A short-oligonucleotide microarray that allows improved detection of gastrointestinal tract microbial communities

Background  

The human gastrointestinal (GI) tract contains a diverse collection of bacteria, most of which are unculturable by conventional microbiological methods. Increasingly molecular profiling techniques are being employed to examine this complex microbial community. The purpose of this study was to develop a microarray technique based on 16S ribosomal gene sequences for rapidly monitoring the microbial population of the GI tract.     

Testing Stem Cell Therapy in a Rat Model of Inflammatory Bowel Disease: Role of Bone Marrow Stem Cells and Stem Cell Factor in Mucosal Regeneration

Background

The gastrointestinal (GI) mucosal cells turnover regularly under physiological conditions, which may be stimulated in various pathological situations including inflammation. Local epithelial stem cells appear to play a major role in such mucosal renewal or pathological regeneration. Less is clear about the involvement of multipotent stem cells from blood in GI repair. We attempted to explore a role of bone marrow mesenchymal stromal cells (BMMSCs) and soluble stem cell factor (SCF) in GI mucosa regeneration in a rat model of inflammatory bowel diseases (IBD).

Methods

BMMSCs labelled with the fluorescent dye PKH26 from donor rats were transfused into rats suffering indomethacin-induced GI injury. Experimental effects by BMMSCs transplant and SCF were determined by morphometry of intestinal mucosa, double labeling of PKH26 positive BMMSCs with endogenous proliferative and intestinal cell markers, and western blot and PCR analyses of the above molecular markers in the recipient rats relative to controls.

Results

PKH26 positive BMMSCs were found in the recipient mucosa, partially colocalizing with the proliferating cell nuclear antigen (PCNA), Lgr5, Musashi-1 and ephrin-B3. mRNA and protein levels of PCNA, Lgr5, Musashi-1 and ephrin-B3 were elevated in the intestine in BMMSCs-treated rats, most prominent in the BMMSCs-SCF co-treatment group. The mucosal layer and the crypt layer of the small intestine were thicker in BMMSCs-treated rats, more evident in the BMMSCs-SCF co-treatment group.

Conclusion

BMMSCs and SCF participate in but may play a synergistic role in mucosal cell regeneration following experimentally induced intestinal injury. Bone marrow stem cell therapy and SCF administration may be of therapeutic value in IBD.     

Analysis of a post-translational steroid induction system for GIGANTEA in Arabidopsis

Background  

To investigate the link between the flowering time gene GIGANTEA (GI) and downstream genes, an inducible GI system was developed in Arabidopsis thaliana L. Heynh. Transgenic Arabidopsis plant lines were generated with a steroid-inducible post-translational control system for GI. The gene expression construct consisted of the coding region of the GI protein fused to that of the ligand binding domain of the rat glucocorticoid receptor (GR). This fusion gene was expressed from the constitutive cauliflower mosaic virus 35S promoter and was introduced into plants carrying the gi-2 mutation. Application of the steroid dexamethasone (DEX) was expected to result in activation of the GI-GR protein and its relocation from the cytoplasm to the nucleus.     

Number and type of guideline implementation tools varies by guideline,clinical condition,country of origin,and type of developer organization: content analysis of guidelines

Background

Guideline implementation tools (GI tools) can improve clinician behavior and patient outcomes. Analyses of guidelines published before 2010 found that many did not offer GI tools. Since 2010 standards, frameworks and instructions for GI tools have emerged. This study analyzed the number and types of GI tools offered by guidelines published in 2010 or later.

Methods

Content analysis and a published GI tool framework were used to categorize GI tools by condition, country, and type of organization. English-language guidelines on arthritis, asthma, colorectal cancer, depression, diabetes, heart failure, and stroke management were identified in the National Guideline Clearinghouse. Screening and data extraction were in triplicate. Findings were reported with summary statistics.

Results

Eighty-five (67.5%) of 126 eligible guidelines published between 2010 and 2017 offered one or more of a total of 464 GI tools. The mean number of GI tools per guideline was 5.5 (median 4.0, range 1 to 28) and increased over time. The majority of GI tools were for clinicians (239, 51.5%), few were for patients (113, 24.4%), and fewer still were to support implementation (66, 14.3%) or evaluation (46, 9.9%). Most clinician GI tools were guideline summaries (116, 48.5%), and most patient GI tools were condition-specific information (92, 81.4%). Government agencies (patient 23.5%, clinician 28.9%, implementation 24.1%, evaluation 23.5%) and developers in the UK (patient 18.5%, clinician 25.2%, implementation 27.2%, evaluation 29.1%) were more likely to generate guidelines that offered all four types of GI tools. Professional societies were more likely to generate guidelines that included clinician GI tools.

Conclusions

Many guidelines do not include any GI tools, or a variety of GI tools for different stakeholders that may be more likely to prompt guideline uptake (point-of-care forms or checklists for clinicians, decision-making or self-management tools for patients, implementation and evaluation tools for managers and policy-makers). While this may vary by country and type of organization, and suggests that developers could improve the range of GI tools they develop, further research is needed to identify determinants and potential solutions. Research is also needed to examine the cost-effectiveness of various types of GI tools so that developers know where to direct their efforts and scarce resources.

     

ACC deaminase‐containing plant growth‐promoting rhizobacteria protect Papaver somniferum from downy mildew

Aims

Analysis of the stability and safety of Sulfolobus monocaudavirus 1 (SMV1) during passage through the mammalian GI tract.

Methods and Results

A major challenge of using nano‐vectors to target gut microbiome is their survival during passage through the extremely acidic and proteolytic environment of the mammalian GI tract. Here, we investigated the thermo‐acidophilic archaeal virus SMV1 as a candidate therapeutic nano‐vector for the distal mammalian GI tract microbiome. We investigated the anatomical distribution, vector stability and immunogenicity of this virus following oral ingestion in mice and compared these traits to the more classically used Inovirus vector M13KE. We found that SMV1 particles were highly stable under both simulated GI tract conditions (in vitro) and in mice (in vivo). Moreover, SMV1 could not be detected in tissues outside the GI tract and it elicited a nearly undetectable inflammatory response. Finally, we used human intestinal organoids (HIOs) to show that labelled SMV1 did not invade or otherwise perturb the human GI tract epithelium.

Conclusion

Sulfolobus monocaudavirus 1 appeared stable and safe during passage though the mammalian GI tract.

Significance and Impact of the Study

This is the first study evaluating an archaeal virus as a potential therapeutic nanoparticle delivery system and it opens new possibilities for future development of novel nanoplatforms.     

Fecal Protease Activity Is Associated with Compositional Alterations in the Intestinal Microbiota

Objective

Intestinal proteases carry out a variety of functions in the gastrointestinal (GI) tract. Studies have reported that elevated enteric proteases in patients with GI disease can alter intestinal physiology, however the origin (human vs. microbial) of elevated proteases in patients with GI disease is unclear.

Aim

The aim of this study was to investigate the association between protease activity and the microbiota in human fecal samples.

Design

In order to capture a wide range of fecal protease (FP) activity stool samples were collected from 30 IBS patients and 24 healthy controls. The intestinal microbiota was characterized using 454 high throughput pyro-sequencing of the 16S rRNA gene. The composition and diversity of microbial communities were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. FP activity levels were determined using an ELISA-based method. FP activity was ranked and top and bottom quartiles (n=13 per quartile) were identified as having high and low FP activity, respectively.

Results

The overall diversity of the intestinal microbiota displayed significant clustering separation (p = 0.001) between samples with high vs. low FP activity. The Lactobacillales, Lachnospiraceae, and Streptococcaceae groups were positively associated with FP activity across the entire study population, whilst the Ruminococcaceae family and an unclassified Coriobacteriales family were negatively associated with FP activity.

Conclusions

These data demonstrate significant associations between specific intestinal bacterial groups and fecal protease activity and provide a basis for further causative studies investigating the role of enteric microbes and GI diseases.     

Extranodal natural killer/T-cell lymphoma involving the gastrointestinal tract: analysis of clinical features and outcomes from the Asia Lymphoma study group

Background

The gastrointestinal (GI) tract is one of the most common extranasal sites in extranodal NK/T-cell lymphoma (ENKTL). However, data regarding ENKTL involving the GI tract are relatively scarce. Thus, we performed a multicenter, multinational retrospective study to analyze clinical features and treatment outcomes of ENKTL involving the GI tract.

Patients and methods

Patients with ENKTL involving the GI tract diagnosed in twelve participating centers between 1991 and 2012 were retrospectively analyzed from five Asian countries.

Results

The analysis of 81 patients with ENKTL involving the GI tract revealed that more than 60% of patients presented as advanced disease with B symptoms. 55 patients (68%) had GI manifestations including abdominal pain (n = 26, 32%), GI tract bleeding (n = 17, 21%) and bowel perforation (n = 12, 15%). The most common GI site was the small intestine, including the jejunum and ileum (n = 57, 70.3%). There were 34 patients (42%) who received systemic chemotherapy while 33 patients (41%) underwent surgery plus chemotherapy. However, 35 patients (43%) died due to disease progression, and treatment-related mortality including sepsis occurred in 17 patients (21%). Thus, the median overall survival was 7.8 months (95% Confidence interval: 3.9 – 11.7 months). Patients who could undergo surgery plus chemotherapy showed a trend of better survival than those treated with chemotherapy alone.

Conclusion

Overall, the data indicated that ENKTL involving the GI tract has a dismal prognosis despite active treatment including chemotherapy and surgery. Thus, more effective treatment strategies are required for this disease entity.

     

Hospital Physicians' Influence on Gastrointestinal Protection during Treatment with Non-Steroidal Anti-Inflammatory Drugs and Acetylsalicylic Acid and the Impact on Prescribing in Primary Care

Background

The aim of this study was to describe the use of gastrointestinal (GI) protection before, during and after hospitalisation for elderly patients using NSAID or low-dose ASA.

Methods

This study included all elderly patients (75+) admitted to hospital in the period of 1^st April 2010 to 31^st March 2011 at Odense University Hospital, Denmark, who were regular users of NSAID or low-dose ASA before hospital admission, or had one of these drugs initiated during hospital stay. By using pharmacy dispensing data and a hospital-based pharmacoepidemiological database, the treatment strategy for the individual patients was followed across hospital stay.

Results

In total, 3,587 patients were included. Before hospital admission, 93 of 245 NSAID users (38.0%) and 597 of 1994 user of low-dose ASA (29.9%) had used GI protection. During hospital stay, use of GI protection increased to 75% and 33.9%, respectively. When hospital physicians initiated new treatment with NSAID or with low-dose ASA, 305 of 555 (55.0%) and 647 of 961 (67.3%) were initiated without concomitant use of GI protection. When hospital physicians initiated GI protection, 26.8–51.0% were continued in primary care after discharge.

Conclusions

During hospital stay, the use of GI protection increases, but when new treatment with NSAIDs or low-dose ASA is initiated in hospital, the use of gastrointestinal protection is low. The low use of GI protection is carried on in primary care after discharge.     

Sequence analysis of percent G+C fraction libraries of human faecal bacterial DNA reveals a high number of Actinobacteria

Background  

The human gastrointestinal (GI) tract microbiota is characterised by an abundance of uncultured bacteria most often assigned in phyla Firmicutes and Bacteroidetes. Diversity of this microbiota, even though approached with culture independent techniques in several studies, still requires more elucidation. The main purpose of this work was to study whether the genomic percent guanine and cytosine (%G+C) -based profiling and fractioning prior to 16S rRNA gene sequence analysis reveal higher microbiota diversity, especially with high G+C bacteria suggested to be underrepresented in previous studies.     

Impact of a Glaucoma Severity Index on Results of Trabectome Surgery: Larger Pressure Reduction in More Severe Glaucoma

Purpose

To stratify outcomes of trabectome-mediated ab interno trabeculectomy (AIT) by glaucoma severity using a simple and clinically useful glaucoma index. Based on prior data of trabectome after failed trabeculectomy, we hypothesized that more severe glaucoma might have a relatively more reduced facility compared to mild glaucoma and respond with a larger IOP reduction to trabecular meshwork ablation.

Methods

Patients with primary open angle glaucoma who had undergone AIT without any other same session surgery and without any second eye surgery during the following 12 months were analyzed. Eyes of patients that had less than 12 months follow up or were diagnosed with neovascular glaucoma were excluded. A glaucoma index (GI) was created to capture glaucoma severity based on visual field, number of preoperative medications, and preoperative IOP. Visual field (VF) was separated into 3 categories: mild, moderate, and advanced (assigned 1, 2, and 3 points, respectively). Preoperative number of medications (meds) was divided into 4 categories: ≤1, 2, 3 or ≥4, and assigned with a value of 1 to 4. Baseline IOP (IOP) was divided into 3 categories: <20 mmHg, 20–29 mmHg, and greater than 30 mmHg and assigned with 1 to 3 points. GI was defined as IOP × meds × VF and separated into 4 groups: <6 (Group 1), 6–12 (Group 2), >12–18 (Group 3) and >18 (Group 4). Linear regression was used to determine if there was an association between GI group and IOP reduction after one year or age, gender, race, diagnosis, cup to disc (C/D) ratio, and Shaffer grade.

Results

Out of 1340 patients, 843 were included in the analysis. The GI group distribution was GI1 = 164, GI2 = 202, GI3 = 260, and GI4 = 216. Mean IOP reduction after one year was 4.0±5.4, 6.4±5.8, 9.0±7.6, 12.0±8.0 mmHg for GI groups 1 to 4, respectively. Linear regression showed that IOP reduction was associated with GI group after adjusting for age, gender, race, diagnosis, cup to disc ratio, and Shaffer grade. Each GI group increase of 1 was associated with incremental IOP reductions of 2.95±0.29 mmHg. Success rate at 12 months was 90%, 77%, 77%, and 71% for GI groups 1 to 4. The log-rank test suggested significant differences between GI groups.

Conclusion

A simple glaucoma index, GI, was created to capture glaucoma severity and a relative resistance to treatment. A higher GI was associated with a larger IOP reduction in trabectome surgery. This indicates that there is a role for AIT beyond mild glaucoma and ocular hypertension.     

A High Dietary Glycemic Index Increases Total Mortality in a Mediterranean Population at High Cardiovascular Risk

Objective

Different types of carbohydrates have diverse glycemic response, thus glycemic index (GI) and glycemic load (GL) are used to assess this variation. The impact of dietary GI and GL in all-cause mortality is unknown. The objective of this study was to estimate the association between dietary GI and GL and risk of all-cause mortality in the PREDIMED study.

Material and Methods

The PREDIMED study is a randomized nutritional intervention trial for primary cardiovascular prevention based on community-dwelling men and women at high risk of cardiovascular disease. Dietary information was collected at baseline and yearly using a validated 137-item food frequency questionnaire (FFQ). We assigned GI values of each item by a 5-step methodology, using the International Tables of GI and GL Values. Deaths were ascertained through contact with families and general practitioners, review of medical records and consultation of the National Death Index. Cox regression models were used to estimate multivariable-adjusted hazard ratios (HR) and their 95% CI for mortality, according to quartiles of energy-adjusted dietary GI/GL. To assess repeated measures of exposure, we updated GI and GL intakes from the yearly FFQs and used Cox models with time-dependent exposures.

Results

We followed 3,583 non-diabetic subjects (4.7 years of follow-up, 123 deaths). As compared to participants in the lowest quartile of baseline dietary GI, those in the highest quartile showed an increased risk of all-cause mortality [HR = 2.15 (95% CI: 1.15–4.04); P for trend  = 0.012]. In the repeated-measures analyses using as exposure the yearly updated information on GI, we observed a similar association. Dietary GL was associated with all-cause mortality only when subjects were younger than 75 years.

Conclusions

High dietary GI was positively associated with all-cause mortality in elderly population at high cardiovascular risk.     

Comprehensive molecular profiling of the B7 family in gastrointestinal cancer

Objectives

B7 family has been identified as co‐stimulatory or co‐inhibitory molecules on T‐cell response and plays an important role in tumour mortality and malignancy. In this study, the expression pattern of B7 family in gastrointestinal (GI) cancer was examined. Its upstream regulating mechanism, downstream targets and association with clinical parameters were also studied.

Materials and methods

The expression level of B7 members was analysed by FIREHOUSE. The gene mutation, DNA methylation, association with clinical parameters and downstream network of B7 members were analysed in cBioportal. The mutation frequency was analysed by Catalogue of Somatic Mutations in Cancer (COSMIC) analysis. The phylogenetic tree was constructed in MEGA7. The interaction protein domain analysis was performed by Pfam 31.0.

Results

Differential expression of B7 family molecules was detected in different kinds of GI cancer. High‐frequency gene alteration was found in tumour samples. There was negative correlation of promoter methylation and mRNA expression of B7 family members in tumour samples, suggesting the epigenetic basis of B7 family gene deregulation in GI cancer. The overexpression of B7‐H1 in pancreatic cancer, B7‐H5 in oesophageal cancer and B7‐H6 in liver cancer were significantly associated with worse overall survival. Finally, by network analysis, we identified some potential interacting proteins for B7‐1/2 and B7‐H1/DC.

Conclusions

Overall, our study suggested that B7 member deregulation was strongly involved in GI cancer tumorigenesis.

     

Complete Biallelic Insulation at the H19/Igf2 Imprinting Control Region Position Results in Fetal Growth Retardation and Perinatal Lethality

Background

The H19/Igf2 imprinting control region (ICR) functions as an insulator exclusively in the unmethylated maternal allele, where enhancer-blocking by CTCF protein prevents the interaction between the Igf2 promoter and the distant enhancers. DNA methylation inhibits CTCF binding in the paternal ICR allele. Two copies of the chicken β-globin insulator (ChβGI)₂ are capable of substituting for the enhancer blocking function of the ICR. Insulation, however, now also occurs upon paternal inheritance, because unlike the H19 ICR, the (ChβGI)₂ does not become methylated in fetal male germ cells. The (ChβGI)₂ is a composite insulator, exhibiting enhancer blocking by CTCF and chromatin barrier functions by USF1 and VEZF1. We asked the question whether these barrier proteins protected the (ChβGI)₂ sequences from methylation in the male germ line.

Methodology/Principal Findings

We genetically dissected the ChβGI in the mouse by deleting the binding sites USF1 and VEZF1. The methylation of the mutant versus normal (ChβGI)₂ significantly increased from 11% to 32% in perinatal male germ cells, suggesting that the barrier proteins did have a role in protecting the (ChβGI)₂ from methylation in the male germ line. Contrary to the H19 ICR, however, the mutant (mChβGI)₂ lacked the potential to attain full de novo methylation in the germ line and to maintain methylation in the paternal allele in the soma, where it consequently functioned as a biallelic insulator. Unexpectedly, a stricter enhancer blocking was achieved by CTCF alone than by a combination of the CTCF, USF1 and VEZF1 sites, illustrated by undetectable Igf2 expression upon paternal transmission.

Conclusions/Significance

In this in vivo model, hypomethylation at the ICR position together with fetal growth retardation mimicked the human Silver-Russell syndrome. Importantly, late fetal/perinatal death occurred arguing that strict biallelic insulation at the H19/Igf2 ICR position is not tolerated in development.     

Gingival Inflammation Associates with Stroke – A Role for Oral Health Personnel in Prevention: A Database Study

Objectives

Gingival inflammation is the physiological response to poor oral hygiene. If gingivitis is not resolved the response will become an established lesion.We studied whether gingivitis associates with elevated risk for stroke. The hypothesis was based on the periodontitis–atherosclerosis paradigm.

Methods

In our prospective cohort study from Sweden 1676 randomly selected subjects were followed up from 1985 to 2012. All subjects underwent clinical oral examination and answered a questionnaire assessing background variables such as socio-economic status and pack-years of smoking. Cases with stroke were recorded from the Center of Epidemiology, Swedish National Board of Health and Welfare, Sweden, and classified according to the WHO International Classification of Diseases. Unpaired t-test, chi-square tests, and multiple logistic regression analyses were used.

Results

Of the 1676 participants, 39 subjects (2.3%) had been diagnosed with stroke. There were significant differences between the patients with stroke and subjects without in pack-years of smoking (p = 0.01), prevalence of gingival inflammation (GI) (p = 0.03), and dental calculus (p = 0.017). In a multiple regression analysis the association between GI, confounders and stroke, GI showed odds ratio 2.20 (95% confidence interval 1.02–4.74) for stroke.

Conclusion

Our present findings showed that gingival inflammation clearly associated with stroke in this 26-year cohort study. The results emphasize the role of oral health personnel in prevention.     

Alterations in the Colonic Microbiota in Response to Osmotic Diarrhea

Background & Aims

Diseases of the human gastrointestinal (GI) tract are often accompanied by diarrhea with profound alterations in the GI microbiota termed dysbiosis. Whether dysbiosis is due to the disease itself or to the accompanying diarrhea remains elusive. With this study we characterized the net effects of osmotic diarrhea on the composition of the GI microbiota in the absence of disease.

Methods

We induced osmotic diarrhea in four healthy adults by oral administration of polyethylene glycol 4000 (PEG). Stool as well as mucosa specimens were collected before, during and after diarrhea and 16S rDNA-based microbial community profiling was used to assess the microbial community structure.

Results

Stool and mucosal microbiotas were strikingly different, with Firmicutes dominating the mucosa and Bacteroidetes the stools. Osmotic diarrhea decreased phylotype richness and showed a strong tendency to equalize the otherwise individualized microbiotas on the mucosa. Moreover, diarrhea led to significant relative shifts in the phyla Bacteroidetes and Firmicutes and to a relative increase in the abundance of Proteobacteria on the mucosa, a phenomenon also noted in several inflammatory and diarrheal GI diseases.

Conclusions

Changes in microbial community structure induced by osmotic diarrhea are profound and show similarities to changes observed in other GI diseases including IBD. These effects so must be considered when specimens from diarrheal diseases (i.e. obtained by stratification of samples according to diarrheal status) or conditions wherein bowel preparations like PEG (i.e. specimens obtained during endoscopy) are used.     

Selective serotonin reuptake inhibitors and gastrointestinal bleeding: a case-control study

Background

Selective serotonin reuptake inhibitors (SSRIs) have been associated with upper gastrointestinal (GI) bleeding. Given their worldwide use, even small risks account for a large number of cases. This study has been conducted with carefully collected information to further investigate the relationship between SSRIs and upper GI bleeding.

Methods

We conducted a case-control study in hospitals in Spain and in Italy. Cases were patients aged ≥18 years with a primary diagnosis of acute upper GI bleeding diagnosed by endoscopy; three controls were matched by sex, age, date of admission (within 3 months) and hospital among patients who were admitted for elective surgery for non-painful disorders. Exposures to SSRIs, other antidepressants and other drugs were defined as any use of these drugs in the 7 days before the day on which upper gastrointestinal bleeding started (index day).

Results

581 cases of upper GI bleeding and 1358 controls were considered eligible for the study; no differences in age or sex distribution were observed between cases and controls after matching. Overall, 4.0% of the cases and 3.3% of controls used an SSRI antidepressant in the week before the index day. No significant risk of upper GI bleeding was encountered for SSRI antidepressants (adjusted odds ratio, 1.06, 95% CI, 0.57–1.96) or for whichever other grouping of antidepressants.

Conclusions

The results of this case-control study showed no significant increase in upper GI bleeding with SSRIs and provide good evidence that the magnitude of any increase in risk is not greater than 2.     

Toxicity outcome in patients treated with modulated arc radiotherapy for localized prostate cancer

Aim

This study evaluates the acute toxicity outcome in patients treated with RapidArc for localized prostate cancer.

Background

Modern technologies allow the delivery of high doses to the prostate while lowering the dose to the neighbouring organs at risk. Whether this dosimetric advantage translates into clinical benefit is not well known.

Materials and methods

Between December 2009 and May 2012, 45 patients with primary prostate adenocarcinoma were treated using RapidArc. All patients received 1.8 Gy per fraction, the median dose to the prostate gland, seminal vesicles, pelvic lymph nodes and surgical bed was 80 Gy (range, 77.4–81 Gy), 50.4 Gy, 50.4 Gy and 77.4 Gy (range, 75.6–79.2 Gy), respectively.

Results

The time between the last session and the last treatment follow up was a median of 10 months (range, 3–24 months). The incidence of grade 3 acute gastrointestinal (GI) and genitourinary (GU) toxicity was 2.2% and 15.5%, respectively. Grade 2 acute GI and GU toxicity occurred in 30% and 27% of patients, respectively. No grade 4 acute GI and GU toxicity were observed. Older patients (>median) or patients with V60 higher than 35% had significantly higher rates of grade ≥2 acute GI toxicity compared with the younger ones.

Conclusions

RapidArc in the treatment of localized prostate cancer is tolerated well with no Grade >3 GI and GU toxicities. Older patients or patients with higher V60 had significantly higher rates of grade ≥2 acute GI toxicity. Further research is necessary to assess definitive late toxicity and tumour control outcome.