Biomedical subjects

R A Rogers

Publications and source records attributed to R A Rogers.

At least 19 recordsLinked to original sources

PubMed2000

T-tropic sequence of the V3 loop is critical for HIV-1 infection of CXCR4-positive colonic HT-29 epithelial cells.

Some colonic and neuronal cells which are CD4- but galactosyl ceramide-positive are susceptible to infection with HIV-1. We have previously shown that the T-cell tropic V3 loop of HIV-1 gp120 serves as a primary viral determinant for infectivity of CD4- neuronal cells. However, the nature of the V3 loop of HIV-1 needed for infection and the V3 loop's interaction with coreceptors on colonic epithelial cells have not been fully analyzed. By using HIV-1 molecular clones, we show that the T-cell tropic V3 domain is critical for HIV-1 infection of colonic HT-29 epithelial cells. Because T-cell tropic HIV-1 can use CXCR4 as a coreceptor in T cells, we set out to determine the role of CXCR4 during infection of HT-29 cells. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunostaining, we show that these epithelial cells of colonic origin express the chemokine receptor CXCR4. Importantly, antibody against CXCR4 or a neutralizing antibody against HIV-1 gp120 V3 loop blocks T-cell tropic HIV-1 entry into HT-29 cells. These data indicate that the V3 loop of HIV-1 and the chemokine receptor CXCR4 are both critical for HIV-1 infection of colonic HT-29 epithelial cells. An HIV-1 T-tropic virus may be responsible for the infection of human colonic epithelial cells in vivo.

Amino Acid Sequence↗

PubMed2000

CD1c molecules broadly survey the endocytic system.

The ability of antigen-presenting cells to sample distinct intracellular compartments is crucial for microbe detection. Major histocompatibility complex class I and class II molecules sample the cytosol or the late endocytic compartment, allowing detection of microbial peptide antigens that arise in distinct intracellular compartments. In contrast, CD1a and CD1b molecules mediate the presentation of lipid and glycolipid antigens and differentially sample early recycling endosomes or late endocytic compartments, respectively, that contain distinct sets of lipid antigens. Here, we show that, unlike the other CD1 isoforms or major histocompatibility complex molecules that each sample restricted only intracellular compartments, CD1c is remarkable in that it distributes broadly throughout the endocytic system and is expressed in both recycling endosomes and late endocytic compartments. Further, in contrast to CD1b, which requires an acidic environment to function, antigen presentation by CD1c was able to overcome dependence on vesicular acidification. Because CD1c is expressed on essential antigen-presenting cells, such as epidermal Langerhans cells (in the absence of CD1b), or on B cells (without CD1a or -b), we suggest that CD1c molecules allow a comprehensive survey for lipid antigens throughout the endocytic system even in the absence of other CD1 isoforms.

Antigen Presentation↗

PubMed2000

Human CD1b and CD1c isoforms survey different intracellular compartments for the presentation of microbial lipid antigens.

CD1b and CD1c are antigen-presenting molecules that mediate recognition of bacterial lipids by T cells, but it is currently not known whether these two molecules are redundant or are specialized to perform different immunological functions. Here, we show that the distribution of CD1c in human dendritic cells was characterized by a high ratio of cell surface to intracellular molecules, whereas CD1b showed a reciprocal pattern of distribution. In contrast to the accumulation of CD1b in lysosomal major histocompatibility complex class II compartments, intracellular CD1c molecules accumulated in other endocytic compartments, most likely early and late endosomes. Deletion of the cytoplasmic tail of CD1c, containing a tyrosine-based internalization motif, abolished most of its intracellular localization. Functional studies using T cells specific for defined lipid antigens revealed that in contrast to CD1b-mediated antigen presentation, antigen presentation by CD1c was resistant to drugs inhibiting endosomal acidification and was independent of endosomal localization of CD1c. Taken together, these results support the hypothesis that CD1b and CD1c are specialized to survey the lipid content of different intracellular compartments.

Antigen Presentation↗

PubMed2000

A soluble mannose receptor immunoadhesin enhances phagocytosis of Pneumocystis carinii by human polymorphonuclear leukocytes in vitro.

Pneumocystis carinii is an opportunistic pathogen that causes pneumonia in immunocompromised hosts. In the normal host, P. carinii is susceptible to an array of first line host defense mechanisms that are operative in the lung. Alveolar macrophages play a central role in the clearance of inhaled organisms. The macrophage mannose receptor (MR) appears to be sufficient for P. carinii phagocytosis. In individuals infected with the human immunodeficiency virus, MR expression on alveolar macrophages and P. carinii phagocytosis are decreased, however, Fc-receptor mediated phagocytosis remains intact. In this study, we demonstrate that a recombinant soluble MR immunoadhesin, consisting of the essential carbohydrate binding MR ectodomain and the Fc-region of human immunoglobulin (Ig)G1, binds P. carinii and leads to an 8.2-fold increased uptake of P. carinii by phagocytic cells. Our results suggest that the soluble MR immunoadhesin may have therapeutic potential in the treatment of P. carinii infections.

Animals↗

PubMed2000

Inducible expression of claudin-1-myc but not occludin-VSV-G results in aberrant tight junction strand formation in MDCK cells.

Occludin and 18 distinct members of the claudin family are tetra-span transmembrane proteins that are localized in cell-specific tight junctions (TJs). A previous study showed that expression of chick occludin in Madin-Darby canine kidney (MDCK) cells raised transepithelial electrical resistance (TER) and, paradoxically, increased mannitol flux. In the present study, we employed epitope tagged canine occludin expression, under the control of the tetracycline repressible transactivator, to determine the extent to which the unexpected parallel increase in TER and mannitol flux was related to a structural mismatch between avian and canine occludins, which are only 50% identical. To determine whether the paradoxical changes in permeability was specific to occludin, we assessed the effect of over-expressing epitope tagged murine claudin-1. Our data revealed that over-expression of either of the epitope tagged mammalian tight junction proteins increased TER, mannitol and FITC-dextran flux. We observed a 2- and up to 5.6-fold over-expression of occludin-VSV-G and claudin-1-myc, respectively, with no change in ZO-1, endogenous occludin or claudin-1 expression. Confocal microscopy revealed that occludin-VSV-G, claudin-1-myc and ZO-1 co-localized at the TJ. In addition, claudin-1-myc formed aberrant strands along the lateral cell surface without an underlying ZO-1 scaffold. In fracture labeled replicas these strands consisted of claudin-1-myc with little accompanying occludin. These observations suggest that in epithelial cells claudin-1 can assemble into TJ strands without the participation of either ZO-1 or occludin. The proximity of the myc tag to the COOH-terminal YV sequence of claudin-1 appeared to interfere with its interaction with ZO-1, since over-expression of non-tagged claudin-1 increased TER but had a minimal effect on solute flux and no aberrant strands formed. From our data we conclude that differences in structure between avian and mammalian occludin do not account for the observed paradoxical increase in mannitol flux. Levels of ZO-1 remained unchanged despite substantial increases in induced TJ integral protein expression, suggesting that an imbalance between levels of ZO-1 and occludin or claudin-1 leads to altered regulation of pores through which non-charged solute flux occurs. We suggest that ion and solute flux are differentially regulated at the TJ.

Animals↗

PubMed1999

Low molecular weight group IIA and group V phospholipase A(2) enzymes have different intracellular locations in mouse bone marrow-derived mast cells.

The subcellular location of the enzymes of eicosanoid biosynthesis is critical for their co-ordinate action in the generation of leukotrienes and prostaglandins. This activity is thought to occur predominantly at a perinuclear location. Whereas the subcellular locations of cytosolic phospholipase (PL) A(2) and each of the pathway enzymes of eicosanoid generation have been defined, the distribution of the low molecular weight species of PLA(2) has remained elusive because of the lack of antibodies that distinguish among homologous family members. We have prepared affinity-purified rabbit antipeptide IgG antibodies that distinguish mouse group IIA PLA(2) and group V PLA(2). Immunofluorescence staining and immunogold electron microscopy reveal different subcellular locations for the enzymes. Group IIA(2) PLA(2) is present in the secretory granules of mouse bone marrow-derived mast cells, consistent with its putative role in facilitating secretory granule exocytosis and its consequent extracellular action. In contrast, group V PLA(2) is associated with various membranous organelles including the Golgi apparatus, nuclear envelope, and plasma membrane. The perinuclear location of group V PLA(2) is consistent with a putative interaction with translocated cytosolic PLA(2) in supplying arachidonic acid for generation of eicosanoid products, while the location in Golgi cisternae may also reflect its action as a secreted enzyme. The spatial segregation of group IIA PLA(2) and group V PLA(2) implies that these enzymes are not functionally redundant.

Amino Acid Sequence↗

PubMed1999

Characterization of human and murine PMP20 peroxisomal proteins that exhibit antioxidant activity in vitro.

We have isolated the cDNAs encoding human and mouse homologues of a yeast protein, termed peroxisomal membrane protein 20 (PMP20). Comparison of the amino acid sequences of human (HsPMP20) and mouse (MmPMP20) PMP20 proteins revealed a high degree of identity (93%), whereas resemblance to the yeast Candida boidinii PMP20A and PMP20B (CbPMP20A and CbPMP20B) was less (30% identity). Both HsPMP20 and MmPMP20 lack transmembrane regions, as do CbPMP20A and CbPMP20B. HsPMP20 mRNA expression was low in human fetal tissues, especially in the brain. In adult tissues, HsPMP20 mRNA was expressed in the majority of tissues tested. HsPMP20 and MmPMP20 contained the C-terminal tripeptide sequence Ser-Gln-Leu (SQL), which is similar to the peroxisomal targeting signal 1 utilized for protein import into peroxisomes. HsPMP20 bound directly to the human peroxisomal targeting signal 1 receptor, HsPEX5. Mutagenesis analysis showed that the C-terminal tripeptide sequence, SQL, of HsPMP20 is necessary for its binding to HsPEX5. Subcellular fractionation of HeLa cells, expressing epitope-tagged PMP20, revealed that HsPMP20 is localized in the cytoplasm and in a particulate fraction containing peroxisomes. Double-staining immunofluorescence studies showed colocalization of HsPMP20 and thiolase, a bona fide peroxisomal protein. The amino acid sequence alignment of HsPMP20, MmPMP20, CbPMP20A, and CbPMP20B displayed high similarity to thiol-specific antioxidant proteins. HsPMP20 exerted an inhibitory effect on the inactivation of glutamine synthetase in the thiol metal-catalyzed oxidation system but not in the nonthiol metal-catalyzed oxidation system, suggesting that HsPMP20 possesses thiol-specific antioxidant activity. In addition, HsPMP20 removed hydrogen peroxide by its thiol-peroxidase activity. These results indicate that HsPMP20 is imported into the peroxisomal matrix via PEX5p and may work to protect peroxisomal proteins against oxidative stress. Because some portion of PMP20 might also be present in the cytosol, HsPMP20 may also have a protective effect in the cytoplasm.

Amino Acid Sequence↗

PubMed1999

Rapid reduction of MDCK cell cholesterol by methyl-beta-cyclodextrin alters steady state transepithelial electrical resistance.

The role of plasma membrane lipids in regulating the passage of ions and other solutes through the paracellular pathway remains controversial. In this study we explore the contribution of cholesterol (CH) in maintaining the barrier function of an epithelial cell line using the CH-solubilizing agent methyl beta-cyclodextrin (MBCD) to stimulate CH efflux. Inclusion of 20 mM MBCD in both apical and basolateral media reduced CH levels by 70-80% with no significant effect on cell viability. Most of that decrease occurred during the first 30 min of incubation. Recovery of CH content to initial values was nearly complete 22 h after removal of MBCD. Within 30 min of adding MBCD to the culture medium, transepithelial electrical resistance (TER) increased, reaching maximum values 30-40% above controls. This early rise in TER occurred when MBCD was added to either side of the monolayer. The later rapid decline in TER was observed only when MBCD bathed the basolateral surface from which, coincidentally, CH efflux was most rapid. Freeze fracture replicas and transmission electron microscopy of monolayers exposed to MBCD for only 30 min revealed no increase in either the average tight junction (TJ) strand number or the dimensions of the lateral intercellular space. There was a statistically significant increase in the number of TJ particles associated with the E fracture face at this time. This raises the interesting possibility that during CH efflux there is a change in the interaction between TJ particles and underlying cytoskeletal elements. There was no change in staining for occludin and ZO-1. After exposing the basolateral surface to MBCD for 2 h, TER fell below control levels. The accompanying increase in mannitol flux suggests strongly that the decrease in TER resulted from an increase in the permeability of the paracellular and not the transcellular pathway. A decrease in immuno-staining for occludin and ZO-1 at TJs, a striking accumulation of actin at tri-cellular areas as well as a decline in the number of parallel strands, as seen in freeze fracture replicas, suggest that changes in cytoskeletal organization during long incubations with MBCD had physically disrupted the TJ network. Data are presented which suggest that the observed changes in paracellular permeability during CH efflux may be related to increased levels of lipid-derived second messengers, some of which may trigger changes in the phosphorylation status of TJ proteins.

Animals↗

PubMed1999

Separate pathways for antigen presentation by CD1 molecules.

The ability to sample relevant intracellular compartments is necessary for effective antigen presentation. To detect peptide antigens, MHC class I and II molecules differentially sample cytosolic and endosomal compartments. CD1 constitutes another lineage of lipid antigen-presenting molecules. We show that CD1b traffics deeply into late endosomal compartments, while CD1a is excluded from these compartments and instead traffics independently in the recycling pathway of the early endocytic system. Further, CD1b but not CD1a antigen presentation is dependent upon vesicular acidification. Since lipids and various bacteria are known to traffic differentially, either penetrating deeply into the endocytic system or following the route of recycling endosomes, these findings elucidate efficient monitoring of distinct components of the endocytic compartment by CD1 lipid antigen-presenting molecules.

Antigen Presentation↗

PubMed1999

Confocal imaging of peripheral regions of intact rat lungs following intratracheal administration of 6-carboxyfluorescein, FITC-insulin, and FITC-dextran.

PURPOSE: This study compared the pulmonary disposition of 3 structurally diverse probe molecules following their intratracheal (i.t.) administration to anesthetized rats. METHODS: Following administration of 6-carboxyfluorescein (CF), FITC-insulin (FI) and FITC-dextran (FD), lungs were removed, inflated, perfused with a marker dye, and peripheral elements examined with confocal microscopy (CLSFM). RESULTS: At 5 min most of each probe remained within airspaces; the remainder distributed to interstitium, capillaries, Type II cells, and macrophages. At 60 min disposition differed significantly among probes. The smallest (CF, 376 Da) had almost completely exited air-spaces and was found primarily in extracellular interstitial spaces, often behind Type II cells. Disposition was consistent with both entry into peripheral lymphatics and association with peripheral fibers. FI and FD (6 and 10 kDa, respectively) were retained substantially longer within airspaces. In contrast to CF, FI appeared to localize along septal and peripheral fibers, but its disposition was inconsistent with the involvement of peripheral lymphatics. CONCLUSIONS: While all probes were < or = 10 kDa, there was considerable disparity among both their rates of absorption and subsequent disposition within peri-alveolar elements. CLSFM appears to be a useful ancillary tool for studying the pulmonary absorption of drugs and macromolecules.

Absorption↗

PubMed1999

Characterization of Mayven, a novel actin-binding protein predominantly expressed in brain.

The cytoskeleton plays an important role in neuronal morphogenesis. We have identified and characterized a novel actin-binding protein, termed Mayven, predominantly expressed in brain. Mayven contains a BTB (broad complex, tramtrack, bric-a-brac)/POZ (poxvirus, zinc finger) domain-like structure in the predicted N terminus and "kelch repeats" in the predicted C-terminal domain. Mayven shares 63% identity (77% similarity) with the Drosophila ring canal ("kelch") protein. Somatic cell-hybrid analysis indicated that the human Mayven gene is located on chromosome 4q21.2, whereas the murine homolog gene is located on chromosome 8. The BTB/POZ domain of Mayven can self-dimerize in vitro, which might be important for its interaction with other BTB/POZ-containing proteins. Confocal microscopic studies of endogenous Mayven protein revealed a highly dynamic localization pattern of the protein. In U373-MG astrocytoma/glioblastoma cells, Mayven colocalized with actin filaments in stress fibers and in patchy cortical actin-rich regions of the cell margins. In primary rat hippocampal neurons, Mayven is highly expressed in the cell body and in neurite processes. Binding assays and far Western blotting analysis demonstrated association of Mayven with actin. This association is mediated through the "kelch repeats" within the C terminus of Mayven. Depolarization of primary hippocampal neurons with KCl enhanced the association of Mayven with actin. This increased association resulted in dynamic changes in Mayven distribution from uniform to punctate localization along neuronal processes. These results suggest that Mayven functions as an actin-binding protein that may be translocated along axonal processes and might be involved in the dynamic organization of the actin cytoskeleton in brain cells.

Amino Acid Sequence↗

PubMed1999

Application of laser scanning confocal microscopy in the analysis of particle-induced pulmonary fibrosis.

Laser scanning confocal microscopy (LSCM) allows us to simultaneously quantitate the degree of lung fibrosis and distinguish various pathological lesions of intact lung tissue. Lucifer Yellow has been shown an ideal fluorescent stain to examine the connective tissue matrix components of embedded lung tissue with LSCM. We evaluated the use of LSCM in quantitating lung fibrosis and compared this procedure with the more traditional method of assessing fibrosis by measuring hydroxyproline, a biochemical assay of collagen. CD/VAF rats were intratracheally dosed with silica (highly fibrogenic), Fe2O3 (non-fibrogenic), and saline (vehicle control) at a high dose of 10-mg/100 g body weight. At 60 days post-instillation, the left lung was dissolved in 6 M HCl and assayed for hydroxyproline. Silica induced increases of 58% and 94% in hydroxyproline content over the Fe2O3 and control groups, respectively. The right lung lobes were fixed, sectioned into blocks, dehydrated, stained with Lucifer Yellow (0.1 mg/ml), and embedded in Spurr plastic. Using LSCM and ImageSpace software, the tissue areas of ten random scans from ten blocks of tissue for each of the three groups were measured, and three-dimensional reconstructions of random areas of lung were generated. The silica group showed increases of 57% and 60% in the lung areas stained by Lucifer Yellow over the Fe2O3 and control groups, respectively. Regression analysis of hydroxyproline vs. lung tissue area demonstrated a significant positive correlation (p < 0.05) with a correlation coefficient of 0.91. Histological analysis of right lung tissue revealed a marked degree of granulomatous interstitial pneumonitis for the silica group, which was absent in the Fe2O3 and control groups. No significant differences (p < 0.05) in hydroxyproline content and measured tissue area were observed between the Fe2O3 and control groups. LSCM, and its associated advanced image analysis and three-dimensional capabilities, is an alternative method to both quickly quantitate and examine fibrotic lung disease without physical disruption of the tissue specimen.

Animals↗

PubMed1999

Recombinant human Monocyte/Neutrophil elastase inhibitor protects rat lungs against injury from cystic fibrosis airway secretions.

Human monocyte/neutrophil elastase inhibitor (M/NEI) is a fast-acting stoichiometric inhibitor of neutrophil elastase (NE), cathepsin-G, and proteinase-3. Recombinant M/NEI (rM/NEI) was evaluated with a rat model of NE-induced lung damage. rM/NEI was found to protect against pulmonary injury caused by instilled human NE or by a preparation from airway secretions (sputum) of cystic fibrosis patients (CF sol). Human NE instilled into rat lungs produced dose-dependent hemorrhage and increased epithelial permeability, whereas NE incubated in vitro with rM/NEI did neither. Similarly, hemorrhage was induced by CF sol, but not by CF sol incubated in vitro with rM/NEI. To examine its distribution and survival time in airways, rM/NEI was labeled with the fluorochrome Texas Red (rM/NEI-TR) and instilled into rat lungs. Confocal microscopy showed that rM/NEI-TR could be detected on large airways (300 microm) at 5 min, 1 h, 4 h, and 24 h after instillation. Pretreating rats with rM/NEI was found to provide extended protection upon subsequent NE challenge, reducing hemorrhage by 98, 96, and 73%, respectively, at 1, 4, and 24 h after rM/NEI pretreatment. Pretreating rats with rM/NEI similarly conferred protection against subsequent exposure to CF sol, reducing hemorrhage by 95, 86, and 87%, respectively, at 1, 4, and 24 h after pretreatment. The findings that rM/NEI (1) mitigates protease-induced lung injury and (2) remains present and active in the lungs for 24 h after instillation strongly support its potential for treating patients with neutrophil protease-induced inflammatory lung damage, such as occurs in CF and other diseases.

Adult↗

PubMed1999

Alveolar cell stretching in the presence of fibrous particles induces interleukin-8 responses.

Inhalation of fibrous particulates is strongly associated with lung injury, but the molecular and cellular mechanisms that could explain the fiber-induced pathogenesis are not fully understood. We hypothesized that the physical stress exerted on the alveolar epithelium by the deposited fibers is greatly enhanced by the tidal cyclic motion of the epithelial cells that is associated with breathing, and that this initial mechanical interaction triggers a subsequent cell response. To test this hypothesis, we developed a dynamic model of fiber-induced cell injury using a cell-stretcher device. We exposed a cyclically stretched monolayer of the human alveolar epithelial cell line A549 to glass or crocidolite asbestos fibers for 8 h and then measured the production of the proinflammatory cytokine interleukin (IL)-8 as a readout of fiber-induced cell injury. Cyclic stretching significantly increased IL-8 production in the fiber-treated cultures, suggesting that the physical stress on the cells caused by the fibers was indeed enhanced by the motion. Coating of the asbestos fibers with fibronectin, a glycoprotein abundant in the alveolar lining fluid, further increased the fiber-induced cell response when the cells were cyclically stretched. This response was, however, significantly reduced by introducing into the culture medium, before fiber treatment, soluble RGD (Arg-Gly-Asp)-containing peptides, which specifically block binding to integrin receptors upon RGD attachment. These results suggested that adhesive interactions between protein-coated fibers and cell surface molecules are involved in the fiber-induced pathogenic process. Our novel findings indicate the importance of physical insults in fiber-induced cell stress, and bring to the forefront the need to study the mechanisms involved in this process.

Asbestos, Crocidolite↗

PubMed1999

In situ microscopic analysis of asbestos and synthetic vitreous fibers retained in hamster lungs following inhalation.

Hamsters breathed, nose-only, for 13 weeks, 5 days/week, 6 hr/day, either man-made vitreous fiber (MMVF)10a, MMVF33, or long amosite asbestos at approximately 300 World Health Organization (WHO) fibers/cc or long amosite at 25 WHO fibers/cc. [World Health Organization fibers are longer than 5 microm and thicker than 3 microm, with aspect ratio >3.] After sacrifice, fiber burden was estimated (left lungs) by ashing and scanning electron microscopy (ashing/SEM) or (right middle lobes) by confocal laser scanning microscopy (CLSM) in situ. In situ CLSM also provided three-dimensional views of fibers retained, undisturbed, in lung tissue. Fibers of each type were lodged in alveoli and small airways, especially at airway bifurcations, and were seen fully or partly engulfed by alveolar macrophages. Amosite fibers penetrated into and through alveolar septa. Length densities of fibers in parenchyma (total length of fiber per unit volume of lung) were estimated stereologically from fiber transsections counted on two-dimensional optical sections and were 30.5, 25.3, 20.0, and 81.6 mm/mm3 for MMVF10a, MMVF33, and low- and high-dose amosite, respectively. Lengths of individual fibers were measured in three dimensions by tracking individual fibers through series of optical sections. Length distributions of amosite fibers aerosolized, but before inhalation versus after retention in the lung were similar, whether determined by ashing/SEM or in situ CLSM. In contrast, the fraction of short MMVF10a and MMVF33 fibers increased and the geometric mean fiber lengths of both MMVFs decreased by approximately 60% during retention. Most likely due to fiber deposition pattern and differences in sampling, fiber burdens [MMVF10a, MMVF33, and amosite (high dose; 269 WHO fibers/cc)] determined by ashing/SEM were 1.4, 1. 5, and 3.5 times greater, respectively, than those calculated from in situ CLSM data. In situ CLSM is able to provide detailed information about the anatomic sites of fiber retention and also fiber lengths and burdens in good agreement with ashing/SEM results.

Administration, Inhalation↗

PubMed1998

Reduced binding and phagocytosis of Pneumocystis carinii by alveolar macrophages from persons infected with HIV-1 correlates with mannose receptor downregulation.

The macrophage mannose receptor, a pattern recognition molecule and component of innate immunity, mediates binding and phagocytosis of Pneumocystis carinii and likely represents an important clearance mechanism in the lungs of immunocompetent hosts. The purpose of this study was to examine the ability of alveolar macrophages from HIV-infected individuals to bind and phagocytose P. carinii, and to investigate the role of the macrophage mannose receptor in mediating this interaction. Compared with healthy individuals, alveolar macrophage phagocytosis of P. carinii from HIV+ persons was reduced up to 74% (P = 0.02), primarily reflecting a reduction in the number of organisms associated with each macrophage (P = 0.019). Furthermore, macrophages from HIV+ individuals demonstrated up to an 80% (P < 0.05) reduction in mannose receptor surface expression and endocytosis. Mannose receptor affinity was unaltered, and mRNA levels were modestly reduced (P < 0.05). Cells from HIV+ individuals with CD4(+) counts < 200 cells/mm3 (representing individuals at high clinical risk for P. carinii pneumonia) demonstrated the lowest levels of P. carinii phagocytosis and mannose receptor endocytosis. In vitro HIV infection of alveolar macrophages from healthy individuals reduced mannose receptor endocytosis to 53.2% (P < 0.05) and P. carinii binding and phagocytosis to 67.4% (P < 0.05) of control. Our studies suggest that HIV infection may alter innate immunity in the lungs, and that impaired alveolar macrophage mannose receptor-mediated binding and phagocytosis of P. carinii may contribute to the susceptibility of HIV-infected individuals to this opportunistic pulmonary pathogen.

Bronchoalveolar Lavage Fluid↗

PubMed1998

Shared antigenic epitopes on the V3 loop of HIV-1 gp120 and proteins on activated human T cells.

Proliferation of HIV-1 in the infected host is characterized by a progressive loss of CD4+ T lymphocytes and consequent dysregulation of the immune system. Both direct and indirect mechanisms have been proposed. We show here that proteins with molecular weights 35, 48, and 110 kDa on stimulated primary human T cells are recognized by neutralizing antibodies against the V3 loop of HIV-1 gp120. Recognition is specific since it can be blocked by a recombinant HIV-1 gp120. Furthermore, these V3 monoclonal antibodies, as well as sera from AIDS patients that recognized these V3-like proteins, induced killing of HIV-1-infected as well as uninfected T cells. This killing was also inhibited by HIV-1 gp120 V3 peptides. These results indicate that the V3 loop shares epitopes with proteins on stimulated T cells. This may be an additional autoimmune mechanism contributing to CD4+ T cell disappearance in AIDS. V3 antibodies have been proposed as potential prophylactic agents. However, if such vaccines were based on certain epitopes, they might induce cross-reacting immune responses with cellular proteins. Vaccine candidates should be evaluated for such potential effects.

Amino Acid Sequence↗

PubMed1998

A novel biotinylated degradable polymer for cell-interactive applications.

We describe the development of a novel biodegradable polymer designed to present bioactive motifs at the surfaces of materials of any architecture. The polymer is a block copolymer of biotinylated poly(ethylene glycol) (PEG) with poly(lactic acid) (PLA); it utilizes the high-affinity coupling of the biotin-avidin system to undergo postfabrication surface engineering. We show, using surface plasmon resonance analysis (SPR) and confocal microscopy that surface engineering can be achieved under aqueous conditions in short time periods. These surfaces interact with cell surface molecules and generate beneficial responses as demonstrated by the model study of integrin-mediated spreading of endothelial cells on polymer surfaces presenting RGD peptide adhesion sequences.

Animals↗