Tumor cells exhibit abnormal actin remodeling profiles, which involve the altered expressions of several important actin-binding proteins. Profilin1 (Pfn1), originally identified as an actin-associated protein, has been linked to several human malignancies. Our recent studies suggested that Pfn1 facilitates apoptosis in pancreatic cancer cells. Here, we investigated the exact role of Profilin1 (Pfn1) in pancreatic adenocarcinoma (PDAC) and the underlying mechanisms.in vitro and in vivo. Immunoprecipitation and mass spectrometry were used to identify the Pfn1-associated proteins and potential pathways.Pfn1 protein expression in PDAC specimens was analyzed by immunohistochemistry using a tissue microarray (TMA) containing PDAC tumor tissue and corresponding normal tissue samples from 72 patients. The effect of Pfn1 expression on cancer proliferation was assessed in cells by up- and down-regulation of Pfn1 in vivo decreased the tumor volume in orthotopic xenograft nude mice models. Pfn1 upregulated the expression of SIRT3, leading to HIF1α destabilization. This data revealed that aberrant Pfn1 expression contributes to pancreatic cancer progression.Pfn1 was downregulated in clinical pancreatic adenocarcinoma specimens compared with the surrounding benign tissues. Univariate survival analysis of the PDAC cohorts showed that low expression of Pfn1 was significantly correlated with shortened patient survival . Restoration of Pfn1 in pancreatic cancer cells with low endogenous Pfn1 expression resulted in a nontumorigenic phenotype, suggesting that Pfn1 may be a negative regulator of pancreatic cancer progression. Overexpression of Pfn1 Our data suggest that Pfn1 is a tumor suppressor in pancreatic cancer that acts via a novel mechanism of regulating the SIRT3-HIF1α axis, independently of its cytoskeleton-related activity.The online version of this article (doi:10.1186/1476-4598-13-187) contains supplementary material, which is available to authorized users. Pancreatic cancer is a devastating disease, with the highest fatality rate among all cancers and a 5-year overall survival rate of less than 5%. Although some progress has been made in surgery, chemotherapy and radiotherapy in recent decades, the incidence of pancreatic cancer remains equal to its mortality rate. Pancreatic cancer is characterized by uncontrolled growth and rapid progression, and is highly resistant to chemoradiotherapy , 2. DespThe dynamic remodeling of the actin cytoskeleton is involved in multiple cellular functions, such as motility, division, and endocytosis , 4. MoreQuantitative real-time PCR was used to analyze Pfn1 expression profiles of 40 paired pancreatic cancer tissues. Pfn1 expression was downregulated in more than half of the tumors examined (29/40) when compared with their adjacent non-cancerous tissue  cells . The nuclear sirtuins, SIRT1, SIRT6 and SIRT7, regulate numerous metabolic pathways through deacetylating the key transcription factors, cofactors and regulators. The mitochondrial sirtuins, SIRT3, SIRT4 and SIRT5, regulate the activity of important mitochondrial enzymes to maintain energy homeostasis . Among tBy GST pull-down and coimmunoprecipitation assays, we confirmed that Pfn1 interacts specifically with SIRT3. Overexpression of Pfn1 resulted in increased SIRT3 protein levels. We also showed that loss of SIRT3 decreased the ability of Pfn1 to destabilize HIF1α, providing direct evidence of Pfn1’s coupling with SIRT3 signaling in the regulation of HIF1α. Future experiments will determine which motif Pfn1 might act to regulate SIRT3 expression.Pancreatic cancer is characterized by low vascular density and prominent stroma, which is different to most other cancers. This specificity severely affects intratumoral perfusion and causes high levels of hypoxia in pancreatic cancer. In the past decade, it has been revealed that under hypoxic conditions, cancer cells switch their glucose metabolism pathway away from the oxygen-dependent tricarboxylic acid (TCA) cycle to oxygen-independent glycolysis, which is known as “Warberg effect” , 35. ThiGLUT1 and LDHA were inhibited remarkably after Pfn1 upregulation, thereby contributing to glycolytic pathways that are crucial for pancreatic cancer progression.In our study, one of the most interesting findings was the changes in HIF1α target genes involved in the process of glucose metabolism. The expressions of These results highlight how differences in protein expression that leads to distinct outcomes in tumors depend on the expression of collaborating proteins. Thus, the assessment of certain biomarkers for clinical outcomes may require analysis of the interacting proteins that together regulate a biological process. In conclusion, we described a novel regulatory mechanism whereby pancreatic cancer proliferation is inhibited by the interaction of Pfn1 and SIRT3 to destabilize HIF1α, which results in reduced expression of HIF1α target genes that coordinate aerobic glucose consumption.2, Sigma, St. Louis, MI, USA) for the indicated times. All cell culture media contained 100 U/mL penicillin and 100 mg/mL streptomycin.Human pancreatic cancer cell lines MIA PaCa-2, SW1990, BxPC-3, CFPAC-1, and Capan-1 were purchased from American Type Culture Collection. BxPC-3-LN cells with lymph node metastasis properties were established previously in our institute . SW1990-Clinical tissue samples used in this study were diagnosed histopathologically and clinically at Fudan University Shanghai Cancer Center from 2010 to 2012. The histological characterization and clinicopathological staging of the samples were determined according to the current International Union Against Cancer (UICC). For the use of these clinical materials for research purposes, prior patients’ consents and approval from the Institutional Research Ethics Committee were obtained. The samples’ clinical information is presented in Table To overexpress Pfn1 in pancreatic cancer cells, a lentivirus mediated transfection method was employed. In brief, DNA encoding FLAG tagged Pfn1 was cloned into the lentiviral vector pCDH-CMV-MCS-EF1-puro and pCDH-CMV-MCS-EF1-copGFP  to generate the pCDH-Pfn1-puro and pCDH-Pfn1-GFP constructs. Lentiviral particles were generated by co-transfection of pCDH-Pfn1 constructs with psPAX2 and pMD2.G into HEK-293 T cells. Pancreatic cancer cells stably expressing FLAG tagged Pfn1 were obtained by lentiviral infection and puromycin screening or flow cytometry sorting.Cloning vector pLKO.1-TRC (Addgene: 10878) was used to express shRNAs targeting Pfn1 and SIRT3. In brief, oligonucleotides containing 21 bp targeting Pfn1 (5′- GCATGGATCTTCGTACCAAGA -3′) and SIRT3 (5′- CCCAACGTCACTCACTACTTT -3′) were ligated into the pLKO.1-TRC cloning vector to generate the pLKO.1-shPfn1 and pLKO.1-shSIRT3 constructs. pLKO.1-shScr (Addgene:1864) containing a scrambled non-target shRNA was used as the control. To produce lentiviral particles, pLKO.1-shPfn1, pLKO.1-shSIRT3, and pLKO.1-shScr were transfected into HEK-293 T cells with lentiviral packaging vectors psPAX2 and pMD2.G. Lentiviral particles were harvested by collecting media from HEK-293 T cells. Pancreatic cancer cell lines were infected with lentiviral particles, and stable shRNA-expressing cell lines were obtained by screening with puromycin.The Cell Counting Kit-8  was used to measure cell proliferation, as previously described , 40.Total RNA was prepared using The TRIzol reagent . A Takara PrimeScript RT reagent kit was used to obtain cDNA by reverse transcription. Quantitative real-time PCR (qPCR) was used to determine the expression status of candidate genes and GAPDH, using an ABI 7900HT Real-Time PCR system . All reactions were run in triplicate. The primers sets are listed in Additional file Total proteins (20 μg) were separated by polyacrylamide gel electrophoresis (PAGE) and blotted onto polyvinyl-difluoride membranes (Millipore). After blocking with 5% nonfat milk in PBST at room temperature for 30 minutes, membranes were probed with primary antibodies. Antibodies against Pfn1, SIRT3, HIF1α, and Glut1 were all purchased from Epitomics (USA). FLAG, HA, and α-tubulin antibodies were purchased from Sigma (USA). All secondary antibodies were obtained from Jackson ImmunoResearch (USA). After incubation with primary antibodies, washing with PBST and incubating with secondary antibodies, immunoblots were visualized using the ECL detection kit (Millipore) and a ImageQuant LAS 4000 mini was used to quantify the immunoreactive protein bands.Whole cell extracts from HEK-293 T cells transiently expressing FLAG-Pfn1 were subjected to affinity purification with anti-FLAG antibody that was immobilized on agarose beads . The purified protein complexes were resolved on SDS-PAGE silver stained, and the bands retrieved for subsequent mass spectrometry.6 cells in a volume of 50 μl were injected orthotopically into the body of the pancreas. The peritoneum and skin were closed with a 4.0 surgical suture. A bioluminescent imaging method was used to visualize and monitor the dynamic growth of orthotopically xenografted pancreatic cancer cells. For the subcutaneous (s.c.) tumor model, 5 × 106 cells in 100 μl PBS were injected s.c. into the right or bilateral flanks. Twenty-eight days post-implantation, the mice were euthanized and tumors were surgically dissected. The tumor specimens were weighted and fixed in 4% paraformaldehyde. Samples were then processed for histopathological examination.BALB/c-nu mice  were housed in sterile filter-capped cages. Our Institutional Animal Care and Use Committee approved the animal studies. For intrapancreatic injections, mice were anesthetized with 2.5% sodium phenobarbitol (50 mg/kg i.p.) and a 0.5- to 1-cm incision was made in the left subcostal region. 2 × 10t-tests. The chi-square test was used to analyze the relationship between Pfn1 expression and the clinicopathological characteristics. The survival curve was plotted using the Kaplan-Meier method and compared by using the log-rank test. All statistical analyses were carried out using the SPSS 19.0 statistical software package. P values < 0.05 were considered statistically significant.All experiments were carried out at least three times. Data were expressed as the mean ± SD, and the differences between any two groups were compared using (A) Western blot analysis of Pfn1 expression in eight pairs of pancreatic cancer tissues (T) and their corresponding adjacent non-cancerous tissues (ANT). α-tubulin was used as a loading control. (B) Representative micrographs showing negative, weak, intermediate, and strong positive expressions of Pfn1 in pancreatic cancer tissues and adjacent non-cancerous tissues . (TIFF 1 MB)Additional file 1: Figure S1:(A) Construction of MIA PaCa-2 transfectants stably expressing Pfn1 and SW1990 transfectants with shRNAs against Pfn1 using lentivirus infection. (B) Construction of BxPC-3-LN and SW1990-HM transfectants stably expressing Pfn1 using lentivirus infection. (C) IHC staining for proliferation-related proteins  in slices of implanted tumors formed by indicated cells . (TIFF 1 MB)Additional file 2: Figure S2:Additional file 3: Table S1: Pfn1 interacting proteins identified by mass spectrometry. (DOC 114 KB)Additional file 4: Figure S3: A dual-luciferase assay was carried out to determine the influence of increased Pfn1 expression on the HRE promoter in HEK293T cells. (PDF 38 KB)Additional file 5: Figure S4: QPCR analysis of HIF1α expression in indicated cells. (PDF 51 KB)(A) QPCR analysis of HIF1α downstream target genes during glycolysis. (B) Western blot analysis of Glut1 expression in the indicated cells. α-tubulin was used as a loading control. (C) IHC staining for Glut1 expression in slices of implanted tumors formed by the indicated cells . (D) Glucose uptake in Pfn1-WT and -OE MIA PaCa-2 cells. (E) Lactate production in Pfn1-WT and -OE MIA PaCa-2 cells. Error bars represent mean ± SD from three independent experiments. *: p < 0.05. (PDF 129 KB)Additional file 6: Figure S5: Pfn1 downregulates downstream target genes of HIF1α during glycolysis. Additional file 7: Table S2: Sequences of qRT-PCR primers. (DOC 52 KB)


Portugal has high rates of healthcare associated infection and antimicrobial resistance. In February of 2013 the National Program on Prevention and Infection Control and Antimicrobial Resistance (PPCIRA) was restructured. In November of 2013 the PPCIRA determined implementation of Antibiotic Stewardship Programs (ASP) in all healthcare facilities.Implementation of a ASP, with mandatory validation of all the prescriptions of carbapenems and quinolones in the first 96 hours, medical education, carbapenems and quinolones consumption reduction and increase of antibiotic free days.Establishment of Antibiotic Stewardship Group in a 400 bed acute care hospital, consisting of Infectious Diseases specialists, who advise and validate the prescription, and Microbiologists, with consulting function. A database network was built to monitor the prescription of carbapenems and quinolones, with alerts sent via email to the group. This database was linked to a validation system, allowing monitoring, auditing and information to the prescriber. The result of the audit appears in warning messages when opening the prescription (adequate or inadequate). If it’s “inadequate”, the infectious diseases specialists contacts prescribing physician for advice. The ASP began in October 2014 and had the involvement of the institutional leaderships.During the first 3 months of the implementation of ASP the prescriptions decreased 38% and the seeking of advice pre-antibiotic prescription increased. From 2013 to 2014 there was a decrease in the consumption of quinolones from 41,5 to 36,9 DDD/100 patients and of carbapenems from 47,8 to 46,3, respectively. The antibiotic free days increased from 43,5 in 2013 to 50,1 in 2014.Implementation of a ASP in an acute care hospital allowed reduction of consumption of carbapenems and quinolones, as well as an increase of antibiotic free days. Its success is dependent on the involvement of the leadership, coordinated multidisciplinary approaches and education.None declared.


Tomato spotted wilt virus (RB-TSWV) through the graft between an old Apulian  tomato variety, denoted Sl-Ma, used as a rootstock and commercial tomato varieties used as scions. In tests with non-grafted plants, Sl-Ma showed resistance to the RB-TSWV infection as viral RNA accumulated at low levels and plants recovered from disease symptoms by 21 days post inoculation. The resistance trait was transmitted to the otherwise highly susceptible tomato genotypes grafted onto Sl-Ma. The results from the analysis of small RNAs hallmark genes involved in RNA silencing and virus-induced gene silencing suggest that RNA silencing is involved in the resistance showed by Sl-Ma against RB-TSWV and in scions grafted on this rootstock. The results from self-grafted susceptible tomato varieties suggest also that RNA silencing is enhanced by the graft itself. We can foresee interesting practical implications of the approach described in this paper.RNA silencing controls endogenous gene expression and drives defensive reactions against invasive nucleic acids like viruses. In plants, it has been demonstrated that RNA silencing can be transmitted through grafting between scions and silenced rootstocks to attenuate virus and viroid accumulation in the scions. This has been obtained mostly using transgenic plants, which may be a drawback in current agriculture. In the present study, we examined the dynamics of infection of a resistance-breaking strain of  In plants, RNA silencing  drives multiple regulatory and defensive reactions triggered by either endogenous or invasive double-stranded RNAs (dsRNA), which are diced into 21 to 24 –long ribonucleotide fragments by Dicer-like (DCL) endoribonucleases .Potato spindle tuber viroid (PSTVd) moved through the graft junction into a non-transgenic scion where they counteracted a de novo PSTVd infection. Similarly, Ali et al. [NtTOM1 and NtTOM3 genes, required for tobamovirus replication, moved into grafted scions of non transgenic tobacco plants that in turn became resistant to tobamovirus infection through the sRNA-induced silencing of NtTOM1 and NtTOM3. Grafting thus may offer an additional or alternative, ready-to-use and flexible solution to control plant diseases caused by viruses in those cases where resistance (gene) strategies are limited or start failing.It is now well established that in virus-infected plants virus-specific sRNAs, like the virus, also follow the phloem transport and that in grafted plants they can be transmitted from a silenced rootstock to a non-silenced scion and vice-versa to trigger antiviral defense in recipient cells , 9. Withi et al.  reportedTomato spotted wilt virus (TSWV) is the representative of the Tospovirus genus within the arthropod-born Bunyaviridae [Thripidae, Genera Frankliniella and Thrips) [In vitro TSWV NSs protein inhibits cleavage of long dsRNA by Dicer enzymes while in vivo the protein is able to interfere in the miRNA pathway as shown by suppression of miRNA-induced silencing of a GFP (eGFP) miRNA sensor construct [aviridae  and one aviridae . TSWV is Thrips) , which,  Thrips) , 16. The Thrips) . The tos Thrips) . In vitronstruct . These donstruct .R) genes are applicable for commercial resistance breeding against tospoviruses, i.e. Tsw and Sw-5. The first one originates from distinct Capsicum chinense accessions [Tsw has been shown to be triggered by the TSWV RNA silencing suppressor protein NSs [Sw-5 [Solanum peruvianum and S. lycopersicum [Sw-5 dominant gene has been transferred in commercial tomato cultivars, making them resistant to common TSWV strains but not to the Sw-5 resistance-breaking (RB) isolates that emerged recently in several tomato cropping areas  grown in the past for its high tolerance to drought and used mainly for winter consumption in the Apulia region  due to its incredibly long shelf-life. In preliminary screening tests, plants of this tomato variety showed resistance to the infection of an RB isolate of TSWV and recovered from disease symptoms but the underlying mechanism of this resistance, is not yet known. In this study we show that RNAi is involved in the resistance to a Sw-5 RB strain of TSWV and that such resistance can be induced in otherwise highly susceptible tomato scions when grafted onto a non-transgenic tomato rootstock from Sl-Ma. Resistance to TSWV infection seems to be the result of a combination of the ability of the rootstock to activate and hold a strong defense response based on RNAi and the graft itself as some levels of resistance were observed also in self-grafted susceptible genotypes. Since other documented cases of RNAi-mediated resistance to virus infection through grafting involved transgenic rootstocks, this non-transgenic approach presents an attractive practical application to limit the occurrence of RB strains of TSWV in tomato crops. Interestingly resistance to RB strains of TSWV was observed also in (the otherwise susceptible) genotypes of commercial tomato varieties carrying the Sw-5 gene, when grafted onto Sl-Ma.In the framework of a regional project on biodiversity, we rescued seeds of the Manduria tomato cultivar , S. integrifolium, S. nigrum (black nightshade), S. torvum (Turkey berry), S. lycopersicum cv Manduria (Sl-Ma) and S. lycopersicum cv Regina (Sl-Re). Messapico (Sl-Me) and Faino (Sl-Fa) were selected as commercial tomato varieties carrying the Sw-5 gene, while Pullrex (Sl-Pu) and UC82 (Sl-UC) were used as susceptible controls. Upon infection with TSWV-CiPz, an Sw-5 resistance breaking (RB) strain, Solanum nigrum and Sl-UC showed symptoms ranging from severe mosaic to leaf and stem necrosis and plant death, while those of Sl-Ma, Sm-Mo, S. torvum and S. integrifolium mostly consisted of mild mosaic  and 1.8-fold increased root development (1.203 ± 0.575 g from six grafted infected plants versus 0.660 ± 0.34 g from six non grafted plants). Analysis of the viral RNA load in samples of all graft combinations collected at 21 dpi revealed a substantial correlation with the severity of disease symptoms displayed by the scion. Compared to non grafted infected plants, approx 99% reduction in viral RNA accumulation was detected in the Sl-Me scion grafted on Sl-Ma rootstock as well as in all self-grafted plants  produced and purified according to the protocol of Feldhoff et al. [Tomato yellow ring virus (TYRV), grown and purified according the protocol of Hassani-Mehraban et al. [-Ma plants was stronger than the signals obtained with the sRNA probe from Sl-UC plants  was estimated by quantitative real-time PCR (qPCR) in leaves and roots of grafted and non-grafted tomato plants, in TSWV-CiPz-challenged and not challenged conditions. Three groups of plants were analyzed: i) non-grafted plants , ii) plants grafted on Sl-Ma  and iii) self-grafted plants . RNA preparations extracted from leaves or roots of mock-inoculated and infected plants collected at 21 dpi were prepared separately from three biological replicates for each condition tested and a technical replicate was included in the test for each biological replicate. Melting curves of each reaction showed a single peak, demonstrating high specificity for primer pairs, while PCR efficiency ranged from 95 to 103%. From the comparison of plants infected by TSWV-CiPz with respective mock-inoculated controls, analysis of the results from qPCR was focalized on genes with fold change ≥ 2  and in the roots (7-fold) of infected Sl-Ma genotype, compared to mock-inoculated controls.In infected non-grafted plants no significant upregulation was observed for any of the genes, with the exception of AGO1 mRNAs was detected in Sl-UC grafted on Sl-Ma rootstock as well as in self-grafted genotypes Sl-Ma/Sl-Ma (4.7-fold) and Sl-UC/Sl-UC (2.4-fold), compared to uninfected controls. A 2.5-fold increase in transcription of AGO1 was observed also in self-grafted Sl-Me over its non-grafted counterpart, meaning that in this genotype the AGO1 overexpression was due to the graft itself rather than to a response to viral infection. AGO2 was overexpressed (2-fold) only in the self-grafted SL-UC/SL-UC genotype and AGO4 in Sl-UC grafted on Sl-Ma (2.2-fold). A significant overexpression of DCL2 (3.3-fold) was observed in self-grafted Sl-UC and in Sl-UC grafted on Sl-Ma (2.3-fold) in relation to mock-inoculated control plants and non-grafted plants infected by TSWV-CiPz. Finally, an enrichment of DCL4 transcripts (4.8-fold) was detected in the infected leaves of self-grafted Sl-UC genotype, while RDR1 was upregulated significantly in those of self-grafted Sl-Ma (2.4-fold) and Sl-Me (2.5-fold) genotypes infected by TSWV-CiPz.In leaves of grafted plants infected by TSWV-CiPZ, a 5.2-fold increase of AGO1 was over-expressed (6-fold) in self-grafted Sl-UC genotype and transcripts of AGO2 and AGO4 were enriched 3- and 4.5-fold respectively, in Sl-UC grafted on Sl-Ma. No upregulation of the enzymes was observed in self-grafted plants. DCL2 showed a 2-fold overexpression in Sl-Ma and Sl-Me grafted on Sl-Ma, while PAZ was upregulated (3.3-fold) in Sl-Me/Sl-Ma plants and up to 4.4-fold in Sl-Ma self-grafted plants. Increases in PAZ transcription were also observed in Sl-UC/Sl-Ma (4.7-fold) as well as in Sl-UC (2.3-fold) and Sl-Me (2.3-fold) combinations when compared to not-infected controls. Finally, transcription of RDR1 was upregulated in Sl-UC/Sl-Ma (8-fold) and Sl-Me/Sl-Ma (3.5-fold) as well as in self-grafted Sl-Ma (7.3-fold), while the upregulation of RDR6 was observed only in Sl-UC/Sl-Ma (12.5-fold) and in self-grafted Sl-Ma (12-fold) plants infected by TSWV-CiPz.In infected root samples from grafted plants, Tobacco rattle virus (TRV) was used to induce silencing of the tomato endogenous phytoene desaturase (PDS), a gene commonly used as a visual marker in VIGS and resulting in a characteristic photo-bleached phenotype that can be easily monitored in plants [-UC plants photo-bleaching appeared not earlier and between 7 and 10 dpa in the top leaves of all the three plants. In comparison to Sl-Ma  test. To this end, a recombinant viral vector based on n plants , 34. As to Sl-Ma  photo-blto Sl-Ma , which rto Sl-Ma .RDR1 or RDR6 fragments to obtain the pTRV2-RDR1 and pTRV2-RDR6 plasmids, respectively. Plasmids were used for the agro-infiltration of Sl-Ma and Sl-UC genotypes, using three plants for each inoculum. At 14 dpa with pTRV2-RDR1 or pTRV2-RDR6, all plants showed a slightly stunted phenotype in comparison to mock-inoculated controls. The abundance of RDR1 and RDR6 transcripts was estimated by quantitative PCR (qPCR) of reverse-transcribed RNA preparations extracted from samples collected at 14 dpa from three distinct plants of Sl-Ma and Sl-UC as well as from their respective controls. Compared to mock-agroinfiltrated plants the results revealed a partial down-regulation in the expression of both genes in the two genotypes that was not significant  can be obtained in tomato by grafting the otherwise susceptible commercial varieties carrying the Sw-5 gene onto an old tomato variety, denoted Sl-Ma. In tests with non-grafted plants, Sl-Ma resisted the TSWV-CiPz infection by limiting viral RNA accumulation and recovering from mild disease symptoms by 21 dpi. The resistance trait was transmitted to susceptible tomato genotypes grafted onto Sl-Ma suggesting that a mobile signal moving through the graft junction could be implicated. The RNAi seemed a good candidate since: i) its signal travels along the vascular system [Sw-5 resistance gene and is susceptible to resistance-breaking strains of TSWV [Sw-5 is of the gene-for-gene type with Sw-5 triggering a hypersensitive reaction around the TSWV infection foci [The results of our study show that suitable levels of resistance against an r system , iii) mur system –38 and ir system . From thr system , enhance of TSWV . The resion foci  and the ion foci . How sel-Ma plants was higher than that obtained with the sRNA probe from Sl-UC plants. The stronger RNAi response in Sl-Ma than in Sl-UC plants was in good agreement with the 3-fold reduction of viral RNA accumulation in Sl-Ma plants compared to Sl-UC plants. It has been pointed out that a somewhat lower efficiency in the activation of RNAi in the scion could be due to differences in the developmental stage of the plants [PAZ, there was no significant upregulation of the other genes in non-grafted infected plants compared to mock-inoculated controls. On the contrary, more enzymes were clearly upregulated in grafted plants challenged with TSWV-CiPz, and among them differences were observed between leaves and roots. Excluding from the analysis enzymes that were upregulated in leaves and/or roots of the self-grafted genotypes, we can conclude that the overexpression of AGO1, AGO4 and DCL2 in the leaves of Sl-UC grafted on Sl-Ma was very likely in response to viral infection. Similarly, AGO2, AGO4 and PAZ were overexpressed in the roots of Sl-UC/Sl-Ma and DCL2 and PAZ in those of Sl-Me/Sl-Ma, while RDR1 and RDR6 were both overexpressed in the roots of Sl-UC/Sl-Ma and RDR1 also in those of Sl-Me/Sl-Ma. Therefore the increased level of transcription of all of these enzymes in Sl-UC and Sl-Me genotypes when grafted on Sl-Ma seemed to be genuinely due to a viral challenge in the grafted setting  the reduced number of plants transplanted in the field and ii) the major production and better fruit quality of each plant [Altogether, the results presented here provide a potentially useful approach to mitigate economical losses due to infections of ng areas , 16. We ch plant .Tomato spotted wilt virus (TSWV-CiPz) was collected in Apulia  from chicory plants with necrotic symptoms characteristic of tospoviral infection. The virus was transferred and propagated in N. benthamiana, by rubbing leaves with sap obtained from leaf tissues of naturally infected chicory plants ground in 100 mM phosphate buffer, pH 7.2, containing 1 mM sodium sulphite. TSWV-CiPz was characterized for the ability to overcome the resistance gene Sw-5 by mechanical inoculation onto Solanum lycopersicum cv Messapico, a commercial tomato variety carrying the Sw-5 resistance gene but susceptible to TSWV-RB strains [A resistance-breaking isolate of  strains .Solanum spp and local ecotypes of tomato and eggplant were provided by an Apulian nursery Plant and tested for susceptibility/resistance to TSWV-CiPz: Solanum melongena cv molfettese (Sm-Mo), S. integrifolium, S. nigrum (black nightshade), S. torvum (Turkey Berry), S. lycopersicum cv manduria (Sl-Ma) and S. lycopersicum cv regina (Sl-Re). The commercial tomato varieties Messapico (Sl-Me) and Faino (Sl-Fa), carrying the Sw-5 gene and the susceptible Pullrex (Sl-Pu) and UC82 (Sl-UC) varieties were used in different graft combinations, including self-grafting. Grafting was carried out when the tomato seedlings had two to four true-leaves and the stems were 1.5 to 3 millimeters in diameter. To grow different cultivars to the same developmental stage, sowing time was adjusted according to the germination time required from each plant species/variety. The graft, also denoted top grafting [N. benthamiana plants infected systemically with TSWV-CiPz. All the plants were grown and maintained in a temperature-controlled glasshouse at 24±2°C with 16 h photoperiod and monitored daily for symptom appearance.The following wild grafting , was mad2 of DIG Easy Hyb Granules solution  containing 100 ng/ml of DIG-labeled RNA probe of TSWV M RNA synthesized as described previously [Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as housekeeping gene for normalization [Samples for dot blot analysis were collected from inoculated and mock-inoculated plants at 7, 14, 21, 28 days post inoculation (dpi), unless reported otherwise and ground in the presence of 6 vol (w/v) of 50 mM NaOH, 2.5 mM EDTA. The extract was incubated at room temperature for 5 min, then 5 μl were spotted onto a positively charged nylon membrane  and nucleic acids cross-linked for 5 min under UV light. Membranes were hybridized overnight at 58°C in 150 μl/cmeviously . After hlization .For tissue-print hybridization, sections of roots, stems and leaves of infected and mock-inoculated tomato plants were collected at 19 dpi and cut surfaces gently pressed onto positively charged nylon membrane  pre-wetted with 50 mM NaOH, 2.5 mM EDTA and air-dried for 5 min before printing. Viral RNA distribution and accumulation was estimated by hybridization and densitogram analysis as described above.AGO1, AGO2, AGO4, DCL1, DCL2, DCL4, PAZ, RDR1, RDR6 transcripts was estimated by reverse-transcription real-time quantitative PCR (qPCR) using the comparative cycle threshold (2-ΔΔCt) method corrected for PCR efficiencies [DCL2, DCL4, PAZ, RDR1 and RDR6 gene expression, while β-Tubulin (TUB) was used for the normalization of AGO1, AGO2 AGO4 and DCL1. First-strand cDNA was synthesized from 1 μg of total RNA preparations extracted from samples collected from grafted and non-grafted genotypes at 21 dpi with TSWV-CiPz or buffer (mock-inoculated controls). High Capacity cDNA Reverse Transcription kit  and 10 pmol random hexamers were used, following the protocol of the manufacturer. qPCR was set up in 10 μl of 2X Fast SYBR Green PCR Master Mix (Applied Biosystems), containing 100 ng of first strand cDNA template, and 200 nM each of the forward and reverse primer pairs listed in AGO1, AGO2 AGO4, DCL1, DCL2, DCL4, PAZ, RDR1, RDR6 with that of the respective endogenous mRNA. The experiment was repeated twice and statistical significance of the RQ values was assessed by factorial analysis of variance (ANOVA)  with Tukey post-hoc test (P ≤ 0.05), for each target gene, separately.Relative abundance of ciencies . GAPDH wA. tumefaciens cultures containing recombinant plasmids pTRV1 and pTRV2-PDS derived from Tobacco rattle virus (TRV) were prepared and co-agroinfiltrated in cotyledons of the selected tomato genotypes as described below. For the construction of pTRV2-RDR derivatives, specific primers were designed on conserved regions of RDR1 (GeneBank Acc. AK321474.1) and RDR6 (GeneBank Acc. AP009260.1) . Electroporated cells were plated on Luria-Bertani (LB) medium, and selected with standard ampicillin and white/blue colony screening. After sequencing to confirm their identity, RDR1 and RDR6 clones were used to generate recombinant vectors for VIGS as described by Liu and colleagues [attB primer pairs for BP clonase. The cDNA fragments were subsequently transferred to the destination vector pTRV2 by LR clonase and used to transform E. coli DH5α competent cells. Vectors pTRV2-RDR1 and pTRV2-RDR6 were electroporated into competent cells of A. tumefaciens GV3101, positive clones were identified by PCR and used for agroinfiltration.Two 09260.1) . First-s-columns , cloned lleagues , using t4, containing antibiotics 25 μg·ml-1 rifampicin, 50 μg·ml-1 kanamycin). Cell culture was collected by centrifugation at 3000 g for 20 min, washed once with cold 10 mM MgCl2, resuspended in infiltration medium  to an optical density at 600 nm of 1.5 and incubated at 28°C for 4 h. pTRV1and pTRV2-derivatives were mixed with a 1:1 ratio and used to infiltrate lower epidermis of tomato cotyledons using a 2.5 ml syringe.Cultures of pTRV1 and pTRV2-derivatives  collected from individual colonies were grown overnight at 28°C in 5 ml of LB3 medium  in the presence of random primer hexamer mix and then amplified with 2 min denaturation at 95°C followed by 40 cycles of 5 sec denaturation at 95°C, 15 sec annealing and synthesis at 60°C in SensiFAST SYBR-HiROX mix  containing 20 μM primers.Silencing of endogenous 32P) ATP by T4 polynucleotide kinase, according to the manufacturer’s instructions . TSWV-CiPz RNA was extracted from virus particles purified from systemically infected leaves of N. rustica according to the protocol of Feldhoff et al. [Tomato yellow ring virus (TYRV) was prepared from virus particles purified from leaves of N. benthamiana following the protocol of Hassani-Mehraban et al., [® reagent  and resuspended in RNase-free water. Equimolar amounts (0.5 μg) of TSWV-CiPz and TYRV RNA were resolved on 1% agarose gel in 0.5X TBE , blotted to Hybond-N membrane  by a TurboBlotter™ s  and cross-linked by UV light. One μg of total nucleic acid and 1 μg of sRNA preparations were also run on the gel and used as positive controls. Four replicates of filters were, respectively, hybridized overnight at 48°C with the Sl-Ma and Sl-UC γ-32P-labeled sRNAs purified from healthy and infected tomato plants in 360 mM Na2HPO4, 140 mM NaH2PO4, containing 7% (w/v) SDS and 1 mM EDTA. After hybridization, membranes were washed three times at 48°C with 2X SSC , followed by three washes in 2X SSC, containing 0.2% SDS. To detect hybridization signals, membranes were exposed to a phosphor screen and visualized by phosphoimaging .Total nucleic acid was extracted from 1 g of leaves collected from Sl-Ma and Sl-UC tomato plants mock-inoculated and at 21 dpi with TSWV-CiPz, according to the method of Bucher et al. . Low-molf et al.  while RN


Sub-Sahara Africa hosts up to 71 % of all HIV infected people in the world. With this high incidence of Human immunodeficiency virus ( HIV) comes the burden of co-morbidities such as malignant and premalignant lesions. Aids defining malignancies have been listed as Kaposi’s sarcoma, Non-Hodgkin’s lymphoma and invasive squamous cell carcinoma of the cervix. People with HIV/AIDS(PLWAS) have a higher risk of developing these neoplasms than the rest of the population. The pathogenesis of these neoplasms in people with HIV has been linked to immune suppression, persistent antigenic stimulation and cytokine dysregulation.Current study analyzes and presents the patterns and trends in the presentation of HIV related malignancies in patients diagnosed through histopathology at Kenyatta National Hospital.To describe the patterns of AIDS- defining and non-AIDS- defining malignancies and premalignant lesions 10 years pre- and post HAART period at Kenyatta National hospital, Kenya.This was a hospital based descriptive cross sectional study. The Formalin fixed paraffin embedded (FFPE) blocks and histological reports of patients diagnosed between 2000 and 2011 were traced from archives. The patients’ demographic data and clinical presentation was entered in an excel spreadsheet and the diagnosis and coding confirmed by a histopathologist. The data was then cleaned and analyzed using SSPS version 17.0 Ink.A total of 173 lesions were reviewed and analyzed. Of these 118 (68 %) were from females and 55 from males (32 %). The male to female ratio was 1:2. The age range was from two to 56 years with a median of 36 years. Kaposi sarcoma is the leading AIDS defining malignancy in Kenya while invasive squamous cell carcinoma of the conjunctiva is the leading non-AIDS defining malignancy. This is closely followed by invasive squamous cell carcinoma of the cervix and NHL.Kaposi sarcoma is the leading AIDS associated neoplasm in Kenya. Physicians and caretakers managing and following up on HIV/AIDS patients should look out for Kaposi sarcoma as a form of IRIS following the institution of HAART in all HIV/AIDS patients. The incidence of invasive squamous cell carcinoma of the conjunctiva is increasing in PLWAS in Kenya. There is therefore a need to introduce early screening programs for squamous intraepithelial neoplasm of the conjunctiva in HIV/AIDS patients. Sub-Saharan Africa hosts up to 71 % of all adults and children living with HIV, 70 % of adults and children newly infected, and 75 % of adults and children dying from AIDS . AIDS deHaving obtained approval from the KNH/UON ethics and research committee. The data clerk at the AIDS and Cancer Specimen Resource-Kenya (ACSR K) retrieved all the reports indicated to be HIV positive on the histological request forms between 2000 and 2011. This was done by checking for clinical information on the form indicating p24 marker positive (p24 is a marker of HIV antigenicity in serum), retroviral disease, if patient was on ARV or HAART treatment, or patient on follow-up at the comprehensive care clinic (CCC). Using the histological reports, blocks for the respective cases were then retrieved from the routine block storage and archived. 4 The criteria for inclusion as HIV positive case included information from the clinician in the patient’s histological request form indicating follow up at the CCC, HAART treatment, ARV treatment, immunosuppression, p24 marker reactive, retrovirus disease, and HIV positive.The patients’ demographic and clinical data from the histological request form and partly from the hospital medical records were entered into Microsoft Excel 2010. Ink spreadsheet. The data was cleaned and analyzed using SPSS statistics 17.0 Ink.A total of 173 lesions were reviewed and analyzed by the study histopathologists . Of these 118 (68 %) were females and 55 males 32 %). The male to female ratio was 1:2. The age range was from 2 to 56 years with a median of 36 years . The female gender is at a higher risk of acquiring HIV infection due to many factors such as the anatomy and the immunity of the reproductive system and socio-economic factors . Over thThe data in this study reveals a progressive increase in the number and variety of malignancies and premalignant lesions diagnosed since the introduction of HAART in 2003. The trend is such that Kaposi’s sarcoma and squamous cell carcinoma of the cervix and vulva predominated between 2000 and 2003, the pre HAART period in Kenya. Kaposi’s sarcoma is still leading, but incidence of squamous cell carcinoma of the conjunctiva is surpassing that of the cervix and vulva. Later in 2003, majority of HIV patients started receiving ARV from MEDS . Following this, in the same year, the Kenya government announced the inception of HAART treatment through public hospitals which were implemented through funding by GLOBAL and PEPFAR funds in 2004 . Over thReports from Kampala, Uganda show that HIV related Kaposi’s sarcoma affects mainly the younger and middle age groups, and the median age in 1990s being 32.0 years and 27.1 years for men and women respectively. This distribution is similar to the median age of AIDS cases recorded for the country in 1996 of 34 years and 30 years in men and women respectively . KS can Invasive squamous cell carcinoma of the cervix is classified as one of AIDS defining malignancies. The occurrence of this neoplasm in HIV represents a progression to AIDS. The neoplasm is directly linked to HPV, a virus that has been shown to transform squamous cells through the E6 and E7 proteins. These proteins act via the cyclin dependent kinases E/CDK2 to phosphorylate and inactivate RB gene. Proposals have been put forth suggesting that HIV may upregulate E6 and E7 through Tat . WhereasIn general aggressive B cell lymphomas are one of the nightmares in management of HIV/AIDS. These neoplasms are usually non-responsive to routine protocols. Diffuse large B cell lymphomas form the largest percentage followed by Burkitt lymphoma. Non Hodgkin’s lymphoma put together accounted for 4.6 % of all the neoplasms and premalignant lesions in this study. In the United States, the relative risk of NHL in subjects with AIDS is 100 or more and much higher than the relative risk in an African series reporting that shows Rwanda leads at 13   and SoutBurkitt lymphoma in sub-Saharan Africa is mainly a childhood disease accounting for up to 82 % of all pediatric and adolescent lymphomas in the region . HIV relRecent data reported from several hospitals in East Africa reveal that Hodgkin’s Lymphoma is a disease of children and adolescents , the onlAlthough Burkitt lymphoma is one of the well reported high grade lymphomas seen in HIV/AIDS, the cases reported in this study were very few. Of note is that during the period under review, the main diagnostic method for Burkitt lymphoma was largely through FNAC, and may account for the low incidence of archived tissue of BL in this series.Kaposi sarcoma is the leading AIDS associated neoplasm in Kenya. The incidence of invasive squamous cell carcinoma of the conjunctiva is increasing in PLWAS in Kenya. There is therefore a need to introduce early screening programs for squamous intraepithelial neoplasm of the conjunctiva in HIV/AIDS patients.


In this study, we identified the mRNA expression pattern of Th2 and regulatory T-cell (Treg) cytokines in IgG4-RD and in IgG4-associated MZL and IgG4-negative MZL using real-time polymerase chain reaction analysis. Ocular IgG4-RD and IgG4-associated MZL exhibited significantly higher expression ratios of interleukin (IL)-4/β-actin, IL-10/β-actin, IL-13/β-actin, transforming growth factor (TGF) β1/β-actin, and FOXP3/β-actin than did IgG4-negative MZL (p < 0.05). This finding further supports our prior observations that a significant subset of ocular MZLs arises in the setting of IgG4-RD. Furthermore, the presence of a different inflammatory background in IgG4-negative MZLs suggests that IgG4-associated MZLs may have a different pathogenesis.We previously suggested a relationship between ocular immunoglobulin (Ig)G4-related disease (IgG4-RD) and marginal zone lymphomas (MZLs). However, the cytokine background associated with these disorders and whether it differs between ocular adnexal MZLs with (IgG4-associated MZL) and without (IgG4-negative MZL) numerous IgG4 IgG4-RD can affect almost any organ, including the pancreas, hepatobiliary duct, lacrimal and salivary glands, lung, kidney, retroperitoneum, aorta, and lymph nodes12345678910Immunoglobulin (Ig)G4-related disease (IgG4-RD) is a systemic syndrome characterized by dense lymphoplasmacytic infiltrates that are rich in IgG4Recent reports have described upregulation of T-helper-2 cells (Th2) and regulatory T-cell (Treg) cytokines in tissues with IgG4-RD, suggesting that the immune reaction mediated by these cytokines is responsible for the lesions121415+ plasma cells that fulfilled the histological diagnostic criteria for IgG4-RD; therefore, we suggested that MZLs can arise in a background of IgG4-RD+ plasma cells has not been clarified.Previously, we reported cases of ocular adnexal MZLs with numerous IgG4+ plasma cells, we aimed to identify the mRNA expression patterns of Th2 and Treg cytokines and to determine the inflammatory background associated with benign and neoplastic ocular lymphoplasmacytic proliferations with numerous IgG4+ plasma cells that is distinct from that associated with ocular IgG4-negative MZL.In this study of ocular IgG4-RD and MZLs with (IgG4-associated MZL) and without (IgG4-negative MZL) numerous IgG4Formalin-fixed excisional biopsies from the ocular adnexal region of patients were selected, including 11 patients with IgG4-RD, 11 with IgG4-negative MZL, and 6 with IgG4-associated MZL . All MZLThe following evaluations were carried out in accordance with the approved guidelines. All experimental protocols were approved by the Institutional Review Board at Okayama University. Total RNA was extracted from the paraffin-embedded sections of all samples using an miRNeasy FFPE Kit . cDNA was prepared using a SuperScript VILO MasterMix kit . Multiplex real-time PCR was performed for quantitative analysis, according to a standard protocol, using TaqMan Gene Expression Assays , a Step One Plus Real-Time PCR System (Applied Biosystems), and specific primers and probes for FOXP3 (Hs01085834_m1), transforming growth factor (TGF)β1 (Hs00998133_m1), interleukin (IL)-4 (Hs00174122_m1), IL-5 (Hs00174200_m1), IL-10 (Hs00961622_m1), IL-13 (Hs00174379_m1), and β-actin (Hs99999903_m1) (Applied Biosystems). The PCR cycling conditions were as follows: 20 s at 95 °C, 50 cycles of 1 s at 95 °C, and 20 s at 60 °C. The expression of each target was normalized to that of β-actin, which was used as an endogenous control.+ and IgG+ cells were estimated in areas with the highest density of IgG4+ cells. In accordance with the consensus statement on the pathological features of IgG4-RD+ cells per HPF and the IgG4+/IgG+ cell ratio. In situ hybridization was also performed for κ and λ-light chains (Leica Biosystems) using a Bond Max stainer.Serial sections (4 μm) were cut from the block of paraffin-embedded tissue, stained with hematoxylin and eosin, and used for the following immunohistochemical stains: CD20 , CD3 , CD5 , CD10 , CyclinD1 , Ki-67 , IgG , and IgG4 . Following immunostaining using an automated Bond Max stainer , the numbers of IgG4PCR molecular genetic analysis for immunoglobulin heavy chain gene rearrangements was performed as previously described18191819U-test with SPSS software .All statistical analyses were performed using the Mann–Whitney + cells/HPF, and the IgG4+/IgG+ cell ratio was >40% . All cases revealed few, if any, IgG4+ cells, and the IgG4+/IgG+ cell ratio was <40%.The 11 IgG4-negative MZLs showed dense infiltration by small- to medium-sized CD20dex <10% . Fibrosi+ cells, and fibrosis was present in 3 cases  . Based on the distribution of the IgG4+ cells and non-dominant light chain staining, the IgG4+ cells appeared to be polytypic. In all 6 cases, >100 IgG4+ cells/HPF were detected; therefore, these cases fulfilled the histological diagnostic criteria for IgG4-RD. Furthermore, in 5 cases, the IgG4+/IgG+ cell ratio was >40%. In case 28, the IgG4+/IgG+ cell ratio was not estimated because the lymphoma cells present were IgG positive, and the much fewer IgG4+ cells appeared to reflect a polytypic IgG+ population that could not be quantitated  , Fig. 3.ntitated .Serum IgG4 and IgG levels were determined in 7 patients with ocular IgG4-RD, in 2 patients with IgG4-negative MZL, and in 4 patients with IgG4-associated MZL . Serum IThe expressions of IL-4, IL-5, IL-10, IL-13, TGFβ1, and β-actin in the samples were quantitatively analyzed using real-time PCR. Ocular IgG4-RD and the IgG4-associated MZL samples exhibited significantly higher expression ratios of IL-4/β-actin, IL-10/β-actin, IL-13/β-actin, and TGFβ1/β-actin than did the IgG4-negative MZL samples (p < 0.05) . No signThe ocular IgG4-RD and IgG4-associated MZL biopsies exhibited significantly higher expression ratios of FOXP3/β-actin than did the IgG4-negative MZL specimens (p < 0.05) .+, whereas an ocular MZL was the only non-cutaneous IgG4+ MZL found in the same studyThe Th1/Th2 balance is considered important for healthy immune responses, and a Th1/Th2 imbalance can cause immune-mediated disease. Th1-dominant immune responses are noted in patients with rheumatoid arthritis, type 1 diabetes, and multiple sclerosis, whereas Th2-dominant immune responses are observed in patients with type 1 hypersensitivity disorders, such as allergies or asthma22242514+ plasma cells that were polytypic and not part of the neoplasm. An explanation for this difference remains to be established, and the heavy chain expressed by the ocular lymphomas is uncertain.Recently, high levels of Th2 cytokines (IL-4 and IL-13) and Treg cytokines (TGFβ1 and IL-10) have been detected in tissues with IgG4-RDChlamydia psittaci infection is associated with the development of ocular adnexal MZL2829+ cells, suggesting that they arose from IgG4-RD+ plasma cells. Because the two lesions were adjacent to each other, the MZL may have arisen in the setting of the IgG4-RD lesion. These reports and the results of the present study suggest that a subset of ocular MZLs may arise in the setting of IgG4-RD.The mechanism for the development of ocular adnexal MZL is not clear. However, recent reports indicated that Patients with IgG4-RD are considered to be at high risk of MZLs as well as other malignancies, with malignancy considered a possible complication of IgG4-RD. Lung and colon cancers, as well as malignant lymphomas, are reported to occur in 10.4% of patients with IgG4-RD, an incidence that is approximately 3.5 times higher than that in the general population+ cells in IgG4-RD+ Tregs may infiltrate lesions associated with IgG4-RD and further suppress tumor immunity, also promoting the growth of neoplasms.In the current study, upregulation of FOXP3 was detected in cases of IgG4-RD and IgG4-associated MZL lesions. This is consistent with an earlier report describing the presence of numerous FOXP3Although steroid therapy is effective in IgG4-RD, the most effective treatment for MZLs arising in the setting of IgG4-RD is uncertain. Although evaluation of additional cases of IgG4-associated MZLs is needed to determine the treatment and prognosis, radiation or chemotherapy may be necessary. Therefore, when IgG4-RD is diagnosed, the lesion should be assessed to determine if it includes an MZL component. The possible tumor-promoting environment of IgG4-RD supports treatment for these patients before an MZL arises, in addition to ongoing follow-up.IgG4-associated MZL is characterized by the upregulation of Th2 and regulatory cytokines, as is the case in IgG4-RD, but unlike the cytokine background of IgG4-negative MZLs. The current results further suggest that a subset of MZLs may arise in an IgG4-RD setting and that this subset of MZLs may have a different pathogenesis than IgG4-negative MZLs.How to cite this article: Ohno, K. et al. A subset of ocular adnexal marginal zone lymphomas may arise in association with IgG4-related disease. Sci. Rep.5, 13539; doi: 10.1038/srep13539 (2015).


IgG4-related disease is a relatively novel clinical entity whose gastrointestinal manifestations include type 1 autoimmune pancreatitis (AIP) and IgG4-associated sclerosing cholangitis. The presence of elevated serum IgG4 is suggestive but not essential for the diagnosis of type 1 AIP and is a pervasive feature of the proposed diagnostic criteria. The differential diagnosis of type 1 AIP includes malignant conditions, emphasizing the importance of a deliberate, comprehensive evaluation. Management of patients with a suggestive clinical presentation, but without serum IgG4 elevation, is difficult. Here we present three cases of IgG4-seronegative AIP and sclerosing cholangitis that responded to empiric steroid therapy and discuss approach considerations. These cases demonstrate the value of meticulous application of existing diagnostic algorithms to achieve a clinical diagnosis and avoid surgical intervention. Type 1 autoimmune pancreatitis (AIP) and immunoglobulin G4- (IgG4-) associated sclerosing cholangitis (IgG4-SC) are pancreatic and biliary manifestations of IgG4-related disease, respectively. Recently, IgG4-related disease has emerged as a novel clinical entity characterized by multiorgan infiltration of IgG4-positive cells, storiform fibrosis, and elevated serum IgG4 levels. Several diagnostic criteria have been proposed in the last decade based on radiology, histopathology, serology, and response to steroids . HoweverCurrent clinical diagnostic criteria for type 1 AIP/IgG4-SC involve elevated serum IgG4 concentrations (>135 mg/dL). The reported sensitivity of increased serum IgG4 levels in AIP ranges from 68% to 81% [An 80-year-old Caucasian man with a history of resected prostate adenocarcinoma and hyperlipidemia presented with complaints of pruritus, anorexia, fatigue, 20 pound weight loss, and painless jaundice. He denied abdominal pain, melena, or hematochezia. His vital signs were within normal limits and physical examination revealed only jaundice. There was no history of significant alcohol use.μ/L, and lipase 54 μ/L. His platelet count was 258 and International Normalized Ratio (INR) 0.9. Carbohydrate antigen (CA) 19-9 was 175 U/mL and carcinoembryonic antigen (CEA) level 1.1 ng/mL. Total IgG was 1,550 mg/dL and IgG4 levels were 51.8 (reference range 2.4–121). Other serologic tests included anti-neutrophil antibody (ANA), anti-smooth muscle antibody (ASMA), and anti-mitochondrial antibody (AMA), all of which were negative. A computed tomography (CT) of the abdomen demonstrated diffuse intrahepatic biliary ductal dilatation. Magnetic resonance imaging (MRI) revealed an area of enhancement concerning for neoplasm at the biliary confluence, narrowing of the central right and left intrahepatic bile ducts and intrahepatic biliary duct dilation  299 IU/L, aspartate aminotransferase (AST) 143 IU/L, amylase 70 dilation . DiffuseSix months after initial presentation, a repeat MRI was obtained which demonstrated a new 2.7 cm mass at the posterior aspect of the head of the pancreas extending into the uncinate process . An endoAfter a thorough negative workup for malignancy, based on the available clinical, radiological, and extrapancreatic presentation, a diagnosis of type 1 AIP and IgG4-SC was suspected. The patient was started on 40 mg of prednisone and continued for 4 weeks and gradually tapered. His symptoms abated and a follow-up MRI performed demonstrated complete resolution of the pancreatic mass and biliary strictures Figures . With thA 45-year-old gentleman with prior history of small duct primary sclerosing cholangitis and Crohn's disease presented with symptoms of abdominal pain, pruritus, dark urine, and jaundice. Laboratory analysis showed an AST of 152, ALT of 158, alkaline phosphatase of 531, total bilirubin of 5.6, direct bilirubin of 3.8, total protein of 7.4, albumin of 4.0, lipase of 157, amylase of 82, CA of 19-9 194, CEA of 2.9, total IgG of 1620, and IgG4 of 53.3. MRI of the abdomen demonstrated diffuse enlargement of the body and the tail of the pancreas. No focal enhancing pancreatic mass was identified. MRCP revealed new irregular narrowing of the common hepatic duct, upper common bile duct beaded dilatation, and narrowing of the intrahepatic bile ducts Figures . SeveralAn ERCP was performed and revealed mild intrahepatic biliary strictures and moderately severe common bile duct stricture, most consistent with a type 2 cholangiogram . SphinctA 52-year-old gentleman presented to the gastroenterology clinic with a history of acute pancreatitis and complaints of abdominal pain. He denied abnormal weight loss or alcohol use. Laboratory analysis showed an amylase of 495, lipase of 1305, bilirubin of 2.8, AST of 658, ALT of 533, and alkaline phosphatase of 65. He was diagnosed with acute pancreatitis and treated conservatively. Abdominal MR imaging revealed segmental biliary dilatation from the bifurcation proximally to the right and left system with CBD stricture. The common hepatic duct was 10 mm and the CBD distally was 2.5 mm. The pancreatic duct was unremarkable. Additionally, there was significant retroperitoneal adenopathy and diffuse pulmonary nodules on the imaging.An ERCP was performed and revealed a 2.5 cm high-grade stricture in the distal common bile duct, consistent with type 1 cholangiogram [IgG4-related disease is a multiorgan fibroinflammatory disorder characterized by organ infiltration with lymphoplasmacytic cells rich in IgG4, storiform fibrosis, obliterative phlebitis, elevated serum IgG4 levels, and response to steroids. Two types of AIP have been described in the literature. Type 1 AIP is related to IgG4-related disease, while type 2 (idiopathic duct centric pancreatitis) is associated with a distinct characteristic histological pattern and is usually not accompanied by extrapancreatic manifestations . Type 1 In our current series, we describe three patients with type 1 AIP and IgG4-SC with normal serum IgG4 levels in whom steroid treatment was successful and surgery was entirely avoided. Each of these cases provides lessons in the diagnosis of IgG4-SC in patients with normal IgG4 levels. In Case 1, cholangiocarcinoma was suspected on the basis of clinical findings and compatible radiologic findings on CT, MR, and ERCP. However, a thorough and measured approach to diagnosis led to four ERCPs with biliary brushings and FISH, which were negative for malignancy. This allowed the patient to defer needless surgery. Follow-up MRI after six months revealed a new pancreatic mass which would not be expected with cholangiocarcinoma, and pulmonary nodules and mediastinal lymphadenopathy were seen on CT imaging, suggesting extrapancreatic involvement. When an EUS with FNA of the pancreatic mass did not reveal malignancy, but rather a chronic stromal infiltrate, the combination of pathologic findings and extrapancreatic involvement led to a trial of prednisone. With dramatic clinical and radiologic response to steroids, the diagnosis of type 1 AIP was made and surgery was avoided.In Case 2, the patient presented with abdominal pain and jaundice. Imaging showed diffuse enlargement of the pancreas, as well as biliary strictures and pulmonary nodules. Serological tests were negative for IgG4, and histology and FISH were negative from ERCP brushings. Based on clinical and radiological presentation, a diagnosis of AIP was considered and empiric treatment was initiated with steroids. The patient responded well, with resolution of pancreatic enlargement and biliary strictures on imaging.In our 3rd case, the patient presented with a diagnosis of acute pancreatitis of unknown etiology. His abdominal imaging, however, revealed biliary strictures which raised the question of an uncommon cause of pancreatitis. By carefully pursuing this clinical clue, misdiagnosis was avoided. Two ERCPS with biliary brushings and FISH were negative for malignancy, and EUS demonstrated pancreatic masses in the head and the body of the pancreas, biopsies of which were also negative for malignancy. The subsequent detection of an enlarged submandibular gland allowed a diagnostic biopsy that demonstrated chronic inflammatory infiltrate with numerous IgG4 positive plasma cells. This patient also was treated successfully with steroids, and continues on Imuran with no relapse.The diagnosis of AIP is challenging and a number of diagnostic criteria  have been proposed in the recent years. The HISORt criteria proposed by the Mayo Clinic to diagnose AIP include histology, imaging findings, elevated serum IgG4 levels, other organs involvement, and response to steroids . In ordeThe ICDC criteria can still be successfully applied in cases of seronegative IgG4-associated AIP and IgG4-SC. In Case 1, MRI pancreatography findings were indeterminate for AIP. A thorough evaluation for possible malignancy was negative. Pancreatic ductal imaging was normal. ERCP revealed level 1 findings, but serology was negative and no additional organ involvement (OOI) was noted. Two level 1 criteria were not met, so EUS-guided pancreatic biopsy was performed and was inconclusive. Because of the presence of a single level 1 OOI, steroid trial was pursued and resulted in radiographic and symptomatic resolution, supporting a diagnosis of type 1 AIP. In Case 2, initial MR pancreatography was typical for type 1 AIP. There was a single level 1 OOI criterion ; thus, a steroid trial was pursued and resulted in substantial improvement, confirming the diagnosis. In Case 3, neither pancreatic parenchymal imaging nor ductal imaging was typical for AIP. However, level 1 OOI findings were seen on ERCP. A pancreatic biopsy was thus performed and yielded inconclusive findings. At this point, a steroid trial would have been reasonable; however, the interval development of submandibular gland enlargement allowed for establishment of definitive histological diagnosis.The typical histological findings in type 1 AIP are comprised of abundant infiltration of lymphocytes, IgG4 positive plasma cells (greater than 10 IgG4 positive cells per high power field), fibrosis in periductal and interlobular areas, and obliterative phlebitis . Tissue Increased serum IgG4 levels >135 mg/dL) have been frequently described in patients with type 1 AIP and have been included as a part of diagnostic criteria. Hamano and colleagues first described an association between elevated IgG4 and AIP in 2001 and reported the sensitivity and specificity of elevated IgG4 for AIP as 95 and 97%, respectively . However5 mg/dL hIt has also been suggested that IgG4-seronegative AIP may behave as a different clinicopathologic entity. When compared to IgG4-seropositive patients, those with negative serology are more likely to be female, present with abdominal pain or acute pancreatitis, and demonstrate segmental pancreatic body and/or tail enlargement , 16. TheIn Cases 1 and 3, pancreatic imaging was not suggestive of typical features for AIP, while cholangiography suggested the presence of IgG4-SC. While AIP and IgG4-SC often coexist, it is important to recognize that they are likely distinct, if closely related, manifestations of the single overarching clinical entity of IgG4-related disease. Several studies have reported histologically proven IAC  in the absence of any clinical or radiographic evidence of AIP –20. As wThe diagnosis of isolated IgG4-SC is challenging and IgG4 serology alone is inadequate to establish the diagnosis, as has been previously discussed. The sensitivity of IgG4 for IgG4-SC diagnosis has been estimated as 74%, a level comparable to estimates in AIP . SeveralAlthough our goal is early identification and treatment of benign IgG4-related disease and avoidance of surgery, it should be emphasized that the misdiagnosis of AIP/IgG4-SC in the setting of pancreatic cancer or cholangiocarcinoma must be strictly avoided . In a reType 1 AIP/IgG4-SC typically responds dramatically to steroids. In our series we treated all patients with prednisone 40 mg daily for 4 weeks and tapered by 5 mg/week with close clinical follow-up, liver function tests, and imaging studies. In addition, all patients required biliary drainage procedures. Even though disease relapse is common with type 1 AIP, (30%–50%) none of our patients had clinical or biochemical relapse , 26. HowIn summary, our case series highlights the benefit of early recognition and medical treatment of IgG4-negative type 1 AIP and IgG4-SC. Despite normal IgG4 levels, a diagnosis of IgG4-related AIP and IgG4-SC should be considered when there is a typical clinical presentation, the presence of characteristic pancreatobiliary imaging findings, and extrapancreatic involvement. When IgG4-related AIP is recognized without elevation of IgG4, the patient can be spared morbidity associated with unnecessary surgical treatment by initiating a steroid trial and monitoring patients carefully.


Antibiotic abuse can lead to antibiotic resistance, which is a severe problem in China. The purpose of this study is to evaluate the short-term effects of antimicrobial stewardship strategies, including formulary restriction, preauthorization, perioperative quinolone restriction, and control of total antibiotic consumption in the ICU at a tertiary hospital in China. After implementation of antimicrobial stewardship, the total antibiotic consumption in the ICU significantly decreased. The defined daily doses (DDDs) per 100 patient-days decreased from 197.65 to 143.41; however, the consumption of cephalosporins increased from 53.65 to 63.17 DDDs. Significant improvements in resistance to amikacin, gentamicin, ciprofloxacin, ofloxacin, ceftriaxone, ceftazidime, and piperacillin in Enterobacteriaceae and resistance to ceftazidime, imipenem, and meropenem in non-fermenting Gram-negative rods were observed. In addition, the initial use of no antibiotics or of a single antibiotic significantly increased (P<0.001) and the use of two antibiotics in combination significantly decreased (P<0.001). Our results demonstrate that implementation of antimicrobial stewardship in a short period in the ICU dramatically reduced antibiotic consumption and significantly improved antibiotic resistance, which leads to more reasonable antibiotic selections by ICU physicians. Since antibiotics were first applied in clinical settings, more varieties have emerged, resulting in the recovery of numerous critically infected patients The Chinese government is aware of the harm caused by antibiotic overuse and has developed national policies to regulate its use. From April to August in 2011, according to the policy of the Ministry of Health of China and the Bureau of Health of Shandong Province, all public hospitals in Shandong province began to follow strict antimicrobial stewardship strategies. It has been reported that antimicrobial stewardship is an efficient way to improve antibiotic resistance and to reduce patient cost by optimizing antimicrobial prescriptions This study was conducted with the permission and supervision of the Ethics Committee of the Affiliated Hospital of Taishan Medical College, Taishan, Shandong Province, China. Oral consent was obtained from patients and documented in our research notebook for use of their clinical records in this study. Written consent was not obtained because of illiteracy problem of many patients. The ethics committee mentioned above approved the oral consent protocol. All patient records/information was anonymized prior to analysis.The Affiliated Hospital of Taishan Medical College is a tertiary teaching hospital with 700 beds. The ICU of our hospital is comprehensive with 12 beds and annually handles 550 patients. From April to August in 2011, antimicrobial stewardship strategies were put into practice, including formulary restrictions, preauthorization requirements, perioperative quinolone restriction and control of total antibiotic consumption. The specific strategies and classification standards are described in the flow-chart in To evaluate the effects of antimicrobial stewardship in antibiotic consumption, data on antibiotic resistance, clinical outcomes, demographic and clinical data of patients, antibacterial use density (AUD), antibiotic resistance level, and initial antibiotic selection were collected and compared before and after the implementation of antimicrobial stewardship.Demographic and clinical data included age, gender, APACHE II score, primary (infectious or non-infectious) disease when admitted to the ICU, the application of antibiotics within the 30 days prior to the transfer, the application of glucocorticoid therapy within the 30 days prior to the transfer, whether the patient accepted the treatment of mechanical ventilation in the ICU, days of treatment with mechanical ventilation, length of ICU stay, death, and whether the death was correlated with infection. Previous application of glucocorticoid therapy referred to the application of glucocorticoids, oral or intravenous, within 30 days, excluding aerosol inhalation.Antibiotic consumption was calculated based on the WHO standards Data on antibiotic use before admission to the ICU were collected, including intravenous and oral antibiotic application 30 days before admission to the ICU, use, antibiotic varieties and how many varieties used. Statistics on the initial antibiotic use after admission to the ICU were also collected, including whether used, the antibiotic varieties used, and whether monotherapy or combination therapy was used.2 analysis. Continuous variables are presented as the mean ± standard deviation and were compared using one-way ANOVA.Data were analyzed using SPSS 11.0. All tests were two-sided, and significance was defined as a P-value≤0.05. Categorical variables are presented as a percentage and were compared using χThere was no significant difference in sex ratio, age, APACHE II score, primary disease, proportion of infectious diseases, and prior glucocorticoid use between, before, and after the implementation of antimicrobial stewardship. No significant differences in ICU length of stay, the number of days on mechanical ventilation, mortality, or infection-related mortality was observed between these two groups. However, we found a higher proportion of no antibiotic therapy or single antibiotic therapy in the ‘after’ period, which may be explained by the hospital-wide implementation of antimicrobial stewardship strategies .Our studies demonstrated a significant decrease in the hospital-wide total antimicrobial consumption . Concomitantly, the ICU total antibiotic consumption also decreased significantly after implementation of the antimicrobial stewardship strategies. The DDDs per 100 patient-days decreased from 197.65 to 143.41, which is a decrease of 27.44%. The consumption of quinolones, imidazoles, other β-lactams, and macrolides decreased significantly. Particularly, the DDDs of quinolone per 100 patient-days decreased sharply from 49.82 to 7.74. The DDDs of glycopeptide and carbapenem did not decrease but increased slightly. Accompanied by the increase in the consumption of cephalosporins, the DDDs per 100 patient-days increased from 53.65 to 63.17, which is an increase of 17.74% .In the ICU, antibiotic resistance for Gram-negative bacilli improved significantly after the implementation of antimicrobial stewardship strategies. The percentage of Enterobacteriaceae isolates with resistance to amikacin, gentamicin, ciprofloxacin, ofloxacin, ceftriaxone, ceftazidime and piperacillin noticeably decreased. In addition, the percentage of isolate resistance to ceftazidime, imipenem and meropenem also decreased for non-fermenting Gram-negative rods. The percentage of isolates with resistant Gram-positive cocci did not change significantly .2 = 3.339, P = 0.297), whereas in the ‘after’ period, the initial selection between the two groups was significantly different , with a higher proportion of no antibiotic and single antibiotic use  and a lower proportion of two antibiotic use (13.2% vs 31.8%) in the non-infectious group , and the initial selection of two antibiotics were significantly lower (16.1% vs 74.1%). In the ‘before’ period, between the infectious and the non-infectious group, there was no significant difference in initial selection concerning whether to use and how many varieties used  were higher than for other departments, the Antimicrobial Stewardship Committee gave a monthly statistical analysis report to the director of the ICU, who was required to improve antibiotic control with the goal of progressively lowered use, especially when the AUD was higher. Such administrative pressure would ideally be transmitted to each ICU physician. ICU physicians would then have to implement the antimicrobial stewardship strategies strictly, so that the increase of alternative antibiotic consumption and related rise in resistance would not occur. In addition, at the same time, hospital-wide, regional and even national implementations of antimicrobial stewardship strategies are much more likely to have been the key to the positive results achieved. The majority of the ICU patients were transferred from other departments or other hospitals. Patients transferred from so-called infection departments that have heavy antibiotic use and high resistance rates accounted for 45.54% of individuals with infectious disease patients accounting for 37%. These patients might already have resistant strains before they are transferred to the ICU; therefore, if antimicrobial stewardship strategies are only implemented in the ICU, and not included in other departments, the effect of the implementation in the ICU would be seriously affected.In this study, there was a significant difference (P = 0.011) in the ‘before’ and ‘after’ periods of antibiotic use before admission to the ICU . There wPseudomonas aeruginosa resistance to imipenem. By limiting the use of quinolone antibiotics, P. aeruginosa resistance to imipenem and meropenem can be reduced in addition to decreasing the second-generation carbapenem antibiotic selection pressure P. aeruginosa to quinolones and carbapenems, such as a loss or decrease in levels of the outer membrane porin protein, OprD, or the overexpression of the MexAB-OprM efflux system. Such mechanisms may explain why an improvement of quinolone resistance in P. aeruginosa was accompanied by an improvement of carbapenem resistance We noted that compared with the ‘before’ period, the consumption of carbapenems did not decline; instead, an increase was seen in their use. Interestingly, there was a decline in the resistance to imipenem and meropenem for non-fermenting Gram-negative rods. This phenomenon may be associated with strict quinolone prescription regulations and the rigid decrease of quinolone use. Many studies have demonstrated that excessive quinolone use is an independent risk factor of We noted that, in addition to the decrease of antibacterial use density and the improvement of resistance, the implementation of antimicrobial stewardship also had impacts on the antibiotic selections of ICU physicians There are some limitations to this study. This was a passive intervention study with only a single-center and a small sample size. Encouragingly, 33 tertiary hospitals, including ours, have formed a collaborative network for monitoring ICU antimicrobial resistance. The effect of the implementation of antimicrobial stewardship strategies is now being analyzed under the assessment of this collaboration. In addition, the present study only investigated the short-term effects of antimicrobial stewardship but the long-term effects should also be considered in future studies.In summary, implementation of antimicrobial stewardship strategies, including formulary restriction, preauthorization, restriction of perioperative antimicrobial prophylaxis, and control of total antibiotic consumption, significantly reduced antibiotic use in a relatively short period. The resistance of Gram-negative bacteria to some antibiotics was improved to some extent. Simultaneously, the selection of antibiotics in the ICU became more reasonable. Abuse of antibiotics and resulting antibiotic resistance has become a major clinical and public health problem in China. Our results further support the national implementation of antimicrobial stewardship strategies; however, the long-term effects of these strategies require continuous tracking, monitoring and assessment.Appendix S1Antimicrobial Stewardship.(DOCX)Click here for additional data file.Appendix S2Antimicrobial Classification.(DOCX)Click here for additional data file.


The standard treatment pathway for locally advanced rectal cancer is neoadjuvant chemoradiotherapy (CRT) followed by surgery. Neoadjuvant CRT has been shown to decrease physical fitness, and this decrease is associated with increased post-operative morbidity. Exercise training can stimulate skeletal muscle adaptations such as increased mitochondrial content and improved oxygen uptake capacity, both of which are contributors to physical fitness. The aims of the EMPOWER trial are to assess the effects of neoadjuvant CRT and an in-hospital exercise training programme on physical fitness, health-related quality of life (HRQoL), and physical activity levels, as well as post-operative morbidity and cancer staging.The EMPOWER Trial is a randomised controlled trial with a planned recruitment of 46 patients with locally advanced rectal cancer and who are undergoing neoadjuvant CRT and surgery. Following completion of the neoadjuvant CRT (week 0) prior to surgery, patients are randomised to an in-hospital exercise training programme  or a usual care control group . The primary endpoint is oxygen uptake at lactate threshold . Sponsor: University Hospital Southampton NHS Foundation Trust.ClinicalTrials.gov  Chemfunction . Furtherfunction . Neoadjufunction . This defunction . Moreovefunction . In thisfunction .CPET is a good pre-operative risk assessment tool as it provides an objective global assessment of the integrative responses of the pulmonary, cardiovascular and musculoskeletal systems rather than evaluating the function of individual organ systems in isolation . FurtherExercise training can stimulate skeletal muscle adaptations such as increased mitochondrial content and improved oxygen uptake capacity , both ofIn this manuscript, we describe the design of a parallel group randomised controlled multi-centre trial comparing an in-hospital exercise training programme to a usual care control group in patients undergoing neoadjuvant CRT and elective surgery for locally advanced rectal cancer. To the best of our knowledge, the EMPOWER trial is the first RCT of such an intervention in a neoadjuvant setting.The aims of this study are to evaluate the following hypotheses:-1.min−1) assessed using CPET, following neoadjuvant CRT prior to elective cancer surgery.A 9-week, in-hospital, exercise training programme compared with a usual care control group  will result in a clinically significant difference in oxygen uptake at lactate threshold  will result in an improvement in psychological health benefits as assessed using semi-structured interviews and questionnaires such as the EORTC QLQ-30 and EQ-5D following neoadjuvant CRT prior to cancer surgery.Neoadjuvant CRT will result in a reduction in A 9-week in-hospital exercise training programme compared with a usual care control group  will be associated with 1) greater physical activity, assessed using SenseWear accelerometer, following neoadjuvant CRT prior to cancer surgery; 2) lower day 5 surgical morbidity (using POMS); and 3) improved Magnetic Resonance (MR)-defined local rectal cancer staging assessed using the TNM classification system  and tumour regression grading (mrTRG).The EMPOWER Trial is a parallel group randomised controlled trial in locally advanced rectal cancer patients undergoing both neoadjuvant CRT and elective surgery. The trial is funded by the National Institute for Health Research - Research for Patient Benefit Programme (PB-PG-0711-25093), approved by North West Centre for Research Ethics Committees (13/NW/0259) and registered with clinicaltrials.gov (NCT01914068).Five NHS hospitals are currently recruiting to the trial: the University Hospital Southampton (UHS) NHS Foundation Trust, Aintree University Hospitals NHS Foundation Trust, Royal Hampshire County Hospital, South Tees Hospitals NHS Foundation Trust and Royal Bournemouth Christchurch Hospitals.Eligibility criteria for inclusion at cancer diagnosis include the following: age ≥ 18 years, with MR-defined, locally advanced , resectable rectal cancer (≥ T2N + M0), undergoing standardised neoadjuvant CRT; with no distant radiological defined metastasis. Exclusion criteria include the following: inability to give informed consent, non-resectable disease, distant metastasis, inability to perform CPET or bicycle exercise, and patients who declined surgery or neoadjuvant CRT or who received non-standard neoadjuvant CRT. Figure The EMPOWER trial is currently recruiting . All potentially eligible patients are being identified at multi-disciplinary meetings and approached with written information about the trial at the oncology/surgical outpatient appointment. Patients are contacted by telephone to provide additional information about the trial and to confirm their eligibility. If the patient chooses to participate in this trial, the first research visit is organised where written informed consent is obtained and all baseline measurements are undertaken. Following neoadjuvant CRT (week 0), patients are allocated to the in-hospital exercise training group or usual care control group. Patients are randomised (1:1) to either an in-hospital exercise training programme or usual care control group using the Trans European Network for patient randomisation in clinical trials system (TENAELA System).The usual care control group  receive routine care throughout their cancer pathway from diagnosis to surgical resection. No specific advice about exercise training is offered.The exercise training programme was designed to improve physical fitness in the time interval between end of neoadjuvant CRT and surgery. Exercise training begins on the first week following completion of neoadjuvant CRT, and the patients exercise in pairs for camaraderie. The exercise training programme is described below.The delivery of the exercise training programme is described using the FITT principle ) detailed below.Patients are requested to attend three in-hospital exercise training sessions per week for 6 to 9 weeks .The exercise training is an aerobic interval exercise training programme incorporating moderate and severe intensities. Exercise training intensities are derived from each individual CPET derived at Week 0 (immediately post-CRT). Moderate-intensity exercise is at a power output equivalent to 80 % of the oxygen uptake at lactate threshold  see Fig. .Fig. 2ExAll patients undergo a series of outcome measure assessments throughout the period of the trial: baseline (pre-neoadjuvant CRT), mid-neoadjuvant CRT and at several time points following completion of the neoadjuvant CRT  prior to surgery . In UHS only, based on the clinical pathway, some patients are treated with neoadjuvant chemotherapy (four cycles) followed by neoadjuvant CRT. In this instance, an additional research visit is scheduled halfway between the completion of chemotherapy and commencing CRT). The outcome measures used in the EMPOWER trial are summarised in Table CPET is used to assess physical fitness over a series of time points : baseline (pre-neoadjuvant CRT); mid chemotherapy-CRT (in UHS only); and post-neoadjuvant CRT (week 0), week 3, week 6 and week 9. All CPETs are performed in the hospital by trained and experienced staff. Every effort is made to coordinate research visits with other clinical appointments. Each CPET is conducted at a similar time of day. Participants are asked to refrain from caffeine and strenuous exercise prior to the test. All CPETs are performed using an electromagnetically-braked cycle ergometer (Ergoline 2000), a 12-lead ECG, non-invasive blood pressure measurement and pulse oximetry, and a metabolic cart . The incremental rise in work rate is pre-determined using the equation derived by Wasserman and colleagues  in whichEach participant receive instructions on how to rate the Borg Scale of perceived exertion , which is a subjective rating of breathlessness and for leg fatigue (assessed every 2 minutes during the test). Additionally, blood pressure is recorded every 2 minutes during the test. Saddle height is measured and recorded at the first CPET and remains constant for all other CPETs. Once the patient is comfortable on the bike, the mask is fitted. The patients are asked to perform an incremental ramp test to the limit of tolerance and to maintain a cycling cadence at 55–65 revolutions per minute (rpm) throughout the test. CPET allows for the derivation of lactate threshold using the modified V-Slope method , 24. Theexercise . The intexercise . Two indHRQoL is assessed in two ways: 1) Semi-structured interviews are conducted at week 0 (following neoadjuvant CRT) and at weeks 6 to 9 (prior to surgery) to explore the patients’ perceptions of quality of life, which allows the patients to focus on personal accounts of their HRQoL within a specific context . 2) HRQoL questionnaires are administered over several time points during the study: baseline (pre-neoadjuvant CRT), mid-chemotherapy CRT (in UHS only), mid-neoadjuvant CRT, post-neoadjuvant CRT (week 0), week 3, week 6, week 9 and 30 days post-surgery. The two questionnaires administered at these time points are the EORTC QLQ-30 and EQ-5D. EORTC QLQ-30 is specific to cancer patients and is validated to assess generic aspects of HRQoL. EQ-5D is a simple descriptive profile and a single index value for health status.Daily physical activity is measured over three consecutive days and nights at a series of time points: baseline (pre-neoadjuvant CRT), mid-chemotherapy CRT (in UHS only), post-neoadjuvant CRT (week 0), week 3, week 6 and week 9. Physical activity is measured using a multi-sensory accelerometer ; BodyMedia, Inc., Pittsburgh, PA, USA)), which records daily movements, including total energy expenditure, physical activity duration, lying-down time, active energy expenditure, number of steps, sleep duration, total energy expenditure, degree of physical activity, average metabolic equivalent threshold (MET), duration of sensor on body, sleep efficiency, and physical activity level .Post-operative outcomes will be objectively recorded using POMS on days 3, 5, 8 and 15. The POMS is a validated 18-item tool that addresses nine domains of morbidity relevant to the post-surgical patient: pulmonary, infection, renal, gastrointestinal, cardiovascular, neurological, wound complications, haematological and pain , 27. ForCancer down-staging will be assessed using the TNM classification. Rectal cancer staging will be assessed using an abdominal-pelvic CT scan and pelvic MRI scan pre-neoadjuvant CRT and at 9 weeks following completion of the neoadjuvant CRT. The TNM classification of malignant tumours is a cancer staging notation system that gives codes to describe the stage of a solid tumour .All adverse events are recorded in the relevant case report form. Adverse events during CPET are reported to the chief investigator, and adverse effects during exercise training (pain and muscle soreness) are recorded in the relevant case report form by the research physiologist/nurse. Fatal or life-threatening serious adverse events (SAEs) are reported within 24 hours of the local site becoming aware of the event. The SAE form documents the nature of the event, date of onset, severity, corrective therapies given, outcome and causality . Questions concerning adverse event reporting are directed to the chief investigator in the first instance.-1.min−1−1·min−1 and is inflated to allow for 20 % patient drop-out [A sample of 46 patients is required to detect a difference between groups of 2.0 mL.kgdrop-out . This esData will be double data entered, and data validation will take place according to the procedures set out in the data management plan and data validation plan. Prior to any statistical analysis, all variables will be checked for the number of missing values, impossible values and improbable values. Impossible and improbable values will be defined by clinical opinion. Improbable values will also include values that are outside three standard deviations of the mean value. Any questions regarding the data will go back to the data manager. Descriptive statistics will be calculated for all variables, and distributional assumptions will be checked.Descriptive analyses will be carried out to summarise patient characteristics. Continuous variables will be reported as mean (range), mean (SD) or median and inter-quartile range (IQR), depending on distribution, and categorical variables as frequency (%). The effect of neoadjuvant CRT and the exercise intervention on physical fitness will be assessed using a two-sample t-test, and a 95 % confidence interval on the mean difference between the two groups will be presented. In order to assess the sensitivity of results to covariates, a further ‘adjusted’ effect will be calculated using linear regression where up to three candidate covariates will be identified . Basic eThe Standard Protocol Items-Recommendations for Interventional Trials (SPIRIT) table provides an overview of the study conduct, review, reporting and interpretation and is presented in Table The cancer treatment pathway for locally advanced rectal cancer carries some risk. Neoadjuvant CRT and surgery are associated with side effects and post-operative complications , 31. ThePre-operative physical fitness is associated with post-operative outcome, with the less fit patients having a greater prevalence of adverse outcomes. Neoadjuvant CRT decreases physical fitness in this patient group, and this decrease is associated with increased post-operative complications . The EMPStrengths of the EMPOWER trial include the study design with parallel group 1:1 randomisation; the two independent physiological data assessors who are blind to group allocation and independent of the intervention; and that the multi-disciplinary team caring for the patients are not provided with any information regarding predictive measures  ensuring a low risk of confounding by indication . Also, pIn summary, the EMPOWER trial will investigate changes in objectively measured physical fitness and HRQoL with a structured pre-operative exercise training programme. Furthermore, it will also explore changes in physical activity with exercise and any impact on post-operative outcome measures. Additionally, it may inform the development of generalisable exercise programmes for major surgical patients who are identified as high risk  following cancer treatment.The trial started patient enrolment in August 2013 and is expected to be completed by the end of December 2015.