Comparability of ELISA and toxin neutralization to measure immunogenicity of Protective Antigen in mice, as part of a potency test for anthrax vaccines.

Complexities of lethal challenge models have prompted the investigation of immunogenicity assays as potency tests of anthrax vaccines. An ELISA and a lethal toxin neutralization assay (TNA) were used to measure antibody response to Protective Antigen (PA) in mice immunized once with either a commercial or a recombinant PA (rPA) vaccine formulated in-house. Even though ELISA and TNA results showed correlation, ELISA results may not be able to accurately predict TNA results in this single immunization model.

Decreased transferrin receptor expression by neuromelanin cells in restless legs syndrome.

BACKGROUND: Restless legs syndrome (RLS) is a sensory-movement disorder affecting 5 to 10% of the population. Its etiology is unknown, but MRI analyses and immunohistochemical studies on autopsy tissue suggest the substantia nigra (SN) of patients with RLS has subnormal amounts of iron. METHODS: Neuromelanin cells from the SN of four RLS and four control brains were isolated by laser capture microdissection, and a profile of iron-management protein expression was obtained by immunoblot analysis. Binding assays for iron regulatory protein activity were performed on cell homogenates. RESULTS: Ferritin, divalent metal transporter 1, ferroportin, and transferrin receptor (TfR) were decreased in RLS neuromelanin cells compared with control. Transferrin was increased in RLS neuromelanin cells. This protein profile in RLS neuromelanin cells is consistent with iron deficiency with the exception that TfR expression was decreased rather than increased. The concentration and activity of the iron regulatory proteins (IRP1 and IRP2) were analyzed to determine whether there was a functional deficit in the post-transcriptional regulatory mechanism for TfR expression. Total IRP activity, IRP1 activity, and IRP1 protein levels were decreased in RLS, but total IRP2 protein levels were not decreased in RLS. CONCLUSION: Restless legs syndrome may result from a defect in iron regulatory protein 1 in neuromelanin cells that promotes destabilization of the transferrin receptor mRNA, leading to cellular iron deficiency.

Neuropathological examination suggests impaired brain iron acquisition in restless legs syndrome.

OBJECTIVE: To assess neuropathology in individuals with restless legs syndrome (RLS). METHODS: A standard neuropathologic evaluation was performed on seven brains from individuals who had been diagnosed with RLS. The substantia nigra was examined in greater detail for iron staining and with immunohistochemistry for tyrosine hydroxylase and proteins involved in iron management. Five age-matched individuals with no neurologic history served as controls. RESULTS: There were no histopathologic abnormalities unique to the RLS brains. Tyrosine hydroxylase staining in the major dopaminergic regions appeared normal in the RLS brains. Iron staining and H-ferritin staining was markedly decreased in the RLS substantia nigra. Although H-ferritin was minimally detected in the RLS brain, L-ferritin staining was strong. However, the cells staining for L-ferritin in RLS brains were morphologically distinct from those in the control brains. Transferrin receptor staining on neuromelanin-containing cells was decreased in the RLS brains compared to normal, whereas transferrin staining in these cells was increased. CONCLUSIONS: RLS may not be rooted in pathologies associated with traditional neurodegenerative processes but may be a functional disorder resulting from impaired iron acquisition by the neuromelanin cells in RLS. The underlying mechanism may be a defect in regulation of the transferrin receptors.

Distribution of divalent metal transporter 1 and metal transport protein 1 in the normal and Belgrade rat.

Iron accumulation in the brain occurs in a number of neurodegenerative diseases. Two new iron transport proteins have been identified that may help elucidate the mechanism of abnormal iron accumulation. The Divalent Metal Transporter 1 (DMT1), is responsible for iron uptake from the gut and transport from endosomes. The Metal Transport Protein 1 (MTP1) promotes iron export. In this study we determined the cellular and regional expression of these two transporters in the brains of normal adult and Belgrade rats. Belgrade rats have a defect in DMT1 that is associated with lower levels of iron in the brain. In the normal rat, DMT1 expression is highest in neurons in the striatum, cerebellum, thalamus, ependymal cells lining the third ventricle, and vascular cells throughout the brain. The staining in the ependymal cells and endothelial cells suggests that DMT1 has an important role in iron transport into the brain. In Belgrade rats, there is generalized decrease in immunodetectable DMT1 compared to normal rats except in the ependymal cells. This decrease in immunoreactivity, however, was absent on immunoblots. The immunoblot analysis indicates that this protein did not upregulate to compensate for the chronic defect in iron transport. MTP1 staining is found in most brain regions. MTP1 expression in the brain is robust in pyramidal neurons of the cerebral cortex but is not detected in the vascular endothelial cells and ependymal cells. MTP1 staining in Belgrade rats was decreased compared to normal, but similar to DMT1 this decrease was not corroborated by immunoblotting. These results indicate that DMT1 and MTP1 are involved in brain iron transport and this involvement is regionally and cellularly specific.

Iron and iron management proteins in neurobiology.

The ability of the brain to store a readily bioavailable source of iron is essential for normal neurologic function because both iron deficiency and iron excess in the brain have serious neurologic consequences. The blood-brain barrier presents unique challenges to timely and adequate delivery of iron to the brain. The regional compartmentalization of neurologic function and a myriad of cell types provide additional challenges. Furthermore, iron-dependent events within the central nervous system (CNS) are age dependent (e.g., myelination) or region specific (e.g., dopamine synthesis). Thus the mechanisms for maintaining the delicate balance of CNS iron concentration must be considered on a region-specific and age-specific basis. Confounding factors that influence brain iron acquisition in addition to age-specific and region-specific requirements are dietary factors and disease. This article raises and addresses the novel concept of regional regulation of brain iron uptake by reviewing the developmental patterns of iron accumulation and expression of proteins responsible for maintaining iron homeostasis in a region-specific and cell-specific manner. Understanding these mechanisms is essential for generating insights into diseases such as Hallervorden-Spatz syndrome, in which excess iron accumulation in the brain plays a significant role in the disease process, and should also unveil windows of opportunity for replenishing the brain in a state of iron deficiency.

Cellular distribution of iron in the brain of the Belgrade rat.

In this study, we investigated the cellular distribution of iron in the brain of Belgrade rats. These rats have a mutation in Divalent Metal Transporter 1, which has been implicated in iron transport from endosomes. The Belgrade rats have iron-positive pyramidal neurons, but these are fewer in number and less intensely stained than in controls. In the white matter, iron is normally present in patches of intensely iron-stained oligodendrocytes and myelin, but there is dramatically less iron staining in the Belgrade rat. Those oligodendrocytes that stained for iron did so strongly and were associated with blood vessels. Astrocytic iron staining was seen in the cerebral cortex for both normal rats and Belgrade rats, but the iron-stained astrocytes were less numerous in the mutants. Iron staining in tanycytes, modified astrocytes coursing from the third ventricle to the hypothalamus, was not affected in the Belgrade rat, but was affected by diet. The results of this study indicate that Divalent Metal Transporter 1 is important to iron transport in the brain. Iron is essential in the brain for basic metabolic processes such as heme formation, neurotransmitter production and ATP synthesis. Excess brain iron is associated with a number of common neurodegenerative diseases. Consequently, elucidating the mechanisms of brain iron delivery is critical for understanding the role of iron in pathological conditions.

Changes in iron histochemistry after hypoxic-ischemic brain injury in the neonatal rat.

Iron can contribute to hypoxic-ischemic brain damage by catalyzing the formation of free radicals. The immature brain has high iron levels and limited antioxidant defenses. The objective of this study was to describe the early alterations in nonheme iron histochemistry following a hypoxic-ischemic (HI) insult to the brain of neonatal rats. We induced a HI insult to the right cerebral hemisphere in groups of 7-day-old rats. Rats were anesthetized, then their brains were perfused and fixed at 0, 1, 4, 8, 24 hr, and 1, 2, and 3 weeks of recovery. Forty-micron-thick frozen sections were stained for iron using the intensified Perls stain. Increased iron staining was first detected within the cytoplasm of cells with pyknotic nuclei at 4 hr of recovery. Staining increased rapidly over the first 24 hr in regions of ischemic injury. By 7 days recovery, reactive glia and cortical blood vessels also stained. Increased staining in gray matter persisted at 3 weeks of recovery, whereas white matter tracts had fewer iron-positive cells compared to normal. The early increase in iron staining could be caused by an accumulation of iron posthypoxicischemic injury or a change in iron from nonstainable heme iron to stainable nonheme iron. Regardless of the source, our results indicate that there is an increase in iron available to promote oxidant stress in the neonatal rat brain following hypoxia-ischemia.

Relationship of iron to oligodendrocytes and myelination.

Oligodendrocytes are the predominant iron-containing cells in the brain. Iron-containing oligodendrocytes are found near neuronal cell bodies, along blood vessels, and are particularly abundant within white matter tracts. Iron-positive cells in white matter are present from birth and eventually reside in defined patches of cells in the adult. These patches of iron-containing cells typically have a blood vessel in their center. Ferritin, the iron storage protein, is also expressed early in development in oligodendrocytes in a regional and cellular pattern similar to that seen for iron. Recently, the functionally distinct subunits of ferritin have been analyzed; only heavy (H)-chain ferritin is found in oligodendrocytes early in development. H-ferritin is associated with high iron utilization and low iron storage. Consistent with the expression of H-ferritin is the expression of transferrin receptors (for iron acquisition) on immature oligodendrocytes. Transferrin protein accumulation and mRNA expression in the brain are both dependent on a viable population of oligodendrocytes and may have an autocrine function to assist oligodendrocytes in iron acquisition. Although apparently the majority of oligodendrocytes in white matter tracts contain ferritin, transferrin, and iron, not all of them do, indicating that there is a subset of oligodendrocytes in white matter tracts. The only known function of oligodendrocytes is myelin production, and both a direct and indirect relationship exists between iron acquisition and myelin production. Iron is directly involved in myelin production as a required co-factor for cholesterol and lipid biosynthesis and indirectly because of its requirement for oxidative metabolism (which occurs in oligodendrocytes at a higher rate than other brain cells). Factors (such as cytokines) and conditions such as iron deficiency may reduce iron acquisition by oligodendrocytes and the susceptibility of oligodendrocytes to oxidative injury may be a result of their iron-rich cytoplasm. Thus, the many known phenomena that decrease oligodendrocyte survival and/or myelin production may mediate their effect through a final common pathway that involves disruptions in iron availability or intracellular management of iron.

Cellular management of iron in the brain.

All organs including the brain contain iron, and the proteins involved in iron uptake (transferrin and transferrin receptor) and intracellular storage (ferritin). However, because the brain resides behind a barrier and has a heterogeneous population of cells, there are aspects of its iron management that are unique. Iron management, the timely delivery of appropriate amounts of iron, is crucial to normal brain development and function. Mismanagement of cellular iron can result not only in decreased metabolic activity but increased vulnerability to oxidative damage. There is regional specificity in cell deposition of iron and the iron regulatory proteins. However, the sequestration of iron in the brain seems primarily the responsibility of oligodendrocytes, as these cells contain most of the stainable iron in the brain. Transferrin, the iron-mobilizing protein, is also found predominantly in these cells. The transferrin receptor is abundantly expressed on blood vessels, large neurons in the cortex, striatum, and hippocampus, and is also present on oligodendrocytes and astrocytes. Ferritin, the intracellular iron storage protein, consists of 2 subunits which are functionally distinct, and we provide evidence in this report that the cellular distribution of the ferritin subunits is also distinct. In addition, changes in the cellular distribution of iron and its associated regulatory proteins occur in Alzheimer's disease. Neuritic plaques contain relatively large amounts of stainable iron, and the surrounding cells robustly immunostain for ferritin and the transferrin receptor. Analysis of the cellular distribution of iron indicates the different levels of requirement of iron in the brain by different cell types and should ultimately elucidate how cells acquire and maintain this essential component of oxidative metabolism. In addition, changes in the ability of cells to deliver and manage iron may provide insight into altered metabolic activity with age and disease as well as identify cell populations at risk for iron-induced oxidative stress.

A histochemical study of iron-positive cells in the developing rat brain.

The establishment of normal iron levels in the neonatal brain is critical for normal neurological development. Studies have shown that both iron uptake and iron concentration in the brain are relatively high during neonatal development. This histochemical study was undertaken to determine the pattern of iron development at the cellular level in the rat forebrain. Iron-stained cells were observed as early as postnatal day (PND) 3, which was the earliest time point examined. At PND 3, there were four major foci of iron-containing cells: the subventricular zone and three areas within the subcortical white matter. These latter foci are associated with myelinogenic regions. The blood vessels were prominently stained for iron throughout the brain. At PND 7, as in PND 3, the majority of the iron-containing cells were in white matter. However, there were also patches of iron staining located specifically in the layer IV of the somatosensory cortex. These cortical patches were no longer visible by PND 14. At PND 14, numerous iron-stained cells were dispersed throughout white matter regions and the tanycytes aligning the third ventricle were prominently stained. The blood vessel staining was less prominent than at earlier time periods. By PND 28, the adult pattern of iron staining was emerging. Iron-stained cells were aligned in rows in white matter and had an apparent preference for a location near blood vessels. This clustering of iron-positive cells around blood vessels gave the white matter a "patchy" appearance. The pattern of development, cell distribution, and morphological appearance of the iron-stained cells are consistent with that reported for oligodendrocytes. That iron-positive cells in the neonate may be oligodendrocytes is consistent with the reports for iron staining in adult brains. The recent reports that oligodendrocytes are highly susceptible to oxidative damage would be consistent with the high iron levels found in these cells. These results indicate that oligodendrocytes play a major role in the development of iron homeostasis in the brain. The role of iron in oligodendrocytes may be associated with metabolic demands of myelinogenesis, including cholesterol and fatty acid synthesis. However, these cells may be a morphologically similar but functionally distinct subset of oligodendrocytes whose function is to regulate the availability of iron in the brain.

Isoforms of ferritin have a specific cellular distribution in the brain.

Ferritin is the major iron storage protein and accounts for the majority of the iron in the brain. Thus, ferritin is a key component in protecting the brain from iron induced oxidative damage. The high lipid content, high rate of oxidative metabolism, and high iron content combine to make the brain the organ most susceptible to oxidative stress. The role of oxidative damage and disruption of brain iron homeostasis is considered clinically important to normal aging and a potential pathogenic component of a number of neurologic disorders including Alzheimer's disease and Parkinson's disease. Little is known, however, of the mechanism by which the brain maintains iron homeostasis at either the whole organ or cellular level. In this study we report the cellular distribution of the two isoforms of ferritin in the brain of adult subhuman primates. A subset of neurons immunolabel specifically for the H-chain ferritin protein, whereas cells resembling microglia are immunolabeled only after exposure to the L-chain ferritin antibody. Only one cell type immunostains for both H- and L-chain ferritin; these cells are morphologically similar and have the same distribution pattern as oligodendrocytes. Neither ferritin isoform is usually detected in astrocytes. These data indicate considerable differences in iron sequestration and use between neurons and glia and among neuronal and glial subtypes. This information will be essential in determining the role of each of these cells in maintaining general brain iron homeostasis and the relative abilities of these cells to withstand oxidative stress.

Transferrin in the central nervous system of the shiverer mouse myelin mutant.

Transferrin, the iron mobilization protein, and its mRNA are normally present in oligodendrocytes. Previous reports using myelin mutants have shown both a decrease in transferrin protein and mRNA when the oligodendrocyte population is compromised. In this study the shiverer mouse mutant in which the oligodendrocyte population is numerically normal, but has both quantitatively diminished and qualitatively abnormal myelin was used. This animal model was chosen to address the question whether expression of the transferrin message and/or protein correlated more closely to the number of oligodendrocytes (normal) or the amount of myelin (abnormally low). A 1/2 to 2/3 decrease in transferrin protein occurred in all brain regions examined except for the spinal cord in the shiverer group compared to both heterozygous littermates and wild type controls. Levels of transferrin transcripts in the brain are not affected by the shiverer mutation. These results taken with previous reports from this laboratory indicate that the presence of oligodendrocytes is a requirement for normal expression of transferrin mRNA in brain but is not sufficient for normal values of the protein. The level of Tf protein correlates more closely with the amount of myelin present than it does with the numbers of oligodendrocytes present. These data are consistent with previous reports from our laboratory that transferrin accumulation by oligodendrocytes is associated with myelin production by these cells. These data further suggest transferrin mRNA may be constitutively expressed by oligodendrocytes and that the protein expression is regulated at the level of translation.

A histochemical study of iron, transferrin, and ferritin in Alzheimer's diseased brains.

Immunohistochemical and histochemical staining were performed on Alzheimer's diseased brain tissue obtained at autopsy. The iron-regulatory proteins transferrin and ferritin as well as iron are, in general, found predominantly in oligodendrocytes similar to that previously reported for normal brain tissue. However, in the vicinity of senile plaques, the staining pattern is altered for both proteins and iron. Transferrin is homogenously distributed around the senile plaques and is apparently extracellular. In addition, transferrin is found in astrocytes in the cerebral cortical white matter of the Alzheimer's tissue rather than its normal distribution in oligodendrocytes. A robust ferritin immunoreaction accompanies senile plaques and many blood vessels in the Alzheimer's brain tissue. Although many ferritin-positive oligodendrocytes are present in the Alzheimer's tissue, most of the ferritin-containing cells associated with senile plaques and blood vessels are microglia. Iron can also be demonstrated in the senile plaques. The iron reaction product is observed both diffusely in proximity of the plaques and in cells associated with the plaques. These data strongly suggest a disruption in brain iron homeostasis in Alzheimer's disease as demonstrated by alterations in the normal cellular distribution of iron and the proteins responsible for iron regulation. These data will contribute to understanding both the potential for oxidative damage and the potential for metal neurotoxicity in Alzheimer's disease.

Cellular distribution of transferrin, ferritin, and iron in normal and aged human brains.

The iron transport protein, transferrin, and the iron storage protein ferritin were examined immunohistochemically along with iron in a number of brain regions from normal and aged humans. Two age groups were examined: a middle-aged group (28-49 years), and an older group (60-90 years). Transferrin, ferritin, and iron are found throughout all brain regions examined, predominantly in the perikaryal cytoplasm of cells that are small and round, fitting the description of oligodendrocytes. These cells are present in the optic nerve and in both the gray and white matter of the cerebral cortex, cerebellum, and olfactory bulb in both age groups. Ferritin is also found in microglial cells in the gray matter of most of these brain regions. In the subcortical regions examined (corpus striatum, hippocampus, amygdala), in addition to oligodendrocytes, astrocytes can frequently be observed that contain transferrin, ferritin, and iron. There is an age-related alteration in cell labeling: astrocytes in both gray and white matter contained transferrin in the oldest age group, whereas in the younger group the subcortical transferrin immunoreactivity was confined mostly to oligodendrocytes. Ferritin in the subcortical brain regions is also present in astrocytes but is primarily confined to those in the gray matter, even in the oldest age group. Iron is found predominantly in oligodendrocytes, although a few iron-positive astrocytes and microglia can be identified. These results indicate that (1) normally oligodendrocytes contain much of the iron and iron-binding proteins found in the brain; and (2) an increase in age is associated with altered cellular distribution of iron-binding proteins, but the altered distribution is specific to glial cells. These results suggest glial cells may have previously undescribed functions related to metal regulation and sequestration.

Altered cellular distribution of iron in the central nervous system of myelin deficient rats.

Under normal conditions, iron is found predominantly in oligodendrocytes, the myelin producing cell, in the rat brain. A genetic mutant strain of rats known as myelin deficient rats is examined in the present study because their number of oligodendrocytes is decreased and those oligodendrocytes present are structurally abnormal. The levels of iron in the liver (major site of iron storage) and in the pons-cerebellum did not differ statistically between the myelin deficient rats and the littermate control rats, whereas only half of the iron normally found in the cerebrum-midbrain was present in the myelin deficient rat. Histologically, iron was found predominantly in oligodendrocytes in the littermate control rats, as expected. In the myelin deficient rat, iron staining was confirmed to astrocytes and microglia. The results of this study strongly suggest that iron uptake into the brain continues in the absence of normal oligodendrocytes and myelin. Furthermore, these data suggest that iron metabolism can be substantially altered, as indicated by the accumulation of iron in astrocytes and microglia, when normal or near normal levels of iron are quantitatively demonstrated. The response of astrocytes and microglia to sequester the iron (presumably through phagocytosis) in the absence of invasive damage represents, to our knowledge, a new functional observation for these cells. Based on these observations it is clear that iron histochemistry in combination with quantitative analysis is necessary to interpret data regarding iron physiology, at least in neurobiology, and iron accumulation by astrocytes and microglia may provide clues of altered iron metabolism despite normal iron levels.