Decoding murine cytomegalovirus.

The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor ≈170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundred viral ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 365 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include >200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral upstream ORFs (uORFs) tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being ≈5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model.

Dissecting Herpes Simplex Virus 1-Induced Host Shutoff at the RNA Level.

Herpes simplex virus 1 (HSV-1) induces a profound host shutoff during lytic infection. The virion host shutoff (vhs) protein plays a key role in this process by efficiently cleaving host and viral mRNAs. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in host transcriptional activity. To dissect relative contributions of both mechanisms and elucidate gene-specific host transcriptional responses throughout the first 8 h of lytic HSV-1 infection, we used transcriptome sequencing of total, newly transcribed (4sU-labeled) and chromatin-associated RNA in wild-type (WT) and Δvhs mutant infection of primary human fibroblasts. Following virus entry, vhs activity rapidly plateaued at an elimination rate of around 30% of cellular mRNAs per hour until 8 h postinfection (p.i.). In parallel, host transcriptional activity dropped to 10 to 20%. While the combined effects of both phenomena dominated infection-induced changes in total RNA, extensive gene-specific transcriptional regulation was observable in chromatin-associated RNA and was surprisingly concordant between WT and Δvhs infections. Both induced strong transcriptional upregulation of a small subset of genes that were poorly expressed prior to infection but already primed by H3K4me3 histone marks at their promoters. Most interestingly, analysis of chromatin-associated RNA revealed vhs-nuclease-activity-dependent transcriptional downregulation of at least 150 cellular genes, in particular of many integrin adhesome and extracellular matrix components. This was accompanied by a vhs-dependent reduction in protein levels by 8 h p.i. for many of these genes. In summary, our study provides a comprehensive picture of the molecular mechanisms that govern cellular RNA metabolism during the first 8 h of lytic HSV-1 infection.IMPORTANCE The HSV-1 virion host shutoff (vhs) protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in vhs activity, as well as virus-induced global loss of host transcriptional activity, during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were dominated by these two global effects. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and Δvhs infections, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection, and identified vhs-dependent transcriptional downregulation of the integrin adhesome and extracellular matrix components. The latter explained seemingly gene-specific effects previously attributed to vhs-mediated mRNA degradation and resulted in a concordant loss in protein levels by 8 h p.i. for many of the respective genes.

Clinical Reality and Treatment for Local Recurrence of Rectal Cancer: A Single-Center Retrospective Study.

Background and Objectives: Despite advances in treatment, local recurrence remains a great concern in patients with rectal cancer. The aim of this study was to investigate the incidence and risk factors of local recurrence of rectal cancer in our single center over a 7-year-period. Materials and Methods: Patients with stage I-III rectal cancer were treated with curative intent. The necessity for radiotherapy and chemotherapy was determined before surgery and/or postoperative histopathological results. Results: Of 365 rectal cancer patients, 76 (20.8%) developed recurrent disease. In total, 27 (7.4%) patients presented with a local tumor recurrence (isolated in 40.7% of cases). Radiotherapy was performed in 296 (81.1%) patients. The most often used schema was 5 × 5 Gy followed by immediate surgery (n = 214, 58.6%). Local recurrence occurred less frequently in patients treated with 5 × 5 Gy radiotherapy followed by surgery (n = 9, 4%). Surgical procedures of relapses were performed in 12 patients, six of whom were operated with radical intent. Only two (7.4%) patients lived more than 5 years after local recurrence treatment. The incidence of local recurrence was associated with primary tumor distal location and worse prognosis. The median overall survival of patients after local recurrence treatment was 19 months. Conclusions: Individualized rectal cancer patient selection and systematic treatment algorithms should be used clinical practice to minimize likelihood of relapse. 5 × 5 Gy radiotherapy followed by immediate surgery allows good local control in resectable cT2N+/cT3N0 patients. Radical resection of isolated local recurrence offers the best chances of cure.

HSV-1-induced disruption of transcription termination resembles a cellular stress response but selectively increases chromatin accessibility downstream of genes.

Lytic herpes simplex virus 1 (HSV-1) infection triggers disruption of transcription termination (DoTT) of most cellular genes, resulting in extensive intergenic transcription. Similarly, cellular stress responses lead to gene-specific transcription downstream of genes (DoG). In this study, we performed a detailed comparison of DoTT/DoG transcription between HSV-1 infection, salt and heat stress in primary human fibroblasts using 4sU-seq and ATAC-seq. Although DoTT at late times of HSV-1 infection was substantially more prominent than DoG transcription in salt and heat stress, poly(A) read-through due to DoTT/DoG transcription and affected genes were significantly correlated between all three conditions, in particular at earlier times of infection. We speculate that HSV-1 either directly usurps a cellular stress response or disrupts the transcription termination machinery in other ways but with similar consequences. In contrast to previous reports, we found that inhibition of Ca2+ signaling by BAPTA-AM did not specifically inhibit DoG transcription but globally impaired transcription. Most importantly, HSV-1-induced DoTT, but not stress-induced DoG transcription, was accompanied by a strong increase in open chromatin downstream of the affected poly(A) sites. In its extent and kinetics, downstream open chromatin essentially matched the poly(A) read-through transcription. We show that this does not cause but rather requires DoTT as well as high levels of transcription into the genomic regions downstream of genes. This raises intriguing new questions regarding the role of histone repositioning in the wake of RNA Polymerase II passage downstream of impaired poly(A) site recognition.

Effects of feeding intact, ground and/or pelleted rapeseed on nutrient digestibility and growth performance of broiler chickens.

In this study, the effect of grinding or pelleting of rapeseed on the digestibility, apparent metabolisable energy (AMEN), growth performance and gizzard weight of broiler chickens was evaluated. In Exp. 1, four treatments were tested (240 broiler chickens, 20 replications, 3 birds per cage). Birds received either a basal diet or were fed with rapeseed of different sized particles (fine, medium and coarse ground, respectively). In Exp. 2, 10 treatments were assessed (360 chickens, 12 replications, 3 birds per cage), where five different diets (a basal diet and four diets with intact rapeseed, fine ground, medium ground or coarse ground rapeseed, respectively) were fed non-pelleted or pelleted. In Exp. 3, two treatments were compared (224 broiler chickens, 14 replications, 8 birds per floor pen): fine ground rapeseed (FG) and intact rapeseed (IS). Compared with medium and coarse ground rapeseed, fine grinding increased in Exp. 1 the AMEN of rapeseed and the precaecal digestibility (PD) of dry matter and ether extract (EE) (p < 0.05). In Exp. 2, the content of AMEN and the PD of EE and crude protein (CP) increased as the degree of grinding increased when diets were not pelleted (p < 0.01). In contrast, the significantly highest values for these parameters were measured after pelleting without effect of grinding, resulting in a significant interaction between pelleting and grinding (p < 0.001). In Exp. 3, performance of chickens was not affected by dietary treatments, but gizzard weight and the PD of CP were higher when IS were included in the diet (p < 0.05). The results suggested that fine grinding may be used to increase the feeding value of full-fat rapeseed for broiler chickens. Nevertheless, pelleting of diets with rapeseed seemed to be much more effective in the reduction of particle size than grinding.

Neoadjuvant chemotherapy with or without oxaliplatin after short-course radiotherapy in high-risk rectal cancer: A subgroup analysis from a prospective study.

AIM: To evaluate the role of oxaliplatin in neoadjuvant chemotherapy delivered after short-course irradiation. BACKGROUND: Using oxaliplatin in the above setting is uncertain. PATIENTS AND METHODS: A subgroup of 136 patients managed by short-course radiotherapy and 3 cycles of consolidation chemotherapy within the framework of a randomised study was included in this post-hoc analysis. Sixty-seven patients received FOLFOX4 (oxaliplatin group) while oxaliplatin was omitted in the second period of accrual in 69 patients because of protocol amendment (fluorouracil-only group). RESULTS: Grade 3+ acute toxicity from neoadjuvant treatment was observed in 30% of patients in the oxaliplatin group vs. 16% in the fluorouracil-only group (p = 0.053). The corresponding proportions of patients having radical surgery or achieving complete pathological response were 72% vs. 77% (odds ratio [OR] = 0.88; 95% confidence interval [CI]: 0.39-1.98; p = 0.75) and 15% vs. 7% (OR = 2.25; 95% CI: 0.83-6.94; p = 0.16), respectively. The long-term outcomes were similar in the two groups. Overall and disease-free survival rates at 5 years were 63% vs. 56% (p = 0.78) and 49% vs. 44% (p = 0.59), respectively. The corresponding numbers for cumulative incidence of local failure or distant metastases were 33% vs. 38% (hazard ratio [HR] = 0.89; 95% CI: 0.52-1.52; p = 0.68) and 33% vs. 33% (HR = 0.78; 95% CI: 0.43-1.40; p = 0.41), respectively. CONCLUSION: Our findings do not support adding oxaliplatin to three cycles of chemotherapy delivered after short-course irradiation.

Exome scale map of genetic alterations promoting metastasis in colorectal cancer.

BACKGROUND: Approximately 90% of colorectal cancer (CRC) deaths are caused by tumors ability to migrate into the adjacent tissues and metastase into distant organs. More than 40 genes have been causally linked to the development of CRC but no mutations have been associated with metastasis yet. To identify molecular basis of CRC metastasis we performed whole-exome and genome-scale transcriptome sequencing of 7 liver metastases along with their matched primary tumours and normal tissue. Multiple, spatially separated fragments of primary tumours were analyzed in each case. Uniformly malignant tissue specimen were selected with macrodissection, for three samples followed with laser microdissection. RESULTS: > 100 sequencing coverage allowed for detection of genetic alterations in subpopulation of tumour cells. Mutations in KRAS, APC, POLE, and PTPRT, previously associated with CRC development, were detected in most patients. Several new associations were identified, including PLXND1, CELSR3, BAHD1 and PNPLA6. CONCLUSIONS: We confirm the essential role of inflammation in CRC progression but question the mechanism of matrix metalloproteinases activation described in other work. Comprehensive sequencing data made it possible to associate genome-scale mutation distribution with gene expression patterns. To our knowledge, this is the first work to report such link in CRC metastasis context.

The gentamicin-collagen implant and the risk of distant metastases of rectal cancer following short-course radiotherapy and curative resection: the long-term outcomes of a randomized study.

PURPOSE: A previous randomized study conducted by our group showed that application of gentamicin-collagen implant (GCI) into the pelvic cavity after total mesorectal excision (TME) reduced the incidence of distant metastases. Therefore, we decided to conduct a confirmatory study. METHODS: Patients with rectal cancer were included in the study if they met the following criteria: adenocarcinoma of the rectum, preoperative short-term radiotherapy (5 × 5 Gy), and WHO performance score 0-1. RESULTS: One hundred seventy-six patients were randomly assigned either to an experimental group in which GCI was applied (n = 81) or to a control group without GCI (n = 81). Median follow-up was 80 months. Cumulative incidence of distant metastases at 5 years was higher in the control group compared to the experimental group: 23.5 vs 8.6% (HR 2.4 [95% CI 1.1-5.5], P = 0.005). Overall survival (OS), disease-free survival (DFS), and cancer-specific survival (CSS) did not differ between the experimental group and the control group: HR 0.95 [95% CI 0.55-1.70], P = 0.864; HR 0.85 [95% CI 0.50-1.45], P = 0.548, and HR 0.5 [95%CI 0.22-1.22], P = 0.093, respectively. The predefined by the protocol subgroup analysis for yp stage III disease showed better DFS in the experimental group compared to the control group; HR 0.47 [95%CI 0.23-0.97], P = 0.042). CONCLUSIONS: The results confirmed our previous finding that GCI applied in the pelvis significantly reduced the rate of distant metastases in patients after radical rectal cancer resection.

The safety of low-molecular-weight heparins in the prevention of venous thromboembolism in surgically-treated cancer patients: results of a multicentre observational study.

AIM OF THE STUDY: Despite widespread use of pharmacological prophylaxis, venous thromboembolism (VTE) still constitutes a common complication in cancer patients. The aim of the study was to analyse the safety of low-molecular-weight heparins (LMWH) in the prevention of VTE in surgically-treated cancer patients. MATERIAL AND METHODS: A total of 5207 cancer patients (44.5% men and 55.5% women) aged 16-97 years participated in a prospective observational study conducted in 13 Polish cancer centres in 2005-2008. This cohort included 4782 subjects who were treated surgically and received LMWH as a pharmacological prophylaxis for VTE prior to or after the surgery. The incidence of haemorrhagic complications and thrombocytopaenia was analysed in this cohort, along with intra-hospital mortality. RESULTS: Mean duration of LMWH administration was 9.4 ±7.8 days. Haemorrhagic complications: heavy (n = 15) or light bleeding (n = 299), were observed in 314 patients (6.5%). A total of 314 patients (6.5%) presented with haemorrhagic complications: heavy (n = 15, 0.3%) or light bleeding (n = 299, 6.3%). Four cases of heavy bleeding: gastrointestinal bleeding (n = 2), retroperitoneal bleeding (n = 1), and central nervous system bleeding (n = 1), were classified as definitely related to LMWH. No significant association was found between the incidence of haemorrhagic complications and the type of administered LWMH (p = 0.523). No cases of thrombocytopaenia or deaths related to administration of LMWH were reported. CONCLUSIONS: LMWH seems to be a safe form of pharmacological prophylaxis for VTE in surgically-treated cancer patients.

Effect of extrusion on the nutritional value of peas for broiler chickens.

The study was conducted to investigate the nutritional value of five samples of raw and extruded pea seeds (Pisum sativum L., Tarachalska cv.) from different experimental fields. The study included 150 male 1-day-old Ross 308 chickens, which were randomly assigned to three dietary treatments (50 replications each) and kept in individual cages. From days 1 to 16, all birds received only the basal diets. From days 17 to 21, the control group received still the basal diet, but for the two other groups, 20% of basal diet was replaced by raw or extruded peas. Furthermore, the groups receiving raw or extruded peas were divided into five subgroups of 10 animals each, where the diets contained one of the five pea samples of the same cultivar grown at different locations, respectively. On days 19 and 20, excreta were individually collected, and then all chickens were sacrificed and ileal digesta were sampled for determination of ileal digestibility, which was calculated by the difference method. Extrusion of pea seeds decreased the contents of crude fibre, acid and neutral detergent fibre, trypsin inhibitor activity (TIA), phytic P and resistant starch (RS) (p ≤ 0.05), but increased the contents of apparent metabolisable energy (AMEN) by approximately 2.25 MJ/kg dry matter (DM). Furthermore, extrusion improved the DM and crude protein digestibility significantly by about 21.3% and 11.6%, respectively. Similar results were observed for the digestibility of all analysed amino acids. In conclusion, extrusion markedly influenced the chemical composition of peas, reduced their contents of phytic P, TIA and RS and consequently had a positive impact on nutrient digestibility and AMEN values.

The nutritional value of narrow-leafed lupine (Lupinus angustifolius) for fattening pigs.

The aim of this study was to determine the nutrient digestibility of seeds of four varieties of narrow-leafed lupines (Lupinus angustifolius) and the possibility of soya bean meal (SBM) substitution by lupine seeds alone and in combination with rapeseed meal (RSM) in the diets of pigs. The seeds of the lupine varieties Kalif, Sonet, Zeus and Boruta were analysed. The apparent total tract digestibility (ATTD) was determined on 50 cross-bred pigs using the difference method with titanium dioxide as a marker. The substitution of SBM by lupine seeds alone (at 0 - 100%) was tested on 60 pigs (20-105 kg body weight (BW)) and by a combination of lupine seeds and RSM on 180 fattening pigs (35-80 kg BW). The chemical composition of lupine seeds differed considerably, especially in terms of crude protein and mineral content. All seeds contained less than 0.05% alkaloids and 9.3% oligosaccharides in dry matter. The ATTD of protein ranged from 70% to 74%, those of ether extract from 36% to 55% and those of gross energy from 77% to 84%. The entire replacement of SBM by lupine seeds (var. Sonet) did not have a negative effect on the performance of grower and fattener pigs. The substitution of SBM by a combination of lupines and RSM reduced the performance of growing and finishing pigs significantly.

The matrix metalloproteinase-7 and pro-enzyme of metalloproteinase-1 as a potential marker for patients with rectal cancer without distant metastasis.

The aim of this study was the evaluation of clinical usage of metalloproteinase (MMP): proMMP-1, MMP-2, MMP-7, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in serum of patients with rectal cancer, as well as the selection of parameters of the greatest diagnostic sensitivity and the determination of their relation with clinicopathological features, what is more, the demonstration whether their concentrations may have a prognostic value in the assessment of disease-free survival (DFS) and overall survival (OS). The study comprised 100 patients with rectal cancer including 29 women and 71 men. The tested group was comprised of qualified patients without distant metastasis (M0). It was demonstrated that in patients with rectal cancer, the concentrations of MMP-9, MMP-7, and proMMP-1 as well as TIMP-1 were significantly higher in comparison to the reference group. On the basis of ROC curves, the greatest diagnostic sensitivity of MMP-9 was demonstrated. When evaluating the correlation of tested parameters with the size of the tumor (T1-T2 vs T3-T4), essential differences were shown for proMMP-1 concentrations. The highest percentage of patients with progression had an increased concentration of MMP-7 and TIMP-1. During a 5-year follow-up, univariate log-rank analysis had shown an essential dependence between the concentration of MMP-7 in men and DSF which was confirmed in Cox multivariate analysis. It was demonstrated that the pretreatment concentration of proMMP-1 may be clinically useful when evaluating the mass of the tumor, whereas MMP-7 may be a prognostic factor for DFS in men with rectal cancer without distant metastasis.

Deciphering the modulation of gene expression by type I and II interferons combining 4sU-tagging, translational arrest and in silico promoter analysis.

Interferons (IFN) play a pivotal role in innate immunity, orchestrating a cell-intrinsic anti-pathogenic state and stimulating adaptive immune responses. The complex interplay between the primary response to IFNs and its modulation by positive and negative feedback loops is incompletely understood. Here, we implement the combination of high-resolution gene-expression profiling of nascent RNA with translational inhibition of secondary feedback by cycloheximide. Unexpectedly, this approach revealed a prominent role of negative feedback mechanisms during the immediate (≤60 min) IFNα response. In contrast, a more complex picture involving both negative and positive feedback loops was observed on IFNγ treatment. IFNγ-induced repression of genes associated with regulation of gene expression, cellular development, apoptosis and cell growth resulted from cycloheximide-resistant primary IFNγ signalling. In silico promoter analysis revealed significant overrepresentation of SP1/SP3-binding sites and/or GC-rich stretches. Although signal transducer and activator of transcription 1 (STAT1)-binding sites were not overrepresented, repression was lost in absence of STAT1. Interestingly, basal expression of the majority of these IFNγ-repressed genes was dependent on STAT1 in IFN-naïve fibroblasts. Finally, IFNγ-mediated repression was also found to be evident in primary murine macrophages. IFN-repressed genes include negative regulators of innate and stress response, and their decrease may thus aid the establishment of a signalling perceptive milieu.

Effects of glyceryl polyethylene glycol ricinoleate on nutrient utilisation and performance of broiler chickens.

A completely randomised design study with a 2 × 2 factorial arrangement was conducted to observe effects of an emulsifier (glyceryl polyethylene glycol ricinoleate (GPR)) on nutrient utilisation and performance of broiler chickens. A total of 384 male broiler chickens were used to determine the influence of GPR (without addition or added at 0.04% of diet) and two levels of apparent metabolisable energy (AMEN) (according to standard requirements (Diets SE) or energy reduced by 0.4 MJ/kg diet (Diets LE)) on birds' performance and apparent total tract digestibility (ATTD). ATTD of ether extract (EE) and AMEN were measured on d 14 and 35, and ATTD of neutral detergent fibre (NDF) was measured on d 35. All diets were based on wheat-maize-soybean meal with rapeseed oil and lard (blend of 50:50) as fat sources. During the grower period and the whole trial, birds fed Diets supplemented with GPR were characterised by higher body weight gain (BWG) and lower feed conversion ratio (FCR) compared to chicken receiving diets without GPR (p < 0.05). At the end of experiment, birds fed Diet LE without GPR were characterised by lower BWG and higher FCR (p < 0.05). Supplementation with GPR caused a higher ATTD of EE for diets SE and LE at d 14 and 35 (p < 0.05). Moreover, the GPR addition to Diet LE improved ATTD of NDF at d 35 (p < 0.05). In conclusion, the findings suggest that GPR effects the digestion of a blend of animal fat/rapeseed oil positively, even in a practical maize-wheat-based broiler diet with decreased AMEN level.

The impact of oncological package implementation on the treatment of rectal cancer in years 2013-2019 in Poland - multicenter study.

<b><br>Introduction:</b> In 2015, in Poland, the oncological package (OP) was established. This law constituted a fast track of oncological diagnosis and treatment and obligatory multidisciplinary team meetings (MDT).</br> <b><br>Aim:</b> The aim of this study was to analyze the impact of OP on rectal cancer treatment.</br> <b><br>Methods:</b> The study was a multicenter, retrospective analysis of data collected from five centers. It included clinical data of patients operated on due to rectal cancer between 2013 and 2019. For most analyses, patients were categorized into three groups: 2013-2014 - before OP (A), 2015-2016 - early development of OP (B), 2017-2019 - further OP functioning (C).</br> <b><br>Results:</b> A total of 1418 patients were included. In all time intervals, the majority of operations performed were anterior resections. There was a significantly lower local tumor stage (T) observed in subsequent time intervals, while there were no significant differences for N and M. In period C, the median of resected nodes was significantly higher than in previous periods. Four of the centers showed an increasing tendency in the use of preoperative radiotherapy. The study indicated a significant increase in the use of short-course radiotherapy (SCRT) and a decrease in the number of patients who did not receive any form of preoperative therapy in subsequent periods. In the group that should receive radiotherapy (T3/4 or N+ and M0), the use of SCRT was also significantly increasing.</br> <b><br>Conclusions:</b> In the whole cohort, there was a significant increase in the use of preoperative radiotherapy and a decrease in the T stage, changing with the development of OP. Nevertheless, this relation is indirect and more data should be gathered for further conclusions.</br>.

Which tumour factors preclude organ preservation in patients with rectal cancer?

BACKGROUND: cT3cdT4, cN2, mesorectal nodes > 8 mm, clinically positive lateral nodes, extramural vascular invasion (EMVI) and mesorectal fascia threatening (MRF+) have been utilized as exclusion criteria in several studies on the watch-and-wait (w&w) strategy. Here, our aim was to validate these criteria through a post hoc analysis of two pooled prospective studies on w&w following routine radio(chemo)therapy. METHODS: A review of baseline magnetic resonance imaging was performed in a subgroup of 223 patients treated at a single institution. Of these, 17.9 % started w&w, 12.6 % achieved clinical complete response (cCR) and 9.0 % sustained cCR during median follow-up of 54 months. RESULTS: The multivariable logistic analysis showed that the proportion of circumferential bowel involvement and EMVI significantly influenced the chance of sustained cCR; odds ratios were 0.063 (95 % confidence interval [CI] 0.008-0.489, p = 0.008), and 0.109 (95 % CI 0.014-0.850, p = 0.034), respectively. Sustained cCR was observed in none of the 57 patients with 90 %-100 % circumferential bowel involvement and in only one of the 89 patients with EMVI. In contrast, cT3cdT4, cN2, mesorectal nodes > 8 mm, clinically positive lateral nodes or MRF+ were not independently associated with sustained cCR. Among the subgroups of patients with these features but without (near-)circular tumour or EMVI+, sustained cCR was observed in 12 %-25 % of patients. CONCLUSION: Sustained cCR after routine preoperative radio(chemo)therapy is unlikely in patients with (near-)circular tumour or EMVI, whereas patients with cT3cdT4, cN2, mesorectal nodes > 8 mm, clinically positive lateral nodes and MRF+ should not be denied w&w.

Regulation of autologous immunity to the mouse 5T4 oncofoetal antigen: implications for immunotherapy.

Effective vaccination against tumour-associated antigens (TAA) such as the 5T4 oncofoetal glycoprotein may be limited by the nature of the T cell repertoire and the influence of immunomodulatory factors in particular T regulatory cells (Treg). Here, we identified mouse 5T4-specific T cell epitopes using a 5T4 knock out (5T4KO) mouse and evaluated corresponding wild-type (WT) responses as a model to refine and improve immunogenicity. We have shown that 5T4KO mice vaccinated by replication defective adenovirus encoding mouse 5T4 (Adm5T4) generate potent 5T4-specific IFN-γ CD8 and CD4 T cell responses which mediate significant protection against 5T4 positive tumour challenge. 5T4KO CD8 but not CD4 primed T cells also produced IL-17. By contrast, Adm5T4-immunized WT mice showed no tumour protection consistent with only low avidity CD8 IFN-γ, no IL-17 T cell responses and no detectable CD4 T cell effectors producing IFN-γ or IL-17. Treatment with anti-folate receptor 4 (FR4) antibody significantly reduced the frequency of Tregs in WT mice and enhanced 5T4-specific IFN-γ but reduced IL-10 T cell responses but did not reveal IL-17-producing effectors. This altered balance of effectors by treatment with FR4 antibody after Adm5T4 vaccination provided modest protection against autologous B16m5T4 melanoma challenge. The efficacy of 5T4 and some other TAA vaccines may be limited by the combination of TAA-specific T regs, the deletion and/or alternative differentiation of CD4 T cells as well as the absence of distinct subsets of CD8 T cells.

Is the 1-cm rule of distal bowel resection margin in rectal cancer based on clinical evidence? A systematic review.

BACKGROUND: Distal intramural spread is present within 1 cm from visible tumor in a substantial proportion of patients. Therefore, ≥ 1 cm of distal bowel clearance is recommended as minimally acceptable. However, clinical results are contradictory in answering the question of whether this rule is valid. The aim of this review was to evaluate whether in patients undergoing anterior resection, a distal bowel gross margin of <1 cm jeopardizes oncologic safety. METHODS: A systematic review of the literature identified 17 studies showing results in relation to margins of approximately <1 cm (948 patients) versus >1 cm (4626 patients); five studies in relation to a margin of ≤ 5 mm (173 patients) versus >5 mm (1277 patients), and five studies showing results in a margin of ≤ 2 mm (73 patients). In most studies, pre- or postoperative radiation was provided. RESULTS: A multifactorial process was identified resulting in selection of favorable tumors for anterior resection with the short bowel margin and unfavorable tumors for abdominoperineal resection or for anterior resection with the long margin. In total, the local recurrence rate was 1.0% higher in the <1-cm margin group compared to the >1-cm margin group (95% confidence interval [CI] -0.6 to 2.7; P = 0.175). The corresponding figures for ≤ 5 mm cutoff point were 1.7% (95% CI -1.9 to 5.3; P = 0.375). The pooled local recurrence rate in patients having ≤ 2 mm margin was 2.7% (95% CI 0 to 6.4). CONCLUSIONS: In the selected group of patients, <1 cm margin did not jeopardize oncologic safety.

The Effects of Protease Supplementation and Faba Bean Extrusion on Growth, Gastrointestinal Tract Physiology and Selected Blood Indices of Weaned Pigs.

The aim of the study was to investigate how the extrusion of faba bean seeds (var. Albus) and/or the addition of protease to pigs' diets affected the animals' growth parameters, digestibility of nutrients, selected physiological parameters of the digestive tract, and biochemical blood parameters. A 28-day experiment was conducted on 32 pigs weighing 9 ± 0.2 kg. The animals were allocated to four treatments in a 2 × 2 factorial arrangement with the main effects of extrusion (raw or extruded) and effects of protease supplementation (0 and 0.05%). Extrusion reduced the levels of neutral detergent fibre, trypsin inhibitor, phytate-P, and resistant starch but did not improve the digestibility of protein and dry matter in faba bean seeds. The pigs' growth performance, ileal digestibility, enzyme activity, and morphometric parameters of the ileum were not significantly affected by extrusion, except for a higher feed intake between the 15th and 28th day of the experiment. The protease supplementation gave comparable results as the diet without protease, except the feed conversion ratio (in the periods of 15-28th day and 0-28th day), which was higher than in the groups without protease. The extrusion and protease increased acetate and acetoacetate contents in the cecal digesta, but propionate, butyrate, and isovalerate concentrations in the digesta of the pigs in this group were lower. Thanks to protease supplementation, protein and oil levels in the diet were reduced, which cut the cost of feed mixtures. The extrusion and protease additive combined together did not improve the pigs' growth performance in this experiment.