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Nilofar Daneshi,1,2 Elizabeth Holliday,3,4 Stephen Hancock,3,4 Jennifer J Schneider,1,2 Rodney J Scott,1,2,5 John Attia,3,4 Elizabeth A Milward1,2 1Faculty of Health and Medicine, School of Biomedical Sciences and Pharmacy, 2Faculty of Health and Medicine, Centre for Bioinformatics, Biomarker Discovery and Information-Based Medicine, The University of Newcastle, Callaghan, 3Clinical Research Design, IT and Statistical Support Unit, Hunter Medical Research Institute, 4Centre for Clinical Epidemiology and Biostatistics, School of Medicine and Public Health, Faculty of Health, University of Newcastle, 5Hunter Area Pathology Service, John Hunter Hospital, Newcastle, NSW, Australia Abstract: This study analyzed clinically actionable pharmacogenotypes for clopidogrel, warfarin, statins, thiopurines, and tacrolimus using microarray data for 2121 participants (55–85 years) from the Australian Hunter Community Study (HCS). At least 74% of participants (95% confidence interval [CI]: 72%–76%) had strong level evidence for at least one medium- or high-risk actionable genotype that would trigger a change in standard therapy under current international recommendations. About 14% of these participants (95% CI: 12%–16%) were taking medication potentially affected by the genotype in question. Furthermore, ~2.6% of all participants with medication data (95% CI: 1.4%–3.8%) had a high-risk clinically actionable genotype for a medication to which they were exposed. This represents a considerable number of people at the population level. Although relationships between genotype and health outcomes remain contentious, pharmacogenotyping of multiple variants simultaneously may have considerable potential to improve medication safety and efficacy for older people in the community. Keywords: actionable genotype, community, older adults, pharmacogenomics, pre-emptive genotyping, single-nucleotide polymorphism

Iron abnormalities are observed in the brains of Alzheimer's disease (AD) patients, but it is unclear whether common disorders of systemic iron overload such as hemochromatosis alter risks of AD. We used microarrays and real-time reverse transcription-PCR to investigate changes in the brain transcriptome of adult Hfe-/- mice, a model of hemochromatosis, relative to age- and gender-matched wildtype controls. Classification by functional pathway analysis revealed transcript changes for various genes important in AD. There were decreases of up to 9-fold in transcripts for amyloid-β protein precursor, tau, apolipoprotein E, presenilin 1, and various other γ-secretase components, as well as Notch signaling pathway molecules. This included decreased transcripts for 'hairy and enhancer of split' Hes1 and Hes5, downstream targets of Notch canonical signaling. The reductions in Hes1 and Hes5 transcripts provide evidence that the changes in levels of transcripts for γ-secretase components and Notch signaling genes have functional consequences. The effects appeared relatively specific for AD in that few genes pertaining to other important neurodegenerative diseases, notably Parkinson's disease and Huntington's disease, or to inflammation, oxidative stress, or apoptosis, showed altered transcript levels. The observed effects on AD-related gene transcripts do not appear to be consistent with increased AD risk in HFE hemochromatosis and might, if anything, be predicted to protect against AD to some extent. As Hfe-/- mice did not have higher brain iron levels than wildtype controls, these studies highlight the need for further research in models of more severe hemochromatosis with brain iron loading. [ABSTRACT FROM AUTHOR]

Maternal iron deficiency occurs in 40-50% of all pregnancies and is associated with an increased risk of respiratory disease and asthma in children. We used murine models to examine the effects of lower iron status during pregnancy on lung function, inflammation and structure, as well as its contribution to increased severity of asthma in the offspring. A low iron diet during pregnancy impairs lung function, increases airway inflammation, and alters lung structure in the absence and presence of experimental asthma. A low iron diet during pregnancy further increases these major disease features in offspring with experimental asthma. Importantly, a low iron diet increases neutrophilic inflammation, which is indicative of more severe disease, in asthma. Together, our data demonstrate that lower dietary iron and systemic deficiency during pregnancy can lead to physiological, immunological and anatomical changes in the lungs and airways of offspring that predispose to greater susceptibility to respiratory disease. These findings suggest that correcting iron deficiency in pregnancy using iron supplements may play an important role in preventing or reducing the severity of respiratory disease in offspring. They also highlight the utility of experimental models for understanding how iron status in pregnancy affects disease outcomes in offspring and provide a means for testing the efficacy of different iron supplements for preventing disease.

Accumulating evidence highlights links between iron regulation and respiratory disease. Here, we assessed the relationship between iron levels and regulatory responses in clinical and experimental asthma.We show that cell-free iron levels are reduced in the bronchoalveolar lavage (BAL) supernatant of severe or mild-moderate asthma patients and correlate with lower forced expiratory volume in 1 s (FEV 1 ). Conversely, iron-loaded cell numbers were increased in BAL in these patients and with lower FEV 1 /forced vital capacity (FVC) ratio. The airway tissue expression of the iron sequestration molecules divalent metal transporter 1 ( DMT1 ) and transferrin receptor 1 ( TFR1 ) are increased in asthma, with TFR1 expression correlating with reduced lung function and increased Type-2 (T2) inflammatory responses in the airways. Furthermore, pulmonary iron levels are increased in a house dust mite (HDM)-induced model of experimental asthma in association with augmented Tfr1 expression in airway tissue, similar to human disease. We show that macrophages are the predominant source of increased Tfr1 and Tfr1 + macrophages have increased Il13 expression. We also show that increased iron levels induce increased pro-inflammatory cytokine and/or extracellular matrix (ECM) responses in human airway smooth muscle (ASM) cells and fibroblasts ex vivo and induce key features of asthma in vivo , including airway hyper-responsiveness (AHR) and fibrosis, and T2 inflammatory responses.Together these complementary clinical and experimental data highlight the importance of altered pulmonary iron levels and regulation in asthma, and the need for a greater focus on the role and potential therapeutic targeting of iron in the pathogenesis and severity of disease., Competing Interests: Conflict of interest: M.K. Ali has nothing to disclose. Conflict of interest: R.Y. Kim has nothing to disclose. Conflict of interest: A.C. Brown has nothing to disclose. Conflict of interest: J.R. Mayall has nothing to disclose. Conflict of interest: R. Karim has nothing to disclose. Conflict of interest: J.W. Pinkerton has nothing to disclose. Conflict of interest: G. Liu has nothing to disclose. Conflict of interest: K.L. Martin has nothing to disclose. Conflict of interest: M.R. Starkey has nothing to disclose. Conflict of interest: A.L. Pillar has nothing to disclose. Conflict of interest: C. Donovan has nothing to disclose. Conflict of interest: P.S. Pathinayake has nothing to disclose. Conflict of interest: O.R. Carroll has nothing to disclose. Conflict of interest: D. Trinder has nothing to disclose. Conflict of interest: H.L. Tay has nothing to disclose. Conflict of interest: Y.E. Badi has nothing to disclose. Conflict of interest: N.Z. Kermani has nothing to disclose. Conflict of interest: Y-K. Guo has nothing to disclose. Conflict of interest: R. Aryal has nothing to disclose. Conflict of interest: S. Mumby has nothing to disclose. Conflict of interest: S. Pavlidis has nothing to disclose. Conflict of interest: I.M. Adcock reports grants from EU-IMI, during the conduct of the study. Conflict of interest: J. Weaver has nothing to disclose. Conflict of interest: D. Xenaki has nothing to disclose. Conflict of interest: B.G. Oliver has nothing to disclose. Conflict of interest: E.G. Holliday has nothing to disclose. Conflict of interest: P.S. Foster has nothing to disclose. Conflict of interest: P.A. Wark has nothing to disclose. Conflict of interest: D.M. Johnstone has nothing to disclose. Conflict of interest: E.A. Milward has nothing to disclose. Conflict of interest: P.M. Hansbro has nothing to disclose. Conflict of interest: J.C. Horvat has nothing to disclose., (Copyright ©ERS 2020.)

Alzheimer's disease, the most common form of dementia, was first formally described in 1907 yet its etiology has remained elusive. Recent proposals that Aβ peptide may be part of the brain immune response have revived longstanding contention about the possibility of causal relationships between brain pathogens and Alzheimer's disease. Research has focused on infectious pathogens that may colonize the brain such as herpes simplex type I. Some researchers have proposed the respiratory bacteria Chlamydia pneumoniae may also be implicated in Alzheimer's disease, however this remains controversial. This review aims to provide a balanced overview of the current evidence and its limitations and future approaches that may resolve controversies. We discuss the evidence from in vitro, animal and human studies proposed to implicate Chlamydia pneumoniae in Alzheimer's disease and other neurological conditions, the potential mechanisms by which the bacterium may contribute to pathogenesis and limitations of previous studies that may explain the inconsistencies in the literature., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Iron is essential for many biological processes, however, too much or too little iron can result in a wide variety of pathological consequences, depending on the organ system, tissue or cell type affected. In order to reduce pathogenesis, iron levels are tightly controlled in throughout the body by regulatory systems that control iron absorption, systemic transport and cellular uptake and storage. Altered iron levels and/or dysregulated homeostasis have been associated with several lung diseases, including chronic obstructive pulmonary disease, lung cancer, cystic fibrosis, idiopathic pulmonary fibrosis and asthma. However, the mechanisms that underpin these associations and whether iron plays a key role in the pathogenesis of lung disease are yet to be fully elucidated. Furthermore, in order to survive and replicate, pathogenic micro-organisms have evolved strategies to source host iron, including freeing iron from cells and proteins that store and transport iron. To counter these microbial strategies, mammals have evolved immune-mediated defence mechanisms that reduce iron availability to pathogens. This interplay between iron, infection and immunity has important ramifications for the pathogenesis and management of human respiratory infections and diseases. An increased understanding of the role that iron plays in the pathogenesis of lung disease and respiratory infections may help inform novel therapeutic strategies. Here we review the clinical and experimental evidence that highlights the potential importance of iron in respiratory diseases and infections., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The 'neurodegeneration with brain iron accumulation' (NBIA) disease family entails movement or cognitive impairment, often with psychiatric features. To understand how iron loading affects the brain, we studied mice with disruption of two iron regulatory genes, hemochromatosis (Hfe) and transferrin receptor 2 (Tfr2). Inductively coupled plasma atomic emission spectroscopy demonstrated increased iron in the Hfe -/- × Tfr2 mut brain (P=0.002, n ≥5/group), primarily localized by Perls' staining to myelinated structures. Western immunoblotting showed increases of the iron storage protein ferritin light polypeptide and microarray and real-time reverse transcription-PCR revealed decreased transcript levels (P<0.04, n ≥5/group) for five other NBIA genes, phospholipase A2 group VI, fatty acid 2-hydroxylase, ceruloplasmin, chromosome 19 open reading frame 12 and ATPase type 13A2. Apart from the ferroxidase ceruloplasmin, all are involved in myelin homeostasis; 16 other myelin-related genes also showed reduced expression (P<0.05), although gross myelin structure and integrity appear unaffected (P>0.05). Overlap (P<0.0001) of differentially expressed genes in Hfe -/- × Tfr2 mut brain with human gene co-expression networks suggests iron loading influences expression of NBIA-related and myelin-related genes co-expressed in normal human basal ganglia. There was overlap (P<0.0001) of genes differentially expressed in Hfe -/- × Tfr2 mut brain and post-mortem NBIA basal ganglia. Hfe -/- × Tfr2 mut mice were hyperactive (P<0.0112) without apparent cognitive impairment by IntelliCage testing (P>0.05). These results implicate myelin-related systems involved in NBIA neuropathogenesis in early responses to iron loading. This may contribute to behavioral symptoms in NBIA and hemochromatosis and is relevant to patients with abnormal iron status and psychiatric disorders involving myelin abnormalities or resistant to conventional treatments.

We previously demonstrated elevated brain iron levels in myelinated structures and associated cells in a hemochromatosis Hfe (-/-) xTfr2 (mut) mouse model. This was accompanied by altered expression of a group of myelin-related genes, including a suite of genes causatively linked to the rare disease family 'neurodegeneration with brain iron accumulation' (NBIA). Expanded data mining and ontological analyses have now identified additional myelin-related transcriptome changes in response to brain iron loading. Concordance between the mouse transcriptome changes and human myelin-related gene expression networks in normal and NBIA basal ganglia testifies to potential clinical relevance. These analyses implicate, among others, genes linked to various rare central hypomyelinating leukodystrophies and peripheral neuropathies including Pelizaeus-Merzbacher-like disease and Charcot-Marie-Tooth disease as well as genes linked to other rare neurological diseases such as Niemann-Pick disease. The findings may help understand interrelationships of iron and myelin in more common conditions such as hemochromatosis, multiple sclerosis and various psychiatric disorders.

As recognised by the National Institutes of Health (NIH) Precision Medicine Initiative (PMI), microarray technology currently provides a rapid, inexpensive means of identifying large numbers of known genomic variants or gene transcripts in experimental and clinical settings. However new generation sequencing techniques are now being introduced in many clinical genetic contexts, particularly where novel mutations are involved. While these methods can be valuable for screening a restricted set of genes for known or novel mutations, implementation of whole genome sequencing in clinical practice continues to present challenges. Even very accurate high-throughput methods with small error rates can generate large numbers of false negative or false positive errors due to the high numbers of simultaneous readings. Additional validation is likely to be required for safe use of any such methods in clinical settings. Custom-designed arrays can offer advantages for screening for common, known mutations and, in this context, may currently be better suited for accredited, quality-controlled clinical genetic screening services, as illustrated by their successful application in several large-scale pre-emptive pharmacogenomics programs now underway. Excessive, inappropriate use of next-generation sequencing may waste scarce research funds and other resources. Microarrays presently remain the technology of choice in applications that require fast, cost-effective genome-wide screening of variants of known importance, particularly for large sample sizes. This commentary considers some of the applications where microarrays continue to offer advantages over next-generation sequencing technologies.

Aberrant brain iron deposition is observed in both common and rare neurodegenerative disorders, including those categorized as Neurodegeneration with Brain Iron Accumulation (NBIA), which are characterized by focal iron accumulation in the basal ganglia. Two NBIA genes are directly involved in iron metabolism, but whether other NBIA-related genes also regulate iron homeostasis in the human brain, and whether aberrant iron deposition contributes to neurodegenerative processes remains largely unknown. This study aims to expand our understanding of these iron overload diseases and identify relationships between known NBIA genes and their main interacting partners by using a systems biology approach. We used whole-transcriptome gene expression data from human brain samples originating from 101 neuropathologically normal individuals (10 brain regions) to generate weighted gene co-expression networks and cluster the 10 known NBIA genes in an unsupervised manner. We investigated NBIA-enriched networks for relevant cell types and pathways, and whether they are disrupted by iron loading in NBIA diseased tissue and in an in vivo mouse model. We identified two basal ganglia gene co-expression modules significantly enriched for NBIA genes, which resemble neuronal and oligodendrocytic signatures. These NBIA gene networks are enriched for iron-related genes, and implicate synapse and lipid metabolism related pathways. Our data also indicates that these networks are disrupted by excessive brain iron loading. We identified multiple cell types in the origin of NBIA disorders. We also found unforeseen links between NBIA networks and iron-related processes, and demonstrate convergent pathways connecting NBIAs and phenotypically overlapping diseases. Our results are of further relevance for these diseases by providing candidates for new causative genes and possible points for therapeutic intervention., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Interventions to delay or slow Alzheimer's disease (AD) progression are most effective when implemented at pre-clinical disease stages, making early diagnosis essential. For this reason, there is an increasing focus on discovery of predictive biomarkers for AD. Currently, the most reliable predictive biomarkers require either expensive (brain imaging) or invasive (cerebrospinal fluid collection) procedures, leading researchers to strive toward identifying robust biomarkers in blood. Yet promising early results from candidate blood biomarker studies are being refuted by subsequent findings in other cohorts or using different assay technologies. Recent evidence suggests that univariate blood biomarkers are not sufficiently sensitive or specific for the diagnosis of disorders as complex, multifactorial, and heterogeneous as AD. To overcome these present limitations, more consideration must be given to the development of 'biomarker panels' assessing multiple molecular entities. The selection of such panels should draw not only on traditional statistical approaches, whether parametric or non-parametric, but also on newer non-statistical approaches that have the capacity to retain and utilize information about all individual study participants rather than collapsing individual data into group summary values (e.g., mean, variance). These new approaches, facilitated by advances in computing, have the potential to preserve the context of interrelationships between different molecular entities, making them amenable to the development of panels that, as a multivariate collective, can overcome the challenge of individual variability and disease heterogeneity to accurately predict and classify AD. We argue that the AD research community should take fuller advantage of these approaches to accelerate discovery.

While Illumina microarrays can be used successfully for detecting small gene expression changes due to their high degree of technical replicability, there is little information on how different normalization and differential expression analysis strategies affect outcomes. To evaluate this, we assessed concordance across gene lists generated by applying different combinations of normalization strategy and analytical approach to two Illumina datasets with modest expression changes. In addition to using traditional statistical approaches, we also tested an approach based on combinatorial optimization. We found that the choice of both normalization strategy and analytical approach considerably affected outcomes, in some cases leading to substantial differences in gene lists and subsequent pathway analysis results. Our findings suggest that important biological phenomena may be overlooked when there is a routine practice of using only one approach to investigate all microarray datasets. Analytical artefacts of this kind are likely to be especially relevant for datasets involving small fold changes, where inherent technical variation-if not adequately minimized by effective normalization-may overshadow true biological variation. This report provides some basic guidelines for optimizing outcomes when working with Illumina datasets involving small expression changes.

Iron abnormalities within the brain are associated with several rare but severe neurodegenerative conditions. There is growing evidence that more common systemic iron loading disorders such as hemochromatosis can also have important effects on the brain. To identify features that are common across different forms of hemochromatosis, we used microarray and real-time reverse transcription polymerase chain reaction (RT-PCR) to assess brain transcriptome profiles of transferrin receptor 2 mutant mice (Tfr2(mut)), a model of a rare type of hereditary hemochromatosis, relative to wildtype control mice. The results were compared with our previous findings in dietary iron-supplemented wildtype mice and Hfe(-/-) mice, a model of a common type of hereditary hemochromatosis. For transcripts showing significant changes relative to controls across all three models, there was perfect (100%) directional concordance (i.e. transcripts were increased in all models or decreased in all models). Comparison of the two models of hereditary hemochromatosis, which showed more pronounced changes than the dietary iron-supplemented mice, revealed numerous common molecular effects. Pathway analyses highlighted changes for genes relating to long-term depression (6.8-fold enrichment, p=5.4×10(-7)) and, to a lesser extent, long-term potentiation (3.7-fold enrichment, p=0.01), with generalized reductions in transcription of key genes from these pathways, which are involved in modulating synaptic strength and efficacy and are essential for memory and learning. The agreement across the models suggests the findings are robust and strengthens previous evidence that iron loading disorders affect the brain. Perturbations of brain phenomena such as long-term depression and long-term potentiation might partly explain neurologic symptoms reported for some hemochromatosis patients., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

The advent of real-time macromolecular sequencing is opening the way for new levels of understanding of dynamic biological states and new approaches to point-of-care diagnosis and drug discovery. This fast-growing area promises unprecedented capacity to develop detailed descriptions of biosystems in health and disease that integrate features from both inherent trait analysis (e.g. by genotyping) and dynamic analysis of transient states (e.g. by transcriptome or proteome typing). This evolving, integrated 'pharmacomics' will facilitate the characterization and monitoring of disease states and drug responses in terms of perturbations of biosystems from optimal states. This review considers how the latest generation of advances in ultra-rapid macromolecular sequencing will accelerate the evolution of personalized medicine and more systematic, rational drug discovery., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: up to 25% of older people in the USA and other Western countries are anaemic by World Health Organization (WHO) criteria. The objective of this study was to examine the long-term relationships of haemoglobin concentration with all-cause and cause-specific mortality in a community-based sample of Australian adults surveyed in 1978., Methods: a community survey of 2,194 adults aged 40+ years in Busselton, Western Australia in 1978 with mortality follow-up to 2001. Cox regression models were used to investigate the relationships of haemoglobin as a continuous measure and anaemia by WHO criteria (women <12 g/dl (7.5 mmol/l); men <13 g/dl (8.1 mmol/l)) with all-cause, cardiovascular and cancer mortality., Results: anaemia was predominantly mild (>10 g/dl) and normocytic. There was an increased risk of death from all causes and from cancer for men with low haemoglobin. Cancers were predominantly of the prostate and genito-urinary organs, and to a lesser extent the gastrointestinal tract. There was no increased risk of all cause or cancer death in women., Conclusion: mild, normocytic anaemia is associated with survival reductions in middle-aged and older men, where it often occurs with prostate, gastrointestinal and other cancers, and should be investigated to exclude treatable causes.

Severe disruption of brain iron homeostasis can cause fatal neurodegenerative disease, however debate surrounds the neurologic effects of milder, more common iron loading disorders such as hereditary hemochromatosis, which is usually caused by loss-of-function polymorphisms in the HFE gene. There is evidence from both human and animal studies that HFE gene variants may affect brain function and modify risks of brain disease. To investigate how disruption of HFE influences brain transcript levels, we used microarray and real-time reverse transcription polymerase chain reaction to assess the brain transcriptome in Hfe(-/-) mice relative to wildtype AKR controls (age 10 weeks, n≥4/group). The Hfe(-/-) mouse brain showed numerous significant changes in transcript levels (p<0.05) although few of these related to proteins directly involved in iron homeostasis. There were robust changes of at least 2-fold in levels of transcripts for prominent genes relating to transcriptional regulation (FBJ osteosarcoma oncogene Fos, early growth response genes), neurotransmission (glutamate NMDA receptor Grin1, GABA receptor Gabbr1) and synaptic plasticity and memory (calcium/calmodulin-dependent protein kinase IIα Camk2a). As previously reported for dietary iron-supplemented mice, there were altered levels of transcripts for genes linked to neuronal ceroid lipofuscinosis, a disease characterized by excessive lipofuscin deposition. Labile iron is known to enhance lipofuscin generation which may accelerate brain aging. The findings provide evidence that iron loading disorders can considerably perturb levels of transcripts for genes essential for normal brain function and may help explain some of the neurologic signs and symptoms reported in hemochromatosis patients., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Recent Alzheimer's disease (AD) research has focused on finding biomarkers to identify disease at the pre-clinical stage of mild cognitive impairment (MCI), allowing treatment to be initiated before irreversible damage occurs. Many studies have examined brain imaging or cerebrospinal fluid but there is also growing interest in blood biomarkers. The Alzheimer's Disease Neuroimaging Initiative (ADNI) has generated data on 190 plasma analytes in 566 individuals with MCI, AD or normal cognition. We conducted independent analyses of this dataset to identify plasma protein signatures predicting pre-clinical AD., Methods and Findings: We focused on identifying signatures that discriminate cognitively normal controls (n = 54) from individuals with MCI who subsequently progress to AD (n = 163). Based on p value, apolipoprotein E (APOE) showed the strongest difference between these groups (p = 2.3 × 10(-13)). We applied a multivariate approach based on combinatorial optimization ((α,β)-k Feature Set Selection), which retains information about individual participants and maintains the context of interrelationships between different analytes, to identify the optimal set of analytes (signature) to discriminate these two groups. We identified 11-analyte signatures achieving values of sensitivity and specificity between 65% and 86% for both MCI and AD groups, depending on whether APOE was included and other factors. Classification accuracy was improved by considering "meta-features," representing the difference in relative abundance of two analytes, with an 8-meta-feature signature consistently achieving sensitivity and specificity both over 85%. Generating signatures based on longitudinal rather than cross-sectional data further improved classification accuracy, returning sensitivities and specificities of approximately 90%., Conclusions: Applying these novel analysis approaches to the powerful and well-characterized ADNI dataset has identified sets of plasma biomarkers for pre-clinical AD. While studies of independent test sets are required to validate the signatures, these analyses provide a starting point for developing a cost-effective and minimally invasive test capable of diagnosing AD in its pre-clinical stages.

Lewy bodies are made from insoluble, phosphorylated α-synuclein, but the earliest changes that precipitate such pathology still remain conjecture. In this study, we quantify and identify relationships between the levels of the main pathologic form of phosphorylated α-synuclein over the course of Parkinson's disease in regions affected early through to end-stage disease. Brain tissue samples from 33 cases at different disease stages and 13 controls were collected through the Australian Network of Brain Banks. 500 mg of frozen putamen (affected preclinically) and frontal cortex (affected late) was homogenized, fractionated and α-synuclein levels evaluated using specific antibodies (syn-1, BD Transduction Laboratories; S129P phospho-α-synuclein, Elan Pharmaceuticals) and quantitative western blotting. Statistical analyses assessed the relationship between the different forms of α-synuclein, compared levels between groups, and determined any changes over the disease course. Soluble S129P was detected in controls with higher levels in putamen compared with frontal cortex. In contrast, insoluble α-synuclein occurred in Parkinson's disease with a significant increase in soluble and lipid-associated S129P, and a decrease in soluble frontal α-synuclein over the disease course. Increasing soluble S129P in the putamen correlated with increasing S129P in other fractions and regions. These data show that soluble non-phosphorylated α-synuclein decreases over the course of Parkinson's disease, becoming increasingly phosphorylated and insoluble. The finding that S129P α-synuclein normally occurs in vulnerable brain regions, and in Parkinson's disease has the strongest relationships to the pathogenic forms of α-synuclein in other brain regions, suggests a propagating role for putamenal phospho-α-synuclein in disease pathogenesis.

Background: Hereditary hemochromatosis resulting either from homozygosity for the C282Y polymorphism of the HFE gene, or compound heterozygosity for C282Y and H63D, manifests with liver disease and hypogonadism. However, it is unclear whether men who are heterozygotes for C282Y or H63D exhibit subtle abnormalities of sex hormone status., Aims: To evaluate whether heterozygosity for either of the HFE gene polymorphisms C282Y or H63D is associated with circulating testosterone and SHBG in men., Subjects and Methods: We performed a cross-sectional analysis of 388 community-dwelling men. Men were genotyped for C282Y and H63D. Sera were analysed for testosterone and SHBG, and insulin resistance was estimated using a homeostatic model (HOMA2-IR)., Results: Mean age of men in the cohort was 56.9 yr. Men who were heterozygous for the C282Y polymorphism in the HFE gene had higher SHBG levels than men who did not carry this polymorphism (mean ± SE, 38.2 ± 1.64 vs 32.8 ± 0.71 nmol/l, p=0.006). Total and free testosterone levels did not differ in the two groups. In multivariate analysis adjusting for potential confounders including age, waist circumference, testosterone, and HOMA2-IR, C282Y heterozygosity remained associated with SHBG levels (p<0.001)., Conclusion: The C282Y polymorphism is associated with SHBG levels in men who do not manifest iron overload. Further studies are needed to clarify potential mechanisms and determine the clinical relevance of this finding.

Unlabelled: Iron and cholesterol are both essential metabolites in mammalian systems, and too much or too little of either can have serious clinical consequences. In addition, both have been associated with steatosis and its progression, contributing, inter alia, to an increase in hepatic oxidative stress. The interaction between iron and cholesterol is unclear, with no consistent evidence emerging with respect to changes in plasma cholesterol on the basis of iron status. We sought to clarify the role of iron in lipid metabolism by studying the effects of iron status on hepatic cholesterol synthesis in mice with differing iron status. Transcripts of seven enzymes in the cholesterol biosynthesis pathway were significantly up-regulated with increasing hepatic iron (R(2) between 0.602 and 0.164), including those of the rate-limiting enzyme, 3-hydroxy-3-methylglutarate-coenzyme A reductase (Hmgcr; R(2) = 0.362, P < 0.002). Hepatic cholesterol content correlated positively with hepatic iron (R(2) = 0.255, P < 0.007). There was no significant relationship between plasma cholesterol and either hepatic cholesterol or iron (R(2) = 0.101 and 0.014, respectively). Hepatic iron did not correlate with a number of known regulators of cholesterol synthesis, including sterol-regulatory element binding factor 2 (Srebf2; R(2) = 0.015), suggesting that the increases seen in the cholesterol biosynthesis pathway are independent of Srebf2. Transcripts of genes involved in bile acid synthesis, transport, or regulation did not increase with increasing hepatic iron., Conclusion: This study suggests that hepatic iron loading increases liver cholesterol synthesis and provides a new and potentially important additional mechanism by which iron could contribute to the development of fatty liver disease or lipotoxicity.

Iron is essential in the brain, yet too much iron can be toxic. Tight regulation of iron in the brain may involve intrinsic mechanisms that control internal homeostasis independent of systemic iron status. Iron abnormalities occur in various neurological disorders, usually with symptoms or neuropathology associated with movement impairment or behavioral disturbances rather than cognitive impairment or dementia. Consistent with this, polymorphisms in the HFE gene, associated with the iron overload disorder hemochromatosis, show stronger associations with the movement disorder amyotrophic lateral sclerosis (motor neuron disease) than with cognitive impairment. Such associations may arise because certain brain regions involved in movement or executive control are particularly iron-rich, notably the basal ganglia, and may be highly reliant on iron. Various mechanisms, including iron redistribution causing functional iron deficiency, lysosomal and mitochondrial abnormalities or oxidative damage, could underlie iron-related neuropathogenesis. Clarifying how iron contributes causatively to neurodegeneration may improve treatment options in a range of neurodegenerative disorders. This review considers how modern molecular genetic approaches can be applied to resolve the complex molecular systems and pathways by which brain iron homeostasis is regulated and the molecular changes that occur with iron dyshomeostasis and neuropathogenesis.

The effects of systemic iron overload on the brain are unclear. Microarray analysis of brain gene expression in mice following short-term iron supplementation revealed altered expression of 287 genes, although most changes were small. Transcripts for the iron storage protein ferritin light chain increased 20% (p=0.002) and transcripts for iron regulatory protein 1, which negatively regulates ferritin translation, decreased 28% (p=0.048). There were expression changes for genes involved in important brain functions such as neurotransmission and nitric oxide signaling, which is dependent on iron. Few changes related to reactive oxygen species, inflammation or apoptosis, however expression changes were observed for genes causatively linked to neurological disorders, including Charcot-Marie-Tooth disease, neuronal ceroid lipofuscinosis and mucolipidosis. The latter involve intralysosomal lipofuscin build-up that may reflect lysosomal iron accumulation. The findings suggest that high iron intake may cause subtle brain effects of clinical relevance in some circumstances., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Studies of iron overload in humans and animals suggest that brain iron concentrations may be related in a regionally specific way to body iron status. However, few quantitative studies have investigated the associations between peripheral and regional brain iron in a normal elderly cohort. To examine these relationships, we used MRI to measure the proton transverse relaxation rate (R(2)) in 13 gray and white matter brain regions in 18 elderly men (average age, 75.5 years) with normal cognition. Brain R(2) values were compared with liver iron concentrations measured using the FerriScan MRI technique and serum iron indices. R(2) values in high-iron gray matter regions were significantly correlated (positively) with liver iron concentrations (globus pallidus, ventral pallidum) and serum transferrin saturation (caudate nucleus, globus pallidus, putamen) measured concurrently with brain R(2), and with serum iron concentrations (caudate nucleus, globus pallidus) measured three years before the current study. Our results suggest that iron levels in specific gray matter brain regions are influenced by systemic iron status in elderly men.

The relationship of iron status with cognition and dementia risk in older people is contentious. We have examined the longitudinal relationship between serum ferritin and cognition in 800 community-dwelling Australians 60 years or older. Iron studies (serum iron, transferrin saturation, serum ferritin) were performed in 1994/5 and 2003/4 and clinical and cognitive assessments were conducted in 2003/4 for 800 participants of the Busselton Health Study. All participants completed the Cambridge Cognitive test (CAMCOG). Those with CAMCOG scores <84 underwent expert clinical review for cognitive disorders, including the Clinical Dementia Rating scale. Mean serum iron (18.3 micromol/l) and transferrin saturation (28.5%) in 2003/4 did not differ significantly from 1994/5 whereas mean serum ferritin decreased from 162 microg/l in 1994/5 to 123 microg/l in 2003/4, possibly reflecting aging or dietary changes. No relationships were observed between serum iron or transferrin saturation and presence or absence of dementia (p> 0.05). In participants without dementia (n=749), neither serum ferritin in 1994/5 or 2003/4 nor change in serum ferritin between these times was related to total CAMCOG or executive function scores, with or without adjustment for gender, age, National Adult reading test, or stroke history (all p> 0.05). No relationships were observed between ferritin and cognition for participants with possible or probable dementia (n=51). All participants identified as HFE C282Y homozygous or with serum ferritin >1,000 ng/ml had normal CAMCOG scores. We conclude abnormal body iron stores (low or high) are unlikely to have clinically significant effects on cognition or dementia risk in community-dwelling older people.

Background: Lower testosterone levels are associated with anaemia in older men and women. The relation between testosterone and haemoglobin (Hb) in younger and middle-aged men is less well defined. The aim of the study was to examine the association between testosterone and Hb levels in men spanning middle to older ages., Methods: A cross-sectional analysis of 492 men aged 30.7-94.5 years from the Busselton Health Survey, Western Australia, was carried out. Haemoglobin (Hb), early-morning serum total testosterone and sex hormone-binding globulin (SHBG) were measured. Free testosterone was calculated using mass action equations., Results: Haemoglobin correlated to total and free testosterone concentrations (r= 0.13, P= 0.003 and r= 0.20, P < 0.001, respectively). Hb and SHBG were inversely correlated (r=-0.14, P= 0.001). Hb increased across lowest to highest quartiles of total testosterone (P= 0.02) and free testosterone (P < 0.001), but not SHBG. After adjusting for age, waist circumference, smoking status, alcohol consumption, renal function and ferritin, total testosterone was associated with Hb (beta= 0.037, P= 0.003) as was free testosterone (beta= 2.32, P < 0.001), whereas SHBG was not associated., Conclusion: Testosterone concentration modulates Hb levels in community-dwelling men across a wide age range. Further studies are needed to clarify implications of this association between testosterone and Hb in men.

Hereditary nonpolyposis colorectal cancer (HNPCC) is characterized by germline mutations in DNA mismatch repair genes; however, variation in disease expression suggests that there are potential modifying factors. Polymorphisms of the HFE gene, which cause the iron overload disorder hereditary haemochromatosis, have been proposed as potential risk factors for the development of colorectal cancer (CRC). To understand the relationship between HNPCC disease phenotype and polymorphisms of the HFE gene, a total of 362 individuals from Australia and Poland with confirmed causative MMR gene mutations were genotyped for the HFE C282Y and H63D polymorphisms. A significantly increased risk of developing CRC was observed for H63D homozygotes when compared with combined wild-type homozygotes and heterozygotes (hazard ratio = 2.93, p = 0.007). Evidence for earlier CRC onset was also observed in H63D homozygotes with a median age of onset 6 years earlier than wild type or heterozygous participants (44 vs. 50 years of age). This effect was significant by all tests used (log-rank test p = 0.026, Wilcoxon p = 0.044, Tarone-Ware p = 0.035). No association was identified for heterozygosity of either polymorphism and limitations on power-prevented investigation of C282Y homozygosity or compound C282Y/H63D heterozygosity. In the Australian sample only, women had a significantly reduced risk of developing CRC when compared with men (hazard ratio = 0.58, p = 0.012) independent of HFE genotype for either single nucleotide polymorphisms. In conclusion, homozygosity for the HFE H63D polymorphism seems to be a genetic modifier of disease expression in HNPCC. Understanding the mechanisms by which HFE interrelates with colorectal malignancies could lead to reduction of disease risk in HNPCC.

Objective: To investigate whether citrus fruit, noncitrus fruit, and other dietary factors act as environmental modifiers of iron status in the absence or presence of hemochromatotic HFE gene mutations., Participants and Methods: Iron studies, HFE genotypic analyses, and dietary data from a survey conducted from March 21, 1994, through December 15, 1995, were analyzed for a group of 2232 residents (1105 men, 1127 women) aged 20 to 79 years recruited from the community electoral roll of Busselton in Western Australia. Data were analyzed by linear regression analysis and analysis of covariance., Results: Higher levels of fresh fruit intake (excluding citrus fruits and citrus juices) had a significant protective effect (P=.002) against high body iron status as gauged by ferritin levels in men, irrespective of HFE genotype. Consumption of 2 or more pieces of fruit per day on average reduced mean serum ferritin levels by 20% compared with average consumption of less than 1 piece of fruit per day. This effect was not observed in women. Consumption of citrus fruits and citrus juices had no significant effects in either sex. No protective effects were observed for tea consumption or any other dietary factors studied. Red meat and alcohol consumption correlated with high body iron stores (P<.05), consistent with previous studies, but did not interact with fruit with regard to effects on serum ferritin (P>.05)., Conclusion: Noncitrus fruits are environmental modifiers of iron status independent of HFE genotype. This could have important implications for the provision of evidence-based dietary advice to patients with other iron-storage disorders.

Hereditary hemochromatosis (HH) comprises a group of inherited disorders of iron metabolism that can result in progressive iron overload, morbidity, and mortality, generally in adulthood. HFE-related HH is the most common type of HH and will form the core of this discussion. The discovery of new proteins and gene mutations has defined other types of HH, termed non-HFE HH. The regulatory protein hepcidin has a central role in iron homeostasis in these disorders. While the liver is the predominant organ of iron deposition and iron-overload-related disease in HFE-related HH, involvement of extrahepatic tissue can also result in morbidity and mortality if the disorder is not diagnosed before organ damage develops. This review traverses the road from HFE genotype to phenotype with a focus on clinical penetrance, modifier factors for disease expression, and current thoughts and controversies on HH diagnosis and screening.

The matrix metalloproteinases (MMPs) are expressed in response to pro-inflammatory stimuli and other triggers. The MMPs cleave numerous substrates including extracellular matrix components, cytokines and growth factors. In the CNS, while most studied in the context of disease, the many physiological functions of the MMPs are now becoming appreciated. This review provides an overview of the growing body of evidence for physiological roles of MMPs both in CNS development and in CNS plasticity in normal brain functioning, including learning and memory, as well as in CNS repair and reorganization as part of the neuroimmune response to injury.

The MYH gene has recently been shown to be associated with a recessive form of colorectal adenomatous polyposis. Two common mutations in the MYH gene have been identified that lend themselves to rapid screening. We have examined a series of 302 individuals comprising 120 control subjects, 120 patients diagnosed with adenomatous polyposis but without germline mutations in the APC gene and 62 patients diagnosed with familial adenomatous polyposis all harbouring confirmed causative APC germline mutations. The results reveal that MYH accounts for 16 percent of polyposis patients without germline mutations in the APC gene and that it does not appear to be a modifier gene in FAP patients diagnosed with APC germline mutations., ((c) 2005 Wiley-Liss, Inc.)

Insoluble alpha-synuclein plays a central role in Lewy body diseases, with considerable controversy as to whether it plays a similar role in Alzheimer's disease (AD). We assessed the tissue location and solubility of cortical alpha-synuclein in AD (without Lewy body formation) compared with controls, using sequential extraction procedures and Western immunoblotting to quantify different alpha-synuclein species in their different solubility states. Controls had no insoluble cortical alpha-synuclein and a ratio of soluble:lipid-associated alpha-synuclein of 1.2-/+0.1. Total alpha-synuclein protein was significantly increased in AD and concentrated within the lipid-associated fraction (soluble:lipid ratio 0.9-/+0.05, soluble:insoluble 1.5-/+0.1, lipid:insoluble 1.7-/+0.1) which proved difficult to localize in paraffin-embedded tissue. Tissues prepared without lipid extraction revealed alpha-synuclein-immunoreactivity in the amorphous components of mature cored AD plaques. This lipid-association of alpha-synuclein in mature AD plaques links this protein with other lipid changes thought to be important in disease pathogenesis.

Excess iron accumulation in the brain is a consistent observation in Alzheimer's Disease. Iron affects amyloid precursor protein (AbetaPP) processing and promotes deposition of Abeta. Iron is also among the most potent biological toxins because of its ability to react with oxygen to form reactive oxygen species. Consequently, elucidation of the mechanisms associated with maintaining brain iron homeostasis is fundamentally important to understanding the underlying pathogenesis in AD. The iron overload disorder, Hemochromatosis, is the most common genetic disorder (1:200) so a significant percentage of AD patients can be expected to carry this mutation. Heterozygotes for this mutation also have an increased, but sub-clinical iron burden. Given the high percentage of the population who are at significant risk for iron overload, we propose that the hemochromatosis mutation be considered as a confounding factor when evaluating the contribution of genetic associations with AD and treatment strategies and efficacy. Two recent papers and new evidence presented here that the protein associated with hemochromatosis is expressed on blood vessels, choroid plexus and the ependymal cells in the brain are offered as support for this proposal.

This aim of the present study was to identify whether apoptotic features relate to the degree of cortical neuronal loss in cases with variable cortical degeneration. Neuronal apoptosis was assessed using histochemical and morphological criteria in cases with Alzheimer's disease (AD, n=7) or Lewy bodies (n= 11) compared with controls (n=11). AD cases had both significant plaque and tangle formation but no Lewy bodies, while cases with Lewy bodies had significant plaque formation but no tangles. Cortical sections were stained using (TdT)-mediated UTP nick end labelling (TUNEL), propidium iodide, and cell and pathology-specific labels. Cells identified as non-neuronal were excluded. Large cortical nuclei were classified as abnormal if they were TUNEL-positive with DNA condensation across the nucleus and no visible nucleolus, and further subdivided according to the presence or absence of visible neuronal cytoplasm. Nuclei were considered morphologically normal regardless of TUNEL staining if they possessed a clear nucleolus. Cortical fields containing the greatest density of TUNEL-positive nuclei were regionally sampled and the proportion of all classified nuclei calculated. Analysis of variance was used to identify any significant relationships. Only AD cases had significant numbers of abnormal nuclei (23+/-6%, P=0.002) and a corresponding decrease in normal neurons (28+/-7% loss, P= 0.004). Absolute neuronal density was also decreased in AD (AD density 64+/-11% of other cases, P=0.02). Abnormal nuclei were not associated with plaque or tangle pathology. Our results suggest that nuclear abnormalities appear restricted to AD cases with substantial tau deposition and are related to the degree of neuronal degeneration.

Transplantation repair of demyelinating lesions is restricted because relatively few cells can be introduced at only a limited number of sites. Repair could be enhanced by stimulating division of transplanted cells and by directing migration to multiple or distant lesions. This article demonstrates that transplanted oligodendroglial progenitors proliferate more when co-grafted with growth factor-secreting cells, yet retain the capacity to form myelin. Transplanted glial cells also migrate preferentially toward the growth factor-secreting cells when the two are implanted at separate sites. This opens avenues to examine growth factor actions on glia in vivo and improves the prospects for human remyelination therapies., (Copyright 2000 Wiley-Liss, Inc.)

The present investigation aimed to examine associations of anaemia with dementia. Analysis of community-dwelling, elderly subjects characterized for different dementias failed to confirm a previously reported association of anaemia with Alzheimer's disease (AD) but revealed instead a significant association with vascular dementia (VAD). Nearly 45% of VAD subjects were anaemic, compared with 17% of controls. Close to one-third of all subjects with haemoglobin levels > 0.5 g/dl below reference anaemia levels had VAD. Co-existing VAD may explain previous links between AD and anaemia. The association was independent of age, dementia severity and a range of other factors including vitamin B 12 and folate levels. Anaemia can exacerbate focal cerebral ischaemia and could precipitate or amplify VAD symptoms in elderly subjects with vasculopathy.

Glial cell transplantation into myelin-deficient rodent models has resulted in myelination of axons and restoration of conduction velocity. The shaking (sh) pup canine myelin mutant is a useful model in which to test the ability to repair human myelin diseases, but as in humans, the canine donor supply for allografting is limited. A solution may be provided by self-renewing epidermal growth factor (EGF)-responsive multipotential neural progenitor cell populations ("neurospheres"). Nonadherent spherical clusters, similar in appearance to murine neurospheres, have been obtained from the brain of perinatal wildtype (wt) canine brain and expanded in vitro in the presence of EGF for at least 6 months. Most of the cells in these clusters express a nestin-related protein. Within 1-2 weeks after removal of EGF, cells from the clusters generate neurons, astrocytes, and both oligodendroglial progenitors and oligodendrocytes. Transplantation of lacZ-expressing wt neurospheres into the myelin-deficient (md) rat showed that a proportion of the cells differentiated into oligodendrocytes and produced myelin. In addition, cells from the neurosphere populations survived at least 6 weeks after grafting into a 14-day postnatal sh pup recipient and at least 2 weeks after grafting into an adult sh pup recipient. Thus, neurospheres provide a new source of allogeneic donor cells for transplantation studies in this mutant.

Oligodendrocytes originate in different neural tube domains, within boundaries of expression of a series of patterning genes which condition the diverse morphogenetic programme of each area. Although neuronal and astrocyte heterogeneity are widely accepted, and despite accumulating evidence for oligodendrocyte heterogeneity in vivo, oligodendrocytes in vitro are currently considered as a homogeneous cell population. The present investigation demonstrates that oligodendrocyte diversity can be detected in vitro and characterizes a novel morphological class of O4-positive oligodendrocyte which is consistently identifiable in rat central nervous system cultures. These cells have a very characteristic epithelioid, unbranched and often lobulated morphology which enables their identification within 2 h of plating. Immunostaining shows that this morphological type is sometimes positive for GD3, A2B5 and vimentin, and most of the time positive for Ranscht antibody, O1 and Rip but negative for glial fibrillary acidic protein, OX-42, neuron-specific enolase, nestin and erbB2. The apparent levels and/or distributions of (i) microtubules, (ii) surface glycolipids recognized by O4, O1 and Ranscht antibody, and (iii) the less specific marker carbonic anhydrase II, typically differ from those of nearby classical, branched oligodendrocytes. Cells with this epithelioid morphology also express myelin basic protein and O10 (a proteolipid protein epitope), both of which are markers for mature oligodendrocytes. Conversely, O4+/O1- cells with this membranous appearance were also seen. Although these atypical oligodendrocytes were most abundant in spinal cord cultures (representing >10% of the O4+ population), they were not exclusive to this region and occurred at a low frequency in neonatal optic nerve cultures.

Transplantation of cells into the CNS of human patients with neurodegenerative disorders offers a radical new approach to the treatment of previously incurable diseases. Considerable success has been achieved in Parkinson's disease following transplantation of human fetal dopaminergic neurons. Disorders of myelination of the brain, of either inherited or acquired origin, might also be treated by glial cell transplantation although there are additional challenges. Cells of the oligodendrocyte lineage have been found to be capable of myelinating axons on transplantation into numerous experimental pathological environments, including the CNS of myelin mutants and focal areas of demyelination in normal animals made by injection of myelinotoxic chemicals. In general, primary cells and progenitors are likely to have the greatest myelinating capacity. Cell lines can also be used, but those driven by oncogenes may produce little myelin, and tumor formation is likely. Schwann cells are also a potential source of cells, possibly as a homograft, and may be primed by treatment ex vivo with glial growth factors. The variable CNS milieu seen in human myelin disease will mean that transplanted cells must be able to migrate appropriately and myelinate axons in an adult, pathological environment, and this awaits experimental confirmation. Physiological analysis of transplants in such situations in adult animals will provide the functional data which may expedite clinical trials.

The major component of the amyloid deposition that characterizes Alzheimer's disease is the 4-kDa beta A4 protein, which is derived from a much larger amyloid protein precursor (APP). A procedure for the complete purification of APP from human brain is described. The same amino terminal sequence of APP was found in two patients with Alzheimer's disease and one control subject. Two major forms of APP were identified in human brain with apparent molecular masses of 100-110 kDa and 120-130 kDa. Soluble and membrane fractions of brain contained nearly equal amounts of APP in both humans and rats. Immunoprecipitation with carboxyl terminus-directed antibodies indicates that the soluble forms of APP are truncated. Carboxyl terminus truncation of membrane-associated forms of human brain APP was also found to occur during postmortem autolysis. The availability of purified human brain APP will facilitate the investigation of its normal function and the events that lead to its abnormal cleavage in patients with Alzheimer's disease.

The beta A4 protein, the major component of the amyloid deposition characterizing Alzheimer's disease, derives from the amyloid protein precursor (APP), an integral membrane protein with soluble derivatives. The function of APP is unknown. Both soluble and membrane-associated human brain APP (10(-10) M) significantly increased (P less than 0.025) neurite length and branching in pheochromocytoma PC12 cells, but did not affect the number of neurites per cell. At higher concentrations, APP was cytotoxic, with a half-maximal concentration of 5 x 10(-9) M. Nerve growth factor (NGF) is known to affect APP expression in vivo and in vitro. Antibodies to APP specifically diminished the effects of NGF on neurite length and branching. Thus APP may act to mediate neurite outgrowth promotion by NGF.

Previous studies have established that, under appropriate conditions, fetal rat hepatocytes will differentiate in culture. This is characterized by the acquisition and loss of enzyme markers which are observed during liver development in vivo. The expression of alpha-fetoprotein (AFP), which declines during normal development, is examined in cultured hepatocytes derived from 15- and 19-day gestation rats. Secretion of AFP and relative levels of AFP mRNA and gene transcription were measured. Initially, AFP expression was greater in 15-day gestation hepatocytes, and in both instances AFP secretion and AFP mRNA decreased during culture. The decline in AFP expression by 15-day gestation fetal hepatocytes in vitro was not significantly altered by various manipulations of the culture conditions. It is proposed that cultured fetal hepatocytes continue to differentiate in vitro by repressing AFP expression while the expression of other liver-specific genes is being initiated. The fetal hepatocyte culture model therefore adequately reflects in vivo changes in developmentally regulated liver-specific genes.