Transport of human immunodeficiency virus type 1 pseudoviruses across the blood-brain barrier: role of envelope proteins and adsorptive endocytosis

Blood-borne human immunodeficiency virus type 1 (HIV-1) crosses the blood-brain barrier (BBB) to induce brain dysfunction. How HIV-1 crosses the BBB is unclear. Most work has focused on the ability of infected immune cells to cross the BBB, with less attention devoted to the study of free virus. Since the HIV-1 coat glycoprotein gp120 can cross the BBB, we postulated that gp120 might be key in determining whether free virus can cross the BBB. We used radioactive virions which do (Env+) or do not (Env-) bear the envelope proteins to characterize the ability of HIV-1 to be taken up by the murine BBB. In vivo and in vitro studies showed that the envelope proteins are key to the uptake of free virus and that uptake was enhanced by wheat germ agglutinin, strongly suggesting that the envelope proteins induce viral adsorptive endocytosis and transcytosis in brain endothelia. Capillary depletion showed that Env+ virus completely crossed the vascular BBB to enter the parenchyma of the brain. Virus also entered the cerebrospinal fluid, suggesting passage across the choroid plexus as well. About 0.22% of the intravenously injected dose was taken up per g of brain. In vitro studies showed that postinternalization membrane cohesion (membrane binding not reversed with acid wash or cell lysis) was a regulated event. Intact virus was recovered from the brain endothelial cytosol and was effluxed from the endothelial cells. These results show that free HIV-1 can cross the BBB by an event related to adsorptive endocytosis and mediated by the envelope proteins.     

Human immunodeficiency virus type 1 infection increases the in vivo capacity of peripheral monocytes to cross the blood-brain barrier into the brain and the in vivo sensitivity of the blood-brain barrier to disruption by lipopolysaccharide

Human immunodeficiency virus type 1 (HIV-1), introduced into the brain by HIV-1-infected monocytes which migrate across the blood-brain barrier (BBB), infects resident macrophages and microglia and initiates a process that causes HIV-1-associated neurocognitive disorders. The mechanism by which HIV-1 infection circumvents the BBB-restricted passage of systemic leukocytes into the brain and disrupts the integrity of the BBB is not known. Circulating lipopolysaccharide (LPS), which can compromise the integrity of the BBB, is significantly increased in HIV-1-infected individuals. We hypothesized that HIV-1 infection increases monocyte capacity to migrate across the BBB, which is further facilitated by a compromise of BBB integrity mediated by the increased systemic LPS levels present in HIV-1-infected individuals. To investigate this possibility, we examined the in vivo BBB migration of monocytes derived from our novel mouse model, JR-CSF/EYFP mice, which are transgenic for both a long terminal repeat-regulated full-length infectious HIV-1 provirus and ROSA-26-regulated enhanced yellow fluorescent protein. We demonstrated that JR-CSF/EYFP mouse monocytes displayed an increased capacity to enter the brain by crossing either an intact BBB or a BBB whose integrity was partially compromised by systemic LPS. We also demonstrated that the JR-CSF mouse BBB was more susceptible to disruption by systemic LPS than the control wild-type mouse BBB. These results demonstrated that HIV-1 infection increased the ability of monocytes to enter the brain and increased the sensitivity of the BBB to disruption by systemic LPS, which is elevated in HIV-1-infected individuals. These mice represent a new in vivo system for studying the mechanism by which HIV-1-infected monocytes migrate into the brain.     

Effects of wheatgerm agglutinin and aging on the regional brain uptake of HIV-1GP120.

HIV-1 is associated with infection and altered functions of the CNS, especially in the elderly. Most studies indicate that HIV-1 is not evenly distributed throughout the CNS but is concentrated in deep brain nuclei. This study examined whether regional or age-related differences in the permeability of the blood-brain barrier to gp120, the viral coat of HIV-1, exist. The initial concentration of gp120 in 10 brain regions correlated with vascular content in young and old mice. Susceptibility to wheatgerm agglutinin (WGA)-induced uptake of gp120, which relates to endothelial cell internalization, varied regionally, with no induction of uptake into the striatum or hypothalamus but with large increases in the cerebellum, cortex, and midbrain. Transport across the BBB, as measured by the unidirectional influx rate (Ki), also varied regionally with the hypothalamus, hippocampus, and pons-medulla showing the highest values for Ki and the striatum the lowest. These regional variations in the permeability of the BBB to gp120 could contribute to the inhomogeneous distribution of HIV-1 within the CNS whereas the failure to see differences with aging suggests other causes underlie the susceptibility of the elderly to the CNS manifestations of AIDS.     

Contribution of proteoglycans to human immunodeficiency virus type 1 brain invasion

As a neurotropic virus, human immunodeficiency virus type 1 (HIV-1) invades the brain and causes severe neuronal, astrocyte, and myelin damage in AIDS patients. To gain access to the brain, HIV-1 must migrate through brain microvascular endothelial cells (BMECs), which compose the blood-brain barrier (BBB). Given that BMECs lack the entry receptor CD4, HIV-1 must use receptors distinct from CD4 to enter these cells. We previously reported that cell surface proteoglycans serve as major HIV-1 receptors on primary human endothelial cells. In this study, we examined whether proteoglycans also impact cell-free HIV-1 invasion of the brain. Using an artificial BBB transmigration assay, we found that both heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) are abundantly expressed on primary BMECs and promote HIV-1 attachment and entry. In contrast, the classical entry receptors, CXCR4 and CCR5, only moderately enhanced these processes. HSPGs and CSPGs captured HIV-1 in a gp120-dependent manner. However, no correlation between coreceptor usage and transmigration was identified. Furthermore, brain-derived viruses did not transmigrate more efficiently than lymphoid-derived viruses, suggesting that the ability of HIV-1 to replicate in the brain does not correlate with its capacity to migrate through the BBB as cell-free virus. Given that HIV-1-proteoglycan interactions are based on electrostatic contacts between basic residues in gp120 and sulfate groups in proteoglycans, HIV-1 may exploit these interactions to rapidly enter and migrate through the BBB to invade the brain.     

Human dendritic cells transduced with herpes simplex virus amplicons encoding human immunodeficiency virus type 1 (HIV-1) gp120 elicit adaptive immune responses from human cells engrafted into NOD/SCID mice and confer partial protection against HIV-1 challenge

Small-animal models are needed to test human immunodeficiency virus (HIV) vaccine efficacy following viral challenge. To this end, we examined HIV-1-specific immune responses following immunization of nonobese diabetic-severe combined immunodeficient mice that were repopulated with human peripheral blood lymphocytes (hu-PBL-NOD/SCID mice). Autologous dendritic cells (DC) were transduced ex vivo with replication-defective, helper virus-free, herpes simplex virus type 1 (HSV-1) amplicons that expressed HIV-1 gp120 and were then injected into the hu-PBL-NOD/SCID mice. This resulted in primary HIV-1-specific humoral and cellular immune responses. Serum samples from vaccinated animals contained human immunoglobulin G that reacted with HIV-1 Env proteins by enzyme-linked immunosorbent assay and neutralized the infectivity of HIV-1 LAI and ADA strains. T cells isolated from the mice responded to viral antigens by producing gamma interferon when analyzed by enzyme-linked immunospot assay. Importantly, exposure of the vaccinated animals to infectious HIV-1 demonstrated partial protection against infectious HIV-1 challenge. This was reflected by a reduction in HIV-1(ADA) and by protection of the engrafted human CD4(+) T lymphocytes against HIV-1(LAI)-induced cytotoxicity. These data demonstrate that transduction of DC by HSV amplicon vectors expressing HIV-1 gp120 induce virus-specific immune responses in hu-PBL-NOD/SCID mice. This mouse model may be a useful tool to evaluate human immune responses and protection against viral infection following vaccination.     

Glycosylation is necessary for the correct folding of human immunodeficiency virus gp120 in CD4 binding.

Conflicting results have been reported regarding the role of carbohydrate on human immunodeficiency virus (HIV) envelope glycoprotein gp120 in CD4 receptor binding. Glycosylated, deglycosylated, and nonglycosylated forms of HIV type 1 (HIV-1) and HIV-2 gp120s were used to examine CD4 receptor-binding activity. Nonglycosylated forms of gp120 generated either by deletion of the signal sequence of HIV-1 gp120 or by synthesis in the presence of tunicamycin failed to bind to CD4. In contrast, highly mannosylated gp120 bound to soluble CD4 molecules well. Enzymatic removal of carbohydrate chains from glycosylated gp120 by endoglycosidase H or an endoglycosidase F/N glycanase mixture had no effect on the ability of gp120 to bind CD4. An experiment which measured the ability of gp120 to bind to CD4 as an assay of the proper conformation of gp120 showed that carbohydrate chains on gp120 are not required for the interaction between gp120 and CD4 but that N-linked glycosylation is essential for generation of the proper conformation of gp120 to provide a CD4-binding site.     

Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors

We have obtained, for the first time, a quantitative protein expression profile of membrane transporters and receptors in human brain microvessels, that is, the blood-brain barrier (BBB). Brain microvessels were isolated from brain cortexes of seven males (16-77 years old) and protein expression of 114 membrane proteins was determined by means of a liquid chromatography-tandem mass spectrometric quantification method using recently established in-silico peptide selection criteria. Among drug transporters, breast cancer resistance protein showed the most abundant protein expression (8.14 fmol/μg protein), and its expression level was 1.85-fold greater in humans than in mice. By contrast, the expression level of P-glycoprotein in humans (6.06 fmol/μg protein) was 2.33-fold smaller than that of mdr1a in mice. The organic anion transporters reported in rodent BBB, that is, multidrug resistance-associated protein, organic anion transporter and organic anion-transporting polypeptide family members, were under limit of quantification in humans, except multidrug resistance-associated protein 4 (0.195 fmol/μg protein). Among detected transporters and receptors for endogenous substances, the glucose transporter 1 level was similar to that of mouse, while the L-type amino acid transporter 1 level was fivefold smaller than that of mouse. These findings should be useful for understanding human BBB function and its differences from that in mouse.     

Molecular mimicry between the human immunodeficiency virus type 1 gp120 V3 loop and human brain proteins.

Immunologically cross-reactive proteins in the human brain that resemble the V3 loop of human immunodeficiency virus type 1 (HIV-1) gp120 have been identified. When several homogenized tissues from normal brains were used, a monoclonal antibody raised against amino acids 308 to 320 of the V3 loop reacted with three prominent human brain proteins (HBP) of 35, 55, and 110 kDa. Among the three, the 55-kDa HBP appears to be specific to the central nervous system. These results indicate that the V3 loop of HIV-1 gp120 shares an epitope with HBP. An immune response to the V3 loop that generates cross-reactive antibodies to cellular proteins may be an autoimmune mechanism by which HIV-1 can damage the central nervous system.     

Human immunodeficiency virus type 1 gp120 induces abnormal maturation and functional alterations of dendritic cells: a novel mechanism for AIDS pathogenesis

Dendritic cells (DCs) play a crucial role in bridging innate and acquired immune responses to pathogens. In human immunodeficiency virus type 1 (HIV-1) infection, immature DCs (iDCs) are also main targets for HIV-1 at the mucosal level. In this study, we evaluated the effects of HIV-1-DC interactions on the maturation and functional activity of these cells. Exposure of human monocyte-derived iDCs to either aldrithiol-2-inactivated HIV-1 or gp120 led to an upmodulation of activation markers indicative of functional maturation. Despite their phenotype, these cells retained antigen uptake capacity and showed an impaired ability to secrete cytokines or chemokines and to induce T-cell proliferation. Although gp120 did not interfere with DC differentiation, the capacity of these cells to produce interleukin-12 (IL-12) upon maturation was markedly reduced. Likewise, iDCs stimulated by classical maturation factors in the presence of gp120 lacked allostimulatory capacity and did not produce IL-12, in spite of their phenotype typical of activated DCs. Exogenous addition of IL-12 restores the allostimulatory capacity of gp120-exposed DCs. The finding that gp120 induces abnormal maturation of DCs linked to profound suppression of their activities unravels a novel mechanism by which HIV can lead to immune dysfunction in AIDS patients.     

Circulating Brain Microvascular Endothelial Cells (cBMECs) as Potential Biomarkers of the Blood–Brain Barrier Disorders Caused by Microbial and Non-Microbial Factors

Despite aggressive research, central nervous system (CNS) disorders, including blood-brain barrier (BBB) injury caused by microbial infection, stroke, abused drugs [e.g., methamphetamine (METH) and nicotine], and other pathogenic insults, remain the world''s leading cause of disabilities. In our previous work, we found that dysfunction of brain microvascular endothelial cells (BMECs), which are a major component of the BBB, could be caused by nicotine, meningitic pathogens and microbial factors, including HIV-1 virulence factors gp41 and gp120. One of the most challenging issues in this area is that there are no available cell-based biomarkers in peripheral blood for BBB disorders caused by microbial and non-microbial insults. To identify such cellular biomarkers for BBB injuries, our studies have shown that mice treated with nicotine, METH and gp120 resulted in increased blood levels of CD146+(endothelial marker)/S100B+ (brain marker) circulating BMECs (cBMECs) and CD133+[progenitor cell (PC) marker]/CD146+ endothelial PCs (EPCs), along with enhanced Evans blue and albumin extravasation into the brain. Nicotine and gp120 were able to significantly increase the serum levels of ubiquitin C-terminal hydrolase 1 (UCHL1) (a new BBB marker) as well as S100B in mice, which are correlated with the changes in cBMECs and EPCs. Nicotine- and meningitic E. coli K1-induced enhancement of cBMEC levels, leukocyte migration across the BBB and albumin extravasation into the brain were significantly reduced in alpha7 nAChR knockout mice, suggesting that this inflammatory regulator plays an important role in CNS inflammation and BBB disorders caused by microbial and non-microbial factors. These results demonstrated that cBMECs as well as EPCs may be used as potential cell-based biomarkers for indexing of BBB injury.     

Discontinuous, conserved neutralization epitopes overlapping the CD4-binding region of human immunodeficiency virus type 1 gp120 envelope glycoprotein.

Monoclonal antibodies have been isolated from human immunodeficiency virus type 1 (HIV-1)-infected patients that recognize discontinuous epitopes on the gp120 envelope glycoprotein, that block gp120 interaction with the CD4 receptor, and that neutralize a variety of HIV-1 isolates. Using a panel of HIV-1 gp120 mutants, we identified amino acids important for precipitation of the gp120 glycoprotein by three different monoclonal antibodies with these properties. These amino acids are located within seven discontinuous, conserved regions of the gp120 glycoprotein, four of which overlap those regions previously shown to be important for CD4 recognition. The pattern of sensitivity to amino acid change in these seven regions differed for each antibody and also differed from that of the CD4 glycoprotein. These results indicate that the CD4 receptor and this group of broadly neutralizing antibodies recognize distinct but overlapping gp120 determinants.     

Probing the structure of the V2 domain of human immunodeficiency virus type 1 surface glycoprotein gp120 with a panel of eight monoclonal antibodies: human immune response to the V1 and V2 domains.

We have analyzed a panel of eight murine monoclonal antibodies (MAbs) that depend on the V2 domain for binding to human immunodeficiency virus type 1 (HIV-1) gp120. Each MAb is sensitive to amino acid changes within V2, and some are affected by substitutions elsewhere. With one exception, the MAbs were not reactive with peptides from the V2 region, or only poorly so. Hence their ability to bind recombinant strain IIIB gp120 depended on the preservation of native structure. Three MAbs cross-reacted with strain RF gp120, but only one cross-reacted with MN gp120, and none bound SF-2 gp120. Four MAbs neutralized HIV-1 IIIB with various potencies, and the one able to bind MN gp120 neutralized that virus. Peptide serology indicated that antibodies cross-reactive with the HxB2 V1 and V2 regions are rarely present in HIV-1-positive sera, but the relatively conserved segment between the V1 and V2 loops was recognized by antibodies in a significant fraction of sera. Antibodies able to block the binding of V2 MAbs to IIIB or MN gp120 rarely exist in sera from HIV-1-infected humans; more common in these sera are antibodies that enhance the binding of V2 MAbs to gp120. This enhancement effect of HIV-1-positive sera can be mimicked by several human MAbs to different discontinuous gp120 epitopes. Soluble CD4 enhanced binding of one V2 MAb to oligomeric gp120 but not to monomeric gp120, perhaps by inducing conformational changes in the oligomer.     

HIV-1 gp120 and tobacco smoke synergistically disrupt the integrity of the blood-brain barrier

In the United States, the Centers for Disease Control and Prevention (CDC) terms HIV and tobacco use among the ten most important public health challenges we face today. In the last decade, there has been a remarkable decrease in the number of deaths due to HIV/AIDS, especially after the widespread availability and use of combination antiretroviral therapy (cART). However, people living with HIV/AIDS have a heightened risk of chronic complications and comorbidities, including neurological disorders. Around 40–60 % of HIV-infected individuals progress to NeuroAIDS, a group of disorders caused primarily by HIV-mediated damage to the central and peripheral nervous systems, despite receiving cART. The detrimental effects of chronic smoking on the cerebrovascular system are also well studied and reported. Addictive behavior, such as smoking, is more common in HIV patients compared to the general population. In this context, given the existing immune suppression, smoking can pose a significant risk for the progression of the disease to NeuroAIDS by disrupting the integrity of the blood-brain barrier (BBB). Here we show that co-treatment with Tobacco Smoke Extract (TSE) and HIV-1 gp120 (HIV envelope glycoprotein) in primary cultures of human brain microvascular endothelial cells promoted heightened cellular stress responses compared to control and individual treatments. Our findings suggest that a potential synergistic effect between smoke exposure and gp120 can worsen the loss of BBB viability, possibly exacerbating NeuroAIDS progression.     

Acute ethanol administration downregulates human immunodeficiency virus-1 glycoprotein 120-induced KC and RANTES production by murine Kupffer cells and splenocytes

Glycoprotein 120 from HIV-1, HIV-2 and SIV is known to stimulate secretion of chemokines by mononuclear cells. Thus, this work tests the hypothesis that acute ethanol intoxication suppresses HIV-1 gp120-induced chemokine production by murine Kupffer cells and splenocytes. Male Balb/c mice were given ethanol (1.70 g/Kg) by intragastric gavage in 0.1 ml volume of saline. Five minutes after ethanol administration, mice received an intravenous injection of HIV-1 gp120 (5 microg/Kg). After 24 hr, serum samples, splenocytes and Kupffer cells were obtained. Isolated cells were cultured in DMEM for 24 hr to determine production of chemokines and cytokines in vitro. Chemokines (MIP-2, KC, RANTES, MIP-1 alpha and MCP-1) and cytokines (IL-1 beta, TNF alpha, IL-10, gamma-IFN) were measured by ELISA. M-RNA abundance of these mediators was determined by RT-PCR. Results show that HIV-1 gp120 treatment was associated with significant elevations in serum KC and RANTES. No changes were observed with regard to other chemokines and cytokines. Oral administration of ethanol significantly suppressed HIV-1gp120-induced KC and RANTES release. KC and RANTES-mRNA expression and protein release by splenocytes and Kupffer cells were up-regulated by HIV-1 gp120. Such up-regulation was attenuated by ethanol treatment. These data show that acute ethanol administration attenuates HIV-1 gp120-induced chemokine release in vivo by isolated splenocytes and Kupffer cells. Through this mechanism, previous in vivo ethanol use may compromise the ability of HIV-1 gp120 to induce chemokine-mediated inhibition of HIV-1 entry into target cells.     

Epithelial Uptake and Transport of Cell-Free Human Immunodeficiency Virus Type 1 and gp120-Coated Microparticles

Cell-free human immunodeficiency virus type 1 (HIV-1) can be taken up and released by a monolayer of primary human gingival cells and remain infectious for CD4⁺ cells. Virus-sized latex particles covalently coated with purified native HIV-1 envelope glycoprotein gp120 are also transported through the primary epithelial cells. This process is significantly stimulated by increasing the intracellular cyclic AMP (cAMP) concentration. Inhibition experiments with mannan and α-methyl-mannopyranoside indicated that mannosyl groups are involved in the interaction between gp120 and gingival cells. An increase of cellular oligomannosyl receptors by incubation with the mannosidase inhibitor deoxymannojirimycin augmented transcellular transport of the gp120-coated particles. The results suggest that infectious HIV can penetrate gingival epithelia by a cAMP-dependent transport mechanism involving interaction of the lectin-like domain of gp120 and mannosyl residues on glycoproteins on the mucosal surface. Penetration of HIV could be inhibited by soluble glycoconjugates present in oral mucins.     

Elicitation of neutralizing antibodies by intranasal administration of recombinant vesicular stomatitis virus expressing human immunodeficiency virus type 1 gp120

Recombinant viral vectors are useful tools for AIDS vaccine development. However, expression of HIV-1 envelope genes using viral vectors has not been successful in the induction of potent neutralizing antibodies in vivo. We took advantage of the strong immunogenicity of vesicular stomatitis virus (VSV)-based vector and expressed HIV-1 HXB2 gp120 gene in the recombinant VSV. Our results showed that HIV-1 gp120 protein expressed by the recombinant VSV retained the native conformation of the protein to some degree and was recognized by two well-characterized broad anti-HIV-1 neutralizing monoclonal antibodies b12, 2G12. We further showed that only one time intranasal immunization with the recombinant VSV led to production of anti-HIV-1 anti-sera in mice. In addition, we found that the anti-sera had the ability to neutralize not only HXB2 envelope-pseudotyped HIV-1 viruses but also HIV-1 pseudotyped viruses with JRFL envelopes. These results suggest that HIV-1 gp120 expressed by the recombinant VSV, in combination with the route of intranasal administration, is an effective strategy to evaluate the immunogenicity of HIV-1 envelope protein and its variants in mice.     

Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1.

We have isolated and characterized human monoclonal antibody 2G12 to the gp120 surface glycoprotein of human immunodeficiency virus type 1 (HIV-1). This antibody potently and broadly neutralizes primary and T-cell line-adapted clade B strains of HIV-1 in a peripheral blood mononuclear cell-based assay and inhibits syncytium formation in the AA-2 cell line. Furthermore, 2G12 possesses neutralizing activity against strains from clade A but not from clade E. Complement- and antibody-dependent cellular cytotoxicity-activating functions of 2G12 were also defined. The gp120 epitope recognized by 2G12 was found to be distinctive; binding of 2G12 to LAI recombinant gp120 was abolished by amino acid substitutions removing N-linked carbohydrates in the C2, C3, V4, and C4 regions of gp120. This gp120 mutant recognition pattern has not previously been observed, indicating that the 2G12 epitope is unusual. consistent with this, antibodies able to block 2G12 binding to recombinant gp120 were not detected in significant quantities in 16 HIV-positive human serum samples.     

Modeling HIV-1 Induced Neuroinflammation in Mice: Role of Platelets in Mediating Blood-Brain Barrier Dysfunction

The number of HIV-1 positive individuals developing some form of HIV-associated neurocognitive disorder (HAND) is increasing. In these individuals, the integrity of the blood-brain barrier (BBB) is compromised due to an increase in exposure to pro-inflammatory mediators, viral proteins, and virus released from infected cells. It has been shown that soluble CD40L (sCD40L) is released upon platelet activation and is an important mediator of the pathogenesis of HAND but the underlying mechanisms are unclear, emphasizing the need of an effective animal model. Here, we have utilized a novel animal model in which wild-type (WT) mice were infected with EcoHIV; a derivative of HIV-1 that contains a substitution of envelope protein gp120 with that of gp80 derived from murine leukemia virus-1 (MuLV-1). As early as two-weeks post-infection, EcoHIV led to increased permeability of the BBB associated with decreased expression of tight junction protein claudin-5, in CD40L and platelet activation-dependent manner. Treatment with an antiplatelet drug, eptifibatide, in EcoHIV-infected mice normalized BBB function, sCD40L release and platelet activity, thus implicating platelet activation and platelet-derived CD40L in virally induced BBB dysfunction. Our results also validate and underscore the importance of EcoHIV infection mouse model as a tool to explore therapeutic targets for HAND.     

Tyrosine sulfation of human antibodies contributes to recognition of the CCR5 binding region of HIV-1 gp120

Sulfated tyrosines at the amino terminus of the principal HIV-1 coreceptor CCR5 play a critical role in its ability to bind the HIV-1 envelope glycoprotein gp120 and mediate HIV-1 infection. Here, we show that a number of human antibodies directed against gp120 are tyrosine sulfated at their antigen binding sites. Like that of CCR5, antibody association with gp120 is dependent on sulfate moieties, enhanced by CD4, and inhibited by sulfated CCR5-derived peptides. Most of these antibodies preferentially associate with gp120 molecules of CCR5-utilizing (R5) isolates and neutralize primary R5 isolates more efficiently than laboratory-adapted isolates. These studies identify a distinct subset of CD4-induced HIV-1 neutralizing antibodies that closely emulate CCR5 and demonstrate that tyrosine sulfation can contribute to the potency and diversity of the human humoral response.     

Influence of breast cancer resistance protein (Abcg2) and p-glycoprotein (Abcb1a) on the transport of imatinib mesylate (Gleevec) across the mouse blood-brain barrier

Imatinib, a protein tyrosine kinase inhibitor, may prevent the growth of glioblastoma cells. Unfortunately, its brain distribution is restricted by p-glycoprotein (p-gp or multidrug resistance protein Mdr1a), and probably by breast cancer resistance protein (Bcrp1), two efflux pumps expressed at the blood-brain barrier (BBB). We have used in situ brain perfusion to investigate the mechanisms of imatinib transport across the mouse BBB. The brain uptake of imatinib in wild-type mice was limited by saturable efflux processes. The inhibition of p-gp, by valspodar and zosuquidar, increased imatinib uptake (2.5-fold), as did the deficiency of p-gp in Mdr1a/1b(-/-) mice (5.5-fold). Perfusing imatinib with the p-gp/Bcrp1 inhibitor, elacridar, enhanced the brain uptake of imatinib in wild-type (4.1-fold) and Mdr1a/1b(-/-) mice (1.2-fold). However, the brain uptake of imatinib was similar in wild-type and Bcrp1(-/-) mice when it was perfused at a non-saturating concentration. The brain uptake of CGP74588, an active metabolite of imatinib, was low. It was increased by perfusion with elacridar (twofold), but not with valspodar and zosuquidar. CGP74588 uptake was 1.5 times greater in Bcrp1(-/-) mice than in wild-type mice. These data suggest that imatinib transport at the mouse BBB is limited by p-gp and probably by Bcrp1, and that CGP74588 transport is restricted by Bcrp1.