Thesetein p29 . While Pinitrite ,8, it al(1)2O2 or other oxidant substrate. The PRX 1–5 family members express a second (resolving) Cys that, in conjunction with thioredoxin, reduces the peroxidatic Cys and restores the physiologically active form. Prdx6, however, expresses only a single conserved Cys and uses glutathione (GSH) plus GSH S-transferase (GST) for reduction and resolution of its oxidized peroxidatic Cys [Catalytic mechanism: All peroxiredoxins express a conserved cysteine (Cys) residue, called the peroxidatic Cys, that is oxidized by interaction with Hatic Cys ;(2)Substrate binding: Unlike other PRXs, Prdx6 can bind to phospholipids . This is(3)Phospholipid hydroperoxide reductase activity: Prdx6 is able to bind and to reduce phospholipid hydroperoxides that may be produced as a result of oxidative stress . This ph(4)sn-2 position indicating a phospholipase A2 (PLA2) activity [Phospholipid hydrolysis: Phospholipids bound to Prdx6 can be hydrolyzed at the activity ;(5)2 and LPCAT activities of Prdx6 represents a major mechanism for phospholipid remodeling through hydrolysis followed by re-acylation at the sn-2 position [Lysophosphosphatidylcholine acyltransferase (LPCAT) activity: Prdx6 is able to acylate lysophospholipids (lysophosphatidylcholine is the primary substrate) by a transferase reaction to generate a phospholipid (phosphatidylcholine) .The coupposition ,12.(6)Subcellular localization: Like several other PRXs, Prdx6 is localized primarily to cytosol, but it is also the only member of the PRX family to be present in both lysosomes and lysosomal related organelles such as the lung lamellar bodies that are a site for synthesis and storage of the lung surfactant .The special characteristics that differentiate Prdx6 from the other PRXs include:These six special characteristics of Prdx6 allow this protein to play specific and important roles in normal physiology and pathobiology including the scavenging of oxidants, the repair of peroxidized cell membranes, the turnover of lung surfactant phospholipids, and cellular signaling as mediated by reactive oxygen and nitrogen species (ROS/RNS) ,16,17,182 activities of Prdx6 using mice with C47S-Prdx6 and D140A-Prdx6 mutations. The second article by Bannitz-Fernandes et al. describes original research that, for the first time, shows the presence of PLA2 activity in several non-mammalian Prdx6 enzymes [2 activity at cytosolic pH; this effect was unrelated to the formation of multimers [2•−) generation by NADPH oxidase (NOX2) and its important role in cellular communication [2 activity, that results in the downstream activation of Rac, a required co-factor for the activation of NOX2. The following original research article by Fisher et al. identifies several peptides derived from the naturally occurring protein surfactant protein A (SP-A) that can inhibit the PLA2 activity of Prdx6 and prevent the activation of NOX2 [The first article in this special issue is a review by Feinstein that reports on currently available mouse models to evaluate the physiological and pathophysiological roles of Prdx6 . Of spec enzymes . The ori enzymes , similar enzymes . The fouultimers . The fifnication . Prdx6 g of NOX2 . The sev of NOX2 . The aut of NOX2 . The nin of NOX2 . Both ex of NOX2 . This re2 activity following phosphorylation of the protein, although the Thr177 amino acid in Prdx6 has been identified as the phosphorylation site [2O2, hydroperoxides, and peroxynitrite, hydrolyze phospholipids (PLA2 activity), and transfer acyl groups. In many cases, the impact of Prdx6 may relate to its specific tissue expression as seems to be the explanation for the predominant role of Prdx6 to reduce phospholipid hydroperoxides in the lung [Despite the considerable increase during the past 25 years in our knowledge of Prdx6, there remain large gaps in our understanding of its structure-function relationships and (patho)physiological roles. Although a structural mechanism to account for its ability to bind phospholipids was proposed some time ago , there hion site  and a chion site . Anotherthe lung . But, ththe lung ). Alterethe lung ). Howeve


To find the shortest, acceptable stabilization period before recording resting, supine ultra-short-term Ln RMSSD and heart rate (HR).2max) 64.1 ± 6.6 ml·kg-1·min-1) and 30 male students  were recruited. Upon awaking at home, resting, supine RR intervals were measured continuously for 10 min using a Polar V800 HR monitor. Ultra-short-term Ln RMSSD and HR values were calculated from 1-min RR interval segments after stabilization periods from 0 to 4 min in 0.5 min increments and were compared with reference values calculated from 5-min segment after 5-min stabilization. Systematic bias and intraclass correlation coefficients (ICC) including 90% confidence intervals (CI) were calculated and magnitude based inference was conducted.Thirty endurance-trained male athletes  biases for ultra-short-term Ln RMSSD compared with reference values. Stabilization periods of 60 s for athletes and 90 s for students showed trivial biases and ICCs were 0.84; 90% CI 0.72 to 0.91, and 0.88; 0.79 to 0.94, respectively. For HR, biases were trivial and ICCs were 0.93; 0.88 to 0.96, and 0.93; 0.88 to 0.96, respectively.The shortest stabilization period required to stabilize Ln RMSSD and HR was set at 60 s for endurance-trained athletes and 90 s for university students. Analysis of heart rate variability (HRV) has provided a non-invasive method for evaluating cardiac autonomic regulation ,2. In spCoaches who implement HRV analysis as part of a training strategy may struggle with low athlete compliance relating to regular measurement, which should be performed on a daily basis ,15. DailThe root mean square of successive differences between adjacent RR intervals (RMSSD) has been regarded as an index of vagal activity ,14 and cPlews et al.  showed tIt was shown that the stabilization period can be shortened from 5 min to 1 min ,25. HoweTherefore, the aim of this study was to find the shortest, acceptable stabilization period before capturing ultra-short-term Ln RMSSD and HR in the supine position.The study protocol was approved by the Ethics Committee of the Faculty of Physical Culture, Palacký University Olomouc (reference number 14/2015) and was done in accordance with the Declaration of Helsinki. After comprehensive explanation of the study, all participants provided written informed consent. Inclusion criteria were as follows: male, aged 20 to 29 years, non-smoker, and taking no medication. Prior to participation in the study, participants underwent resting 10-lead electrocardiogram (ECG) examination and blood pressure measurement. The exclusion criteria included a pathological ECG pattern and hypertension (> 140/90 mmHg). This study included two groups. The first group consisted of 30 Czech national level endurance male athletes  and the second group consisted of 30 male university students. Characteristics of participants are presented in 2max) were determined in a laboratory a week before HRV recording and were used for descriptive purposes only. Body mass and height were measured using the Soehnle 7307 scale . VO2max was determined during an incremental running test on the treadmill . The protocol consisted of a 4 min warm-up (2 min at 8 km.h-1 with 0% elevation and then 2 min at the same speed with 5% elevation) followed by an increase in speed to 10 km.h-1 with 5% elevation for 1 min. From this point, at each minute, the speed was increased by 1 km.h-1, keeping elevation the same, up to 16 km.h-1. Then the speed was maintained and only the elevation increased by 2.5% per minute until exhaustion. Ventilation and gas exchange were recorded breath by breath and averaged to 30 s by Blue Cherry system . The criteria for attaining VO2max was defined as reaching one of the following criteria: a) respiratory exchange ratio of >1.11, b) VO2 plateau defined as no increase in VO2 in response to an increase in work rate. VO2max was considered the highest VO2 value in the final 30 s of the test.Body mass, body height and maximal oxygen consumption  which was showed as valid RR interval measurement tool . ParticiRR recordings were transferred to a computer using the Polar Flow cloud service and were further analyzed using a custom program written in MATLAB language . Artifacts  were identified by visual inspection of RR intervals and simply deleted because the deletion method provided the best overall performance .Ultra-short-term values of Ln RMSSD, RMSSD, and average values of HR were calculated from 1-min segments using various stabilization periods. Stabilization periods varied from 0 to 4 min with step interval increase of 0.5 min which yielded 9 periods in total. The first segment used no stabilization period (0 min) and included RR intervals between 0 and 1 min after the start of RR recording. The second segment used a stabilization period of 0.5 min and included RR intervals between 0.5 and 1.5 min. This procedure continued stepwise and finally the 9th segment used a stabilization period of 4 min and included RR intervals between 4 and 5 min. Reference values of Ln RMSSD, RMSSD, and HR were calculated from the second half of the 10-min RR recording, resulting in a 5-min segment of RR intervals from 5 to 10 min.diff is standard deviation calculated from differences between ultra-short-term and reference values [Data are presented as arithmetic mean ± standard deviation (SD). Anthropological and physiological variables  of endure values . Agreemee values  and the Because null-hypothesis significance testing (NHST) is not able to distinguish between a trivial effect and an insufficient sample size, we used magnitude-based inference (MBI) . MBI was2max, possibly higher both Ln RMSSD and RMSSD, and very likely lower HR , whilst suitable for athletes, was not valid for university students, because at least 1.5 min was required to stabilize Ln RMSSD in students. It is well known, that endurance athletes exhibit higher autonomic nervous system (ANS) activity presented by greater resting cardiac vagal activity together with a lower resting HR compared with the normal population . TherefoWe prefer using Ln RMSSD rather than RMSSD because the logarithmic transformation enables to correct the skewed probability distribution of RMSSD shifting the probability distribution of Ln RMSSD closer to normal ,18. ParaFor statistical analyses we prefer using MBI to NHST based on arguments presented by Buchheit . NeverthWhen ultra-short-term (60 s) recordings are used to calculate Ln RMSSD and HR in the supine position, the minimal stabilization period required to stabilize both indexes was 60 s for endurance athletes but 90 s for university students.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.


Objective: Tumor necrosis factor-beta (TNF-β), as an inflammatory mediator that has been shown to promote tumorigenesis, induces NF-κB. Natural multi-targeted agent resveratrol in turn shows anti-inflammatory and anti-cancer properties. Epithelial-to-mesenchymal transition (EMT) allows cancer cells to turn into a motile state with invasive capacities and is associated with metastasis and development of cancer stem cells (CSC). However, TNF-β-induced EMT and the anti-invasion mechanism of resveratrol on CRC are not yet completely understood. Methods: We investigated the underlying molecular mechanisms of resveratrol on TNF-β/TNF-βR-induced EMT and migration of CRC cells  in monolayer or 3D alginate cultures. Results: TNF-β, similar to TNF-α, induced significant cell proliferation, morphological change, from an epithelial to a spindle-like mesenchymal shape with the formation of filopodia and lamellipodia associated with the expression of EMT parameters , increased migration/invasion, and formation of CSC in all CRC cells. Interestingly, these effects were dramatically decreased in the presence of resveratrol or anti-TNF-βR with TNF-β co-treatment, inducing biochemical changes to the mesenchymal-epithelial transition (MET), with a planar cell surface and suppressed formation of CSC cells. This was associated with a significant increase in apoptosis. Furthermore, we found that resveratrol suppressed TNF-β-induced NF-κB and NF-κB-regulated gene biomarkers associated with growth, proliferation, and invasion. Finally, TNF-βR interacts directly with focal adhesion kinase (FAK) and NF-κB. Conclusion: These results suggest that resveratrol down-regulates TNF-β/TNF-βR-induced EMT, at least in part via specific suppression of NF-κΒ and FAK in CRC cells. In industrialized countries, colorectal cancer is the third most common cancer diagnosed and ranks second in terms of mortality . It is eChronic inflammation presents a critical component of tumor initiation and progression . The nucWe and others have previously shown that TNF-β can, like TNF-α, modulate proliferation, survival, invasion, migration, and colony formation in colorectal and ovarian cancer ,13. IndeEvidence has shown that inflammation in the tumor microenvironment acts as a potent promoter, converting cells into a motile state by stimulating epithelial-to-mesenchymal-transition (EMT) in cancer cells . The cantrans-3,5,4’-Trihydroxystilben), a natural polyphenol, has been associated with a variety of different medicinal effects, like anti-inflammatory and anti-neoplastic properties [Resveratrol  migration from 3D-alginat cultures and TNF-β has increased chemoresistance in CRC cells to 5-FU. Furthermore, resveratrol could significantly chemosensitize CRC cells to 5-FU in a TNF-β-promoted inflammatory tumor microenvironment . Very reStaphylococcus aureus bacteria were from Sigma–Aldrich Chemie . TNF-β, TNF-α, anti-TNF-β-receptor, and anti-TNF-β were purchased from eBiosciences . In addition, TNF-β and TNF-α, were kindly provided by Genetech, Inc.  [Anti-CXCR4 (CXC-Motiv-Chemokinreceptor 4) was acquired from Abcam PLC . Anti-CD133, -ALDH1, and -CD44 were purchased from Antibodies-online. Anti-phospho-specific p65 (NF-κB) and anti-MMP-9 were from R&D Systems . Anti-E-cadherin, anti-vimentin, and anti-slug were obtained from Santa Cruz Biotechnology . Anti-FAK was purchased from Becton Dickinson . Monoclonal antibodies to Ki-67 and secondary antibodies used for fluorescence labelling were purchased from Dianova . Anti-β-actin, alginate, resveratrol, and CA, USA) . SecondaThe human CRC cell lines  used in this study, were from the European Collection of Cell Cultures . The RKO cell line was from the American Type Culture Collection (ATCC). A whole cell culture growth medium, supplemented with 10% FCS, was prepared and cells cultured as previously described in detail .Human CRC cell  monolayer cultures were washed three times with a serum-starved medium (3% FCS) and incubated for 1 h with the same medium. CRC cells were either left untreated or treated with 10 ng/mL of TNF-β or TNF-α alone for 12 h, or pretreated with 5 µM resveratrol by itself or 5 µM resveratrol for 4 h, followed by co-treatment with 10 ng/mL of TNF-β or TNF-α and 5 µM resveratrol for 12 h.For the invasion assay, serum-starved HCT116, RKO, and SW480 CRC cells in alginate bead culture were left untreated, treated with 5ng/ml or 10ng/mL TNF-β or TNF-α, 5 µM resveratrol, or the combination of resveratrol and TNF-β or TNF-α for 14 days. In an additional approach, HCT116, RKO, and SW480 cells were left untreated, treated with TNF-β, resveratrol, TNF-β and resveratrol, or treated in suspension with an anti-TNF-β-receptor  for 15 min, and then transferred to the alginate, followed by co-treatment with or without 10 ng/mL of TNF-β or resveratrol for 14 days. These investigations were performed in triplicate and the findings are presented as mean values from three independent investigations.CRC cells  in monolayer cultures were treated as described above and were subjected to immunofluorescent labelling for slug, vimentin, and E-cadherin as previously described in detail . In a seCultivation of CRC cells in three-dimensional in vitro culture was performed as alginate bead culture, as previously described ,32,35. Tp < 0.05 are shown by one asterisk (*); p < 0.01 by two asterisks (**).The influence of resveratrol and/or TNF-β/TNF-α on invasion and colony formation capacity of CRC cells was investigated in alginate bead culture, as previously described ,35. CellCell viability of CRC cells in alginate bead culture was assessed by applying the MTT method as previously stated . BrieflysO4 solution. After dehydration in an ascending alcohol series, cells were embedded in Epon and cut ultrathin with a Reichert-Jung Ultracut E . Ultrathin sections were contrasted with uranyl acetate/lead citrate and evaluated with a transmission electron microscope  or a Jeol 1200 EXII . For statistical evaluation of apoptotic cells, 100 cells from 25 microscopic fields were counted manually and all obtained data were compared to the control and statistically significant differences were highlighted with p < 0.05 (*); p < 0.01 (**).Ultrastructural investigations of monolayer cultures were performed to evaluate the effect of resveratrol and/or TNF-β on CRC cells, as previously stated . CRC monsO4 solution for 5 min, cells were dehydrated in a series of ethanol concentrations and finally embedded in Epon. Ultrathin sections were prepared and examined using a Zeiss transmission electron microscope  or a Jeol 1200 EXII .Monolayer cultures of CRC cells were left untreated or treated, as stated above. The effect of resveratrol and/or TNF-β on EMT marker E-Cadherin and vimentin in CRC cells was investigated by an ultrastructural immunoelectron pre-embedding technique, as previously described . BrieflyHCT116, RKO, and SW480 CRC cells in alginate bead culture were left untreated or treated, as described above. Cell lysates for immunoblotting investigations were extracted from CRC cells in alginate bead cultures, as previously described ,35. The Staphylococcus aureus bacteria to pre-clear samples. They were incubated with antibodies against TNF-β-receptor in a wash buffer  for 2 h at 4 °C, followed by 30 min incubation with Staphylococcus aureus bacteria. Finally, obtained immune complexes were washed with a lysis buffer. Samples were reduced with 2-mercaptoethanol, subjected to SDS-PAGE under reducing conditions, and were blotted onto a nitrocellulose membrane, using a transblot apparatus .Immunoprecipitation investigations were performed to elucidate the relationship between TNF-β-receptor and endogenous signaling interactions with focal adhesion kinase (FAK) or with NF-κB, as previously described ,38. CRC p-value < 0.05 was considered statistically significant.The data were expressed as the mean ± SE and statistical significance between the groups was compared by one-way, two-way, or a three-way ANOVA, and the Wilcoxon–Mann–Whitney test. Each investigation was performed at least three times. A The goal of this paper was to investigate the effect of resveratrol on TNF-β-induced tumorigenesis of human colorectal cancer cells (CRC) through regulation of EMT. We used three well-described CRC cell lines  in a monolayer, as well as a 3D alginate tumor microenvironment culture to answer this question. The amount of ethanol or DMSO used in our study had no suppressive effect on the viability of cells.Since EMT plays an important role in tumor invasion and metastasis , we firsTo confirm the morphological results described above and to show more precisely the identity of the epithelial-to-mesenchymal-transition (EMT) biomarker, whole cell extracts were probed for slug, vimentin, and E-cadherin expression. Western blotting analysis showed that in CRC cells, TNF-β, similar to TNF-α, reduced the epithelial marker (E-cadherin) and elevated the mesenchymal markers (vimentin) and EMT-related transcription factor (slug) on the protein level, and resveratrol alone or in co-treatment with TNFs significantly decreased vimentin and slug levels and increased E-cadherin expression B.Furthermore, to confirm the morphological and western blotting results that the CRC cells underwent EMT by TNF-β treatment, which is inhibited by resveratrol, we investigated the expression of EMT markers and the EMT-related transcription factor by immunofluorescence analysis. The untreated control cells demonstrated a minimum amount of E-cadherin, vimentin, and slug expression. On the other hand, the expression of vimentin and slug was markedly elevated, and E-cadherin was reduced in the presence of TNF-β alone C. TreatmIt is known that EMT is the first step in increasing cancer malignancy and the cell membrane ultrastructure plays a crucial role in cell adhesion and invasion ,23,24. TWe next examined the epithelial morphology induced through resveratrol and cell surface proteins of the MET marker (E-cadherin) in all CRC cells by immunoelectron microscopy. HCT116, SW480, and RKO cells were either untreated or treated, as described before, in a monolayer culture. As demonstrated in To examine whether resveratrol has a modulatory effect on CRC cell invasion and migration promoted by TNF-β, alginate invasion tests were performed. As shown in Since it was demonstrated that TNF-β, as cytokine, induced the formation of cancer stem cells (CSCs) in HCT116 cells , we furtA large body of literature indicates that NF-κΒ is a crucial transcriptional mediator of pro-inflammatory cytokines that promote proliferation, invasion, malignancy, and EMT of cancer cells ,41,42,43We found that TNF-β was a potent cytokine for promoting the malignancy of cancer cells. To further validate these findings, we also investigated the role of TNF-βR on the migration of all CRC cells. We then investigated the endogenous expression of TNF-β and TNF-βR in different CRC cell lines by immunofluorescence labeling and immunoblotting analysis. Interestingly, in control cultures, immunofluorescence labeling demonstrated that all three CRC cell lines had a basal expression of TNF-β and of TNF-βR protein A. OveralResults from We further investigated the metastasis effect of TNF-β and TNF-βR on proteins that contribute to cancer metastasis. Cytoskeleton remodeling, cell adhesion, chemotaxis and focal adhesion kinase (FAK)-signaling pathways, and NF-κB were associated with regulating tumor cell survival, invasiveness, and metastasis ,44,45,46Previous reports suggested that inflammatory cytokines produced in the cancer microenvironment, such as TNF-α, act as a major promotor and modulator of tumorigenesis through activation of EMT and different signaling pathways, including NF-κΒ ,48,49,50In this paper, we showed that TNF-β, similar to TNF-α, stimulated distinctive morphological and molecular changes from epithelial to fibroblast-like mesenchymal phenotypes (EMT) with the formation of filopodia and lamellipodia, and this was accompanied by EMT factors  in all three CRC cell lines. We also found, with immunoblotting and immunofluorescence evaluation, clear TNF-β-induced expression of CSC-specific biomarkers  in all three CRC cells. These findings underline the essential impact of the tumor-promoting inflammation factor TNF-β as a dominant agent in promoting and supporting EMT and development of CSCs, thus, cancer progression and metastasis in CRC. In addition, these data are in accordance with findings from other studies, indicating that tumor cells change their morphology and phenotype from epithelial to higher cell motility and malignancy-mesenchymal phenotypes, and synthetize EMT-specific factors during tumorigenesis , accompaIn correlation with the consideration that TNF-β as an inflammatory cytokine is a key decisive factor in cancer promotion, we showed that resveratrol, like anti-TNF-βR with TNF-β co-treatment, significantly decreased cell migration and EMT of CRC cells and promoted biochemical changes to MET with a planar cell surface and suppressed formation of CSC, and this was accompanied by a marked increase in apoptosis. Moreover, to show the anti-cancer impacts of resveratrol and anti-TNF-βR, we performed MTT and migration assays to determine the optimal suppression rate and cytotoxicity in all three CRC cell lines. In addition, pro-inflammatory cytokines have been noted to be synthesized through activated inflammatory cells, like macrophages in the tumor microenvironment , and durWe also showed that resveratrol induced nucleus pyknosis, chromatin condensation, and apoptotic body generation, indicating that resveratrol activated caspase-dependent cell death in CRC cells, which is in correlation with previous studies ,54, highIt has been reported, that TNF-α as an activator and modulator of tumorigenesis induces the invasion abilities of tumor cells ,66, but These data suggest for the first time the impact of TNF-βR on CRC cell EMT and migration, and underlines an important functional coherence between TNF-βR and cancer-stimulating factors, such as FAK, NF-κB, and NF-κB-related proteins.This report examined the down-regulatory effects of resveratrol on TNF-β as a potent inflammatory cytokine, activating EMT in three CRC cell lines. Resveratrol, like anti-TNF-βR, significantly suppressed the TNF-β-induced EMT, EMT-associated factors, CSC formation, and migration of CRC cell lines. We found that TNF-β-promoted TNF-βR physically formed a protein complex with FAK and NF-κB and that these could be upregulated by TNF-β. More interestingly, this is the first study to indicate that FAK and NF-κB serve as a subcellular molecular downstream target of TNF-βR . Thus, o


Protein shells assembled from viral coat proteins are an attractive platform for development of new vaccines and other tools such as targeted bioimaging and drug delivery agents. Virus-like particles (VLPs) derived from the single-stranded RNA (ssRNA) bacteriophage coat proteins (CPs) have been important and successful contenders in the area due to their simplicity and robustness. However, only a few different VLP types are available that put certain limitations on continued developments and expanded adaptation of ssRNA phage VLP technology. Metagenomic studies have been a rich source for discovering novel viral sequences, and in recent years have unraveled numerous ssRNA phage genomes significantly different from those known before. Here, we describe the use of ssRNA CP sequences found in metagenomic data to experimentally produce and characterize novel VLPs.Approximately 150 ssRNA phage CP sequences were sourced from metagenomic sequence data and grouped into 14 different clusters based on CP sequence similarity analysis. 110 CP-encoding sequences were obtained by gene synthesis and expressed in bacteria which in 80 cases resulted in VLP assembly. Production and purification of the VLPs was straightforward and compatible with established protocols, with the only exception that a considerable proportion of the CPs had to be produced at a lower temperature to ensure VLP assembly. The VLP morphology was similar to that of the previously studied phages, although a few deviations such as elongated or smaller particles were noted in certain cases. In addition, stabilizing inter-subunit disulfide bonds were detected in six VLPs and several possible candidate RNA structures in the phage genomes were identified that might bind to the coat protein and ensure specific RNA packaging.Compared to the few types of ssRNA phage VLPs that were used before, several dozens of new particles representing ten distinct similarity groups are now available with a notable potential for biotechnological applications. It is believed that the novel VLPs described in this paper will provide the groundwork for future development of new vaccines and other applications based on ssRNA bacteriophage VLPs.The online version of this article (10.1186/s12951-019-0497-8) contains supplementary material, which is available to authorized users. The single-stranded RNA (ssRNA) bacteriophages of the Levivirdae family are small viruses that infect a variety of Gram-negative bacteria. Their virions consist of a compact, approximately 3500 to 4200 nucleotide-long genome packaged in a small, spherical-looking protein shell about 28 nm in diameter with an underlying T = 3 quasi-equivalent icosahedral symmetry. The capsid is constituted of the major coat protein (CP) and one or two species of minor structural proteins that are involved in recognition and packaging of the genome and are required to adsorb the virion to the bacterial receptor and convey the RNA genome into the cell. At least for the currently studied ssRNA phages, the minor virion proteins are not essential either for assembly or for structural integrity of the protein shell, and recombinant expression of a cloned coat protein gene results in the appearance of virus-like particles (VLPs) that are morphologically very similar to native virions but have spontaneously packaged bacterial RNA inside the particles instead of the genome –4.The ssRNA phage VLPs have found a variety of applications, mostly in the field of vaccine development where various antigens are presented onto the capsid surface to invoke a strong immune response. Phage Qβ VLPs conjugated with various peptide and small-molecule moieties have reached clinical trials against conditions such as hypertension , asthma Escherichia coli and related Enterobacteria, while the remaining few target bacteria of the Pseudomonas, Acinetobacter or Caulobacter genera. The CPs of the Enterobacteria- and Pseudomonas-specific phages share very low, yet still detectable sequence similarity, while those from the Acinetobacter and Caulobacter phages, of which only a single representative of each has been sequenced, have no sequence similarity to other CPs [Up to recently, the number of known ssRNA phages has been rather small. All of the currently identified phages use various Proteobacteria as their hosts; the great majority of these infect ther CPs , 17. TheWhile no novel ssRNA phages have been isolated lately, the increasing metagenomic sequencing efforts in recent years have uncovered a previously unknown diversity of these viruses in nature. In 2015, genomes of two novel Leviviridae phages EC and MB were assembled from San Francisco wastewater , and sooWhile the metagenomic studies have greatly expanded the known ssRNA phage diversity, infectious phages cannot be resurrected from the partial genome sequences, and their host bacteria, along with many other aspects of their biology, remain unknown. However, the CPs of the previously studied ssRNA phages have been able to assemble into VLPs in absence of other phage components, which provides an opportunity to obtain and study ssRNA phage VLPs even if the CP sequence is the only available information. In this study, we acquired 110 putative ssRNA phage CP-encoding ORFs from the metagenomic data using gene synthesis, and here we report the expression, purification and characterization of 80 novel ssRNA phage VLPs.Pseudomonas-infecting phages, the Acinetobacter phage AP205 [Caulobacter phage Cb5 [Based on multiple sequence alignment, the previously known ssRNA phage CPs can be divided into three broad similarity groups represented by the Enterobacteria- and ge AP205  and the hage Cb5 , respectLevivirus and Allolevivirus F-pili specific phage genera, with an adjacent cluster containing the other conjugative-pili specific phages M [Pseudomonas phage PP7 [Caulobacter phage Cb5 and more than 30 related CP sequences from the metagenomic data. These further cluster into five subgroups, all formed by short and rather uniform-length proteins (most around 115–120 residues). One more supergroup with some 30 sequences emerges which is comprised entirely of metagenomic sequences and contains two major recognizable subgroups. The CPs in this “AVE015-like” supergroup, named after a representative phage from the group, are considerably bigger than those of MS2- or Cb5-like phages with an average length around 165 residues, with some of them spanning 180 residues. The three supergroups comprise approximately 80% of all of the known ssRNA phage CP sequences; the rest cluster into several considerably smaller clades ranging from two to five CPs, designated here the ESE017-like, ESE020-like, AP205-like, AVE001-like, AVE002-like, EMS000-like and AC-like groups. Several CP sequences  have no reliable assignment to any of the clusters and were considered orphans.  to determine the average particle size in solution. In a homogeneous sample, the particle size measured using DLS is in good agreement with values determined using other methods such as electron microscopy or X-ray crystallography, while significant deviations are an indicator of particle aggregation. For the majority of VLPs, the determined average particle diameter values Table  lie withCoat protein modifications introduced for vaccine development and related applications have a tendency to destabilize the assembled VLPs, and the experimental success rate appears to positively correlate with the stability of the starting unmodified particles. While in some of the studied ssRNA phages inter-subunit contacts are mediated solely by non-covalent protein–protein interactions, in others coordinated metal ions , 27 and In all of the previously studied ssRNA phage particles where disulfide bonds exist, they are formed between CP loops positioned around the icosahedral threefold and fivefold symmetry axes that results in covalently linked CP pentamers and hexamers in the capsid. It cannot be excluded, however, that in other phages stabilizing disulfide bonds might occur also in other positions. We therefore selected all experimentally available CPs that were able to assemble into VLPs, could be purified to near homogeneity and which contained at least two cysteine residues, and subjected the VLPs to denaturing but non-reducing conditions. In such conditions the disulfide-containing Qβ and AP205 VLPs disassemble into pentameric and hexameric CP species that can be tracked in SDS–polyacrylamide gel electrophoresis Fig. . From thThe thermal stability of a VLP is an important characteristic that positively correlates with the overall robustness of the particle and its performance in downstream applications such as vaccine development or drug delivery. To determine the thermal stability of the novel ssRNA phage VLPs, we subjected the particles to increasing temperatures and visualized their disassembly in native agarose gel electrophoresis. Intact ssRNA phage VLPs migrate as a distinct band in the gel which is detectable both when staining for RNA and for protein, but as the VLPs are heated and gradually disassemble, the VLP band accordingly becomes weaker until it completely disappears. The thermal stability Tm is defined as the lowest tested temperature at which the VLP band can no longer be observed.The novel VLPs have a broad range of thermal stability Table . While tPseudomonas phage PP7 [In a number of the previously studied ssRNA phages, the coat protein recognizes and binds a genomic RNA hairpin at the beginning of the replicase gene which regulates the synthesis of the replicase enzyme and contributes to specific packaging of phage genome into the virions. The hairpin is a stem-loop structure comprised of an approximately eight base pair-long stem with an unpaired adenosine residue and a three- to six-nucleotide-long loop, and is often designated the translational operator (TR) of the replicase gene  high-level CP expression in bacteria; (2) efficient assembly of the CP into VLPs; (3) high stability of the assembled VLPs; (4) simple and effective means of VLP purification and (5) VLP tolerance for chemical and genetic modification. An ability to package substances of interest inside the VLPs is further preferable. In our study we have addressed a number of these points, and the sample size allows for some general conclusions to be made.E. coli in standard conditions which resulted in highly efficient assembly of VLPs. We adopted a similar strategy for the novel ssRNA phage CPs, and the high-level expression part indeed did not present any problems with only a few of the proteins not being produced. Instead, the solubility of the produced CPs proved to be the first roadblock as in the initial conditions approximately 40% of the tested proteins were found in inclusion bodies. In this respect, it can be noted that the reported sources for the metagenomic datasets are extremely diverse, ranging from intestinal contents of warm-blooded animals to deep sea microbial sediments and arctic soils in Svalbard. It is therefore conceivable that at least for some of the proteins, high-level expression at 37 °C is such a dramatic departure from the conditions in their native host bacteria that it adversely affects their folding, stability or assembly into VLPs. This assumption appeared to be correct as lowering the CP expression temperature indeed largely resolved the solubility issues.For all of the previously studied ssRNA phages, the CPs could be cloned and expressed in As the study progressed, it was further established that the solubility of the CP does not necessarily lead to VLP formation. Generally, CPs of the MS2-like supergroup readily formed VLPs although there were some exceptions in particular subclusters. CPs of the AVE015-like supergroup were also generally able to assemble into VLPs, although a higher prevalence of aberrant particles was observed in these samples. In contrast, in the Cb5-like supergroup VLP formation was detected for less than a half of the examined CPs. It can be noted that the Cb5 VLPs are very sensitive to salt , 37 whicEscherichia coli might cause issues for CPs from phages with markedly different original hosts that might be more complex than growth temperature alone. In most of the cases, however, the failure to form VLPs is presumably caused by the absence of other phage components during the assembly process. The assumption that the presence of unspecific RNA is sufficient to promote particle assembly has been built on a small subset of previously studied coat proteins, and there is no particular reason to expect similar properties for all ssRNA phages. Contrary to the large DNA phages, the ssRNA phages do not package their genome into a preformed empty capsid but instead the CP subunits condense around the genomic RNA molecule to form an enclosing protein shell. In the process, the phage maturation protein specifically binds both the genome and the coat protein, and the genomic RNA itself has a highly complex three-dimensional shape that is thought to actively promote its encapsidation [The rather high proportion of assembly-deficient CPs was somewhat unexpected from our prior experience and, besides the possible VLP stability issues discussed above, likely has several additional reasons. The metagenomic data vary significantly in quality and in some cases the failure of a CP to be expressed or to assemble into VLPs might result from an incorrect sequence caused by sequencing or sequence assembly errors. In other cases, the high-level heterologous expression in sidation –41. Conssidation  and refeThe purification of the novel VLPs was generally straightforward and in most cases, a previously established two-step VLP purification procedure using gel filtration and ion exchange chromatography was able to yield an at least 90% pure preparation. In some cases, however, only about 50% pure material was obtained, presumably due to low VLP stability and/or co-aggregation with bacterial proteins. Still, for the great majority of the novel VLPs, purification does not pose any problems and is suitable for biotechnological processes.To further characterize the VLPs and enable rational structure-guided modification of the capsid, efforts are currently underway to determine their high-resolution three-dimensional structures by X-ray crystallography. The tolerance for foreign antigens by chemical and genetic modification is also being tested in our laboratory for a number of VLPs, and preliminary data indicate that several could be of comparable or superior performance to the Ms2, Qβ and AP205 VLPs (to be published).In this study, we have demonstrated that environmental viral sequences uncovered in metagenomic studies can be useful not only for comprehending the diversity of viruses in nature but can also be successfully utilized to reconstruct virus-like particles in a laboratory setting. In this way we have for the first time experimentally characterized 11 new ssRNA phage coat protein types and their ability to assemble into VLPs. The 80 novel ssRNA bacteriophage VLPs that we have obtained and characterized here will be important for development of new vaccines and related applications using the ssRNA phage VLP platform. The results also provide a rich ground for further fundamental studies of ssRNA bacteriophage biology such as their structure and protein–RNA interactions.The metagenomic ssRNA phage genome sequences were fetched from GenBank using accession numbers reported in –20 or soAll available ssRNA phage CP sequences were used to generate a BLAST database against which each sequence was individually queried using the blastp program. The results in CSV format were imported into a Google Sheets document for calculation of BLAST bit score ratios  and creation of a distance matrix using values of 1-BSR as the distance measure. The matrix was used to cluster the sequences with the UPGMA algorithm using the program neighbor from the Phylip package . FigtreeThe CP-encoding sequences were synthesized by General Biosystems and provided by the manufacturer cloned in pET24a (Novagen) as expression-ready constructs.E. coli BL21(DE3) cells were transformed with CP-encoding plasmids, individual colonies were inoculated in 5 ml of LB media supplemented with 30 μg/ml kanamycin and incubated at 37 °C overnight without shaking. The overnight cultures were transferred into 50 ml of 2xTY medium and the cells were grown at 37 °C or 15 °C with aeration until OD600 reached 0.6 to 0.8. IPTG was then added to a final concentration of 1 mM and the cultures were incubated for additional 4 h at 37 °C or 20 h at 15 °C, after which aliquots were harvested by centrifugation for assessment of expression level and solubility by SDS-PAGE. Large-scale production for VLP purification purposes followed the same protocol using 2 l of 2xTY medium. To determine the solubility of the produced CPs, the aliquoted cells were suspended in lysis buffer  in a wet cell weight/buffer volume ratio of 1:4, lysed by sonication, centrifuged for 30 min at 13000 g and the supernatant and pellet analyzed in SDS-PAGE. The same protocol was used for preparation of lysates for VLP purification.For small-scale expression and solubility analysis, For small-scale purification, 1 ml of clarified bacterial lysate was loaded onto a 12 ml, 6.6 × 400 mm Sepharose 4 FF column  equilibrated with PBS. Chromatography was done on an Acta Prime Plus system  with the flow rate set to 0.3 ml/min and fraction size to 1 ml. VLP-containing fractions were detected in SDS-PAGE and those of the highest purity were pooled and applied to a 0.7 ml, 6.6 × 50 mm Fractogel DEAE (M) ion exchange column . The flow-through was collected and the column further washed with 2 ml of PBS. Column-bound proteins were eluted with a linear 10 column volume gradient to PBS containing 1 M NaCl using a flow rate of 1 ml/min and fraction size of 1 ml on an Akta Pure 25 system . The VLPs were usually found in most of the fractions while the contaminating proteins only in some. The fractions of the highest purity were pooled, dialyzed against 20 mM tris–HCl pH 8.0, supplemented with glycerol to a final concentration of 50% and stored at − 20 °C for downstream experiments. The purification protocol was accordingly upscaled if a larger quantity of VLPs was required.For transmission electron microscopy, VLP samples after gel-filtration were adsorbed on carbon-Formvar-coated copper grids and negatively stained with a 1% aqueous solution of uranyl acetate. The grids were examined in a JEM-1230 electron microscope  operated at 100 kV. Electron micrographs were recorded with iTEM software  using a side-mounted Morada digital camera .DLS measurements were performed using Malvern Zetasizer Nano ZS and quartz cuvette ZEN2112, 173 Backscatter according to manufacturer’s instructions.Aliquots of purified cysteine-containing VLPs in storage buffer  were mixed with an equal volume of Laemmli buffer  with or without added 5% 2-mercaptoethanol. The samples were heated for 10 min at 95 °C and run on a 15% polyacrylamide gel using a standard Tris–glycine SDS electrophoresis system.Assessment of thermal stability was done essentially as described before . VLP samThe starting positions of replicase ORFs in the metagenomic ssRNA phage genome sequences were located based either on the available genome annotations or found by examining the sequences manually. A region flanking 20 nucleotides in both directions from the first nucleotide of the replicase ORF was extracted from each genome and the sequences were input to rnafold using the default parameters.Additional file 1: Table S1. Protein sequences used in the study. Accession numbers are provided for the respective genome sequences. Entries without an accession number were sourced from Dataset S1 from Krishnamurthy et al. [y et al. . In caseAdditional file 2: Figure S1. SDS-PAGE analysis of the production level of the metagenomic ssRNA phage CPs. The samples represent total cellular protein content four hours after the CP expression was induced at 37 °C. 1 and M2—protein molecular weight markers . Figure S2. Solubility of the metagenomic ssRNA phage CPs at 37°C and 15°C. The samples represent the soluble (s) and insoluble (d) cellular protein fractions after the CPs were produced at the indicated temperature. The proteins were produced at 15 °C only if they were completely insoluble at 37 °C. M2—lanes with MW marker—14, 18, 25, 35, 45, 66 and 116 kDa. M1 and M2—protein molecular weight markers . Figure S3. Predicted RNA hairpin structures at the beginning of the replicase gene in the metagenomic ssRNA phage genome sequences. A region flanking 20 nucleotides in each direction from the first nucleotide of the replicase gene was used for the prediction.


As wild poliovirus is eradicated and countries switch from oral poliovirus vaccine (OPV) to inactivated poliovirus vaccine (IPV) per World Health Organization recommendations, preventing circulation of vaccine-derived poliovirus (cVDPV) is a top priority. Currently, the impact of prior poliovirus vaccination on OPV shedding is not fully understood.Stool samples from 2 populations were tested for OPV to assess shedding patterns. 505 samples from 43 US children vaccinated with OPV were collected over 42 days post-vaccination. 1,379 samples from 148 Mexican children vaccinated with OPV were collected over 71 days post-vaccination. Prior vaccination history was recorded for both groups.Seventeen (40%) of the US children had never received poliovirus vaccination while the Mexican children had received at least 2 doses of IPV and 116 (78%) had OPV exposure. In total, 84% of US children and 78% of Mexican children shed OPV , with a mean shedding duration of 17.4 days for US children and 9.3 days for Mexican children .Prior vaccination did not affect the likelihood of shedding, as the US and Mexico cohorts had similar shedding proportions. However, prior vaccination affected shedding duration as the Mexican children, who were largely OPV exposed and all of whom had at least 2 IPV vaccinations, shed OPV for half as long as the US cohort. Since different countries maintain different poliovirus vaccination schedules, it is likely that duration of shedding of OPV varies in populations around the world. In 1988, the World Health Organization announced the inception of the Global Polio Eradication Initiative, dedicated to the eradication of wild poliovirus (WPV). Since then, polio eradication efforts have successfully decreased the number of WPV cases by more than 99%, from 350000 worldwide in 1988 to only 22 reported cases in 2017 [Widespread vaccination with Sabin oral poliovirus vaccine (OPV) is largely responsible for the success of polio eradication efforts. OPV is particularly effective in low- and middle-income settings due to its low cost, easy administration, and ability to provide community-level immunization through fecal–oral transmission from vaccinees to household and community contacts . HoweverThe Polio Eradication and Endgame Strategic Plan 2013–2018 established by the Global Polio Eradication Initiative proposes the introduction of IPV into routine childhood vaccination in countries using only OPV and the eventual removal of OPV from global use . This trWith this in mind, understanding the impact of prior polio vaccination and immunity on OPV shedding in OPV-vaccinated children is of critical importance. Immune responses to OPV and IPV have been extensively studied in a variety of settings. OPV has been shown to produce both humoral and mucosal immunity . By contIn order to better understand the impact of different polio vaccination schedules on OPV shedding patterns in vaccinees, we compared stool samples obtained after OPV vaccination in 2 distinct populations: children living in Palo Alto, California, in 1998 before OPV cessation in the United States, and Mexican children from 3 rural communities in Orizaba, Veracruz, during a National Health Week (NHW). We chose to compare OPV shedding patterns in these 2 populations as we have a unique dataset from these populations with distinct poliovirus vaccination schedules.The data used in this analysis were collected for 2 distinct OPV studies . The firThe second study was a prospective cluster-randomized trial in 3 rural indigenous Mexican communities in Orizaba, Veracruz, Mexico  . ApproxiStool samples from both studies were analyzed for OPV isolates using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Samples were run in triplicate and considered positive for OPV if two-thirds of the reactions had a threshold cycle <37. Positive samples were Sanger sequenced, when possible, to confirm results. More details about the protocol for this analysis are provided by Van Hoorebeke et al .P value of <.05 was considered statistically significant.Shedding duration was calculated with the assumption that a vaccinee’s first positive sample was the first day that vaccinees shed OPV, and that their last positive sample was the last day that vaccinees shed OPV. Additionally, shedding durations were only calculated if participants had multiple positive samples with less than 1 week between positive samples. Comparisons of shedding duration, both overall and by OPV serotype, between US and Mexican vaccinees were done for the overall groups, including all vaccinees, and for the IPV-vaccinated, OPV-naive vaccinees. For Mexican vaccinees, the shedding duration of OPV-exposed vaccinees was compared to OPV-naive vaccinees. For US vaccinees, the shedding duration of poliovirus vaccine-naive children was compared to that of IPV-vaccinated children. These comparisons were made using Wilcoxon-Mann-Whitney test. Comparisons of the number of vaccinees who shed OPV, both overall and by each OPV serotype, as well as for the number of vaccinees with multiple positive samples were made using Fisher exact test. A The vaccination history of the vaccinees is described in P = .44). For each serotype, a higher proportion of the US vaccinees were found to shed each serotype: Mexico vs US OPV-1, 59% vs 74%; OPV-2, 66% vs 74%; and OPV-3, 49% vs 79%. Of these differences, only OPV-3 was statistically significant (P = .0005). Most vaccinees in both groups also shed at multiple time points throughout the studies, with 80% of Mexican vaccinees and 94% of US vaccinees shedding at least twice during the study periods. This difference was not statistically significant (P = .07).A total of 116 (78%) Mexican vaccinees and 36 (84%) US vaccinees shed OPV after vaccination . While aP < .0001). By serotype, the US vaccinees also shed longer than the Mexican vaccinees for all serotypes: 7.7 vs 7.1 days for OPV-1, 10.1 vs 7.3 days for OPV-2, and 17.4 vs 11 days for OPV-3. The differences for OPV-2 and OPV-3 were both statistically significant , while the difference in duration for OPV-1 was not significant (P = .62). In the US vaccinees, both overall and across serotypes, the IPV-vaccinated children shed slightly longer than the children with no prior polio vaccination: 19.4 vs 17 days overall, 8.2 vs 5.7 days for OPV-1, 11.7 vs 7.9 days for OPV-2, and 20.4 vs 18.1 days for OPV-3 (P = .0004) overall, 5.7 vs 10.3 days (P = .03) for OPV-1, 5.7 vs 10.3 days (P = .0002) for OPV-2, and 7.8 vs 15.3 days (P = .0015) for OPV-3. Additional subgroups are listed in Shedding durations were calculated for each group overall, as well as for each distinct polio vaccine combination . Generalor OPV-3 . These dMost vaccinees who shed had OPV isolates in their samples by day 4 in both populations. For Mexican vaccinees 41% began to shed 1 day after OPV vaccination, and an additional 48% were shedding OPV by day 4. Similarly, 69% of the US vaccinees were shedding within 3 days of OPV vaccination, with an additional 22% beginning to shed before the end of the first week after vaccination. Additionally, for both populations, most vaccinees who began to shed early in the study period also had multiple positive samples throughout the study. While few vaccinees began shedding after the first week, the Mexican vaccinee data suggest that vaccinees who began to shed later were less likely to shed OPV for extended periods of time, as shown by a decrease in the percent of vaccinees who shed OPV in multiple samples.The serotype shedding patterns appeared to be similar for both populations. OPV-1 peaked the earliest, by days 4 and 3 for the Mexican and US vaccinees, respectively. OPV-1 was the second most isolated serotype until day 7 for the Mexican vaccinees and until day 10 for US vaccinees, at which point it was the least detected OPV serotype. OPV-2 became the dominant isolate found in samples by day 4 and peaked at day 7 and day 9 for the Mexican and US vaccines, respectively. Additionally, OPV-2 was the most commonly found isolate in both groups and was largely responsible for the peak of overall shedding, also at days 7 and 9. After peak shedding, OPV-2 showed a sharp decline, becoming the second most detected serotype. The 2 groups showed the largest difference in OPV-3 shedding. In the Mexican vaccinees, peak OPV-3 shedding occurred at day 7, after which OPV-3 declined slowly for the rest of the study period. In contrast, the US vaccinees demonstrated sustained shedding for OPV-3, peaking at day 4 and declining after day 14. However, in both groups, OPV-3 was found in the lowest quantities for the first 7–10 days of the study and became the dominant serotype isolated from samples by day 14.Here, we present the results of RT-qPCR analysis of 1884 stool samples collected from 2 distinct populations: 1379 samples collected in 2015 from 148 Mexican vaccinees who were primarily IPV vaccinated and had prior OPV exposure and 505 samples collected in 1998 from US vaccinees who were either completely unvaccinated or primarily IPV vaccinated. Prior poliovirus vaccination history did not significantly impact the likelihood of OPV shedding in these populations, as a similar proportion of vaccinees in both countries had OPV isolates in their stool. Prior literature has found OPV vaccination, as well as OPV-IPV combined vaccination, to be protective against shedding after a dose of OPV , 14. HowAlthough prior vaccination did not appear to significantly impact the likelihood to shed, certain vaccination combinations did appear to impact duration of OPV shedding. Overall, the Mexican children had significantly lower overall shedding durations than the US vaccinees and also had significantly lower shedding durations for OPV-2 and OPV-3. This decreased shedding duration indicates that there may be increased mucosal immunity against OPV , 14 and Shedding patterns in both populations were similar for OPV-1 and OPV-2, indicating that the differences in prior polio vaccination may not impact serotype-specific shedding patterns. However, differences in OPV-3 shedding were detected between the 2 populations, as the US vaccinees demonstrated longer sustained shedding of OPV-3 up to 14 days after vaccination, which was not found in the Mexican vaccinees. This difference could reflect the variability in sample collection, which occurred every 3 days for the first 2 weeks of the study among the Mexican vaccinees, as opposed to the daily samples that were collected for the US vaccinees. It is interesting to note that in the original publication of the US samples, bimodal shedding of OPV-3 was identified and significantly associated with an early single-point mutation related to OPV-3 VAPP . An undeOPV-2 was the most commonly found OPV serotype, identified in 71% and 75% of all positive samples from the Mexican and US vaccinees, respectively. Prior OPV studies have also found OPV-2 to be the most commonly found isolate in stool and sewage samples, which likely contributes to the increased transmission of OPV-2 and the high prevalence of cVDPV serotype 2 . With thOur analysis has some key limitations. First, some vaccination groups were too small for us to draw any meaningful conclusions, such as the OPV-only children, which made up 7% of the US vaccinees. Second, we cannot account for covariates that may impact shedding duration or likelihood to shed, such as sociodemographic status or anthropometric data, as they were not collected for the US vaccinees. Third, samples were not collected daily over the entire study period for all vaccinees. As a result, some shedding may have been missed between sample collections.Our approach also has a number of strengths. First, this dataset is novel, incorporating data from 2 OPV studies in distinct populations, allowing for comparisons between unique groups. Second, in the Mexican study, researchers were able to leverage Mexico’s unique vaccination scheme to ensure that all vaccinee shedding was the result of OPV vaccination as part of our study, as no other children were vaccinated with OPV in these Mexican communities until after our study period was complete, and OPV is not used during routine childhood immunization.In summary, we found no significant difference in the proportion of OPV shedding when comparing US and Mexican vaccinees, indicating that the differences in their prior poliovirus vaccinations did not impact their likelihood to shed OPV. However, prior poliovirus vaccination did appear to impact shedding duration, as the Mexican vaccinees, who were largely vaccinated with both IPV and OPV, shed for significantly fewer days than the US vaccinees, who either had no prior polio vaccination or who were IPV vaccinated. This difference appears to be driven by OPV exposure, as no significant differences were found in shedding duration when comparing the IPV-only vaccinees in both populations. The shedding curves for both populations were quite similar, particularly for OPV-1 and OPV-2, indicating that prior vaccination did not strongly alter the way vaccinees shed these viruses. The US children additionally showed a more sustained shedding of OPV-3, which in a previous study was associated with the acquisition of a mutation related to OPV-3 VAPP. Finally, most positive samples contained OPV-2, which may explain why most cases of cVDPVs have been serotype 2 and which supports the transition to bOPV.With the intent to transition global childhood immunization to IPV-only vaccination schedules, more research is needed to understand what shedding patterns to expect in populations with varying plans of polio vaccination, in the event of future cVDPV outbreaks and possible reintroduction of OPV.Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary_Table_S1Click here for additional data file.


Uterine fibroids (UFs) are the most common benign tumors of the female genital tract. The incidence of UFs has been estimated at 25–80% depending on selected population. The pathophysiology of UFs remains poorly understood. The transformation of smooth muscle cells of the uterus into abnormal, immortal cells, capable of clonal division, is the main component of all pathways leading to UF tumor formation and tumor necrosis factor α (TNF-α) is believed to be one of the key factors in this field. TNF-α is a cell signaling protein involved in systemic inflammation and is one of the cytokines responsible for the acute phase reaction. This publication presents current data about the role of tumor necrosis factor α in the biology of UFs and the related symptoms. TNF-α is an extremely important cytokine associated with the biology of UFs, UF-related symptoms and complaints. Its concentration has been proven to be elevated in women with clinically symptomatic UFs. The presented data suggest the presence of an “inflammation-like” state in women with UFs where TNF-α is a potent inflammation inducer. The origin of numerous symptoms reported by women with UFs can be traced back to the TNF-α influence. Nevertheless, our knowledge on this subject remains limited and TNF-α dependent pathways in UF pathophysiology should be investigated further. Uterine fibroids (UFs) are the most common benign tumors of the female genital tract. The incidence of UFs has been estimated at 25–80%, depending on the populations and multiple risk factors ,2,3,4. ADespite intensive research, the pathophysiology of UFs remains poorly understood. The transformation of smooth muscle cells of the uterus into abnormal, immortal cells, capable of clonal division, is the main component of all pathways leading to fibroid tumor formation. The second component is tumor growth through uncontrollable cell division, as well as production and accumulation of the extracellular matrix (ECM) ,7,12,13 According to various authors, UF metabolism is affected through steroid hormones, growth factors, cytokines, and chemokines ,16,17,18Steroid action is mediated through the interactions of estrogen (ER) and progesterone (PR) receptors with different DNA response elements, which regulate the transcription of selected genes ,20. EstrUF growth is determined by the rate of cell proliferation, differentiation, apoptosis, angiogenesis and ECM deposition ,20,32. ACytokines are low-molecular-weight proteins which are produced and released by immune system cells . They haAlthough various molecules are involved in UF biology, it appears that TNF-α may be one of the most important myometrium-associated cytokines . TNF-α iTNF-α is produced mainly by activated macrophages but it can also be produced by other cell types, e.g., lymphocytes and neutrophils ,60. TNF-TNF-α is also found in smooth muscle cells as a response to tissue injury or upon immune responses to various stimuli ,66,67. TTNFR1 is expressed in most tissues, whereas TNFR2 is found primarily in the immune system cells . After bTNFRs form trimers when reached by the ligand. This binding leads to the dissociation of the silencer of death domains (SODD) inhibitory protein . When SOA review of the publications on the role of TNF-α in UF biology and UF-derived symptoms is presented. Authors conducted their search in PubMed of the National Library of Medicine and Google Scholar. Databases were extensively searched for all original and review articles/book chapters using keywords (one or in combinations): uterine fibroid; uterine leiomyoma; tumor necrosis factor α published in English until October 2018. Moreover additional articles in bibliographies of reviewed articles were searched. Overall, most relevant articles were reviewed and included as appropriate.Inflammation plays a major role in tumorigenesis and inflammation-related factors are key players in the development of many benign and malignant neoplasms, especially due to high increase in the occurrence of a mutation and the proliferation rate of the mutated cells ,87. As sMED12 somatic mutation is the most often detected DNA mutation in human UFs 70–80%) ,96,97,980–80% ,96According to the available sources, elevated levels of TGF-β play an important role in UF growth and clinical symptom progression ,29,103, ECM found in UF differs from the normal ECM in a well-formed myometrial tissue ,14,110. TNF-α is produced by macrophages whose significant numbers can be found in UFs ,43,60. IAccording to available data, fibroid tissue and the adjacent unchanged myometrial tissue demonstrated that TNF-α is abundantly present in the cytoplasm of tumor cells ,121, whiIn the work of Kurachi et al., staining for TNF-α in UF tissue obtained in the proliferative phase was more abundant than in the secretory phase of the menstrual cycle . This reThe picture becomes more complex when we bear in mind that TNF-α is a potent stimulator of aromatase activity, what results in enhanced conversion of androstenedione to estrone . TNF-α iThe reciprocal feedback of progesterone and TNF-α seems more complex when considered along with activin A. As mentioned before, activin A is one of the major factors involved in UF pathogenesis, with a direct pro-fibrotic effect on UF cells by the expression of ECM protein (via Smad pathway) induction . The regActivins are produced in the gonads, pituitary gland, placenta, and other organs. In the ovary, activin increases FSH binding and FSH-induced aromatization. Activin A enhances the activity of aromatase enzyme and simultaneously suppresses progesterone production . In a stWang et al., found that TNF-α upregulates the mRNA levels of MMP-2 in UF cell cultures. The same observations were made with protein levels, whereas this effect was insignificant in the normal myometrium . Their fIslam et al., found that activin A expression was increased under the influence of TNF-α , which mObesity is a condition in which excessive body fat may interfere with the maintenance of an optimal state of health. Obesity is predominantly caused by excessive food intake, lack of physical activity, and genetic susceptibility ,139. AccObesity is characterized by a chronic state of inflammation and this might be a cause of abnormal tissue regeneration, as is the case in UFs . ExcessiMacrophages are important components of the adipose tissue, which actively participate in its activities ,60,149. Further studies are necessary to show the effect of body weight change (gain or loss) on TNF-α and other pro-inflammatory factors levels, and whether this can affect the symptoms associated with UFs.TNF-α may be considered as a chemokine which could influence numerous clinical symptoms associated with UFs ,35. AbdoSymptomatic UFs can cause chronic pelvic pain. Interestingly, this pain cannot always be attributed to the presence of the mass or position of the tumor. While the subject of chronic pain in patients with UFs has been examined only to a small extent, in the case of endometriosis this topic is relatively well-researched . TNF-α lThe connection between UFs and infertility, together with the potential impact of TNF-α in this regard, should be highlighted.Infertility is defined as the inability to become pregnant or carry a pregnancy to full term. There are many causes of infertility and many different methods of treatment . The immIt is officially accepted that UFs decrease the fertility potential ,172. AccTNF-α plays a major role in gastroenterology. Not all gastrointestinal symptoms associated with UFs are only related to pressure on the neighboring organs. Some of them may also have a paracrine or endocrine background. There is increasing evidence for the involvement of the immune system and the related cytokines in various gastrointestinal disorders, altered cytokine expression, and abnormal presence of immune cells, resulting in the occurrence of the clinical symptoms. Several diseases and symptoms depend on the TNF-α-related pathways . TNF-α iTherefore, TNF-α targets were proposed as potential treatment options and today anti-TNF-α drugs are the gold standard in inflammatory bowel disease therapy ,182. TheUFs are just one serious health problem worldwide. Pharmacotherapy is an important chapter in UF management. Prophylaxis of UFs is practically non-existent, while treatment is often costly and expensive. According to Soave et al. current anti-UF strategies that preserve uterus and fertility are not capable yet of controlling clinical symptoms and tumor progression and they are mostly ineffective in the long term outcome . Anti-UFIn the case of other anti-UF agents, their poor effectiveness remains the greatest issue. Innovative forms of UF pharmacotherapy are still under intensive investigation. As stated in recent review from 2017 vitamin D, paricalcitol, epigallocatechin gallate, elagolix, aromatase inhibitors (AIs) and cabergoline might find their place in UF therapy as safe and effective alternatives or co-drugs . After rHowever, this is not so obvious due to the failed attempts to use anti-TNF-α drugs (like infliximab) in case of endometriosis-related pain due to the lack of clinical effects ,190. BetNonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat endometriosis-TNF-α-related symptoms. Nevertheless, data proving that they significantly reduce endometriosis-related pain remain insufficient ,195. AccAccording to less known studies, capsaicin and its analogs are able to block TNF-α-induced NF-κB activation in a dose-dependent manner. This substance can therefore play a chemopreventive role in tumor growth and the derived symptoms. However, no studies on that topic are available  Figure .The use of AIs in therapy has given rise to some expectations . This thUPA, one of the main drugs related to the treatment of UFs ,133,188 In our opinion, there is a possibility that TNF-α serum levels may become a marker used for clinical verification in the case of problematic differentiation (UF or different tumor) , or to dAccording to a study by our group, women can be diagnosed with UFs solely using the serum TNF-α level cut-off point . StudiesIf higher levels of TNF-α are confirmed in the serum of patients with uterine sarcoma, as compared to UF-positive patients, TNF-α could be considered as a non-specific marker which could indicate factors such as what type of surgery should be chosen, if morcellation can be performed, and where to refer the patient. This is important, especially in the current state of surgical management of UFs . TNF-α mEven if the concept was wrong, it should also be considered whether, for example, TNF alpha could be used as a potential therapy efficacy marker, as with our idea about TGF-β3 ,28. SincTNF-α is an extremely important cytokine associated with the biology of UFs, UF-related symptoms and complaints. Its concentration has been proven to be elevated in women with clinically symptomatic UFs. The presented data suggest the presence of an “inflammation-like” state in women with UFs where TNF-α is a potent inflammation inducer. The origin of numerous symptoms reported by women with UFs can be traced back to the TNF-α dependent pathways.Nevertheless, our knowledge on this subject remains limited. It seems vital to study the pathophysiological pathways dependent on TNF-α, in particular its associations with progesterone and activin A. Hopefully, the results of that research will be the decisive factor in selecting the appropriate, individually tailored UF treatment methods. It is possible that TNF-α will prove useful as an additional clinical marker for the diagnosis and therapy of UFs. The importance of anti-TNF-α drugs in the treatment of UFs and how drugs with proven anti-UF action affect the TNF-α dependent symptoms should be investigated further.


Synovial fibroblasts (SF) were reported to produce B cell activating factor (BAFF) in response to stimulation with interferon-γ (IFN-γ) or tumor necrosis factor (TNF). However, the influence of these pro-inflammatory cytokines on other receptors/ligands of the TNF superfamily or associated cytokine receptors in SF has not been investigated yet. Here we show the differential regulation of BAFF (CD257), Fn14 (CD266), TACI (CD267), BAFF-R (CD268), BCMA (CD269), CD40 ligand , IFN-γR (CD119), Leptin receptor , VCAM-1 (CD106) and membrane TGF-β in isolated SF and the impact of IFN-γ/TNF co-incubation on proliferation, IL-6 and IL-8 production. In addition, the impact of differentially stimulated SF on B cell survival in co-cultures was assessed. Surface cytokines and cytokine receptors were detected by flow cytometry. Soluble cytokine receptors and cytokines were quantified by ELISA. Proliferation was assessed by cell titer blue. Murine B cell survival in fibroblast/ B cell co-cultures was determined by annexin V/propidium iodide staining and flow cytometry. IFN-γ together with TNF synergistically and significantly increased the cell surface levels of BAFF, Fn14, TACI, BAFF-R, BCMA, CD40L, ObR and IFN-γR in rheumatoid arthritis SF after 72 h incubation. Soluble BAFF was only induced by IFN-γ and inhibited by TNF. Addition of TWEAK had no influence on proliferation or IL-8 production but decreased TNF-induced IL-6 production, whereas APRIL, BAFF and leptin did not modulate TNF or TNF/IFN-γ-induced proliferation or cytokine production. Proliferation was increased by TNF and further enhanced by the addition of IFN-γ. In co-culture experiments, SF stimulated with TNF/IFN but not TNF or IFN-γ alone increased shedding of VCAM-1 and expression of membrane TGFβ, which was associated with reduced survival of murine B cells. IFN-γ and TNF regulate the expression of TNF family member cytokines and associated receptors. Ligation of IFN-γR and Fn14 under pro-inflammatory conditions modulated IL-6/IL-8 production and proliferation. In B cell/SF co-cultures, the combination of TNF/IFN reduced B cell survival possibly via enhanced VCAM-1 shedding and/or increased TGF-β production. IFN-γ is necessary for the observed effects on B cell survival and SF cytokine production and emphasizes its anti-inflammatory role in rheumatoid arthritis. IFN-γ has a dual role in chronic inflammation since it does increase the presentation of antigens via induction of MHC class II molecules in synovial fibroblasts (SF) and foster chemokine production4. Conversely, IFN-γ counteracts matrix metalloprotease production by IL-1β and increases production of the IL-18 scavenger IL-18 binding protein in SF6. In addition, several studies already evaluated IFN-γ as an anti-arthritic therapy and reported a modest beneficial effect on pain and joint swelling10. These findings are further emphasized by the fact that IFN-γ receptor  knockout mice are highly susceptible to the development of arthritis11. In rheumatoid arthritis synovial fibroblasts (RASF), IFN-γ has been found to increase the expression of B cell activating factor 12. BAFF is a member of the TNF superfamily of ligands and receptors and is an important cytokine for the survival of autoreactive B cells13. Besides BAFF, SF express the related molecules B cell maturation antigen , BAFF receptor (CD268), Fn14  transmembrane activator and CAML interactor  and possibly membrane-bound APRIL 16. Although some effects of APRIL and TWEAK (TNF-related weak inducer of apoptosis) on cytokine production and proliferation in SF have been described, it is still unclear how these ligands influence SF function17. In addition, the effects of pro-inflammatory cytokines on the expression of CD266, CD267, CD268, CD269 and CD154 have not been investigated. In this study, we shed light on the regulation of these surface receptors by cytokines, which play an important role in RA (TNF and IFN-γ) and further provide evidence that SF pre-stimulated with these cytokines differentially influence B cell survival. In addition we investigated the surface levels of ObR (leptin receptor) and influence of leptin on the effects of TNF and IFN-γ, since leptin has been shown to enhance IFN-γ signaling by activating JAK/STAT pathways18.In rheumatoid arthritis (RA), interferon-γ (IFN-γ) levels are increased in synovial fluid and concomitantly, synovial fluid mononuclear cells show increased IFN-γ mRNA levelsWe determined the cell surface levels of BAFF, TACI, BCMA, BAFF-R, CD40L, Fn14, INF-γR and ObR under the influence of TNF (10 ng/ml), and IFN-γ (10 ng/ml). Whereas INF-γ alone had no significant effect on the proteins investigated, we found the combination of IFN-γ and TNF to be a strong stimulus that robustly increased membrane (m)BAFF . In contrast, BAFF production was dose-dependently induced by IFN-γ , BAFF (BAFF-R = TACI»BCMA), APRIL (BCMA = TACI), IFN-γ (IFN-γR) and leptin (ObR) influences RASF proliferation and the cytokine-induced production of IL-6 and IL-8. Proliferation of RASF was significantly increased by the addition of TNF  and further enhanced by the addition of IFN-γ as compared to TNF 10 ng/ml alone  and TNF/IFN-γ  induce significant cell surface expression of VCAM-1 ; + 2286% ± 1421%, p < 0.001, Fig. 27. Consequently, we also assessed cell surface levels of this cytokine and found that TNF  and, even more pronounced, TNF/IFN-γ  up-regulated membrane-bound TGF-β  on human fibroblasts for these experiments, we first showed that human BAFF has an impact on murine B cells and found that conditioned medium from RASF stimulated for 48 h with human BAFF (0.1 ng/ml–10 ng/ml) increased murine B cell survival in RASF/B cell co cultures Fig. . ConditiM-1 Fig. . Since VF-β Fig. . TNF/IFNF-β Fig. .Figure 6et al., we detected BAFF-R on the cell surface of RASF which was increased by TNF/IFN-γ28. BCMA and TACI have already been shown to be expressed on RASF and rat arthritic fibroblasts, respectively, but their regulation has not been investigated15. In line with data from Kamijo et al. we detected Fn14 on RASF and found that TNF alone and TNF/IFN-γ are inducers of its expression29. Since we used IFN-γ as one major stimulus, we were also interested in the regulation of its receptor and showed an increase in response to TNF/IFN-γ. This is in line with results from Alvaro-Gracia et al. who demonstrated an increase of IFN-γ binding after TNF stimulation on RASF30. Binding of IFN-γ to its receptor is usually associated with a downregulation of IFN-γR, but this does not occur in fibroblasts32. We also assessed the influence of IFN-γ and TNF on leptin recepter (ObR) expression and found this combination to increase ObR surface levels. Like IFN-γR, ObR signaling is initiated by JAK/STAT activation and some effects of IFN-γ are potentiated by leptin which has been associated with disease activity in RA33. In addition, TNF has been shown to increase surface levels of ObR by the activation of protein kinase C34. Functionally, leptin has already been shown to increase IL-6 levels in SF but we did not observe a potentiation of TNF or TNF/IFN-γ effects in RASF by leptin. In a next step, we investigated the expression of molecules important for B cell survival and activation in RASF. An indispensable co-stimulatory protein to activate B cells in a T cell dependent manner is CD40L, whose expression by RASF in contrast to CD40, has not been demonstrated directly. The ability of RASF to activate B cells and induce antibody class switching, however, suggests CD40L expression37. Indeed, we found CD40L on the surface of RASF and its levels were augmented by TNF/IFN-γ which might support B cell activation by RASF under these conditions. Another important molecule for B cell function is BAFF, which enhances the survival and class switching of (autoreactive) B cells37. We found an increase of membrane-bound BAFF on RASF after stimulation with TNF/IFN-γ, whereas soluble BAFF was induced only by IFN-γ and inhibited by TNF. This somewhat contrasts results from Reyes et al. that showed increased BAFF levels after TNF stimulation but supports data from Alsaleh et al. who demonstrated reduced BAFF expression in response to TNF39. However, reported TNF-induced BAFF levels in the study by Reyes were extremely low (below 10 pg/ml) and we considered such low amounts as baseline level in our study since control values for BAFF in unstimulated RASF were between 0 and 10 pg/ml. Since we found an upregulation of cytokine receptors TACI, BAFF-R, BCMA, ObR and IFN-γR after stimulation with TNF/IFN-γ, we next investigated whether ligands for these receptors have any impact on proliferation or cytokine production by RASF. We found IFN-γ to further enhance TNF-induced proliferation with a concomitant reduction in IL-6 and IL-8 production. With respect to RASF proliferation this contrasts results from Alvaro-Garcia et al., who showed reduced proliferation when TNF was combined with IFN-γ40. This might be due to different culture conditions since we used RPMI medium with only 2% FCS while the above mentioned study used DMEM with 10% FCS. We found only minimal basal proliferation using our culture conditions, with strong induction of proliferation upon addition of cytokines. Using less stringent culture conditions, where basal proliferation is already significant, IFN-γ might exert different effects. Additionally, Alvaro-Garcia et al. demonstrated that IFN-γ reduced collagenase expression in RASF, which supports our observations regarding IL-6 production, since collagenase and IL-6 are both induced by TNF and employ overlapping transcriptional regulation of mRNA synthesis42. In line with the inhibitory effects of IFN-γ on RASF, studies from Schlaak et al. and Ohta et al. showed reduced IL-6 production when IL-4 was combined with IFN-γ and diminished IL-8 production when IFN-γ and TNF were added together, respectively44. Our results demonstrated a dual role of IFN-γ on inflammation: It cooperates with TNF to up-regulate membrane-bound cytokines and receptors on RASF while concomitantly decreasing the release of cytokines IL-6 and IL-8. IFN-γ is usually produced in a TH1-biased environment during the development of the adaptive immune response and in this situation it might be detrimental to attract innate immune cells such as neutrophils that cannot support a T cell/ B cell response. In fact, it has been demonstrated that IFN-γ priming of macrophages before an LPS challenge reduced the production of mediators necessary for leukocyte recruitment45.In this study, we demonstrated that the combination of TNF and IFN-γ up-regulated the surface expression of the cytokine receptors TACI, BCMA, BAFF-R, Fn14, IFN-γR, ObR, CD40L and also the membrane-bound form of the B cell survival cytokine BAFF in RASF. IFN-γ further enhanced TNF-induced proliferation of RASF while inhibiting IL-6 and IL-8 production. On the other hand, TNF reduced IFN-γ-induced BAFF production. Activation of Fn14 with TWEAK increased TNF-induced proliferation while it decreased TNF-induced IL-6 production by RASF. In RASF/murine B cell co-cultures we found RASF to strongly support B cell survival and this was further enhanced by the addition of BAFF. In addition, RASF stimulated with IFN-γ or TNF increased B cell survival while a combination of both reduced survival. Survival might be linked to the expression and increased shedding of VCAM-1 and membrane-bound TGF-β which are up-regulated by TNF/IFN-γ. To our knowledge, this is the first study that investigated the regulation of surface levels of BAFF, BAFF-R, BCMA, TACI, Fn14, CD40L, ObR and IFN-γR on isolated RASF. In contrast to results from Nakajima et al. also found TWEAK to reduce TNF-induced proliferation and IL-6 synthesis in RASF, TWEAK increased TNF-induced proliferation in our study17. In contrast to Yamana et al., we incubated RASF for 72 h instead of 48 h and included different TNF concentrations. In addition, like mentioned above with IFN-γ, we used only 2% FCS in culture medium for proliferation assays, which might change the effects of TWEAK from inhibitory to stimulatory. TWEAKs ability to promote proliferation is in line with in vivo results, where TWEAK has been shown to stimulate wound-healing47. APRIL modestly increased TNF-induced proliferation but not IL-6 or IL-8 production. In contrast, data from Nagatani et al. showed that APRIL fosters IL-6 mRNA expression in RASF, but this study investigated the effects of APRIL without concomitant stimulation with TNF15. In addition, the same study demonstrated significant APRIL mRNA production by RASF, which was not confirmed on protein level of soluble APRIL by our results or by Alsaleh et al.37. However, APRIL might be present in a membrane-bound form, which has been demonstrated on the surface of macrophages48. Overall, the effects of APRIL on RASF are weak and might only be relevant before the adaptive immune response takes over. BAFF exerted no influence on proliferation, IL-6 and IL-8 production of RASF. In HT1080 fibrosarcoma cells however, BAFF serves as an autocrine stimulator of basal NFκB activity by binding to intracellular BAFF-R49. Since we only employed surface staining of BAFF-R, there might still be a receptor pool located intracellularly. Addition of exogenous BAFF likely activates membrane-bound BAFF-R exclusively, which does not exclude a specialized role of intracellular BAFF in RASF, which needs to be defined in future studies.We further examined the impact of TWEAK, APRIL, BAFF and leptin on TNF and TNF/IFN-γ induced proliferation and IL-6/IL-8 production. TWEAK supported TNF but not TNF/IFN-γ induced proliferation and reduced IL-6 production only when induced by TNF but not by TNF/IFN-γ. While Yamana 53. Murine B cells cultured alone showed very little survival after 48 h, but survival was enhanced when conditioned medium of RASF was added, suggesting a pro-survival effect of a RASF-derived soluble factor. When murine B cells were cultured with RASF directly survival rates quadrupled compared to conditioned medium. This confirms results from Alasaleh et al. that showed that cell-cell contact is a prerequisite for class switch recombination in B cell RASF co-cultures and they identified BAFF as a major factor37. We also demonstrated that addition of BAFF alone or to co-cultures increased B cell survival and this was inhibited by the addition of the monoclonal anti-BAFF antibody belimumab. Although belimumab was considered to only block soluble BAFF, subsequent studies found that this antibody also inhibits membrane-bound BAFF54, and there are hints that inhibition of membrane BAFF might also be relevant in a clinical setting55. An effect on membrane bound BAFF might explain its inhibitory effect on B cells cultivated with unstimulated RASF, as they produce no soluble BAFF but do display membrane-bound BAFF under these conditions. We identified IFN-γ as the sole inducer of soluble BAFF in SF and therefore tested whether RASF stimulated with this cytokine increase B cell survival. Indeed, IFN-γ-stimulated RASF enhanced murine B cell survival and this effect was inhibited by belimumab, suggesting an influence of BAFF. Since membrane-bound BAFF was not modified by IFN-γ, soluble BAFF might be important as it is induced by IFN-γ. While TNF-stimulated RASF did not increase B cell survival, the combination of TNF and IFN-γ reduced survival compared to treatment with each cytokine alone. This was puzzling, since this combination was the strongest inducer of membrane-bound BAFF which is supposed to be indispensable for B cell survival12. However, RASF not only support B cell survival by BAFF but they also express other proteins involved in regulating B cell function. Another regulator of B cell survival, vascular cellular adhesion molecule 1 (VCAM-1), which is the cellular receptor for integrin α4β1 on B cells, is expressed in RASF56. Interestingly, we found TNF and to a lesser extent TNF/IFN-γ to up-regulate cell-bound VCAM-1, but only the combination strongly induced soluble VCAM-1. This suggests that IFN-γ when combined with TNF induces the shedding of VCAM-1 possibly by regulating a specific enzyme e.g. ADAM1723. Soluble VCAM-1 might then compete with membrane-bound VCAM-1 on RASF inhibiting cell-cell contact and thereby diminishing the pro-survival effect of RASF. Another cytokine that regulates B cell function is TGF-β and it is produced by RASF57. We detected the membrane-bound form on RASF, which was up-regulated by TNF and even more so by the combination of TNF and IFN-γ. TGF-β regulates many aspects of B cell function from Ig production to apoptosis and an up-regulation on RASF might reduce B cell survival26.Since we found synergistic and antagonistic effects of IFN-γ when combined with TNF on RASF, we were interested how RASF treated with these cytokines regulate B cell survival in co-cultures. We used murine B cells isolated from spleens as they do respond to human BAFF and CD40L, but only minimally to human TNF and not to human IFN-γ minimizing own effects of these cytokines58. Hence, the in vitro observed up-regulation of receptors and cytokines of the TNF family in response to TNF/IFN-γ likely occurs in RASF exclusively.In addition to RASF, we also analyzed SF from patients with osteoarthritis (OA) as a non-chronically inflamed control. In all assays, we didn’t find any difference between OASF and RASF (data not shown). However, in vivo this might be completely different, since high levels of pro-inflammatory cytokines  are only found in RA but not OA synovial tissue11. Interestingly, in lupus erythematodes (SLE), IFN-γ is a prerequisite for disease development and this might be due to the cell type mainly responsible for disease initiation59. While lupus is mainly driven by the activity of autoreactive B cells, RA is dependent on T cell activation and innate mechanisms61. This is also evident in the therapeutic approach of both diseases, where RA profits from TNF inhibition, while SLE is combatted by B cell-depleting agents, like belimumab, whereas anti-TNF treatment exacerbates or even induces SLE. IFN-γ also induced soluble VCAM-1 when combined with TNF, which might, similar to soluble ICAM-1 alter leucocyte recruitment dependent on cell type and inhibit B cell attachment by disrupting VCAM1/integrin α4β1 interactions63. Additionally, increased membrane-bound TGF-β under the same culture conditions might not only alter B cell but also T cell function und support T-reg generation leading to an overall anti-inflammatory phenotype64.In this study we demonstrated synergistic effects of IFN-γ and TNF on the expression of TNF family member receptors, associated cytokine receptors and RASF proliferation. In contrast, cytokine production was negatively regulated by the combination of TNF and IFN-γ: While BAFF production was inhibited by TNF, IL-6 and IL-8 production was reduced by IFN-γ. Similarly, survival was increased by TNF or IFN-γ-stimulated RASF but reduced by the combination of both cytokines. These findings along with the shedding of VCAM-1 and the up-regulation of TGF-β by the combination of IFN-γ and TNF confirm a partial anti-inflammatory effect of IFN-γ in a TNF-driven pro-inflammatory environment in RA. In fact, it has already been shown that IFN-γR knockout mice show exacerbated arthritis65 were included in this study. The RA group comprised of 21 females and 7 males with a mean age of 63.6 years ± 7.6 years and 67.3 ± 9.8 years, respectively. C-reactive protein was 21.9 mg/dl ± 33.4 mg/dl for females and 67.7 mg/dl ± 61.4 mg/dl for males. 20 out of 28 patients received non-steroidal anti-inflammatory drugs, 20 out of 28 glucocorticoids, 5 out of 28 methotrexate, 1 out of 28 sulfasalazine, 3 out of 28 biologicals and 1out of 28 JAK inhibitor. All patients underwent elective knee joint replacement surgery, and they were informed about the purpose of the study and gave written consent. The study was approved by the Ethics Committees of the University of Düsseldorf  and Regensburg . We confirm that all experiments were performed in accordance with relevant guidelines and regulations.28 patients with long-standing RA fulfilling the American College of Rheumatology revised criteria for RAAll antibodies used in this study are listed in Table Antibodies were used in a 1:11 dilution as recommended by the supplier. Cytokines used in this study Table :66. In brief, excised synovial tissue was cut into small fragments, minced and treated with liberase  at 37 °C overnight. The cell suspension was filtered (70 µm) and centrifuged at 300 g for 10 min. After that, the pellet was treated with erythrocyte lysis buffer  for 5 min, recentrifuged for 10 min and then resuspended in RPMI-1640  with 10% FCS. After overnight incubation, RPMI medium was replaced with fresh medium to wash off dead cells and debris.Samples from RA synovial tissue were collected immediately after opening the knee joint capsule and tissue was prepared for cell isolation thereafter6000 cells were seeded onto 96 well microtiter plates, grown for three days and were then stimulated with either TNF (0.1–10 ng/ml), IFN-γ (0.1–10 ng/ml) or a combination (IFN-γ 10 ng/ml + TNF 10 ng/ml) for 72 h . After that, cells or supernatants were used for flow cytometry (cells) and ELISA (supernatants).Cell culture supernatants were used for ELISAs 72 h after addition of respective stimulants. Tests were conducted as described by the supplier  and R&D/Biotechne, Wiesbaden, Germany ). Inter- and intraassay coefficient of variation was below 10%.68 and therefore, we minimized this influence by only using 2% FCS.Proliferation was assessed by the cell titer blue viability assay (promega). We analyzed proliferation in RPMI medium containing only 2% FCS. This was important since FCS contains up to 28 µM lysophosphatidic acid (LPA). LPA has been reported to induce proliferation, calcium mobilization and IL-8 production in a wide variety of cell typesData were acquired on a MACSQuant analyzer 10 . After detachment of RASF with accutase  at 37 °C for 20 min, RASF were incubated with primary labelled antibodies for 10 min at 4 °C in PBS/1% BSA and analyzed thereafter. Cells were single-stained with respective antibodies.Murine B cells were isolated from spleens using the B Cell Isolation Kit mouse (miltenyi #130-104-443) according to the suppliers’ instructions.Male DBA/1 J mice (6–8 weeks old) were purchased from Janvier (France). They had unrestricted access to chow and water. Five animals lived in one cage, and they were acclimated to the environment for one week before commencement of experiments. The University of Düsseldorf approved all experiments according to institutional and governmental regulations for animal use (project: O57/15). All procedures were performed in accordance with the German Animal Welfare Act and the European Directive 2010/63/EU on the protection of animals used for scientific purposes.Co-culture experiments were performed in 48 well plates . In brief, 20.000 RASF were seeded in 750 µl RPMI-1640 (Sigma-Aldrich) and grown for 72 h. Then, 250.000 isolated murine B cells were added for 48 h and cells were stimulated with cytokines as indicated. After that, B cell survival and apoptosis were assessed by Annexin V/propidium iodide staining.B cell survival and apoptosis were assessed using the FITC Annexin V assay kit  according to manufacturers’ instructions.Statistical analysis was performed with SigmaPlot 13  and SPSS 25 . The statistic tests used are given in the figure legends. When data are presented as box plots, the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the 10th and 90th percentiles. When data are presented as line plots, the line represents the mean and error bars are depicted as standard error of mean. The level of significance was p < 0.05.