As most proteoglycans exert their biological activities in the pericellular region, circulating Endocan has appeared since its discovery as an atypical dermatan sulfate proteoglycan, with distinctive structural and functional properties. Endocan is naturally expressed by endothelial cells, highly regulated in presence of proinflammatory and proangiogenic molecules, binds to matrix proteins, growth factors, integrin, and cells, and may be then considered as an accurate marker of endothelial activation. Consequently, Endocan expression has been associated with a growing number of pathological conditions where endothelium gets challenged and notably in highly vascularized cancers. In this context, Endocan has indeed been rapidly emerging as a promising tissue- and blood-based marker of the vascular growth and neoangiogenesis during cancer progression. Furthermore, very recent studies have reported an expression of Endocan by the tumor cells themselves. This highlights Endocan as a multifaceted molecule with a great interest for researchers and clinicians to better understand tumor development, from the bench to the clinics. With promising perspectives of clinical applications, Endocan thus appears as an exciting model for on going and future developments of proteoglycan-based approaches in cancer diagnostics and/or therapy. During the past 20 years, proteoglycans (PGs) have emerged as critical modulators of most major cellular processes, including cell proliferation, adhesion, and migration, and have been involved in many pathological conditions, such as inflammation, cancer, or infection –5. Thesesensu stricto, (ii) in contrast with most ECM PGs, which are usually large, modular proteins that harbor several GAG chains, Endocan comprises a relatively small polypeptide associated to one single polysaccharide chain, and (iii) characterization of Endocan DS chain revealed uncommon structural features and saccharide composition [The history of Endocan originates from the discovery in 1996 of a soluble protein produced by endothelial cells: endothelial cell-specific molecule-1 (ESM-1) . Posttraposition . Interesposition , 23–28. esm, located in the proximal region of chromosome 5 long arm (5q11.2) [esm gene is organized in 3 exons separated by 2 introns, encoding an 552 bp open reading frame. Transcriptional control is ensured by a 3888 bp promoter at the 5′ flanking region of esm, which features a typical TATA box and a number of putative transcriptional binding sites, including Ets, Hhex, and CRE-like motifs [In vivo, Endocan is detected with an average concentration of ~1 ng/mL in the serum healthy patients [Endocan is the product of one single gene, (5q11.2) . The esme motifs , 30. Note motifs , 29, 30.e motifs , but expe motifs . Surprise motifs , 31, suge motifs . In vivopatients  However,patients  or cancepatients , kidney patients , or colopatients  cancers.α (TNF-α), interleukin-1 (IL-1), transforming growth factor-β1 (TGF-β1), fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor (VEGF) have been shown to induce Endocan expression in vitro [γ (IFN-γ) inhibits TNF-α induced upregulation of Endocan [κB and PI3 K/AKT/FKHRL1 signaling pathways, respectively. This was further supported by Rennel and collaborators, who also reported that VEGF-A induction of Endocan expression in cultured endothelial cells was regulated via the PI3 K pathway. Furthermore, they showed that PI3 K inhibitor LY294002 alone caused an increase in Endocan transcripts to levels higher than those induced by VEGF [Expression of Endocan is regulated by a number of cytokines and growth factors. Tumor necrosis factor-in vitro –40, whil Endocan . Signali Endocan . Using p Endocan . VEGF in by VEGF . Notewor by VEGF , 44. How by VEGF . This va by VEGF , 31. 1-K111) that includes an EGF-like domain (Y46-M90), a phenylalanine-rich region (F113-Y118), and a C-terminal region (S119-R165) that comprises the S137G138 dipeptide, where attachment of the DS chain occurs. Surprisingly, Endocan shares no obvious homologies with other extracellular members of the PG family. Endocan does not include leucin-rich repeats, which are the signature of small leucine-rich protein (SLRP) PGs, nor C-type lectin-like domains that are typically found in hyalectans [Endocan is one of the rare circulating PGs that have been identified, along with bikunin  and macrβ2 integrin, fibrillin-1, laminin-β2, and procollagen α2 [In contrast, 15–28% of sequence identity can be found between the cystein-rich N-terminal region of Endocan and members of the insulin-like growth factor binding protein (IGFBP) family. However, unlike these proteins, Endocan does not bind to IGF , 47. In lagen α2 . Finallylagen α2 . PG25 halagen α2 , 29, 48.N-substituted hexosamine and an uronic acid. In galactosaminoglycans, the hexosamine is an N-acetylgalactosamine, which is associated with either glucuronic acid (GlcA) for CS or GlcA and a variable proportion of its C5-epimer iduronic acid (IdoA) for DS. In addition, this saccharide backbone is modified further by addition of O-sulfate groups: at C-2 of the uronic acid and at C-4/C6 of the galactosamine. Resulting sulfation patterns have led to a classification of CS disaccharide units as 0 units standing for GlcA-GalNAc, A units for GlcA-GalNAc,4S, B units for GlcA,2S-GalNAc,4S, C units for GlcA-GalNAc,6S, D-units for GlcA,2S-GalNAc,6S, and E-units for GlcA-GalNAc,4,6S (The saccharide moiety of human Endocan is represented by as single chain of dermatan sulfate (DS) . DS is aNAc,4,6S . For DS In contrast with most extracellular PG, one single GAG chain is found attached to Endocan protein core. Early studies reported partial susceptibility to chondroitinase B, thereby identifying Endocan chain as of the DS type . Since tIn vitro, Endocan protein core binds to lymphocytes and monocytes through a high affinity interaction with the integrin CD11a/CD18, also called lymphocyte function-associated antigen-1 (LFA-1) [in vitro [Kd of ~3–6 nM for HGF/Endocan interaction [in vivo. As many PGs, biological properties of Endocan depend on ligand interactions involving either its protein core or its DS chain.  (LFA-1) . Endocan (LFA-1) , 58. Endin vitro , 22. HGFin vitro , 60. Impin vitro . Furthereraction , which ieraction , 61, 62.eraction –65. Throin vitro [β, Il-8, MCP-1, IFN-γ, and PF-4, and growth factors Midkine, Pleiotrophin FGF-2, and -7 [In addition, Endocan has been shown to promote the mitogenic and migratory activities of VEGF-A and -C in vitro . Finally, and -7 , 57, leaAngiogenesis is a key event in many cancer types. This process gives rise to a new blood supply so that an emerging tumor shifts from a localized stage to an aggressive behavior , 68. Perin vitro [The release of Endocan by cultured endothelial cells has been largely described to be strongly upregulated in presence of proangiogenic molecules such as VEGF, FGF-2, and VEGF-C that are critical mediators involved in angiogenesis , lymphangiogenesis , and cancer progression (spreading and metastasis) , 39, 66.in vitro . In animin vitro . A link in vitro , 71. TheData obtained from cancer tissue samples strengthen all that experimental evidence of Endocan as a relevant biomarker for clinics. By immunohistochemistry, Endocan is visualized in the cytoplasm of endothelial cells of the newly growing tumor vessels . In the Endocan detection may be today the most pertinent tool to discriminate the “good” , the “bad” (the emerging vessel dedicated tumor angiogenesis), and the “ugly” (the proliferating multilayered capillary as seen in highly aggressive tumor of the brain). Indeed, Endocan is detected in the vessels primarily activated by VEGF , 74. GliThe immunodetection of Endocan in tissues may help to reveal an aggressive behavior and/or recurrence according to the type of tumors. For instance, Endocan immunoreactivity within endothelial cells clearly correlates with a shorter survival in glioblastoma patients . SimilarSince Endocan expression reflects endothelial activation occurring during neoangiogenesis of tumors, dosage of the secreted protein in the blood circulation could also provide a valuable read out of tumor progression and of patient response to drugs. Secreted Endocan is naturally found at low levels in the sera from healthy subjects . Levels Other PGs have been proposed as potential biomarkers of tumor progression, such as the proteolytically shed ectodomains of Syndecan-1 and Glypican-3. For instance, the level of shed Syndecan-1 is increased in sera of patients with lung cancer, myeloma, or liver cancer and was associated with bad prognosis –81. HoweStudy of Endocan in cancer samples took another twist, when expression of the PG by the tumor cells themselves has been reported by immunohistochemistry . SurprisThe question was then raised about the role of Endocan expression in tumor cells themselves. Going through the literature, an early study had already presented Endocan as a protumorigenic molecule when expressed in tumor epithelial cells . When coesm gene encoding Endocan appears as a signature of the angiogenic switch from dormant to aggressive phenotype of breast, brain, osteosarcoma, and liposarcoma tumor cell lines [in vitro Endocan as a DSPG [α and of proangiogenic growth factors such as FGF-2 as previously described for endothelial cultured cells [Further investigations will be needed to clarify the role(s) of Endocan expression in the tumor cells, but the first hints arose when Endocan was described in cultured tumor cell lines as one of the genes associated with two of the major events in tumor progression: the angiogenic switch and the vascular mimicry of tumor cells. First, the angiogenic switch has been described as the key step that determines the development from a dormant and localized tumor into an aggressive tumor able to disseminate and spread over the body . The esmll lines , 85. Intll lines , 85. Incll lines . Interess a DSPG , 84 and ed cells .in vitro (44-fold increase) in highly aggressive skin and uveal melanoma cells compared to poorly aggressive melanoma cells [The expression of Endocan in tumor cells could also be explained by the mechanism described as the vasculogenic mimicry. Vasculogenic mimicry is defined as the unique capability of highly aggressive tumor cells—but not of poorly aggressive cells—to express endothelium-associated genes, to form patterned, tubular networks in three-dimensional culture, which could convey blood plasma and red blood cells and therefore mimics the vasculogenic networks formed by endothelial cells . When inma cells , 88. Thema cells , 90. Thema cells , 91–93. ma cells , 84, 85.Endocan is therefore a unique circulating PG that appears today as a molecule of versatile interest in the study of tumor progression . First,


A 24-year-old man was diagnosed with klinefelter’s syndrome (KS) and his wife wasfound to have an inversion on chromosome 9-46, XX, inv (9) (p11q21)- because of infertility.Intracytoplasmic sperm injection (ICSI) was performed for fertilization after fluorescencein-situ hybridization (FISH) was used to analyze the aneuploidy rate of the Xand Y chromosomes of the ejaculated sperms of the patient, and 99 sperms were haploidamong 100 sperms that were to be analyzed. A twin pregnancy was achieved. The chromosomesof the two fetuses were identified as 46, XY and 46, XY, inv (9)(p11q21) after aprenatal diagnosis at 18 weeks gestation. Two healthy twins were born through caesareansection at 32 weeks gestation because of premature rupture of membranes (PROM). Klinefelter’s syndrome (KS) is the most frequentsex chromosomal abnormality and is usually pathologicallycharacterized by spermatogenic dysfunction. It inclPericentric inversion of chromosome 9 is one of thefrequent chromosomal rearrangements, considereda normal variant with a prevalence of 1-3% -10.AlthHere, we report a case of KS, in which fluorescencein-situ hybridization (FISH) was used to analyzethe aneuploidy rate of the ejaculated spermof the patient, while identifying an inversion inchromosome 9 in his wife. For the first time, intracytoplasmicsperm injection (ICSI) was performedto treat the patient, who fathered a twinpregnancy successfully.A married couple (a 24-year-old man and a22-year-old woman) were treated at our center fora diagnosis of primary infertility. A physical examinationfound the following in the man: height,178 cm; span height, 178 cm; sitting height, 98cm; weight, 79 kg; a history of mumps; normalsecondary sexual characteristics; and a negativeresponse to anti-sperm antibody.6/ ml . His peripheral blood karyotype wasnon-mosaic 47, XXY after we analyzed 200 peripherallymphocytes. The karyotyping results of100 oral exfoliated cells were also analyzed andthe diagnosis was confirmed. No AZF deletionwas found after multiplex PCR analysis. Basedon a previously published method ,bilateral epididymitis, vas deferens, and prostate,but no antheridiogen varicose veins and bilateraltestes of a small size (~5 ml). A semen examination revealed severe asthenozoospermia, of which thesperm showed a volume of 0.8 ml and a densityof 0.7×10d method , FISH wad method .His horHis wife had a menstrual cycle of 4/37 and mild dysmenorrhea,normal vulva and vagina, smooth cervixwithout masts or lacerations, a normal-sized posterioruterus with medium texture, no active tenderness; andnormal bilateral attachments with no lesions or tenderness.The basal endocrine index was as follows:FSH, 6.8 mIU/mL; LH, 5.2 mIU/mL; E2, 19.6 pg/mL;thyroid hormone (T1), 17.7 ng/dL; PRL, 20.7 ng/mL;testo, 0.94 ng/mL and P, 0.41 ng/mL. Multiple immaturefollicles were observed in both of ovaries bytransvaginal ultrasound (TVS) , thus, sKaryotype examination of 200 peripheral bloodcells showed pericentric inversion of one chromosome9, with the breakpoint in the short arm at 9p11and in the distal region of the long arm at 9q21 . The womBefore the couple agreed to ‘assisted reproductivetreatment, they received detailed geneticcounseling and chose the ICSI treatment.Furthermore, the patient and his wife rejecteda preimplantation genetic diagnosis, but werewilling to undergo second trimester prenatal diagnoses.The wife received a short controlledovarian hyperstimulation (COH), and the protocolwas as follows: 1. at menstrual day 3, anintramuscular injection of Decapeptyl  at adose of 0.65 mg was carried for down-regulation;2. from day 5, Gonal-F  was continually used at a dose of 150IU/d to promote ovulation; 3. at day 9, the doseof Gonal-F was reduced to 75 IU/d, at the sametime, Luveris (recombinant LH: rLH) was injectedat a dose of 75 IU/d; 4. serial ultrasoundexaminations and the evaluation of serum E2,LH, and P levels were used to monitor the follicularmaturation. Pregnyl (hCG) was employedfor ovulation induction at a dose of 10000 IUIM when at least two follicles achieved a meandiameter of 18 mm; 5. oocyte aspiration wasperformed at 35th hour after hCG administration.Controlled ovarian stimulation, followedby TVS-guided follicle aspiration, was performedand resulted in the recovery of 11 matureMII stage eggs.On the same day as the oocyte aspiration, 0.5mL of semen was obtained from the patient . Aftercentrifugation, the pellet of the semen was mixedwith 0.1 mL of culture medium, and sperm wasacquired to carry out the ICSI procedure.Eleven mature MII stage eggs were selectedand fertilized by ICSI. At 72 hours post-eggacquisition, two of eleven 8-cell embryos wererandomly selected for embryo transfer, and theother 9 embryos were frozen. A twin pregnancywas established and at 18 weeks, amniotic fluidcells were obtained through transabdominal amnioticfluid puncture to perform prenatal diagnosis.The results showed that the karyotypes of thetwo embryos were 46, XY and 46, XY, inv(9)(p11q21), respectively. Twin boys were deliveredat 34 weeks of gestation because of PROMand admitted to the neonatal intensive care unit(NICU). After 7 days, they were discharged. Todate, the twin brothers have developed normallywith no signs of impaired nervous system functionor cognition.The gold standard of genetic diagnosis forKS remains karyotyping of metaphase spreadsfrom cultured peripheral blood lymphocytes,yet karyotype analysis of other additional tissues could providemore precise diagnosis . KS is cBefore the development of ICSI, patients withKS could only acquire offspring by either adoptionor artificial insemination with another’s sperm.However, in some cases, sperm could still be occasionallyfound in the patient’s semen or througha testicular biopsy. Hence, development of testicularsperm retrieval technology can improve thechance that KS patients can father their own biologicaloffspring .Before ICSI, it is necessary for patients to receiveadequate genetic counseling. Analysis ofthe chromosome aneuploidy rate of the patient’sejaculated sperm can provide direct evidenceof the need for genetic counseling, for whichFISH is the most rapid and accurate method. BergereChromosome 9 has the highest degree of morphologicalvariation , as inveIn summary, the application of FISH in the investigationof the sperm aneuploidy rate can facilitateevaluating the risk of patients with KS and generatinggenetically normal offspring. For those patientsthat still can provide sperm, ICSI treatmentcan be performed after receiving adequate geneticcounseling and obtaining informed consent. Further,ICSI can be performed even when the femalepartner has an inversion of chromosome 9. Lastly,a follow-up study will be conducted on the patient’soffspring.


Pseudomonas aeruginosa, and particularly its predominant metalloproteinase, LasB. For this, we used the secretome of the LasB-expressing pseudomonal strain, PAO1, and compared it with that from the isogenic, LasB-deficient strain (PAO1∆lasB), as well as with purified LasB. Secretomes were tested for apoptotic activity on cultured human endothelial cells derived from the umbilical vein or from the cerebral microvasculature. We found that the PAO1 secretome readily induced endothelial cell anoikis, as did secretomes of LasB-positive clinical pseudomonal isolates, while the PAO1∆lasB secretome had only a limited impact on endothelial adherence and viability. Notably, purified LasB reproduced most of the effects of the LasB-containing secretomes, and these were drastically reduced in the presence of the LasB-selective inhibitor, phosphoramidon. A precocious and extensive LasB-dependent degradation of several proteins associated with the endothelial extracellular matrix, fibronectin and von Willebrand factor, was observed by immunofluorescence and/or immunoblotting analysis of cell cultures. Moreover, the PAO1 secretome, but not that from PAO1∆lasB, specifically induced rapid endoproteolysis of two major interendothelial junction components, VE-cadherin and occludin, as well as of the anti-anoikis, integrin-associated urokinase receptor, uPAR. Taken as a prototype for exogenous haemorrhagic proteinases, pseudomonal LasB thus appears to induce endothelial anoikis not only via matrilysis, as observed for many pro-apoptotic proteinases, but also via cleavage of some essential cell-to-cell and cell-to-matrix adhesion receptors implicated in the maintenance of the endothelial barrier.Within the vasculature, uncontrolled pericellular proteolysis can lead to disruption of cell-to-cell and cell-to-matrix interactions and subsequent detachment-induced cell apoptosis, or anoikis, contributing to inflammatory vascular diseases, with the endothelium as the major target. Most studies so far have focused on endogenous proteinases. However, during bloodstream infections, bacterial proteinases may also trigger endothelial anoikis. We thus investigated the potential apoptotic activity of the proteinases secreted by the haematotropic opportunistic pathogen,  Proteinases associated with focal or systemic inflammation in the vessel wall play a prominent role in modulating permeability of the endothelial barrier, endothelial cell (EC) proliferation and migration, and the balance between EC survival and apoptosis. These include serine-proteinases of the plasma coagulation and fibrinolytic systems, or secreted by activated leukocytes, as well as metalloproteinases, either secreted or membrane-associated ,2. A notPseudomonas aeruginosa is such an opportunistic human pathogen, and a major agent for bloodstream infections, particularly in immunocompromised patients, leading to focal and/or systemic severe vascular diseases [P. aeruginosa [The vascular endothelium is a major target for many human pathogenic bacteria and their virulence factors. Upon entering the bloodstream, they can dampen and escape from the immune defenses, and, in order to disseminate into the body, they alter or even disrupt the endothelial barrier, thus inducing severe pathological events within the vasculature -12. The diseases -15. Thisdiseases ,15-18. Tdiseases ,19. Partdiseases ,20. It iruginosa , is wideruginosa ,23, and ruginosa ,19,24,25via secreted effector proteins is well established. However, investigations have mainly focused on toxins that can be transferred into the cytoplasm of target cells, as shown for P. aeruginosa [P. aeruginosa to produce severe infections within the vasculature and its capacity to induce programmed cell death of cultured ECs [via both matrilysis and receptor proteolysis. Thus, in addition to proteolysis of fibronectin (Fn), LasB readily degrades the EC matrix-specific protein von Willebrand factor (vWf). Furthermore, LasB specifically and directly degrades interendothelial junctional proteins such as VE-cadherin and occludin, as well as uPAR, an important integrin-associated membrane protein involved in cell adhesion to matrix and cell survival, all these proteolytic events being thus likely to participate in endothelial anoikis.The aptitude of pathogenic bacteria to alter the viability of host cells, including ECs, ruginosa ,17. The ruginosa ,27. Thusured ECs , the impured ECs . In the ured ECs  to barriRabbit polyclonal and mouse monoclonal antibodies  were from the following sources: anti-Fn pAb F3648, from Sigma-Aldrich ; anti-vWf pAb A0082, from DakoCytomation ; anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mAb 2D4A7, from Abcam ; anti-VE-cadherin pAb BMS158, from BenderMedSystems ; anti-occludin pAb 71-1500, and anti-claudin-5 pAb 34-1600, from Invitrogen Corp. ; anti-uPAR domain 2 mAb #3932, from American Diagnostica , or a gift from Dr. Viktor Magdolen . Control antibodies were mAb MOPC-21 and rabbit polyclonal serum IgGs, from Sigma-Aldrich and DakoCytomation, respectively, while horseradish peroxidase-, or Alexa Fluor 555-conjugated IgGs were from Jackson Immunoresearch  and Invitrogen, respectively.Streptomycestanashiensis) and aprotinin  were from Sigma-Aldrich, while batimastat (BB-94) was from BioVision Research Products . Polymyxin B sulfate  was from Sigma-Aldrich.Proteinase inhibitors phosphoramidon  was stored at -20°C at 1 mg/ml (≈ 3.6 μM based on a vWf monomer molecular mass ≈ 280 kDa). Purified LasB  was kept at -80°C at 0.5 mg/ml (≈ 15 μM based on a molecular mass ≈ 33 kDa). Its purity and enzymatic activity have been previously described .P. aeruginosa strain PAO1 and its LasB-deficient isogenic mutant PAO1∆lasB [lasB-Sec, respectively) in Luria-Bertani broth (LB) liquid medium (Invitrogen) was performed exactly as previously described [P. aeruginosa clinical isolates derived from patients with infective endocarditis subsequent to implantation of a pacemaker [The previously characterized LasB-producing AO1∆lasB ,31 were escribed ,29, and acemaker ,29,32,33Batches of human umbilical vein endothelial cells (HUVECs) from single donors at first passage were obtained from Promocell Bioscience , as were cell culture medium  and supplements. Cells were seeded on plastic dishes  precoated with 0.1 mg/mL purified rat tail collagen type I , grown to confluence, and passaged after brief trypsinization in the continuous presence of 5 U/ml penicillin, 0.5 μg/ml streptomycin, and 25 ng/ml amphotericin B . Experiments were performed on cell cultures between second and fifth passage.via trypsinization. Experiments were performed on cultures at passages 31 to 35.The human adult brain microvascular endothelial cell line hCMEC/D3 is an immortalized cell line that was established through lentiviral transduction of hTERT and SV40 large T antigen into a primary culture of adult brain endothelial cells , and wasPurified vWf was adjusted to 5 μg/mL (≈ 20 nM) in Tris 50 mM, NaCl 200 mM, pH 7.4 (Tris/NaCl) containing 0.2% FCS, and 100 ng fractions were exposed for 24 h at 37°C to purified LasB (100 nM), to bacterial secretomes or to LB medium in the range of 1 to 10%, in a final volume of 20 μl. The reaction was stopped by addition of sodium dodecyl sulfate (SDS) and heating, before SDS-polyacrylamide gel electrophoresis (SDS-PAGE) coupled to immunoblotting (IB) analysis.2, and maintained at 37°C for variable times. Cells were observed by phase contrast light microscopy and photographed at the end of treatment. In some experiments, incubation of cells was performed in parallel in the presence of the proteinase inhibitors PA (50 μM), BB-94 (40 nM), or Apro (10 μM), or of the antibiotic PMB (20 μg/mL).Confluent human EC cultures were rinsed with culture medium containing low (i.e. 0.2%) FCS, then cells were exposed to either LB medium or bacterial secretomes in the range of 1 to 20%, to purified LasB in the range of 50 to 200 nM, or to 5 μM staurosporine , all diluted in low FCS culture medium for a final volume of ≥ 0.15 ml/cmg for 5 min at 20°C. Cell-free media were further centrifuged at 18,000g for 30 min at 4°C to remove debris, and supernatant then stored at -80°C until analysis. Residual adherent cells were lyzed for 30 min on ice with a radioimmunoprecipitation assay (RIPA) medium containing a cocktail of proteinase inhibitors , and cell lysates were processed as previously detailed [Following exposure of cell cultures to tested substances, treatment media were collected, and detached floating cells were pelleted by centrifugation at 2,000detailed  for furtIn Situ Cell Death Detection kit; Roche Applied Science, Mannheim, Germany) and cell nucleus counterstaining with 4',6-diamidino-2-phenylindole , or Trypan blue exclusion, or total cell protein measurement  were all performed exactly as previously described [Cell quantity assay using Crystal violet  staining, cell viability assay using tetrazolium dye  metabolism, cell death assays using either the terminal deoxynucleotidyl transferase dUTP nick end labeling  were exposed to tested substances, and residual adherent cells were fixed with 3.7% (w/v) paraformaldehyde. Immunostaining of ECM proteins or cell receptors was performed as reported , and as t test. P values are provided in the figure legends.Unless otherwise stated, quantitative data are presented as means ± standard error of the mean (SEM) of the indicated number of independent determinations, and were statistically analyzed by the unpaired, 2-tailed Student’s lasB-Sec) of its isogenic, experimentally-derived LasB-deficient strain or to purified LasB. As previously reported [lasB-Sec is totally devoid of LasB, although still containing active AprA and PrpL , corresponding to LasB concentrations in the range of 25 to 30 nM, produced little morphological changes of cells within 6 h, but cell retraction, rounding and detachment became evident at 24 h  for exposures of 24 h or longer, cell detachment remained significantly lower compared to that induced by PAO1-Sec (P ≤ .02) , as well as with HUVECs exposed to 5% Psa1-Sec or Psa2-Sec, two secretomes derived from clinical P. aeruginosa blood isolates that contained ≈ 450 nM LasB [Upon exposure of cultures to the bacterial secretomes, residual adherent cells were quantified using the Crystal violet colorimetric assay, confirming that, on a time course basis, the number of adherent ECs, both hCMEC/D3 cells and HUVECs, sharply decreased beyond 6 h of exposure to 10% PAO1-Sec, being reduced, at 24 h, by ≈ 50% as compared to control culture wells . FurtherP ≤ .02) . When exlasB-Sec . By contlasB-Sec . FinallylasB-Sec . Similar nM LasB , with orlasB-Sec for 24 h, residual adherent ECs displayed DAPI-stained nuclei with normal appearance, as compared with control LB-treated cells  or 5% (HUVECs) of PAO1-Sec for 24 h  from EC cultures exposed to 10% PAO1-Sec for a period as short as 1 h, or to 1 to 5% secretome for 24 h, that was accompanied by the occurrence of cell-associated fragments with Mr in the range of ≈ 47 to 57 kDa , α5β1 (a receptor for Fn), and αVβ3 (a receptor for Vn and vWf) [r ≈ 57 kDa, and a truncated two-domain (D2D3) species with Mr ≈ 42 kDa that is devoid of the amino terminal D1 domain  ,29,38, t1 domain . Exposur1 domain , while mane uPAR . IntereslasB-Sec . As far P. aeruginosa is an important opportunistic bacterium, inducing focal and systemic vascular diseases [P. aeruginosa produces extracellular proteinases that are also virulence factors [P. aeruginosa isolates from bacteraemic patients are particularly prone to express these proteinases [per se.diseases ,13,14. Idiseases -12, sincdiseases ,28,62,63 factors ,19,20,24 factors ,29,31; , two zymogens constitutively produced by ECs in culture [per se induce EC detachment and death [We establish here that, among the extracellular secreted pseudomonal toxins, LasB can severely affect the adherence and thus the survival of human ECs in culture. Massive changes in cell morphology and loss of attachment seen within 24 hours of exposure of EC cultures to PAO1-Sec, but not to PAO1∆ukocytes ,44,64. Tukocytes . By contitor, PA , drastic culture  and acti culture ,32, and nd death . It mustnd death , and thuAn important feature is that residual adherent ECs remaining over a 24-hour exposure to a LasB-containing secretome do not exhibit an apoptotic phenotype and appear fully viable, a feature that corresponds to anoikis, with detachment of ECs prior to cell death ,8, contrlasB-Sec for up to 24 h. Because the massive apoptosis induced by staurosporine was not accompanied by degradation of ECM-associated Fn and vWf, one can assume that matrilysis is not the mere consequence of proteinases released by dying cells, but rather a LasB-specific process. Interestingly, cleavage of plasma vWf by bacterial metalloproteinases was previously previously reported to be associated with anti-haemostatic and pro-haemorrhagic properties of these enzymes in vivo [P. aeruginosa [Proteinase-induced vascular cell anoikis has generally been assumed to result mostly from matrilysis leading to the loss of cell anchorage and/or to the release of ECM degradation products with anti-adhesive and thus, pro-apoptotic activities -8,35,44. in vivo , and it ruginosa .via interendothelial gap formation [A salient finding of this investigation is that an efficient cleavage of adhesion cell receptors appears to combine with matrilysis to promote EC anoikis. One striking feature is the early and extensive cleavage of two major components of endothelial cell-to-cell junctions, namely VE-cadherin at adherens junctions and occludin at tight junctions. Many inflammatory proteinases, such as neutrophil serine-proteinases and endothelial metalloproteinases, cleave the extracellular domains of VE-cadherin, possibly to facilitate transendothelial emigration of circulating leukocytes ormation ,50,69. Cormation ,70. Howeormation , as oppoormation . It is tormation ,52,53. F2β1, α5β1 and αVβ3 are receptors among those most implicated in EC attachment to ECM components such as collagens, Fn, or Vn and vWf, respectively, providing cells with major survival, and particularly anti-anoikis, signaling pathways [5 and/or β1 has been reported to accompany cell detachment and death, including that induced by bacterial proteinases [via its intrinsic capacity to bind Vn, and as an integrin-associated protein that stimulates cell adherence, migration, and survival [Integrins αpathways ,54-57,67teinases ,46,61. Tteinases . In contsurvival -58. uPA-survival . Since isurvival ,56.P. aeruginosa strains can secrete, under particular microenvironmental conditions, a phospholipase/sphingomyelinase complex that was recently shown to induce EC cytotoxicity and to have anti-angiogenic activity [Taken together, our present results indicate that rapid and extensive, LasB-triggered cleavage of junctional proteins such as VE-cadherin and occludin, accompanied by that of uPAR and of major ECM components, would undoubtedly alter cell adherence and survival, and engage ECs in anoikis. It is however obvious that the pseudomonal secretome contains substance(s), that, in addition to LasB, can alter the morphology of confluent ECs by acting on intercellular junctions. LasB-deficient secretomes still contain proteinases such as AprA and PrpL, but because they induce very limited proteolytic events within the ECM and adhesion receptors, it can be assumed that these proteinases have low, if any, activity in this context. This assumption also applies to LPS, a cell wall component released in the secretomes from Gram-negative bacteria, and a potent virulence factor particularly active on the endothelium ,15, but activity . These mactivity ,62,63. Bactivity ,73.P. aeruginosa extracellular proteasome is one of the most extensively studied for interaction with host proteins and cells [In conclusion, although the nd cells ,29,31,33nd cells . Our prend cells ,68.File S1This file contains Protocol S1 and Figure S1 through Figure S4.Protocol S1, Preparation of extracellular bacterial secretomes and proteinase activity assays. Figure S1, Profile of proteinases in the secretome of wild type and LasB-deficient strains of P. aeruginosa. Figure S2, Clinical pseudomonal secretomes drastically alter adherence of human endothelial cells in culture in a LasB-dependent fashion. Figure S3, Residual adherent endothelial cells in the presence of pseudomonal secretomes are fully viable. Figure S4, The LasB-containing secretome does not proteolytically alter the major endothelial integrins.(DOC)Click here for additional data file.


Lysinibacillus sphaericus strain OT4b.31 is a native Colombian strain having no larvicidal activity against Culex quinquefasciatus and is widely applied in the bioremediation of heavy-metal polluted environments. Strain OT4b.31 was placed between DNA homology groups III and IV. By gap-filling and alignment steps, we propose a 4,096,672 bp chromosomal scaffold. The whole genome  revealed differences in comparison to the L. sphaericus C3-41 genome, such as syntenial relationships, prophages and putative mosquitocidal toxins. Sphaericolysin B354, the coleopteran toxin Sip1A and heavy metal resistance clusters from nik, ars, czc, cop, chr, czr and cad operons were identified. Lysinibacillus sphaericus OT4b.31 has applications not only in bioremediation efforts, but also in the biological control of agricultural pests. Lysinibacillus sphaericus as an insect pathogen, studies have shown mosquitoes to be the major target of this bacterium  analysis. In the first 40 Kbp of contig 1, we found dnaX, recR, and holB, while dnaA, recG and recA were found at the end (after 290 Kbp) of contig 13. This may suggest that contig 13 should be allocated immediately before contig 1. Besides, there was no evidence of multiple dnaA boxes around the potential origin. The replication termination site of the chromosomal scaffold is believed to be localized near 2.5 Mbp in the contig 18, according to GC skew analysis, and the coding bias for the two strands of the chromosome is for the majority of CDSs to be on the outer strand from 0 to ~2.5 Mbp and on the inner strand from ~2.5 Mbp to the end of the chromosomal scaffold  and parE (H131_12183), which encode the subunits of the chromosome-partitioning enzyme topoisomerase IV [L. sphaericus C3-41 genome [rtp (replication terminator protein-encoding gene) in the chromosomal assembly of OT4b.31.The origin of replication of the chromosome of erase IV . Similar1 genome , we did Lactobacillus phage C5 (intact), Bacillus phage φ105, Clostridium phage c-st, Bacillus Phage SPP1 and Bacillus phage Wβ predicted regions were allocated at contigs 34, 8, 15, 18 and 37, respectively. Only lysis proteins were predicted in phages C5 and c-st regions. The only genes remaining in the phage φ105 region are those for coat proteins, integrase, and hypothetical and phage-like coding sequences. This is probably the remnant of phage invasion and genome deterioration during evolution. In addition, any previously reported phages in the genome of L. sphaericus C3-41 are in the genome of OT4b.31.A total of 42 hypothetical protein coding sequences were assigned as putative transposable elements, with the most frequent families being IS66, IS110, IS1272 and IS3. In addition, five prophage regions were identified, of which one region is intact and 4 regions are incomplete. Listeria pathogenicity island LIPI-1, functionally assigned as a thiol-activated cytolysin and a phosphatidylinositol phospholipase C. The first was confirmed to correspond to the L. sphaericus B354 sphaericolysin coding gene in contig 18 (H131_12483). Sphaericolysin B354 has been reported to be widespread across L. sphaericus DNA homology groups not only including IIA, IIB, IV and V [Bacillus toxin from the family Mtx2 (PFam PF03318) was found and described as a hypothetical Sip1A toxin coding sequence (H131_12498). Purified from Bacillus thuringiensis strain EG2158, Sip1A is a secreted insecticidal protein of 38 KDa having activity against Colorado Potato beetle (Leptinotarsa decemlineata) [L. sphaericus OT4b.31 was isolated from beetle larvae, we suggest potential coleopteran larvicidal activity. To our knowledge, strain OT4b.31 is the first report of a predicted Sip1A-like toxin in a native Lysinibacillus sphaericus. Unexpectedly, mtx or bin mosquito pathogenic genes were not found in the OT4b.31 genome, despite a previous report showing positive evidence of BinA/B toxins with no larvicidal activity [Two elements contain conserved domains from the IV and V  but alsolineata) . Consideactivity .sllB (H131_05299) previously reported in L. sphaericus JG-A12 [Bacillus sp. B-14905 was the most similar sequence for the majority of S-layer protein domains. In addition, a putative glycoprotein (H131_22117), a bifunctional periplasmic precursor (H131_05993) and an S-layer fusion (H131_05409) coding sequence associated with S-layer proteins were recognized. On the other hand, a cluster of spore germination genes were determined near the termination of the replication site (including genes from the ger and ype operons) among other genes widespread in the genome. Three clusters of sporulation genes were allocated at contigs 1, 10 and 13 .A total of 32 CDSs were described as surface (S) layer proteins or S-layer homologs (SLH). The putative S-layer gene s JG-A12  was founL. sphaericus OT4b.31 could be controlled by efflux pumps related genes in clusters found in contigs. Putative coding sequence order is as follows: yozA→czcD→csoR→copZA ; nikABC→oppD→nikD ; cadC-like→cadA ; arsRBC – putative extracellular secreted protein CDS – arsR-like→arsR-like→ putative excinuclease CDS . The function of YozA is still unknown [nik cluster, we could not find transcriptional regulators. In summary, L. sphaericus OT4b.31 has protein encoding sequences probably involved in the resistance against Cd, Zn, Co, Cu, Ni, Cr, and As. In fact, prior reports of resistance to toxic metals [L. sphaericus OT4b.31 may be explained due to participation of heavy-metal resistance proteins.Responses against toxic metal(oid)s in  unknown , but is c metals ,17 in L.bceBASR and yvcPQRS; multidrug resistance, MATE (multidrug and toxin extrusion) family efflux pump genes ydhE/norM and acrB; antibiotics resistance, genes vanRSW, tetP-like group II, fusA (elongation factor G), fosB, blaZ and ampC-like. Based in the KEGG analysis, some predicted proteins might participate in peripheral pathways for the degradation of benzoate, aminobenzoate, quinate, toluene, naphthalene, geraniol, limonene, pinene, chloroalkane, chloroalkene, styrene, ethilbenzene, caprolactam and atrazine compounds, and biosynthesis of streptomycin, novobiocin, zeatin, ansamycins, penicillin and cephalosporins.Strain OT4b.31 probably has a diverse defense repertoire according to the following responses and predicted genes: bacitracin stress responses, genes Lysinibacillus sphaericus OT4b.31, isolated from beetle larvae, is classified between DNA similarity groups III and IV. A comparison of the chromosomal sequences of strain OT4b.31 and its closest complete genome sequence, L. sphaericus C3-41, demonstrates the presence of only a few similar regions with syntenial rearrangements, and no prophage or putative mosquitocidal toxins are shared. Sphaericolysin B354 and the coleopteran toxin Sip1A were predicted in the strain OT4b.31, a finding which may be useful not only in bioremediation of polluted environments, but also for biological control of agricultural pests. Finally, Cd, Zn, Co, Cu, Ni, Cr and As resistances probably are supported by efflux pumps genes.The native Colombian strain


APC mutations, APC methylation or chromosomal loss of the APC locus on chromosome 5q, suggesting the involvement of other Wnt-pathway genes. The present study investigated promoter methylation of several Wnt-pathway antagonists in both nonpolypoid and polypoid adenomas.Nonpolypoid adenomas are a subgroup of colorectal adenomas that have been associated with a more aggressive clinical behaviour compared to their polypoid counterparts. A substantial proportion of nonpolypoid and polypoid adenomas lack SFRP2, WIF-1, DKK3 and SOX17 in 18 normal colorectal mucosa samples, 9 colorectal cancer cell lines, 18 carcinomas, 44 nonpolypoid and 44 polypoid adenomas. Results were integrated with previously obtained data on APC mutation, methylation and chromosome 5q status from the same samples.Quantitative methylation-specific PCR (qMSP) was used to evaluate methylation of four Wnt-antagonists, WIF-1 and DKK3 showed a significantly lower level of methylation in nonpolypoid compared to polypoid adenomas (p < 0.01). Combining both adenoma types, a positive trend between APC mutation and both WIF-1 and DKK3 methylation was observed (p < 0.05).Increased methylation of all genes was found in the majority of cell lines, adenomas and carcinomas compared to normal controls. Methylation of Wnt-pathway antagonists represents an additional mechanism of constitutive Wnt-pathway activation in colorectal adenomas. Current results further substantiate the existence of partially alternative Wnt-pathway disruption mechanisms in nonpolypoid compared to polypoid adenomas, in line with previous observations. Colorectal cancer (CRC) results from the accumulation of multiple alterations in the (epi) genome of the epithelial cells that line the large intestine. These events first give rise to an adenoma that, in a minority of cases progresses into an invasive and potentially metastasizing adenocarcinoma.et al. described a lesion in the large intestine that was termed ‘small flat adenoma’ [The terms polyp and adenoma have long been used as synonyms. However, more recently it was recognized that other phenotypes exist besides the traditional polypoid colorectal adenomas. Already in 1985 Muto adenoma’ ,3. Curreadenoma’ -7. NonpoTP53, and constitutive activation of KRAS and the Wnt-pathway [APC[APC mutations and simultaneously more frequent chromosome 5q loss (locus of APC) compared to polypoid adenomas [APC silencing by promoter hypermethylation occurred at similar frequencies in both phenotypes . However, in a substantial part of adenomas of both phenotypes no direct APC disruption was observed. Next to activation of the Wnt-signalling pathway via inactivation of the APC gene , methylation-mediated silencing of other upstream Wnt-signal regulating genes may present an alternative mechanism of constitutive Wnt-pathway activation in CRC [Well known events during the progression of adenoma to carcinoma are the loss of tumor suppressor -pathway . Wnt-pathway [APC,12. Receadenomas ,14. APC n in CRC ,16. MethAPC in both nonpolypoid and polypoid adenomas. To this end, four genes were selected known to have an antagonistic effect on the Wnt-pathway, which have been described before to be frequently methylated in CRC; Secreted Frizzled-Related Protein-2 (SFRP2), Wnt Inhibitory Factor-1 (WIF-1), Dickkopf-3 (DKK3) and SRY-Box-17 (SOX17) [APC mutation, APC promoter methylation and genomic loss of the APC locus in the same adenomas.We aimed to investigate the contribution of methylation of a number of Wnt-regulators other than  (SOX17) . Promote (SOX17)  in a welA panel of 9 CRC cell lines  was used in this study. Colo205, Colo320, HCT116, HT29, SW480, SW620, LS174T and LS513 were cultured in DMEM  supplemented with 10% fetal calf serum (FCS) . Caco2 was cultured in RPMI1640 (Lonza Biowhittaker) supplemented with 20% FCS. Both cell culture media were supplemented with 2 mM L-Glutamine, 100 IU/ml sodium-penicillin  and 100 μg/ml streptomycin , Italy). The cervical cancer cell line CaSki was used as positive control and cultured as described before . All celhttp://www.fmwv.nl and http://www.federa.org). This study was approved by the VU University medical center (2011-03), the Leeds University (CA02/014 Leeds (West)) and the Hospital Vitkovice (EK/140/10).Collection, storage and use of archival tissue and patient data were performed in compliance with the “Code for Proper Secondary Use of Human Tissue in the Netherlands” . The IRB waived the need for consent for use of the archive samples, and the samples were analyzed anonymously.Formaldehyde-fixed, paraffin-embedded (FFPE) colorectal tissue samples were collected at three different institutes; Leeds General Infirmary in Leeds, UK, Hospital Vitkovice in Ostrava, Czech Republic and VU University medical center in Amsterdam, The Netherlands ,13,27. PDNA and RNA from cell lines was isolated using TRIzol Reagent  according to the manufacturers’ instructions . DNA froSFRP2, WIF-1, DKK3 and SOX17 was performed using quantitative methylation specific PCR (qMSP) as described before [DNA methylation analysis of d before ,30. All -ΔCt method [In addition, the modified, unmethylated sequence of the housekeeping gene B-actin (ACTB) was amplified as a reference to verify sufficient DNA quality and successful DNA modification . Samplest method . The uppt method .CaSki cells were incubated with 0.2 and 5 μM 5-aza-2′-deoxycytidine  diluted in PBS for five days. All incubations were performed in duplicate, and cells were directly harvested for DNA and RNA isolation. Gene expression was evaluated by RT-PCR as previously described .Statistical analysis was performed using SPSS 20. We used a significance level of p < 0.013 (0.05/4 genes), to adjust for multiple testing according to the correction suggested by Bonferroni . CompariWIF-1 methylation and the independent variables phenotype, location, APC methylation, 5q loss and APC mutation. First the univariate relationships between gene methylation and the dependent variables were examined. Multivariate analyses were performed including all variables with a univariate p-value of less than 0.1. Next, we used a stepwise procedure and removed the variable with the largest p-value in each step, until only variables with a p-value < 0.05 remained in the multivariate model.Logistic regression was used to examine the relationship between SFRP2, WIF-1, DKK3 and SOX17 promoter methylation levels between nine CRC cell lines and eighteen normal colon mucosa samples revealed significantly elevated methylation levels in CRC cells for all four genes . For SFRP2, WIF-1 and DKK3 increased methylation levels were observed in all nine CRC cell lines, whereas SOX17 showed higher methylation levels in all but two cell lines (LS513 and LS174T) .DKK3 and WIF-1 methylation frequencies were significantly higher in polypoid adenomas , WIF-1; 87% (34/39)) compared to nonpolypoid adenomas , WIF-1; 57% (24/42); p = 0.005 and p = 0.003, respectively). WIF-1 methylation was also significantly higher in polypoid adenomas compared to carcinomas , p = 0.003).To dichotomize the qMSP results into positive or negative for methylation, a cut off was calculated for each gene based on the 99% confidence interval of the normal controls Figure . SignifiWhen the methylation results of the four Wnt-antagonists were combined into one value that was positive if all four markers were methylated 79% 31/39) of the polypoid adenomas were positive in comparison to only 40% (16/40) of the nonpolypoid adenomas (p = 0.0004), indicating a lower level of Wnt-antagonist methylation in nonpolypoid adenomas in general  compared to the right colon 56% (18/32), p = 0.01  and chromosome 5q loss [APC methylation as well as for chromosome 5q loss, no relation was found with the promoter methylation results for SFRP2, WIF-1, DKK3 and SOX17 when combining both adenomas types or in nonpolypoid adenomas or polypoid adenomas, separately  showed WIF-1 methylation and of the APC wild type adenomas 61% (25/41) showed WIF-1 methylation (p = 0.048). For DKK3, 95% (38/40) of the APC mutated samples showed DKK3 methylation whereas only 78% (32/41) showed DKK3 methylation in the APC wild type adenomas (p = 0.026) , a multivatiate analysis was performed for WIF-1 methylation. For the genes SFRP2, DKK3 and SOX17, we were unable to perform a valid multivariate analysis, due to the limited number of unmethylated samples[To investigate the possible interaction between the different variables , location  and APC mutation (p = 0.035) were related to WIF-1 methylation. In the multivariate analysis, location and APC mutation were removed from the model , leaving only phenotype in the model. This suggests that phenotype is the major contributor to the observed difference in WIF-1 methylation in our samples.For the SFRP2, WIF-1, DKK3 and SOX17), in polypoid and nonpolypoid adenomas, and its possible association with other molecular events that can play a role in Wnt-pathway activation. All four Wnt-antagonists showed significant increased methylation in CRC cell lines, carcinomas as well as in nonpolypoid and polypoid adenomas compared to normal colon mucosa. A functional relation between methylation and gene silencing was shown for SFRP2, DKK3 and SOX17.The present study focussed on promoter methylation of four known Wnt-pathway antagonists  but not compared to nonpolypoid adenomas. Lower levels of methylation in carcinomas compared to adenomas have been described before for WIF-1[ESR1[WIF-1 is less important in carcinomas or that silencing of these genes in carcinomas is achieved by other changes to the DNA [WIF-1, indicating that WIF-1 gene expression might be regulated by more complex regulatory mechanisms, potentially including histone modification.Interestingly, we found lower for WIF-1 but alsofor WIF-1 and ESR1IF-1[ESR1. This ma the DNA . We did DKK3 methylation a positive relation with higher CRC stages was described [WIF-1 methylation no relation with CRC stage was observed by either Aguilera et al.[For escribed . This cora et al. or us.APC disrupting events  revealed a positive trend between WIF-1 and DKK3 methylation and APC mutation. Although, the role of WIF-1 and DKK3 in the Wnt-signaling pathway is still poorly understood, these data may suggest that methylation of these Wnt-antagonists is complementing APC disruption and acts synergistically [Analysis of the relation of methylation of all four genes with previously published results on stically ,38.WIF-1 methylation in left-sided adenomas compared to right-sided adenomas. All other three genes were location-independent.Left and right CRCs have been suggested to be different clinicopathological entities ,40 RightWIF-1 methylation was more frequent in adenomas from the left colon, WIF-1 methylation was also higher in polypoid adenomas compared to nonpolypoid adenomas. This could introduce a bias in our analysis, since it is reported that nonpolypoid adenomas occur more frequently in the right colon compared to the left colon [APC mutation, APC methylation and chromosome 5q loss. From this analysis it became clear that phenotype was the main contributor to the observed difference between polypoid and nonpolypoid adenomas.Next to the above mentioned observation that ft colon . To furtIn the current study we had to restrict our analysis to a candidate gene approach, given the fact that the nonpolypoid adenomas studied are very small and concerned FFPE material, as of which only a few methylation events could be studied. A genome-wide methylation profiling approach may reveal further distinctions between both types of adenomas.SFRP2, WIF-1, DKK3 and SOX17 was significantly higher in carcinomas as well as both types of adenomas compared to normal colorectal mucosa. We found higher levels of methylation for WIF-1 and DKK3 in polypoid adenomas compared to nonpolypoid adenomas. These results further substantiate differences in Wnt-pathway disruption as already observed previously for APC mutation rate and APC loss in nonpolypoid adenomas compared to polypoid adenomas.Methylation of APC: Adenomatous polyposis coli; CRC: Colorectal cancer; FFPE: Formaldehyde-fixed, paraffin-embedded; qMSP: Quantitive methylation specific PCR; MSP: Methylation specific PCR.The authors declare that they have no competing interests.Study concept and design, BC, RS, CM, MvE, GM; acquisition of data, QV, JJ, MT, SM, SS; analysis and interpretation of data, QV, JJ, SS; drafting of the manuscript, QV, JJ; critical revision of the manuscript, BC, RS, GM, MvE, HG; statistical analysis, QV, JJ, SS; obtained funding; CM, MvE, GM; technical support, MT, SM, SS; material support, NvG, MK, HG, BR; study supervision, GM, BC, RS. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/603/prepubDKK3, SFRP2 and SOX17 promoter methylation is associated with reduced expression. We evaluated whether SFRP2, WIF-1, DKK3 and SOX17 DNA methylation was inversely correlated with its gene expression. It was shown before that all four genes were methylated in CaSki cells [SFRP2, WIF-1 and SOX17 and to a somewhat lesser extent for DKK3. As shown in panel B, the decreased methylation after DAC treatment was correlated to an increase in SFRP2, DKK3 and SOX17 mRNA expression. The housekeeping gene SnRNP was used as control [WIF-1 mRNA expression was found after DAC treatment. Hence, methylation of SFRP2, DKK3 and SOX17 affects its gene expression.ki cells  and ther control . No effeClick here for fileSFRP2, WIF-1, DKK3 and SOX17 in polypoid adenomas, nonpolypoid adenomas and carcinomas. Table S2. Promoter methylation frequencies of SFRP2, WIF-1, DKK3 and SOX17 in relation with an APC disrupting event. Results are shown for all adenomas, divided into samples that harbor an APC disrupting event or lack an APC disrupting event.Promoter methylation frequencies of Click here for file


Orthomyxoviridae. Influenza can pose a serious risk to the health of mainly the elderly, the very young, and to people suffering from medical conditions (e.g. weak immune system). For example, seasonal influenza strains are fatal to more than 50,000 people annually in the United States alone [Influenza, or “flu”, is an upper respiratory tract infection caused by a virus belonging to the family of es alone . The moses alone .Influenza viruses take advantage of the host cell metabolism to replicate their genetic material and to synthesize viral proteins. The influenza virus particle consists of three major parts: the ribonucleocapsid, the matrix protein M1, and the envelope, which is derived from the plasma membrane of the host cell. The lipid bilayer contains the ion channel protein M2 and the immunogenic glycoproteins hemagglutinin and neuraminidase . The rep3) in a CO2 incubator at 37 °C and 5 % CO2 to the stationary phase  [All chemicals and enzymes were purchased from Sigma  or Roche . Adherent MDCK cells obtained from the ECACC No. 84121903) were routinely cultured in 6-well plates containing 4 mL of GMEM-based medium  fetal calf serum, 2 g/L peptone, 48 mM NaHCO4121903 w6 cells  to 10348.06±1663.65 nmol/min/106 cells  in H1N1 and mock-infected cells, respectively. Highest activities (>1000 nmol/min/106 cells) were found for TPI, pyruvate kinase, lactate dehydrogenase, and malate dehydrogenase, while the other activities were in the range of 1 to 500 nmol/min/106 cells. Very low enzyme activities, which indicate possible rate-limiting steps in the respective metabolic pathway, were found for PDH, pyruvate carboxylase (PC), NAD+-dependent isocitrate dehydrogenase (NAD-ICDH), and glutamine synthetase (<1 nmol/min/106 cells). Significant differences in catalytic activity between infected cells and mock-infected MDCK cells were found for 9 enzymes: glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from the pentose phosphate pathway, glutaminase and malic enzyme from glutaminolysis, PC, citrate synthase, citrate lyase and NAD-ICDH from the citric acid cycle, and acetate-CoA-ligase , i.e. an up-regulation of key metabolic enzyme activities in MDCK cells. This shift in metabolism is most likely required to compensate for the metabolic imbalance caused by viral replication. Interestingly, activity of some key enzymes producing NADPH and acetyl-CoA, a precursor needed for lipid and cholesterol biosynthesis, were increased by at least 20 % in infected MDCK cells. Hence, fatty acid synthesis might play a crucial role for the replication of influenza viruses as they acquire lipid envelopes from their host cells.


Lymphedema (LE) is a chronic medical condition characterized by lymphatic fluid retention, resulting in tissue swelling. Cancer treatments involving lymph nodes can damage lymph drainage routes, causing accumulation of lymph fluid in the interstitial tissue of related limbs and body areas and secondary LE. Basically, the LE has a negative impact on physical and mental quality of life. Moreover, 0.07–0.04% of long term survivors (most patients undergoing mastectomy) can develop the Stewart-Treves syndrome, a rare and aggressive multifocal lymphangiosarcoma arising within the LE region. Here we describe the case of a 45-year-old woman with a massive LE of the left arm, as a consequence of previous breast cancer, who was diagnosed after 4 years of stage IV mantle cell lymphoma (MCL). The patient, after obtaining complete remission with chemotherapy and autologous hematopoietic stem cell transplant, had a relapse of MCL in the lymphedema site. Lymphedema (LE) is a chronic medical condition characterized by lymphatic fluid retention, resulting in tissue swelling. Cancer treatments involving lymph nodes can damage lymph drainage routes, causing accumulation of lymph fluid in the interstitial tissue of related limbs and body areas and secondary LE. Recently, Paskett et al. reviewed risk factors, diagnosis and treatment of cancer related LE.2 day 1, cyclophosphamide 750 mg/m2 iv day 1, doxorubicin 50 mg/m2 iv day 1, vincristine 1.4 mg/m2 ev days 1 and prednisone 1mg/m2 days 1–5), followed by three courses of R-MiCMA2 day 1, Mitoxantrone 10 mg/m2 iv day 1, Carboplatin 100 mg/m2 iv days 1–4; Aracytin 2000 mg/m2 iv day 5 and Methylprednisolone 500 mg/m2 iv days 1–5) with haematopoietic stem cells (HSC) harvesting. The patient achieved complete remission and underwent autologous HSC transplantation as consolidation therapy. The post-transplant follow-up was uneventful for one year, when patient was again admitted to our department with a massive dissemination of purplish cutaneous nodules, in part ulcerated and infected, over the entire surface of the lymphedematous arm (A 45-year-old woman with stage IV mantle cell lymphoma (MCL) was recently admitted to our hematology department. She had a massive LE of the left arm, as a consequence of previous breast cancer, treated four years before with radical mastectomy, radiation and chemotherapy. The patient received four courses of R-CHOP (Rituximab 350 mg/mtous arm . She reftous arm . A totaltous arm  that at There are few cases reported in the literature on malignant lymphomas arising in a lymphoedematous arm, which can sometimes simulate the Stewart-Treves syndrome.9In conclusion, our experience emphasizes that, in assessing the response to chemotherapy of lymphoma patient with chronic LE, in addition to routine examinations, it should be performed a careful investigation of the involved extremity tailored to exclude residual tumour localizations.


In contrast, M2 macrophages express a wide array of anti-inflammatory molecules, such as IL-10, TGF-β, and arginase1. In most tumors, the infiltrated macrophages are considered to be of the M2 phenotype, which provides an immunosuppressive microenvironment for tumor growth. Furthermore, tumor-associated macrophages secrete many cytokines, chemokines, and proteases, which promote tumor angiogenesis, growth, metastasis, and immunosuppression. Recently, it was also found that tumor-associated macrophages interact with cancer stem cells. This interaction leads to tumorigenesis, metastasis, and drug resistance. So mediating macrophage to resist tumors is considered to be potential therapy.Macrophages are widely distributed innate immune cells that play indispensable roles in the innate and adaptive immune response to pathogens and in-tissue homeostasis. Macrophages can be activated by a variety of stimuli and polarized to functionally different phenotypes. Two distinct subsets of macrophages have been proposed, including classically activated (M1) and alternatively activated (M2) macrophages. M1 macrophages express a series of proinflammatory cytokines, chemokines, and effector molecules, such as IL-12, IL-23, TNF- Macrophages were initially described by Elie Metchnikoff, who won the Nobel prize in 1905 because of his identification of phagocytes and his phagocytosis theory [Macrophages exist in almost all tissues and play important roles in the maintenance of tissue homeostasis. In mature adults, macrophages differentiate from peripheral blood monocytes, which develop from common myeloid progenitor cells. These cells are identified as granulocyte/macrophage colony-forming units (GM-CFUs) in the bone marrow. In response to a macrophage colony-forming factor, GM-CFUs sequentially give rise to macrophage colony-forming units (M-CFUs), monoblasts, and pro-monocytes. Subsequently, they move into the peripheral blood and differentiate into monocytes. Finally, the monocytes migrate into different tissues and replenish the populations of long-lived tissue-specific macrophages, such as alveolar macrophages and kupffer cells , 3. Howeγ (IFN-γ) alone or in concert with bacterial moieties, such as lipopolysaccharide (LPS) or cytokines ) [β (TGF-β), or glucocorticoids (GCs) [α, IL-1, IL-6, IL-12,andIL-23) and increase their concentrations of superoxide anions, oxygen radicals, and nitrogen radicals [Macrophages, like other immune effector cells, can have multiple subtypes and take on various phenotypes depending on the microenvironment. By analogy to the Th1/Th2 classification, two distinct states of polarized activation for macrophages have been proposed: the classically activated (M1) macrophage and the alternatively activated (M2) macrophage subsets . M1 macr(TNF-α))     ligand 9 (CXCL9), CXCL10, and CXCL5 through the activation of the transcription factor IFN regulatory factor-3 (IRF-3), which results in IFN-β expression and subsequent STAT1  activation. These proinflammatory chemokines can promote the recruitment of Th1, Tc1, and NK cells, which can improve their capacity for intracellular pathogen killing [κB and STAT1 [Differentially polarized macrophages can also express different chemokines. For instance, LPS and IFN-nd STAT1 , 22. M2aHeterogeneity and plasticity are important features of macrophages. Under different stimuli, macrophages can polarize into different phenotypes. However, these phenotypes are not stable. Several in vivo studies have demonstrated that the phenotype of an activated macrophage population can change over time. For example, during tumor progression, the macrophage phenotype changes from classically activated to alternatively activated . In contAs we mentioned above, macrophages in different microenvironments play different roles. Next, we will discuss the role of TAMs in tumor progression.A tumor, as defined by Wills, is “an abnormal mass of tissue, the growth of which is uncoordinated with that of the normal tissues and persists after the cessation of the stimuli which evoked the change.” Tumors are composed of proliferating tumor cells and stromal cells, including endothelial cells, inflammatory cells, and fibroblasts . In the γ, IL-1, and IL-6, which activate type-1 T-cell associated with antitumor immune responses [The role of TAMs in tumors is still controversial. It has been reported that in colorectal tumors TAMs are proinflammatory, and play an antitumor role, which leads to a good prognosis , 28. Oneesponses . Howeveresponses . Therefoβ, and IL-10, all of which lead to the adoption of an M2 phenotype [TAMs exhibit an M2-like phenotype because they express a series of markers, such as CD163, the Fc fragment of IgG, C-type lectin domains, and heat shock proteins –32. On thenotype . TAMs or+ myeloid cells [TAMs are differentiated from monocytes by a number of chemoattractants that are produced by tumor cells and stromal cells. For instance, tumor-derived chemokine CCL2, formerly known as monocyte chemotactic protein (MCP), is critical for the recruitment of macrophages , 35. CCLid cells .Tumors do not grow beyond 2-3 mm³ and cannot metastasize unless they are vascularized . It is wβ (TGF-β), and a member of the FGF family, which can promote angiogenesis in many tumors, such as gliomas, squamous cell carcinomas of the esophagus, and breast, bladder, and prostate carcinomas [How do the TAMs regulate angiogenesis? It has been reported that TAMs are able to modulate and induce neovascularization and support functions. When TAMs are activated, they can express a broad repertoire of substances  to promote angiogenesis. For instance, TAMs release growth factors such as VEGF, PDGF, transforming growth factor rcinomas , 36, 45.rcinomas . Lin andrcinomas , 48. Morrcinomas , 50. MMPrcinomas .TAMs have been found to accumulate in hypoxic regions of tumors, which are characterized by low-oxygen tension. As TAMs adapt to the hypoxic microenvironment, they can express more proangiogenic genes, such as VEGF, pFGF, CXCL8, and glycolytic enzymes, whose transcription is controlled by the transcription factors HIF-1 and HIF-2 . In addiLymphangiogenesis is the initial step in the generalized spread of tumor cells, which predicts a poor clinical prognosis. TAMs promote the lymphangiogenesis mediated by VEGF-C and VEGF-D via VEGFR3 . It has + macrophages physically contribute to lymphangiogenesis under pathological conditions and that bone marrow-derived CD11b+ macrophages express lymphatic endothelial markers, such as LYVE-1 and Prox-1, under inflamed conditions in the corneal stroma of mice [Recently, Maruyama and colleagues found that CD11b of mice . These f of mice .In addition to promoting angiogenesis and lymphangiogenesis, TAMs also play a pivotal role in tumor growth. It has been demonstrated that TAM infiltration is positively correlated with the proliferation of tumor cells in several tumors, such as breast cancer, endometrial cancer, and renal cell cancer , 60. MacMMP9, which was mentioned earlier as a primary factor promoting angiogenesis, also plays an important role in tumor growth. The cytokine IL-23 is considered to promote tumor incidence and growth by upregulating MMP9, thereby stimulating inflammatory responses , 63. MorRecently, a notable paper reported that in melanomas, TAM-derived adrenomedullin is involved in angiogenesis and tumor growth. It was found that the adrenomedullin derived from TAMs interacts with its receptors on endothelial cells to promote tumor growth via a paracrine loop through the activation of the eNOS signaling pathway similar to the angiogenesis cytokine VEGF . On the Tumor metastasis is an important marker in determining the severity of cancer. Tumor cells metastasize through the blood and lymphatic vessels, which leads to the formation of ectopic tumors. These tumors present a great therapeutic challenge and result in a poor prognosis . More thCoffelt et al. found that both TAM and tumor cells migrated frequently when they were in close proximity by multiphoton microscopy. Further study revealed that the epidermal growth factor (EGF) released by TAMs interacted with the CSF-1 released by tumor cells to promote the migration of the tumor cells . Accumulα-dependent manner [κB ligand (RANKL) [β, cathepsin B, Wnt5a, and semaphorin 4D (Sema4D), have also been reported to promote tumor metastasis [On the other hand, TAMs influence the microenvironment, which can also promote tumor cell invasion. Hagemann and colleagues demonstrated that coculturing TAMs with tumor cells can promote the expression of MMPs, especially MMP2 and MMP9, in TNF-t manner . Both MMt manner . In addi (RANKL) , 73. Othtastasis .β-catinen pathway [Recently, it has been reported that macrophage-derived microRNA (miRNA) also regulated tumor invasion. Yang et al. found that oxesomes containing miR-233 shuttle between macrophages and breast tumor cells. miR-233-regulating tumor invasion is considered to work through the mef2c- pathway . This reβ, IL-10, and arginase 1, make a significant contribution to immunosuppression [β has a crucial immunosuppressive role in both the innate and the adaptive arms of the immune response. In the innate immune response, TGF-β promotes tumor-associated macrophage polarization to an M2-versus-M1 phenotype, which further promotes TGF-β production and deepens immunosuppression [β also inhibits the cytolytic activity of natural killer (NK) cells expressing the activating receptor NKG2D, further resulting in a poor antitumor response [β decreases dendritic cells (DCs) migration and increases apoptosis, which decreases antigen presentation and dampens the adaptive immune response [β promotes CD4+ T cells differentiation into Th2 cells rather than Th1 cells, which promotes a less efficient antitumor immune response [β also inhibits the CD8+ T cells antitumor activity by suppressing the expression of several cytolytic genes, including the genes encoding granzyme A, granzyme B, IFN-γ, and FAS ligand [β promotes tumor growth by the maintenance of Treg cell differentiation, which inhibits the antitumor response [Tumor immunosuppression is a well-established mechanism for the regulation of tumor growth. Several studies have reported that TAM-derived cytokines and proteases, such as TGF-pression –77. For pression . TGF-β aresponse , 80. In response , 82. In response . TGF-β aS ligand , 84. Furresponse .+ T-cells, and tumor cells. IL-10 is commonly regarded as an anti-inflammatory, immunosuppressive cytokine that favors tumor escape from immune surveillance. TAM-derived IL-10 acting in an autocrine circuit suppresses the expression of IL-12, a potential antitumor cytokine [γ, which is the main factor that stimulates naïve T-cell differentiation, to promote immune evasion [+/IL-10+ cells there are, the longer the overall survival will be [IL-10, an important cytokine in the tumor microenvironment, is expressed by TAMs, CD8cytokine . Severalcytokine , 86. IL- evasion . Further evasion . However evasion –91. For  will be . So the + T cell unresponsiveness [2O2 production by myeloid-derived suppressor cells (MDSCs), which present class I-restricted epitopes directly to CD8+ T cells and inhibit their release of IFN-γ through the contact-dependent production of H2O2 [2O2 generation following arginase 1 activation is not clear and may be linked to the synchronous activation of a different NOS isoform [ Arginase 1, the molecular marker for M2 macrophages, is highly expressed in tumors. In recent years, it has been demonstrated that arginase 1, which primarily metabolizes L-arginine into polyamine and proline, causes dysregulation of the T cell receptor (TCR) signal and subsequently induces CD8siveness , 92. In  of H2O2 . HoweverκB, but the specific mechanism still needs to be explored [Finally, several studies have found that chemokines also play an important role in immunosuppression. Chemokines, such as CCL17 and CCL22, can prevent the infiltration of cytotoxic T-cells but promote that of Treg and Th2 cells , 94. TAMexplored .Over the past 5 to 10 years, it has been found that a specific subpopulation of tumor cells has distinct stem cell properties in tumors. These cells are defined as cancer initiating cells or cancer stem cells (CSCs). A CSC has the ability to initiate tumorigenesis by undergoing self-renewal and differentiation , 96. Howβ1, IL-10, and IL-23. In addition, M2 macrophages could induce T-cell anergy and therefore immunosuppression in agreement with what we mentioned previously [TAMs, as the dominant immune cell components, are considered to be closely related to CSCs in position. Several studies have reported that TAMs are always found distributed around CSCs, and the number of infiltrating TAMs has been positively correlated with the histological grade of the malignancy and the number of CSCs found , 99. Fureviously . Both ofRecently, other articles have demonstrated that macrophages can also influence the characteristics of CSCs, which promote tumorigenesis and metastasis. For example, Jinushi and colleagues found that CSCs could specifically stimulate TAMs to express the downstream factor milk-fat globule epidermal growth factor VIII (MFG-E8), which has been identified as a growth factor involved in phagocytosis, angiogenesis, and immune tolerance. MFG-E8 induces CSCs to form tumors and develop antitumor drug resistance through the STAT3 and hedgehog signaling pathways . Okuda eAll of these studies indicate that macrophages promote CSC proliferation and metastasis. However, the investigation into the interaction between macrophages and CSCs is still at an early stage. More in-depth research requires our joint efforts. Accumulating studies have demonstrated that the density of TAMs is associated with a poor prognosis, suggesting macrophages as a target for clinical therapy –107. As Ecteinascidia  turbinata, has a specific cytotoxic effect on human macrophages and TAMs in vitro [Several studies reporting the use of “antimacrophage” approaches have primarily focused on counteracting monocyte chemokines and receptors as anticancer targets , 36, 42.in vitro . Recentlin vitro . Other pin vitro , 113. 3 unless they are vascularized, so inhibiting angiogenesis is also a good therapeutic approach. Several studies revealed that anti-VEGF-A with Avastin/bevacizumab or other neutralizing antibodies can both inhibit the infiltration of macrophages and enhance the activity of antiangiogenic therapies by preventing TAMs from secreting additional pro-angiogenic factors [As we mentioned earlier, tumors do not grow beyond 2-3 mm factors , 115. As M1 macrophages induce proinflammatory response which protects body from injury, converting the M2 macrophages into M1 is also considered to be a better potential therapy. Several articles have reported that activation of TLRs stimulates M1-polarized macrophage response, which induce the activation of proinflammatory program . In a mo+ and CD8+ cells and reduce tumor growth and metastasis [α, and IL-1β. Through the increased levels of these substances, paclitaxel can enhance tumor cell cytotoxicity and restore IL-12 production by macrophages in tumor-bearing mice [ In addition, accumulating studies report using macrophages as natural vectors to deliver therapeutic molecules to the neoplastic site , 41, 119tastasis . Moreoveing mice . Recentling mice , 122. PeHeterogeneity is one of the most important characteristics of macrophages. In different diseases, macrophages can be polarized into different phenotypes. In most tumors, macrophages are considered to be polarized into the M2 phenotype. TAMs express a series of cytokines, chemokines, and proteases to promote angiogenesis, lymphangiogenesis, tumor growth, metastasis, and immunosuppression. Recently, it has also been reported that TAMs interact with CSCs, which facilitate tumorigenicity, metastasis, and drug resistance. Taken together, these findings indicate that targeting macrophages in the tumor microenvironment may provide more efficacious novel therapies for future tumor management.


The allergic reaction occurring primarily in the nasal mucosa can induce a secondary conjunctival response of an immediate (SICR), late (SLCR), or delayed (SDYCR) type in some patients with allergic conjunctivitis (AC).4, LTC4, LTE4), myeloperoxidase (MPO), interferon-γ (IFN-γ), and interleukins  in tears during the SICR, SLCR, and SDYCR.To investigate the concentration changes of histamine, tryptase, eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), leukotrienes , 13 SLCR (p<0.001), and eight SDYCR (p<0.01) to nasal challenges with allergens (NPTs), the NPTs and 32 control tests with PBS were repeated and supplemented with the determination of these factors in tears.4, and IL-4. The SLCRs were accompanied by significant changes in concentrations of histamine, ECP, LTB4, LTC4, MPO, IL-4, and IL-5. The SDYCRs were associated with significant concentration changes in tears (p<0.05) of LTB4, MPO, IFN-γ, and IL-2. No significant changes in these factors were recorded in tears during the 32 PBS controls (p>0.1) or in the ten control patients (p>0.1).The SICRs were associated with significant concentration changes in tears (p<0.05) of histamine, tryptase, ECP, LTCThese results provide evidence for causal involvement of nasal allergy in some patients with AC, inducing secondary conjunctival response of immediate (SICR), late SLCR, or delayed SDYCR type, associated with different mediator, cytokine, and cellular profiles in the tears, suggesting involvement of different hypersensitivity mechanisms. These results also emphasize the diagnostic value of nasal allergen challenge combined with monitoring of the conjunctival response in some patients with AC. Allergic conjunctivitis (AC) includes five clinical entities -4, a seaVarious hypersensitivity mechanisms, such as immediate type (IgE-mediated type I), late (type III), or delayed (cell-mediated type IV), may be involved in both forms of AC -22. The Thirty-two of the 81 patients suffering from allergic conjunctivitis, 14 with seasonal allergic conjunctivitis (SAC) and 18 with perennial allergic conjunctivitis (PAC), for more than 3 years, showing insufficient compliance with the standard topical ophthalmologic treatment, referred to our Department of Allergology & Immunology  during 1998–1999 for more extensive analysis of their AC complaints, and developing the secondary conjunctival response (SCR) to nasal provocation tests with allergens (NPTs), volunteered to participate in this study. These patients, 13 men and 19 women, 20–43 years of age , had pre2HPO4 , 0.2 g KH2 PO4, distilled water ad 1000 μl; pH=7.4) were performed using rhinomanometry combined with simultaneous recording of the ocular signs and subjective symptoms.The routine diagnostic procedure performed in these 32 patients revealed positive or suspect history for nasal allergy (93%), positive skin tests with various inhalant allergens (100%), hyperaemic /livid and edematic nasal mucosa (97%), increased eosinophil and neutrophil counts in nasal secretions (87%), conjunctival hyperaemia and tearing to a slight degree (100%), appearance of incidental eosinophils and conjunctival epithelial cells in the tear specimens (78%), increased blood eosinophil counts (22%), and positive specific IgE in the serum (ImmunoCAP) for some inhalant allergens 19%; . In thesThe ocular signs and relevant subjective symptoms were evaluated with Pelikan’s scoring (grading) system -10. The Dialyzed and lyophilized allergen extracts  were diluted in PBS and used for skin tests in concentrations of 100–500 BU/ml and for NPTs in concentrations of 1000–5000 BU/ml , as recoSkin prick tests (SPTs) with allergenic extracts in concentrations of 500 BU/ml were performed and evaluated after 20 min. The intracutaneous tests in concentrations of 100 BU/ml and 500 BU/ml were then performed in all patients and evaluated 20 min and 6, 12, 24, 36, 48, 56, 72, and 96 h after the intradermal injection. A skin wheal (>7.0 mm in diameter) occurring after 20 min was qualified as a positive immediate skin response, skin infiltration appearing between 6 and 12 h as a late skin response, and skin induration recorded later than 48 h as a delayed skin response [2O. NPTs were performed using the following schedule: (1) baseline values recorded at 0, 5, and 10 min; (2) PBS control values recorded at 0, 5, and 10 min after a 3-min application of PBS to the nasal mucosa of the non-intubated nasal cavity by a saturated wad of cotton wool on a nasal probe inserted under the middle turbinate; (3) the post-challenge values recorded after a 3-min challenge with the allergen, performed in the same manner as the challenge with PBS, at 0, 5, 10, 20, 30, 45, 60, 90, and 120 min, then every hour up to the 12th h, and every second hour during the time periods between the 24th–38th and 48th–56th (60th) h  h  [5-10,24(60th) h .2O  regarding the baseline values, recorded at least at three consecutive time intervals [The nasal response was assessed as positive when the post-challenge mean NPG values increased by at least 2.0 cm Hntervals -10,24. Tntervals -10,24.The control nasal challenge with PBS was performed in each patient studied by the same schedule as that used for the NPTs with allergen.The objective conjunctival signs and subjective symptoms were registered before and during all NPTs with allergens and PBS at the same time points as the nasal NPG values . The feath–38th and 48th–56th (60th) h after the allergen challenge (g for 1 min at 4○ C. The supernatants were removed and stored at −8 °C.The tear specimens (80–150 μl) were gently collected with a sterilized micropipette from each eye separately from the inferior conjunctival fornix and/or lacus lacrimalis at the following time intervals: (a) at 10, 5, and 0 min before the NPT and (b) at 0, 5, 10, 20, 30, 60, and 120 min and every second hour up to 12 h and between the 24hallenge . The tea-ImmunoCAP , DL: 1.0 μg/L; (c) Eosinophil cationic protein (ECP) –ImmunoCAP (Pharmacia), DL: 2 μg/l; (d) Eosinophil-derived neurotoxin (EDN/EPX)- enzyme-linked immunoassay (ELISA) kit , DL: 0.64 ng/ml; (e) Leukotrienes B4, C4, E4 -EIA kits , DL: LTB4=4.8 pg/ml, LTC4=2.1 pg/ml, LTE4=3.7 pg/ml; (f) Myeloperoxidase (MPO) -ELISA kit , DL: 25 ng/ml; (g) Interferon-gamma (IFN-γ)-ELISA kit , DL:1.0 pg/ml; (h) Interleukin 4 (IL-4) - ELISA kit (Bender), DL: 0.7 pg/ml; (i) Interleukin 2 (IL-2) - ELISA kit , DL:<3.0 pg/ml; (j) Interleukin 5 (IL-5) - ELISA kit (R & D System), DL: 3.2 pg/ml.The factors in tears were measured by commercial kits, according to the manufacturer’s recommendations. The measurements were performed in tear samples from each eye separately on each occasion, in duplicate and by a double-blind schedule. The results were then calculated as the mean of both eyes. The intra-assay and the inter-assay coefficients of variations for the assay kits were less than 10%. The detection limits measured by the author are abbreviated as DL. The following factors were recorded: (a) Histamine concentrations, so-called blanks, were measured by Siraganian’s fluorometric method , DL: 1.0The tear specimens were spread out on the slide surface with a glass probe, air-dried, fixed with polyethylene glycol, and stained with May-Grünwald-Giemsa, which we modified ,23,29,30Ten randomly selected adults suffering from allergic rhinitis without history of ocular disease and with normal ophthalmologic findings volunteered to participate as control subjects. In these patients, 10 positive NPTs with inhalant allergens  were repeated and supplemented with registration of the conjunctival and subjective symptoms and a single determination of these mediators in tears .(1) Nasal and ocular responses  and the PBS control challenges in individual patients were analyzed by the Wilcoxon matched-pair signed rank test, comparing the post-challenge values at each time point with the mean prechallenge values. (2) The mean NPG values and the mean total ocular score values were compared with the corresponding PBS control values at each time point and analyzed by the Mann–Whitney U test. (3) The post-challenge mediator values measured at each time point during the repeated NPT and PBS controls in each patient were compared with their prechallenge values and analyzed by the Wilcoxon matched-pair signed rank test. (4) The mean post-challenge mediator values were also compared with corresponding PBS values and evaluated by the Mann–Whitney U test. Statistical evaluation was performed separately for each eye, and then the mean values from both eyes were calculated. A p value <0.05 was considered statistically significant.The 32 patients in whom 61 NPTs with various inhalant allergens  and 32 PNo significant differences were found in the appearance of the particular NR types regarding the individual allergens (p>0.1). The repeated NPTs resulted in the development of similar NR types . No statThe 32 positive NRs were associated with significantly positive secondary conjunctival responses (SCR), 11 immediate , 13 late , and eight delayed , whereas no CR was observed during the 29 NNRs (p>0.2). The SCRs were represented by significant changes in the objective conjunctival signs (p<0.01) as well as subjective symptoms (p<0.05). The 32 repeated NPTs induced similar and statistically significant SCRs, when the post-challenge values were compared with the prechallenge (baseline) values (p<0.01) and with the PBS control challenge (p<0.01). .No significant differences in the conjunctival features recorded during the initial and the repeated SCRs were observed between the right and left eye (p>0.1). No significant corneal signs were recorded in any SCR type. No conjunctival changes were recorded during the 32 PBS control challenges (p>0.1).4, and IL-4 in the tears  of histamine, ECP, LTB4, LTC4, MPO, IL-4, and IL-5  of LTB4, MPO, IFN-γ, and IL-2 in the tears  of histamine, tryptase, ECP, LTChe tears , whereasand IL-5 , but thehe tears , while ton limit , whereasThe prechallenge cell counts in tears were low compared with the primary types of AC ,7 The SIThe ten control subjects demonstrated neither conjunctival signs nor significant concentration changes in the factors in tears during the ten repeated positive nasal responses (p>0.2).The conjunctiva and the nose have a manifold anatomic and functional relationship ,3,7-10. Allergic reactions taking place primarily in the nasal mucosa due to intranasal exposure to an inhalant allergen may affect the conjunctiva and other ocular tissues, such as the cornea, in various ways and upon involvement of various mechanisms ,20,31-344, LTC4, MPO, cytokines, such as IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IFN-γ, tumor necrosis alpha (TNF-α), and chemokines, such as monokine-induced by gamma interferon (MIG),interferon gamma-induced protein ( IP-10), interferon inducible T-cell alpha chemoattractans ( I-TAC), regulated on activation normal T cell expressed and secreted (RANTES) and eotaxins. In most of these studies, a single determination of these factors was performed in the tears of patients with the primary forms of allergic conjunctivitis or keratoconjunctivitis. Studies following the concentration changes in mediators in tears during particular types of conjunctival response, such as immediate or late CR, due to the conjunctival provocation tests with allergens (CPTs), are not numerous [The occurrence and possible role of various mediators and cytokines in patients with various forms of allergic conjunctivitis has already been extensively investigated ,33,35-54numerous ,52,54,554, LTC4, MPO, PGD2, and TAME-esterase in tears [4, and LTC4 in tears [4 were recorded in tears during SICRs, whereas MPO has been detected in tears during SLCRs [The primary form of immediate CR has been reported to be accompanied by increased concentration of histamine, tryptase, ECP, LTBin tears ,52,54,55in tears ,36,45,52in tears ,35,56,57in tears ,23,44,56ng SLCRs ,35,38,44Generally, there is a dearth of structural data demonstrating the course of mediator and cytokine profiles in tears during the particular types of the primary conjunctival response to the CPT. Data illustrating the appearance and concentration changes of mediators and cytokines in tears during the secondary forms of conjunctival response to NPTs are, unfortunately, not available. Moreover, few data are also available on the mediator and cytokine appearance and profiles in the tears of healthy subjects, which may serve as reference values ,59.4, IL-4, and IL-5 in tears during the SICR would suggest the involvement of mast cells, eosinophils, and probably of Th2-lymphocytes in the mechanism underlying this response type. The concentration changes in histamine, ECP, LTB4, LTC4, MPO, IL-4, and IL-5 in tears during the SLCR may be suggestive of the role of basophils, eosinophils, neutrophils, and Th2-lymphocytes in SLCR, whereas the concentration changes of LTB4, MPO, IFN-γ, and IL-2 in tears during the SDYCR may be indicative of the involvement of neutrophils, monocytes, and Th1-lymphocytes in this response type. However, this presumption has also some limitations since various mediators and cytokines can be produced by various cell types. Moreover, these mediators have most probably been released by cells participating in the primary allergic reactions in the nasal mucosa. This hypothesis is supported by our previous results, demonstrating only limited numbers of these cell types, in a non-activated condition, in tears during the secondary conjunctival response types [Our results demonstrating increased concentrations of histamine, tryptase, ECP, LTCse types -10,23,56Another important aspect concerns the participation of the cells in an immunologic process. The appearance of a certain cell type in a medium, in this case in the tears, does not necessarily signify its activation and active participation in that immunologic process but can also be a consequence of another, proceeding, mechanism or its part. Vice versa, the activation of a particular cell type and its active participation in an immunologic process may not necessarily result in the changes of its count .4), Th2-lymphocytes ; (b) SLCR: basophils (histamine), eosinophils , neutrophils , Th2-lymphocytes ; (c) SDYCR: neutrophils , Th1-lymphocytes , and monocytes (LTB4) [The simultaneous recording of the concentration changes of particular mediators and some cytokines in the tears and of the changes in the counts of individual cell types in the tears, performed in the current study, has revealed activation and active participation of the relevant cell types in the mechanism(s) underlying the particular SCR types. The active participation of these cells in the particular SCR can be summarized as follows: (a) SICR: mast cells , eosinophils  . The acts (LTB4) ,23,56,61However, the results of the current study are limited only to the tears and cannot serve as an indication of the cellular and mediator changes and processes in the conjunctival mucosal membrane. These changes can be demonstrated only by biopsies ,14,16. TThe results of this study additionally stress the importance of provocation tests with allergens. The CPTs, performed directly on the conjunctiva, confirm the role of allergic reaction taking place in the conjunctiva due to the direct exposure of conjunctival tissue to an inhalant allergen ,52,54,55Finally, the results of the current study may also have an important impact on the therapeutic management of these ocular disorders, regarding not only the choice of the most suitable drugs but also the administration route ,23,61-63


Neoromicia nanus), captured near Mouyassué village in Côte d'Ivoire, West Africa, in June 2011. Phylogenetic analysis of partial L-segment sequences using maximum-likelihood and Bayesian methods revealed that the newfound hantavirus, designated Mouyassué virus (MOUV), was highly divergent and basal to all other rodent- and soricomorph-borne hantaviruses, except for Nova virus in the European common mole . Full genome sequencing of MOUV and further surveys of other bat species for hantaviruses, now underway, will provide critical insights into the evolution and diversification of hantaviruses.Recently identified hantaviruses harbored by shrews and moles (order Soricomorpha) suggest that other mammals having shared ancestry may serve as reservoirs. To investigate this possibility, archival tissues from 213 insectivorous bats (order Chiroptera) were analyzed for hantavirus RNA by RT-PCR. Following numerous failed attempts, hantavirus RNA was detected in ethanol-fixed liver tissue from two banana pipistrelles ( Discovery of phylogenetically divergent hantaviruses in shrews and moles  -13 raise2, 1 U AmpliTaq polymerase  and 0.25 μ oligonucleotide primers. Initial denaturation at 94°C for 5 min was followed by two cycles each of denaturation at 94°C for 40 s, two-degree step-down annealing from 48°C to 38°C for 40 s, and elongation at 72°C for 1 min or 1 min 20 s, then 32 cycles of denaturation at 94°C for 40 s, annealing at 42°C for 40 s, and elongation at 72°C for 1 min, in a GeneAmp PCR 9700 thermal cycler . Amplicons were purified using the QIAQuick Gel Extraction Kit , and DNA sequencing was performed using an ABI Prism 377XL Genetic Analyzer .As in our previous investigations on the spatial and temporal distribution of hantaviruses in soricomorphs -13, we rNeoromicia nanus Peters 1852), captured during June 2011 near Mouyassué village  in Aboisso District, 130 km from Abidjan, in the extreme southeastern region of Côte d'Ivoire in West Africa , amplified by PCR as previously described [Pipistrellus nanulus), collected in Parc National du Mont Péko, 700 km northwest of Mouyassué, in February 1992, and ethanol-fixed liver tissue of three tiny pipistrelles, collected in December 2009 in Azagny, where a hantavirus was previously found in the West African pygmy shrew (Crocidura obscurior) [After innumerable failed attempts, hantavirus RNA was detected by RT-PCR in ethanol-fixed liver tissues from two of 12 banana pipistrelles  [A 423-nucleotide region of the RNA-dependent RNA polymerase-encoding L segment, amplified using a hemi-nested primer set , was aligned and compared with hantavirus sequences available in GenBank, using ClustalW   and tranuropaea) . InteresMOUV formed a uniquely divergent lineage, distant from all other hantaviruses identified to date, except for NVAV Figure , in phyl® RNA Stabilization Reagent [Despite the overall success of our brute-force RT-PCR approach at identifying previously unrecognized hantaviruses in frozen tissues -7,10-13  Reagent ,8, desigApodemus agrarius) [Dating to the seminal discovery of Hantaan virus in lung tissue of the striped field mouse (grarius) , lung hagrarius) -30. Howegrarius) ,31. In ograrius) ,5,12,13.Neoromicia of the family Vespertilionidae and subfamily Vespertilioninae. Like other vesper bats, the banana pipistrelle is insectivorous. Unlike large fruit bats, such as the straw-colored fruit bat (Eidolon helvum) and hammer-headed bat (Hypsignathus monstrosus), which are sold as bush meat, the banana pipistrelle, weighing approximately 3 g, is not consumed as food. However, because banana pipistrelles occasionally roost within houses or reside near human habitation, rare human encounters raise the possibility of hantavirus exposure.Having their fossil origins in the Eocene epoch, approximately 50 million years before present, bats occur on every continent except Antarctica and are among the most speciose orders of mammals, with more than 1,100 extant species . The banEptesicus serotinus) and greater horseshoe bat (Rhinolophus ferrumequinum) captured in Korea [Previously, serological evidence of hantavirus infection was reported in the common serotine (in Korea , but genin Korea , suggestin Korea ,10, withThe authors declare that they have no competing interests.LS and SHG independently performed RNA extraction, RT-PCR and DNA sequencing reactions. HJK and JWS provided primer design and phylogenetic analysis. BK, BKL and JAC provided tissues and information about bats. RY conceived the project and provided overall scientific oversight. All authors contributed to the preparation of the final manuscript.


Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is an autosomal dominant autoinflammatory disease characterized by periodic fever, accompanying with attacks of abdominal pain, arthralgia, myalgia, erythematous rashes, periorbital edema and conjunctivitis. Mutations in the extracellular domain of the 55-kD tumor necrosis factor receptor (TNFRSF1A) has been shown to be responsible for the TRAPS syndrome.A sixty two year old Turkish female presented with fever, periorbital edema, erythematous skin rash on the face, neck and arms at age 53 on her first admission. She had history of arthritic attacks on wrists, fingers, knees, elbows lasting 10-15 days, once or twice a year, which began at the age of twelve. Since the age of 50 she has experienced febrile attacks accompanying with abdominal pain, lasting 3-4 days in every 3-4 months. After the age of 53 she had attacks of pruritic erythematous rash on the neck, arms, legs and face, and bilateral periorbital edema as well lasting 10-14 days, once or twice a year. She had no family history of periodic fever. At age 55, she was diagnosed as primary Sjögren’s syndrome, with the findings of parotitis, dry eyes, positive Schirmer’s test and serum ANA and Anti-SSA positivity. Methyl prednisolon (MP) 8 mg/day and hydroxy chloroquine 400 mg/day was started. One year later, at age 56, her erythematous, pruritic skin lesions repeated with periorbital edema. MP was given 32 mg/day. ESR and CRP levels were elevated during attacks . Her skin lesions disappeared after commencement of MP. In attack free period, ESR and CRP returned to normal levels . MEFV mutation analysis of Exons 2 and 10 were negative by sequencing. For her periodic fever symptoms such as 39ºC fever accompanying with abdominal pain and skin lesions, TNFRS1 mutations were analyzed. Exon 2,3,4,5,6,7 and intron 2-3, 4-5 and 6-7 mutations were analyzed by polymerase chain reaction/sequence based typing technique. A novel mutation on exon 7  was identified.This patient represents a novel mutation of TNFRSF1A in a Turkish patient with signs and symptoms of TRAPS syndrome, accompanying with primary Sjögren’s syndrome. As reported in Japanese cases previously, this patient with a novel mutation, has milder disease and her attacks of fever and rash respond well to glucocorticoid therapy. Coexistence of Sjögren’s syndrome may have masked the clinical manifestations of TRAPS in this patient. Without any family history, in this particular patient de novo TNFRSF1A mutation is possible.None declared.