Mechanistic modeling of DNA double strand break (DSB) rejoining is important for quantifying and medically exploiting radiation-induced cytotoxicity (e.g. in cancer radiotherapy). Most radiation-induced DSBs are quickly-rejoinable and are rejoined within the first 1–2 hours after irradiation. Others are slowly-rejoinable , and yet others are essentially unrejoinable (persist for >24 hours). The dependences of DSB rejoining kinetics on radiation dose and dose rate remain incompletely understood. We hypothesize that the fraction of slowly-rejoinable and/or unrejoinable DSBs increases with increasing dose/dose rate. This radiation-dependent (RD) model was implemented using differential equations for three DSB classes: quickly-rejoinable, slowly-rejoinable and unrejoinable. Radiation converts quickly-rejoinable to slowly-rejoinable, and slowly-rejoinable to unrejoinable DSBs. We used large published data sets on DSB rejoining in yeast exposed to sparsely-ionizing  and densely-ionizing (α-particles) radiation to compare the performances of the proposed RD formalism and the established two-lesion kinetic (TLK) model. These yeast DSB rejoining data were measured within the radiation dose range relevant for clonogenic cell survival, whereas in mammalian cells DSB rejoining is usually measured only at supra-lethal doses for technical reasons. The RD model described both sparsely-ionizing and densely-ionizing radiation data much better than the TLK model: by 217 and 14 sample-size-adjusted Akaike information criterion units, respectively. This occurred because: the RD (but not the TLK) model reproduced the observed upwardly-curving dose responses for slowly-rejoinable/unrejoinable DSBs at long times after irradiation; the RD model adequately described DSB yields at both high and low dose rates using one parameter set, whereas the TLK model overestimated low dose rate data. These results support the hypothesis that DSB rejoining is progressively impeded at increasing radiation doses/dose rates. Mechanistic quantitative modeling of DNA double strand break (DSB) rejoining kinetics is important for predicting radiation-induced cytotoxicity and for exploiting it (e.g. in cancer radiotherapy) –5, as weMechanistic quantitative analysis of DSB rejoining  is often performed using kinetic models which describe the rates of change of the average number of DSBs per cell during and/or after radiation exposure. Many such models have been proposed, some of which attempt very detailed descriptions of molecular machinery involved in DSB repair , 30–33. Here, we hypothesize that the understanding of how DSB rejoining depends on radiation dose and dose rate can be enhanced by incorporating into kinetic models a new mechanism, whereby the fraction of slowly-rejoinable and/or unrejoinable DSBs increases with increasing dose and/or dose rate. The hypothetical mechanism can occur, for example, due to a gradually increased fraction of spatial DSB clustering along chromosomes . Such clSaccharomyces cerevisiae) exposed to sparsely-ionizing  2 by the term 2/ Oi,(j)2, reduced GOF for both the RD and TLK models because the data points at low doses, which had small values of Oi,(j), affected the fit more strongly than other points.Additional exploratory calculations showed that an alternative assumption of error magnitudes proportional to O2 behavior of the log likelihood distribution.Uncertainties (95% CIs) for best-fit model parameter values were estimated by profile likelihood  as follo2 or R2 [i-th data set  is given below, where KM is the number of adjustable parameters and LLM,i is the maximized log-likelihood value . AICc has gained popularity for this purpose in various fields , 52. For2 or R2 –56. One ERM,i), can be expressed as:The likelihood of the M-th model relative to other tested model(s), called the evidence ratio  for sparsely-ionizing radiation was much larger than the number of LDR points (29), the RD model reasonably described DSB yields at both high and low dose rates using one set of parameters . In contThese visually-apparent differences in model performance were quantified by relative and absolute GOF assessments Tables  and 2. T2 was higher and 95% CIs for the intercept and slope of linear regression of model predictions vs. observed data point values were often narrower for the RD model vs. the TLK model for HDR data  at best-fit values, thereby reducing the number of adjustable parameters to the same number as in the RD model (7), the RD model would remain the best-ranked model by a still very comfortable margin of 210 AICc units for sparsely-ionizing and 7.1 units for densely-ionizing radiation data.The AICc scale being logarithmic, the relative support from the sparsely-ionizing data for the TLK model vs. the RD model .However, it is important to note that the TLK model performed much better than its predecessor–the LPL formalism . When apectively . On densq1×R and q2×R in q1 and q2, respectively), model performance plummets by 210 and 7 AICc units for sparsely-ionizing and densely-ionizing radiation, respectively. In other words, without radiation-induced conversion of DSB classes, the RD model performs equivalently to the TLK model.These results suggest that the TLK formalism, which contains three DSB classes, represents an important improvement over the LPL model with two classes. However, the RD model, which contains radiation-induced conversion of DSB classes, yields a further major improvement for the yeast data at hand. The improved performance of the RD model relative to the TLK model is caused specifically by the dose/dose rate dependence of conversion of DSB classes: if the conversion is allowed to occur without influence by radiation (i.e. the terms k1/k2). The direct yield of unrejoinable DSBs was not detectable: parameter k3 had a best-fit value of zero. Consequently, the suggested interpretation is that unrejoinable DSBs were produced only indirectly by conversion of slowly-rejoinable DSBs with a rate proportional to the radiation dose rate and to parameter q2. Conversion of quickly-rejoinable DSBs  was an important indirect source of slowly-rejoinable DSBs. The rejoining rates for these two DSB classes differed by a factor of 24 (based on the parameter ratio v1/v2).The best-fit RD model parameter values  suggestek1 and k3 had best-fit values of zero . However, their rejoining rate (parameter v2) was the same for both sparsely-ionizing and densely-ionizing radiation  exposed to sparsely-ionizing and densely-ionizing radiation [We analyzed large published data sets on DSB rejoining in yeast  data. Yeast data are useful because DSB rejoining was measured in the dose range relevant for clonogenic cell survival [S. cerevisiae relies on homologous recombination to a greater extent than many other organisms [The strengths of the current study include rigorous information theoretic comparison of the performances of two models with different assumptions about the mechanisms of DSB rejoining, using extensive data sets which include different radiation types, doses, dose rates, and rejoining times. The weaknesses include the use of survival , but somrganisms , 59, andrganisms . The numrganisms , 61. Howrganisms , radiatirganisms . In addirganisms , than atDespite these potential drawbacks, we believe that the current study provides potentially clinically-relevant insight into mechanistic modeling of DSB rejoining. In particular, because the TLK and RD models are based on different mechanistic assumptions, comparison of their performances on the same data enabled us to make the following observations about which putative mechanisms are most (or least) useful for describing DSB rejoining kinetics.1, η2, and η1,2) for quadratic interactions between different DSBs within/between DSB classes . In other words, the TLK model relied on unrejoinable DSBs (produced directly by radiation and/or by fixation of rejoinable DSBs) to describe the HDR data for both sparsely-ionizing and densely-ionizing radiation  was too high to describe LDR data, leading to overestimation of these data  can be visualized in Our analysis suggests that the proposed mechanism of radiation-dependent conversion of DSB classes strongly enhanced the ability of kinetic models to describe DSB rejoining in yeast. In contrast, the analyzed data did not support the notion (represented in the TLK model) that interactions between different DSBs constitute an important route of DSB removal. However, interactions between DSBs which lead to transition into a different class of DSB would still be compatible with the data. Our analysis also suggests that the time needed to rejoin a particular DSB depends mainly on the properties of this DSB and/or on radiation damage to nearby chromatin. These factors can be represented by the generic term “DSB complexity”, which depends not only on radiation type, but also on dose and dose rate.The previously mentioned GLOBLE model , 28 provIn contrast, the RD model can be viewed as a more generalized approach where the nature of DSB classes and the mechanisms of conversion between them are not specifically tied to chromatin structure. For example, when repair enzymes encounter a complex DSB, and/or are oxidatively damaged during the repair process (which is more likely to occur at higher radiation doses/dose rates), rejoining of the DSB may become stalled. Conceptually similar explanations were considered by the authors of the data sets analyzed here, but were never implemented as a mathematical model . Such exk3). The quadratic component is composed of the product of the yield of slowly-rejoinable DSBs (parameter k2) and the conversion coefficient for slowly-rejoinable to unrejoinable DSBs (parameter q2). This quadratic component is absent at low dose rates. Thus, lowering the dose rate reduces the quadratic dose response term, but the mechanistic interpretation of this phenomenon is not the same as in other kinetic models: in other models the quadratic term is reduced because, at low dose rates, most DSBs are rejoined before they can interact with each other; whereas in the RD model the quadratic term is reduced because, at low dose rates, most quickly-rejoinable DSBs are rejoined before radiation can convert them to slowly-rejoinable ones.The LQ approximation to RD model solutions for the number of DSBs which remain unrejoined at long (effectively infinite) rejoining times Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.


Objectives. This study evaluated the influence of cavity surface finishing with diamond burs of different grit mounted on high-speed turbine and ultrasound on the roughness and microshear bond strength (MBS) of a lithium silicate glass-ceramic to enamel and dentin. Methods. Enamel and dentin specimens were divided into seven groups, according to the type of surface finishing: 1200-grit sandpaper (control), two different brands of medium-grit and fine-grit diamond burs in a high-speed turbine; medium-grit and fine-grit CVD  tips in an ultrasonic device. Roughness parameters (n = 5) and MSBS to a glass-ceramic (n = 10) were determined. Data were analyzed using ANOVA and Tukey's test (α = 5%). Results. Control group showed lower mean roughness readings and groups that used medium-grit diamond burs showed the highest mean roughness values. Regarding MSBS, there was no statistical difference when comparing the groups gritted with the same brand of medium- and fine-grit burs and tips. Conclusions. Cavity surface finishing influenced the roughness parameters and MSBS of a glass-ceramic to enamel and dentin. Medium-grit diamond burs in high-speed turbine showed the highest mean roughness values. Fine-grit CVD tips in ultrasound presented the highest MSBS values for both enamel and dentin. High-speed turbines and diamond burs have been the primary choice for tooth preparation to receive direct and indirect dental restorations . Their uNew methods have been suggested for the preparation of cavities in an attempt to preserve healthy dental tissue and improve the quality of its interface with restorative materials. Lasers are one of such methods. However, they are generally expensive and are not suitable for all types of preparations, especially for indirect restorations, such as crowns and laminate veneers. Ultrasonic devices on the other hand can be considered a good alternative to conventional high-speed turbines, since they produce less noise and are less expensive than lasers .Recently, chemical vapor deposition (CVD) technology was used to produce new diamond tips to be used with ultrasonic devices . This teLittle is known about the influence of different surface textures resulting from cutting with rotary and ultrasonic instruments on enamel bond strength , 7. SevgThe literature reports that the use of CVD diamond tips in ultrasonic device, when compared to diamond rotary instruments, can produce different morphological characteristics in the dentin surface, more specifically in relation to the thickness of the smear layer and the surface roughness . MoreoveSurface roughness of dental hard tissues plays an important role in operative dentistry, since it affects how well the restorative material will adapt to the dental structure. This aspect is generally overlooked especially when indirect procedures are considered. In such procedures, the smoothness of the surface plays an important role in the impression and cementation procedures. While a smooth surface is important during the impression, rougher surfaces could provide better substrates for adhesion.While dental surface finish characteristics may have been in a way neglected, new reinforced ceramic materials for esthetic purposes have been developed. Glass-ceramics are indicated for anterior and posterior esthetic restorations because of their optical and mechanical properties. Recently, a zirconia-reinforced, higher strength lithium silicate-reinforced glass-ceramic that features a fine-grained and homogenous microstructure and that supports a wide range of applications, including anterior and posterior restorations, has become available. Thus, it is necessary to study the adhesive property of this new lithium silicate glass-ceramic to tooth structure and the influence of textures on the ceramic material.Ceramic laminate veneers have high survival rates when bonded to enamel and provide a safe and predictable treatment option that preserves tooth structure. In a retrospective study evaluating porcelain laminate veneers up to 12 years, survival rates of 99% for veneers with preparations confined to enamel and 94% for veneers with enamel-only margins were observed . DespiteHence, it is important to evaluate the effects of surface roughness on enamel and dentin surfaces created with different types and granulations of diamond burs and tips on bond strength using new glass-ceramic systems used for indirect laminate veneers. The objectives of the present study were to evaluate the influence of enamel and dentin surface finishing with medium and fine-grit diamond burs and tips mounted on a high-speed turbine handpiece and ultrasonic device on the surface roughness and influence of surface roughness on the microshear bond strength of a lithium silicate glass-ceramic to enamel and dentin. Two null hypotheses were tested: (1) there are no differences in the surface roughness parameters evaluated on enamel and dentin surfaces finished with medium and fine-grit diamond burs and tips mounted on a high-speed turbine handpiece and ultrasonic device, and (2) there are no differences in the microshear bond strength of a glass-ceramic luted to enamel and dentin surfaces finished with medium and fine-grit diamond burs and tips mounted on a high-speed turbine handpiece and ultrasonic device.Thirty-five bovine incisors were selected and stored in chloramine T 0.5% at 4°C until use. The crowns were separated from the roots at the enamel-cemental junction and each crown was sectioned at the incisal third using a slow-speed diamond saw  so enamel and dentin specimens could be obtained. The incisal third was used for enamel specimens and the remaining two thirds were used for dentin specimens. Dentin specimens were standardized using only mid-coronal sections.The tooth fragments were embedded in PVC cylinders with acrylic resin . A semiautomatic polishing machine  with 600-grit sandpaper under water-cooling was used to expose flat enamel and dentin surfaces.G1: 1200-grit sandpaper, under refrigeration, in semiautomatic polishing machine (Control);G2: medium-grit diamond burs  in a water-cooled high-speed turbine;G3: medium-grit diamond burs (#4138), followed by fine-grit diamond burs  in a water-cooled high-speed turbine;G4: medium-grit diamond burs  in a water-cooled high-speed turbine;G5: medium-grit diamond burs (TR-26), followed by fine-grit diamond burs  in a water-cooled high-speed turbine;G6: medium-grit CVD tip  in a piezoelectric ultrasonic device;G7: medium-grit CVD tip (CR1), followed by fine-grit CVD tip  in a piezoelectric ultrasonic device.The specimens were then randomly divided into seven groups, according to the type of surface finishing of dentin and enamel:The diamond burs were mounted in a high-speed turbine  and the grinding was performed using low pressure and intermittent cutting, with a water flow rate of 45 mL/min. For ultrasonic-gritted specimens, CVD diamond tips were adapted to a piezoelectric ultrasonic device , operating according to the parameters recommended by manufacturer .For standardization purposes, diamond burs and tips were passed 10 times in the same direction on the enamel and dentin surfaces, as uniformly as possible by using light pressure and keeping the active part of the burs and tips always in contact with the tooth surface.n = 5) of each group were analyzed with a roughness tester  equipped with a diamond needle (radius of 5 μm) at a constant speed of 0.5 mm/s. Before the readings, the roughness tester was calibrated according to the manufacturer's recommendations with a roughness standard . For each reading, a length of 2.5 mm was analyzed with a cutoff of 0.25 mm. Three measurements were made for each specimen and the average of these three readings was used as the roughness value for each specimen. The Ra, Rz, and Rq roughness parameters were evaluated [(i)Ra (roughness average) is the mathematical average height of roughness irregularities measured from a mean line within the sampling length.(ii)Rz (low-point height) is the average distance between the 5 highest peaks and 5 deepest valleys within the sampling length.(iii)Rq (root mean square) is the geometric average of roughness component irregularities measured from the mean line within the sampling length.For the roughness test, the specimens Ra For the microshear bond strength test, zirconia reinforced lithium silicate glass-ceramic  cylinders with 1 mm in diameter and 1 mm in height were bonded to the enamel and dentin specimens. The glass-ceramic surface was treated with 10% hydrofluoric acid  for 20 s and rinsed and air-dried. A thin layer of silane  was applied with a microbrush for 1 min. The tooth surfaces were etched with 37% phosphoric acid,  30 s for enamel and 15 s for dentin, rinsed with water for 20 s, and blot-dried using absorbent paper. An etch-and-rinse adhesive system  was applied according to the manufacturer's recommendations and air-dried for 5 s at an approximate distance of 20 cm to allow solvent evaporation.2 irradiance. The output irradiance was measured with a radiometer . The specimens were then stored for 24 hours in distilled water at 37°C.Glass-ceramic cylinders were luted to enamel and dentine with a light-cured resin cement . The excess of cement was removed and each cylinder was light-cured for 40 s, with a LED light curing device  operating on standard mode and emitting 800 mW/cmThe microshear bond strength tests were performed with a universal testing machine  at a crosshead speed of 0.5 mm/min until fracture. A stainless steel wire-loop (0.2 mm diameter) was used and the specimens were carefully aligned to allow the load to be applied as close as possible to the bonded interface.The fractured interfaces were examined in a light microscope under 57x magnification  to determine the failure mode .Ra with microshear bond strength for enamel and dentin.Data were statistically analyzed using one-way ANOVA and Tukey's HSD test with a significance level of 5%. Pearson's product moment coefficient was used to correlate p < 0.0001). For the three parameters analyzed, G1 (Control) showed the lowest mean roughness readings. G2 and G4 (medium-grit diamond burs) showed the highest mean roughness readings. Groups in which the tooth surface was finished with fine-grit diamond burs on a high-speed turbine or with medium versus fine-grit CVD tips in ultrasonic device showed intermediate values of surface roughness and were statistically similar.Means and standard deviations of the roughness parameters for enamel and dentin are shown in Tables p = 0.0035 and p = 0.012, resp.). In all groups, the microshear bond strength values for enamel were higher than for dentin.Means and standard deviations of enamel and dentin microshear bond strengths are presented in In enamel groups, G7 (fine-grit CVD tip) presented the highest microshear and was statistically different from G1 but statistically similar to G6 (medium-grit CVD tip), G3 (KG fine-grit diamond bur in high-speed turbine), G2 (KG medium-grit diamond bur in high-speed turbine), and G5 (Mani fine-grit diamond bur in high-speed turbine). G1 (1200-grit sandpaper) and G4 (Mani medium-grit diamond bur in high-speed turbine) presented the lowest microshear bond strength values.For dentin, G7 (fine-grit CVD tip in ultrasound) also presented the highest microshear bond strength, being statistically similar to G1 (1200-grit sandpaper), G6 (medium-grit CVD tip), G4, and G2 (Mani and KG medium-grit diamond burs in high-speed turbine). Both groups whose surface was completed with fine-grit diamond burs in high-speed turbine (G3 and G5) presented the lowest microshear bond strength values, which were also statistically different from G1. For both enamel and dentin, there was no statistical difference when comparing the groups gritted with the same brand of medium- and fine-grit burs and tips .Ra and microshear bond strength for enamel and dentin   .The results from failure analysis are graphically summarized in Bonding ceramic restorations to tooth structure rely on a number of factors, including the treatment of the ceramic surface, selection of a suitable resin luting agent, and appropriate treatment of prepared tooth structure –16. VariThe preparation technique, the cavity surfaces characteristics, and the bond strength of ceramics to enamel and dentin are key factors to the clinical longevity of laminate veneers. Whenever possible, laminate veneer preparation should be made meticulously and maintained completely in enamel. This is more often achieved in minimum thickness veneers. In these cases, while better adhesion to enamel is achieved, decreased resistance of the ceramic material due to the lower thickness is expected. Thus, the technique of minimum intervention relies on a good bonding between both resin cement and enamel and resin cement and ceramics. On the other hand, the exposure of considerable amounts of dentin is sometimes inevitable during preparation, especially at the cervical and proximal areas . It is aBonding of dental ceramics to enamel is superior to bonding to dentin. Öztürk et al., demonstrated that the type of preparation surface had a significant effect on the shear bond strength of porcelain laminate veneers. When the tooth surfaces were compared, there was no significant difference between the groups bonded to enamel and enamel-dentin complex substrates . HoweverLaminate veneer preparations are often finished with fine-grit diamond burs or with polishing discs. In theory, smoother surfaces would be more favorable to impression taking, but it has been described that higher bond strength can be obtained by increasing the roughness surface, since it enhances the surface area to be bonded . In the It is expected that all the superficial dissimilarities produced by different preparation techniques would influence bond strength to dentin. Within the dentin groups, according to the results of the present study, although microshear bond strength values of fine-grit diamond bur group tended to be lower than medium-grit diamond bur group, these variations were not significantly different . LiteratA two-step etch-and-rinse adhesive was used in the present study. It is well known that this type of adhesive removes the smear layer due to the previous etching with phosphoric acid. As mentioned above, the amount and quality of smear layer produced by different surface finishing procedures are different. Thus, the results of the present study could be different if a smear layer-modifier dentin adhesive such as self-etching ones was employed. Moreover, universal adhesives containing silane-coupling agents could have significant effects on bonding outcomes . The lutRa values. However, Ra alone may not be sufficient to discriminate among surfaces. Under certain conditions, other parameters  may be important and should be used in addition to Ra to properly describe the surface characteristics [Ra, Rz, and Rq were determined, but the ranking of the groups was practically the same for the three parameters, for enamel and dentin.Regarding the surface roughness parameters analyzed, the majority of the studies report only eristics , 14. In A number of new materials are available for CAD-CAM restorations. These materials include esthetic and high-strength ceramics, composite resins, and hybrid ceramics . Each one has unique characteristics and is indicated for specific clinical applications . LaminatThe authors did not find information on the use of ultrasonic diamond tips for the preparation of ceramic laminate veneers. CVD diamond tips mounted in ultrasonic devices can be used to perform the finishing procedures on the finishing line of indirect restorations, particularly in ceramic laminate veneers in anterior teeth. It is well known that the subgingival dental preparation as well as retraction cords and hemostatic solutions may cause injury to gingival tissues. Diamond burs produced by CVD technology may be used in ultrasonic handpieces; thus the process is slower to avoid injury to the gingival tissues , 5, whic in vitro study and some limitations need to be addressed, such as the number of specimens per group and the use of microshear bond strength test. A larger sample size could influence the results, lowering the standard deviations and assuring an adequate power to detect statistical significance. Microtensile bond strength is usually regarded as a more reliable bond strength test; however, in the present study, the microshear bond strength test was chosen because it also allowed small areas to be tested, is relatively easy to perform, and does not need sectioning procedures to obtain specimens, since these procedures may induce early microcracking within the specimens.This is anWithin the limitations of this study, it can be concluded that the cavity surface finishing influenced the roughness parameters and the microshear bond strength of a glass-ceramic to enamel and dentin. Medium-grit diamond burs in high-speed turbine showed the highest mean roughness values. Fine-grit CVD tips in ultrasound presented the highest microshear bond strength values for both enamel and dentin. However, when comparing between the same manufacturer/product categories, there were no significant differences in the microshear bond strength.


Background/Aims. Antitumor necrosis factor antibodies and calcineurin inhibitors have shown good therapeutic efficacy for steroid-refractory ulcerative colitis (UC). Although some studies have compared the efficacy of infliximab (IFX) and cyclosporin A, there are no published studies comparing IFX and tacrolimus (Tac). This study aimed to compare therapeutic efficacies between IFX- and Tac-based strategies for steroid-refractory UC. Methods. Between July 2009 and August 2013, 95 patients with steroid-refractory UC received either IFX (n = 48) or Tac (n = 47) in our hospital. In the IFX group, the patients continued to receive maintenance treatment with IFX. In the Tac group, patients discontinued Tac treatment up to 3 months and subsequently received thiopurine. We retrospectively compared the therapeutic outcomes between the groups. Results. There was no significant difference in the colectomy-free rate, clinical remission rate, and clinical response rate at 2 months between the groups. However, relapse-free survival was significantly higher in the IFX group than in the Tac group . The proportions of serious adverse events did not differ between the groups. Conclusion. The findings of our study showed that IFX and Tac have similar short-term therapeutic efficacy for steroid-refractory UC. Maintenance treatment with IFX, however, yields better long-term outcomes than Tac-thiopurine bridging treatment. Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease of unknown etiology. 5-Aminosalicylates or topical treatments are usually the first-line treatment, and corticosteroids are the second-line treatment. Owing to the excellent efficacy of systemic administration of corticosteroids, most patients achieve remission even if they required second-line treatment. However, 20–40% of patients fail to respond to corticosteroids (steroid-refractory UC) or fail to maintain remission without them (steroid-dependent UC) –5. PreviThiopurines such as azathioprine and 6-mercaptopurine are commonly used to maintain remission and are also suitable for reducing the dose of corticosteroids in patients with steroid-dependent UC. On the other hand, anti-TNF antibodies, such as infliximab (IFX) or adalimumab, and calcineurin inhibitors, such as cyclosporin A (CsA) or tacrolimus (Tac), have shown good salvage therapeutic efficacies for inducing remission in steroid-refractory UC.Anti-TNF antibodies are usually administered for both inducing and maintaining remission consecutively. However, calcineurin inhibitors are used mainly for inducing remission. Other drugs such as thiopurines are used for maintaining remission when the remission was achieved with calcineurin inhibitors because of the absence of long-term treatment data. To understand the difference in efficacy between treatment with anti-TNF antibodies and calcineurin inhibitors, some studies have compared the therapeutic outcomes with IFX-based treatment versus CsA-based treatment –20. HoweTac is a newly developed calcineurin inhibitor that inhibits the transcription of interleukin-2 and interferon-gamma in T lymphocytes, similar to the mechanism of CsA. The utility of Tac for treating refractory UC has been reported in some studies: the short-term response rates range from 55 to 98%, with less severe adverse events than with CsA , 21–24. In order to make an appropriate therapeutic choice for steroid-refractory UC, it is important to make evidence comparing the outcomes between an IFX-based strategy and a Tac-based strategy. Therefore, the aim of this study was to compare the short- and long-term efficacy of IFX-based strategy and Tac-thiopurine bridging strategy for the treatment of steroid-refractory UC. We retrospectively reviewed data regarding the clinical courses of patients with steroid-refractory UC in our hospital who were treated with either IFX or Tac and then evaluated the comparable therapeutic outcomes in both groups. To the best of our knowledge, this is the first report to compare the short- and long-term effectiveness and safety of IFX-based treatment and Tac-based treatment for steroid-refractory UC.Between July 2009 and August 2013, a total of 95 patients with corticosteroid-refractory UC received either IFX or Tac in our hospital. All consecutive 95 patients were included in this study. Of those, 48 patients received IFX (IFX group) and 47 patients received Tac (Tac group), respectively. In all cases, the diagnosis of UC and disease activity was confirmed according to standardized criteria by prior clinical assessment, radiology, endoscopy, and histology. As for the definition of the response to corticosteroids, UC was regarded as steroid-refractory if the patient was in either steroid-refractory or steroid-dependent state defined as follows. Steroid-refractory state was defined as lack of a meaningful clinical response to oral or intravenous prednisolone more than 30 mg/day over at least two weeks. Steroid-dependent state was defined as occurring if prednisolone cannot be tapered to less than 10 mg/day without recurrent disease or if relapse occurs within three months of stopping prednisolone.We retrospectively reviewed the medical records of all 95 patients and compared the short- and long-term therapeutic efficacy between the IFX group and Tac group. All patients provided their informed consent for the present study.Criteria for choosing IFX or Tac depended on the treatment policy in our hospital. The important factor is current situation of thiopurine administration. Basically, IFX is mainly chosen for thiopurine-refractory or thiopurine-intolerant patient, and Tac is mainly chosen for thiopurine-naïve patient. Most of the patients enrolled in this study were treated under this treatment strategy. However, not all the patients followed this strategy. We selected another treatment arm when we could not obtain the informed consent from the patients or the patients had complications at baseline that could possibly get worse, such as renal dysfunction in Tac-based strategy.μL in a manner similar to that previously reported [Patients in the IFX group were treated with a 5 mg/kg infusion at weeks 0, 2, and 6. When clinical response was observed, maintenance treatment with IFX (5 mg/kg every 8 weeks) was consecutively initiated. Concomitantly, thiopurines were administered if the patients tolerated the initial regimen well. The dose of thiopurines was adjusted to achieve white blood cell counts between 3000 and 5000 cells/reported . GeneralPatients in the Tac group were treated with adjusted oral doses of Tac. When starting Tac treatment, inpatient therapy was the standard approach in our hospital. The initial dose of Tac was 0.1 mg/kg per day twice daily. Blood was collected to determine the tacrolimus whole-blood trough concentrations at 12 hours, 24 hours, 2–5 days, and 7–10 days and every 7 days after providing the initial dose during the early period of therapy. The dosage was then adapted to achieve a trough level between 10 and 15 ng/mL to induce remission. The equation (previous dose × 12.5 mg/mL/the blood trough level) was used for the dose adjustment during the induction periods. After inducing clinical remission, Tac trough concentrations were maintained at a lower level, between 5 and 10 ng/mL. The equation (previous dose × 7.5 mg/mL/the blood trough level) was used for the dose adjustment during the maintenance periods. The patients were hospitalized until their clinical conditions had stabilized and their tacrolimus levels were within the therapeutic range. Thereafter, the patients were regularly seen as outpatients at least every four weeks.μL in a manner similar to that previously reported [Thiopurines were also administered to the thiopurine-naïve patients within the first 4 weeks of Tac induction if the patients tolerated the regimen. The patients using thiopurine at the start of Tac therapy concomitantly continued to take them. The thiopurine dose was adjusted to achieve white blood cell counts between 3000 and 5000 cells/reported . MaintenThe short-term therapeutic efficacy was evaluated at 2 months after starting each treatment. In this study, we evaluated the patients' clinical disease activity using the Rachmilewitz Clinical Activity Index (CAI) . ClinicaU test. Kaplan-Meier plots and log-rank tests were performed to analyze relapse-free survival in the two groups. A Cox proportional hazard model was used to identify the risk factors for relapse in the two groups. A p value < 0.05 was regarded as statistically significant for between-group comparisons. All statistical analyses were performed using JMP Pro 11.0.0. .Proportions between two groups were compared by using the Fisher exact probability test, and continuous variables were compared with the Mann-Whitney p = 0.0004). In the IFX group, 14 out of 48 patients had previous Tac treatment history. In these 14 patients, 6 patients were Tac-refractory and 8 patients were the relapse cases after withdrawal of Tac. In the Tac group, 5 out of 47 patients had previous IFX treatment history. In these 5 patients, 4 patients were IFX-refractory and 1 case was IFX-intolerant.A total of 95 patients were analyzed in this study: 48 in the IFX group and 47 in the Tac group. Baseline patients' characteristics are shown in The patient with inactive colitis was not included in the study. However, some patients with mild clinical activity were included. Precisely, in the IFX group, 2 patients with CAI 2 and 1 patient with CAI 4 were included. In the Tac group, 3 patients with CAI 4 were included. However, all 6 patients were steroid-refractory cases and had endoscopic active colitis (moderate activity). According to the laboratory parameters  and CAI score at treatment onset, the Tac group had higher disease severity than the IFX group.The median follow-up period was 24.5 months  in the IFX group and 5.8 months  in the Tac group.n = 48), colectomy was performed in 4 patients within 2 months after starting IFX treatment. At 2 months, clinical response was achieved in 39 patients, and clinical remission was achieved in 33 patients, respectively. Maintenance treatments with IFX were administered in 32 patients. In these 32 patients, 17 (53.1%) were treated with a combination of IFX and thiopurine and 15 (46.9%) were treated with IFX monotherapy. The maintenance treatments with IFX were continued in 27 patients, of whom 8 experienced relapse during the observation periods. Endoscopic remission  was achn = 47), the median days of achieving high trough level was 5 days (range: 1–20 days). Colectomy was performed in 7 patients within 2 months after starting Tac treatment. At 2 months, clinical response was achieved in 32 patients, and clinical remission was achieved in 26 patients, respectively. Maintenance treatments with Tac were administered in 36 patients. In these 36 patients, thiopurines were administered in 31 patients (86.1%); five patients (13.9%) were treated with Tac monotherapy because of intolerance to thiopurines. In 32 of these 36 patients, Tac administration was withdrawn at 3 months followed by bridging to thiopurine (if tolerant), and 21 patients experienced relapse during the observation periods. In these 32 cases, endoscopic evaluations were performed in 23 cases before stopping Tac. Endoscopic remission (defined as a Rachmilewitz Endoscopic Index of ≤4 [In the Tac group (The colectomy-free rate at 2 months was 91.7% in the IFX group and 85.1% in the Tac group . There wn = 32) and 90%, 57%, 37%, and 37% in the Tac group (n = 36), respectively. The relapse-free survival rate was significantly higher in the IFX group than in the Tac group . We also performed the Kaplan-Meier analysis and log-rank analysis excluding the thiopurine-intolerant cases in the Tac group .The Kaplan-Meier plots for relapse-free survival in both groups are shown in ac group . Even thp = 0.012). Neither concomitant use of thiopurine nor withdrawal of IFX treatment was related to relapse.Risk factors for relapse in long-term in each group were identified by using the Cox proportional hazard model. The result of the Cox model analysis for relapse in the IFX group is shown in p = 0.027). The patients who did not continue to use Tac beyond 3 months had a significantly higher risk for relapse .The results of the Cox model for relapse in the Tac group are shown in n = 32) and 96%, 92%, and 82% in the Tac group (n = 36), respectively. There was no significant difference between both groups.The Kaplan-Meier plots for colectomy-free survival in both groups are shown in The adverse events observed in both groups were shown in In the present study, we evaluated the short- and long-term therapeutic efficacies of IFX-based strategy and Tac-based strategy for steroid-refractory UC. To the best of our knowledge, this is the first report comparing the efficacy between these strategies.Firstly, our results showed that there were no significant differences in the colectomy-free rate, clinical remission rate, and clinical response rate at 2 months between the IFX group and the Tac group. These results indicated that IFX and Tac have similar short-term therapeutic efficacy for the treatment of steroid-refractory UC. When interpreting these results, we should pay special attention to the methods of Tac administration. In this study, we tightly adjusted the Tac dose to maintain the appropriate trough concentration. During the initial 2 weeks, the patients in the Tac group were treated by maintaining the high trough Tac concentration 10–15 ng/mL). A randomized controlled trial published by Ogata et al. showed that Tac has a trough concentration-dependent effect [ ng/mL. AIn the present study, we also evaluated whether switching therapies (IFX therapy in patients previously treated with Tac or Tac therapy in those previously treated with IFX) has an adequate therapeutic efficacy or not. The results showed that clinical remission rate at 2 months was 57.1% in the IFX treatment in patients previously treated with Tac. On the other hand, none of the 5 patients previously treated with IFX reached the clinical remission at 2 months by Tac treatment. These results indicate that IFX therapy could be meaningful even in patients with Tac treatment failure. However, Tac therapy in patients with IFX treatment failure might not have adequate therapeutic efficacy. Because the number of patients who experienced a switching treatment in our study was quite small, further investigations are needed to clarify this issue.Secondly, the results of the long-term outcomes in the present study showed remarkable differences between the IFX-based strategy and Tac-based strategy. The IFX-based strategy showed obviously better outcomes than the Tac-based strategy when comparing the relapse-free survival rates by using the Kaplan-Meier method. Even though the 5 thiopurine-intolerant cases were excluded from the Tac group, the relapse-free survival rate was still significantly higher in the IFX group than in the Tac group. Previously, a few studies have reported the differences in long-term outcomes between IFX and CsA treatment –20. Crofα inhibitors in combination with thiopurines, but not with TNF-α inhibitors alone [Here, it is quite important to discuss the risk factors that lead to relapse during the IFX-based strategy and Tac-based strategy. In the IFX-based strategy, disease duration at treatment was identified as an independent risk factor for relapse. The patients with disease duration of less than 5 years had a significantly higher risk for relapse. Neither concomitant use of thiopurine nor withdrawal of IFX was related to relapse. Over the recent years, combination therapy with IFX and azathioprine has shown to be superior to IFX monotherapy in both short-term  and longrs alone . TherefoIn the Tac-based strategy, the absence of concomitant use of thiopurines and discontinuing the use of Tac beyond 3 months were identified as independent risk factors for relapse. As described above, in the IFX group, concomitant thiopurine use was not related to relapse independently in the IFX group. However, the absence of concomitant use of thiopurine was significantly related to relapse in the Tac group. Here, it is quite important to recognize that the role of thiopurine for each treatment strategy is different. In the IFX group, thiopurine was administrated to intensify the efficacy of IFX. On the other hand, in the Tac group, thiopurine mainly plays a role in the maintenance treatment after withdrawal of Tac. We suppose that the mechanisms underlying the efficacy in thiopurine treatment might be different between thiopurine monotherapy and combination treatment with IFX. When initiating thiopurine monotherapy as maintenance treatment, upfront administration should be needed considering a lag time of action. On the other hand, in combination treatment with IFX, thiopurine might have different additional mechanisms of action, such as inhibiting antibody production to IFX, which could quickly work from the initiation of drug administration. Therefore, we suggest that initiating thiopurine at the same time with IFX should be adequate to intensify the short-term efficacy of IFX in combination therapy. Thus, we must comprehend that the results of efficacies of thiopurine administrations in the IFX and Tac treatment should be interpreted independently and should not be compared directly, because the role of thiopurine is different. In most of our patients in the Tac group, Tac treatment was generally withdrawn at 3 months after the first administration if clinical remission was achieved; the patients were subsequently transferred to thiopurine monotherapy. The results of the Kaplan-Meier curve analysis revealed that relapses frequently occurred especially after the withdrawal of Tac at 3 months. All 4 patients who continued Tac beyond 3 months did not experience relapse during the observation periods. According to these results, induction treatment using Tac up to 3 months followed by thiopurine monotherapy is not sufficient to maintain long-term remission in patients with steroid-refractory UC. In many cases, administration of Tac beyond 3 months might be needed to sustain long-term remission, but the efficacy of Tac as long-term maintenance treatment is not well established yet. Thus far, there have been only a few reports describing the efficacy and safety of long-term maintenance therapy using Tac , 24, 25.When considering the relapse after stopping Tac, we should also focus on the achievement of endoscopic remission before the drug withdrawal. Recent studies have identified mucosal healing on endoscopy as a key prognostic parameter in the management of inflammatory bowel diseases . HoweverAs for the long-term colectomy-free survivals, there was no difference between the IFX group and Tac group. However, the result should be carefully interpreted. In the Kaplan-Meier analysis and log-rank test for colectomy-free survival, data collections were ended when the patients relapsed in order to evaluate the pure long-term efficacy of each treatment strategy. When relapses were observed, the patients were treated by changing the agents. If we collected the data even after the relapse, the colectomy-free survival would be strongly affected by the efficacies of newly switched drugs. As shown in In this study, the frequency of adverse events seemed to be much higher in the Tac group than in the IFX group. However, most of the events observed in the Tac group were clinically mild and the frequency of the severe adverse events was similar in IFX group and Tac group. Therefore, the results suggest that clinicians might not focus much on the frequency of adverse events when selecting the appropriate treatment. Here, we must describe a limitation when interpreting the frequency of adverse events in this study. Although the present study was retrospectively designed, we could successfully collect precise data, such as renal function, liver function, and anemia because they were numeric data easily reviewed in medical records. On the other hand, as for the clinical symptoms such as tremor, there was a possibility that we failed to collect some events that were not described in the records. This is an important limitation when evaluating the adverse events in the study.Limitations of our study include the selection bias, sample size, differences of the timing among the patients in evaluating the short-time efficacies, differences of the proportions of administrated thiopurine, and single-center experience. In particular, the most important limitations include the selection bias when choosing one of the regimens (IFX- or Tac-based strategy) and the imbalance in the baseline patient backgrounds in each group. In this study, criteria for choosing IFX or Tac depended on the treatment policy in our hospital. Basically, IFX was mainly chosen for thiopurine-refractory or thiopurine-intolerant patients, and Tac was chosen for thiopurine-naïve patients. Because the treatment period with Tac is limited up to 3 months, maintenance treatment with thiopurine is necessary after withdrawing Tac administration. Therefore, Tac treatment is not preferable for a thiopurine-refractory or thiopurine-intolerant patient. On the other hand, IFX is preferable for such patients because the long-term maintenance treatment with IFX is available. The same treatment strategy is recommended in the review article by Naganuma et al.  and manyIn conclusion, this report presents the findings of a single-center retrospective study of the relative efficacies of IFX- or Tac-based strategy for steroid-refractory UC. Our study showed that IFX and Tac have similar short-term therapeutic efficacy for the treatment of steroid-refractory UC. Maintenance treatment with IFX, however, yields better long-term outcomes than Tac-thiopurine bridging treatment. Because our data reflected the therapeutic outcomes under the certain selective treatment strategy, further randomized controlled trials are required to discern whether there is a clear discrepancy between the efficacies of these agents.


Previously described transgenic tobacco lines express the full length infectious Tobacco mosaic virus (TMV) genome under the 35S promoter . Through their young stages these plants exhibit strong resistance against both the endogenously expressed and exogenously inoculated TMV, but at the age of about 7–8 weeks they break into TMV infection, with typical severe virus symptoms. Infections with some other viruses  induce the breaking of the TMV resistance and lead to synergistic proliferation of both viruses. To deduce the gene functions related to this early resistance, we have performed microarray analysis of the transgenic plants during the early resistant stage, and after the resistance break, and also of TMV-infected wild type tobacco plants. Comparison of these transcriptomes to those of corresponding wild type healthy plants indicated that 1362, 1150 and 550 transcripts were up-regulated in the transgenic plants before and after the resistance break, and in the TMV-infected wild type tobacco plants, respectively, and 1422, 1200 and 480 transcripts were down-regulated in these plants, respectively. These transcriptome alterations were distinctly different between the three types of plants, and it appears that several different mechanisms, such as the enhanced expression of the defense, hormone signaling and protein degradation pathways contributed to the TMV-resistance in the young transgenic plants. In addition to these alterations, we also observed a distinct and unique gene expression alteration in these plants, which was the strong suppression of the translational machinery. This may also contribute to the resistance by slowing down the synthesis of viral proteins. Viral replication potential may also be suppressed, to some extent, by the reduction of the translation initiation and elongation factors eIF-3 and eEF1A and B, which are required for the TMV replication complex. Viruses are obligate intracellular molecular parasites which depend on host's cellular machinery and on multiple host factors to complete their infectious life cycle. They utilize a large variety of host-encoded proteins and molecular structures as components of their replication complex or cell-to-cell movement machinery Viruses can also alter the functions and composition of their host cells to benefit their own proliferation. For instance, they can enhance the expression of their needed host factors, bind or suppress various resistance factors, induce changes in the lipid composition of infected cells, and interfere with host's hormonal pathways Viruses can initiate infection process only in susceptible host species that provide compatible host factors, needed for the viral replication and spread. However, many of these potential host species also recognize the invading viruses and mount different defense mechanisms to stop their proliferation or spread. For instance, activation of the R-gene mediated resistance leads to hypersensitive reaction (HR) and virus localization, enhanced expression of various defense-related genes and induction of systemic acquired resistance (SAR) The virus-host interactions thus consist of a very complex molecular interplay. It involves depletion of various host factors and energy compounds through viral parasitism, altered expression of the viral-induced host factors, active defense mechanisms mounted by the host, active viral counter-defense mechanisms, disturbance of the silencing-mediated regulatory network, and the general infection related stress-reaction in the host Now, different system biology approaches are available to investigate the plant responses induced in different stages of virus infections, or in transgenic plants that express individual viral genes. Expression levels of many hundreds or even several thousands of genes have been found to be altered in these plants, and although these alterations have some common features (enhanced expression of defense- and stress-related genes), they are mostly unique and specific to each virus/host combination Through the last few decades, Tobacco mosaic virus (TMV) or its constituent genes have often been used as a model system to investigate the plant-virus interactions and to dissect the details of viral replication, movement, host resistance and physiological alterations in situ immunolabeling, first appeared as isolated infection foci in the vascular tissues of the upper leaves , and of the same plants after the resistance break (ARB), and of three wt TMV-infected (TMVi) plants were analyzed by the microarray approach, and compared each to the transcriptomes of healthy control plants of corresponding age. The observed transcriptome alterations were compared between these three types of plants to reveal gene functions that would be specific to each condition.The microarray analysis was performed using Tobacco 4×44K microarray (Agilent), according to Agilent's standard procedures see . Our preThe microarray analyses indicate that total of 1362 transcripts were up- and 1422 transcripts were down-regulated in the BRB transgenic plants , total oMany viruses infecting eukaryotic hosts use different mechanisms to reduce the host-specific protein synthesis, and to increase the synthesis of the virus-specific proteins In BRB transgenic plants, the largest group of down-regulated genes  was composed of translation-related transcripts . Most ofInterestingly, nearly opposite expression pattern of translation-related genes was observed in the ARB transgenic plants. In these plants, only 120 translation-related transcripts were down-regulated, and a total of 135 translation-related transcripts were up-regulated . Among tIn the TMVi plants, the translation and protein processing related transcripts were predominantly up-regulated, with a total of 197 translation-related transcripts being up-regulated and 44 being down-regulated  & S5. MoIn compatible plant-virus interactions the virus must be able to infect the host plant without mounting excessive or fast defense reactions, or to suppress the basal host defense mechanisms In the BRB transgenic plants, total of 174 stress response-related transcripts were up-regulated and 76 were down-regulated  & S6. MaEven more  of stress-related genes were up-regulated in the ARB transgenic plants , includiIn TMVi plants, only total of 61 of stress-related transcripts were up-regulated . These iPlant hormones play a major role in various defense signaling pathways, and some viruses interact with these signaling pathways to enhance their infection process In ARB plants, a total of 59 transcripts related to defence hormones (ethylene and jasmonic acid) or coding for development-related embryo-specific proteins were up-regulated . The jasIn TMVi plants, a total of 33 transcripts related to hormones and development were down-regulated , includiSeveral studies indicate that virus infections or expression of virus-derived genes in transgenic plants reduce the photosynthesis process and cause alterations of the carbohydrate metabolism and translocation in the plants Contrastingly, total of 239 photosynthesis-related genes were down-regulated in the ARB transgenic plants . Many ofSimilar to the ARB transgenic plants, photosynthesis related transcripts were predominantly down-regulated in TMVi plants . Total oThe gene expression related to cell cycle and cell organization was differentially altered in BRB and ARB transgenic plants and in TMVi plants. Specific to the BRB transgenic plants, 30 and 18 transcripts related to cell cycle and organization were up- and down- regulated, respectively  & S12. SIn the ARB transgenic plants total of 36 transcripts related to cell cycle and cell organization were down-regulated , includiVery few of cell division and organization-related transcripts were altered in the TMVi plants. Only 5 transcripts, coding for the CDC kinase and annexin were down-regulated , and 5 tSimilar to the cell cycle and organization gene expression pattern, also the transcripts for histones and DNA repair proteins were affected differently in BRB and ARB transgenic plants and in TMVi plants. In the BRB transgenic plants, a total of 54 histone-encoding transcripts and 28 DNA binding and repair protein-encoding transcripts were down-regulated , while 1In TMVi plants, only four transcripts coding for DNA modifying proteins, i.e. one coding for a transposase, one coding for nuclease and two coding for DNA binding proteins were down-regulated, and only one NAP1-related transcript was up-regulated  & S13.Potato virus YAfrican cassava mosaic virus Potato virus × To reveal the gene expression alterations that were unique to the resistant stage of the BRB transgenic plants, their RNA expression profile was compared with that of the same transgenic plants at the ARB stage, with TMVi plants and with our previously published transcriptomes of transgenic plants expressing various viral silencing suppressors, i.e. HC-Pro from Similarly, comparison of the total of 1462 transcripts that were down-regulated in the BRB transgenic plants against the 2884 down-regulated transcripts of other plants revealed that 1232 of these were unique to the BRB plants . Most ofTo find out how the strong reduction of the proteins synthesis-related transcripts, and of the increase of protein degradation-related transcripts in the BRB-plants affects the total protein content of these plants, their total soluble protein was extracted, quantitated by the Lowry method, and compared to the soluble protein content of healthy control plants of the same age. This revealed that the total soluble protein content of the BRB-TMV transgenic plants was 28% lower that that of the wt plants . From prThe total protein composition of the three types  of test plants was analysed by using the 1D and 2D-PAGE, and compared with their corresponding wild type controls. The analyses were repeated with three biological replicates for each plant type. Upon loading of equal amounts of protein samples, the 2D-PAGE analysis showed that the protein profile of the BRB samples contained multiple altered (either enhanced or reduced) bands or spots as compared to the wild type control plants, but the altered spots were not identified . Also thDifferential photosynthetic gene expression and appearance of the chlorotic TMV symptoms at different stages of plant growth indicated that photosynthetic activity was differentially altered in BRB and ARB transgenic plants and in TMVi plants. To analyze this, we measured their oxygen evolution per ug chlorophyll, and compared this against the oxygen evolution rate of control plants of the same age. The results indicated that oxygen evolution was not changed much in the BRB transgenic plants , whereas.To check whether the observed TMV resistance in the young TMV-transgenic plants was TMV-specific, or active against a broader range of viruses, the plants were inoculated with PVX, PVY and PVA. ELISA results indicated that these viruses reacted somewhat differently to theTMV- transgenic host. The PVY level was somewhat increased, and PVX level somewhat decreased in the inoculated leaves of the transgenic plants, as compared to the inoculated leaves of the wt plants at 7 days post inoculation (dpi). Any of these viral levels did not differ significantly in the systemically infected leaves of TMV-transgenic and wt plants at 7 dpi, but PVA infection was strongly enhanced in the transgenic plants at 10 dpi . MeanwhiTypically, viruses can infect and complete their life cycle in a susceptible host and also easily spread systemically in such a host plant. This kind of compatible interaction involves effective use of necessary host factors and suppression of the host defense mechanisms Our transgenic tobacco plants represent an artificial inoculation system, where the infectious TMV genome is expressed under the constitutive 35S promoter, supposedly already in all cells of the germinating embryo. This leads to a very strong resistance condition that prevails in the plants until they reach a certain stage of maturity (about seven weeks from germination). This resistance was mostly specific to its endogenous inducer virus, although it provided low level transient protection also against two exogenously inoculated viruses (PVY and PVA), maybe due to the multiple defence genes that are activated in these tissues . HoweverThis suggests that the resistance may be related to the TMV-specific RNA silencing, and similar in nature to the resistance surrounding the DGIs in the young systemically infected leaves. Still, this was not supported by our earlier results, which showed that the TMV-derived transgene was not silenced by methylation, and that virus-specific siRNAs were not detected in the resistant tissues. The methylation level increased and the siRNAs became detectable only after the resistance break, indicating that the RNA silencing became activated at this stage 10 as compared to normal productive infection (data not shown). No viral CP was detected during this period in the plants. Still, the transgene expression, and the resistance reaction against the expressed infectious viral RNA caused a severe stress condition of the plants, manifested by the strong reduction of the plant growth  Similar, but not identical, virus resistance has been reported in transgenic To identify physiological and molecular processes that are associated with the resistance status of the young plants, we investigated the transcriptome of the transgenic plants before and after resistance break. The microarray analysis revealed multiple gene expression alterations in these plants, and several of them - for example the strong suppression of the translational machinery, enhanced biotic stress responses including the activation of the SAR and HR pathways, hormonal changes or cell division alterations - may all contribute to the resistance.An interesting feature in the transcript profile was the strong down-regulation of the multiple (more than 700) transcripts coding for different components of the translation machinery. The strong reduction of the 40S and 60S ribosomal RNAs, and of other ribosomal genes in the BRB plants, compared to their strong up-regulation in the TMV infected wt plants, and also to their normal expression in the ARB transgenic plants, suggest that the availability of host translational machinery is actively restricted in the BRB transgenic plants. This may directly suppress the accumulation of viral proteins. Furthermore, the reduction of the eIF3 and eEF1A and eEF1B translation initiation and elongation factors, which are known to be needed for the TMV-specific replicase complex Many viruses modify the host translational machinery to increase the viral protein synthesis but not host protein synthesis Many protein degradation pathways and several proteases were induced at the resistant stage but reduced in the TMVi plants, indicating that the BRB transgenic plants may promote the resistance also through enhanced turnover of the viral proteins. The reduced protein synthesis, and enhanced protein degradation were reflected in the reduced soluble protein content, and also in the altered protein profile of the BRB plants.Chloroplasts are the main center for many important metabolic functions, and many biotic/abiotic stresses, including virus infections, influence their environment In the TMVi plants the transcriptome was altered much less than in the transgenic BRB and ARB plants. Interestingly, while induction of defense-related genes is not typical to TMVi plants due to the compatible interaction, this reaction was quite opposite in the BRB and ARB transgenic plants. Induced expression of SAR- and HR-associated proteins in the BRB transgenic plants, and their down-regulation after resistance break or virus infection (ARB and TMVi plants) indicates the incompatible host-virus interactions and induction of the active resistance pathways Several animal studies have revealed that viruses can hijack the host cell division machinery to control the anti-cancer mechanisms Many cyclins (A and B type) were strongly down-regulated in the BRB transgenic plants. Cyclins are involved in the cell cycle regulation by the cyclin dependent protein kinases phosphorylation process (CDKs) The microarray analysis reveals that the expression of the infectious TMV genome in the germinating and developing tobacco tissue induces strong alterations in an unique pool of transcripts, many of which may contribute to the TMV resistance status of the plants. One important factor in this condition appears to be the strong enhancement of the SAR- and HR-type defence pathways. Another interesting response is the suppression of the translational machinery, which is a totally unique reaction observed in these plants. This may slow down the synthesis of viral proteins, and also deprive the cells of different host factors (e.g. translation factors eIF-3 and eEF1A and B) which are needed for TMV replication, but the role of these reactions in terms of the virus resistance remains unclear. How all these responses are induced and mutually integrated in the young transgenic plants remains to be solved.Nicotiana tabacum cv. xanthi nn) and transgenic tobacco plants, which express the whole TMV genome (strain U1) were grown in greenhouse at 60% relative humidity and in 22°C temperature, with a light/night regime of 16 h light (150 µmol photons m−2s−1) and 8 h dark. Three replicate samples were collected from the selected transgenic tobacco line, from three BRB plants at 6 weeks, and from the same ARB plants at 8 weeks after germination. At the same time, corresponding sets of the control samples were collected from wild type (wt) plants, which were grown in the same conditions and of the same age as the test plants at the time of sampling. During this collection period all plants remained in the vegetative growth stage. Always the third leaf from the apex  was collected, and the collection took place always at the same time of the day (11 am). Wt tobacco plants were mechanically inoculated with TMV at 8 weeks after germination, and samples of systemically infected leaves were collected from these plants at 8 days after inoculation, in parallel with corresponding control samples from healthy wt plants. All the leaf samples were directly frozen in liquid nitrogen and used for RNA extraction. Positive expression of transgene (TMV) RNA was detected in the leaf samples by qRT-PCR prior to the array analysis; TMV RNA was not detectable in these samples by Northern blotting The wild type tobacco , BRB and ARB transgenic, and TMVi wild type tobacco plants by using TRIsure reagent  according to manufacturer's instructions. The extracted total RNA was purified with the RNA purification kit  and then subjected to DNaseI treatment (Promega RQ1 RNase free-DNaseI) according to the manufacturer's recommendations. Subsequently, the total RNA was concentrated with Amicon Ultra-0.5 centrifugal filter devices. The cDNA labeling, and Agilent 4×44k microarray hybridization were done according to the manufactures instructions  and the raw numerical data handling and its statistical analysis were done by using Chipster  program http://mapman.gabipd.org/web/guest/mapman-annotationexperts website, with additional information obtained from the websites like JCVI http://plantta.jcvi.org/ and BLAST http://blast.ncbi.nlm.nih.gov/Blast.cgi. The functional grouping of probes was also attained from the mapman.gabipd.org website, with some additional manual adjustments.The probe information provided by the manufacturer for the Agilent 4×44k microarray was limited and mainly based on EST and cDNA sequences. Therefore, most of this descriptive annotation information was obtained from the The microarray results were verified by using the quantitative real-time PCR (RT-qPCR) method by following the MIQE guidelines Sample sections (7 µm) were prepared from the shoot apical domains of BRB/ARB TMV transgenic plants at 7 weeks after germination, i.e. just at the time of resistance break, by excising and immediate fixing as described previously 2, 1 mM EDTA, and 1% BSA) with ice cold mortar and pestle. The ground mixture was filtered through the Miracloth and 2 ml of the filtrate was taken and centrifuged at 12000×g for 2 minutes. The chlorophyll concentration measurements were done according to the procedure stated in 2 and 1 mM KH2PO4). The oxygen evolution measurements were carried out by a Clark type electrode by using 0.5 mM DCBQ as electron donor.Equal amount of leaf samples (1.0 g) from the healthy control, BRB and ARB transgenic plants, and from TMVi tobacco plants were taken and ground in 4 ml of thylakoid isolation buffer . Subsequently, supernantent was isolated and used for protein quantity measurement by using lowry assay.Protein extraction for the measurement of the total soluble protein content from the BRB plants and healthy control plants was done from by 0.5 g of leaf tissue, ground inthe ice cold oxygen electrode buffer  according to the manufacture recommendations with some adaptive steps from TRIzol protocol (Invitrogen Inc. USA). The concentrations of isolated protein samples were measured by using Lowry assay. A total of 20 µg protein samples were loaded and run on 1D SDS-page electrophoresis to verify the equal loading of the protein samples. 250 µg of protein samples were taken and mixed with rehydration buffer . The protein samples were incubated for 2 hours at room temperature and separated by isoelectric focusing using Bio-Rad 7 cm IPG pH 3–10 strips. Subsequently, the IPG strips were subjected to the second dimension separation with the 15% PAGE gels by using protein II apparatus (Bio-Rad). The protein gels were fixed with isopropanol and acetic acid treatments for 15 minutes each and incubated overnight with coomassie blue stain . In the next morning, gels were destained and photographed. Optionally, the gels were stained again with silver staining kit  according to manufacturer's instructions to analyze even the low abundance proteins.Different viruses used in this study  were detected by using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) according to the manufacturers guidelines  with slightly modified protocol. The commercial polyclonal, alkaline phosphatases conjugated antibodies  against all viruses were diluted to 1∶10000 for use. The ELISA reactions were developed by using the p-Nitrophenyl phosphate as a substrate, and measured at 405 nm absorbance by using the ELISA plate reader . 100 ng of purified virions of the corresponding viruses were used as internal standards.Figure S1Graphic presentation of the altered protein synthesis-related transcripts , as detected and portrayed by the MapMan software from the microarray data of the BRB-, ARB-TMV transgenic and TMVi plants. The blue- and red squares and bars represent the up- and down-regulated transcripts, respectively, and the picture frame depicts their functional location in the cell. The boxes portrayed inside the nuclear circles indicate the alterations in the transcription- and mRNA processing-related transcripts, respectively. Alterations of the translation-related transcripts are portrayed in the cytoplasm, separately for the plastidic, mitochondrial and cytoplasmic ribosomes.(TIF)Click here for additional data file.Figure S2Graphic presentation of the altered stress-related transcripts  as detected and portrayed by using the MapMan software from the microarray data of the BRB- and ARB-TMV transgenic and TMVi plants. The blue and red squares represent the up- and down-regulated transcripts, respectively, that are involved in the different signaling pathways and in different stress responces.(TIF)Click here for additional data file.Figure S31D-SDS-PAGE gels (left panel), and 2D-polyacrylamide gel electrophoresis (2D-PAGE) (right panel) showing, respectively, the equal loading of samples and the levels of various individual proteins. Upper two panels show BRB-TMV transgenic plants protein samples analysis at before resistance break stage, the middle two panels show the ARB-TMV transgenic plants protein samples analysis at after resistance break stage, and the bottom two panels show samples from the TMV-infected wild type tobacco plants (TMVi). All gels were stained with coomassie blue. Molecular weight ladder (Thermo Scientific) contains markers for the 250, 130, 100, 70, 55, 35, 25, 15, and 10 kDa proteins – the most important ones are marked in the panels. The TMV CP is indicated with the * in the ARB and TMVi gels.(TIF)Click here for additional data file.Table S1Normalized microarray data showing both down- (sheet1) and up-regulated (sheet 2) transcripts in the leaves of BRB-TMV transgenic tobacco plants.(XLSX)Click here for additional data file.Table S2Normalized microarray data showing both down- (sheet1) and up-regulated (sheet 2) transcripts in the leaves of ARB-TMV transgenic tobacco plants.(XLSX)Click here for additional data file.Table S3Normalized microarray data showing both down- (sheet1) and up-regulated (sheet 2) transcripts in the leaves of TMVi wild type tobacco plants.(XLS)Click here for additional data file.Table S4Down-regulated transcripts related to the protein synthesis, degradation and amino acid metabolism in the leaves of BRB-, ARB- transgenic and TMVi plants.(DOCX)Click here for additional data file.Table S5Protein synthesis, degradation and amino acid metabolism related up-regulated transcripts detected in the leaves of BRB-, ARB- transgenic and TMVi plants.(DOCX)Click here for additional data file.Table S6Biotic stress related down-regulated transcripts detected in the leaves of BRB-, ARB- transgenic and TMVi plants.(DOCX)Click here for additional data file.Table S7Biotic stress related up-regulated transcripts detected in the leaves of BRB-, ARB- transgenic and TMVi plants.(DOCX)Click here for additional data file.Table S8Hormones and development related down-regulated detected in the leaves of BRB-, ARB- transgenic and TMVi plants.(DOCX)Click here for additional data file.Table S9Hormones and development related up-regulated detected in the leaves of BRB-, ARB- transgenic and TMVi plants.(DOCX)Click here for additional data file.Table S10Photosynthesis and carbohydrate metabolism related down-regulated transcripts detected in the leaves of BRB-, ARB- transgenic and TMVi plants.(DOCX)Click here for additional data file.Table S11Photosynthesis and carbohydrate metabolism related up-regulated transcripts detected in the leaves of BRB-, ARB- transgenic and TMVi plants.(DOCX)Click here for additional data file.Table S12Cell division and DNA-binding related down-regulated transcripts detected in the leaves of BRB-, ARB- transgenic and TMVi plants.(DOCX)Click here for additional data file.Table S13Cell division and DNA-binding related up-regulated transcripts detected in the leaves of BRB-, ARB- transgenic and TMVi plants.(DOCX)Click here for additional data file.Table S14Different up-regulated transcripts in the BRB-TMV transgenic plants after subtracting the up-regulated transcripts from other ARB-TMV, TMVi and different VRS expressing transgenic tobacco plants .(DOCX)Click here for additional data file.Table S15Different down-regulated transcripts in the BRB-TMV transgenic plants after subtracting the up-regulated transcripts from other ARB-TMV, TMVi and different VRS expressing transgenic tobacco plants .(DOCX)Click here for additional data file.Table S16Primers used in RT-qPCR experiment for validation of microarray data.(DOCX)Click here for additional data file.


Trypanosoma brucei variant surface glycoproteins (VSG) are glycosylated by both paucimannose and oligomannose structures which are involved in the formation of a protective barrier against the immune system. Here, we report that the stinging nettle lectin (UDA), with predominant N-acetylglucosamine-binding specificity, interacts with glycosylated VSGs and kills parasites by provoking defects in endocytosis together with impaired cytokinesis. Prolonged exposure to UDA induced parasite resistance based on a diminished capacity to bind the lectin due to an enrichment of biantennary paucimannose and a reduction of triantennary oligomannose structures. Two molecular mechanisms involved in resistance were identified: VSG switching and modifications in N-glycan composition. Glycosylation defects were correlated with the down-regulation of the TbSTT3A and/or TbSTT3B genes  responsible for glycan specificity. Furthermore, UDA-resistant trypanosomes exhibited severely impaired infectivity indicating that the resistant phenotype entails a substantial fitness cost. The results obtained further support the modification of surface glycan composition resulting from down-regulation of the genes coding for oligosaccharyltransferases as a general resistance mechanism in response to prolonged exposure to carbohydrate-binding agents. Trypanosoma brucei, the causative agent of African trypanosomiasis, is covered by glycosylphosphatidylinositol-anchored glycoproteins, which shield parasites from effectors of the host immune system. The most abundant protein is the variant surface glycoprotein (VSG), which plays an essential role in antigenic variation and the ability of the parasite to evade the immune system. VSGs are N-glycosylated in a site-specific manner by different oligosaccharyltransferases giving rise to complex and mannose-rich N-glycans. In this study, we report that the carbohydrate binding agent, stinging nettle agglutinin , exhibits trypanocidal activity, impairs endocytosis, and cytokinesis and induces parasite lysis. We have investigated the mechanisms conferring resistance in order to understand UDA mode of action and found that resistant strains present changes in the expression of oligosaccharyltransferases, and modifications in the nature of the predominant VSG, therefore resulting in important changes in N-glycan composition. The modification in surface glycans gives rise to reduced infectivity in vivo further underscoring the role of glycosylation in parasite virulence. Our findings show that carbohydrate binding agents that bind effectively to surface glycoproteins can provide a novel avenue for design of drugs to combat African trypanosomiasis. Trypanosoma brucei spp are protozoan parasites that cause the neglected disease known as African trypanosomiasis or sleeping sickness in human and nagana in animals, which exhibit a fatal impact on health and economy of the affected countries. So far, the treatments are insufficient and unsatisfactory; therefore discovery of new approaches to design novel drugs is a critical challenge to combat these diseases.N-glycans of the VSGs  pentasaccharide of complex N-glycans treated with sialidase, β-galactosidase, and β-hexosaminidase and to the exposed ManGlcNAc2 (Manβ1–4GlcNAcβ1–4GlcNAc) trisaccharide core of oligomannose N-glycans treated with α-mannosidase [5GlcNAc2 is not a TL ligand [N-acetyl lactosamine, or abbreviated core structures derived from paucimannose N-glycans   . The exitemplate .TbSTT3 genes  and their corresponding 5’UTRs and 3’UTRs were determined in order to verify their integrity and are shown in TbSTT3 genes.In addition, the nucleotide sequence of canonical 9 parasites/mL, UDA15a trypanosomes were not able to infect and kill mice during the monitored period (2 months) (7 parasites/mL) that eventually led to animal death.To evaluate the effect of altered OST expression on VSG glycosylation and the subsequent capacity of trypanosomes to survive and grow in the mammalian host, Balb/C mice were inoculated with parental, UDA15a or UDA15b strains and the course of the infection was followed by monitoring parasitaemia. While mice infected with the parental line died at 6 days post-infection when parasitaemia reached 10 months) . In the TbSTT3 mRNA levels and reevaluate the resistance to UDA. The isolated parasites, renamed UDA15b/M4 , expressed a novel set of VSGs that appeared as three discrete bands on SDS-PAGE. MALDI-TOF analysis identified bands 1 and 2 as a mixture of VSG832 and VSG629, while band 3 consisted exclusively of VSG832 Manα1  as probeN-glycans of oligomannose-type structures, in UDA15b parasites, such defects result in an increase in complex paucimannose-derived N-glycans.In UDA15a parasites, the repertoire of glycoproteins detected by TL was different to that of the parental line although the overall signal intensity was similar in both lines . Taken tA detailed study on lectin binding and endocytosis was performed to provide detail on the mechanism of UDA killing and parasite adaptation to UDA. Parasites were labeled with UDA-FITC, transferrin-FITC, ConA-AlexaFluor 594 conjugate or Alexa Fluor 488-labeled dextran. After exhaustive washing, the fluorescence signal was measured by FACS or analyzed by 3D microscopy. In the first place, parental and UDA-resistant strains were labeled with UDA-FITC for 30 min as described in Materials and Methods. Notably, all UDA-resistant strains, with the exception of UDA3.5a and UDA3.5b, had lost most of their capacity to bind the lectin, which remained between 5% and 20% with regard to the UDA-binding capacity of the parental line . Additio50 for 6 h , and subsequently, to the subtelomeric VSG MITat 1.1. To understand the nature of this observation, a variable region located downstream of the active VSG promoter was amplified by PCR using cDNA from resistant and parental lines. Sequencing of the amplified region showed no differences between parental and resistant lines , thus co50 values for each compound were determined , whereas the remaining CBAs produced R-index values between 9.7 and 26.9. Indeed, the high cross-resistance to EHA further supports the conclusion that glycoproteins in resistant cell lines are enriched in paucimannose-derived N-glycans in disadvantage of oligomannose residues.In order to determine whether UDA-resistant mutants exhibited cross-resistance to other CBAs with different substrate specificities, UDA15a and UDA15b trypanosomes were cultured with increasing concentrations of HHA, EHA, GNA and NPA and the ECtermined . Cross-rT. brucei [Urtica dioica agglutinin), one of the smallest monomeric plant lectins, binds irreversibly to the surface coat of T. brucei with a cytotoxic activity at concentrations close to the EC50 (0.225 μM). As an antiviral agent, nanomolar concentrations of UDA have been shown to strongly bind to HIV-1 gp120, thus inhibiting virus infection by blocking virus entry and viral transmission upon co-cultivation of persistently HIV-infected and uninfected cells [The present study aimed to examine the properties of carbohydrate-binding agents as trypanocidal agents, their mode of action and the potential pathways that these organisms may employ to generate resistance. In contrast with previous studies showing that a variety of lectins do not affect viability of bloodstream forms of . brucei , here weed cells ,39,40. Ted cells . FurtherHippeastrum hybrid agglutinin (HHA) [Trypanosome exposure to UDA induces profound defects in endocytosis, cytokinesis stalling, morphological cell abnormalities characterized by the appearance of big and rounded cells, accumulation of the lectin and enlargement of the flagellar pocket and of cell vesicles; a phenotype largely reminiscent of that induced by treatment with the /-mannose-specific in (HHA) . A stallin (HHA) . In the in (HHA) ,43. We hN-glycosylated with paucimannose-type structures at the expense of oligomannose structures. Similar alterations in glycosylation have been previously reported for TbGlcaseIIα null mutants [TbUAP conditional null mutant cells [TbALG3 null mutants [T. brucei glycosylates VSGs in a site-specific manner by the action of two catalytic OSTs. The STT3A activity transfers the Man5GlcNAc2-PP-Dol to asparagines flanked by an acidic sequence yielding paucimannose structures, whereas STT3B catalyzes the transfer of Man9GlcNAc2-PP-Dol to any remaining asparagine giving rise to oligomannose N-glycans [TbSTT3B and/or a switch to a different VSG with novel glycosylation sites are the genetic factors involved in the emergence of parasites with surface glycoproteins rich in paucimannose N-glycans instead of oligomannose structures. It is worth emphasizing at this point that regardless the type of structure, the reason behind the drastic reduction in affinity is likely the nature or the accessibility of the novel glycan residues covalently attached to the VSGs. Importantly, whereas N-glycosylation defects did not affect proliferation in vitro, resistant parasites either were not infective or virulence was significantly attenuated in a murine model of infection. The vital role of VSG-glycosylation in host-parasite interactions has been demonstrated in previous work where RNAi-mediated or HHA-induced TbSTT3B knock-downs exhibited reduced virulence [In order to gain insight into the molecular basis of UDA’s mode of action we generated resistant parasite strains. The resistance phenotype was characterized by a reduced capacity of VSGs to bind UDA either due to defective glycosylation or to VSG switching events that led to the predominant expression of novel VSGs with low affinity for the lectin. Because this phenotype was stable and remained in the absence of UDA’s selective pressure, we concluded that the resistance phenotype is genetically encoded. Evidence obtained from different approaches including glycosidase digestion, lectin blotting and cross-resistance assays suggests that resistance to UDA is specifically conferred by the presence of VSGs which are primarily  mutants , TbUAP cnt cells , TbALG3  mutants , TbSTT3B mutants , GDP-Man mutants  and HHA- mutants . T. bruc-glycans . Therefoirulence ,19.3-oligomers in addition to high mannose N-glycans such as Man9, Man8, Man6 and Man 5 [The differences in resistance modalities observed for UDA when compared with HHA  can be dnd Man 5 . Such diIt should also be emphasized that lectins in general may also bind to mammalian glycoproteins and can display pronounced mitogenic effects. However, the binding specificities and potential toxic side-effects may differ considerably between different lectins. For example, whereas the α-mannose-specific lectin cyanovirin N is highly mitogenic and induces a wide variety of chemokines, microvirin, targeting α-mannose oligomers as well, was shown to have a much higher safety profile . It shouUrtica dioica agglutinin on the bloodstream form of T. brucei. We present evidence indicating that the lectin binds mainly to surface VSGs, although other glycoproteins cannot be discarded. However, the exact molecular mechanism responsible for the resulting cell cycle defects and switching events remains to be established. Prolonged exposure to UDA can generate resistant mutants through different strategies whose ultimate goal is to alter the glycosylation status of the parasite surface and thus, reduce lectin-binding. However, a major drawback of the adaptation to UDA for the parasite is the loss of infectivity and virulence. Taken together, these observations suggest that carbohydrate binding agents that interact with VSG glycans could be explored as a novel avenue to design antitrypanosomal agents.In the present study, we have conducted a detailed investigation of the cytotoxic activity and mode of action of S1 FigTbSTT3 genes and primers used in the study. B) PCR analysis of the full-length and chimeric TbSTT3 genes. Target sequences were amplified from UDAa and UDAb genomic DNAs with different primer combinations according to the scheme shown in panel A.A) Scheme of (TIF)Click here for additional data file.S2 FigThe alignment was performed with ClustalW2 (EMBL-EBI).(TIF)Click here for additional data file.S1 TableChanges were referred to the amino acid sequences of the parental line obtained in our laboratory.(DOCX)Click here for additional data file.S2 TableChanges were referred to the sequences of the parental line obtained in our laboratory.(DOCX)Click here for additional data file.