A new direction for tackling phosphorus inefficiency in the UK food system.

The UK food system is reliant on imported phosphorus (P) to meet food production demand, though inefficient use and poor stewardship means P is currently accumulating in agricultural soils, wasted or lost with detrimental impacts on aquatic environments. This study presents the results of a detailed P Substance Flow Analysis for the UK food system in 2018, developed in collaboration with industry and government, with the key objective of highlighting priority areas for system interventions to improve the sustainability and resilience of P use in the UK food system. In 2018 the UK food system imported 174.6 Gg P, producing food and exportable commodities containing 74.3 Gg P, a P efficiency of only 43%. Three key system hotspots for P inefficiency were identified: Agricultural soil surplus and accumulation (89.2 Gg P), loss to aquatic environments (26.2 Gg P), and waste disposal to landfill and construction (21.8 Gg P). Greatest soil P accumulation occurred in grassland agriculture (85% of total accumulation), driven by loadings of livestock manures. Waste water treatment (12.5 Gg P) and agriculture (8.38 Gg P) account for most P lost to water, and incineration ashes from food system waste (20.3 Gg P) accounted for nearly all P lost to landfill and construction. New strategies and policy to improve the handling and recovery of P from manures, biosolids and food system waste are therefore necessary to improve system P efficiency and reduce P accumulation and losses, though critically, only if they effectively replace imported mineral P fertilisers.

Biomarker changes associated with the development of resistance to aromatase inhibitors (AIs) in estrogen receptor-positive breast cancer.

BACKGROUND: The purpose of this study was to identify any differences in key biomarkers associated with estrogen action between biopsies taken at diagnosis and at recurrence or progression during treatment with an aromatase inhibitor (AI). PATIENTS AND METHODS: Patients were retrospectively identified from a clinical database as having relapsed or progressed during AI treatment. Immunohistochemistry was carried out against estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), insulin-like growth factor type-1 receptor (IGF1R), insulin receptor substrate-1 (IRS-1), stathmin, phosphatase and tensin homolog and Ki67. RESULTS: Fifty-five pairs of samples were identified with ER- and/or PgR-positive diseases. Four (7%) patients were ER-negative at progression. Overall, PgR levels were lower in the recurrence sample, but 35% of cases remained positive. IGF1R levels decreased significantly. There were no substantial changes in HER2, IRS-1 or stathmin levels to indicate a role in resistance. Higher Ki67 levels at resistance indicate more proliferative disease. CONCLUSIONS: The phenotype of AI-recurrent lesions shows high between-tumour heterogeneity. There is evidence of an increase in Ki67, a reduction in IGF1R and a loss of ER expression in some individuals and some activation of growth factor signalling pathways that may explain resistance in individuals and merit treatment targeted to those pathways. Biopsy at recurrence will be necessary to identify the relevant target for individuals.

HER2 staining intensity in HER2-positive disease: relationship with FISH amplification and clinical outcome in the HERA trial of adjuvant trastuzumab.

BACKGROUND: Trastuzumab treatment improves survival of HER2-positive primary breast cancer. HER2 staining intensity varies widely in HER2-positive tumours. PATIENTS AND METHODS: We investigated whether differences in immunohistochemical (IHC) staining intensity for HER2 in HER2-positive tumors (IHC 3+ or FISH ratio ≥2.0) was associated with prognosis or benefit from trastuzumab treatment in patients randomized to 1 year or no trastuzumab in the HERceptin Adjuvant (HERA) trial. Median follow-up was 2 years. The nested case-control analysis, included 425 patients (cases) with a disease-free survival (DFS) event and two matched controls (no DFS event) per case. Tissue sections stained for HER2 were assessed for HER2 staining intensity by image analysis. RESULTS: HER2 staining intensity varied widely and correlated with HER2 gene copy number (Spearman, r = 0.498, P < 0.001) or less closely with HER2/CEP17 FISH ratio (r = 0.396, P < 0.001). We found no significant difference in DFS in the observation arm according to staining intensity (odds ratio [OR] change per 10 unit change in intensity: 1.015, 95% confidence interval [CI] 0.930-1.108) and no impact of staining intensity on benefit derived from 1-year trastuzumab (OR: 1.017, 95% CI 0.925-1.120). CONCLUSIONS: Variability in HER2 staining in HER2-positive tumours has no role in clinical management with adjuvant trastuzumab. HERA TRIAL NO: NCT00045032.

Positive and negative affect in individuals with spinal cord injuries.

STUDY DESIGN: Participants with spinal cord injuries (SCIs) and healthy controls completed standardized questionnaires assessing depression level, positive and negative affect, and personality traits. OBJECTIVES: To identify the specific characteristics of emotional experiences affected by spinal cord injury. SETTING: A Canadian rehabilitation center. Individuals with SCIs were recruited from a list of patients who had volunteered to participate in studies being conducted by the SCI clinic. Healthy controls were recruited from the community, but tested in the SCI clinic. METHODS: Thirty-six individuals with complete (ASIA A) SCIs and 36 age-, gender- and education-matched controls participated in this study. SCI participants were classified as cervical (C1-C7), upper thoracic (T1-T5) or lower thoracic/upper lumbar (T6-L2). All participants completed the Beck Depression Inventory, the Positive and Negative Affect Schedules, the NEO Neuroticism Questionnaire, and the harm avoidance scale of the Tridimensional Personality Questionnaire. Data were analyzed using independent-samples t-tests (when contrasting SCI and controls) and analysis of variance (when comparing across SCI groups). RESULTS: Participants with SCIs experienced significantly less positive affect than controls. The two groups did not differ in their experience of negative affect. Participants with SCIs also reported greater levels of depression. Depression scores improved with an increasing number of years post injury. CONCLUSION: Individuals with SCIs are characterized by specific emotional dysfunction related to the experience of positive emotions, rather than a tendency to ruminate on negative emotions. The results suggest that these individuals would benefit from rehabilitation programs that include training in positive psychology.

Enhanced expression of the PDGFR/Abl signaling pathway in aromatase inhibitor-resistant breast cancer.

BACKGROUND: We have found that the platelet-derived growth factor receptor (PDGFR)/Abl signaling pathway is up-regulated as a determinant of the acquisition of resistance to estrogen deprivation in vitro. We aimed to determine its clinical relevance in aromatase inhibitor (AI)-resistant breast cancer. PATIENTS AND METHODS: We identified a cohort of 45 patients with estrogen receptor-positive breast cancer who had been treated with an AI, subsequently relapsed and had biopsy material available from both the presentation and post-AI recurrent lesion. PDGFRα, PDGFRß and Abl expression was assessed in formalin-fixed paraffin-embedded sections. RESULTS: Tumor protein expression of PDGFRα (1.39-fold, P=0.0065), PDGFRß (4.32-fold, P=0.006) and Abl (1.8-fold, P=0.001) was increased at the point of relapse. Tumor and stromal expression of PDGFRα as well as PDGFRß was significantly correlated in pre-treatment and relapse samples. High post-treatment tumor and stromal PDGFRß levels were associated with a short time to treatment failure (TTF). Expression of PDGFRα in relapsing tumor specimens was correlated with Abl expression and Ki67 levels. Furthermore, changes in Abl correlated significantly with changes in ER expression. CONCLUSIONS: These clinical data support a role for enhanced PDGF/Abl signaling in AI-resistant disease and provide a rationale for targeting the pathway in endocrine-resistant breast cancer.

Changes in breast cancer biomarkers in the IGF1R/PI3K pathway in recurrent breast cancer after tamoxifen treatment.

Development of resistance to the antioestrogen tamoxifen occurs in a large proportion of patients with oestrogen receptor-positive (ER+) breast cancer and is an important clinical challenge. While loss of ER occurs in c.20% of tamoxifen-resistant tumours, this cannot be the sole explanation for tamoxifen treatment failure. PI3K pathway activation, including by insulin-like growth factor receptor 1 (IGF1R), has been implicated in some resistance models. The primary aim was to determine whether evidence exists in clinical breast cancer for a role of IGF1R and/or the PI3K pathway, in acquisition of resistance to tamoxifen. Invasive primary and recurrent tamoxifen-resistant tumours from the same patient (n=77) were assessed for changes in ER, progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), IGF1R, stathmin, PTEN expression and PIK3CA mutations where possible. ER and PgR levels were significantly reduced at recurrence with 22 and 45%, respectively, showing negative status at this time. Acquisition of HER2 overexpression occurred in 6% of cases. IGF1R expression was significantly reduced in both ER+ and ER- recurrences and stathmin levels increased. A positive association between stathmin and IGF1R emerged in recurrent samples, despite their opposing relationships with ER, suggesting some coalescence of their activities may be acquired. The data confirm loss of ER and PgR and gain of HER2 in some tamoxifen-resistant tumours. There is no evidence for IGF1R gain in tamoxifen resistance; increases in stathmin levels suggest that activation of the PI3K pathway may have contributed, but PTEN loss and PIK3CA hotspot mutations were relatively rare.

High-efficiency genotype analysis from formalin-fixed, paraffin-embedded tumor tissues.

Single-nucleotide polymorphisms (SNPs) can be assayed using DNA isolated from archival formalin-fixed, paraffin-embedded (FFPE) samples, making retrospective pharmacogenetic studies possible. In this study, we describe methods that significantly increase the number of SNP determinations possible using FFPE samples. Quantifying the amount of DNA amenable to PCR (amplification-quality DNA, AQ-DNA) allows a significant reduction in the amount of sample required for Taqman-based SNP assays. Optimizing AQ-DNA input increases PCR amplification efficiency and SNP determination accuracy. DNA was extracted from 39 FFPE tumor sections and matched tumor and stromal cores, which were of the type used to generate tissue microarrays. Sections and tumor cores yielded sufficient AQ-DNA for more than 1000 SNP determinations. Seven SNPs were assessed following individual assay optimization for minimal AQ-DNA. Genotypes from tumor cores for single SNPs were 92.3-100% concordant with those obtained from sections. Using these methods, the number of SNP genotypes that can be determined from single FFPE samples is greatly increased expanding the genetic association studies possible from limited archival specimens. The use of tumor cores is of particular importance as the harvesting of tumor cores has minimal impact on the utility of the donor blocks for other purposes.

Variability in bleeding phenotype in Amish carriers of haemophilia B with the 31008 C-->T mutation.

The aim of this study was to characterize the variability of bleeding phenotype and its association with plasma factor IX coagulant activity (FIX:C) in haemophilia B carriers in a large Amish pedigree with a unifying genetic mutation, C-to-T transition at base 31008 of the factor IX gene (Xq27.1-27.2). A cross-sectional survey of haemophilia B carriers included a multiple choice questionnaire evaluating symptoms of mucocutaneous bleeding, joint bleeding and bleeding after haemostatic stress [menstruation, postpartum haemorrhage (PPH), dental extractions and invasive surgeries]. Severity of bleeding was graded as 0 to 4, 0 being no bleeding whereas 4 being severe bleeding. Association between total bleeding scores and the FIX:C was evaluated. Sixty-four haemophilia B carriers participated in this study. Median age: 18 years (range 1-70 years); median bleeding score: 1 (range 0-8). Besides PPH, isolated symptoms of bruising, epistaxis, menorrhagia and postsurgical bleeding including dental extraction were not associated with lower FIX:C. Bleeding score >/=3 was associated with involvement of at least two bleeding sites and a lower mean FIX:C of 42 +/- 10.3% (95% CI 36.4-47.7) while a score >3 had involvement of /=3. Phenotypic variability existed among the carriers of haemophilia B who belonged to a single pedigree carrying a single unifying mutation. The utility of bleeding scores to define bleeding phenotype precisely in haemophilia B carriers needs further evaluation.

Clinical trial: effects of tegaserod on gastric motor and sensory function in patients with functional dyspepsia.

BACKGROUND Tegaserod, a serotonin receptor type-4 partial agonist, stimulates gastrointestinal motility and has been shown to increase gastric volumes before and after a meal in healthy volunteers. Its effect on gastric motor and sensory function in patients with functional dyspepsia is unclear. AIM To evaluate the effects of tegaserod on gastric compliance, accommodation and gastric sensory function in patients with functional dyspepsia and healthy volunteers. METHODS Sixteen patients with functional dyspepsia and 12 healthy volunteers were studied on two occasions, each after a 7-day treatment with either placebo or tegaserod 6 mg b.d. using a double-blind, randomized, crossover design. After each treatment period a gastric barostat study was performed fasting and during intraduodenal lipid infusion. RESULTS Tegaserod increased postprandial gastric compliance in functional dyspepsia patients (P = 0.04). Healthy volunteers showed enhanced postprandial gastric compliance after placebo (P = 0.03). Between-treatment analysis of gastric accommodation revealed a significant increase in intrabag volumes after tegaserod in healthy volunteer (P = 0.04); no difference could be seen in functional dyspepsia patients. Tegaserod had no effect on gastric sensation. CONCLUSIONS Tegaserod enhances postprandial gastric compliance in functional dyspepsia patients and gastric accommodation in healthy volunteers. The improvement of proximal gastric motor function suggests a beneficial role of tegaserod in patients with functional dyspepsia.

Microarray-based comparative genomic hybridisation of breast cancer patients receiving neoadjuvant chemotherapy.

We analysed the molecular genetic profiles of breast cancer samples before and after neoadjuvant chemotherapy with combination doxorubicin and cyclophosphamide (AC). DNA was obtained from microdissected frozen breast core biopsies from 44 patients before chemotherapy. Additional samples were obtained before the second course of chemotherapy (D21) and after the completion of the treatment (surgical specimens) in 17 and 21 patients, respectively. Microarray-based comparative genome hybridisation was performed using a platform containing approximately 5800 bacterial artificial chromosome clones (genome-wide resolution: 0.9 Mb). Analysis of the 44 pretreatment biopsies revealed that losses of 4p, 4q, 5q, 12q13.11-12q13.12, 17p11.2 and 17q11.2; and gains of 1p, 2p, 7q, 9p, 11q, 19p and 19q were significantly associated with oestrogen receptor negativity. 16q21-q22.1 losses were associated with lobular and 8q24 gains with ductal types. Losses of 5q33.3-q4 and 18p11.31 and gains of 6p25.1-p25.2 and Xp11.4 were associated with HER2 amplification. No correlations between DNA copy number changes and clinical response to AC were found. Microarray-based comparative genome hybridisation analysis of matched pretreatment and D21 biopsies failed to identify statistically significant differences, whereas a comparison between matched pretreatment and surgical samples revealed a statistically significant acquired copy number gain on 11p15.2-11p15.5. The modest chemotherapy-driven genomic changes, despite profound loss of cell numbers, suggest that there is little therapeutic selection of resistant non-modal cell lineages.

Benefit from adjuvant tamoxifen therapy in primary breast cancer patients according oestrogen receptor, progesterone receptor, EGF receptor and HER2 status.

BACKGROUND: Most women with oestrogen receptor (ER) positive primary breast cancer receive adjuvant tamoxifen after surgery. The measurement of tumour biomarkers should allow better selection of patients for such treatment or for therapies such as aromatase inhibitors. PATIENTS AND METHODS: Histopathological blocks of primary breast cancer patients who had been randomized to receive 2-years tamoxifen or no adjuvant therapy in two mature randomised clinical trials were retrieved. Immunohistochemical staining for ER, progesterone receptor (PgR), HER2 and epidermal growth factor receptor (EGFR) was undertaken. The primary endpoint was relapse free survival. RESULTS: 813 patients were included in the study. Benefit from tamoxifen was seen in ER-positive patients [Relative risk (rr) 0.77, ci 0.63-0.93]. ER-negative patients also showed a strong trend to benefit from tamoxifen (rr 0.73, ci 0.52-1.02) which was largely confined to the PgR-positive group. Amongst the ER-positive group, PgR-positive and PgR-negative patients showed similar benefit (rr 0.81; ci 0.65-1.02 and 0.70; ci 0.49-0.99, respectively). Patients positive for HER2 did not benefit significantly (rr 1.14; ci 0.75-1.73) but this group was small. CONCLUSIONS: Measurement of PgR status in ER-negative patients defines a group of patients that benefit from tamoxifen but would be excluded from tamoxifen therapy on the basis of ER status alone. The data are consistent with HER2 positive tumours being resistant to tamoxifen.

Growth factor signalling and response to endocrine therapy: the Royal Marsden Experience.

De novo resistance to endocrine therapy is a near-universal feature of oestrogen receptor (ER)- negative breast cancer. Although many ER-positive breast cancers also show no response to tamoxifen or aromatase inhibitors on objective clinical grounds the large majority show reduced proliferation indicating that some oestrogen dependence is present in almost all ER-positive breast cancer. In neoadjuvant studies HER2 positivity is associated with poor response rates to tamoxifen but not aromatase inhibitors, consistent with preclinical models. Acquired resistance to tamoxifen is associated with decreases in ER positivity but most recurrent lesions remain ER-positive. A small proportion of these show increased HER2 expression and in these patients increased phospho-p38 may contribute to the tamoxifen-resistant phenotype. There is an unfortunate paucity of clinical and biological data on acquired resistance to aromatase inhibitors.

Studies of the potential utility of Ki67 as a predictive molecular marker of clinical response in primary breast cancer.

INTRODUCTION: Objectives were to characterise the relationship of the proliferation marker Ki67 with response to systemic treatment in early breast cancer and to assess its clinical utility, using fine needle aspirates. MATERIALS AND METHODS: Hundred and six women were treated with primary tamoxifen (n = 33), chemotherapy (n = 33) or chemotherapy and tamoxifen (n = 40). Treatment was not randomised and response was assessed clinically. Ki67 was evaluated prior to treatment and at Day 14 or 21 after commencing treatment. To assess reproducibility, Ki67 was evaluated in repeat FNAs taken from 37 untreated patients. RESULTS: The percentage change in Ki67 in first 21 days was different between responders and non-responders for patients treated with tamoxifen (p = 0.007) and chemotherapy (p = 0.005) but not for chemoendocrine treatment (p = 0.062). The reproducibility study indicated that a decrease to 36% or less of the pre-treatment Ki67 value in an individual patient was required for it to be regarded as a statistically significant change. A significant decrease in Ki67 was seen in responding patients treated with chemotherapy (p = 0.026) and chemoendocrine treatment (p = 0.041). Positive and negative predictive values for response were 85 and 59% for chemotherapy patients and 88 and 54% for chemoendocrine patients, respectively. CONCLUSION: Ki67 is unlikely to be useful as a predictive marker in individual patients. Further molecular markers that predict lack of response continue to be required.

Influence of tegaserod on proximal gastric tone and on the perception of gastric distension.

BACKGROUND: Tegaserod, a 5-hydroxytryptamine-4 receptor agonist, enhances gastric emptying, but its effects on proximal stomach function have not been studied. AIM: To study the effect of tegaserod on gastric compliance, accommodation and perception of distension in humans. METHODS: Nineteen healthy volunteers (10 females; mean age, 23.9 years) were studied on three separate occasions after 7 days of treatment with placebo, tegaserod 2 mg b.d. or tegaserod 6 mg b.d. in a double-blind, randomized, three-way cross-over design. After the introduction of a barostat bag, stepwise distensions were performed to determine gastric compliance and sensitivity, and a mixed liquid meal was administered in isobaric mode to assess accommodation. RESULTS: Tegaserod had no effect on the pressures or volumes inducing first perception or discomfort. Tegaserod 6 mg b.d. enhanced fasting gastric compliance compared with placebo. Pre-prandial and post-prandial intra-balloon volumes were significantly higher after 6 mg b.d. than after placebo. Both tegaserod 2 and 6 mg b.d. shortened the time to maximum post-prandial intra-balloon volume. The amplitude of meal-induced gastric relaxation (post-prandial minus pre-prandial volumes) did not differ between the treatment arms. CONCLUSION: In humans, tegaserod allows for larger intra-balloon volumes both before and after a meal. These findings warrant the investigation of the therapeutic potential of tegaserod in dyspeptic patients with impaired accommodation.

Early changes in apoptosis and proliferation following primary chemotherapy for breast cancer.

Patients undergoing primary chemotherapy for invasive breast cancer consented to a core biopsy of the invasive breast primary pre- and 24 h postchemotherapy. The resulting tissue was analysed for apoptosis, Ki67, ER and HER-2 using immunohistochemical techniques. These data were then used to evaluate the relationship between these biological markers and response to chemotherapy and overall survival. Response rate to chemotherapy in this group was 86%, 16 patients (25%) achieved a clinical complete response and 41 (63%) a partial response. Prechemotherapy there was a significant correlation between Ki67 and apoptotic index (AI), r=0.6, (P<0.001). A significant rise in AI (P<0.001), and fall in Ki67 (P=0.002) was seen 24 h following chemotherapy. No relationship was seen between pretreatment AI and clinical response, but higher Ki67 and growth index (Ki67/AI ratio, GI) did correlate with clinical response (both r=0.31, P<0.025). No correlation was seen between the change in AI or Ki67 at 24 h and clinical response or survival. Significant changes in apoptosis and proliferation can be demonstrated 24 h following chemotherapy, but these changes do not relate to clinical response or outcome in this study. Pretreatment proliferation and GI are however predictive of response to chemotherapy in breast cancer.

Correlation between immunohistochemistry (HercepTest) and fluorescence in situ hybridization (FISH) for HER-2 in 426 breast carcinomas from 37 centres.

Accurate diagnostic assessment of HER-2 is essential for the appropriate application of the humanized anti-HER-2 monoclonal antibody trastuzumab (Herceptin) to the treatment of patients with metastatic breast cancer. The diagnostic test needs to be applicable to archival, fixed tissue removed at excision, in many cases several years earlier. We compared the assessment of HER-2 by immunohistochemistry (IHC; HercepTest) and fluorescence in situ hybridization (FISH) in 426 breast carcinomas from patients being considered for trastuzumab therapy. The tumours were tested in three reference centres having been sent in from 37 hospitals. Only 2/270 (0.7%) IHC 0/1+ tumours were FISH positive. Six of 102 (5.9%) IHC 3+ tumours were FISH negative. Five of the six had between 1.75 and 2.0 HER-2 gene copies per chromosome 17 and the sixth had multiple copies of chromosome 17. Thirteen per cent of tumours were IHC 2+ and overall 48% of these were FISH positive but this proportion varied markedly between the centres. Sixty IHC-stained slides selected to be enriched with 2+ cases were circulated between the three laboratories and scored. There were 20 cases in which there was some discordance in scoring. Consideration of the FISH score in these cases led to concordance in the designation of positivity/negativity in 19 of these 20 cases. These data support an algorithm in which FISH testing is restricted to IHC 2+ tumours in reference centres. The results may not extrapolate to laboratories with less experience or using different methodologies.

HER-2 amplification impedes the antiproliferative effects of hormone therapy in estrogen receptor-positive primary breast cancer.

In experimental models, human epidermal growth factor receptor-2 (HER-2) amplification leads to estrogen independence and tamoxifen resistance in estrogen receptor (ER)-positive human breast cancer cells. Some but not all reports suggest an association between HER-2 positivity and hormone independence in breast cancer patients. This study aimed to evaluate the antiproliferative effects of endocrine therapy in HER-2-positive/ER-positive primary human breast cancer. The effect on proliferation (Ki67) of hormone therapy was assessed at 2 weeks and/or 12 weeks in biopsies from 115 primary breast cancers with ER-positive tumors. The patients took part in one of 3 neoadjuvant trials of hormonal therapy with a SERM (tamoxifen or idoxifene) or an aromatase inhibitor (anastrozole or vorozole). HER-2 status was assessed by immunocytochemistry and fluorescence in situ hybridization (FISH). Fifteen patients were defined as HER-2 positive by both immunohistochemistry and FISH, with the remaining 100 patients HER-2 negative. Geometric mean Ki67 levels were substantially higher in HER-2-positive than HER-2-negative tumors (27.7% versus 11.5%, respectively; P = 0.003). In HER-2-negative patients, Ki67 was reduced by 62 and 71% at 2 and 12 weeks, respectively (P < 0.0001 for both), but HER-2-positive patients showed no significant fall. The proportional change in Ki67 was significantly different between HER-2-positive and -negative patients (P = 0.014 at 2 weeks; P = 0.047 at 12 weeks). Mean ER levels were lower in the HER-2-positive patients (P = 0.06) but the change in Ki67 was impeded even in those with high ER. Apoptotic index was reduced by 30% at 2 weeks in the HER-2-negative group. However, there were no statistically significant differences in apoptotic index between the groups. It is concluded that ER-positive/HER-2-positive primary breast carcinomas show an impeded antiproliferative response to endocrine therapy that nonetheless may vary between individual treatments. This together with high baseline proliferation is likely to translate to poor clinical response.

Platelets modulate ischemia/reperfusion-induced leukocyte recruitment in the mesenteric circulation.

P-selectin-dependent leukocyte-endothelial cell adhesion has been implicated in the pathogenesis of ischemia/reperfusion (I/R) injury in several vascular beds, including the gut. Because platelet-endothelial (P/E) cell adhesion also occurs in postischemic venules, the possibility exists that the expression of P-selectin on the surface of platelets that are adherent to venular endothelial cells may mediate the leukocyte recruitment elicited by I/R. P-selectin expression [dual radiolabeled monoclonal antibody (MAb) technique] and neutrophil accumulation [myeloperoxidase (MPO) activity] were measured in the postischemic small intestine of untreated rats and rats treated with either antiplatelet serum (APS) or MAbs directed against either P-selectin, GPIIb/IIIa, or fibrinogen. The increases in P-selectin expression and tissue MPO normally elicited by I/R were significantly attenuated in the different treatment groups, suggesting that I/R-induced neutrophil recruitment is a platelet-dependent, P-selectin-mediated process. Intravital microscopy was then employed to examine this process relative to leukocyte-endothelial cell adhesion in postischemic rat mesenteric venules. The recruitment of adherent and emigrated leukocytes after I/R was attenuated by pretreatment with a MAb against, either P-selectin, GPIIb/IIIa, or fibrinogen, as well as an Arg-Gly-Asp peptide. Whereas thrombocytopenia greatly blunted leukocyte emigration, it did not alter the leukocyte adherence response to I/R. These findings suggest that platelet-associated P-selectin contributes to the accumulation of leukocytes in postischemic tissue via a mechanism that alters transendothelial leukocyte migration.

Regulation of murine intestinal inflammation by reactive metabolites of oxygen and nitrogen: divergent roles of superoxide and nitric oxide.

Several reports have implicated reactive oxygen and nitrogen metabolites (RONS) in the initiation and/or progression of inflammatory bowel diseases (IBDs). We have investigated the role of three key RONS-metabolizing enzymes (inducible nitric oxide synthase [iNOS], superoxide dismutase [SOD], nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) in a murine model of IBD. Mice genetically deficient ((-/-)) in either iNOS or the p47phox subunit of NADPH oxidase, transgenic (Tg) mice that overexpress SOD, and their respective wild-type (WT) littermates were fed dextran sulfate sodium (DSS) in drinking water for 7 days to induce colitis. In addition, the specific iNOS inhibitor 1400W was used in DSS-treated WT and p47phox(-/-) mice. WT mice responded to DSS feeding with progressive weight loss, bloody stools, elevated serum NO(X) and colonic mucosal injury with neutrophil infiltration. Both the onset and severity of colitis were significantly attenuated in iNOS(-/-) and 1400W-treated WT mice. While the responses to DSS did not differ between WT and p47phox(-/-) mice, enhanced protection was noted in 1400W-treated p47phox(-/-) mice. Interestingly, SOD(Tg) mice exhibited more severe colitis than their WT littermates. These findings reveal divergent roles for superoxide and iNOS-derived NO in intestinal inflammation.

Role of intercellular adhesion molecule 1 in indomethacin-induced ileitis.

Adhesion molecules have been implicated in the pathogenesis of inflammatory bowel diseases. We investigated their expression and contribution to leukocyte recruitment in experimental intestinal inflammation. Ileitis was induced in Sprague-Dawley rats by two injections of indomethacin (7.5 mg/kg), given 24 h apart. Endothelial intercellular adhesion molecule-1 (ICAM-1) expression was quantified using the dual radiolabeled monoclonal antibody technique and Mac-1 (CD11b/CD18) expression on leukocytes by flow cytometry. Leukocyte infiltration was monitored by tissue myeloperoxidase (MPO) activity. The first indomethacin injection induced a time- and site-dependent increase of ICAM-1 expression in ileal mucosa and muscularis. The second injection resulted in a reduction of ICAM-1 expression below constitutive levels whereas Mac-1 was upregulated. MPO changes paralleled lesion development over 48 h. ICAM-1 and MPO values were correlated for the first 24 h. Immunoneutralization of either ICAM-1 or Mac-1 attenuated mucosal injury. We conclude that (i) indomethacin-induced ileitis is associated with a temporally disassociated upregulation of ICAM-1 and (ii) despite a reduction in ICAM-1 after 24 h, ICAM-1, in concert with Mac-1, contributes to mucosal injury and leukocyte infiltration elicited by indomethacin.