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The distinction between nevoid melanoma and a mitotically active nevus can be challenging at the microscopic level. In this study, we performed cytogenetic testing on a cohort of 25 mitotically active melanocytic proliferations resembling common melanocytic nevus from 25 patients. Based on cytogenetic findings, the lesions were classified as "nevoid melanoma" (n = 13) or "mitotically active nevus" (n = 12). Subsequently, we compared the clinicopathological features between these 2 groups. Nevoid melanomas occurred in older patients (P = 0.007); however, there were no significant differences in gender, size, or anatomical distribution between the 2 groups. Histologically, deep/marginal mitoses (P = 0.006), lack of maturation with depth (P = 0.036), and pseudo-maturation (P = 0.006) were significantly more common in nevoid melanomas. Immunohistochemically, complete loss of p16 was an important divisive feature (P = 0.0004), seen in 70% of nevoid melanomas, and highly correlated with loss of CDKN2A gene (chromosome 9p21). Our findings suggest that such reproducible immunomorphological differences can be of value in distinguishing nevoid melanoma from mitotically active nevus. Nevoid melanomas demonstrated a spectrum of chromosomal aberrations similar to those seen in common subtypes of melanoma, which can serve as a powerful adjunct diagnostic tool in morphologically challenging lesions.

Increasing evidence points to the presence of low-level de novo T790M mutations in patients with non-small cell lung carcinoma (NSCLC) harboring activating EGFR mutations. We utilized digital PCR (dPCR), a highly sensitive gene mutation detection method, to detect pre-treatment T790M mutations in NSCLC tumor samples and correlated the T790M status with clinical features and patient outcomes. DNA extracted from pre-treatment NSCLC tumor tissue with known activating EGFR mutations, diagnosed between October 2010 and May 2017 at PathWest laboratory, was used to perform targeted dPCR for quantitative detection of T790M mutations. T790M was detected in 42 of 109 pre-treatment samples (38.5%). Median variant allele frequency was 0.14% (range 0.02–28.5%). Overall response rate to first generation EGFR tyrosine kinase inhibitors (TKI) was 67% regardless of T790M status. The median progression free survival was 10.7 (IQR 5.6–19.9) versus 6.7 (IQR 3.5–20.8) months in T790M negative and positive patients respectively. T790M positivity correlated with increased rate of early disease progression. It also correlated with increased mortality (HR 3.1 95%CI 1.2–8.1, p = 0.022) in patients who did not respond to TKI treatment. We detected a significant rate of low-level pre-treatment T790M mutations in NSCLC using highly sensitive dPCR. Low-level pre-treatment T790M did not impact treatment response rate or overall survival, but was associated with increased rate of early progression on TKI therapy.

Summary Ki-67 proliferative index (PI) has prognostic and predictive value in invasive breast carcinoma (IBC), but clinical uptake has been hampered by suboptimal accuracy, reproducibility and standardisation. Published guidelines have addressed pre-analytical and analytical factors to improve Ki-67 PI utility; however, practicalities of ongoing monitoring of Ki-67 PI quality in IBC reporting have not been established. We aimed to evaluate the internal and external quality of our established digital Ki-67 PI IBC reporting practice at a tertiary institution. In the 5 years since initial validation work, we've completed a series of internal and external quality assurance (QA) projects: (1) an interobserver agreement study, (2) a two site interlaboratory agreement study, (3) determination of the error of our Ki-67 results, (4) an audit of the year-to-year Ki-67 values, (5) an audit of Ki-67 in neoadjuvant chemotherapy (NAC) treated cases, and (6) comparison of our Ki-67 datasets with similar published datasets. There was excellent concordance (intra-class correlation = 0.98) and good agreement [kappa (κ) = 0.76–0.96] between pathologists, excellent concordance [Pearson correlation (R) = 0.94] and very good agreement (κ = 0.80) between laboratories and excellent concordance (R = 0.92–0.95) and good agreement (κ = 0.67–1.0) over time for our Ki-67 results. No significant difference was observed in Ki-67 data from year-to-year. Expected associations with clinico-pathological prognosticators, pathological complete response following NAC and mitotic index were evident. The median Ki-67 values from the overall and NAC treated datasets were within the range reported in other studies, and our data could be separated into similarly proportioned ‘high’ and ‘low’ Ki-67 PI groups when dichotomised as per protocols in other studies. Collectively, our work provides evidence of adequate internal and external quality control for our digital Ki-67 PI IBC reporting protocols. Given the paucity of formal Ki-67 QA programs, our approach could be emulated, and results compared between laboratories as a framework for internal and external Ki-67 QA.

Abstract. Objective: Fecal incontinence and constipation are common disorders in childhood. The enteric nervous system and the central nervous system are highly interactive along the brain-gut axis. The interaction is mainly afferent. These afferent pathways include centers that are involved in the central nervous processing of emotions as the mid/posterior insula and the anterior cingulate cortex. A previous study revealed altered processing of emotions in children with fecal incontinence. The present study replicates these results. Methods: In order to analyze the processing of emotions, we compared the event-related potentials of 25 children with fecal incontinence and constipation to those of 15 control children during the presentation of positive, negative, and neutral pictures. Results: Children with fecal incontinence and constipation showed altered processing of emotions, especially in the parietal and central cortical regions. Conclusions: The main study results of the previous study were replicated, increasing the certainty and validity of the findings.

Summary Distinction between melanocytic naevi and melanoma occasionally poses a diagnostic challenge in ambiguous cases showing overlapping histological features. Melanomas are characterised by the presence of multiple genomic copy number variants (CNVs), while this is not a feature of naevi. We assessed the feasibility and utility of array-based comparative genomic hybridisation (aCGH) to assess CNVs in melanocytic lesions. DNA was extracted from formalin fixed, paraffin embedded (FFPE) sections of unambiguous naevi ( n = 19) and melanomas ( n = 19). The test DNA and gender mismatched human reference DNA were differentially labelled with fluorophores. Equal quantities of the two DNA samples were mixed and co-hybridised to a SurePrint G3 Human CGH 8x60K array, and digitally scanned to capture and quantify the relative fluorescence intensities. The ratio of the fluorescence intensities was analysed by Cytogenomics software (Agilent). Frequent large CNVs were identified in 94.7% of melanoma samples, including losses of 9p (73.6%), 9q (52.6%), 10q (36.8%), 11q (36.8%), 3p (21%), and 10p (21%), and gains of 6p (42.1%), 7p (42.1%), 1q (36.8%), 8q (31.5%) and 20q (21%). Only one naevus showed two large copy number changes. Overall aCGH showed a specificity and sensitivity of 94.7% in separating naevi from melanomas. Based on our results, aCGH can be successfully used to analyse CNVs of melanocytic lesions utilising FFPE derived biopsy samples, providing a potentially useful adjunctive test for the classification of diagnostically challenging melanocytic proliferations.

Invasive lobular carcinoma (ILC) is almost always classified as Nottingham histological grade 2. Despite this, prognosis is markedly varied, with some ILCs behaving more akin to grade 3 invasive ductal carcinoma (IDC). Methods to separate these aggressive ILCs are needed. Digital image analysis (DIA) of the Ki-67 biomarker has potential in this regard; thus, we sought to determine the feasibility of its use for automated evaluation of ILC. An initial pilot study demonstrated no ILC specific changes were required to our Ki-67 DIA algorithm for reproducible results. Subsequently, 42 consecutive cases of ILC were evaluated by visual mitosis counting in HE p

Mitosis counting in HE stained sections is the most informative constituent of the Nottingham histological grade in breast carcinoma prognosis. Phosphohistone H3 (PHH3) immunohistochemistry (IHC) is a highly specific marker of mitoses, with practical application in identifying mitoses in poorly fixed or distorted tissue and is of prognostic significance in breast carcinoma. Our aim was to assess methods of PHH3 IHC mitosis counting in a tissue microarray (TMA) of 2 mm cores from 36 resected breast carcinomas. Mitoses in HE and PHH3 stained slides were manually scored by pathologist consensus and expressed as counts/2 mm. PHH3 stained cores were also evaluated by automated digital image analysis (DIA). Results were compared using Spearman correlation. A strong and significant correlation was observed between manual PHH3 and manual HE mitotic counts (correlation = 0.81; p 0.0001) and between automated PHH3 DIA and manual HE mitotic counts (correlation = 0.79; p 0.0001). More mitoses were identified with PHH3 IHC than with HE. Manual and DIA PHH3 counts were strongly and significantly correlated (correlation = 0.83; p 0.0001) and of similar absolute values. PHH3 DIA is a valid alternative to manual counting with potential application in breast cancer reporting and prognostication.

Hairy cell leukaemia (HCL) is a rare, indolent chronic B-cell leukaemia accounting for approximately 2% of all adult leukaemias. The recent association of the BRAF p.Val600Glu (V600E) mutation in HCL makes it a valuable molecular diagnostic marker. We compared the ability of Sanger sequencing, fluorescent single-strand conformational polymorphism (F-SSCP) and high resolution melting (HRM) analysis to detect BRAF mutations in 20 cases of HCL consisting of four archival Romanowsky stained air-dried peripheral blood and bone marrow aspirate smears, 12 mercury fixed decalcified bone marrow trephine biopsies, three formalin fixed, paraffin embedded (FFPE) splenectomy samples and one fresh peripheral blood sample. DNA was amplified and BRAF mutation status determined by the three methods above. V600E mutation was identified in 94%, 89% and 72% of HCL cases by F-SSCP, HRM and Sanger sequencing, respectively. In one case, in addition to the p.Val600Glu mutation, a p.Lys601Thr (K601T) mutation was identified. DNA from archival slide scrapings, mercury-fixed and FFPE tissue can be used to identify BRAF mutations with high sensitivity, especially using HRM/F-SSCP. The V600E mutation can be used as a supplementary molecular marker to aid in the diagnosis of HCL and the presence of the mutation may provide a target for therapy.

Serosal pathologies including malignant mesothelioma (MM) can show features of osseous and/or cartilaginous differentiation although the mechanism for its formation is unknown. Mesothelial cells have the capacity to differentiate into cells with myofibroblast, smooth muscle and endothelial cell characteristics. Whether they can differentiate into other cell types is unclear. This study tests the hypothesis that mesothelial cells can differentiate into cell lineages of the embryonic mesoderm including osteoblasts and adipocytes. To examine this, a functional assay of bone formation and an adipogenic assay were performed in vitro with primary rat and human mesothelial cells maintained in osteogenic or adipogenic medium (AM) for 0–26 days. Mesothelial cells expressed increasing levels of alkaline phosphatase, an early marker of the osteoblast phenotype, and formed mineralized bone-like nodules. Mesothelial cells also accumulated lipid indicative of a mature adipocyte phenotype when cultured in AM. All cells expressed several key osteoblast and adipocyte markers, including osteoblast-specific runt-related transcription factor 2, and demonstrated changes in mRNA expression consistent with epithelial-to-mesenchymal transition. In conclusion, these studies confirm that mesothelial cells have the capacity to differentiate into osteoblast- and adipocyte-like cells, providing definitive evidence of their multipotential nature. These data strongly support mesothelial cell differentiation as the potential source of different tissue types in MM tumours and other serosal pathologies, and add support for the use of mesothelial cells in regenerative therapies.

Prospective surveillance programmes to monitor the incidence of surgical-site infection (SSI) in patients who have had orthopaedic implant surgery can be difficult to implement due to limited human and technical resources. In addition, prolonged patient follow-up, up to one year, may be required. Traditional methods of surveillance can be enhanced by using administrative databases to assist in case finding and facilitate overall surveillance activities. The aim of this study was to identify the incidence of SSI in patients who had undergone total hip replacement (THR) or total knee replacement (TKR) surgery in all Western Australian (WA) hospitals during 1999 using the Western Australian Data Linkage System. The WA Data Linkage System links several population-based administrative health datasets within the state, including the Hospital Morbidity Data System (HMDS), containing International Classification of Disease-coded discharge information, and mortality records. A total of 1476 THR and 1875 TKR procedures was identified from 21 WA hospitals during 1999. The incidence of SSI after these procedures was 5% (95% CI 4.3-5.7) [THR (4.86%, 95% CI 3.77-5.95) and TKR (5.15%, 95% CI 4.15-6.15)]. The incidence was 33.72 infections per 1000 person-years. Patients aged over 80 years experienced a significantly higher rate of infection after THR compared with patients aged 80 or less (z-test, z = 2.56, P = 0.015), but not for TKR. No patients with an SSI died during follow-up. The WA Data Linkage System provided a unique opportunity to review the incidence of SSIs in patients undergoing THR or TKR surgery in WA hospitals.

Absorption of non-haeme iron occurs mainly in the duodenum. It involves the divalent metal transporter 1 (DMT1) in the uptake of ferrous Fe(II) iron and the basolateral transporter ferroportin/IREG-1/MTP-1/SLC40A1 in its release. Whether ferroportin functions in this process at other sites in the enterocyte is unknown. In this study the effect of a blocking antibody to ferroportin on the uptake and release of iron was evaluated in enterocyte-like cells (IEC-6 and Caco-2) and in freshly isolated duodenal enterocytes from rats.Uptake of 1 microM Fe(II) and its release by cells was studied in the presence of the antibody. Ferroportin expression was determined by western blot analysis of duodenal mucosa enriched microvillus membranes, Caco-2 cells, IEC-6 cells, and freshly isolated enterocytes. Immunofluorescent detection of ferroporitn was performed on frozen sections of duodenum from rats with variations in body iron stores.Ferroportin was expressed in all cell types. In these cells, the antibody significantly reduced (p0.05) uptake of Fe(II) by 40-50% but had no effect on the release of iron. In Caco-2 cells, Fe(II) uptake was reduced only when the antibody was in contact with the apical membrane. Ferroportin protein was enriched in microvillus membrane preparations. In enterocytes from iron deficient rats, ferroportin was expressed along the brush border where it colocalised with lactase. Ferroportin was seen in the basal cytoplasm and along the basolateral membranes. Iron loading markedly reduced intracellular expression of ferroportin. In Caco-2 cells, ferroportin also localised to the microvillus and lateral and basal membranes.In addition to release, ferroportin functions in the uptake of iron at the apical membrane, possibly by modulating the activity of DMT1.

Regulation of iron absorption, which is the primary mechanism for maintaining body iron stores, occurs primarily in the proximal small intestine. Recent identification of proteins that are involved in iron absorption such as the uptake transporter-divalent metal transporter (DMT1), the basolateral transporter, IREG1, and the ferroxidase-hephaestin provide new opportunities to study this process. We evaluated the rat intestinal cell line, IEC-6, as a model of intestinal iron transport. This involved measuring the expression of DMT1 and IREG1 by Western blot analysis and confocal microscopy, and hephaestin by protein-dependent copper oxidase activity. DMT1 and IREG1 were expressed in IEC-6 cells. The uptake of 1 micromol/L ferrous iron [Fe(II)]:ascorbate and its efflux also was associated with the expression of DMT1 under different levels of iron loading. The expression of DMT1 changed inversely with iron levels as did the uptake of Fe(II). However, with different levels of cellular iron, IREG1 expression remained constant, as did the release of iron from the cells, suggesting that they could be related. Ceruloplasmin and apotransferrin did not enhance the rate or extent of iron release. Copper oxidase activity, considered to indicate hephaestin activity, was detected only intracellularly. Confocal microscopy showed DMT1 and IREG1 on the cell membrane of IEC-6 cells at 4 degrees C but at intracellular locations at 37 degrees C, indicating that these proteins can function at the cell membrane and intracellularly. In terms of iron absorption, IEC-6 cells have a villous enterocyte phenotype and are regulated by iron stores as occurs in vivo; therefore, they represent an appropriate cell model with which to study this process.

Regulation of iron absorption is thought to be mediated by the amount of iron taken up by duodenal crypt cells via the transferrin receptor (TfR)-transferrin cycle and the activity of the divalent metal transporter (DMT1), although DMT1 cannot be detected morphologically in crypt cells. We investigated the uptake of transferrin-bound iron by duodenal enterocytes in Wistar rats fed different levels of iron and Belgrade (b/b) rats in which iron uptake by the transferrin cycle is defective because of a mutation in DMT1. We showed that DMT1 in our colony of b/b rats contains the G185R mutation, which in enterocytes results in reduced cellular iron content and increased DMT1 gene expression similar to levels in iron deficiency of normal rats. In all groups the uptake of transferrin-bound iron by crypt cells was directly proportional to plasma iron concentration, being highest in iron-loaded Wistar rats and b/b rats. We conclude that the uptake of transferrin-bound iron by developing enterocytes is largely independent of DMT1.

BACKGROUND—The mechanism of iron absorption by the intestine and its transfer to the main iron storage site, the liver, is poorly understood. Recently an iron carrier was cloned and named DMT1 (divalent metal transporter 1). AIMS—To determine the level of DMT1 gene expression and protein distribution in duodenum and liver. METHODS—A DMT1 cRNA and antibody were produced and used in in situ hybridisation and immunohistochemistry, respectively, in rats in which the iron stores were altered by feeding diets with normal, low, and high iron content. RESULTS—Duodenal DMT1 mRNA was low in crypts and increased at the crypt-villus junction in iron deficient and control rats; it fell in the iron loaded state. Staining for DMT1 protein was not detected in crypts. In villus enterocytes, protein staining was localised to the microvillus membrane in iron deficiency, in the cytoplasm and to a lesser extent in the membrane in controls, and entirely in the cytoplasm of iron loaded animals. Liver DMT1 mRNA was distributed evenly across hepatocytes. DMT1 protein staining was observed on hepatocyte plasma membranes, with highest values in the iron loaded state, lower values in control animals, and none after iron depletion. CONCLUSIONS—Results are consistent with a role for DMT1 in the transmembrane transport of non-transferrin bound iron from the intestinal lumen and from the portal blood. Keywords: DMT1; iron transporters; iron absorption; duodenum; liver

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Purpose: A challenge in ocular preventive medicine is identification of patients with early pathological retinal damage that might benefit from nutritional intervention. The purpose of this study is to evaluate retinal thinning (RT) in early atrophic age-related macular degeneration (AMD) against visual function data from the Zeaxanthin and Visual Function (ZVF) randomized double masked placebo controlled clinical trial (FDA IND #78973). Methods: Retrospective, observational case series of medical center veterans with minimal visible AMD retinopathy (AREDS Report #18 simplified grading 1.4/4.0 bilateral retinopathy). Foveal and extra-foveal four quadrant SDOCT RT measurements were evaluated in n = 54 clinical and ZVF AMD patients. RT by age was determined and compared to the OptoVue SD OCT normative database. RT by quadrant in a subset of n = 29 ZVF patients was correlated with contrast sensitivity and parafoveal blue cone increment thresholds. Results: Foveal RT in AMD patients and non-AMD patients was preserved with age. Extrafoveal regions, however, showed significant slope differences between AMD patients and non-AMD patients, with the superior and nasal quadrants most vulnerable to retinal thinning (sup quad: −5.5 μm/decade thinning vs. Non-AMD: −1.1 μm/decade, P < 0.02; nasal quad: −5.0 μm/decade thinning vs. Non-AMD: −1.0 μm/decade, P < 0.04). Two measures of extrafoveal visual deterioration were correlated: A significant inverse correlation between % RT and contrast sensitivity (r = −0.33, P = 0.01, 2 Tailed Paired T) and an elevated extrafoveal increment blue cone threshold (r = +0.34, P = 0.01, 2 Tailed T). Additional SD OCT RT data for the non-AMD oldest age group (ages 82–91) is needed to fully substantiate the model. Conclusion: A simple new SD OCT clinical metric called “% extra-foveal RT” correlates well with functional visual loss in early AMD patients having minimal visible retinopathy. This metric can be used to follow the effect of repleting ocular nutrients, such as zinc, antioxidants, carotenoids, n-3 essential fats, resveratrol and vitamin D.

Background: The risk of injury or fatality (driver, passenger or pedestrian) associated with motor vehicle accidents has been determined to increase with age, as a result of age-related declines in vision, motor and cognitive functioning. Elderly drivers with age related macular degeneration are particularly vulnerable to sensory visual impairment when driving at night, as they suffer declines in both Contrast sensitivity (CS) and Glare recovery (GR). Objective: This study evaluates the relationship between carotenoid supplementation, CS and GR and the relationship between driving ability and retinal macular pigmentation. Methods: Self-described driving performance is the basis of this report, with data derived from the Zeaxanthin and Vision Function (ZVF) Study (FDA IND #78,973), a 1 year, n=60, 4 visit, prospective randomized controlled clinical trial (RCT) of predominantly elderly male veterans (74.9 SD 10 y) with mild / moderate Age related Macular Degeneration (AMD). The twenty five question - National Eye Institute Visual Functioning Questionnaire (VFQ-25) v. January 2000, Rand Corporation® includes 3 questions assessing driving performance and were completed at baseline and 1 year after nutritional supplementation with approximately equal daily doses of the retinal macular carotenoids: lutein (9 mg) or zeaxanthin (8 mg). Replicate measures of foveal 1 degree estimated retinal macular pigment optical density were evaluated with the QuantifEye® (ZeaVision, Chesterfield, MO) heterochromic flicker photometer. Results: VFQ25 self-described driving ability was notably associated with baseline pre-supplementation macular pigmentation. Linear regression modeling suggests that self-described ability to safely drive a car was strongly associated with final macular re-pigmentation post supplementation (P=0.02). Moreover, the greatest effect was found with zeaxanthin (ns but P = 0.057 for trend) even though lutein had greater effects than zeaxanthin with respect to CS and GR, suggesting that unique zeaxanthin post-receptorial processes may be at play. Discussion: Carotenoid supplementation and subsequent macular repigmentation improved the self-described driving ability of patients with macular degeneration. Older male drivers with AMD should be encouraged to have their foveal macular MP measured at yearly eye examinations. If low macular pigment is found, these patients should attempt macular re-pigmentation via diet and/or supplementation to improve the sensory aspect of driving.

Background Lipofuscin is the most consistent and phylogenically constant morphologic marker of cellular aging. Autofluorescence of the A2E fluorophore within retinal pigment epithelial (RPE) lipofuscin affords the opportunity for noninvasive evaluation of age- and disease-related pathophysiological changes in the human retina. It is being used in National Eye Institute/Age-Related Eye Disease Study II to evaluate age-related macular degeneration (AMD) geographic atrophy expansion. Experiments show lipofuscin can be reversed in cell culture and animal models in heart, brain, spinal cord, and retinal tissues, using an array of antioxidants and iron chelators. Methods An 80-year-old man with a gastric resection presented with complaints of unremitting night driving difficulty despite treatment with lutein and omega III fatty acids. Notable parafoveal deposition of retinal lipofuscin by 50° fundus auto-fluorescence (580 nm excitation/660 barrier filters) and concurrent abnormalities in non-Snellen measures of visual function–Contrast Sensitivity Function, 6.5° large field tritan threshold, 10° threshold visual fields, and deficits in the National Institutes of Health/National Eye Institute Visual Function Questionnaire (VFQ) 25 subjective night driving/mental health subscale questionnaire were obtained. The patient was placed on an over-the-counter daily oral polyphenolic mixture containing resveratrol and re-evaluated 5 months later. Results The data reveal improvements in all measures of visual function, subjective improvement in vision and mental functioning on the VFQ 25, and visible clearing of RPE lipofuscin. Conclusion To our knowledge, we believe this to be the first reported human clinical case of lipofuscin reversal in the human eye correlated with measured clinical and subjective improvement in visual and mental function after nutraceutical intervention.

The haemochromatosis protein (HFE) is an important regulator of body iron stores. In the liver, HFE is required for appropriate expression of hepcidin, a humoral mediator of iron absorption. HFE is also present in enterocytes, though its function in the intestine is unknown; it is not intrinsically required for iron absorption, but can augment iron absorption when over-expressed-independent of hepcidin regulation by the liver. In this study, an antibody was raised against rat HFE and validated by enzyme-linked immunosorbent assay, Western blot and quenching of antibody function by the immunising peptide. The sub-cellular location of HFE in enterocytes of iron-deficient and control rats was determined by double-labelling experiments with markers for the microvillus membrane, terminal web, early endosomes, lysosomes and the transferrin receptor. Parallel studies were performed for the primary iron absorption protein, divalent metal transporter 1 (DMT1). HFE co-localised exclusively with the terminal web of intestinal enterocytes. HFE expression was increased in iron deficiency, consistent with a second regulatory role for HFE in iron absorption, independent of hepcidin from the liver. DMT1 was localised primarily on the microvillus membrane, but did partially co-localise with HFE raising the possibility that the two proteins may interact to regulate iron absorption.

Absorption of iron occurs by duodenal enterocytes, involving uptake by the divalent metal transporter-1 (DMT1) and release by ferroportin. Ferroportin responds to the hepatocyte-produced 25-amino-acid-peptide hepcidin-25 by undergoing internalisation to late endosomes that impair iron release. Ferroportin is also expressed on the apical membrane of polarised Caco-2 cells, rat intestinal cells and in IEC-6 cells (an intestinal epithelial cell line). A blocking antibody to ferroportin also impairs the uptake, but not the release, of iron. In this study IEC-6 cells were used to study the mechanism of impairment or recovery from impairment produced by the blocking antibody and the fate of DMT1 and ferroportin. Uptake of 1 muM Fe(II) was studied by adding the antibody from time 0 and after adding or removing the antibody once a steady state had been reached. Surface binding, maximum iron transport rate V(max) and transporter affinity (K(m)) were measured after impairment of iron uptake. Ferroportin and DMT1 distribution were assessed by immunofluorescence microscopy. Antibody-mediated impairment, or recovery from impairment, of Fe(II) uptake occurred within minutes. Impairment was lost when the antibody was combined with the immunizing peptide. DMT1 and ferroportin undergo internalisation to late endosomes and, in the presence of the antibody, augmented internalisation of DMT1 and ferroportin caused swelling of late endosomes. Surface binding of Fe(II) and iron transport V(max) were reduced by 50%, indicating that the antibody removed membrane-bound DMT1. The ferroportin antibody induced rapid turnover of membrane ferroportin and DMT1 and its internalisation to late endosomes, resulting in impaired Fe(II) uptake.

Dietary iron is present in the intestine as Fe(II) and Fe(III). Since enterocytes take up Fe(II) by the divalent metal transporter (DMT1), Fe(III) must be reduced. Whether other Fe(III) transport processes are present is unknown. Release of iron from the enterocyte into the plasma involves the iron-regulated transporter-1/metal transporter protein-1 (IREG-1/MTP-1, ferroportin) but ferroportin is also found on the apical membrane. We compared the uptake of iron from Fe(II):ascorbate and Fe(III):citrate using the rat intestinal enterocyte cell line-6 (IEC-6), in the presence of ferrous chelators, a blocking antibody to ferroportin, at different pH and during the over-expression of DMT1. Firstly, surface ferrireduction was absent. Secondly, blocking ferroportin partly and totally reduced Fe(II) and Fe(III) uptake, respectively. Thirdly, optimal Fe(II) uptake occurred at pH 5.5 but Fe(III) uptake was unaffected by pH and, fourthly, over-expression of DMT1 increased uptake of Fe(II) and Fe(III). This indicates that an increased extracellular H+ concentration facilitates DMT1-mediated Fe(II) uptake at the cell membrane. However, since Fe(III) uptake required DMT1, but not cell surface ferrireduction, and was independent of variations in extracellular pH, it appears that Fe(III) is internalised before ferrireduction and transport by DMT1. Ferroportin may function as a modulator of DMT1 activity and play a role in Fe(III) uptake, possibly by affecting the number or affinity of citrate binding sites.

Release of iron from enterocytes and hepatocytes is thought to require the copper-dependent ferroxidase activity of hephaestin (Hp) and ceruloplasmin (Cp), respectively. In swine, copper deficiency (CD) impairs iron absorption, but whether this occurs in rats is unclear. By feeding a diet deficient in copper, CD was produced, as evidenced by the loss of copper-dependent plasma ferroxidase I activity, and in enterocytes, CD reduced copper levels and copper-dependent oxidase activity. Hematocrit was reduced, and liver iron was doubled. CD reduced duodenal mucosal iron and ferritin, whereas CD increased iron absorption. Duodenal mucosal DMT1-IRE and ferroportin1 expression remained constant with CD. When absorption in CD rats was compared with that seen normally and in iron-deficient anemic animals, strong correlations were found among mucosal iron, ferritin, and iron absorption, suggesting that the level of iron absorption was appropriate given that the erythroid and stores stimulators of iron absorption are opposed in CD. Because CD reduced the activity of Cp, as evidenced by copper-dependent plasma ferroxidase I activity and hepatocyte iron accumulation, but iron absorption increased, it is unlikely that the ferroxidase activity of Hp is important and suggests another function for this protein in the export of iron from the enterocyte during iron absorption. Also, the copper-dependent ferroxidase activity of Cp does not appear important for iron efflux from macrophages, because Kupffer cells of the liver and nonheme iron levels of the spleen were normal during copper deficiency, suggesting another role for Cp in these cells.

Background: Iron stores in the body are thought to be regulated by a mechanism associated with the rate of iron absorption from the diet, with no significant role played by iron excretion. We report the existence of an iron excretory process that results in the loss of significant amounts of liver iron. Methods and Results: Rats were fed 3% carbonyl iron for 9 weeks, which resulted in a 20-fold increase in liver non-haem iron. When the rats on this iron-loaded diet were switched to a low iron diet for 2 and 7 days, liver non-haem iron levels fell 30% and 45%, respectively. A similar fall in transferrin-bound plasma iron was also seen. As the liver iron had not redistributed to other body compartments, it was concluded that the iron had been excreted and that the excreted iron represented a loss of 22% and 28% in total body non-haem iron over 2 and 7 days, respectively. Ligation of the common bile duct in iron loaded rats that had been switched to the iron-deficient diet was accompanied by a similar loss of liver iron and also hepatocellular damage. In addition, measurement of enterocyte iron levels showed that only approximately 5% of the total iron excreted was found in these cells. Conclusion: Neither bile nor enterocytes play a significant role in iron excretion. The similarity in the degree of fall in transferrin-bound iron levels with a change in diet suggests that iron excretion involves the uptake and excretion of transferrin bound-iron, possibly by goblet cells. The observed hypertrophy of the intestinal mucosa associated with carbonyl iron feeding may facilitate hypersecretion of mucous and the excretion of this iron.

It is not known how the efficiency of intestinal iron absorption is regulated. One hypothesis suggests that an interaction between the transferrin receptor (TfR) and the haemochromatosis protein (HFE) regulates the level of iron loading in crypt cells. The hypothesis goes on to suggest that this determines the amount of transport protein, expressed in villus enterocytes, that is involved in iron absorption. Mice with haploinsufficiency for TfR are iron deficient and this is thought to be caused by reduced iron absorption. This suggests that TfR may play a role in the regulation and/or mechanism of iron absorption. We investigated TfR function and distribution by measuring iron uptake from plasma transferrin and by immunohistochemistry. The uptake of transferrin-bound iron (Tf–Fe2) into mucosal cells subsequently separated along the crypt–villus axis was compared to the presence of TfR, determined by immunohistochemistry using frozen and wax sections. Frozen sections showed TfR staining in crypt and villus epithelial cells. In wax sections TfR was only identified in a supranuclear region commencing in enterocytes at the crypt–villus junction and attaining greatest levels at the villus tip. This indicates that the processing of wax tissue exposes a TfR epitope that otherwise remains undetectable when studied in frozen sections. This appearance in paraffin sections was inversely related to the uptake of Tf–Fe2. Supranuclear TfR was not associated with lysosomes, since there was no difference in the uptake of normal Tf–Fe2 and that of the non-digestible cellobiose Tf–Fe2, and Western blot analysis revealed similar amounts of TfR in crypt and villus cells. Also the uptake of Tf–Fe2 increased linearly with time, albeit less in villus than crypt cells, suggesting that maturation of an efflux system in villus cells is not responsible for this difference. We hypothesise that TfR in the supranuclear region of villus enterocytes may play a role in iron absorption.

The intestinal mucosa is characterized by cell proliferation, commitment, differentiation, digestion and absorption. These processes occur at specified locations along the crypt to villus axis. A technique is reported for the isolation of cells along this axis which allows the study of any one of these processes in an enriched population of cells. As an example, the uptake of transferrin-bound iron by enterocytes was studied. Rats were fed diets normal, high (30% carbonyl iron) or low in iron for 12 days. Cells from either the duodenum or ileum were isolated by incubating in a Ca(2+)-, Mg2+-free, cation chelating solution for varying periods. The incorporation of thymidine into DNA was measured in these cells as a marker of the crypt region, while alkaline phosphatase and sucrase activities marked mature enterocytes. The in vivo uptake of transferrin-bound 59Fe was measured in cells isolated either 2 or 4 h after intravenous injection. This procedure resulted in the isolation of 10 fractions of viable cells. Earlier fractions were enriched at least 10-fold in villus cells and the last fractions in crypt cells. Cells in intermediate fractions were at various stages of development. Uptake of transferrin-bound iron into enterocytes was highest with feeding an iron-loaded diet compared with control or iron-deficient diets. However, with all diets uptake was highest in crypt cells and this fell at the crypt-villus junction to be only 25%, as high at the villus tip as the crypt. A technique for the reproducible isolation of viable enterocytes along a crypt-villus axis is described. Transferrin receptor activity changes with maturation of the enterocyte.

Introduction Primary central nervous system lymphomas (PCNSL) are diffuse large B-cell lymphomas (DLBCL) with clinical and prognostic features distinct from systemic diffuse large B-cell lymphomas. Limited previous studies suggest that PCNSL in the immunocompetent patient shows differential expression of micro-RNAs (miRNAs) distinct from systemic DLBCL, suggesting different pathogenesis. MiRNAs are short non-coding single stranded RNAs with a role in post-transcrip-tional regulation of protein expression, some shown to be associated with cancer development and progression. Methods The expression of a panel of miRNAs was measured by quantitative real-time PCR after isolating RNA from formalin fixed paraffin embedded (FFPE) tissue of primary CNS lymphoma ( n = 57), controlled by normalising to normal brain samples ( n = 12) and endogenous miRNA controls (hsa-SNORD48, hsa-SNORD47, hsa-SNORD44 and hsa-U6). The expression profile was compared to that of systemic DLBCL (n = 30), controlled by normalising to benign tonsil tissue ( n = 12) and the same endogenous miRNA controls. Results Hierarchical clustering revealed a distinct expression profile in PCNSL compared to systemic DLBCL. The most significantly overexpressed miRNAs in PCNSL compared to DLBCL were hsa-miR-155, hsa-miR-146a, hsa-miR-1321 and hsa-miR-200c. The most underexpressed miRNA in PCNSL compared to DLBCL were hsa-miR-145, hsa-miR-122, hsa-miR-497 and hsa-miR-579. Discussion Primary CNS lymphoma is usually associated with a dismal prognosis. The expression of B-cell related miRNAs in PCNSL is distinct from that of systemic DLBCL, suggesting a different biological entity. To our knowledge this is the first genome-wide microRNA profiling conducted to distinguish systemic DLBCL from PCNSL. We have identified a number of miRNAs which appear to differ in expression between the two entities and these findings could provide insights into the clinical behaviour and pathogenesis of PCNSL and aid discovery of potential therapeutic targets.

Background There has been increasing interest in recent years in molecular assessment of proliferative activity (PA) as a means of stratifying prognosis in some subsets of invasive breast carcinoma. In addition to commercial molecular risk stratification tools, which are strongly influenced by proliferative markers, recent consensus guidelines have suggested incorporation of Ki67 assessment of PA by immunohistochemistry into treatment selection decisions in some circumstances. Despite this, there are significant practical obstacles to measurement of PA outside a research setting. In particular, recommended manual methods involve counting a minimum of 1000 cells, which is likely to be too time consuming for ready incorporation into a busy clinical workload. Aims and Methods We conducted a prospective study of 70 cases in routine practice to compare two less time consuming methods of Ki67 assessment: a rapid semi-quantitative manual method and computerised image analysis. The latter has previously been shown to be strongly correlated with formal manual assessment. We also sought to determine whether dual staining with cytokera-tin and Ki67 was necessary to exclude background cells from automated proliferation assessment. Results We found that rapid semi-quantitative manual assessment showed only a fair correlation with automated counting (correlation coefficient 0.7). There was a strong correlation between single stained and dual stained specimens by automated assessment (correlation coefficient 0.9). Conclusions Our results suggest that computerised image analysis may be the best alternative to formal manual Ki67 counting when that method is impractical in busy clinical practice.

Background The purpose of this study is to evaluate whether dietary supplementation with the carotenoid zeaxanthin (Zx) raises macula pigment optical density (MPOD) and has unique visual benefits for patients with early atrophic macular degeneration having visual symptoms but lower-risk National Institute of Health/National Eye Institute/Age-Related Eye Disease Study characteristics. Methods This was a 1-year, n = 60 (57 men, 3 women), 4-visit, intention-to-treat, prospective, randomized controlled clinical trial of patients (74.9 years, standard deviation [SD] 10) with mild-to-moderate age-related macular degeneration (AMD) randomly assigned to 1 of 2 dietary supplement carotenoid pigment intervention groups: 8 mg Zx (n = 25) and 8 mg Zx plus 9 mg lutein (L) (n = 25) or 9 mg L (“Faux Placebo,” control group, n = 10). Analysis was by Bartlett’s test for equal variance, 3-way repeated factors analysis of variance, independent t test ( P ® ), low- and high-contrast visual acuity, foveal shape discrimination (Retina Foundation of the Southwest), 10° yellow kinetic visual fields (KVF), glare recovery, contrast sensitivity function (CSF), and 6° blue cone ChromaTest ® color thresholds were obtained serially at 4, 8, and 12 months. Results Ninety percent of subjects completed ≥ 2 visits with an initial Age-Related Eye Disease Study report #18 retinopathy score of 1.4 (1.0 SD)/4.0 and pill intake compliance of 96% with no adverse effects. There were no intergroup differences in 3 major AMD risk factors: age, smoking, and body mass index as well as disease duration and Visual Function Questionnaire 25 composite score differences. Randomization resulted in equal MPOD variance and MPOD increasing in each of the 3 groups from 0.33 density units (du) (0.17 SD) baseline to 0.51 du (0.18 SD) at 12 m, ( P = 0.03), but no between-group differences (Analysis of Variance; P = 0.47). In the Zx group, detailed high-contrast visual acuity improved by 1.5 lines, Retina Foundation of the Southwest shape discrimination sharpened from 0.97 to 0.57 ( P = 0.06, 1-tail), and a larger percentage of Zx patients experienced clearing of their KVF central scotomas ( P = 0.057). The “Faux Placebo” L group was superior in terms of low-contrast visual acuity, CSF, and glare recovery, whereas Zx showed a trend toward significance. Conclusion In older male patients with AMD, Zx-induced foveal MPOD elevation mirrored that of L and provided complementary distinct visual benefits by improving foveal cone-based visual parameters, whereas L enhanced those parameters associated with gross detailed rod-based vision, with considerable overlap between the 2 carotenoids. The equally dosed (atypical dietary ratio) Zx plus L group fared worse in terms of raising MPOD, presumably because of duodenal, hepatic-lipoprotein or retinal carotenoid competition. These results make biological sense based on retinal distribution and Zx foveal predominance.