ColorQuest  apparatus, on the L*a*b* scale, D65 illuminant and observer 10°, transmitted light. Color of cherry liqueurs was additionally measured after one year of storage.Organoleptic analyses were carried out by a team of five trained tasters, always the same persons to assess flavor and taste.1/2). Degradation of phenolic compounds was considered as first-order reaction. The parameters of degradation kinetics were calculated using the following equations:t is the compound concentration (µg/mL) at time t (min), c0 is the initial concentration (t = 0), k is the reaction rate constant.Degradation kinetics of main phenolic compounds during storage are obtained by determining the rate constants (k) at a given temperature against the time and half-life (tO-glucosyl-rutinoside (C3RG), cyanidin 3-O-rutinoside (C3R) and total anthocyanins (AN), lightness (L*), redness (a*), yellowness (b*) and dominant wavelength (DWL).Correlation analysis and principal component analysis (PCA) was performed using Statistica , version 13.1  on mean values of 20 samples  and 19 variables: time , temperature of storage , sugar addition , antioxidant activity (ABTS), substances reacting with the Folin-Ciocalteu reagent (FC), procyanidin B2 (B2) and total procyanidins and flavan-3-ols (TPC), quercetin 3-rutinoside (Q3R), quercetin (Q), total flavonols (FL), coumaroylquinic acid (CoQA) and total phenolic acids (PA), cyanidin 3-This study is a contribution to knowledge about the phenolic compounds and antioxidant activity changes of sour cherry liqueurs. Their antiradical activity against ABTS is strongly correlated with the contents of flavonols, anthocyanins, flavanols and phenolic acids. Studies after a half-year liqueur storage period showed that their characteristic features are almost unchanged if stored at 15 °C and without added sugar, but organoleptic properties were better in samples stored at 30 °C. It is best to make a cherry liqueur with no added sugar and store it at 15 °C. This gives the product a good color and a high content of active compounds. Long-term  storage and sugar addition reduce the color attributes but increase the organoleptic value without a great influence on antioxidant activity. Our results suggest that it might be interesting to study other technological variables of the production process, as well as different varieties of cherries.


Natural Language Processing (NLP) technologies have received significant attention in the medical domain and have demonstrated numerous successful uses in healthcare applications. The first international workshop on Health Natural Language Processing  provided a dedicated platform for close interactions among scholars, students, and industry professionals who are interested in NLP over health data such as clinical notes, social media, and biomedical literature. The workshop was organized in conjunction with IEEE International Conference on Healthcare Informatics (ICHI), and attracted submissions in the form of research papers, poster abstracts, and demonstration papers. All submissions were subjected to rigorous peer-review, with at least two peer reviews and at least one review by a senior member of the program committee. Selected papers and abstracts were featured as oral / poster presentations at the workshop. High quality research submissions were invited to expand their submitted works for a special issue of BMC Medical Informatics and Decision Making. The special issue on Health Natural Language Processing consists of twelve high quality papers, focusing on novel methods and applications of NLP over heath data. In the rest of this editorial, we highlight the key problems, approaches, and results from the papers included in this issue.One of the major themes emerging from the papers is to develop novel machine learning algorithms and NLP approaches to process clinical notes for specific tasks. Three papers propose deep learning models for clinical text classification, parsing, and information extraction tasks. Yao et al.  proposedAnother focus area for papers in this issue revolves around challenges introduced by inefficient recording and management of clinical metadata in EHR systems. Hanauer et al.  took a cAvailability of shareable clinical corpora is critical for advancing clinical NLP research. Weng et al.  assessedFinally, acknowledging the current trend of availability and popularity of health information online, there were two papers that studied challenges introduced by the advent of health-related social media as a mechanism for patients to communicate with each other. Vydiswaran and Reddy  introducIn conclusion, these papers highlight the plethora of research in health-related natural language processing. While advancements are being made in meaningful use of electronic health records for clinical decision support, these papers represent a snapshot of the broad spectrum of challenges where natural language processing can help advance the methodologies, tools, and applications in healthcare.


Tumor necrosis factor alpha (TNF-α) blockers are recognized as a risk factor for reactivation of granulomatous infections. Leishmaniasis has been associated with the use of these drugs, although few cases have been reported.We performed a retrospective observational study including patients with confirmed leishmaniasis acquired in the Mediterranean basin that were under TNF-α blockers therapy at the moment of the diagnosis. Patients diagnosed in our hospital from 2008 to 2018 were included. Moreover, a systematic review of the literature was performed and cases fulfilling the inclusion criteria were also included.Forty-nine patients were analyzed including nine cases from our series. Twenty-seven (55.1%) cases were male and median age was 55 years. Twenty-five (51%) patients were under infliximab treatment, 20 (40.8%) were receiving adalimumab, 2 (4.1%) etanercept, one (2%) golimumab and one (2%) a non-specified TNF-α blocker. Regarding clinical presentation, 28 (57.1%) presented as cutaneous leishmaniasis (CL), 16 (32.6%) as visceral leishmaniasis (VL) and 5 (10.2%) as mucocutaneous leishmaniasis (MCL). All VL and MCL patients were treated with systemic therapies. Among CL patients, 13 (46.4%) were treated with a systemic drug  while 14 (50%) patients were given local treatment . TNF-α blockers were interrupted in 32 patients (65.3%). After treatment 5 patients (10.2%) relapsed. Four patients with a CL  and one patient with VL treated with L-AmB maintaining TNF-α blockers.This data supports the assumption that the blockage of TNF-α modifies clinical expression of leishmaniasis in endemic population modulating the expression of the disease leading to atypical presentations. According to the cases reported, the best treatment strategy would be a systemic drug and the discontinuation of the TNF-α blockers therapy until clinical resolution. Leishmania infantum is the main causative agent of leishmaniasis in Southern Europe and is prone to produce the visceral form. However, TNF-α has been implicated in the initial events of the infection mediating the disease expression. In our series, we have observed a surprisingly high proportion of cutaneous form (32.6%) and muco-cutaneous form (10.2%). Clinical outcome observed in this series is also unusual. Four cases (14.3%) with cutaneous leishmaniasis who received local therapy relapsed. Among patients with visceral leishmaniasis, one patient who maintained TNF-α blockers therapy relapsed despite etiological treatment. This data supports the assumption that the blockage of TNF-α modifies clinical expression of leishmaniasis leading to atypical presentations. According to the cases reported we proposed as best treatment strategy a systemic drug and the discontinuation of the TNF-α blockers therapy until clinical resolution.Tumor necrosis factor alpha (TNF-α) blockers are widely used in numerous inflammatory diseases such rheumatoid arthritis, psoriasis or inflammatory bowel diseases. They have been recognized as a risk factor for reactivation of granulomatous infections. Although few cases have been reported, Leishmaniasis has been associated with the use of these drugs.  Tumor Necrosis Factor-α (TNF-α) is a crucial cytokine in the inflammatory cascade by activating the type 1 T helper (Th1) immune response, enhancing the activity of the macrophages and essential for the formation and maintenance of granulomas [Since TNF-α has been implicated in numerous immune-mediated disorders, the blockage of this cytokine has been studied as a therapeutic strategy against such diseases. Nowadays, the anti-TNF based therapy is widely used and approved for the treatment of chronic inflammatory conditions as rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, plaque psoriasis and psoriatic arthritis, ankylosing spondylitis and inflammatory bowel diseases . The firSince their first use, the TNF-α blockers were recognized as a risk factor for reactivation of granulomatous infections such as tuberculosis, intracellular infections such as salmonellosis or listeriosis and other opportunistic fungal or viral infections .Leishmaniasis is a parasitic granulomatous infection and it is endemic to South America, South Asia, Africa and South Europe. The protozoon is an obligate intracellular parasite of mononuclear phagocytic system cells. The clinical spectrum of leishmaniasis comprises subclinical (asymptomatic), localized (cutaneous) and disseminated infection . Its clinical expression is determined on one hand by the species and zimodeme of the parasite and on the other hand by host factors and immune response .Leishmaniasis has been associated with the use of TNF-α blockers, but only few cases have been reported in the literature, mainly in the Mediterranean basin ,6. We reLeishmania, leishmaniasis, TNF-α inhibitors, TNF-α blockers, anti-TNF-α, adalimumab, infliximab, etanercept, certolizumab and golimumab. No language or time restrictions were applied. A manual search of the references of the selected manuscript was also performed.We carried out a retrospective observational study including patients with a diagnosis of leishmaniasis in its different forms. All patients were under TNF- α blockers and were diagnosed in our center between 2008 and 2018. We also performed MEDLINE search using the terms Leishmania polymerase chain reaction (qPCR) of blood, bone marrow or skin samples.In order to be included in the analysis, cases had to be diagnosed based on direct observation of amastigotes and/or positive of Leishmania sp and human RNase P gene  were performed using the primers and probe described previously [Leishmania infantum identification was performed by restriction fragment length polymorphism of the internal transcribed spacer regions (ITS-RFLP) in a reference laboratory.DNA extraction from blood and bone marrow samples was carried out from 110 μl with silica–membrane technology (NucliSens easyMAG. Biomerieux. France) and eluted in 110 μl according to the manufacturer´s instructions. The extraction protocol from skin biopsy was made in a Magcore Compact (RBC Bioscience. Taiwan) and eluted in 100 μl according to the manufacturer´s instructions. A duplex qPCR targeted to kinetoplast minicircle DNA eviously . CyclingLeishmania polymerase chain reaction (qPCR). MCL when compatible signs and symptoms and mucose samples had a positive culture or Leishmania qPCR. CL when compatible signs and symptoms and cutaneous samples had a positive culture Leishmania qPCR. We assessed the patients’ complete medical records: demographic data, underlying disease, TNF- α blockers therapy, specific treatment and outcomes were recorded. Cure was defined as an absence of clinical signs after a minimum follow-up of 1 year after leishmanicidal treatment discontinuation. Unrelated death was established when a death with no relation to the infection or its treatment occurred during follow-up. Relapses were defined as reappearance of clinical signs after treatment discontinuation during the first year of follow-up unless relapse occurring after this period can be microbiologically demonstrated. Those cases without relevant information or not well identified were excluded from analysis.Patients were classified based on the clinical form as follows: VL when compatible signs and symptoms and blood or bone marrow samples had a positive culture or t or the Mann-Whitney U test, depending on distribution. All statistical tests were 2-tailed, and significance was set at P < .05. Statistical analyses were performed using SPSS version 20.0 .Categorical variables are expressed as percentages, and numerical data as the mean±SD for variables with a normal distribution or the median (IQR) for those with a skewed distribution. Categorical variables were compared with the chi-square test or Fisher exact test, and continuous variables with the Student Due to its retrospective design, oral consent was obtained by phone contact from the included patients. The study was approved by the Ethics Committee of Vall d’Hebron Research Institute.folliculitis decalvans in one case each other. All patients were diagnosed in European hospitals and probable place of infection was Spain in thirty-three (67.3%) cases followed by Greece in five (10.5%) cases, Italy in four (8.2%) cases, France in two (4.1%) cases, Malta in two (4.1%) cases, Algeria in two (4.1%) cases and Turkey in one (2%) case.A total of 33 publications were retrieved in our search including forty cases that fulfilled inclusion criteria –39. ThusLeishmania RT-PCR on the bone marrow sample. Identification to the level of species could be performed in eighteen patients. L. infantum was identified in seventeen cases and one was reported as L. donovani complex.Regarding the clinical presentation, CL was the most frequent form , eighteen cases presenting as a solitary ulcerative lesion and ten cases including one attended in our hospital who presented multifocal lesions . SixteenAll MCL and VL patients were treated with a systemic therapy. Fifteen (71.4%) of them were treated with liposomal amphotericine B (L-AmB), including one patient treated in combination with intralesional pentavalent antimonials, three (14.3%) patients were treated with parenteral pentavalent antimonials and one (4.8%) patient with miltefosine. Among CL cases, thirteen (46.4%) patients received a systemic treatment; eleven were given L-AmB, one intramuscular antimonials and one miltefosine. Local treatment was given to fourteen (50%) patients, thirteen received intralesional pentavalent antimonials, one combined with surgery and another with cryotherapy and one patient was treated with surgical excision of the lesion. Only one patient defaulted and did not receive any treatment.In thirty-two (65.3%) cases TNF-α blockers therapy was interrupted. After treatment, four (8.2%) patients with CL diagnosis relapsed. Three of these cases were initially treated with local medication and anti-TNF-α was not stopped. After relapse, the three patients received systemic treatment and anti-TNF-α therapy was discontinued in one patient, all achieving clinical cure. Another relapsing patient was initially treated with miltefosine and finally cured with local antimonials therapy. TNF-α blocker therapy was discontinued in both treatment courses. The last relapsing patient was a VL treated with L-AmB and TNF-α blockers were not stopped as her rheumatic disease was active presenting a MCL form 20 months after. When comparing clinical cure of CL patients, although statistical significance could not be reached cure ratios were 92.3% vs. 78.6% (p = 0.6) when patients received systemic treatment or not and 94.1% vs 70% (p = 0.13) when TNF-α blockers therapy could be stopped or not. Finally, the patient who did not receive any treatment was lost to follow up. Two (4.1%) patients died: one patient after a bacterial superinfection in relation to his immunosuppression and the other patient as a result of a fatal arrhythmia during his treatment with systemic antimonials. Case presentation, treatment and outcomes are represented in According to the published data, TNF-α blockers based therapy seems to be associated with a higher risk of infections, at times with a worse outcome . NeverthL. infantum is the major causative agent of leishmaniasis in Southern Europe and the most frequent zymodeme is MON 1 [L. infantum is prone to produce the visceral form of disease and less frequently the cutaneous form. Although different gens have been proposed to contribute to the viscerlalization process, its mechanisms still remains unclear due to the lack of good animal models [is MON 1 . Even thl models .L. infantum endemic areas there is a frequent natural challenge to Leishmania parasites, producing circulating parasitemia in the host during an undefined non-permanent time-span. Some authors have referred to this subclinical form as “asymptomatic carriage”. It means that these patients without symptoms have parasite load only detectable by highly sensitive methods such as RT-PCR [It is also well known that in s RT-PCR ,44.In southern Europe the prevalence of asymptomatic carriers is remarkably variable (from 0.5% to 48%) depending on the test used for detection . Those dLeishmania species may be able to infect and develop within non-hematopoietic cells such as fibroblasts it is mostly inside monocyte/macrophage lineage cells where the parasite replicates and develops long-time survival [Leishmania interferes in an extremely complex manner with the host immunological response [While it seems that in mammalian hosts survival . Since tresponse .The role of TNF-α in the pathogenesis of the disease has been widely studied in both humans and animal models. As innate TNF-α dependant mechanisms drive cell mediated immunity by activation of CD4+ and CD8+ T cells, TNF-α is known to be fundamental in the initial control of the infection together with other cytokines such as IL-12 and IFN-γ . These cIn CL, a polarized Th1 response with low Th2 cytokines has been related to infection resistance and disease outcome. Besides, Th1 and Th2 pathways may also modify disease expression . HoweverHence, TNF-α has been implicated in the initial events of the infection, in direct leishmanicidal activity and thus controlling the multiplication of the parasite, developing effective acquired immunity for long term control of the disease as well as mediating the disease expression .The classical VL in Mediterranean countries has been found primarily in children and HIV/AIDS infected patients with poor immune status. Nowadays VL cases in adults are also diagnosed among non-VIH immunosuppressed patients . MoreoveL. infantum is benign and trends to heal spontaneously. In fact, if lesions are less than 5 cm in diameter and are localized in areas where there is no risk of disfiguring or disabling, only a local wound care is recommended [Clinical outcome observed in this series is unusual. The natural course of CL caused by ommended . Four ouL. infantum, the main acquisition of this infection is through the skin. Besides, in endemic areas there is a high exposure to Leishmania. Although some studies report up to 48% of prevalence [Despite of the natural viscerotropism of evalence , there aLeishmania kinetoplastid sequences which healed spontaneously. Because of the latency between the first episode and the second one and the appearance of a distant lesion far from the initial one, the episode was thus considered as a reactivation in the context of the TNF-α blockers therapy.The reactivation of a latent parasitic form from a granulomatous lesion could be a feasible explanation for those cases. Clinical data from patient number 21 supports this assumption. This patient was diagnosed with a MCL form  4 years L. infantum, a growing number of treatment failures have been published so further investigation is needed before its systematic recommendation [Previous series including patients with Old and New world leishmania species showed no consensus regarding treatment in patients under TNF-α blockers . In thisendation . ConcernL. infantum presenting forms, that otherwise it would have caused a transient parasitemia or an aborted local replication. Likewise, the course and outcome of the disease may be directly affected by the inhibition of TNF- α. Therefore, discontinuation of the anti TNF-α therapy seems to play an important role in the treatment success. Although miltefosine is an excellent option for the New World CL, evidence is scarce for the treatment of L. infantum infection [As mentioned before, TNF-α plays a key role in the process of controlling infection that ranges from limiting the replication of the parasite up to eliciting an effective adaptative response. TNF-α blockers therapy could modulate the immunological response to a less effective control of the parasite, hence allowing the infection reactivation or more evident disease expression of newly acquired infections. To this end, patients under treatment with TNF-α blockers have less effective immune mediated mechanisms to control and eradicate a parasite challenge, ending in a higher chance to develop classical and non-classical nfection . Thus, iAccording to the cases reported, in our opinion the best treatment strategy would be a systemic treatment and the discontinuation of the TNF-α blockers therapy. L-AmB has proved to have the best safety profile and compelling evidence of its efficacy in immunosuppressed patients. One of the major concerns is the reintroduction of TNF-α blockers. Taking into account published information, it seems reasonable reintroducing them once clinical cure has been achieved ensuring close clinical follow-up and blood RT-PCR. Although there is scarce information for its recommendation, etanercep or certolizumab have been suggested as a therapeutical option instead of re-introducing other anti-TNF-α monoclonal antibodies due to its possible lower risk of reactivating leishmaniasis –66.This study has the limitations of any retrospective review study. Although is one of the biggest series published on this topic, the scarce number of patients analyzed limits the strength of the recommendations. We agree with existing reports on the fact that despite of not being a common disease in our area, leishmaniasis is likely underreported . FinallyThe increase in the use of TNF-α antagonist has been associated with the emergence of new cases of leishmaniasis. The blockage of TNF-α favors the reactivation of latent leishmaniasis modulating its expression and worsening its clinical outcome.Once the leishmaniasis is confirmed, systemic drug treatment and the discontinuation of the TNF-α blockers therapy until clinical recovery seems to be the best therapeutic approach when possible.Otherwise, those patients receiving such therapy and coming from endemic areas require a close monitoring in order to detect early forms and start adequate treatment.Prospective studies and more participation on declaring existing cases in the adverse events notification system is required in order to assess the risk of leishmaniasis and other opportunistic diseases related to the use of anti TNF-α treatment more accurately.S1 Checklist(DOCX)Click here for additional data file.


In HA patients, elevated TNF-α levels were significantly associated with the number of hemarthroses, the grade of synovial hypertrophy, and both the clinical World Federation of Hemophilia score and ultrasound score. The expression of TNF-α, TNF-R1, and TNF-R2 was strongly increased in HA synovium (n = 10) compared to the non-inflamed osteoarthritis control synovium (n = 8), as assessed by both immunohistochemistry and Western blotting. Increased protein levels of TNF-α, TNF-R1, and TNF-R2 were retained in vitro by HA fibroblast-like synoviocytes (n = 6) with respect to osteoarthritis control fibroblast-like synoviocytes (n = 6). Stimulation with TNF-α resulted in a significant increase in HA fibroblast-like synoviocyte proliferation quantified by the water-soluble tetrazolium (WST)-1 assay, while it had no relevant effect on osteoarthritis fibroblast-like synoviocytes. Quantification of active/cleaved caspase-3 by ELISA demonstrated that TNF-α did not induce apoptosis either in HA or in osteoarthritis fibroblast-like synoviocytes. The TNF-α/TNF-R system may represent a crucial mediator of proliferative synovitis and, therefore, a new attractive target for the prevention and treatment of joint damage in HA patients. Our findings provide the groundwork for further clinical investigation of anti-TNF-α therapeutic feasibility in hemophiliacs.Hemophilic arthropathy (HA) typically begins with proliferative synovitis that shares some similarities with inflammatory arthritides, in which the proinflammatory cytokine tumor necrosis factor (TNF)-α has a crucial pathogenetic role. Inappropriate release of TNF-α was shown to contribute to arthropathy development following intra-articular bleeding in hemophilic mice. Here, we were interested in determining whether systemic levels of TNF-α and synovial tissue expression of the TNF-α/TNF receptor (TNF-R) system could be increased and related to joint damage in hemophilia A patients with severe HA. Serum levels of TNF-α measured by quantitative enzyme-linked immunosorbent assay (ELISA) were significantly increased in HA patients ( Intra-articular bleeding plays a pivotal role in the pathogenesis of hemophilic arthropathy (HA), a major clinical manifestation of hemophilia characterized by large-joint synovitis evolving into articular cartilage destruction and subchondral bone resorption that frequently results in osteopenia or osteoporosis ,4,5,6,7.The proinflammatory cytokine TNF-α has been extensively implicated in the pathogenesis of proliferative synovitis and bone resorption and is a major target for the treatment of inflammatory arthritis, such as rheumatoid arthritis (RA) ,17,18. OSixty-seven hemophilia A patients attending the Center for Bleeding Disorders of the Careggi University Hospital in Florence, Italy, were consecutively enrolled in the study. All patients had suffered from at least one joint bleeding. Clinicodemographic characteristics and imaging findings of the study population are shown in The median age of patients was 36.3 years (range 16–69 years). All patients (100%) had severe hemophilia A (FVIII:C <1 IU/dL). Thirty-seven out of 67 patients (55.2%) were treated on demand, eight of 67 (12.0%) with primary and secondary prophylaxis, and twenty-two of 67 (32.8%) with tertiary prophylaxis. According to the last guidelines for the management of hemophilia of the World Federation of Hemophilia (WFH) , primaryThe severity of HA was measured using the WFH orthopedic joint scale score consisting of a physical examination and pain scale  . All patg for 15 min. Serum samples were collected and stored in aliquots at −80 °C until used. Serum levels of TNF-α were measured with a commercially available enzyme-linked immunosorbent assay (ELISA) kit , following the manufacturer’s protocol. Optical density was measured by a microtitre plate reader at 450 nm. The serum levels of TNF-α were read off from a standard curve according to the manufacturer’s instructions. The detection range and the sensitivity of the assay were 0.2–10 pg/mL and 0.049 pg/mL, respectively. Each sample was measured in duplicate. The inter-assay and intra-assay variances were <10%.Fresh peripheral venous blood from 67 HA patients and 20 healthy controls  was drawn, allowed to clot for 30 min before centrifugation at 1500× Synovial tissue samples were obtained from 10 HA patients with severe knee arthropathy  who underwent arthroplasty during surgery as described elsewhere . SynoviaFibroblast-like synoviocytes were successfully isolated from 6 HA and 6 OA control synovial biopsy specimens subjected to a mild proteolytic treatment with 0.05% trypsin and 0.5 mM EDTA in phosphate-buffered saline for 10 min at 37 °C under gentle shaking as described elsewhere . Trypsin2O2 in methanol for 15 min to block endogenous peroxidase activity and subjected to immunoperoxidase-based immunohistochemistry using the ready-to-use UltraVision™ Large Volume Detection System Anti-Polyvalent, HRP kit . After blocking non-specific antibody binding sites, tissue sections were incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal anti-TNF-α , rabbit polyclonal anti-TNF receptor 1 (TNF-R1)  and rabbit polyclonal anti-TNF receptor 2 (TNF-R2) . Isotype- and concentration-matched irrelevant rabbit IgG  were used as negative controls. The day after, tissue slides were incubated sequentially with biotinylated secondary antibodies and streptavidin-peroxidase solution (both from Lab Vision) followed by immunoreactivity development using 3,3′-diaminobenzidine tetrahydrochloride  or ImmPACT VIP  peroxidase substrates as chromogens. Immunostained tissue sections were examined with a Leica DM4000 B microscope equipped with a Leica DFC310 FX 1.4-megapixel digital color camera and the Leica software application suite LAS V3.8 .Synovial tissue sections (5 μm thick) were deparaffinized, rehydrated, boiled for 10 min in sodium citrate buffer  for antigen retrieval and subsequently treated with 3% Hhttp://rsbweb.nih.gov/ij) and the values were normalized to α-tubulin.Proteins were extracted from synovial specimens by homogenization for 5 min in ice-cold lysis buffer  supplemented with the protease inhibitor cocktail , followed by sonication. Proteins were also extracted from cultured fibroblast-like synoviocytes at basal conditions or after treatment for 24 h with recombinant human TNF-α . Protein lysates were cleared by centrifugation for 30 min at 4 °C at 15000 rpm and assayed for protein content using Bradford’s method. Fifty micrograms of proteins were electrophoresed in SDS 12%–13.5% polyacrylamide gel under reducing conditions and then blotted to a nitrocellulose transfer membrane . The membranes were blocked in 5% non-fat dry milk with 0.05% Tween-20 in phosphate buffered saline for 1 h at room temperature, and then incubated overnight at 4 °C with the following rabbit polyclonal antibodies: anti-TNF-α, anti-TNF-R1, and anti-TNF-R2 . After incubation with horseradish peroxidase-conjugated anti-rabbit IgG  for 1 h at room temperature, immune complexes were detected with the enhanced chemiluminescence detection system and the membranes were exposed to autoradiographic films (Amersham Biosciences). The membranes were stripped and reprobed with rabbit monoclonal anti-α-tubulin antibodies  as the loading control. Densitometric analysis of the bands was performed using the free-share ImageJ software 64-bit Java 1.8.0_112 Windows version  in RPMI 1640 with 15% FCS and were left to adhere overnight. Cells were then washed three times with serum-free medium and incubated in RPMI 1640 with 0.2% FCS for an additional 24 h. Fibroblast-like synoviocytes were incubated for 24 h in RPMI 1640 with 0.2% FCS containing 10 ng/mL recombinant human TNF-α (PeproTech). Cell viability was determined by a cell proliferation assay using the water-soluble tetrazolium (WST)-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) reagent  according to the manufacturer’s protocol. All measurements were performed in triplicate, and the results were expressed as the percentage increase/decrease in cell viability over the basal response.HA and OA fibroblast-like synoviocytes were seeded onto 96-well plates . Positive apoptosis controls were obtained by incubation of cells with staurosporine (Sigma-Aldrich). Cleaved (active) caspase-3 levels in fibroblast-like synoviocyte lysates were determined with a specific quantitative colorimetric sandwich ELISA kit designed to specifically detect human active caspase-3 protein . Fibroblast-like synoviocytes were collected, centrifuged, and then the pellet was incubated in ice-cold cell extraction buffer for 30 min and treated according to the manufacturer’s instructions. All measurements were performed in triplicate.HA and OA fibroblast-like synoviocytes were plated in culture dishes at a concentration of 1–1.5 × 10t-test was used to compare two independent groups for normally distributed parameters, while the Mann–Whitney U-test was used to compare two independent groups for non-normally distributed parameters. Spearman’s rank correlation coefficient (ρ) was used to analyze the relationship between two continuous variables. Values of p < 0.05 were considered statistically significant.Statistical analyses were performed using the SPSS software for Windows, version 25.0 . Data are expressed as the mean ± standard error of the mean (SEM) or median and interquartile range (IQR), as appropriate. Student’s p < 0.0001)  compared to healthy controls  ( 0.0001) A. No sign = 7 patients and n = 17 patients, respectively). Therefore, these patients were grouped (n = 24 patients) for further comparison with the group with >50 episodes of hemarthrosis (n = 43 patients). Circulating levels of TNF-α in the >50 group  were significantly higher than in the <10/10–50 groups  (p < 0.0001) (p = 0.002) (p = 0.02)  B. As far= 0.002) C. Furthe = 0.02) D.p < 0.0001) and US score  was found (p = 0.058).In HA patients, a significant positive correlation between the circulating levels of TNF-α and both clinical WFH score .The ex vivo expression of TNF-α, TNF-R1, and TNF-R2 in knee synovial tissue sections from HA patients and OA controls was investigated by immunoperoxidase-based immunohistochemical staining procedures. TNF-α, TNF-R1, and TNF-R2 immunostainings were strongly increased either in the hyperplastic synovial lining layer or the synovial sublining layer of HA compared to non-inflamed OA control synovial specimens, particularly in synoviocytes, the vascular endothelium, and perivascular cells A. As disp < 0.05 vs. OA fibroblast-like synoviocytes for all comparisons)  A.p < 0.05 vs. the basal condition)  resulted in a significant increase in the proliferation of HA fibroblast-like synoviocytes (ndition) B. Converndition) B. Quantindition) C.This study was undertaken based on the evidence that (i) HA usually begins with proliferative synovitis that shares some similarities with inflammatory arthritis in which the TNF-α cytokine is recognized as a crucial pathogenetic orchestrator, and (ii) inappropriate release of TNF-α contributes to arthropathy development following intra-articular bleeding in hemophilic FVIII-deficient mice ,4,8,9,19The findings that systemic levels of TNF-α were strongly increased and correlated with disease severity in our patients indicate that this cytokine may be tightly related to the evolution of HA. Indeed, we found significant associations between circulating levels of TNF-α and multiple clinical parameters of HA, including the number of hemarthroses, the grade of synovial hypertrophy, and the clinical WFH score and US score, which altogether suggest a role for TNF-α in the progression of blood-induced arthropathy and its possible suitability in the future as a biomarker of disease severity that could potentially be used to identify patients with progressing joint disease. Moreover, our observation that local synovial expression of TNF-α, TNF-R1, and TNF-R2 is robustly increased in HA and retained in vitro by cultured HA fibroblast-like synoviocytes provides strong support to the notion that TNF-α/TNF-R signaling may be dysregulated in hemophiliacs as previously reported in hemophilic FVIII-deficient mice . This asConsistent with recent observations in a mouse model for HA , the datAlthough our novel findings highlight a non-negligible participation of the TNF-α/TNF-R system to the pathobiology of human HA, we are aware that further in-depth molecular studies will be required to better decipher the mechanisms of action of this pleiotropic pathway and to identify potential downstream therapeutic targets. In fact, it is well known that TNF-α activates complex signaling cascades and induces many other proinflammatory mediators, such as IL-6, whose signaling is currently attracting much attention as a target for the treatment of chronic arthritis and, possibly, also to protect against HA ,37,38,39In summary, our results add to recent data on murine HA  and prov


EGFR突变的晚期肺腺癌患者预后生存，但治疗及异质性等因素可导致初次和疾病进展时EGFR基因状态发生改变。为了探讨真实世界中EGFR基因突变在疾病进展和基线时的差异，我们开展了此项研究。表皮生长因子受体靶向治疗能够显著提高EGFR基因检测的61例配对标本数据并进行分析。标本取材时间为治疗前和疾病进展时，所有标本均经病理学或细胞学证实，标本来源为肿瘤组织﹑恶性胸腔积液和血浆，患者为初治，一线接受化疗或靶向治疗，采用扩增阻滞突变系统法对29种EGFR基因突变进行检测。收集2015年1月-2017年12月在吉林省肿瘤医院进行n=61)，肿瘤组织、恶性胸腔积液和血浆标本所占的比例分别为90.2% vs 88.5%、6.6% vs 6.6%和3.2% vs 4.9%，其中标本类型前后一致的患者(n=50)EGFR突变差异率为72.0%，标本类型不一致患者(n=11)为36.3%；治疗前EGFR突变率为95.1%，治疗后为91.8%，二者的差异率为63.9%；化疗患者(n=13)治疗前EGFR突变率为69.2%，治疗后为92.3%，二者差异率为46.1%；靶向治疗患者(n=48)治疗前EGFR突变率为100%，治疗后为91.7%，二者差异率为70.8%。EGFR基因变化类型有4类：野生型变为突变型(4.9%)、突变消失(8.2%)、敏感突变类型互变(1.6%)和突变种类增加(49.1%)。初次和再次活检相比患者由于标本取材部位和类型及治疗的影响，治疗前后EGFR基因突变具有较大差异性，动态检测并明确 EGFR)突变是中国非小细胞肺癌患者最常见的驱动基因，针对其敏感突变采用EGFR酪氨酸激酶抑制剂进行治疗已成为晚期NSCLC一线标准治疗方案-3。经EGFR-TKI治疗8个月-14个月后多数患者发生耐药，其中约50%是EGFR外显子20出现T790M突变，其他突变还包括c-met扩增、HER-2突变和小细胞肺癌转化等。除了治疗药物的影响因素之外，新发病灶带来的肿瘤异质性也会造成EGFR基因突变的改变-8。因此，动态检测疾病进展时EGFR基因突变状态对于指导患者后续治疗和实现全程精准治疗具有重要的意义。为了阐明临床实践中治疗前和疾病进展时EGFR基因突变的差异情况，本研究以一线治疗的晚期NSCLC患者为研究对象，对比了61例治疗前(基线)和疾病进展时配对标本的EGFR基因突变数据，为验证再次活检且进行分子病理检测的必要性提供参考数据。表皮生长因子受体和疾病进展时，均经病理学或细胞学证实为NSCLC，所有患者均为一线治疗，治疗方案为EGFR-TKI或标准化疗。其中男性18例，女性43例；中位年龄55岁(范围34岁-79岁)，平均年龄52岁；不吸烟者患者高于吸烟患者，45例vs 16例；腺癌为主要病理类型，腺癌、腺鳞癌和鳞癌患者分别为63例、1例和1例；Ⅳ期、Ⅲ期和Ⅱ期患者分别为63例、1例和1例；标本类型为组织、恶性胸腔积液及血浆，其中初次和再次检测的例数分别为55例vs54例，4例vs4例，2例vs3例。回顾性收集2015年1月-2017年12月在吉林省肿瘤医院进行1.2EGFR基因检测试剂盒购自厦门艾德生物医药科技有限公司(AmoyDx)，DNeasy Blood and Tissue Kit购自QIAGEN，Nanodrop2000C购自Thermo公司，LightCycler 480购自罗氏公司。红细胞裂解液购自BD公司，1.3260/OD280比值为1.8-2.1。按照说明书进行EGFR基因扩增和突变分析：每个反应管中加入模板DNA 2 ng，PCR扩增条件为95 ℃ 5 min，15×；31×，60 ℃时收集FAM和HEX信号。判定标准为：Ct值< 26为EGFR突变阳性，Ct值≥29为EGFR突变阴性，Ct值在26-29之间判断样本△Ct值，如果△Ct值小于对应的Cut-off值，样本为EGFR突变弱阳性。按照说明书提取不同标本来源的DNA，DNA质量控制标准为浓度 > 10 ng/μL，OD1.4患者治疗后2个月进行首次评价，以后每1个月住院复查或门诊、电话随访。按照实体瘤近期疗效评价标准评估病灶变化情况，分为完全缓解、部分缓解、疾病稳定和疾病进展，有效率=(CR+PR)/总例数×100%；临床获益率=(CR+PR+SD)/总例数×100%；所有患者均签署知情同意书。1.5chi-square test检验，采用McNemar检验对比分析接受治疗前和接受治疗后EGFR基因突变状态的改变情况，采用Cochran-Armitage趋势检验来分析EGFR基因突变状态的改变同PR、SD和PD等临床治疗结果的相关性，以P < 0.05为差异有统计学意义。采用SPSS 17.0统计软件进行数据分析，计数资料的组间比较采用22.1EGFR基因突变状态与性别有关，疾病进展时再次活检标本中EGFR基因突变状态与吸烟有关(P=0.039)，其他临床相关因素如年龄、分期等无显著相关性(P > 0.05)(初次活检标本中P > 0.05)。2.2EGFR突变率均有差异，且与标本类型无关，初次和再次活检相比，送检的标本种类相同，包括组织、恶性胸腔积液和血液，但各种标本所占比例有变化，分别为90.2%(55/61)vs88.5%(54/61)、6.6%(4/61)vs6.6%(4/61)和3.2%(2/61)vs4.9%(3/61)。两次活检标本类型相同患者的EGFR基因突变不一致率为72.0%(36/50)，两次标本不同患者EGFR基因突变不一致率为36.3%(4/11)。初次和再次活检相比，EGFR突变率为95.1%(58/61)，突变类型主要为19Del(n=28)、L858R(n=27)和其他类型突变(n=3)；再次活检标本EGFR突变率为91.8%(56/61)，突变类型主要为19Del(n=12)、L858R(n=12)和其他类型突变(n=5)，其中，新出现T790M突变27例，均为伴随突变，包括T790M-19Del(n=15)、T790M-L858R(n=10)、T790M-G719X(n=1)。治疗前和疾病进展相比，EGFR基因突变不一致率为63.9%(39/61)。EGFR基因突变变化类型可分为4种：野生型变为突变型(n=3)、基因突变消失(n=5)、敏感突变类型转换(n=1，L858R和19Del相互转换1例)和突变类型增加(n=29，伴随T790M的复合突变27例，伴随其他类型复合突变3例)，再次活检中T790M突变最常见(44.2%)(McNemar检验，P > 0.05；治疗前EGFR突变率为76.9%(10/13)，治疗后为92.3%(12/13)，二者的不一致率为38.5%(5/13)，差异类型分别为S768I转变成G719X+S768I复合突变(n=1)、野生型变成19Del(n=2)、G719X(n=1)、19Del变成EGFR野生型(n=1)。行靶向治疗患者治疗前EGFR突变率为100.0%(48/48)，治疗后为91.7%(44/48)，二者的差异率为70.8%(34/48)。差异类型分别为出现T790M突变(n=27)、EGFR突变型变为野生型(n=4)、L858R转换为19Del(n=1)、L858R转换为L858R+19Del复合突变(n=1)和19Del转换为L858R+19Del复合突变(n=1)。靶向治疗后EGFR突变状态的改变具有统计学差异、25.0%(1/4)、33.3%(1/3)、77.7%(21/27)，CBR分别为100.0%(14/14)、50.0%(2/4)、100.0%(3/3)、96.3%(26/27)，4组之间的疗效差异具有显著相关性，基因突变频率同较好的靶向临床反应显著相关。48例患者一线接受靶向治疗，治疗有效率为72.9%(35/48)，临床获益率为93.7%(45/48)；按治疗前后2.4EGFR T790M突变状态之间没有相关性(P > 0.05)(48例患者接受一线靶向治疗，其中27例患者在二次活检中检测到T790M突变，比例为56.2%。年龄、性别、吸烟状态和组织学亚型等在内的临床因素同P > 0.05)。3EGFR敏感突变患者可从一代EGFR-TKIs治疗中获益-11，三代EGFR-TKI(奥希替尼)针对T790M突变的患者具有显著的疗效。研究表明，不同靶向药物发生耐药的机制不同，如一线EGFR-TKI的主要耐药机制是T790M，而三代EGFR-TKI的耐药机制包括C797S等。由于治疗药物引起的时间异质性和可能有新发病灶的原因，指南均建议在靶向药物耐药时对患者进行再次活检，明确耐药机制，根据耐药机制选择精准的后续治疗。IPASS、OPTIMAL、NEJ002、WJTOG3405、EURTAC等多项临床试验已表明，EGFR突变阳性的患者可以从EGFR-TKI治疗中获益, 15。本研究发现，再次活检中肿瘤组织标本仍然是主要的标本来源，这可能与本研究单位计算机断层扫描引导下的细针穿刺项目的开展有关，也与临床医生对于肿瘤组织是金标准的认知有关。再次活检中，血液标本送检的例数增加，这与采用ctDNA检测T790M的可及性和临床研究发现相关，BENEFIT、AURA等多项临床试验已证明血液EGFR突变率略高于再次活检标本的EGFR突变率(约为4%)，分析原因可能在于携带EGFR突变的细胞对EGFR-TKI治疗敏感，治疗导致基因突变阳性细胞数量减少。此外也可能是由于再次活检肿瘤标本取材困难，采用ctDNA进行EGFR基因检测的敏感性较差，存在假阴性的情况, 17。本研究的数据与以往文献报道相似，王洁等采用ARMS法分析了264例患者NSCLC患者的血浆和组织标本，其中54例(20.5%)患者在化疗前为阳性突变，化疗后转变为了野生型；24例(9.1%)野生型的患者，在接受化疗后转变为了突变型。这与我们的研究结果相似，在本研究中5例患者在接受化学治疗前后EGFR突变状态发生改变。差异类型分别为S768I转换为G719X+S768I(n=1)、野生型转换为突变型(n=3，2例19Del、1例G719X)和19Del转换为野生型(n=1)。综上所述说明化疗对EGFR突变状态是有影响的，因此在化学治疗后，动态监测EGFR基因状态是十分重要的。再次活检与治疗前标本相比，基因突变的阳性率有变化，治疗前采用数字PCR对使用EGFR-TKI药物的NSCLC患者的ctDNA进行检测，发现在117例EGFR-TKI继发耐药的患者中，有47%(55/117)的患者发生了EGFR T790M突变，并且在EGFR T790M阳性和阴性的继发耐药患者中，年龄、性别、组织学、吸烟史及EGFR-TKI作为几线药物治疗等方面均无明显差异。在另一项Ⅱ期临床试验中，共招募60例患者，分别于2个月及肿瘤进展时抽取患者外周血，使用数字PCR检测EGFR 19del、L858R及T790M的突变状态，最终有44例患者的肿瘤发生进展，其中行再次活检的35例患者中，发现有66%(23/35)的标本发生了EGFR T790M突变；有39例患者通过数字PCR检测ctDNA，有23%(9/39)的患者也发现了EGFR T790M突变。在本研究中，一线接受靶向治疗的患者RR和CBR均高于一线接受化疗的患者。这个结果与以往研究相一致。综上所述，靶向治疗对EGFR突变状态是有影响的，EGFR突变的患者接受不同治疗方案的临床获益也有差异，因此在患者治疗进展后，监测EGFR基因状态的改变，根据EGFR基因状态的改变对患者的治疗方案进行调整是很重要的。再次活检中，主要的突变类型为T790M，比例为56.2%，这一结果与以往研究相一致，Zheng等本实验具有一定的局限性，如标本数量较少，对于突变影响因素分析时纳入的指标少，未来还需要更多的样本和更细致的分析，发现真实世界再次活检和初次活检标本中的最重要影响因素。EGFR基因突变状态差异较大，因此为了更精准地克服耐药，了解耐药后的肿瘤驱动基因状态，有必要在耐药时进行再次活检，明确EGFR基因突变状态。综上所述，一线采用化疗或EGFR-TKI治疗的患者，在真实世界的临床实践中，治疗前和疾病进展时


To document the underlying science of how the Pan American Health Organization (PAHO) adapted the Hanlon method, which prioritizes disease control programs, to its wider range of program areas and used it to implement the PAHO Strategic Plan 2014 – 2019.In 2014, PAHO established a Strategic Plan Advisory Group (SPAG) with representatives from 12 Member States to work closely with the PAHO Technical Team to adapt the Hanlon method to disease and non-disease control programs. Three meetings were held in 2015 – 2016 during which SPAG reviewed existing priority-setting methods, assessed the original Hanlon method and subsequent revisions, and developed the adapted method. This project was initiated by Member States, facilitated by PAHO, and conducted jointly in transparent and horizontal technical cooperation.From the original Hanlon equation, the PAHO-adapted method maintains components A (size of problem), B (seriousness of problem), and C (effectiveness of intervention), drops component D , and adds component E (inequity) and F . The PEARL score was dropped because it serves a purpose for pre-screening process, but not in the priority-setting process for PAHO.The PAHO-adapted Hanlon method provides a refined approach for prioritizing public health programs that include disease and non-disease control areas. The method may be useful for the World Health Organization and country governments with similar needs. Priority-setting is an important component of strategic planning . Given tThis article summarizes the findings of a research project conducted by PAHO and 12 Member States to adapt the Hanlon method for use in developing and implementing the PAHO Strategic Plan 2014 – 2019 . The purIn 2013, during the development of the Strategic Plan , a ProgrIn 2014, a Strategic Plan Advisory Group (SPAG) was established to refine the Framework. Its purpose was to: (a) review other published priority-setting methods; (b) assess the strengths and weaknesses of the original Hanlon method and subsequent revisions; and (c) develop a PAHO-adapted Hanlon method. Given the limitations of the original Hanlon method, the objective of this study was to refine the methodology to suit the breadth of PAHO programs.This research project was conducted during the development of the PAHO Strategic Plan 2014 – 2019. More than 15 priority-setting methods were reviewed in search of a reasonably objective and potentially useful method to prioritize PAHO programs . SeveralThis study combined expert consultation by an advisory group with methodology development by a technical group. Because the final, adapted method would affect all countries and territories of the Americas, the experts needed to represent the entire Region. The expert team, the SPAG, included representatives from 12 Member States hailing from all four sub-regions of the Americas ; namely, the Bahamas, Brazil, Canada, Chile, Costa Rica, Ecuador, El Salvador, Jamaica, Mexico, Paraguay, Peru, and the United States of America.In order to facilitate the process, the SPAG established a technical core group of experts, led by Canada, that included several technically-oriented members of the SPAG and the PAHO Technical Team. The aim of this technical group was to review published priority-setting methods and to develop an adapted Hanlon method. This group also provided technical inputs to address issues and challenges identified by SPAG. This collaborative approach proved to be an effective way to achieve the project objectives. SPAG worked with the PAHO Technical Team via virtual and face-to-face meetings.The technical core group presented findings to three SPAG face-to-face meetings for consideration and approval. The first meeting was held in May 2015 in Washington, D.C. It established an action plan to address Member States’ request to improve the Framework, and produced a revised PAHO-adapted Hanlon equation with refined definitions of several variables. The second meeting was held in August 2015 in Mexico City. It pilot-tested an improved version of the PAHO-adapted Hanlon method and developed a process and timeline for application of the new method by Member States. Pilot testing was conducted during the meeting by two separate groups: the 12 country representatives of the SPAG and 20 senior managers of the Mexican Secretariat of Health. The third meeting was held in April 2016 in Washington, D.C. It validated the final version of PAHO-adapted Hanlon method through a final pilot test by SPAG members.There are a wide variety of methods for priority-setting . Simple The original Hanlon equation was published in 1984 , calculated as the sum of four factors, each worth 0 – 10 points ; C is the effectiveness of the intervention (0 – 10 points); D is its feasibility (0 or 1), determined by five factors (each 0 or 1) that are referred to as “PEARL” . Because the obtainable product of the four components  has a range of 0 – 300 points, dividing by 3 gives the BPR a range of 0 – 100.Vilnius and Dandoy  made sevwhere B ÷ C is benefit-cost ratio (0 – 10 points). With the five components, dividing by 6 gives the BPR a range of 0 – 100.Neiger and colleagues  made a sEarlier versions of the BPR model suggested that analysis of the PEARL criterion should occur after all data had been collected and translated to BPR scores. Since the function of PEARL is to determine whether stakeholders should proceed with or eliminate a health problem, this criterion should be analyzed prior to data collection and analysis .In other words, they consider PEARL to be a pre-screening process to identify the health problems for ranking and not part of the priority-setting process:The PAHO-adapted Hanlon method was designed to rank the breadth of PAHO programs, both disease-oriented and non-disease-oriented . The oriThe PAHO context also needs two new components: E (inequity) and F :where A is the size of the problem (0 – 10 points), meaning prevalence or incidence of the disease (for disease-oriented programs) or extent of system or program deficiencies (for non-disease-oriented programs); B is the seriousness of problem (0 – 20 points), derived from the sum of four factors: B1 (urgency), B2 (severity of consequences), B3 (economic loss), and B4 (negative impact on others), each with 0 – 5 possible points. For non-disease-oriented programs, this also considers seriousness associated with deficiencies of the system or program and consequences of inaction; C is the effectiveness of intervention (0 – 10 points) based on the availability of cost-effective interventions to address the problem or deficiencies in systems or programs. It is the product of 2 factors: C1 (efficacy) and C2 (reach or coverage), each with a range of 0% – 100%. For non-disease-oriented programs, this is a qualitative assessment of effectiveness. E is inequity (0 – 5 points), measured by the degree of differential occurrence of disease or access to health programs between socially-determined population sub-groups  deemed to be unjust . F is the institutional positioning (a factor from 0.67 – 1.50), and refers to the extent to which an institution, such as PAHO, is uniquely positioned to assist a Member State address a program need from its own perspective. Initially, the trial range of F was 0.50  to 2.00 (a doubling effect), but during pilot testing these values were found to be too overwhelming. The maximum value was reduced from 2.00 to 1.50 and found to be satisfactory during further testing. As F is a multiplier, if the maximum is 1.50, the minimum is its reciprocal, or 0.67. A score of 1.00 means a Member State believes PAHO could maintain its technical cooperation at the current level. A score greater than 1.00 signifies that PAHO should increase its technical cooperation, while a score less than 1.00 means PAHO should decrease support because the Member State can deal with the problem or has another strategic partner assisting. Dividing by 5.25 gives the BPR a range of 0 – 100.For component A (size of problem), various measures are used, e.g., the percent of the population exposed to the problem. For diseases, size of the problem refers to the prevalence/incidence. Points: 0 – 3 = low prevalence/incidence; 4 – 6 = medium; and 7 – 10 = high. For non-diseases , size of problem refers to the extent of the problem . It can be measured by percent of the population affected by the problem , or degree of limitations in response capacity. Points: 0 – 3 = low percent of population adversely affected; 4 – 6 = medium; and 7 – 10 = high.Component B (seriousness) has four factors. Urgency (B1) means whether a problem is worsening, stabilizing, or improving, or whether progress is made towards achieving the target, based on previous 5-year trend data. Points: 0 – 1 = problem improving or good progress; 2 – 3 = problem remains the same; and 4 – 5 = worsening. Severity of consequences (B2) is measured by the extent of premature mortality and disability, loss of quality of life, or burden to health services caused by the problem. Points: 0 – 1 = low; 2 – 3 = medium; and 4 – 5 = high. Economic loss (B3) is the social costs, both direct and indirect, associated with the problem. While factor B4, originally defined as “involvement of other people” . In an eEffectiveness (C) is the degree of success of an intervention in producing a desired outcome under usual circumstances. Following the original definition , effectiComponent D (PEARL) is deleted from the equation, consistent with the justifications provide by Neiger and colleagues  and the Component E (inequity) is a new feature in the PAHO-adapted equation. Health inequities are defined as “observable differences in health between two or more socially-determined groups that are judged to be unjust; that is, arbitrary, unnecessary, and avoidable” . InequitComponent F  is another new feature of the PAHO-adapted method. This component was based on the concept first suggested by Musgrove  that higThis PAHO project shows the advantages and effectiveness of the expert consensus approach. Three face-to-face meetings over a total of 8 days with an average of 20 experts (or 160 person-days), plus virtual meetings and individual time invested by members of the SPAG and the PAHO Technical Team, provided the basis for the development of the PAHO-adapted Hanlon method. The final product arising from this expert consensus approach would have been impossible for one expert to complete despite working for 160 days. A single expert would not have been able to identify and resolve the issues from the various sub-regions and multi-factor issues related to the wide range of PAHO programs. The expert group approach also fostered a team environment, as experts from different areas of the Region tackled a common problem together. At the conclusion of this project, the SPAG members had developed strong links that would enhance future country collaboration on PAHO endeavors. Such collaboration has been considered instrumental to developing new strategic frameworks for the Region, as shown by the involvement of more Member States on the new SPAG for the PAHO Strategic Plan 2020 – 2025. Expert consensus is a methodology that has been used successfully in other PAHO prioritization exercises .The PAHO-adapted Hanlon method was approved by the PAHO Directing Council for implementation across the Region. To implement, PAHO first makes an official request to the national health authority of each Member State/Territory for one template (a fillable spreadsheet) with the results for that country. Each country template is based on national and, where appropriate, Regional considerations. PAHO supports a group approach for data collection, following the recommendations of Vilnius and Dandoy . In eachThere were several limitations to this study. First, the participatory process assumed that the only actors with the capacity to contribute to developing the adapted method were among the representatives of the 12 Member States of the SPAG. This might have led to bias as academic institutions were excluded. However, PAHO invited academic professors to provide subject matter expertise at SPAG face-to-face meetings. In addition, several of the SPAG members were also university professors. Second, the range of points for the new components (inequity [E] and positioning [F]) was based on expert consensus and may need to be further adjusted and fine-tuned in the field. Third, there were issues with the operational definitions of some terms, e.g., effectiveness, coverage, and equity. Fourth, limited information for several programs and the subjective nature of criteria assessment may have led to inconsistent application of the methodology across countries in the Region. Fifth, lack of public health experts familiar with the national health situation and the work of PAHO may have been an issue.The PAHO-adapted Hanlon method has many advantages. First, the proposed new Hanlon method applies to both a "disease-oriented (pathogenic) vision” and a "health-oriented  vision" . Second, it has a positive impact in the Region by focusing available PAHO resources on high priority programs. Third, the priority-setting process is transparent, objective, and applied in a systematic manner. Fourth, it is a participatory process that strengthens collaboration between PAHO and its Member States. Fifth, the PAHO-adapted Hanlon method has been validated against the ranking results from a more time consuming Delphi exercise among a group of senior health managers in Mexico, conducted by the Mexican Secretariat of Health independent of the SPAG process. Sixth, the proposed new method allows inclusion of important societal values, such as equity. Seventh, adjustment of the final priority score is enabled by an institutional positioning factor based on political consideration. This prevents the priority-setting exercise from becoming a purely mechanical process, and thereby increases its practical value in a real-world setting.There are other methods for health-related decision making that involve scores, including the EVIDEM framework based on seven modules , and a MThe PAHO-adapted Hanlon method has made a significant contribution to the science and practice of strategic planning. It has expanded the Hanlon method to encompass and align with new regional and global contexts by covering both disease- and non-disease-oriented programs and by considering equity and politics in priority-setting. The adapted method is more relevant and useful to the wider scope of health and public health. It will inform priority-setting as specified in the WHO 2016 Guide for Country Cooperation Strategy . The metBCKC, RAM, and OJM developed and implemented the project methodology. OJM and RAM with the PAHO Technical Team and PAHO SPAG (including BCKC) developed the initial PAHO adaptation of the Hanlon method and applied it with PAHO Member States. BCKC and RAM collected and responded to issues raised by the SPAG. OJM developed data collection and analysis tools for pilot testing. The PAHO Technical Team (including RAM and OJM) provided technical data required by the SPAG, and analytical support on pilot testing. SPAG members (including BCKC) collected issues from Member States and guided progress of the project. All authors contributed to the interpretation of results. BCKC wrote the first draft and RAM and OJM co-wrote the second draft of the manuscript. All authors contributed to subsequent revisions of various drafts, and final approval of the manuscript.Members of the PAHO Strategic Plan Advisory Group who contributed as authors: Martha Leticia Caballero Abraham and Laura E. Gloria Hernández (Chairpersons), the Secretariat of Health, Mexico City, Mexico; Cristina Luna Ribadeneira and Peter N. Skerrett Guanoluisa (Vice Chairpersons), Ministry of Public Health, Quito, Ecuador; Keva S. Thompson, Ministry of Health, Nassau, Bahamas; Thaisa Santos Lima and Juliana V. Borges Vallini, Ministry of Health, Brasilia, Brazil; Bernard C.K. Choi, Public Health Agency of Canada, Ottawa, Ontario, Canada; Odette A. Urrutia Pumeyrau and Raquel Child Goldenberg, Ministry of Health, Santiago, Chile; María Rosibel Vargas Gamboa, Ministry of Health, San José, Costa Rica; Matías Villatoro, Nadia P.R. Villalta, and Maria Elena Marroquin, Ministry of Health, San Salvador, El Salvador; Michele Roofe and Karen Webster-Kerr, Ministry of Health, Kingston, Jamaica; Patricia A. Giménez León and Juan Carlos Coronel Zarate, Ministry of Public Health and Social Welfare, Asunción, Paraguay; Victor Raul Cuba Oré and Martin Yagui, Ministry of Health, Lima, Peru; and Jay McAuliffe, United States Centers for Disease Control and Prevention, Atlanta, Georgia, United States.Members of the PAHO Technical Team who contributed as authors: Federico Gerardo de Cosio, Blanca Cousiño, José A. Escamilla-Cejudo, Travis High, Rony A. Maza, Guillermo Mendoza, Andrea Morales, Oscar J. Mujica, Donna-Lisa Peña, Antonio Sanhueza, Maria Teresa Villen, and Daniel J. Walter, Pan American Health Organization, Regional Office of the World Health Organization for the Americas, Washington, DC, United States.The authors would like to express deep appreciation to PAHO for providing the webinar platform for the virtual meetings and travel funds for many members of the Strategic Plan Advisory Group attending face-to-face meetings. We gratefully acknowledge PAHO and the Secretariat of Health of Mexico for providing the meeting facility in Washington and Mexico City. Our gratitude to PAHO and Member States for their commitment and support of the project, and thanks to the external reviewers for their thoughtful and constructive suggestions on the manuscript.Funded by the Pan American Health Organization, Washington, DC, United States. The funders had no role in the study design, data collection or analysis, decision to publish, or preparation of the manuscript.RPSP/PAJPH and/or PAHO.Authors hold sole responsibility for the views expressed in the manuscript, which may not necessarily reflect the opinion or policy of the The views expressed in this paper are those of the authors and do not necessarily reflect those of the Public Health Agency of Canada (PHAC) and the Government of Canada.The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention (CDC)/the Agency for Toxic Substances and Disease Registry.


We are pleased to report the increase of the impact factors of MDPI journals during 2007 and 2008 see . In 2005Sensors and we thus see a continuous increase of the impact factor in recent years. The 2008 impact factor of the journal Sensors is 1.870. On the other hand, the two tier publication system (Open Access and non-Open Access) was only briefly applied to the journal Molecules papers were retracted because they had also been published elsewhere. Nevertheless, these withdrawn papers were cited elsewhere, while the non-Open Access papers were not. We have now added a watermark to the original PDF files of withdrawn papers, clearly identifying their status, in order to prevent further citation of such papers [We also observed an interesting phenomenon: two h papers . www.mdpi.com. Just recently we published the 5000th paper in a MDPI journal and Molecules alone has published over 2000 papers. As we wrote in our editorial [Molecules and for all the other MDPI journals, including several newly launched journals.Since October 2008, all MDPI journals have been published at the new server ditorial , the yea