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.

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.

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.

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.

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.

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.

Changes in estrogen receptor, progesterone receptor, and pS2 expression in tamoxifen-resistant human breast cancer.

Changes in estrogen receptor (ER) expression and function may explain the development of tamoxifen resistance in breast cancer. ER expression was measured by an immunohistochemical assay, validated for use in tamoxifen-treated tumors against a biochemical enzyme immunoassay, in 72 paired biopsies taken before treatment and at progression or relapse on tamoxifen. Progesterone receptor (PgR) and pS2 gene expression were also measured immunohistochemically as an indicator of ER function. Overall the frequency of ER expression was reduced from 37 of 72 (51%) pretamoxifen to 21 of 72 (29%) at progression or relapse, with a significant reduction in the quantitative level of ER (P < 0.0001; Wilcoxon signed rank sum test). Tumors treated with primary tamoxifen that responded but then developed acquired resistance frequently remained ER positive (ER+) at relapse: 16 of 18 (89%) were ER+ pretamoxifen (75% of these expressed either PgR or pS2) and 11 of 18 (61%) were ER+ at relapse (82% continued to express PgR or pS2). In contrast, only 3 of 20 (15%) tumors that progressed on primary tamoxifen with de novo resistance were ER+ pretamoxifen, and all tumors were ER- at progression. At progression, 6 of 20 (30%) of these tumors expressed high levels of PgR (mean H-score, 98) and/or pS2 (mean, 50% cells positive), despite being ER-. In tumors that recurred during adjuvant tamoxifen therapy, including locoregional and metastatic lesions, ER expression was significantly reduced from 18 of 34 (53%) in the original primary tumor to 10 of 34 (29%) at relapse (P = 0.002). PgR expression was likewise significantly reduced in this group (P = 0.001). This study confirms that expression of a functional ER in breast cancer is a strong predictor for primary response to tamoxifen. Although ER was reduced in tamoxifen-resistant tumors overall, the development of acquired resistance was associated with maintained ER expression and function in many tumors, whereas de novo resistance remained related to lack of ER expression. Recurrence during adjuvant tamoxifen was associated with development of an ER/PgR-negative phenotype in some tumors. These data imply that separate mechanisms of resistance may occur in these different clinical subgroups.

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.

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.

Antiproliferative effects of idoxifene in a placebo-controlled trial in primary human breast cancer.

Idoxifene is a novel selective estrogen receptor modulator. It has reduced agonist activity on breast and uterine cells compared with tamoxifen and antiproliferative effects in tamoxifen-resistant breast cancer cells. Previous studies have shown that a short course of treatment with other antiestrogens prior to surgery caused a significant reduction of the growth fraction when measured by immunohistological staining using the mouse monoclonal antibody Ki67. In this study, we assessed the effect of idoxifene on biological markers of cell proliferation (Ki67) and apoptosis (TdT-mediated dUTP-biotin nick end labeling), and estrogen and progesterone receptor (ER/PR) expression was also evaluated. Core-cut biopsies were obtained in 77 postmenopausal patients with primary breast cancer at diagnosis. Patients were randomized to 40 mg/day idoxifene or placebo for 14-21 days prior to obtaining a second biopsy sample at surgical resection. The percentage of Ki67-positive cells fell from a mean 19.7 +/- 2.7% (SE) to 13.4 +/- 3.4% in idoxifene-treated ER-positive tumors (n = 30; P = 0.0043), but there was no significant effect in placebo-treated ER-positive tumors (n = 27). No effect was seen on ER-negative tumors in either group. Idoxifene had no significant effect on apoptotic index but produced a statistically significant fall in idoxifene-treated ER immunohistochemical score and a small increase in PR that did not reach statistical significance (0.05 < P < 0.10). Idoxifene was well tolerated in all patients. Idoxifene has an antiproliferative effect in ER-positive but not ER-negative breast cancers, and no significant effect on apoptosis in the short-term.

Clinical studies of apoptosis and proliferation in breast cancer.

The interaction between cell death and cell proliferation determines the growth dynamics of all tissues. Studies are described here which relate the changes in proliferation and apoptosis that occur in human breast cancer during medical therapeutic manoeuvres. Xenograft studies strongly support the involvement of increased apoptosis as well as decreased proliferation after oestrogen withdrawal, and limited studies in clinical samples confirm the involvement of both processes. Cytotoxic chemotherapy induces increases in apoptosis within 24 h of starting treatment. However, after 3 months therapy the residual cell population shows apoptotic and proliferation indices much below pretreatment levels. Further molecular studies of this "dormant" population are important to characterise the mechanism of their resistance to drug therapy. The early changes in proliferation and apoptosis may provide useful intermediate response indices.

Identification of a cDNA encoding an active asparaginyl endopeptidase of Schistosoma mansoni and its expression in Pichia pastoris.

Asparaginyl endopeptidases, or legumains, are a recently identified family of cysteine-class endopeptidases. A single gene encoding a Schistosoma mansoni asparaginyl endopeptidase (a.k.a. Sm32 or schistosome legumain) has been reported, but by sequence homology it would be expected to yield an inactive product as the active site C197 had been replaced by N. We now describe a new S. mansoni gene in which C197 is present. Both gene products were expressed in Pichia pastoris. Autocatalytic processing to fully active C197 Sm32 occurred at acid pH. In contrast, N197 Sm32 was not processed and this is consistent with the hypothesis that C197 is essential for catalysis. This was confirmed by mutation of N197 to C and re-expression in Pichia. The availability of recombinant active Sm32 allows detailed analysis of its catalytic mechanism and its function(s) in the biology of this important human parasite.

Modulation of Bcl-2 and Ki-67 expression in oestrogen receptor-positive human breast cancer by tamoxifen.

The expression of the bcl-2 proto-oncogene, which is associated with prolonged cell survival and prevention of programmed cell death, was investigated in human primary breast carcinomas prior to and following endocrine therapy with the anti-oestrogen, tamoxifen. Using the BCL-2-100 antibody, a 26-kD protein was detected by western immunoblot in the cytosols of oestrogen receptor (ER)+ve human breast cancers. In a cross-sectional study, the immunohistochemical expression of Bcl-2 was observed in 32% of invasive breast cancers, but in 65% of tumours treated with tamoxifen (P = 0.009). There was a significant association of Bcl-2 with ER status, with 64% of untreated and 88% of tamoxifen-treated Bcl-2-positive tumours being ER+ve. A significantly lower Ki-67 score was found in tamoxifen-treated tumours which were Bcl-2-positive compared with Bcl-2-negative (9.3 versus 24.6%, P = 0.01). In a separate series of sequential Trucut biopsies from 18 patients, the frequency of Bcl-2 expression was increased in ER+ve tumours from 3/12 to 8/11 following tamoxifen (P = 0.04). This was also associated with a significant reduction in mean Ki-67 score from 32 to 12% (P = 0.0004). The observations from this study clearly indicate that Bcl-2 in human breast cancer is associated with ER status, and that expression is enhanced in ER+ve tumours following tamoxifen, in association with reduced cell proliferation.

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.

Comparison of new immunohistochemical assay for oestrogen receptor in paraffin wax embedded breast carcinoma tissue with quantitative enzyme immunoassay.

AIM: To validate the use of a new mouse monoclonal antibody (1D5) directed against the N-terminal domain (A/B region) of the oestrogen receptor in an immunohistochemical assay (ER-IHA) for paraffin wax embedded tissue. METHODS: Breast cancer specimens were surgically obtained from 119 previously untreated patients. For comparison, oestrogen receptor was measured from cytosol fractions using an established oestrogen receptor enzyme immunoassay (ER-EIA) method. Oestrogen receptor "H-scores" were obtained from the ER-IHA after antigen retrieval using microwave treatment. Where discrepancies occurred between the two methods, further immunohistochemistry was performed using the H222 antibody from the Abbott Laboratories ER-ICA kit. RESULTS: The correlation between the two methods was non- linear, but despite this there was an 86% concordance between ER-EIA and ER-IHA using the 1D5 ER antibody. Fifty four per cent of tumours (64/119) were oestrogen receptor positive and 32% (38/119) were negative by both assays. A mismatch between the ER-EIA and ER-IHA occurred in 17 cases. Seven tumours were IHA positive but EIA+, but five of these were borderline negative by EIA, having values of > 5 and < 10 fmol/mg protein. Ten tumours were IHA negative and EIA+; four of these tumours were completely negative by IHA in the section studied. A further IHA assay, carried out on the 17 tumour mismatches with H222 antibody, showed that three tumours remained substantially discordant. These three tumours were strongly positive with the 1D5 antibody and negative with the H222 antibody. Two of these discordant tumours were of the rare ER negative and PgR positive phenotype and may contain oestrogen receptor that is of biological interest but which lacks the hormone binding epitope. CONCLUSIONS: The concordance between the classic enzyme immunoassay technique and the new immunohistochemical method on paraffin wax embedded sections was good. Moreover, the IHA technique using the 1D5 antibody against the N-terminal was easily reproducible. This technique may allow oestrogen receptor content to be determined in large cohorts of patients in whom archival tumour material is available.

Immunohistochemical and biochemical analysis of the oestrogen regulated protein pS2, and its relation with oestrogen receptor and progesterone receptor in breast cancer.

AIMS: (i) To assess the validity of an immunocytochemical technique for detecting pS2 protein in paraffin wax embedded tissue; (ii) to provide further data on the relation between pS2 protein and oestrogen receptor (ER) and progesterone receptor (PgR). METHODS: Breast cancer excision biopsy specimens were obtained from 35 previously untreated patients. An immunoradiometric assay was compared with an immunohistochemical method for measuring pS2 protein. ER and PgR were measured in cytosol fractions by enzyme immunoassay and the relation between the presence of these receptors and pS2 protein was assessed before and after subdivision of the women into groups of over or under 50 years of age. RESULTS: A good correlation was seen between the immunoradiometric and immunohistochemical methods for pS2 protein measurement (r = 0.84; p = 0.0001). Log-transformed data showed a significant correlation between increasing values of ER and pS2 protein (r = 0.45; p = 0.006) and to a lesser extent between pS2 protein and PgR (r = 0.38; p = 0.03). Correlations were also shown between pS2+ and PgR+ status (p = 0.01), and between ER and PgR positivity (p = 0.05; Fisher's exact test). pS2+ protein status was only associated with ER+ status in patients aged 50 years or less. CONCLUSIONS: The two methods for pS2 analysis are virtually interchangeable. This provides strong support for using immunohistochemistry for pS2 in paraffin wax embedded tissue. The association with ER positivity and pS2+ protein status only in the premenopausal patients may be due to the higher levels of oestrogenic stimuli in that group.

Ocular and regional cerebral blood flow in aging Fischer-344 rats.

Vascular remodeling and changes in vascular responsiveness occur in the rat cerebrum with old age. This includes reductions in cerebral arteriolar numerical density, cross-sectional area, distensibility, the relative proportion of distensible elements in the cerebral arteriolar wall, and reduced endothelium-dependent relaxation. The purpose of this study was to test the hypothesis that old age results in an increase in vascular resistance and, correspondingly, a decrease in blood flow to ocular, regional cerebral, and spinal tissue in the rat. Blood flow was measured in the eye, olfactory bulb, left and right cerebrum, pituitary gland, midbrain, pons, cerebellum, medulla, and spinal cord of juvenile (2-mo-old, n = 6), adult (6-mo-old, n = 7), and aged (24-mo-old, n = 7) male Fischer-344 rats. Arterial pressure and blood flow were used to calculate vascular resistance. Vascular resistance in the eye of aged rats (6.03 +/- 1.08 mmHg . ml-1 . min . 100 g) was higher than that in juvenile (3.83 +/- 0.38 mmHg . ml-1 . min . 100 g) and adult rats (3.12 +/- 0.24 mmHg . ml-1 . min . 100 g). Similarly, resistance in the pons of older rats (2.24 +/- 0.55 mmHg . ml-1 . min . 100 g) was greater than in juvenile (0.66 +/- 0.06 mmHg .ml-1 . min . 100 g) and adult rats (0.80 +/- 0.11 mmHg . ml-1 . min . 100 g). In contrast, vascular resistance in the pituitary gland was lower in the aged rats (juvenile, 3.09 +/- 0.22; adult, 2.79 +/- 0.42; aged, 1.73 +/- 0.32 mmHg . ml-1 . min . 100 g, respectively). Vascular resistance was not different in other cerebral tissues or in the spinal cord in the aged rats. These data suggest that regional cerebral and spinal blood flow and vascular resistance remain largely unchanged in conscious aged rats at rest but that elevations in ocular vascular resistance and, correspondingly, decreases in ocular perfusion with advanced age could have serious adverse effects on visual function.