The aim of this study was to analyze current scientific impact of Ss Cyril and Methodius University of Skopje, Republic of Macedonia in the Scopus Database (1960-2014).Affiliation search of the Scopus database was performed on November 23, 2014 in order to identify published papers from the Ss Cyril and Methodius University of Skopje (UC&M), Republic of Macedonia. A total number of 3960 articles  were selected for analysis (1960-2014). SCImago Journal Rank (SJR), Source Normalized Impact per Paper (SNIP) and h-index were calculated from the Scopus database.The number of published papers was sharply increased with maximum of 379 papers in 2012 year. The largest number of papers has been published in Macedonian Journal of Medical Sciences, Journal of Molecular Structure, Lecture Notes in Computer Science, Acta Pharmecutica, and Macedonian Journal of Chemistry and Chemical Engineering. The biggest SJR and SNIP has journal Nephrology Dialysis Transplantation. First three places of the top ten authors belong to Dimirovski GM, Gavrilovska L, and Gusev M. Top three places based on Scopus h-index  belong to Kocarev L, Stafilov T, and Polenakovic M. The majority of papers originate from UC&M, but significant numbers of papers are affiliated to Faculty of Medicine, Faculty of Pharmacy, and Institute of Chemistry as members of UC&M, as well as Macedonian Academy of Sciences and Arts. Articles are the most dominant type of documents followed by conference papers, and review articles. Medicine is the most represented subject.Officials of the Ss Cyril and Methodius University of Skopje should undertake more effective and proactive policies for journal publishers and their Editorial Boards in order to include more journals from UC&M in the Scopus database. Scopus is the world’s largest abstract and citation database of peer-reviewed literature with smart tools that track, analyze and visualize research. Easy to use and comprehensive, Scopus is designed to quickly find the information researchers’ need. Scopus indexes over 20,500 titles from 5,000 publishers worldwide; contains 49 million records, 78% with abstracts; includes over 5.3 million conference papers; and provides 100% Medline coverage  and one in Trnava, Slovakia [AF-ID (“University of SS Cyril and Methodius Trnava” 60021677)] which was excluded from analysis.A total number of 3960 articles, all from Ss Cyril and Methodius University , were selected for analysis (1960-2014). SCImago Journal Rank (SJR), Source Normalized Impact per Paper (SNIP) and h-index were calculated from the Scopus database [The number of publication in the period of 1960-1991 year was very small (1-29 papers per year). Starting of 1991 year, the number of published papers was sharply increased with maximum of 379 papers in 2012 year .The largest number of papers from Ss Cyril and Methodius University of Skopje have been published in Macedonian Journal of Medical Sciences (136 papers), followed by Journal of Molecular Structure (79 papers), Lecture Notes in Computer Science (46 papers), Acta Pharmecutica (38), and Macedonian Journal of Chemistry and Chemical Engineering (37 papers). The rest of journals published less than 36 papers  top. TheThe number of papers published by top ten authors is shown in Scopus Hirsh index (h-index) for the top ten authors affiliated to Ss Cyril and Methodius University of Skopje was presented as calculated by Scopus. We can see from the The biggest number of papers belongs to Ss Cyril and Methodius University (3051 papers), but significant number of papers belongs to Faculty of Medicine, Ss Cyril and Methodius University (860 papers), Faculty of Pharmacy, Ss Cyril and Methodius University (145 papers), Institute of Chemistry, Cyril and Methodius University (138 papers), as well as Macedonian Academy of Sciences and Arts (105 papers)  left. MoDocuments by type and documents by subject area are published by Ss Cyril and Methodius University of Skopje, shown in In this paper analysis of documents affiliated to Ss Cyril and Methodius University of Skopje deposited in the Scopus database (1960-2014) is presented. Starting of 1991 year, the number of published papers was sharply increased with maximum of 379 papers in 2012 year. The largest number of papers from Ss Cyril and Methodius University of Skopje has been published in Macedonian Journal of Medical Sciences, Journal of Molecular Structure, Lecture Notes in Computer Science, Acta Pharmecutica, and Macedonian Journal of Chemistry and Chemical Engineering. The biggest SCImago journal rank per year (SJR) and Source Normalized Impact per Paper (SNIP) has journal Nephrology Dialysis Transplantation. First three places of the top ten authors belongs to Dimirovski GM, Gavrilovska L, and Gusev M. Top three places based on Scopus h-index  belongs to Kocarev L, Stafilov T, and Polenakovic M.The majority of papers originate from Ss Cyril and Methodius University of Skopje , but significant number of papers are affiliated to Faculty of Medicine, Faculty of Pharmacy, and Institute of Chemistry as a members of Ss Cyril and Methodius University of Skopje, as well as Macedonian Academy of Sciences and Arts. Most of the papers affiliated to Ss Cyril and Methodius University of Skopje originate from Macedonia, Yugoslavia, United States, and Germany. Articles are the most dominant type of documents followed by conference papers, and review articles. Medicine is the most represented subject. Engineering, computer science, chemistry, biochemistry, genetics and molecular biology, physics and astronomy are represented in more than 10%.From the top 10 journals participating in the Ss Cyril and Methodius University of Skopje publishing, only two of them are published in Republic of Macedonia . The biggest SCImago journal rank per year and the biggest Source Normalized Impact per Paper have the journal Nephrology Dialysis Transplantation, published outside the Republic of Macedonia. Thus, the impact of journals from the Ss Cyril and Methodius University of Skopje participating in Scopus database is very small. Other journals supported, attached or affiliated to mention UC&M as a publisher are not participating at all in the Scopus database. Officials of the UC&M should undertake more effective and proactive policies for journal publishers and their Editorial Boards in order to include more journals from UC&M in the Scopus database.http://www.researcherid.com/) [http://orcid.org/) [Analysis of author rank is very sensitive issue. In this analysis total number of published papers in Scopus database was bigger than the number of papers affiliated to Ss Cyril and Methodius University of Skopje in the same database. These differences are caused by affiliation changes of the top ten authors. For example, Dimirovski GM was affiliated with 121 papers to Ss Cyril and Methodius University of Skopje, and the rest of 139 papers are affiliated to Dogus Universitesi Istanbul, School of Engineering, Istanbul, Turkey. Similarly, Soptrajanov B, Polenakovic M, and Kocarev L have additional profiles  as a result of their affiliation to Macedonian Academy of Sciences and Arts. Additional problems arise when author impact analysis search is used: i) some authors have used different names in different periods of time; ii) some authors transliterate their names in several different ways; iii) some authors add or do not add “v”; iv) some authors use only one of their two surnames; v) there are some identical names and surnames in the world for different researchers . All of id.com/)  and in tid.org/) . IdentifFor the first time in Republic of Macedonia, current scientific impact of academic staff employed at the Institutes of the Faculty of Medicine, Ss Cyril and Methodius University of Skopje, Republic of Macedonia, was analyzed and published. Based on different citation values and citation indexes, it was concluded that Institutes of the Faculty of Medicine are very heterogeneous with three groups of institutes: institutes with higher scientific impact; institutes with intermediate scientific impact, and institutes with low scientific impact . ScientiRecently we presented the results of SCImago Institutions Rankings for R. Macedonia (2009-2012) and show that only Ss Cyril and Methodius University of Skopje were included in the SIR with world rank of 1726 to 1821. Regional rank in Eastern Europe was gradually increased from 156th in 2009 to 127th in 2013 year [In summary, analysis of documents affiliated to Ss Cyril and Methodius University of Skopje deposited in the Scopus database (1960-2014) have shown that the number of published papers was sharply increased with maximum of 379 papers in 2012 year. The largest number of papers from Ss Cyril and Methodius University of Skopje has been published in Macedonian Journal of Medical Sciences. The biggest SCImago journal rank per year and Source Normalized Impact per Paper has journal Nephrology Dialysis Transplantation. First three places of the top ten authors belong to Dimirovski GM, Gavrilovska L, and Gusev M. Top three places based on Scopus h-index  belong to Kocarev L, Stafilov T, and Polenakovic M. Officials of the UC&M should undertake more effective and proactive policies for journal publishers and their Editorial Boards in order to include more journals from UC&M in the Scopus database.


These results suggest that SIM may, independent of its cholesterol-lowering effect, suppresses the progression of vascular diseases through the inhibition of the inflammation mediators TNF-α and TNFR1.Simvastatin (SIM) is anti-inflammatory. We used low density lipoprotein receptor knockout (LDLR-/-) mice and human aortic smooth muscle cells (HASMCs) as model systems to study the effect of SIM on arterial calcification and to explore the potential mechanisms contributing to this protective effect. High-fat diet (HFD) caused the LRLR -/- to develop dyslipidemia, diabetics, atherosclerosis and aortic smooth muscle calcification. SIM, N-acetyl cysteine  and apocynin  did not significantly retard the development of dyslipidemia or diabetic. However, those treatments were still effective in attenuating the HFD-induced atherosclerosis and aortic smooth muscle calcification. These findings suggest that the protective effect of SIM against aortic calcification is not contributed by the cholesterol lowering effect. SIM, NAC and APO were found to attenuate the HFD induced elevation of serum TNF-α, soluble TNFR1 (sTNFR1), 3-nitro-tyrosine. We hypothesized that the pro-inflammatory cytokine, oxidative stress and TNFR1 played a role in inducing aortic calcification. We used HASMC to investigate the role of TNF-α, oxidative stress and TNFR1 in inducing aortic calcification and to elucidate the mechanism contributes the protective effect of SIM against aortic calcification. We demonstrated that treating HASMC with TNF-α induced cell Ca deposit and result in an increase in ALP, NADPH oxidase activity, NF-kB subunit p65, BMP2, MSX2, and RUNX2 expression. SIM suppressed the TNF-α induced activation of NADPH oxidase subunit p47, the above-mentioned bone markers and TNFR1 expression. Furthermore, p65, p47 and TNFR1 siRNAs inhibited the TNF-α-mediated stimulation of BMP-2, MSX2, RUNX2 expression. SIM, APO, and NAC either partially inhibit or completely block the TNF-α induced H After centrifugation at 500xg for 10 min at 4°C, the cells were resuspended in 80 μl of buffer B (buffer A containing 0.1% Triton X-100) by gentle pipetting. The cell lysates were allowed to stand on ice for 10 min and then centrifuged at 12,000xg for 10 min at 4°C. Nuclear pellets were re-suspended in 70 μl of ice-cold buffer C , incubated on ice for 30 min with intermittent mixing, and then centrifuged at 15,000xg for 30 min at 4°C.Nuclear protein extracts were prepared as previously described . In brie3VO4, 1 μg/mL leupeptin, and 1 mM PMSF, pH 7.5). The protein concentrations were determined with BioRad Protein Assay Reagent , and the samples were stored at -70°C.Cell membrane fractions were prepared as described previously , with mophox, the translocation of cytosolic NF-kB p65 to the nucleus, as well as the level of MSX-2, a transcription factor in the nucleus of HASMCs stimulated with TNF-α. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes . The membranes were blocked with 5% milk and then probed with anti-p47phox, anti-TR1, anti-p65, goat anti-MSX-2, BMP-2, and RUNX2 (1:1000) antibodies. Then, they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. The proteins were visualized with an enhanced chemiluminescence detection kit . Anti-β-actin (1: 5000), anti-caveolin-1 (1:1000) and anti-hnRNA c1/c2 (1:1000) antibodies were used as loading controls. Protein expression levels were quantified as optical densities using ImageQuant software v. 5.2.Western blot analysis was used to determine the changes in the cell-surface levels of the NADPH oxidase component p47NADPH oxidase activity was determined with superoxide-dependent lucigenin chemiluminescence, as previously described . Cell me2O2 of nonfluorescent DCFH-DA to fluorescent 2’,7’- dichlorofluorescin (DCF). Confluent HASMCs in 24-well plates will be pretreated with various concentrations of antioxidant reagents. The cells were washed with PBS, and then serum-free M231 containing 10 μM DCFH-DA was added and incubation continued for 45 min at 37°C. The fluorescence intensity (relative fluorescence units) was measured at 485 nm excitation and 530 nm emission using a Fluorescence Microplate Reader.The effect of antioxidant reagents on ROS production in HASMCs was determined by a fluorometric assay using DCFH-DA as the probe. This method is based on the oxidation by HSmall interfering RNA (siRNA) oligonucleotides against TNFR1, p65, and p47 were suspended at a concentration of 10 μM. Cells were seeded for transfection in medium without antibiotics at one day before transfection to ensure that they were 85%–95% confluent on the day of transfection. For transfection, regular medium was replaced with serum-free medium without antibiotics. The cells were transfected with siRNA using oligofectamine at a ratio of 1 siRNA: 2 oligofectamine (μg:μl) at a final concentration of 25–50 nM siRNA. The cells were incubated with the siRNA-oligofectamine complex for 5 h, the serum-free medium was replaced with normal medium (containing 10% FBS) without antibiotics, and the cells were incubated for a total of 48 h before further analysis.t test or one-way ANOVA. A p<0.05 was considered statistically significant. SPSS 9.0  software package was used for all statistical analyses.All data were expressed as the mean±SEM for the continuous variables and as the number (percent) for the categorical variables. Intergroup comparisons were performed by Student's LDLR-/- mice were fed a normal diet (n = 6) or an HFD (n = 10) for 6 months. As predicted, HFD-induced artery atherosclerotic plaque formation was detected by H&E staining . It appeHFD induced dyslipidemia and diabetic condition and dyslipidemia and diabetic condition which are believed to contribute to aortic calcification . SIM, APin vitro and in vivo [Tables  in vivo , 17. TheHFD drastically increased the serum TNF-α level in the LDLR -/- mice and SIM, NAC and APO treatment effectively attenuate HFD fed LDLR-/- mice pro-inflammatory cytokine expression  and oxidWe hypothesized that TNF-α activation lead to ROS production and the increase in ROS stimulates activate MSX2 program to cause a pro-calcific response. This hypothesis explains why antioxidant reagents like NAC and APO would attenuate HFD induced oxidation stress, elevation of TNFR1 and aortic calcification.Our working hypothesis postulates that TNF-α stimulates HASMCs differentiation into osteoblast type of cell linage to induce calcium deposition. If this working hypothesis is true, then treating HASMC with TNF-α should result in increases HASMC calcium deposition, increases in osteogenic marker proteins expression and ALP activity. HASMC were cultured in osteogenic differentiation medium in the presence or absence of non-toxic concentrations of TNF-α (5 and 10 ng/mL) for 4 days. We found that TNF-α induced HASMC calcium deposition, upregulate ALP activity and increase in BMP2, MSX2, and RUNX2 expression . Data arWe culture HASMCs in an osteogenic differentiation medium in the presence or absence of non-toxic concentrations of TNF-α (5 and 10 ng/mL) for 3 days, followed by SIM (1 μM) treatment for 1 day. Treating HASMC with 2 μM of SIM for 24 hours did not produce any adverse effect on cell vitality as indicated by MTT assay . If SIM 2O2 (2O2 by TNF-α yet both treatments significantly abolish the effect of TNF-α induced elevation of NADPH activity (Our hypothesis postulates that activation of TNFR by TNF-α lead to oxidative stress (ROS production) and ROS promotes VSMC to differentiate into osteogenic cell linage which leads to aortic calcification . Accordi2O2 . SIM andactivity . These ractivity .NADPH oxidase consists of membrane-bound (gp91phox and p22 phox) and cytosolic components . NADPH oxidase activation requires the translocation of the cytosolic components p47 phox to cell membrane . WesternIt is known that TNF-α can induce MSX2 expression via the NF-κB pathway . P65 is It is known TNFR1 plays a crucial role in TNF-α-induced ROS generation  and in BWe further investigated the effects of the combined use of siRNAs against TNFR1, p65 and p47 on the expression of osteogenic markers because the use of a single siRNA did not have significant effects . The knoFinally, we confirmed the effect of SIM on TNFR1 accumulation in the media layer of the aorta of the HFD-fed mice by IHC staining. The results showed that TNFR1 was significantly increased in the HFD-fed mice and could be attenuated by SIM . We nextThere are several types of vascular calcification, such as atherosclerosis and medial calcification, which are often observed in elderly people and patients with diabetes mellitus and/or chronic kidney disease , 17. In APO, NAC or SIM reduced the HFD-induced elevation of serum TNF-α  and nitr2O2 production NADPH oxidase activity  calcification . In precof NF-κB , 44. NF-of NF-κB . Furtherof NF-κB , 47.in vivostudy, SIM attenuated the HFD induced elevation of serum TNF-α  blocking the activation of cell membrane surface NADPH, and 2) down regulation of TNF-alpha and TNFR1. The reduction in the inflammation mediators TNF-α and TNFR1 suggest that SIM may suppress the progression of vascular diseases.Our findings indicate that SIM lowers inflammatory mediator TNF alpha induced TNFR1 to lower the oxidative stress in HASMC and animal models. As a result of this anti-inflammatory effect, SIM retard the progression of artery calcification in LDLR -/- mice. These results provide novel evidence that SIM treatment attenuates arterial calcification and these effects are demonstrated through potent anti-inflammatory and antioxidant properties of SIM. Our findings provide novel evidence that SIM treatment may attenuate arterial calcification. In addition, ample evidence exists in support of the potent anti-inflammatory properties and antioxidant effects of SIM . HoweverIn summary, in addition to the known roles of statins in influencing endogenous cholesterol levels by inhibiting HMG-CoA reductase and other pleiotropic effects, such as anti-inflammatory, anti-proliferative, and anti-thrombotic effects , 64, we S1 Figp<0.05 compared to the control group, and #p<0.05 compared to the TNF-α groups. N = 6 for each set of experiments.HASMCs were cultured in osteogenic differentiation medium for 3 day in the presence or absence of TNF-α (10 ng/mL) concomitantly with apocynin (A) or NAC (B). *(TIF)Click here for additional data file.S2 FigWestern blotting assay the effects of the knock-down of the TNFR1, p65 and p47 proteins by these siRNAs indicated that they were functioning. Compared with the TNF-α-stimulated cells in the presence of scrambled siRNAs, any of the TNFR1, p65 or p47 siRNAs, didn’t dramatically abolished TNF-α-stimulated bone marker expression in the HASMCs. N = 6 for each set of experiments.(TIF)Click here for additional data file.


In recent years emphasis has been placed on evaluation studies and the publication of scientific papers in national and international journals. In this regard the publication of scientific papers in journals in the Institute for Scientific Information (ISI) databaseis highly recommended. The evaluation of scientific output via articles in journals indexedin the ISI database will enable the Iranian research authorities to allocate and organizeresearch budgets and human resources in a way that maximises efficient science production. The purpose of the present paper is to publish a general and valid view of scienceproduction in the field of stem cells. In this research, outputs in the field of stem cell research areevaluated by survey research, the method of science assessment called Scientometricsin this branch of science. A total of 1528 documents was extracted from the ISI databaseand analysed using descriptive statistics software in Excel. The results of this research showed that 1528 papers in the stem cell field in the Webof Knowledge database were produced by Iranian researchers. The top ten Iranian researchers in this field have produced 936 of these papers, equivalent to 61.3% of the total. Amongthe top ten, Soleimani M. has occupied the first place with 181 papers. Regarding internationalscientific participation, Iranian researchers have cooperated to publish papers with researchers from 50 countries. Nearly 32% (452 papers) of the total research output in this field hasbeen published in the top 10 journals.These results show that a small number of researchers have publishedthe majority of papers in the stem cell field. International participation in this fieldof research unacceptably low. Such participation provides the opportunity to importmodern science and international experience into Iran. This not only causes scientific growth, but also improves the research and enhances opportunities for employment and professional development. Iranian scientific outputs from stem cell researchshould not be limited to only a few specific journals. One criterion ofa country’s stage of development and evolution is its research potential and scientific capacity, and one of the most important indicators of this is the number of indexed scientific papers in valid and creditable databases. Hereby, through measurement of scientific productions, it is possible to provide a view of the scientific activity status of a country. Today, these citation databases form the base of several studies that evaluate the scientific level of countries of the world using various scientometric methods.Stem cell research is considered to be one of themost important fields in Iranian medical research,and one which has improved significantly in recentyears. According to the 2014 obtained results fromthe Institute for Scientific Information (ISI) database,the United State of America has occupiedfirst place in the field of stem cell research by producing104306 scientific papers. Among countriesin the region, only Turkey, which is 26Scientometric experts believe that each researcher in any area or field is and should be able to engage with scientometric activities so that, he/she can clearly present the current state of research to peers and officials, and thereby help them make more effective decisions . The firThe history of introducing "scientometrics" as a term refers to 1969, when a Russian scientist named Vasili Vasilevich Nalimov used the term Naukometrija in an article which means scientometrics in English. In a short time, this term was translated into English under the term of "scientometrics" .Although it is several decades since scientometrics emerged internationally and a journal with this title (SCIENTOMETRICS) has been successfully published since 1978, scientometrics is almost new in Iran, although it has appeared in recent years under different heading, most frequently bibliometrics.As it mentioned before, researchers in the field of scientometrics believe that researchers and experts in all fields should be involved with scientometrics. It is with this in mind that the present study has been conducted. Using information from ISI and other databases a number of different studies have been done, some of which are mentioned here.A study in the same field with the title "evaluation of studies related to stem cells", which included four countries; Taiwan, Hong Kong, Singapore and Korea, based on the ISI database was conducted outside our country. Findings and results of this study showed that, although Taiwan is the first country to have done primary scientific research in this field, scientific output from South Korea has outstripped the other countries. Hong Kong and Taiwan showed a good growth too. The results of this study also showed that seven industrial countries, named Group 7 , produced more than 70% of the scientific research in this field from 1981 to 2001 .The other research done by Li et al.  under thAnother study evaluated scientific output in the field of medical science based on indexed papers in scientific journals in selected databases from 2005 to 2009. The results of this study showed that the number of publications in the field of medical science was lacking, such that during the years of the evaluation, Iranian scientific output in the field of medicine was developing .An evaluation was conducted of 625 papers in sciences websites from the Medical Science of Iran University up to the end of 2007. The results of this study showed that in five case studies made on immunology, circulatory system, neuroscience, surgery and pharmacology, only three authors wrote the article individually and this fact indicated the high tendency of many writers in scientific collaboration and co-authorship. According to this research, the highest level of scientific cooperation is found in the field of immunology (Poris has investigated the status of acquired immune deficiency syndrome (AIDS) scientific output in South Africa and has compared it with other countries from 1996 to 2006. The findings showed that the total number of AIDS papers that were onthe ISI website during the studied period indicated a42% increase. It includes approximately 5% of AIDSresearch at global level. Also the subject fields of immunology,public health and virology respectively,have obtained the highest share of AIDS research ofthis country. The pattern of international cooperationamong the authors showed that about 24% of AIDSpapers have been composed with the cooperation ofAmerican researchers. England, Canada and France,with a total of 2%, are also significant collaboratorsin South African research. In relation to institutions,Harvard and California University have cooperatedthe most with South Africa Organizations for accomplishingresearch projects .With this introduction and due to the importantrole of research in all fields, this study intendsto investigate the scientific output of Iranian researchersin the field of stem cells according to theISI databases. The study will identify the strengthsand weaknesses of research in this field sp practitionersand researchers can take appropriate actionto remedy the weaknesses as well as further developingthe strengths.This survey research includes all papers by Iranianresearchers in the field of stem cell research that havebeen reviewed from 1992 to July of 2014, based onthe Web of Science citation index, which is a sub databaseof ISI institution in Philadelphia of USA.According to the Journal Citation Reports (JCR),more than 13000 core journal in the ISI database areindexed. For doing this research the key word "stemcell" was entered. By entering this key word, all theitems such as: "stem cell" , "stem cells", "stem cellcontaining", "stem cell transplant" , "stem cellulose"were retrieved. Also, a general search about stemcells was done to identify pioneer countries in scientificoutputs in this field as well as other countries inthe region. All the records found were investigatedregarding the following items: international participation,organization affiliation, the years of printing thepapers, the format of papers, the name of journals thathave printed the most papers, and the subjects that arestudied by researchers. The found data was analyzedand evaluated Excel software. To ensure the accuracyof the data, the same searches were undertaken againin the ISI database at the end of the analyses. The resultof this second searching process was exactly thesame as that obtained from the first search.According to the According to According to According to According to According to table 6, all Iranian scientific papersin this field have been published in form of 8 typesof papers among which ARTICLE with 1123 papersequal to 73.49 percent is in first place and MEETINGABSTRACT with 252 papers and REVIEW with 107papers are in second and third places respectively.The results of this research showed that, overall, Iranian researchers’ have produced 1528 papers in the field of stem cell research. In terms of institutions, Tehran Medical Science University, with 396 papers equal to 25.92% of all the scientific output, is in first place. Tarbiat Modares Univ with 304 papers and Tehran University with 163 papers and the Royan Institute with 162 papers are in second, third and fourth places respectively. Overall, 320 organizations have participated in scientific output in this field. The most active researchers in this field, Solimani M, with 181 papers equal to 11.846% of the total scientific output in this field is in first place and Baharvand H, with 174 papers and Gavamzadeh A, with 163 papers are in second and third places respectively.In general, the 10 top authors in this field, altogether have participated in producing 936 papers equal to 61.25% of the total scientific output. Regarding scientific participation all scientific productions in this field have involved co-operation  and not Considering the journals that have printed the most scientific productions in this field, Yakhteh (Cell J) belonging to the Royan Institute, has published the most papers in this area in Iran has occupied first place in this regard. The journal of Bone Marrow Transplantation with 87 papers equal to 5.69% is in second place and Cell Biology International with 34 papers equal to 2.23% is in third place. As a whole, top the 10 journals have printed 452 papers equal to 31.9% of the total scientific output in this field. Iranian researchers' scientific papers in this area have been published in 201 journals.Taking the presented papers format into consideration, all of the Iranian contributions to the field of stem cell research have been printed in the form of 8 types of papers among which ARTICLE with 1123 papers equal to 73.49% is in first place and MEETING ABSTRACT with 252 papers and REVIEW with 107 papers are in second and third places respectively.The contribution of Iranian publications to the field of stem cell research was evaluated according to papers cited on the ISI database. During the period covered by this research, from 1996 to 2013, Iranian contributions have followed a growing trend and rapid growth and development of scientific output in this field is clearly evident. In terms of international collaboration perhaps more could be done. Regarding the journals that have printedscientific papers in this field, the results show thatthe researchers of this field have limited their scientificproductions only to specific journals. Thiscase is also not desirable and by recognizing otherjournals of this field, this problem should be resolved.


Vast numbers of scientific articles are published each year, some of which attract considerable attention, and some of which go almost unnoticed. Here, we investigate whether any of this variance can be explained by a simple metric of one aspect of the paper's presentation: the length of its title. Our analysis provides evidence that journals which publish papers with shorter titles receive more citations per paper. These results are consistent with the intriguing hypothesis that papers with shorter titles may be easier to understand, and hence attract more citations. ColosTwitter –9, Wikipikipedia –13, Goog [Google –17, news [Google  and even [Google –21 can n [Google ,14,16, h [Google  and natu [Google ,23.Scopus contains citation records of papers, offering remarkable insights into academic conversation. Recently, advances have been made in quantifying scientific output based on publication statistics [Scientific endeavours also generate extensive written communication, in the form of papers. We define a paper to be more successful than others if it has received a greater number of citations. The online database atistics –28. A nuatistics ,30. Furtatistics –33 as weatistics . In addiatistics .Here, we consider whether we can find any evidence that the style in which a paper is written may relate to its success. Specifically, we consider the length of the article title chosen by the authors and investigate whether the length bears any relation to the number of citations. Previous studies have explored different characteristics of scientific paper titles –41. A su2.Scopus, one of the leading bibliometric platforms. A Scopus user can search and export data on journal articles in batches of 20 000 records, including data on how often each article has been cited since publication. We download data on the 20 000 most cited papers in each year between 2007 and 2013.We analyse data provided by a). Upon visual inspection, there appears to be a high concentration of papers with short titles and many citations, as well as a high concentration of papers with long titles and few citations. We find that for the top 20 000 most highly cited papers published in 2010, papers with shorter titles receive more citations . We apply the same analysis to each year in our sample and find that papers from all years exhibit this relationship between their title length and citations  correction).We determine the number of characters in each paper's title, including spaces and punctuation. Using the year 2010 as an example, we rank the papers' title length and citations a. Upon vc; τ=−0.020, N=15 395, p<0.001, Kendall's τ correlation). Again, we run parallel analyses for the 20 000 most cited papers in each year between 2007 and 2013. For years 2007–2010, we find that papers with shorter titles receive more citations, whereas papers published during 2011–2013 do not . These smaller τs suggest that the journal in which a paper is published may help explain the relationship between paper title length and the number of citations the paper receives.Some journals may attract a greater number of citations for their papers owing to their reputation. To remove any potential influence of the journal in which a paper is published on the relationship between citations received and paper title length, we rank all of the papers in terms of the number of citations received and transform these ranks into percentiles. We calculate percentiles in terms of the length of papers' titles in the same fashion. In this transformed dataset, for papers published in 2010, we find that papers with shorter titles receive more citations . Parallel analyses for papers published in each year between 2007 and 2013 show that this relationship holds for papers published in all 7 years in our sample . Finally, we carry out a complementary aggregated analysis across all years of data in our sample. We rank all papers published in a given year by citations received and by title length, and transform these ranks into percentiles for that year. Again, we find that journals which publish papers with shorter titles also tend to receive more citations per paper .To investigate this hypothesis further, we group papers by their journal. Again, using 2010 as an example, we calculate the median number of citations and median title length for each journal. We find that journals which published papers with shorter titles also tend to receive more citations per paper . The values of the slope L are given for all years in Our primary analysis is based on rank-based statistics. To complement our analysis, we fit a mixed-effects model to the log of the number of citations a paper receives as a function of its title length controlling for the journal in which each paper is published. A mixed-effects models allows us to control for the journal in which each paper is published. We define our model ascj is the median number of citations received by papers published in journal j. The intercept is I, and there is a slope L for the median number of characters in the titles of papers lj published in journal j. Again, we log the citation counts, so that the distribution of the residuals of our model, ϵ, is closer to a Gaussian distribution. We fit the model for each year. We find that journals which publish papers with shorter titles also tend to receive more citations per paper . The values of the slope L are given for all years in Again, we investigate if this relationship exists when aggregating papers by the journal in which they are published. We fit a linear regression model to the median number of citations papers receive per journal as a function of the median title length. We define our model as3.In this study, we investigate whether the length of a scientific paper's title is related to the number of citations it receives. We analyse the 20 000 most highly cited papers for the years 2007–2013, representing a sample size between 1.12% and 1.53% of all papers published in each of these years. Previous studies analysing much smaller sets of papers have reported conflicting evidence, suggesting either that the length of a paper's title bears no relation to its scientific impact , or thatOur analysis suggests that papers with shorter titles do receive greater numbers of citations. However, it is well known that papers published in certain journals attract more citations than papers published in others. When citation counts are adjusted for the journal in which the paper is published, we find that the strength of the evidence for the relationship between title length and citations received is reduced. Our results do however reveal that journals which publish papers with shorter titles tend to receive more citations per paper.We propose three possible explanations for these results. One potential explanation is that high-impact journals might restrict the length of their papers' titles. Similarly, incremental research might be published under longer titles in less prestigious journals. A third possible explanation is that shorter titles may be easier to understand, enabling wider readership and increasing the influence of a paper.Our findings provide evidence that elements of the style in which a paper is written may relate to the number of times it is cited. Future analysis will investigate whether further stylistic attributes of the language used in a paper can be related to the number of citations it receives.4.Scopus (http://www.scopus.com) between 21 October 2014 and 14 November 2014. To obtain data on the 20 000 most cited papers published in each of the 7 years from 2007 to 2013, we search for any papers that are marked by Scopus as an ‘article’ with the following search query:We retrieve bibliometric data from DOCTYPE(ar) ANDPUBYEAR = {year},year} is replaced by each of the years 2007–2013. In total, we retrieve 140 000 records. In 2007, Scopus reports 1 302 973 published papers which increases to 1 788 065 papers in 2013. The top 20 000 most cited papers published in each year represent a sample of 1.53% in 2007, decreasing to 1.12% in 2013.where {Analyst’ and ‘The Analyst’). For this reason, we clean the dataset from Scopus by deleting leading ‘The’s from each journal's title, and converting the title to lower case. We also identify all journals which have fewer than 10 papers in the most cited 20 000 papers for a given year, and remove the papers in such journals for that year. The basic characteristics of our dataset before and after cleaning are depicted in the electronic supplementary material, figure S1.Some journals are referred to with multiple variations of their name (for example, ‘


Mycobacterium tuberculosis (M. tuberculosis) which involves the activation of innate immune cells and structure maintenance of granulomas. However, the contribution of TNF, in particular neuron-derived TNF, in the control of cerebral M. tuberculosis infection and its protective immune responses in the CNS were not clear.Tuberculosis (TB) affects one third of the global population, and TB of the central nervous system (CNS-TB) is the most severe form of tuberculosis which often associates with high mortality. The pro-inflammatory cytokine tumour necrosis factor (TNF) plays a critical role in the initial and long-term host immune protection against −/−) mice and compared outcomes of disease against TNFf/f control and global TNF−/− mice. Mycobacterial burden in brains, lungs and spleens were compared, and cerebral pathology and cellular contributions analysed by microscopy and flow cytometry after M. tuberculosis infection. Activation of innate immune cells was measured by flow cytometry and cell function assessed by cytokine and chemokine quantification using enzyme-linked immunosorbent assay (ELISA).We generated neuron-specific TNF-deficient (NsTNFM. tuberculosis infection of TNF−/− mice rendered animals highly susceptible, accompanied by uncontrolled bacilli replication and eventual mortality. In contrast, NsTNF−/− mice were resistant to infection and presented with a phenotype similar to that in TNFf/f control mice. Impaired immunity in TNF−/− mice was associated with altered cytokine and chemokine synthesis in the brain and characterised by a reduced number of activated innate immune cells. Brain pathology reflected enhanced inflammation dominated by neutrophil influx.Intracerebral Our data show that neuron-derived TNF has a limited role in immune responses, but overall TNF production is necessary for protective immunity against CNS-TB. Tuberculosis of the central nervous system (CNS-TB) is associated with significant mortality and high distressing levels of neurological morbidity, the majority of survivors suffering permanent neurologic complications –3. It diMycobacterium tuberculosis from the primary pulmonary infection, or rupture of an adjacent parenchymal focus [M. tuberculosis can cross the BBB as a free organism or via infected peripheral myeloid cells [M. tuberculosis [CNS-TB results from a rupture of subpial or subependymal foci which has been deposited earlier following lympho-haematogenous dissemination of al focus , 9. It iid cells . Howeverrculosis , howeverrculosis , 13.M. tuberculosis in the central nervous system [M. tuberculosis in a permissive extracellular niche [M. tuberculosis infection in the CNS. Here, we generated neuron-specific TNF-deficient (NsTNF−/−) mice and investigated outcomes after intracerebral M. tuberculosis infection in comparative studies with TNFf/f control and global TNF−/− mice.Tumour necrosis factor (TNF) has been shown to be critical in the pathogenesis of s system –16. TNF s system –20. The s system , 22 and s system  have shoar niche , 23, 24.f/f (TNF-floxed wild-type [http://jaxmice.jax.org/strain/003966.html), NsTNF−/− (neuron-derived TNF-deficient) and TNF−/− , Syn1-Ceficient ) mice, wf/f mice was previously described [−/− mouse were genotyped for Cre expression using the primer sets IMR1084 (5′ GCG GTC TGG CAG TAA AAA CTA TC 3′) and IMR1085 (5′ GTG AAA CAG CAT TGC TGT CAC TT 3′), and the internal controls IMR7338 (5′ CTA GGC CAC AGA ATT GAA AGA TCT 3′) and IMR7339 (5′ GTA GGT GGA AAT TCT AGC ATC ATC C 3′). For the presence of the TNF loxP gene in NsTNF−/− mice, primers KO41 (5′ TGA GTC TGT CTT AAC TAA CC 3′) and KO42 (5′ CCC TTC ATT CTC AAG GCA CA 3′) were used [Genotyping of mouse strains was performed by PCR analysis of DNA extracted from tail biopsies. Genetic analysis of TNFescribed . Syn1-Crere used .M. tuberculosis with green fluorescent expressing protein  was grown in Middlebrook 7H9 medium (Difco™ Laboratories) containing 25 μg/ml kanamycin and 0.5 % glycerol and enriched with 10 % OADC. Bacterial cultures were incubated at 37 °C and grown until log phase, aliquoted and stored at −80 °C. A frozen aliquot of M. tuberculosis was thawed, passed 30 times through a 29-G needle and diluted in sterile saline. Mice were infected by aerosol inhalation at a dose of 200–500 colony-forming units (CFUs)/lung under biosafety level 3 conditions using a Glas-Col Inhalation Exposure System Model A4224. For analysis, infected mice were deeply anaesthetised and transcardially perfused with 4 % paraformaldehyde. Brains were sectioned at 40 μm using a vibratome and processed for immunohistochemical staining. Neurons were labelled with polyclonal anti-MAP2  or anti-β-III-tubulin antibody , and anti-rabbit Cy3-conjugated secondary antibody . The microglia/monocytes were labelled with monoclonal anti-CD11b antibody  and anti-rat Cy3-conjugated secondary antibodies . The sections were incubated with nuclear marker DAPI (Sigma) and then mounted in fluorescent mounting medium and images captured using a Zeiss 510LSM unit.M. tuberculosis strain H37Rv was grown at 37 °C in Middlebrook 7H9 broth containing 10 % OADC and 0.5 % Tween 80 until log phase, then aliquoted and stored at −80 °C. A frozen aliquot was thawed, passed 30 times through a 29-G needle and diluted in sterile saline. Intracerebral infection was performed using a stereotaxic approach of directly injecting M. tuberculosis H37Rv into the cerebral cortex. Prior to inoculation, a small burr hole was constructed anterior to the bregma and to the left of the midline in the skull exposing the dura mater. Five mice per strain were inoculated intracerebrally with 1 × 103–1 × 104 CFUs of M. tuberculosis H37Rv using a Hamilton syringe . The burr hole was sealed with bone wax and the skin sutured. Animals received a prophylactic pain killer for 3 days at 24-h intervals. The clinical scoring system was adapted from previous reports [ reports , 27. HerM. tuberculosis. Organs were weighed and homogenised in 0.04 % Tween 80 saline. Tenfold serial dilutions of organ homogenates were plated in duplicates on Middlebrook 7H10  agar plates containing 10 % OADC  and were incubated at 37 °C for 19–21 days. The concentration of M. tuberculosis was then determined by counting the CFUs.Bacterial burdens in the brains, lungs and spleens of infected mice were determined at specific time points after infection with f/f, NsTNF−/− and TNF−/− mice were intracerebrally stimulated with 5 μg/ml of lipopolysaccharide (LPS) for 90 min. Mice were transcardially perfused with 4 % paraformaldehyde. For single-cell suspensions, brains were isolated and tissue passed through a 70-μm nylon cell strainer (Beckton and Dickinson) and washed 2× with phosphate-buffered saline (PBS) and the cell concentration was determined by counting in the presence of trypan blue. TNF expression in neurons was measured by intracellular staining through the addition of saponin buffer to permeabilise the cells, which were then labelled with polyclonal anti-β-III-tubulin antibody  and an anti-rabbit PE-conjugated secondary antibody , and anti-TNF antibody .To determine the expression level of TNF by neurons, TNF3 and incubated with the appropriate antibodies for 20 min in the dark. Excess antibodies were removed by washing cells 2× with PBS/0.1 % BSA/0.01 % NaN3. Pelleted cells were fixed for 18–24 h in 2 % phosphate buffered formaldehyde and analysed on a FACSCalibur (Beckton Dickinson) flow cytometer using CellQuest software. Microglia were defined as CD11b+CD45low, macrophages as CD11b+CD45high and dendritic cells as CD11c+CD45high as previously described [To analyse surface marker expression in microglia, macrophages and dendritic cells, the following antibodies were used: CD11b:PerCP-Cy5-5 ; CD11c:Alexa 700 ; CD45:APC ; CD80:FITC ; CD86:V450  and MHCII/(I-A/I-E):PE . Cells were washed with PBS/0.1 % bovine serum albumin (BSA)/0.01 % NaNescribed , 29.M. tuberculosis infection. The chemokines MCP-1, MIP-1α and RANTES, and the cytokines IL-1β, IL-12p70 and TNF  were measured using commercially available ELISA reagents according to the manufacturer’s instructions. Chemokine and cytokine concentrations were measured by absorbance using a Versamax Microplate Reader  with SoftMax software.Supernatants from brain homogenates were prepared for cytokine and chemokine measurement by enzyme-linked immunosorbent assay (ELISA) after 3 weeks subsequent to intracerebral t test for comparisons amongst the time points. For all tests, a p value of ≤0.05 was considered significant.The data are presented as the mean ± standard error of the mean (SD). Statistical analysis was performed by one-way ANOVA and one-tailed M. tuberculosis dissemination and latent infection [M. tuberculosis dissemination which causes extrapulmonary TB including severe CNS-TB [M. tuberculosis dissemination to the CNS, we challenged wild-type (TNFf/f) and TNF−/− mice by aerosol inhalation with M. tuberculosis H37Rv-GFP. In contrast to TNFf/f mice, TNF−/− mice were unable to control pulmonary TB infection, which resulted in high bacilli burdens in the lungs and spleens  through mating of Syn1-Cre mice [f/f) [Cre/wtTNFf/wt F1 offspring by PCR analysis. Typically, Syn1-CreCre/wtTNFf/wt mice displayed the 100-bp amplification product representative of the Cre gene , and offspring were analysed by tail biopsy analysis. The F2-generation Syn1-CreCre/wt TNFf/f mice displayed the 400-bp amplification fragment representative of the TNF-floxed gene while the normal wild-type TNF gene was absent  , 37. We f/f, TNF−/− and NsTNF−/− mice were inoculated with 3 μl LPS (5 μg/ml) in the left brain hemisphere by intracerebral inoculation. Single-cell suspensions, generated from harvested brains, were labelled and analysed for neurons expressing TNF. We confirmed expression of TNF in neurons of TNFf/f mice and the absence of TNF in both TNF−/− and NsTNF−/− mice through comparative TNF mean fluorescence intensity analysis which were both negligible in TNF−/− and NsTNF−/− mice  in TNF−/− mice  within 16–21 days post-infection in TNF−/− mice  in TNF−/− mice at 2 weeks post-infection which were sustained at 3 weeks post-infection. In contrast, NsTNF−/− mice controlled acute infection at burden levels equivalent to those in TNFf/f mice and were similarly significantly lower (p < 0.01) compared to those in TNF−/− mice  of MIP-1α, MCP-1 and RANTES at 3 weeks post-infection  levels of activated innate immune cells were measured at 3 weeks post-infection.The increased presence of innate immune cells in the absence of TNF supports the potential for improved early control of cerebral D86 Fig. . We obsef/f, NsTNF−/− and TNF−/− mice after 3 weeks of intracranial infection  lower than in TNFf/f and NsTNF−/− mice. Therefore, the data indicates that TNF is required to regulate cytokine synthesised by innate immune cells but that neuron-derived TNF is not important.To further investigate innate immune cell functionality, we measured levels of TNF, IL-1β and IL-12p70 in the brain homogenates of TNFion Fig. . The indrce Fig. . As expeice Fig. . Similarice Fig.  and IL-1ice Fig.  productiM. tuberculosis infection. Susceptibility is promoted either under conditions where excess TNF prevails or low TNF concentrations are present [M. tuberculosis targeting of neurons and its capability to generate reactive immune responses [M. tuberculosis control remains largely untested. In the presence of pathogens, neurons induce specific cytokine signatures, TNF comprising a key component of many such signature responses. The synthesis of TNF by neurons was observed after systemic immune challenge [M. tuberculosis infection. We therefore investigated the overall contribution of TNF and in particular the involvement of neuron-derived TNF in host-mediated immunity directed against M. tuberculosis during central nervous system infection. Several converging lines of study/evidence have indicated a neuroprotective role for TNF and its induction demonstrated to be a key determinant of disease outcome during cerebral infection [M. tuberculosis dissemination to the CNS and regulates immune-mediated brain pathology during infection. Moreover, we demonstrate for the first time that TNF is an absolute requirement to mediate protective immunity against CNS-TB; however, neuron-derived TNF is considered redundant. Thus, host susceptibility to CNS-TB in the absence of TNF corroborates its significance in neuroprotective immunity. Although the findings in this study appear contrasting to those of Tsenova et al., where TNF was reported to promote mycobacterial onset of disease and pathogenesis [M. tuberculosis infection, a shift in balance that favours either excessive or reduced levels being detrimental to the host that promotes conditions for mycobacterial persistence.TNF concentration forms a pivotal point of balance that determines outcome of disease during  present , 23, 24. present , 52, 53. present , 55. We esponses ; howeverhallenge . Upon vihallenge –60. In ohallenge , 61–63. nfection . In thisogenesis , it is nM. tuberculosis replication [M. tuberculosis infection or regulate immune pathology and suggest a far greater role for other resident or recruited cells to produce TNF. Interestingly, others have reported that TNF produced by neurons is redundant for mediating inflammation but that membrane TNF expressed on astrocytes has a functional role [Mycobacterial infection of the CNS generates a robust immune response characterised by both pro-inflammatory and anti-inflammatory immune cellular and cytokine profiles . While mlication . Our finnal role .M. tuberculosis infection [−/− mice also associates with a similar unregulated increase in chemokine synthesis during CNS-TB. Moreover, a lethal neutrophil-driven inflammatory response associated with, amongst others, MIP-1α in the absence of microRNA 223 in pulmonary tuberculosis was reported [Nonetheless, the overall requirement of TNF for control of both mycobacterial replication and immune-mediated pathology was evident and the rapid rate of host susceptibility suggested immune deficiency during early infection. Brain pathology, particularly at end-stage disease, was characterised by increased inflammation of dendritic cells and macrophages, and microglial proliferation which was underpinned by an enhanced pro-inflammatory chemokine environment in the absence of TNF. These observations align with earlier reports of a regulatory role for TNF in inflammation during nfection . In earlnfection . We showreported . Taken t−/− mice was the result of defective activation of innate antigen-presenting immune cells during early infection. Activation of macrophages, dendritic cells and microglia within the first 2 weeks of infection appeared to be independent of TNF with equivalent levels of activated cells evident in TNFf/f, NsTNF−/− and TNF−/− mice; however, the ability to subsequently sustain the required measures of activated cells was TNF dependent. These cells which are critical effectors of early innate immunity were rendered functionally defective in the absence of TNF. Initial cytokine signature responses by innate immune cells include synthesis of IL-12 and IL-1 [M. tuberculosis infection and is mediated largely by the presence of IL-1 and TNF [M. tuberculosis. Moreover, the reduction of MHCII- and CD80-expressing cells indicated reduced capacity to initiate M. tuberculosis-specific Th1 protective immune responses required for control of intracellular bacilli, and hence predisposed the host to a susceptible phenotype.Microglia, macrophages and dendritic cells as the main facilitators of early innate immune protection are activated in a TNF-dependent manner , 70. We and IL-1 , 72, botand IL-1 , 73–75. and IL-1  during M and TNF , 78. MorM. tuberculosis replication within the CNS. We further showed that neuron-derived TNF is not required for this process.In conclusion, the findings in this study corroborate earlier reports of the global importance of TNF in immune-mediated responses against tuberculosis and for the first time demonstrate its importance to control


Orientia (O.) tsutsugamushi-induced scrub typhus is endemic across many regions of Asia and the Western Pacific, where an estimated 1 million cases occur each year; the majority of patients infected with O. tsutsugamushi end up with a cytokine storm from a severe inflammatory response. Previous reports have indicated that blocking tumor necrosis factor (TNF)-α reduced cell injury from a cytokine storm. Since TNF-α production is known to be associated with intracellular Ca2+ elevation, we examined the effect of store-operated Ca2+ entry (SOCE) inhibitors on TNF-α production in O. tsutsugamushi-infected macrophages. We found that 2-aminoethoxydiphenyl borate (2-APB), but not SKF96365, facilitates the suppression of Ca2+ mobilization via the interruption of Orai1 expression in O. tsutsugamushi-infected macrophages. Due to the decrease of Ca2+ elevation, the expression of TNF-α and its release from macrophages was repressed by 2-APB. In addition, a novel role of 2-APB was found in macrophages that causes the upregulation of heat shock protein 70 (HSP70) expression associated with ERK activation; upregulated TNF-α production in the case of knockdown HSP70 was inhibited with 2-APB treatment. Furthermore, elevated HSP70 formation unexpectedly did not help the cell survival of O. tsutsugamushi-infected macrophages. In conclusion, the parallelism between downregulated Ca2+ mobilization via SOCE and upregulated HSP70 after treatment with 2-APB against TNF-α production was found to efficiently attenuate an O. tsutsugamushi-induced severe inflammatory response. Orientia (O.) tsutsugamushi in human skin , 26O. ts2+ influx to interrupt the release and expression of TNF-α and upregulating HSP70 to suppress TNF-α expression via activation of ERK or other pathways .(A) Knocked down HSP70 promotes translocation of NF-κB (green) to nucleus (blue) with pathogenic stimulation by immunofluorescence analysis. (B) Activation of NF-κB quantified by measurement of fluorescent intensity of NF-κB in nucleus area using an Olympus fluorescence microscope with an average fluorescence intensity of more than 1000 cells Click here for additional data file.


Background. Chagas disease (CD) is characterized by parasite persistence and immunological unbalance favoring systemic inflammatory profile. Chronic chagasic cardiomyopathy, the main manifestation of CD, occurs in a TNF-enriched milieu and frequently progresses to heart failure. Aim of the Study. To challenge the hypothesis that TNF plays a key role in Trypanosoma cruzi-induced immune deregulation and cardiac abnormalities, we tested the effect of the anti-TNF antibody Infliximab in chronically T. cruzi-infected C57BL/6 mice, a model with immunological, electrical, and histopathological abnormalities resembling Chagas' heart disease. Results. Infliximab therapy did not reactivate parasite but reshaped the immune response as reduced TNF mRNA expression in the cardiac tissue and plasma TNF and IFNγ levels; diminished the frequency of IL-17A+ but increased IL-10+ CD4+ T-cells; reduced TNF+ but augmented IL-10+ Ly6C+ and F4/80+ cells. Further, anti-TNF therapy decreased cytotoxic activity but preserved IFNγ-producing VNHRFTLV-specific CD8+ T-cells in spleen and reduced the number of perforin+ cells infiltrating the myocardium. Importantly, Infliximab reduced the frequency of mice afflicted by arrhythmias and second degree atrioventricular blocks and decreased fibronectin deposition in the cardiac tissue. Conclusions. Our data support that TNF is a crucial player in the pathogenesis of Chagas' heart disease fueling immunological unbalance which contributes to cardiac abnormalities. Trypanosoma cruzi . Anti-TNF therapy tended to decrease the levels of IL-17A, Il-6 and NOx in the serum in comparison with saline-injected chronically infected mice.Supplementary Figure S1: The high levels of IL-17A, IL-6 and NOSupplementary Figure S2: At 120 dpi, chronically Colombian-infected C57BL/6 mice show CD8-enriched myocarditis [18]. Anti-TNF therapy did not diminish the general inflammatory foci composed of CD8+, CD4+, and F4/80+ macrophages) ) but reduced the number of CD8+ cells infiltrating the cardiac tissue  and S2(c)). Therefore, we tested whether it was a site restricted effect examining the cell composition of a secondary lymphoid tissue. The frequencies of CD4+ and CD8+ T-cells in the spleen persisted unchanged after anti-TNF administration ).


Recent research on airway smooth muscle (ASM) cell biology and function has significantly broadened our understanding of the role these cells play in asthma. Apart from being structural contractile cells, they also have immunomodulatory properties. Dynamic multifunctional behavior of ASM cells, namely phenotype plasticity and functional diversity, is now-recognized to be directly involved with tissue inflammation that often interrelates with hypoxia in orchestrating airway remodeling and hyperresponsiveness characteristic of asthma.Herein, we sought to investigate the effect of the inflammatory mediator TNF-α and/or hypoxia on the expression of the ASM cell contractile markers SMMHC (smooth muscle myosin heavy chain), MLCK (myosin light chain kinase), α2 αctin and SM22.in vitro differentiated human bronchial ASM cell cultures were incubated for 24 hours in the presence or absence of TNF-α under normoxia or hypoxia. Total RNA was extracted, reverse transcribed and mRNA expression levels of SMMHC, MLCK, a2 αctin and SM22 were determined by quantitative real-time PCR.Primary TNF-α remarkably increased SMMHC mRNA expression under both normoxia and hypoxia. Treatment under hypoxia, with TNF-α, or their combination decreased MLCK and SM22 mRNA levels. In contrast, α2 αctin mRNA levels were not significantly affected under any of these conditions.TNF-α and hypoxia appear to alter the expression of human bronchial ASM cell contractile protein genes, providing new insights on the complex role of ASM cells in the pathophysiology of asthma. Remarkable up-regulation of SMMHC mRNA by TNF-α is indicative of the excessive ASM hypertrophy in severe asthma.


The inflammation-dependent adhesion molecule expressions are characterized in cardiovascular diseases and myocardial tissue infiltrations. Several pro-inflammatory cytokines are elevated in the acute myocardial injury and infarction. Tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine, is raised in the injury tissues and inflammatory regions and involved in the pathogenesis of cardiac injury, inflammation, and apoptosis. In fibroblasts, TNF-α-triggered expression of vascular cell adhesion molecule (VCAM)-1 aggravated the heart inflammation. However, the mechanisms underlying TNF-α-mediated VCAM-1 expression in cardiac fibroblasts remain unclear. Here, the primary cultured human cardiac fibroblasts (HCFs) were used to investigate the effects of TNF-α on VCAM-1 expression. The molecular evidence, including protein, mRNA, and promoter analyses, indicated that TNF-α-induced VCAM-1 gene expression is mediated through the TNFR-dependent manner. Activation of TNF-α/TNFR system triggered PKCα-dependent NADPH oxidase (Nox)/reactive oxygen species (ROS) signal linking to MAPK cascades, and then led to activation of the transcription factor, AP-1. Moreover, the results of mRNA and promoter assay demonstrated that c-Jun/AP-1 phosphorylated by TNF-α turns on VCAM-1 gene expression. Subsequently, up-regulated VCAM-1 on the cell surface of TNF-α-challenged HCFs increased the number of monocytes adhering to these cells. These results indicated that in HCFs, activation of AP-1 by PKCα-dependent Nox/ROS/MAPKs cascades is required for TNF-α-induced VCAM-1 expression. To clarify the mechanisms of TNF-α-induced VCAM-1 expression in HCFs may provide therapeutic strategies for heart injury and inflammatory diseases. Cardiovascular diseases are characterized by overexpression of inflammatory genes and infiltration of the inflammatory cells via adhesion molecule induction such as VCAM-1 . PreviouHeart diseases are associated with acute myocardial injury initiating a sequence of events that often lead to cardiac hypertrophy, chamber dilation, interstitial fibrosis, and congestive heart failure . Several⋅, and hydrogen peroxide (H2O2). Low levels of these oxidative-contained molecules are required for maintaining normal physiological functions and protecting against invading microorganisms (Reactive oxygen species (ROS) are generated by several enzymatic reactions and chemical processes or directly inhaled, including rganisms . Among trganisms . It has rganisms . In the rganisms . Accumulrganisms . Thereforganisms . HoweverTNF-α induces expression of several inflammatory genes by activation of TNF receptor (TNFR)-mediated diverse signaling molecules, including PKCs, ROS, MAPKs, and transcription factors . PreviouIn the study, to investigate the molecular mechanisms of TNF-α-mediated responses in HCFs, we found that Nox/ROS-mediated pathways were involved in VCAM-1 induction. These results demonstrated that TNF-α induces TNFR1/PKCα-dependent Nox/ROS/MAPKs cascade in HCFs. Through this pathway, TNF-α promoted the activation of c-Jun/AP-1 to increase VCAM-1 transcription and resulting in the VCAM-1 protein expression in HCFs. Moreover, we found that the increased VCAM-1 expression contributes with monocytes adhesion to TNF-α-challenged HCFs.DMEM/F-12 medium, fetal bovine serum (FBS), TRIzol reagent and PLUS-Lipofectamine were from Invitrogen . Hybond C membrane and enhanced chemiluminescence (ECL) detection system were from GE Healthcare Biosciences . PhosphoPlus PKCα (#9375), p42/p44 MAPK (#9101), p38 MAPK (#9211), JNK1/2 (#4668), and c-Jun (#2361) antibody kits were from Cell Signaling . VCAM-1 (sc-8304), PKCα (sc-208), Nox2 (sc-20782), Nox4 (sc-30141), p42 (sc-154), p38α (sc-535), JNK1 (sc-474), and c-Jun (sc-1694) antibody were from Santa Cruz . Ro31-8220, Gö6976, Edaravone, dipheneyleneiodonium chloride (DPI), U0126, SB202190, SP600125, and tanshinone IIA (TSIIA) were from Biomol . Apocynin (APO) was from Cayman . GAPDH antibody was from Biogenesis . Bicinchoninic acid (BCA) protein assay kit was from Pierce . SDS-PAGE reagents were from MDBio Inc . Enzymes and other chemicals were from Sigma .Human cardiac fibroblasts isolated from human fetal heart were obtained from ScienCell Research Laboratories  and grown in DMEM/F-12 containing 10% FBS, 2 mM glutamine and antibiotics  at 37°C. Reaching confluence, cells were treated with 0.05% trypsin/0.53 mM EDTA and plated onto 12-well culture plates and made quiescent at confluence by incubation in serum-free DMEM/F-12 for 24 h, and then incubated with TNF-α at 37°C for the indicated time intervals. When the inhibitors were used, cells were pretreated with the inhibitors for 1 h before exposure to TNF-α. Treatment of HCFs with DMSO, pharmacological inhibitors, or TNF-α alone had no significant effect on cell viability determined by an XTT assay (data not shown). Experiments were performed using cells from passages 3 to 6.g for 1 h at 4°C to yield the whole cell extract, as previously described  and 7500 Real-Time PCR System  using VCAM-1 primers and probe mixture  and endogenous GAPDH  served as an internal control gene. Relative gene expression was determined by the ΔΔCt method, where Ct meant threshold cycle. All experiments were performed in triplicate. PCR were performed to analysis the expressions of NOX1 , NOX2 , NOX3 , Nox4 , Nox5  and β-actin  was used to be the internal control.Human cardiac fibroblasts were seeded on 10-cm culture dishes and treated with TNF-α. Total RNA were extracted with TRIzol reagent  according to the protocol of the manufacturer. The cDNA obtained from 0.5 μg total RNA was used to be a template for PCR amplification . Real-tiTo construct VCAM-1-Luc plasmids, human VCAM-1 promoter region from -1716 to -119 bp , was inserted into pGL3-basic vector . The AP-1 and VCAM-1-Luc transcription activities were determined as previously described  using a TM RNAiMAX reagent according to the manufacturer’s instructions .HCFs (80% confluence) were washed once with PBS and 0.4 ml of serum-free DMEM/F-12 medium was added to each well. Human siRNAs of scrambled, PKCα (L-003523-00-0020) was from Dharmacon  and Nox2 (SASI_Hs01_00086110), Nox4 (SASI_Hs02_00349918), p42 (SASI_Hs01_00058601), p38α (SASI_Hs01_00176564), JNK1 (SASI_Hs02_00319556), and c-Jun (SASI_Hs02_00333461) were from Sigma . Transient transfection of siRNAs (100 nM) was performed using a LipofectamineThe peroxide-sensitive fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCF-DA) was used to assess the generation of intracellular ROS  with ming for 10 min at 4°C. The cell pellet was resuspended in 35 μl of ice-cold PBS and kept on ice. To a final 200 μl of PBS (pre-warmed at 37°C) containing either NADPH (1 μM) or NADH (1 μM) and lucigenin (20 μM), 5 μl of cell suspension (2 × 104 cells) was added to initiate the reaction followed by measurement of chemiluminescence immediately using an out-of-coincidence mode of Appliskan luminometer (Thermo®). Appropriate blanks and controls were established, and recorded chemiluminescence. Neither NADPH nor NADH enhanced the background chemiluminescence of lucigenin alone (30–40 counts/min). Chemiluminescence was continuously detected for 12 min, and the activity of Nox was expressed as counts per million cells.The Nox activity was examined by lucigenin chemiluminescence assay according to the previous report  with minoC. Cell adhesion assay was modified from the previous report  for 1 h at 37°C to label the THP-1 cells. The BCECF-labeled cells were washed thrice with serum-free DMEM/F-12, resuspended in serum-free DMEM/F-12, kept in the dark at room temperature and then the labeled THP-1 cells were added to HCFs for 1 h. Gently washing the plate with serum-free DMEM/F-12 to remove the non-adherent cells, the number of adherent cells was observed by using a fluorescence microscope .The human monocytic cell line THP-1 cells were obtained from ATCC  and maintained in DMEM/F-12 medium containing 10% FBS at 37s report . In Briet-test for western blot data. All others statistical analyses are comparison of multiple groups, a GraphPad Prism Program  by one-way analysis of variance (ANOVA) followed with Tukey’s post hoc test has been used. A P < 0.05 value was considered significant.All the data were expressed as the mean or mean ± SEM of five individual experiments performed in duplicate or triplicate. All these quantified numbers (mean ± SEM) related to the group data were presented and described in Supplementary Tables Figure 1A). TNF-α-induced VCAM-1 mRNA expression and promoter activity was also inhibited by Gö6976 (1 μM) . Next, the involvement of PKCα in TNF-α-induced responses was confirmed by determining its phosphorylation. TNF-α time-dependently stimulated PKCα phosphorylation with a maximal response within 15–30 min, which was attenuated by transfection with PKCα siRNA . Moreover, to ensure PKCα activity was involved in VCAM-1 expression, VCAM-1 protein was also analyzed in the PKCα siRNA transfected HCFs. We found that PKCα knockdown attenuated TNF-α-induced VCAM-1 expression about 65% . Our data suggested that the activity of PKCα transmits the TNF-α stimulation to induce VCAM-1 expression via enhancing its gene transcriptional activity in HCFs.PKCα has been shown to regulate the expression of several proteins in various cell types . To deteFigure 2A). Besides, both of VCAM-1 mRNA expression and promoter activity induced by TNF-α were attenuated by Edaravone (10 μM) . To evaluate the effect of TNF-α-stimulated ROS generation, as shown in Figure 2C, we found that TNF-α treatment time-dependently stimulated ROS generation and the maximal ROS generation was obtained within 60 min. This TNF-α-stimulated ROS generation was attenuated by pretreatment with Edaravone. These results demonstrated that Edaravone can efficiently scavenge ROS to block the VCAM-1 expression, suggesting that ROS generation contributes to TNF-α-induced VCAM-1 expression in HCFs.Previous report indicates that ROS can induce expression of adhesion molecules in various cell types . To deteFigure 3A, we pointed out that pretreatment with DPI attenuated TNF-α-induced VCAM-1 protein expression. Moreover, both VCAM-1 mRNA expression and promoter activity induced by TNF-α were also inhibited by 1 μM DPI . To further investigate whether TNF-α stimulates Nox activity, we found that TNF-α time-dependently stimulated Nox activity with a maximal response within 60 min in HCFs . We also determined which Nox isoforms are expressed and involved in the TNF-α-mediated responses. RT-PCR was performed to analyze the expression of Nox isoforms in HCFs. We found that Nox1, Nox2, Nox4, and Nox5 mRNA, in particular Nox4, can be detected in HCFs . Next, transfection with siRNAs of Nox isoforms was performed to confirm the roles of Nox(s) involved in the TNF-α-induced responses. As shown in Figure 3E, transfection with either Nox2 or Nox4 siRNA exhibited the consistent results that knockdown of the respective Nox protein expression can attenuate TNF-α-induced VCAM-1 expression in HCFs. Moreover, pretreatment with DPI (an inhibitor of Nox) abolished TNF-α-stimulated Nox activity and ROS generation . To further investigate the role of TNFR-mediated PKC(α) pathway in TNF-α-stimulated Nox activity and ROS generation, pretreatment with TNFR neutralizing antibody (TNFR nAb) or Gö6976 blocked TNF-α-stimulated Nox activity and ROS generation . These results suggested that TNF-α stimulates Nox(2/4)-derived ROS generation via TNFR-dependent PKCα cascade leading to VCAM-1 expression in HCFs.Nox complex is considered to be a major source of cytoplasmic ROS generation implicated in physiological and pathological processes . To inveFigure 4A). U0126 (100 nM) also repressed TNF-α-induced VCAM-1 mRNA expression and transcription activity . To investigate whether TNF-α-induced responses are mediated through activation of p42/p44 MAPK. The results of Figure 4C indicated that TNF-α treatment time-dependently stimulated p42/p44 MAPK phosphorylation with a maximal response within 10–15 min, which was attenuated by U0126 (100 nM). To further ensure the role of p42/p44 MAPK in regulation of VCAM-1-expression, p42 MAPK siRNA transfection was used to knock down p42 protein. We found that knockdown of p42 protein by p42 siRNA transfection also attenuated TNF-α-induced VCAM-1 expression . In addition, pretreatment with not only U0126, but also TNFR nAb (10 μg/ml), Gö6976 (10 μM), Edaravone (10 μM), and DPI (10 μM), could attenuate TNF-α-stimulated p42/p44 MAPK phosphorylation . These results suggested that TNF-α triggers the p42/p44 MAPK phosphorylation leading to VCAM-1 induction via a TNFR-mediated PKCα/Nox/ROS pathway in HCFs.Activation of MAPKs plays a key role in the pathogenesis of heart diseases such as myocardial hypertrophy . To deteFigure 5A). Pretreatment with SB202190 (10 μM) also attenuated TNF-α-induced VCAM-1 mRNA expression and transcription activity . To determine whether the activation of p38 MAPK was required for TNF-α-mediated responses, as shown in Figure 5C, TNF-α time-dependently stimulated phosphorylation of p38 MAPK with a maximal response within 10 min, which was prevented by SB202190 (10 μM). To further confirm the role of p38 MAPK in TNF-α-induced VCAM-1 expression, transfection with p38 MAPK siRNA to knock down p38 protein. We observed that knockdown of p38 protein also attenuated TNF-α-induced VCAM-1 expression . Moreover, phosphorylation of p38 MAPK stimulated by TNF-α was also attenuated by pretreatment with TNFR nAb (10 μg/ml), Gö6976 (10 μM), Edaravone (10 μM), or DPI (10 μM) . These results suggested that TNF-α activates TNFR/PKCα/Nox/ROS linking to p38 MAPK phosphorylation and leading to VCAM-1 expression in HCFs.To investigate the effect of p38 MAPK on VCAM-1 induction, pretreatment with a p38 MAPK inhibitor SB202190 concentration-dependently attenuated TNF-α-induced VCAM-1 expression . TNF-α-induced VCAM-1 mRNA expression and transcription activity was also attenuated by pretreatment with SP600125 (10 μM) . We further determined whether TNF-α-stimulated JNK1/2 activation is required for these responses, the activation of JNK1/2 was determined by Western blot using an anti-phospho-JNK1/2 antibody. As shown in Figure 6C, TNF-α time-dependently stimulated JNK1/2 phosphorylation with a maximal response within 15 min, which was attenuated by pretreatment with SP600125 (10 μM). To ensure the role of JNK1/2 in VCAM-1 expression, as shown in Figure 6D, transfection with JNK1 siRNA knocked down JNK1 protein and also attenuated TNF-α-induced VCAM-1 expression. Moreover, TNF-α-stimulated JNK1/2 phosphorylation was also attenuated by pretreatment with TNFR nAb (10 μg/ml), Gö6976 (10 μM), Edaravone (10 μM), or DPI (10 μM) . These results suggested that TNF-α-induced VCAM-1 expression is mediated through TNFR-dependent PKCα/Nox/ROS pathway linking to JNK1/2 phosphorylation in HCFs.To determine the role of JNK1/2 signaling in VCAM-1 expression, pretreatment with JNK1/2 inhibitor (SP600125) concentration-dependently attenuated TNF-α-induced VCAM-1 expression . Consistently, both of the TNF-α-induced VCAM-1 mRNA expression and promoter activity were also attenuated with pretreatment of TSIIA (1 μM) . We further determine whether TNF-α stimulated AP-1 activation required for VCAM-1 expression, the activated AP-1 was detected by Western blot using an anti-phospho-c-Jun antibody. As shown in Figure 7C, we found that TNF-α obviously stimulated c-Jun phosphorylation with a maximal response within 15 min, which was also inhibited by pretreatment with TSIIA (1 μM). To further ensure c-Jun/AP-1 involved in VCAM-1 expression, c-Jun siRNA transfection was used to knock down c-Jun protein expression. We found that knockdown of c-Jun protein also attenuated TNF-α-induced VCAM-1 expression . Moreover, we also differentiated the signaling components involved in TNF-α-stimulated the phosphorylation of c-Jun/AP-1. The results of Western blot indicated that pretreatment with TNFR nAb, Edaravone or DPI attenuated TNF-α-stimulated c-Jun phosphorylation. Next, to investigate whether activation of MAPKs led to c-Jun phosphorylation, we found that pretreatment with Gö6976 (10 μM), U0126 (1 μM), or SP600125 (3 μM) and p38 siRNA transfection also inhibited TNF-α-stimulated c-Jun phosphorylation . Furthermore, TNF-α time-dependently enhanced AP-1 transcription activity with a maximal response within 2 h , which was also attenuated by pretreatment with TNFR nAb or pharmacological inhibitors including Gö6976, DPI, Edaravone, U0126, SB202190, SP600125, or TSIIA . These results suggested that TNF-α-stimulated c-Jun/AP-1 activation via TNFR-mediated PKCα/Nox/ROS/MAPKs is required for up-regulation of VCAM-1 in HCFs.AP-1, heterodimer of c-Fos and c-Jun, is a well-known transcription factor to participate in the expression of inflammatory genes in various cell types . To deteFigure 8A, upper). As mentioned above, pretreatment with DPI, Gö6976, Edaravone, U0126, SB202190, SP600125, and TSIIA attenuated the TNF-α-induced VCAM-1 expression, consistently, the number of monocytes attached to TNF-α-challenged HCFs was also reduced by these pharmacological inhibitors . These results suggested that up-regulation of VCAM-1 via TNFR-mediated PKCα/Nox/ROS/MAPKs/AP-1 pathway plays a critical role to promote monocytes adhesion to TNF-α-challenged HCFs.Ultimately, we investigated the functional activity of up-regulated VCAM-1 by TNF-α in HCFs. Previous reports indicate that THP-1 monocytes can be used as a model system to mimetic the adhesion of monocytes to upregulated adhesion molecules on cell surface of resident cells induced by various stimuli . In thisUp-regulation of adhesion molecules on the surface of the heart cells plays a key role in the recruitment and infiltration of leukocytes at sites of inflammation in heart inflammatory disorders . TNF-α h2 expression in RA synovial fibroblasts (Excessive production of ROS has been shown to play a causative role in numerous pathologies of heart diseases . ROS alsroblasts . These rAbnormal MAPK regulation might lead to various inflammation and injury in the heart diseases . Among tThe progression of oxidative stress during cellular or tissue injuries not only causes damage to cellular macromolecules by oxidation, but also alters functional activity of transcription factors to modulate the pattern of gene expression. Here we focus on the roles of transcription factor AP-1, the ROS-dependent AP-1 activity which is well-known to modulate gene transcriptions during oxidative stress associated with inflammatory diseases . To inveThe infiltration of immune cells is due to up-regulation of adhesion molecules such as VCAM-1 , which cFigure 8B to depict the implicated components of signaling mechanisms in the TNF-α-treated HCFs. These findings imply that because TNF-α triggers the signal transductions of PKCα-mediated activation of Nox/ROS, MAPKs, and AP-1 to up-regulation of VCAM-1 in HCFs, the expression of adhesion molecules could enhance the attachment and linkage of monocytes to HCFs. The results provide new insights to understand the mechanisms by which TNF-α stimulated VCAM-1 expression in HCFs associated with amplifying the inflammatory responses, supporting the hypothesis that TNF-α may play a key role in the exaggeration of cardiac diseases.Based on the observations from literatures and our findings, we proposed a VCAM-1 gene expression model as shown in CCL, CCY, CYW, HCT, CSP, LDH, and CMY substantially contributed to the conception or design of the work, the acquisition, analysis, and interpretation of data for the work. CCL, CCY, CYW, HCT, CSP, LDH, and CMY drafted the work and revised it critically for important intellectual content. CCL, CCY, CYW, HCT, CSP, LDH, and CMY finally approved the version to be published. CCL, CCY, CYW, HCT, CSP, LDH, and CMY agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.


Electrochemotherapy is a tumour ablation modality, based on electroporation of the cell membrane, allowing non-permeant anticancer drugs to enter the cell, thus augmenting their cytotoxicity by orders of magnitude. In preclinical studies, bleomycin and cisplatin proved to be the most suitable for clinical use. Intravenous administration of cisplatin for electrochemotherapy is still not widely accepted in the clinics, presumably due to its lower antitumor effectiveness, but adjuvant therapy by immunomodulatory or vascular-targeting agents could provide a way for its potentiation. Hence, the aim of the present study was to explore the possibility of adjuvant tumour necrosis factor α (TNF-α) therapy to potentiate antitumor effectiveness of electrochemotherapy with intravenous cisplatin administration in murine sarcoma.In vivo study was designed to evaluate the effect of TNF-α applied before or after the electrochemotherapy and to evaluate the effect of adjuvant TNF-α on electrochemotherapy with different cisplatin doses.A synergistic interaction between TNF-α and electrochemotherapy was observed. Administration of TNF-α before the electrochemotherapy resulted in longer tumour growth delay and increased tumour curability, and was significantly more effective than TNF-α administration after the electrochemotherapy. Tumour analysis revealed increased platinum content in tumours, TNF-α induced blood vessel damage and increased tumour necrosis after combination of TNF-α and electrochemotherapy, indicating an anti-vascular action of TNF-α. In addition, immunomodulatory effect might have contributed to curability rate of the tumours.Adjuvant intratumoural TNF-α therapy synergistically contributes to electrochemotherapy with intravenous cisplatin administration. Due to its potentiation at all doses of cisplatin, the combined treatment is predicted to be effective also in tumours, where the drug concentration is suboptimal or in bigger tumours, where electrochemotherapy with intravenous cisplatin is not expected to be sufficiently effective. Electrochemotherapy is a non-thermal tumour ablation modality that is safe and effective in any solid tumour type. Electrochemotherapy is based on the local delivery of electric pulses that permeabilize the cell membrane, allowing non-permeant or low-permeant anticancer drugs to enter the cell, thus augmenting their cytotoxicity by orders of magnitude. Treatment is safe, very well tolerated by the patients, and its efficacy is very high on the different tumour nodules.11Among the drugs that have been tested in preclinical studies, bleomycin and cisplatin (CDDP) proved to be the most suitable for clinical use of electrochemotherapy. In the clinical use, both can be administered locally in the tumour, but only bleomycin systemically, intravenously, to obtain pronounced antitumor effect. Namely, in preclinical studies it was demonstrated that intratumoural administration of CDDP is more effective than systemic administration.2 at 37°C.Murine fibrosarcoma cell line SA-1  was grown as monolayer in Eagle Minimum Essential Medium (EMEM) with Glutamax , supplemented with 10% foetal calf serum (FCS) (Invitrogen) and gentamicin (30 μg/mL) (Invitrogen). Cells were routinely subcultured twice a week and incubated in an atmosphere with 5% CO2O. For each experiment, a fresh solution of CDDP was prepared. In vitro, the final concentrations of CDDP were prepared in EMEM. CDDP concentrations of 0.1 μg/mL and 0.5 μg/mL were used in the in vitro experiments.CDDP  was dissolved in sterile H3 U/mL, 2.5×104 U/mL, and 2.5×105 U/mL were used in the in vitro experiments.Murine recombinant TNF-α was obtained from Affymetrix eBioscience . The final concentrations of TNF-α were prepared in phosphate buffered saline (PBS) (Invitrogen). TNF-α concentrations 2.5×102HPO4; 2.5 mM KH2PO4; 2 mM MgCl2·6H2O) at 4°C. The final cell suspension (22 × 106 cells/mL) was prepared in electroporation buffer at 4°C. Cells (2 × 106) were mixed with CDDP and/or TNF-α. Half of this mixture (1 × 106 cells) was placed between two parallel stainless steel electrodes with a 2 mm gap in-between and subjected to eight square wave electric pulses (EP) with voltage to distance ratio 1300 V/cm, pulse duration 100 μs and frequency 1 Hz. EP were generated by generator of EP ELECTRO CELL B10 . The other half of the mixture served as a control for CDDP and/or TNF-α treatment alone. After electroporation, cells were incubated at room temperature for 5 min, diluted in 2 mL of growth medium and then plated for the clonogenic assay.Confluent cell cultures were trypsinized, washed in EMEM with FCS for trypsin inactivation and once in electroporation buffer , EP + TNF-α and ECT + TNF-α was determined by clonogenic assay. SA-1 cells were plated at a concentration of 200 cells/dish for exposure to CDDP or TNF-α alone and 500 cells/dish for combination of electroporation and CDDP or TNF-α. After 7 days colonies were fixed, stained with crystal violet and counted. The survival curve for the electrochemotherapy-treated cells was normalised for the cytotoxicity of EP treatment alone. The experiment was repeated twice in triplicates.ad libitum. Animals were subjected to an adaptation period of 7–10 days before the experiments were carried out. All procedures on animals were performed in accordance with the official guidelines of European Union Directive 2010/63/EU and with the permission of the Ministry of Agriculture and the Environment of the Republic of Slovenia (permission no. 34401-4/2012/2) which was granted based on the approval of Ethical committee for animal experimentation of the Republic of Slovenia. At the beginning of the experiment, the animals were 9–11 weeks old. Solid tumours were induced by subcutaneous injections of SA-1 cells (5·105 cells/0.1 mL 0.9% w/v NaCl) in the right flank prepared from the ascites from donor mouse. When the tumours reached approximately 40 mm3 in volume, the animals were randomly divided into experimental groups and subjected to a specific experimental protocol.Murine fibrosarcoma SA-1 cells syngeneic to A/J mice  were used in the study. Mice were maintained in a specific pathogen-free animal colony at constant room temperature (20–24°C), relative humidity (55±10%), and 12 hour light/dark cycle. Food and water were provided EP were delivered by generator of EP ELECTRO CELL B10 using flat parallel electrodes with 8 mm gap. Electrodes were placed percutaneously at the opposite tumour margins. Electroporation of tumours was performed by applying eight square-wave EP with voltage to distance ratio 1300 V/cm, pulse width 100 μs and repetition frequency 1 Hz with a perpendicular change of electrode orientation after 4 pulses. Good contact between the electrodes and the overlying skin was ensured by conductive gel .5 U) was applied intratumourally (i.t.) either 5 min before or after the application of EP. CDDP (4 mg/kg) was injected intravenously (i.v.) 3 min before the application of the electric pulses  and 5 min before the application of electric pulses.In the second part of the a·b·c·π/6, where a, b and c were three mutually orthogonal tumour diameters. From the calculated volume, arithmetic means and standard error of the means were calculated for each experimental group. Tumour doubling time was determined for each individual tumour, and tumour growth delay was calculated from the mean doubling time of experimental groups. When the tumour volume reached 300 mm3, animals were considered incurable and were humanely euthanized. When the tumours became impalpable, the response to treatment was scored as complete response. Mice that were in complete response 100 days after the treatment were considered as cured. In addition, the weight of the mice was followed as a general toxicity index.After the therapy, tumour growth was followed up by measurements of tumour diameters with a digital calliper. Tumour volume was calculated by the formula V=Mice were sacrificed 24 h after treatment with CDDP (4 mg/kg), electrochemotherapy, combination of TNF-α and CDDP, and combination of TNF-α and electrochemotherapy. Tumours were excised and removed from the overlying skin. To determine platinum content in tumours after treatment, tumours were weighed, placed in 15-mL graduated polyethylene tubes and digested in 0.5 mL 65% nitric acid by incubation at 37°C for 2 days to obtain a clear solution. Platinum content in the samples was determined by electrothermal atomic absorption spectrometry on a Hitachi Z-8270 Polarized Zeeman Atomic Absorption Spectrometer, adjusted to a wavelength of 265.9 nm.Mice were sacrificed 24 h after treatment with CDDP, electrochemotherapy, combination of TNF-α and CDDP, and combination of TNF-α and electrochemotherapy. Tumours were excised, removed from the overlying skin and fixed in IHC zinc fixative  for 24 h at room temperature, and embedded in paraffin. Two series of 2 μm thick tissue sections were cut from each paraffin block with step thickens of 20 μm.First series of tissue sections were used for immunohistochemical staining of CD31, a marker for endothelial cells. Sections were incubated with rabbit polyclonal antibodies against murine CD31  at dilution 1:1000. A peroxidase-conjugated streptavidin–biotin system  was used as the colorogenic reagent followed by haematoxylin counterstaining. The immunohistochemically stained sections were examined in blind fashion under light microscopy and 5 images of viable tumour tissue from the same tumour section were captured with a DP72 CCD camera  connected to a BX-51 microscope (Olympus).Second series of tissue sections were stained with haematoxylin and eosin for evaluation of tumour necrosis. These slides were examined under the microscope Olympus BX-51 and visual light images were recorded with a digital camera DP72 CCD (Olympus). Digital images of haematoxylin and eosin stained tumour sections were analysed in blind fashion and percentage of necrosis was estimated by 4 independent observers.SigmaPlot 11.0  was used for statistical analysis. Normal distribution of the data was tested using Shapiro-Wilk test. All pairwise multiple comparisons for normally distributed data were tested with Holm-Sidak test after One Way ANOVA. All pairwise multiple comparisons for data that was not normally distributed were tested using Tukey test or Dunn’s test  after Kruskal-Wallis One-Way ANOVA on ranks. Differences were considered significant when p value was less than 0.05.In vitro sensitivity of SA-1 cells to electrochemotherapy with or without TNF-α was determined by clonogenic assay  on tumour growth was evaluated. Based on the previous experiment, adjuvant TNF-α therapy was given before electrochemotherapy.As predicted, electroporation significantly potentiated CDDP effectiveness, which was CDDP dose dependent . ElectroIn the study, the antitumour effectiveness of TNF-α combined with electrochemotherapy with intravenous CDDP was evaluated in murine fibrosarcoma tumour model. Adjuvant immunotherapy with TNF-α preceding electrochemotherapy, increased antitumor effectiveness of electrochemotherapy with intravenous CDDP. Prolonged tumour growth and increased tumour curability were observed after combined treatment with TNF-α and electrochemotherapy with intravenous CDDP. The underlying mechanism is most likely vascular targeted effect of TNF-α combined with its immunomodulatory effect, as previously proposed for combination with electrochemotherapy with bleomycin.16Electrochemotherapy gained clinical recognition in the treatment of cutaneous tumours or metastases as well as deep-seated tumours.Recently, it was demonstrated that tumours larger than 3 cm in size have lower response rate after single electrochemotherapy treatment than smaller ones.TNF-α is a well-known pro-inflammatory cytokine with a wide range of biological functions, including an immunomodulatory role and antivascular action.in vivo. A synergistic interaction between TNF-α and electrochemotherapy with intravenous CDDP administration was observed. This synergistic interaction cannot be attributed to TNF-α cytotoxicity to tumour cells due to the lack of the effect in vitro. Contrary to early speculations of TNF-α cytotoxic action on malignant cells in animal tumour models, it is now accepted that TNF-α is weakly cytotoxic to malignant cells.in vitro results support this, as no cytotoxic effect of TNF-α alone or in combination with electric pulses on SA-1 fibrosarcoma cells was observed. Similarly, no cytotoxic effect of TNF-α in combination with EP for electrochemotherapy was observed in our previous study.Our results demonstrated potentiation of antitumour effectiveness of electrochemotherapy in combination with TNF-α In vivo, two distinct and successive effects of TNF-α on tumour-associated vasculature are known. Firstly, TNF-α increases vascular endothelium permeability, resulting in reduction of interstitial fluid pressure and improved penetration of chemotherapeutic agents within the tumour tissue, and secondly, TNF-α causes tumour vessel destruction.40Taken together, our results indicate indirect antitumour action of adjuvant TNF-α in SA-1 fibrosarcoma tumours treated with ECT with low CDDP doses. This antitumour action of TNF-α is likely due to hyperpermeability of tumour blood vessels, leading also to increased accumulation of CDDP in the tumours. Based on these results, we speculate that the mechanism of action of adjuvant TNF-α in electrochemotherapy treated tumours is primarily through a vascular action. Nonetheless, especially at higher CDDP doses, at which tumour cures were obtained, involvement of activated immune response should be taken into account. Namely, in clinical settings, induction of inflammatory response after TNF-α based isolated limb perfusion was observed in the tumour tissue.In conclusion, adjuvant intratumoural TNF-α therapy synergistically contributes to electrochemotherapy with intravenous CDDP administration. Due to its potentiation at all doses of CDDP, the combined treatment is predicted to be effective also in less perfused areas of tumours, where the drug accumulation is not optimal, or in bigger tumours, where the effectiveness of electrochemotherapy with intravenous CDDP is not expected to be sufficiently effective. Its synergistic effectiveness can be attributed to actions of TNF-α; vascular and also immunomodulatory, that could be further potentiated by replacing the injection of recombinant TNF-α protein with application of gene therapy with TNF-α.