Brain iron acquisition depends on age and sex in iron-deficient mice.

Adequate and timely delivery of iron is essential for brain development. The uptake of transferrin-bound (Tf) iron into the brain peaks at the time of myelination, whereas the recently discovered H-ferritin (FTH1) transport of iron into the brain continues to increase beyond the peak in myelination. Here, we interrogate the impact of dietary iron deficiency (ID) on the uptake of FTH1- and Tf-bound iron. In the present study, we used C57BL/6J male and female mice at a developing (post-natal day (PND) 15) and adult age (PND 85). In developing mice, ID results in increased iron delivery from both FTH1 and Tf for both males and females. The amount of iron uptake from FTH1 was higher than the Tf and this difference between the iron delivery was much greater in females. In contrast, in the adult model, ID was associated with increased brain iron uptake by both FTH1 and Tf but only in the males. There was no increased uptake from either protein in the females. Moreover, transferrin receptor expression on the microvasculature as well as whole brain iron, and H and L ferritin levels revealed the male brains became iron deficient but not the female brains. Last, under normal dietary conditions, 55 Fe uptake was higher in the developing group from both delivery proteins than in the adult group. These results indicate that there are differences in iron acquisition between the developing and adult brain for FTH1 and Tf during nutritional ID and demonstrate a level of regulation of brain iron uptake that is age and sex-dependent.

Common Mutation in the HFE Gene Modifies Recovery After Intracerebral Hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is characterized by bleeding into the brain parenchyma. During an ICH, iron released from the breakdown of hemoglobin creates a cytotoxic environment in the brain through increased oxidative stress. Interestingly, the loss of iron homeostasis is associated with the pathological process of other neurological diseases. However, we have previously shown that the H63D mutation in the homeostatic iron regulatory (HFE) gene, prevalent in 28% of the White population in the United States, acts as a disease modifier by limiting oxidative stress. The following study aims to examine the effects of the murine homolog, H67D HFE, on ICH. METHODS: An autologous blood infusion model was utilized to create an ICH in the right striatum of H67D and wild-type mice. The motor recovery of each animal was assessed by rotarod. Neurodegeneration was measured using fluorojade-B and mitochondrial damage was assessed by immunofluorescent numbers of CytC+ (cytochrome C) neurons and CytC+ astrocytes. Finally, the molecular antioxidant response to ICH was quantified by measuring Nrf2 (nuclear factor-erythroid 2 related factor), GPX4 (glutathione peroxidase 4), and FTH1 (H-ferritin) levels in the ICH-affected and nonaffected hemispheres via immunoblotting. RESULTS: At 3 days post-ICH, H67D mice demonstrated enhanced performance on rotarod compared with wild-type animals despite no differences in lesion size. Additionally, H67D mice displayed higher levels of Nrf2, GPX4, and FTH1 in the ICH-affected hemisphere; however, these levels were not different in the contralateral, non-ICH-affected hemisphere. Furthermore, H67D mice showed decreased degenerated neurons, CytC+ Neurons, and CytC+ astrocytes in the perihematomal area. CONCLUSIONS: Our data suggest that the H67D mutation induces a robust antioxidant response 3 days following ICH through Nrf2, GPX4, and FTH1 activation. This activation could explain the decrease in degenerated neurons, CytC+ neurons, and CytC+ astrocytes in the perihematomal region, leading to the improved motor recovery. Based on this study, further investigation into the mechanisms of this neuroprotective response and the effects of the H63D HFE mutation in a population of patients with ICH is warranted.

Regulation of brain iron uptake by apo- and holo-transferrin is dependent on sex and delivery protein.

BACKGROUND: The brain requires iron for a number of processes, including energy production. Inadequate or excessive amounts of iron can be detrimental and lead to a number of neurological disorders. As such, regulation of brain iron uptake is required for proper functioning. Understanding both the movement of iron into the brain and how this process is regulated is crucial to both address dysfunctions with brain iron uptake in disease and successfully use the transferrin receptor uptake system for drug delivery. METHODS: Using in vivo steady state infusions of apo- and holo-transferrin into the lateral ventricle, we demonstrate the regulatory effects of brain apo- and holo-transferrin ratios on the delivery of radioactive 55Fe bound to transferrin or H-ferritin in male and female mice. In discovering sex differences in the response to apo- and holo-transferrin infusions, ovariectomies were performed on female mice to interrogate the influence of circulating estrogen on regulation of iron uptake. RESULTS: Our model reveals that apo- and holo-transferrin significantly regulate iron uptake into the microvasculature and subsequent release into the brain parenchyma and their ability to regulate iron uptake is significantly influenced by both sex and type of iron delivery protein. Furthermore, we show that cells of the microvasculature act as reservoirs of iron and release the iron in response to cues from the interstitial fluid of the brain. CONCLUSIONS: These findings extend our previous work to demonstrate that the regulation of brain iron uptake is influenced by both the mode in which iron is delivered and sex. These findings further emphasize the role of the microvasculature in regulating brain iron uptake and the importance of cues regarding iron status in the extracellular fluid.

The Nrf2-mediated defense mechanism associated with HFE genotype limits vulnerability to oxidative stress-induced toxicity.

There is considerable interest in gene and environment interactions in neurodegenerative diseases. The HFE (homeostatic iron regulator) gene variant (H63D) is highly prevalent in the population and has been investigated as a disease modifier in multiple neurodegenerative diseases. We have developed a mouse model to interrogate the impact of this gene variant in a model of paraquat toxicity. Using primary astrocytes, we found that the H67D-Hfe(equivalent of the human H63D variant) astrocytes are less vulnerable than the WT-Hfe astrocytes to paraquat-induced cell death, mitochondrial damage, and cellular senescence. We hypothesized that the Hfe variant-associated protection is a result of the activation of the Nrf2 antioxidant defense system and found a significant increase in Nrf2 levels after paraquat exposure in the H67D-Hfe astrocytes than the WT-Hfe astrocytes. Moreover, decreasing Nrf2 by molecular or pharmaceutical manipulation resulted in increased vulnerability to paraquat in the H67D-Hfe astrocytes. To further elucidate the role of Hfe variant genotype in neuroprotection mediated by astrocytes, we added media from the paraquat-treated astrocytes to differentiated SH-SY5Y neuroblastoma cells and found a significantly larger reduction in the viability when treated with WT-Hfe astrocyte media than the H67D-Hfe astrocyte media possibly due to higher secretion of IL-6 observed in the WT-Hfe astrocytes. To further explore the mechanism of Nrf2 protection, we measured NQO1, the Nrf2-mediated antioxidant, in primary astrocytes and found a significantly higher NQO1 level in the H67D-Hfe astrocytes. To consider the translational potential of our findings, we utilized the PPMI (Parkinson's Progression Markers Initiative) clinical database and found that, consistent with the mouse study, H63D-HFE carriers had a significantly higher NQO1 level in the CSF than the WT-HFE carriers. Consistent with our previous reports on H63D-HFE in disease, these data further suggest that HFE genotype in the human population impacts the antioxidant defense system and can therefore alter pathogenesis.

Transferrin and H-ferritin involvement in brain iron acquisition during postnatal development: impact of sex and genotype.

Iron delivery to the developing brain is essential for energy and metabolic support needed for processes such as myelination and neuronal development. Iron deficiency, especially in the developing brain, can result in a number of long-term neurological deficits that persist into adulthood. There is considerable debate that excess access to iron during development may result in iron overload in the brain and subsequently predispose individuals to age-related neurodegenerative diseases. There is a significant gap in knowledge regarding how the brain acquires iron during development and how biological variables such as development, genetics, and sex impact brain iron status. In this study, we used a mouse model expressing a mutant form of the iron homeostatic regulator protein HFE, (Hfe H63D), the most common gene variant in Caucasians, to determine impact of the mutation on brain iron uptake. Iron uptake was assessed using 59 Fe bound to either transferrin or H-ferritin as the iron carrier proteins. We demonstrate that at postnatal day 22, mutant mice brains take up greater amounts of iron compared with wildtype. Moreover, we introduce H-ferritin as a key protein in brain iron transport during development and identify a sex and genotype effect demonstrating female mutant mice take up more iron by transferrin, whereas male mutant mice take up more iron from H-ferritin at PND22. Furthermore, we begin to elucidate the mechanism for uptake using immunohistochemistry to profile the regional distribution and temporal expression of transferrin receptor and T-cell immunoglobulin and mucin domain 2, the latter is the receptor for H-ferritin. These data demonstrate that sex and genotype have significant effects on iron uptake and that regional receptor expression may play a large role in the uptake patterns during development. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14731.

A Possible Role for Platelet-Activating Factor Receptor in Amyotrophic Lateral Sclerosis Treatment.

Amyotrophic lateral sclerosis (ALS) is the third most prevalent neurodegenerative disease affecting upper and lower motor neurons. An important pathway that may lead to motor neuron degeneration is neuroinflammation. Cerebrospinal Fluids of ALS patients have increased levels of the inflammatory cytokine IL-18. Because IL-18 is produced by dendritic cells stimulated by the platelet-activating factor (PAF), a major neuroinflammatory mediator, it is expected that PAF is involved in ALS. Here we show pilot experimental data on amplification of PAF receptor (PAFR) mRNA by RT-PCR. PAFR is overexpressed, as compared to age matched controls, in the spinal cords of transgenic ALS SOD1-G93A mice, suggesting PAF mediation. Although anti-inflammatory drugs have been tested for ALS before, no clinical trial has been conducted using PAFR specific inhibitors. Therefore, we hypothesize that administration of PAFR inhibitors, such as Ginkgolide B, PCA 4248 and WEB 2086, have potential to function as a novel therapy for ALS, particularly in SOD1 familial ALS forms. Because currently there are only two approved drugs with modest effectiveness for ALS therapy, a search for novel drugs and targets is essential.

HFE Genotype Restricts the Response to Paraquat in a Mouse Model of Neurotoxicity.

Parkinson's disease is marked clinically by motor dysfunction and pathologically by dopaminergic cell loss in the substantia nigra and iron accumulation in the substantia nigra. The driver underlying iron accumulation remains unknown and could be genetic or environmental. The HFE protein is critical for the regulation of cellular iron uptake. Mutations within this protein are associated with increased iron accumulation including in the brain. We have focused on the commonly occurring H63D variant of the HFE gene as a disease modifier in a number of neurodegenerative diseases. To investigate the role of H63D HFE genotype, we generated a mouse model in which the wild-type (WT) HFE gene is replaced by the H67D gene variant (mouse homolog of the human H63D gene variant). Using paraquat toxicity as the model for Parkinson's disease, we found that WT mice responded as expected with significantly greater motor function, loss of tyrosine hydroxylase staining and increase microglial staining in the substantia nigra, and an increase in R2 relaxation rate within the substantia nigra of the paraquat-treated mice compared to their saline-treated counterparts. In contrast, the H67D mice showed a remarkable resistance to paraquat treatment; specifically differing from the WT mice with no changes in motor function or changes in R2 relaxation rates following paraquat exposure. At baseline, there were differences between the H67D HFE mice and WT mice in gut microbiome profile and increased L-ferritin staining in the substantia nigra that could account for the resistance to paraquat. Of particular note, the H67D HFE mice regardless of whether or not they were treated with paraquat had significantly less tyrosine hydroxylase immunostaining than WT. Our results clearly demonstrate that the HFE genotype impacts the expression of tyrosine hydroxylase in the substantia nigra, the gut microbiome and the response to paraquat providing additional support that the HFE genotype is a disease modifier for Parkinson's disease. Moreover, the finding that the HFE mutant mice are resistant to paraquat may provide a model in which to study resistant mechanisms to neurotoxicants.

Reduced Cerebral White Matter Integrity Assessed by DTI in Cognitively Normal H63D-HFE Polymorphism Carriers.

BACKGROUND AND PURPOSE: The H63D-HFE single nucleotide polymorphism (SNP) has been associated with brain iron dysregulation; however, the emergent role of this missense variant in brain structure and function has yet to be determined. Previous work has demonstrated that HFE SNP carriers have reduced white matter magnetic resonance imaging (MRI) proton relaxation rates. The mechanism by which white matter alterations perturb MRI relaxation is unknown as is how these metrics are related to myelin integrity. METHODS: Fifteen subjects heterozygous for the HFE-H63D SNP and 25 controls with wild-type HFE had diffusion-weighted, anatomical MRIs taken, and underwent cognitive assessment. Fractional anisotropy (FA), mean diffusion (MD), and mode of anisotropy (MO) were calculated from the diffusion dataset to investigate the relationship between the H63D-HFE SNP and myelin integrity. RESULTS: A decrease in FA, an increase in MD, and an increase in MO are demonstrated in multiple H63D-HFE polymorphism carrier white matter tracts. Regions with altered diffusion metrics are notably located in heavily myelinated white matter association fibers, such as the anterior corona radiata and longitudinal fasciculi. CONCLUSIONS: The MRI data presented here demonstrate that H63D-HFE polymorphism carriers have diffusivity changes in white matter compared to wild-type subjects. The reduced integrity white matter tracts in H63D-HFE carriers are hypothesized to be related to increased susceptibility of these late-myelinating regions to cellular stress induced by oligodendrocyte iron dyshomeostasis.

A role for sex and a common HFE gene variant in brain iron uptake.

HFE (high iron) is an essential protein for regulating iron transport into cells. Mutations of the HFE gene result in loss of this regulation causing accumulation of iron within the cell. The mutated protein has been found increasingly in numerous neurodegenerative disorders in which increased levels of iron in the brain are reported. Additionally, evidence that these mutations are associated with elevated brain iron challenges the paradigm that the brain is protected by the blood-brain barrier. While much has been studied regarding the role of HFE in cellular iron uptake, it has remained unclear what role the protein plays in the transport of iron into the brain. We investigated regulation of iron transport into the brain using a mouse model with a mutation in the HFE gene. We demonstrated that the rate of radiolabeled iron (59Fe) uptake was similar between the two genotypes despite higher brain iron concentrations in the mutant. However, there were significant differences in iron uptake between males and females regardless of genotype. These data indicate that brain iron status is consistently maintained and tightly regulated at the level of the blood-brain barrier.

Statins accelerate disease progression and shorten survival in SOD1(G93A) mice.

INTRODUCTION: HMG-CoA reductase inhibitors (statins) and H63D HFE polymorphism may modify amyotrophic lateral sclerosis (ALS). We hypothesized that statins worsen phenotype in ALS mice, dependent on HFE genotype. METHODS: Mice harboring SOD1(G93A) heterozygous for H67D Hfe (homologous to human H63D HFE) were administered simvastatin and/or coenzyme Q10, and were allowed to reach end stage. Disease progression was measured by grip strength. A separate group of animals was administered simvastatin and euthanized at the symptomatic 120-day time-point. Mitochondria from gastrocnemius muscle and lumbar spine were analyzed. RESULTS: Simvastatin and H67D Hfe accelerated disease progression. Simvastatin decreased survival. Coenzyme Q10 did not rescue statin-induced effects. Statins did not alter mitochondrial protein levels. CONCLUSIONS: Statins and Hfe genotype alter disease course in the ALS mouse model. Because the H63D HFE polymorphism is present in 30% of patients with ALS, studying disease progression in patients who receive statins, stratified for HFE genotype, may guide therapy. Muscle Nerve, 2016 Muscle Nerve 54: 284-291, 2016.

Reduced white matter MRI transverse relaxation rate in cognitively normal H63D-HFE human carriers and H67D-HFE mice.

Mutations within the HFE protein gene sequence have been associated with increased risk of developing a number of neurodegenerative disorders. To this effect, an animal model has been created which incorporates the mouse homologue to the human H63D-HFE mutation: the H67D-HFE knock-in mouse. These mice exhibit alterations in iron management proteins, have increased neuronal oxidative stress, and a disruption in cholesterol regulation. However, it remains undetermined how these differences translate to human H63D carriers in regards to white matter (WM) integrity. To this endeavor, MRI transverse relaxation rate (R2) parametrics were employed to test the hypothesis that WM alterations are present in H63D human carriers and are recapitulated in the H67D mice. H63D carriers exhibit widespread reductions in brain R2 compared to non-carriers within white matter association fibers in the brain. Similar R2 decreases within white matter tracts were observed in the H67D mouse brain. Additionally, an exacerbation of age-related R2 decrease is found in the H67D animal model in white matter regions of interest. The decrease in R2 within white matter tracts of both species is speculated to be multifaceted. The R2 changes are hypothesized to be due to alterations in axonal biochemical tissue composition. The R2 changes observed in both the human-H63D and mouse-H67D data suggest that modified white matter myelination is occurring in subjects with HFE mutations, potentially increasing vulnerability to neurodegenerative disorders.

H63D HFE genotype accelerates disease progression in animal models of amyotrophic lateral sclerosis.

H63D HFE is associated with iron dyshomeostasis and oxidative stress; each of which plays an important role in amyotrophic lateral sclerosis (ALS) pathogenesis. To examine the role of H63D HFE in ALS, we generated a double transgenic mouse line (SOD1/H67D) carrying the H67D HFE (homologue of human H63D) and SOD1(G93A) mutations. We found double transgenic mice have shorter survival and accelerated disease progression. We examined parameters in the lumbar spinal cord of double transgenic mice at 90days (presymptomatic), 110days (symptomatic) and end-stage. Transferrin receptor and L-ferritin expression, both indicators of iron status, were altered in double transgenic and SOD1 mice starting at 90days, indicating loss of iron homeostasis in these mice. However, double transgenic mice had higher L-ferritin expression than SOD1 mice. Double transgenic mice exhibited increased Iba-1 immunoreactivity and caspase-3 levels, indicating increased microglial activation which would be consistent with the higher L-ferritin levels. Although both SOD1 and double transgenic mice had increased GFAP expression, the magnitude of the increase was higher in double transgenic mice at 110days, suggesting increased gliosis in these mice. Increased hemeoxygenase-1 and decreased nuclear factor E2-related factor 2 levels in double transgenic mice strongly suggest the accelerated disease process could be associated with increased oxidative stress. There was no evidence of TAR-DNA-binding protein 43 mislocalization to the cytoplasm in double transgenic mice; however, there was evidence suggesting neurofilament disruption, which has been reported in ALS. Our findings indicate H63D HFE modifies ALS pathophysiology via pathways involving oxidative stress, gliosis and disruption of cellular functions.

A mutation in the HFE gene is associated with altered brain iron profiles and increased oxidative stress in mice.

Because of the increasing evidence that H63D HFE polymorphism appears in higher frequency in neurodegenerative diseases, we evaluated the neurological consequences of H63D HFE in vivo using mice that carry H67D HFE (homologous to human H63D). Although total brain iron concentration did not change significantly in the H67D mice, brain iron management proteins expressions were altered significantly. The 6-month-old H67D mice had increased HFE and H-ferritin expression. At 12 months, H67D mice had increased H- and L-ferritin but decreased transferrin expression suggesting increased iron storage and decreased iron mobilization. Increased L-ferritin positive microglia in H67D mice suggests that microglia increase iron storage to maintain brain iron homeostasis. The 6-month-old H67D mice had increased levels of GFAP, increased oxidatively modified protein levels, and increased cystine/glutamate antiporter (xCT) and hemeoxygenase-1 (HO-1) expression indicating increased metabolic and oxidative stress. By 12 months, there was no longer increased astrogliosis or oxidative stress. The decrease in oxidative stress at 12 months could be related to an adaptive response by nuclear factor E2-related factor 2 (Nrf2) that regulates antioxidant enzymes expression and is increased in the H67D mice. These findings demonstrate that the H63D HFE impacts brain iron homeostasis, and promotes an environment of oxidative stress and induction of adaptive mechanisms. These data, along with literature reports on humans with HFE mutations provide the evidence to overturn the traditional paradigm that the brain is protected from HFE mutations. The H67D knock-in mouse can be used as a model to evaluate how the H63D HFE mutation contributes to neurodegenerative diseases.

Regional and cellular distribution of mitochondrial ferritin in the mouse brain.

Iron and mitochondrial dysfunction are important in many neurodegenerative diseases. Several iron transport proteins have been identified that are associated with mitochondria, most recently mitochondrial ferritin. Here we describe the cellular distribution of mitochondrial ferritin in multiple regions of the brain in C57/BL6 mice. Mitochondrial ferritin was found in all regions of the brain, although staining intensity varied between regions. Mitochondrial ferritin was detected throughout the layers of cerebral cortex and in the cerebellum, hippocampus, striatum, choroid plexus, and ependymal cells. The cell type in the brain that stains most prominently for mitochondrial ferritin is neuronal, but oligodendrocytes also stain strongly in both gray matter and in white matter tracts. Mice deficient in H-ferritin do not differ in the mitochondrial ferritin staining pattern or intensity compared with C57/BL6 mice, suggesting that there is no compensatory expression of these proteins. In addition, by using inbred mouse strains with differing levels of iron content, we have shown that regional brain iron content does not affect expression of mitochondria ferritin. The expression of mitochondria ferritin appears to be more influenced by mitochondrial density. Indeed, at an intracellular level, mitochondrial ferritin immunoreaction product is strongest where mitochondrial density is high, as seen in the ependymal cells. Given the importance and relationship between iron and mitochondrial activity, understanding the role of mitochondrial ferritin can be expected to contribute to our knowledge of mitochondrial dysfunction and neurodegenerative disease.

Prolyl-peptidyl isomerase, Pin1, phosphorylation is compromised in association with the expression of the HFE polymorphic allele, H63D.

There is substantial interest in HFE gene variants as putative risk factors in neurodegenerative diseases such as Alzheimer disease (AD). Previous studies in cell models have shown the H63D HFE variant to result in increased cellular iron, oxidative stress, glutamate dyshomeostasis, and an increase in tau phosphorylation; all processes thought to contribute to AD pathology. Pin1 is a prolyl-peptidyl cis/trans isomerase that can regulate the dephosphorylation of the amyloid and tau proteins. Hyperphosphorylation of these later proteins is implicated in the pathogenesis of AD and Pin1 levels are reportedly decreased in AD brains. Because of the relationship between Pin1 loss of function by oxidative stress and the increase in oxidative stress in cells with the H63D polymorphism it was logical to interrogate a relationship between Pin1 and HFE status. To test our hypothesis that H63D HFE would be associated with less Pin1 activity, we utilized stably transfected human neuroblastoma SH-SY5Y cell lines expressing the different HFE polymorphisms. Under resting conditions, total Pin1 levels were unchanged between the wild type and H63D HFE cells, yet there was a significant increase in phosphorylation of Pin1 at its serine 16 residue suggesting a loss of Pin1 activity in H63D variant cells. To evaluate whether cellular iron status could influence Pin1, we treated the WT HFE cells with exogenous iron and found that Pin1 phosphorylation increased with increasing levels of iron. Iron exposure to H63D variant cells did not impact Pin1 phosphorylation beyond that already seen suggesting a ceiling effect. Because HFE H63D cells have been shown to have more oxidative stress, the cells were treated with the antioxidant Trolox which resulted in a decrease in Pin1 phosphorylation in H63D cells with no change in WT HFE cells. In a mouse model carrying the mouse equivalent of the H63D allele, there was an increase in the phosphorylation status of Pin1 providing in vivo evidence for our findings in the cell culture model. Thus, we have shown another cellular mechanism that HFE polymorphisms influence; further supporting their role as neurodegenerative disease modifiers.

Comparative study of the influence of Thy1 deficiency and dietary iron deficiency on dopaminergic profiles in the mouse striatum.

Thy-1, a glycosyl-phosphatidylinositol (GPI)-linked integral membrane protein, may play a role in stabilizing synapses. Thy1 was identified in a gene expression analysis as iron responsive, and subsequent cell culture and animal models of iron deficiency expanded this finding to the protein. The importance of Thy1 in influencing neurotransmitter feedback mechanisms led to this study to determine the relative effects of Thy1 deficiency and dietary iron deficiency on the dopaminergic system in the mouse striatum. The model for this analysis was the Thy1 null mutant mouse in the presence or absence of dietary iron deficiency. The results revealed significant differences in dopaminergic profiles associated with Thy1 and iron deficiency and also a sex effect. For example, both iron deficiency and the absence of Thy1 are associated with increased dopamine in both sexes, but the dopamine transporter is increased in these experimental groups only in female mice. In male mice, the increase in dopamine transporter is found only in the Thy1 null mutants. Increases in vesicular monoamine transporter and phosphorylated tyrosine hydroxlyase are found only in iron-deficient mice. In contrast decreased release of dopamine from synaptosomes is found only in the Thy1 null mutant animals. In general, these results indicate that a loss of Thy1 can influence the dopaminergic profile in the striatum. Furthermore, the results reveal consistent differences in the dopaminergic profile in Thy1 knockout mice compared with iron-deficient mice, indicating that the effects of iron deficiency are not due only to a change in Thy1 expression.

An inhibitor of the epidermal growth factor receptor function does not affect the ability of human papillomavirus 11 to form warts in the xenografted immunodeficient mouse model.

Epidermal growth factor receptor (EGFr) has been shown to be induced and activated in cells infected with HPV, suggesting that it may play a physiological role in viral replication or in the formation or maintenance of warts. To investigate this possibility, human foreskin tissue was infected with HPV11 and transplanted onto the renal capsule and the dermis of immunodeficient mice. The animals were treated orally or topically with the potent EGFr inhibitor CP-545130, with treatment starting either immediately following graft attachment, or following a 70 day period to allow development of warts. The rate of appearance of warts, wart size and number were monitored. In addition, we measured intra-lesional HPV replication levels and examined the morphology of the graft tissues. Analysis of the results showed no significant difference between placebo and compound-treated groups, despite high levels of compound present in the graft tissue. We conclude that EGFr kinase activity is not required for the development and maintenance of HPV-11-induced warts in this model.

Metabolic analysis of mouse brains that have compromised iron storage.

Iron is a critical component of the CNS that must be tightly regulated; too little iron can result in energy insufficiency and too much iron can result in oxidative stress. The intracellular iron storage protein ferritin is central to the regulation of iron. In this study, we determined the neurochemical profile of brains of animals deficient in heavy-chain ferritin (H-ferritin) using high-resolution magic angle spin proton magnetic resonance spectroscopy (HR-MAS (1)H MRS). Spectra of 2 mm-thick coronal tissue punches ( approximately 4 mg) were obtained using a CPMG pulse sequence on Bruker Avance 500 and quantified (nmol/mg tissue) using customized LCModel software (16 metabolites). In H-ferritin deficient mice, we found significant increases in striatal glutamate, hippocampal choline, and N-acetyl-aspartyl-glutamate in both the cortex and the hippocampus (t-test, p < 0.05). Neurochemical profiling with principal component analysis (PCA) revealed increased glutamate in the hippocampus, striatum, and ventral tegmental area (VTA) in H-ferritin deficient animals as compared to wild-type. While lactate was increased in the VTA of deficient animals, it was decreased in the striatum. Also, GABA was increased in both cortical and striatal regions of deficient mice. These changes reveal the importance of proper iron management for maintaining neurochemical balance and provide new evidence for region specific differences in neurochemical profiles as a result of compromised ability of neurons to store iron while overall iron status is normal. Because H-ferritin is predominantly expressed in neurons, the neurochemical profile is suggestive of neuronal iron deficiency and may have relevance to the functional consequences associated with brain iron deficiency.

Biochemical analysis of human milk treated with sodium dodecyl sulfate, an alkyl sulfate microbicide that inactivates human immunodeficiency virus type 1.

Reduction of transmission of human immunodeficiency virus type 1 (HIV-1) through human milk is needed. Alkyl sulfates such as sodium dodecyl sulfate (SDS) are microbicidal against HIV-1 at low concentrations, have little to no toxicity, and are inexpensive. The authors have reported that treatment of HIV-1-infected human milk with < or = 1% (10 mg/mL) SDS for 10 minutes inactivates cell-free and cell-associated virus. The SDS can be removed with a commercially available resin after treatment without recovery of viral infectivity. In this article, the authors report results of selective biochemical analyses (ie, protein, immunoglobulins, lipids, cells, and electrolytes) of human milk subjected to SDS treatment and removal. The SDS treatment or removal had no significant effects on the milk components studied. Therefore, the use of alkyl sulfate microbicides to treat milk from HIV-1-positive women may be a simple, practical, and nutritionally sound way to prevent or reduce transmission of HIV-1 while still feeding with mother's own milk.

Inactivation of HIV-1 in breast milk by treatment with the alkyl sulfate microbicide sodium dodecyl sulfate (SDS).

BACKGROUND: Reducing transmission of HIV-1 through breast milk is needed to help decrease the burden of pediatric HIV/AIDS in society. We have previously reported that alkyl sulfates (i.e., sodium dodecyl sulfate, SDS) are microbicidal against HIV-1 at low concentrations, are biodegradable, have little/no toxicity and are inexpensive. Therefore, they may be used for treatment of HIV-1 infected breast milk. In this report, human milk was artificially infected by adding to it HIV-1 (cell-free or cell-associated) and treated with or=0.1%) was virucidal against cell-free and cell-associated HIV-1 in breast milk. SDS could be substantially removed from breast milk, without recovery of viral infectivity. Viral load in artificially infected milk was reduced to undetectable levels after treatment with 0.1% SDS. SDS was virucidal against HIV-1 in human milk and could be removed from breast milk if necessary. Milk was not infectious after SDS removal. CONCLUSION: The proposed treatment concentrations are within reported safe limits for ingestion of SDS by children of 1 g/kg/day. Therefore, use of alkyl sulfate microbicides, such as SDS, to treat HIV1-infected breast milk may be a novel alternative to help prevent/reduce transmission of HIV-1 through breastfeeding.