Dietary n-3 PUFA May Attenuate Experimental Colitis.

BACKGROUND: Inflammatory bowel diseases (IBD) occurred in genetically predisposed people exposed to environmental triggers. Diet has long been suspected to contribute to the development of IBD. Supplementation with n-3 polyunsaturated fatty acids (PUFA) protects against intestinal inflammation in rodent models while clinical trials showed no benefits. We hypothesized that intervention timing is crucial and dietary fatty acid pattern may influence intestinal environment to modify inflammation genesis. The aim of this study was to evaluate the dietary effect of PUFA composition on intestinal inflammation. METHODS: Animals received diet varying in their PUFA composition for four weeks before TNBS-induced colitis. Colon inflammatory markers and gut barrier function parameters were assessed. Inflammatory pathway PCR arrays were determined. RESULTS: n-3 diet significantly decreased colon iNOS, COX-2 expression, IL-6 production, and LTB4 production but tended to decrease colon TNFα production (P = 0.0617) compared to control diet. Tight junction protein (claudin-1, occludin) expressions and MUC2 and TFF3 mRNA levels were not different among groups. n-9 diet also decreased colon IL-6 production (P < 0.05). CONCLUSIONS: Dietary n-3 PUFA influence colitis development by attenuating inflammatory markers. Further research is required to better define dietary advice with a scientific rationale.

Evaluation of ubiquitinated proteins by proteomics reveals the role of the ubiquitin proteasome system in the regulation of Grp75 and Grp78 chaperone proteins during intestinal inflammation.

The ubiquitin proteasome system (UPS) is the major pathway of intracellular protein degradation and may be involved in the pathophysiology of inflammatory bowel diseases or irritable bowel syndrome. UPS specifically degrades proteins tagged with an ubiquitin chain. We aimed to identify polyubiquitinated proteins during inflammatory response in intestinal epithelial HCT-8 cells by a proteomic approach. HCT-8 cells were incubated with interleukin 1ß, tumor necrosis factor-α, and interferon-γ for 2 h. Total cellular protein extracts were separated by 2D gel electrophoresis and analyzed by an immunodetection using antiubiquitin antibody. Differential ubiquitinated proteins were then identified by LC-ESI MS/MS. Seven proteins were differentially ubiquitinated between control and inflammatory conditions. Three of them were chaperones: Grp75 and Hsc70 were more ubiquitinated (p < 0.05) and Grp78 was less ubiquitinated (p < 0.05) under inflammatory conditions. The results for Grp75 and Grp78 were then confirmed in HCT-8 cells and in 2-4-6-trinitrobenzen sulfonic acid induced colitis in rats mimicking inflammatory bowel disease by immunoprecipitation. No difference was observed in irritable bowel syndrome like model. In conclusion, we showed that a proteomic approach is suitable to identify ubiquitinated proteins and that UPS-regulated expression of Grp75 and Grp78 may be involved in inflammatory response. Further studies should lead to the identification of ubiquitin ligases responsible for Grp75 and Grp78 ubiquitination.

An α-linolenic acid-rich formula reduces oxidative stress and inflammation by regulating NF-κB in rats with TNBS-induced colitis.

We have previously shown that α-linolenic acid (ALA), a (n-3) PUFA exerts in vitro antiinflammatory effects in the intestine. In this study, we aimed to evaluate its effect on inflammatory and oxidative stress in a colitis model. Colitis was induced in 2 groups at d 0 by intrarectal injection of 2-4-6-trinitrobenzen sulfonic acid (TNBS), whereas the control group received the vehicle. Rats we fed 450 mg . kg(-1) . d(-1) of ALA (TNBS+ALA) while the other colitic group (TNBS) and the control group were fed an isocaloric corn oil formula for 14 d (from d -7 to d 7). RBC fatty acid composition was assessed. Oxidative stress was studied by measuring urinary 8-isoprostanes (8-IP) and colon glutathione (GSH) concentration and inducible nitric oxide synthase (iNOS) expression. Colitis was assessed histologically, by production of proinflammatory mediators, including cytokines, leukotrienes B(4) (LTB(4)), and cyclooxygenase-2 (COX-2) and by nuclear factor-κB (NF-κB) activation. The ALA-rich diet significantly increased the RBC levels of ALA, eicosapentaenoic acid, and docosapentaenoic acid (n-3) compared with the TNBS group (P < 0.01 for all). The beneficial effect of ALA supplementation on oxidative stress was reflected by lower urinary 8-IP levels (P < 0.05), a normalized colon GSH concentration (P < 0.01), and reduced colon iNOS expression (P < 0.05) compared with the TNBS group. ALA also protected against colon inflammation as assessed by lower tumor necrosis factor-α secretion and mRNA level (P < 0.05), reduced NF-κB activation (P = 0.01), and lower colon lipid mediator concentrations such as LTB(4) and COX-2 (P < 0.05) compared with the TNBS group. These findings show that an ALA-rich formula is beneficial to TNBS-induced colitic rats via inhibition of oxidative and inflammatory stress.

The association of intraprostatic calcifications and dosimetry parameters with biochemical control after permanent prostate implant.

PURPOSE: The objective of this study was to evaluate the impact of intraprostatic calcifications (IC) on long-term tumor control in patients treated with permanent implant prostate brachytherapy (PIPB). MATERIALS AND METHODS: Data from 609 I-125 patients treated with PIPB were retrospectively reviewed. The presence of IC was determined by reviewing postimplant CT images. Doses delivered were determined using the Monte Carlo (model-based) calculations and the TG43 approach. Biochemical relapses at 7 and 10 years were determined according to Phoenix definition. Long-term biochemical relapse-free survival (bRFS) was determined using Kaplan-Meier estimates with log rank test. Cox proportional hazard models were used for analysis of predictor factors of biochemical recurrence. RESULTS: IC were observed for 11.1% of patients. Clinical stage, PSA, Gleason score, D'Amico risk group, and ADT use were comparable between IC and no IC groups. The 7- and 10-year bRFS for the entire cohort were 94.1% and 90.6%, respectively. The bRFS at 7 years was 90.5% (with IC) vs. 94.5% (without IC) (p = 0.198); the corresponding values at 10 years were 78.8% vs. 91.8% (p = 0.046). On Cox model, only prostatic calcifications were a significant risk factor for biochemical relapse (HR: 2.30, IC 95%: 1.05-5.00, p = 0.037; and HR: 3.94; IC 95%: 1.00-15.38; p = 0.049 for univariate and multivariate analysis, respectively). CONCLUSION: The presence of IC in patients treated with PIPB decreases V100 and D90 for postimplant Monte Carlo dosimetry (compared with TG43); correspondingly, IC are associated with a lower 10-y bRFS. Model-based dose calculations are critical to evaluate potential cold spots due to calcifications.

Does Seed Migration Increase the Risk of Second Malignancies in Prostate Cancer Patients Treated With Iodine-125 Loose Seeds Brachytherapy?

PURPOSE: To evaluate the risk of second malignancies after migration of seeds (MS) in prostate cancer patients treated with 125I loose seeds brachytherapy. METHODS AND MATERIALS: Data from 2802 prostate cancer patients treated with 125I loose seeds brachytherapy in 3 Canadian centers were reviewed. After seeds implant, all patients underwent postimplant pelvic radiography and computed tomography scan for postimplant dosimetry. These images were used to assess whether seed migration occurred. The incidence of second malignancies was determined through the review of patient charts. The 7- and 10-year cumulative incidences of second malignancies and their 95% confidence intervals (CIs) were calculated. Fine and Gray competing risk regression analysis was used to assess the factors associated with the development of second malignancies. RESULTS: Mean age and median follow-up were 63.5 years and 74 (range, 12-246) months, respectively. Migration of seeds occurred in 263 of 2802 patients (9.4%). Second malignancy occurred in 87 patients (3.1%) for the entire cohort and was not different between patients who experienced MS (9, 3.4%) and those who did not (78, 3.1%) (P = .755). The 7-year cumulative incidence rates of second malignancies were 2.95% (95% CI 1.20%-6.00%) (with MS) versus 2.82% (2.10%-3.70%) (without MS) (P = .756). The corresponding values at 10 years were 6.16% (2.20%-12.3%) versus 4.51% (3.20%-5.50%) (P = .570). Migration of seeds did not seem to be a significant predictive factor for second malignancies development (adjusted hazard ratio 1.27 [95% CI 0.63-2.55]; P = .510). In both models, only advanced age was significantly associated with second malignancies development. CONCLUSIONS: These results did not show an increased risk of second malignancies associated with MS after 125I loose seeds brachytherapy for prostate cancer patients. Longer follow-up and more events are required to better correlate MS and second malignancies.

High-dose-rate brachytherapy boost for prostate cancer treatment: Different combinations of hypofractionated regimens and clinical outcomes.

PURPOSE: To report the outcomes of our high-dose-rate brachytherapy (HDR-BT) boost experience in localized prostate cancer treated with different combinations of radiation doses and fractionation. MATERIAL AND METHODS: Between 1999 and 2011, 832 patients were treated with different regimens of external beam radiotherapy (EBRT) and HDR-BT. These regimens were converted into three biologically effective dose (BED) groups. The biochemical failure-free survival (BFFS), reported with the phoenix definition and prostate-specific antigen (PSA) >0.2ng/ml at 5-year, genitourinary (GU) and gastrointestinal (GI) toxicities were compared between the groups. RESULTS: The 5-, 10-year BFFS for the entire cohort were 94.6% and 92.5%, for overall survival (OS) 96.1% and 80.3% and for prostate cancer-specific survival (PCSS) 99.5% and 97.8%. The percentage of patients with a 5-year PSA level <0.2ng/ml was 68.6%, 78.7% and 86.7% in the BED group of <250, 250-260 and >260Gy (p=0.005) while the 5-year BFFS rates according to phoenix definition were 97.3%, 94.3% and 94.9% for BED group <250, 250-260 and >260Gy (p=0.453). On multivariate logistic regression, patients in the BED>260Gy group were significantly more likely to remain free from 5-year PSA values ≥0.2ng/mL compared with those in the BED<250Gy group (OR: 0.350, p=0.011). Grade≥3 acute GU toxicity was reported in 2 patients (4.7%) for BED>260Gy while grade≥3 late GU toxicity was reported in 6 (1.7%) and 9 (4.9%) patients for 250-260Gy and >260Gy BED groups. CONCLUSIONS: The increase in BED with the hypofractionated regimens correlates with an improvement in biochemical control with of urinary toxicity. This increase in urinary toxicity is small and clinically acceptable.

Image-Guided High-Dose-Rate (HDR) Boost Localization Using MRI/MR Spectroscopy: A Correlation Study with Biopsy.

PURPOSE: The purpose of this study is to compare the blind interpretations of magnetic resonance imaging (MRI) sequences, diffusion-weighted imaging (DWI), apparent diffusion coefficient (ADC), mapping, and magnetic resonance spectroscopy (MRS) of the prostate, in comparison to prostate biopsy to identify a valid dominant intraprostatic lesion (DIL) for dose escalation using high-dose rate brachytherapy. METHODS: MRI/MRS were performed on 20 patients with intermediate risk adenocarcinoma of the prostate. T1W, T2W, DWI-ADC, and MRS sequences were performed at 1.5 T with pelvic and endorectal coils. An experienced radiologist rated the presence of cancer in each sextant by using a dichotomic approach, first on MR standard acquisitions (T1W and T2W), then on DWI-ADC mapping, and later on MRS images. Areas under the receiver's operating characteristic curve were calculated using a sextant as the unit of analysis. The transrectal ultrasonography-guided biopsy results were used as the reference standard. A table summarizing the MRI/MRS findings was made and compared to the corresponding area in the prostate biopsy report. A perfect match was defined to be the presence of cancer in the same sextant of the MRI/MRS exam and the prostate biopsy. RESULTS: The interpretation of the MRI/MRS exams per sextant was compared to the diagnostic biopsy report. MRI readings were compared with the biopsy as a surrogate for the complete pathology specimen of the prostate. A sensitivity (Sn) of 98.6% (95% confidence interval, 92.2% - 99.9%) and specificity (Sp) of 60.8% (46.1% - 74.2%) were found. The positive and negative predictive values (PPV, NPV) were 77.3% (67.1% - 85.5%) and 96.9% (83.8% - 99.9%), respectively. When MRS readings were compared with biopsy, we found a Sn of 96.4% (87.7% - 99.6%) and Sp of 54.8% (38.7% - 70.2%). The PPV and NPV were 74% (62.4% - 83.6%) and 92% (74% - 99%), respectively. DWI-ADC mapping results were also compared with biopsy. We found a Sn and Sp of 93.7% (84.5% - 98.2%) and 82.1% (66.5% - 92.5%), respectively, and a PPV and NPV of 89.4% (79.4% - 95.6%) and 88.9% (73.9% - 96.9%), respectively. Finally, after combining MRI, MRS, and DWI-ADC mapping, compared with biopsy, we obtained a Sn, Sp, PPV, and NPV of 100% (94.8% - 100%), 49% (34.8% - 63.4%), 72.6% (62.5% - 81.3%), and 100% (86.3% - 100%), respectively. CONCLUSIONS: The combination of MRI/MRS is a sensitive tool for both the structural and metabolic evaluation of prostate cancer location. MRI/MRS exams are useful to delineate a DIL for high-dose-rate (HDR) intraprostatic boost.

Does prostate volume has an impact on biochemical failure in patients with localized prostate cancer treated with HDR boost?

PURPOSE: To compare biochemical failure free survival (BFFS) of patients with small and large prostate glands treated with external beam radiation therapy (EBRT) and HDR (high dose rate) brachytherapy boost. MATERIALS AND METHODS: Between 2002 and 2012, 548 patients were treated with EBRT followed by HDR boost. The effect of covariates and prostate volume on biochemical failure was analyzed by survival analysis and Cox regression model. RESULTS: The median follow-up and age were not different between the two groups. The mean prostate gland volume at the time of CT planning was 48.1 and 76.0cc in small (<60cc) and large (⩾ 60cc) prostate volume, respectively (p<0.001). When PSA bounces were excluded, there was no significant difference between the two groups with a 5-years BFFS of 95.8% vs 92.3%, p=0.094. There were no significant differences between the two groups for urinary symptoms (IPSS) as well as acute and late GI toxicities. CONCLUSIONS: This study showed that a HDR brachytherapy boost in large prostate gland cases is feasible at the price of increased PSA bounces. When the benign bounces are excluded, there is no significant difference between the two groups for tumor control and toxicity. Therefore, in our experience, there is no rational precluding the use of HDR boost in patients with a prostate size of 60 cc or more so long as an adequate dosimetry is achievable.

Image-guided high-dose-rate brachytherapy boost to the dominant intraprostatic lesion using multiparametric magnetic resonance imaging including spectroscopy: Results of a prospective study.

PURPOSE: To evaluate the long-term outcomes of image-guided high-dose-rate (HDR) brachytherapy boost to the dominant intraprostatic lesion (DIL) using multiparametric magnetic resonance imaging (MRI), including spectroscopy (MRI/magnetic resonance spectroscopy [MRS]). METHODS AND MATERIALS: Between December 2009 and March 2011, 20 patients with intermediate-risk prostate cancer underwent multiparametric MRI/MRS protocol before treatment. All patients were treated with an external beam radiotherapy dose of 40 Gy, combined with an HDR brachytherapy boost of 15 Gy. Concurrently, the DIL received a boost of 18 Gy. Missing data during followup were handled with multiple imputations. RESULTS: The median followup was 62 months (range, 23-71 months). Six patients (31%) were classified as favorable intermediate risk and 13 patients (69%) as unfavorable intermediate risk. One patient experienced a prostate-specific antigen biochemical failure, and the 5-year biochemical failure-free survival rate was of 94.7%. The mean International Prostate Symptom Score rose from 7, with respect to baseline, to 10.42 1 month after treatment, and rapidly decreased to 6.97 after 3 months. Grade 1, 2, and 3 acute genitourinary toxicities were reported in 13 (68%), 3 (16%), and 1 (5%) patients, respectively. Grade 1 and 2 late genitourinary toxicities were reported in 9 (53%) and 3 (18%) patients, respectively. Only grade 1 acute and late gastrointestinal toxicities were reported in 4 (21%) and 3 (18%) patients, respectively. CONCLUSIONS: Delivering an HDR brachytherapy boost to the DIL using image-guided multiparametric MRI/MRS is feasible with good outcomes for biochemical control, acute and late toxicities, and dosimetric constraints for critical organs.

Adjunct therapy of n-3 fatty acids to 5-ASA ameliorates inflammatory score and decreases NF-κB in rats with TNBS-induced colitis.

5-aminosalicylic acid (5-ASA) is widely used for the treatment of inflammatory bowel disease (IBD). Recent studies have evaluated the potential of nutritional intervention as adjunct therapy to 5-ASA in IBD. N-3 polyunsaturated fatty acids (PUFA) have shown potent anti-inflammatory properties in gut inflammation. Therefore, we aimed to evaluate the efficacy of the dual therapy (n-3 PUFA plus 5-ASA) in rats with 2, 4, 6-trinitrobenzen sulfonic acid (TNBS)-induced colitis. Colitis was induced by intrarectal injection of TNBS while control rats received the vehicle. Rats received by gavage a fish oil-rich formula (n-3 groups) or an isocaloric and isolipidic oil formula supplemented with 5-ASA for 14 days. A dose response of 5-ASA (5-75 mg. suppression mg kg(-1) d(-1)) was tested. Colitis was evaluated and several inflammatory markers were quantified in the colon. COX-2 expression (P<.05) and pro-inflammatory eicosanoids production of prostaglandin E2 (P<.001) and leukotriene B4 (P<.001) were significantly inhibited by n-3 PUFA or 5-ASA therapy. 5-ASA also reduces mRNA levels of tumor necrosis factor α (P<.05). n-3 PUFA or 5-ASA significantly inhibits nuclear factor κB (NF-κB) activation (P<.01 and P<.05, respectively). The dual therapy n-3 PUFA plus 5-ASA also inhibited inflammatory response by lowering NF-κB activation (P<.01) or inducing peroxisome proliferator-activated receptor-γ (PPARγ) expression (P<.05). These results indicate that 5-ASA plus n-3 PUFAs are more effective than a higher dose of 5-ASA alone to reduce NF-κB activation and to induce PPARγ. By contrast, the dual therapy did not improve the effects of individual treatments on eicosanoids or cytokine production. Use of n-3 PUFA in addition to 5-ASA may reduce dose of standard therapy.

Dietary α-linolenic acid-rich formula reduces adhesion molecules in rats with experimental colitis.

OBJECTIVE: The ω-3 polyunsaturated fatty acid therapy in inflammatory bowel disease is focused on the effects on fish oil-derived polyunsaturated fatty acids. We speculated that a vegetal oil rich in α-linolenic acid (ALA) might also inhibit colitis. Therefore, we evaluated whether dietary ALA would decrease the expression of adhesion molecules by inducing the protective enzyme heme oxygenase-1 (HO-1) in a rat colitis model. METHODS: Colitis was induced at day 0 by an intrarectal injection of 2-4-6-trinitrobenzen sulfonic acid (TNBS), whereas control rats received the vehicle. Rats were fed an ALA-rich formula 450 mg · kg⁻¹ · d⁻¹, whereas the other colitic group (TNBS) and the control group were fed an isocaloric corn oil formula for 14 d (from day -7 to day 7). The colonic expressions of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), vascular endothelial growth factor A receptor-2 (VEGFR2), and HO-1 were studied by immunohistochemistry. RESULTS: The ALA-rich diet significantly decreased the expression of ICAM-1, VCAM-1, and VEGFR-2 compared the TNBS group, but it did not affect the expression of HO-1. CONCLUSION: A vegetal ALA-rich formula decreases the expression of ICAM-1, VCAM-1, and VEGFR-2 and independently of HO-1 in rats with TNBS-induced colitis. Further studies are required to evaluate its therapeutic potential in inflammatory bowel disease as an alternative to fish oil.

Alanyl-glutamine restores maternal deprivation-induced TLR4 levels in a rat neonatal model.

BACKGROUND & AIMS: Increased intestinal permeability of Irritable bowel syndrome (IBS) patients has been recently associated with a decreased level of glutamine synthetase. Neonatal maternal deprivation (NMD) is considered as an IBS-like model. The aim of our study was to investigate whether early nutritional intervention with glutamine may attenuate the deleterious impact of early life stress on gut barrier function in NMD. METHODS: 124 rat pups were separated from their dam 3 h daily during postnatal days 2-14, or left undisturbed (NS). Separated rats received by gavage either with alanyl-glutamine (GLN), an isonitrogenous amino acid mix (AA, control) or an equal amount of drinking water (H(2)O). Production of cytokines was measured by multiplex, expression of COX-2, PPARγ, tight junction proteins and TLR4 by western blot. Intestinal permeability was studied by Lactulose/Mannitol test. RESULTS: Treatment of pups with GLN or AA abolished the decrease in body weight observed in NMD. Treatment with GLN decreased in the colon (i) TLR4 expression at D20, (ii) IL-2 and -10 productions at D60 (iii) protein expression of occludin at D20 compared to AA. GLN also decreased colon expression of COX2 and PPARγ at D60 compared to NS. Colon production of IFNγ is significantly reduced by GLN compared to H(2)O. No significant change in intestinal permeability was observed. CONCLUSIONS: These results showed that an early nutritional intervention with alanyl-glutamine specifically abolished the up-regulation of TLR4 expression in NMD. Glutamine may be evaluated as a potential treatment for IBS patients.

Anti-inflammatory and anti-angiogenic effect of long chain n-3 polyunsaturated fatty acids in intestinal microvascular endothelium.

BACKGROUND & AIMS: The role of endothelial cells in inflammatory bowel disease has been recently emphasized. Endothelial activation and expression of adhesion molecules are critical for leukocytes recruitment into the inflammatory wall. Compelling evidence demonstrated anti-inflammatory effects of long chain n-3 PUFA in inflammatory models. We previously showed that long chain n-3 PUFA (EPA and DHA) inhibited inflammatory response in epithelial and dendritic cells. As long chain n-3 PUFA treatment led to a decreased expression of adhesion molecules in endothelial cells from other organs, we have now investigated their effect on intestinal endothelial cells in vitro and in colitic rats. METHODS: In vitro study: Primary culture of human intestinal microvascular endothelial cells (HIMEC) were pre-treated with DHA and then incubated with IL-1ß. In vivo study: Colitis was induced in 2 groups at day0 by intrarectal injection of 2-4-6-trinitrobenzen sulfonic acid (TNBS). Rats received by gavage either fish oil, rich in EPA and DHA (TNBS+n-3) or an isocaloric isolipidic oil formula for 14 days. RESULTS: DHA led to a decreased VCAM-1, TLR4, cyclooxygenase-2 and VEGFR2 expression and a decreased production of IL-6, IL-8 and GM-CSF and a reduced production of PGE(2) and LTB(4) (p < 0.001) in IL-1ß-induced HIMEC. Similarly, dietary intervention with fish oil rich in EPA and DHA significantly decreased colon production of PGE(2) and LTB(4,) endothelial VCAM-1 and VEGFR2 in rats with colitis. CONCLUSIONS: Data obtained from in vitro and in vivo studies reveal a potential anti-angiogenic role of long chain n-3 PUFA in intestinal endothelial cells. This protective effect of long chain n-3 PUFA may partly explain the observed benefit of dietary intake of long chain n-3 PUFA in IBD development.