The MHC class I-like IgG receptor controls perinatal IgG transport,IgG homeostasis,and fate of IgG-Fc-coupled drugs

Abs of the IgG isotype are efficiently transported from mother to neonate and have an extended serum t(1/2) compared with Abs of other isotypes. Circumstantial evidence suggests that the MHC class I-related protein, the neonatal FcR (FcRn), is the FcR responsible for both in vivo functions. To understand the phenotypes imposed by FcRn, we produced and analyzed mice with a defective FcRn gene. The results provide direct evidence that perinatal IgG transport and protection of IgG from catabolism are mediated by FcRn, and that the latter function is key to IgG homeostasis, essential for generating a potent IgG response to foreign Ags, and the basis of enhanced efficacy of Fc-IgG-based therapeutics. FcRn is therefore a promising therapeutic target for enhancing protective humoral immunity, treating autoimmune disease, and improving drug efficacy.     

IgG transcytosis and recycling by FcRn expressed in MDCK cells reveals ligand-induced redistribution

The neonatal Fc receptor (FcRn) transports IgG across epithelial cells and recycles serum IgG. FcRn binds IgG at the acidic pH of endosomes and releases IgG at the basic pH of blood. We expressed rat FcRn in polarized MDCK cells and demonstrated that it functions in transcytosis and recycling of IgG. In the absence of IgG, FcRn is distributed predominantly apically, but redistributes to basolateral locations upon IgG addition, indicating that ligand binding induces a signal that stimulates transcytosis. FcRn transcytoses IgG more efficiently in the apical to basolateral than the reverse direction when IgG is internalized by receptor-mediated endocytosis at acidic pH or by fluid phase endocytosis at basic pH. The PI 3-kinase inhibitor wortmannin disrupts basolateral recycling and transcytosis in both directions, but only minimally reduces apical recycling. Confocal imaging and quantitative IgG transport studies demonstrate that apically-internalized IgG recycles to the apical surface mainly from wortmannin-insensitive apical early endosomes, whereas FcRn-IgG complexes that transcytose to the basolateral surface pass through downstream Rab11-positive apical recycling endosomes and transferrin-positive common endosomal compartments.     

Nonvectorial surface transport, endocytosis via a Di-leucine-based motif, and bidirectional transcytosis of chimera encoding the cytosolic tail of rat FcRn expressed in Madin-Darby canine kidney cells

Transfer of passive immunity from the mother to the fetus or newborn involves the transport of IgG across several epithelia. Depending on the species, IgG is transported prenatally across the placenta and yolk sac or is absorbed from colostrum and milk by the small intestine of the suckling newborn. In both cases apical to basolateral transepithelial transport of IgG is thought to be mediated by FcRn, an IgG Fc receptor with homology to major histocompatibility class I antigens. Here, we analyzed the intracellular routing of chimera encoding the rat FcRn tail fused to the ecto- and transmembrane domain of the macrophage FcgammaRIIb. Newly synthesized chimera were delivered in a nonvectorial manner to the apical and basolateral cell surface, from where the chimera were able to internalize and transcytose. Apical to basolateral and basolateral to apical transcytosis were differently regulated. This intracellular routing of the chimera is similar to that of the native FcRn, indicating that the cytosolic tail of the receptor is necessary and sufficient to endow an unrelated FcR with the intracellular transport behavior of FcRn. Furthermore, the di-leucine motif in the cytosolic domain of FcRn was required for rapid and efficient endocytosis but not for basolateral sorting of the chimera.     

Immunoglobulin G transport increases in an in vitro blood–brain barrier model with amyloid-β and with neuroinflammatory cytokines

Immunotherapies are a promising strategy for the treatment of neurological diseases such as Alzheimer's disease (AD), however, transport of antibodies to the brain is severely restricted by the blood–brain barrier (BBB). Furthermore, molecular transport at the BBB is altered in disease, which may affect the mechanism and quantity of therapeutic antibody transport. To better understand the transport of immunotherapies at the BBB in disease, an in vitro BBB model derived from human induced pluripotent stem cells (iPSCs) was used to investigate the endocytic uptake route of immunoglobulin G (IgG). In this model, uptake of fluorescently labeled IgGs is a saturable process. Inhibition of clathrin-mediated endocytosis, caveolar endocytosis, and macropinocytosis demonstrated that macropinocytosis is a major transport route for IgGs at the BBB. IgG uptake and transport were increased after the addition of stimuli to mimic AD (Aβ_1–40 and Aβ_1–42) and neuroinflammation (tumor necrosis factor-α and interleukin-6). Lastly, caveolar endocytosis increased in the AD model, which may be responsible for the increase in IgG uptake in disease. This study presents an iPSC-derived BBB model that responds to disease stimuli with physiologically relevant changes to molecular transport and can be used to understand fundamental questions about transport mechanisms of immunotherapies in health and neurodegenerative disease.     

Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences

Many biological functions, including control of the homeostasis and maternofetal transfer of serum gamma-globulins, are mediated by the MHC class I-related neonatal FcR (FcRn). A correlation exists in mice between the binding affinity of IgG1/Fc fragments to FcRn at pH 6.0 and their serum t(1/2). To expand this observation, phage display of mutagenized Fc fragments derived from a human IgG1 was used to increase their affinity to both murine and human FcRn. Ten variants were identified that have a higher affinity toward murine and human FcRn at pH 6.0, with DeltaDeltaG (DeltaG(wild type) - DeltaG(mutant)) from 1.0 to 2.0 kcal/mol and from 0.6 to 2.4 kcal/mol, respectively. Those variants exhibit a parallel increase in binding at pH 7.4 to murine, but not human, FcRn. Although not degraded in blood in vitro, accumulated in tissues, nor excreted in urine, their serum concentration in mice is decreased. We propose that higher affinity to FcRn at pH 7.4 adversely affects release into the serum and offsets the benefit of the enhanced binding at pH 6.0.     

Electron tomography of late stages of FcRn-mediated antibody transcytosis in neonatal rat small intestine

The neonatal Fc receptor (FcRn) transports maternal immunoglobulin (IgG) across epithelia to confer passive immunity to mammalian young. In newborn rodents, FcRn transcytoses IgG from ingested milk across the intestinal epithelium for release into the bloodstream. We used electron tomography to examine FcRn transport of Nanogold-labeled Fc (Au-Fc) in neonatal rat jejunum, focusing on later aspects of transport by chasing Au-Fc before fixation. We observed pools of Au-Fc in dilated regions of the lateral intercellular space (LIS), likely representing exit sites where Au-Fc accumulates en route to the blood. Before weaning, the jejunum functions primarily in IgG transport and exhibits unusual properties: clathrin-rich regions near/at the basolateral LIS and multivesicular bodies (MVBs) expressing early endosomal markers. To address whether these features are related to IgG transport, we examined LIS and endocytic/transcytotic structures from neonatal and weaned animals. Weaned samples showed less LIS-associated clathrin. MVBs labeled with late endosomal/lysosomal markers were smaller than their neonatal counterparts but contained 10 times more internal compartments. These results are consistent with hypotheses that clathrin-rich basolateral regions in neonatal jejunum are involved in IgG exocytosis and that MVBs function in IgG transport while FcRn is expressed but switch to degradative functions after weaning, when the jejunum does not express FcRn or transport IgG.     

Neonatal FcR expression in bone marrow-derived cells functions to protect serum IgG from catabolism

The neonatal FcR (FcRn) is a receptor that protects IgG from catabolism and is important in maintaining high serum Ab levels. A major site of expression of FcRn is vascular endothelial cells where FcRn functions to extend the serum persistence of IgG by recycling internalized IgG back to the surface. Because FcRn is expressed in other tissues, it is unclear whether endothelial cells are the only site of IgG protection. In this study, we used FcRn-deficient mice and specific antiserum to determine the tissue distribution of FcRn in the adult mouse. In addition to its expression in the vascular endothelium of several organs, we found FcRn to be highly expressed in bone marrow-derived cells and professional APCs in different tissues. Experiments using bone marrow chimeras showed that FcRn expression in these cells acted to significantly extend the half-life of serum IgG indicating that in addition to the vascular endothelium, bone marrow-derived phagocytic cells are a major site of IgG homeostasis.     

聚乳酸纳米粒穿透血脑屏障的分析电镜研究

观察以聚乳酸 (D ,L-polylacticacid,PLA)为材料制备、经吐温-80(T-80)表面改性的纳米粒对血脑屏障的穿透效果并探讨其机制 ,分别将FITC-Dextran、叶绿素铜作为PLA纳米粒的示踪标记 ,应用荧光显微镜、透射电镜及分析电镜观察经静脉注射入小鼠体内的PLA纳米粒在脑组织中的分布、穿透血脑屏障的特性。荧光显微镜观察到小鼠脑组织中散在及沿毛细血管壁分布的荧光颗粒 ,透射电镜可观察到小鼠脑毛细血管内皮细胞及周围脑组织中圆形或类圆形的外源性纳米粒 ;进一步采用分析电镜对颗粒处组织进行能谱分析证实其为叶绿素铜标记的PLA纳米粒。证实了T-80修饰的PLA纳米粒具有穿透血脑屏障的特性 ,机制可能是毛细血管内皮细胞的胞吞转运作用 ,同时 ,为研究纳米粒在组织内的定位提供了新的标记方法.     

Neutral amino acid transport at the human blood-brain barrier

Transport regulates nutrient availability in the brain, and many pathways of brain amino acid metabolism are influenced by precursor supply. Therefore, amino acid transport through the blood-brain barrier (BBB) plays an important rate-affecting role in brain metabolism. Information on the Km of BBB amino acid transport provides the quantitative basis for understanding the physiological importance of BBB transport competition effects. For example, the uniquely low Km values of BBB amino acid transport as compared to other organs in the rat provides the basis for the selective vulnerability of the rat brain to changes in amino acid supply caused by nutritional factors. The development of amino acid imbalances in the human brain in parallel with amino acid imbalances in blood is likely to occur if the Km of BBB neutral amino acid transport in humans is low, e.g., 25-100 microM, as is the case for the rat. A new model system of the human BBB, the isolated human brain capillary, has been developed. Recent studies with this system indicate that the Km of phenylalanine transport into human brain microvessels is approximately the same as that found during in vivo studies with laboratory rats. These results support the emerging hypothesis that the human brain, like the rat brain, is subject to acute regulation by dietary-related amino acid imbalances, and that the major site of this regulation is the amino acid transport system at the BBB.     

Neonatal FcR overexpression boosts humoral immune response in transgenic mice

The neonatal FcR (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes active part in phagocytosis, and delivers Ag for presentation. We have previously shown that overexpression of FcRn in transgenic (Tg) mice extends the half-life of mouse IgG by reducing its clearance. In this paper, we demonstrate that immunization of these mice with OVA and trinitrophenyl-conjugated human IgG results in a 3- to 10-fold increase of Ag-specific IgM and IgG in serum. The IgM increase was unexpected because FcRn does not bind IgM. Our results showed that the affinity of the Ag-specific IgG was at least as good in Tg mice as in the wild-type (wt) controls, implying appropriate affinity maturation in both groups. Influenza vaccination produced a 2-fold increase in the amount of virus-specific Ab in Tg animals, which proved twice as efficient in a hemagglutination inhibition assay as was the case in wt controls. After immunization, Tg mice displayed significantly larger spleens containing a higher number of Ag-specific B cells and plasma cells, as well as many more granulocytes and dendritic cells, analyzed by ELISPOT and flow cytometric studies. The neutrophils from these Tg mice expressed the Tg FcRn and phagocytosed IgG immune complexes more efficiently than did those from wt mice. These results show that FcRn overexpression not only extends the IgG half-life but also enhances the expansion of Ag-specific B cells and plasma cells. Although both effects increase the level of Ag-specific IgG, the increase in immune response and IgG production seems to be more prominent compared with the reduced IgG clearance.     

Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2

We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood-brain barrier (BBB) in a series of functional in vivo and in vitro studies. The apparent BBB efflux rate constant of [(3)H]DHEAS evaluated by the brain efflux index method was 2.68 x 10(-2) min(-1). DHEAS efflux transport was a saturable process with a Michaelis constant (K:(m)) of 32.6 microM: Significant amounts of [(3)H]DHEAS were determined in the jugular venous plasma by HPLC, providing direct evidence that most of the DHEAS is transported in intact form from brain to the circulating blood across the BBB. This efflux transport of [(3)H]DHEAS was significantly inhibited by common rat organic anion-transporting polypeptide (oatp) substrates such as taurocholate, cholate, sulfobromophthalein, and estrone-3-sulfate. Moreover, the apparent efflux clearance of [(3)H]DHEAS across the BBB (118 microl/min-g of brain) was 10.4-fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 microl/min-g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4), [(3)H]DHEAS uptake by TM-BBB4 cells exhibited a concentration dependence with a K:(m) of 34.4 microM: and was significantly inhibited by the oatp2-specific substrate digoxin. Conversely, [(3)H]digoxin uptake by TM-BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [(3)H]DHEAS at 30 min was significantly increased under ATP-depleted conditions, suggesting that an energy-dependent efflux process may also be involved in TM-BBB4. RT-PCR and sequence analysis suggest that an oatp2 is expressed in TM-BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT-PCR suggest that there is oatp2-mediated DHEAS transport at the BBB.     

A novel Fc gamma R-defined, IgG-containing organelle in placental endothelium

Placental transfer of IgG from maternal circulation to that of the fetus is crucial for fetal and newborn immunity. This process requires that IgG broach two cellular layers of the placenta. IgG transport across the first layer, the syncytiotrophoblast, is almost certainly mediated by the MHC-related FcR for IgG, FcRn. The second layer, the villus endothelium, was until recently thought to allow IgG movement nonspecifically by constitutive transcytosis in caveolae. However, we recently showed that villus endothelium expressed a separate FcR for IgG, the inhibitory motif-bearing Fc gammaRIIb2 seen most notably on macrophages and as a minor fraction of the Fc gammaRIIb expressed on B cells. Now, by quantitative microscopy, we find Fc gammaRIIb2 to be expressed abundantly in an unidentifiable and likely novel organelle of the villus endothelium, unassociated with caveolae. About half of these Fc gammaRIIb2 organelles contain IgG; the remainder lack IgG. The majority fraction (approximately 80%) of IgG-containing organelles is associated with Fc gammaRIIb. No IgG-containing organelles are associated with caveolin. These findings are compatible with Fc gammaRIIb-mediated transfer of IgG across the villus endothelium, independent of caveolae.     

The recycling and transcytotic pathways for IgG transport by FcRn are distinct and display an inherent polarity

The Fc receptor FcRn traffics immunoglobulin G (IgG) in both directions across polarized epithelial cells that line mucosal surfaces, contributing to host defense. We show that FcRn traffics IgG from either apical or basolateral membranes into the recycling endosome (RE), after which the actin motor myosin Vb and the GTPase Rab25 regulate a sorting step that specifies transcytosis without affecting recycling. Another regulatory component of the RE, Rab11a, is dispensable for transcytosis, but regulates recycling to the basolateral membrane only. None of these proteins affect FcRn trafficking away from lysosomes. Thus, FcRn transcytotic and recycling sorting steps are distinct. These results are consistent with a single structurally and functionally heterogeneous RE compartment that traffics FcRn to both cell surfaces while discriminating between recycling and transcytosis pathways polarized in their direction of transport.     

Re-engineering biopharmaceuticals for delivery to brain with molecular Trojan horses

Biopharmaceuticals, including recombinant proteins, monoclonal antibody therapeutics, and antisense or RNA interference drugs, cannot be developed as drugs for the brain, because these large molecules do not cross the blood-brain barrier (BBB). Biopharmaceuticals must be re-engineered to cross the BBB, and this is possible with genetically engineered molecular Trojan horses. A molecular Trojan horse is an endogenous peptide, or peptidomimetic monoclonal antibody (mAb), which enters brain from blood via receptor-mediated transport on endogenous BBB transporters. Recombinant neurotrophins, single chain Fv antibodies, or therapeutic enzymes may be re-engineered as IgG fusion proteins. The engineering of IgG-avidin fusion proteins enables the BBB delivery of biotinylated drugs. The IgG fusion proteins are new chemical entities that are dual or triple function molecules that bind multiple receptors. The fusion proteins are able both to enter the brain, by binding an endogenous BBB receptor, and to induce the desired pharmacologic effect in brain, by binding target receptors in the brain behind the BBB. The development of molecular Trojan horses for BBB drug delivery allows the re-engineering of biopharmaceuticals that, owing to the BBB problem, could not otherwise be developed as new drugs for the human brain.     

Chimeric peptides as a vehicle for peptide pharmaceutical delivery through the blood-brain barrier

A new strategy for peptide delivery through the brain capillary wall, i.e., the blood-brain barrier (BBB), is the synthesis of chimeric peptides which are formed by the covalent coupling of a non-transportable peptide (e.g., beta-endorphin) to a transportable peptide that undergoes receptor- or absorptive-mediated transcytosis at the BBB. beta-endorphin was covalently coupled via disulfide linkage to cationized albumin (pI greater than or equal to 9) which, owing to it's highly basic charge, undergoes rapid absorptive-mediated transport into brain from blood. The [3H]labeled beta-endorphin-cationized albumin chimera was rapidly taken up by isolated brain capillaries in vitro and by rat brain in vivo; conversely, the BBB uptake of native [3H]beta-endorphin was negligible. The synthesis of chimeric peptides is a new strategy for solving the problem of peptide delivery through the BBB.     

Transport of nutrients and hormones through the blood-brain barrier

The transport of circulating nutrients (glucose, amino acids, ketone bodies, choline, and purines) through the brain endothelial wall, i.e., the blood-brain barrier (BBB), is an important regulatory step in several substrate-limited pathways of brain metabolism. The in vivo kinetics of nutrient transport has been well characterized in the rat, and the kinetic constants of saturable (Km, Vmax) and nonsaturable (KD) transport through the BBB are now known for more than 30 circulating nutrients. The kinetic constants can be used to gain insight into the important rate-limiting role played by BBB nutrient transport in the regulation of brain metabolism and function. Unlike most nutrients, steroid and thyroid hormones circulate tightly bound to plasma proteins. However, owing to favorable kinetic relationships among brain capillary transit times and rates of hormone dissociation from plasma proteins and hormone diffusion through the brain endothelia, the BBB is able to strip hormones off circulating plasma proteins. With regard to peptide hormone, no specific BBB transport systems for peptides have been identified thus far. However, peptides are able to rapidly distribute into brain interstitial space at the circumventricular organs. In addition, specific receptors for insulin are located on the BBB. The presence of BBB peptide receptors provides a mechanism by which circulating peptides may rapidly influence brain function without the peptide crossing the BBB.     

MHC class I-related neonatal Fc receptor for IgG is functionally expressed in monocytes, intestinal macrophages, and dendritic cells

The neonatal Fc receptor (FcRn) for IgG, an MHC class I-related molecule, functions to transport IgG across polarized epithelial cells and protect IgG from degradation. However, little is known about whether FcRn is functionally expressed in immune cells. We show here that FcRn mRNA was identifiable in human monocytes, macrophages, and dendritic cells. FcRn heavy chain was detectable as a 45-kDa protein in monocytic U937 and THP-1 cells and in purified human intestinal macrophages, peripheral blood monocytes, and dendritic cells by Western blot analysis. FcRn colocalized in vivo with macrosialin (CD68) and Ncl-Macro, two macrophage markers, in the lamina propria of human small intestine. The heavy chain of FcRn was associated with the beta(2)-microglobulin (beta(2)m) light chain in U937 and THP-1 cells. FcRn bound human IgG at pH 6.0, but not at pH 7.5. This binding could be inhibited by human IgG Fc, but not Fab. FcRn could be detected on the cell surface of activated, but not resting, THP-1 cells. Furthermore, FcRn was uniformly present intracellularly in all blood monocytes and intestinal macrophages. FcRn was detectable on the cell surface of a significant fraction of monocytes at lower levels and on a small subset of tissue macrophages that expressed high levels of FcRn on the cell surface. These data show that FcRn is functionally expressed and its cellular distribution is regulated in monocytes, macrophages, and dendritic cells, suggesting that it may confer novel IgG binding functions upon these cell types relative to typical Fc gamma Rs: Fc gamma RI, Fc gamma RII, and Fc gamma RIII.     

Amelioration of experimental autoimmune myasthenia gravis in rats by neonatal FcR blockade

The neonatal FcR (FcRn) plays a critical role in IgG homeostasis by protecting it from a lysosomal degradation pathway. It has been shown that IgG has an abnormally short half-life in FcRn-deficient mice and that FcRn blockade significantly increases the catabolism of serum IgG in mice. Therefore, reduction of serum IgG half-life may have therapeutic benefits in Ab-mediated autoimmune diseases. We have studied the therapeutic effects of an anti-rat FcRn mAb, 1G3, in two rat models of myasthenia gravis, a prototypical Ab-mediated autoimmune disease. Passive experimental autoimmune myasthenia gravis was induced by administration of an anti-acetylcholine receptor (AChR) mAb, and it was shown that treatment with 1G3 resulted in dose-dependent amelioration of the disease symptoms. In addition, the concentration of pathogenic Ab in the serum was reduced significantly. The effect of 1G3 was also studied in an active model of experimental autoimmune myasthenia gravis in which rats were immunized with AChR. Treatment with 1G3 significantly reduced the severity of the disease symptoms as well as the levels of total IgG and anti-AChR IgG relative to untreated animals. These data suggest that FcRn blockade may be an effective way to treat Ab-mediated autoimmune diseases.     

Blood-brain barrier transport of a novel micro 1-specific opioid peptide,H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA)

The purpose of this study was to clarify the mechanism of the blood-brain barrier (BBB) transport of H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA), which is a novel dermorphin analog with high affinity for the micro 1-opioid receptor. The in vivo BBB permeation influx rate of [125I]TAPA after an i.v. bolus injection (7.3 pmol/g body weight) into mice was estimated to be 0.265 +/- 0.025 microL/(min.g of brain). The influx rate of [125I]TAPA was reduced 70% by the coadministration of unlabeled TAPA (33 nmol/g of brain), suggesting the existence of a specific transport system for TAPA at the BBB. In order to elucidate the BBB transport mechanism of TAPA, a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4) was used as an in vitro model of the BBB. The acid-resistant binding of [125I]TAPA, which represents the internalization of the peptide into cells, was temperature- and concentration-dependent with a half-saturation constant of 10.0 +/- 1.7 microm. The acid-resistant binding of TAPA was significantly inhibited by 2,4-dinitrophenol, dansylcadaverine (an endocytosis inhibitor) and poly-l-lysine and protamine (polycations). These results suggest that TAPA is transported through the BBB by adsorptive-mediated endocytosis, which is triggered by binding of the peptide to negatively charged sites on the surface of brain capillary endothelial cells. Blood-brain barrier transport via adsorptive-mediated endocytosis plays a key role in the expression of the potent opioid activity of TAPA in the CNS.     

Capillary Depletion Method for Quantification of Blood–Brain Barrier Transport of Circulating Peptides and Plasma Proteins

Recent studies indicate that circulating peptides or plasma proteins, such as insulin or transferrin, or modified proteins, such as cationized albumin, undergo receptor-mediated or absorptive-mediated transport through the brain capillary wall, i.e., the blood-brain barrier (BBB). Although morphologic studies such as autoradiography or immunoperoxidase labeling can demonstrate transport of blood-borne protein into brain, there is a need for a rapid, sensitive, and quantifiable physiology-based technique for comparing the relative rates of transport of several different blood-borne peptides or proteins into brain. Therefore, the present investigations describe a carotid arterial infusion technique coupled with a capillary depletion method for quantifying transport of blood-borne cationized albumin, cationized IgG, and acetylated low-density lipoprotein (LDL). Because differentiation of true transcytosis into the postcapillary compartment of brain parenchyma from binding and/or endocytosis to the brain microvasculature is important, the present studies use a dextran density centrifugation step to deplete brain homogenate of the vasculature. In addition, 3H-labeled native albumin is used as a vascular space marker to account for release of capillary contents into the postcapillary supernatant following homogenization of brain. This study demonstrates rapid transport of cationized IgG or cationized albumin into brain, as these compounds achieve a volume of distribution of 20-30 microliters/g within 10 min of arterial perfusion. Conversely, acetylated LDL, although rapidly bound by cerebral microvasculature, is shown not to undergo transport into the post-capillary compartment of brain parenchyma. These studies provide the basis for a sensitive, quantifiable technique for studying transport of radiolabeled blood-borne peptides and proteins across the BBB of anesthetized animals.