Dendritic cells (DCs) are essential antigen-presenting cells for the induction of T cell immunity against pathogens such as human immunodeficiency virus (HIV)-1. At the same time, HIV-1 replication ...is strongly enhanced in DC-T cell clusters, potentially undermining this process. We found that immature CD123(+) plasmacytoid DCs (PDCs) and CD11c(+) myeloid DCs (MDCs) were susceptible to both a CCR5- and a CXCR4-using HIV-1 isolate in vitro and were able to efficiently transfer that infection to autologous CD4(+) T cells. Soon after HIV-1 exposure, both PDCs and MDCs were able to transfer the virus to T cells in the absence of a productive infection. However, once a productive infection was established in the DCs, newly synthesized virus was predominantly spread to T cells. HIV-1 exposure of the MDCs and PDCs did not inhibit their ability to present cytomegalovirus (CMV) antigens and activate CMV-specific memory T cells. As a result, both PDCs and MDCs preferentially transmitted HIV-1 to the responding CMV antigen-specific CD4(+) T cells rather than to nonresponding T cells. This suggests that the induction of antigen-specific T cell responses by DCs, a process crucial to immune defense, can lead to preferential HIV-1 infection and the deletion of responding CD4(+) T cells.
Dendritic cells (DCs) are essential antigen-presenting cells for the induction of T cell immunity against pathogens such as human immunodeficiency virus (HIV)-1. At the same time, HIV-1 replication ...is strongly enhanced in DC-T cell clusters, potentially undermining this process. We found that immature CD123 super(+) plasmacytoid DCs (PDCs) and CD11c super(+) myeloid DCs (MDCs) were susceptible to both a CCR5- and a CXCR4-using HIV-1 isolate in vitro and were able to efficiently transfer that infection to autologous CD4 super(+) T cells. Soon after HIV-1 exposure, both PDCs and MDCs were able to transfer the virus to T cells in the absence of a productive infection. However, once a productive infection was established in the DCs, newly synthesized virus was predominantly spread to T cells. HIV-1 exposure of the MDCs and PDCs did not inhibit their ability to present cytomegalovirus (CMV) antigens and activate CMV-specific memory T cells. As a result, both PDCs and MDCs preferentially transmitted HIV-1 to the responding CMV antigen-specific CD4 super(+) T cells rather than to nonresponding T cells. This suggests that the induction of antigen-specific T cell responses by DCs, a process crucial to immune defense, can lead to preferential HIV-1 infection and the deletion of responding CD4 super(+) T cells.
Plasminogen binding to cell surfaces may be important for tumor invasion and other processes that involve cellular migration. In this investigation, the principal plasminogen-binding protein was ...identified in the plasma membrane fraction of rat hepatocytes. The protein had an apparent mass of 59 kDa, was insoluble in a spectrum of detergents, and was identical to cytokeratin 8 (CK 8) as determined by sequence analysis of nine amino acids at the N terminus of two cyanogen bromide fragments. The 59 kDa protein bound CK 8-specific antibody in western blot analyses. These studies demonstrate that CK 8 or a CK 8-like protein binds plasminogen. Given this newly determined and potentially important CK 8 function, immunofluorescence and immunoelectron microscopy studies were performed to determine whether CK 8 may be present on the external surfaces of unpermeabilized, viable hepatocytes. All of the cells in each preparation were immunopositive with two separate CK 8-specific antibodies. A punctate pattern of immunofluorescence was detected on the cell surface with approximately even intensity from cell to cell. By immunoelectron microscopy, CK 8 was preferentially associated with microvilli. In order to determine whether other epithelial cells express cell-surface CK 8, immunofluorescence and immunoelectron microscopy studies were performed with HepG2 hepatocellular carcinoma cells and with BT20 and MCF-7 breast carcinoma cells. The pattern of antigen expression was equivalent with each cell type and comparable to that observed with hepatocytes. These studies support the hypothesis that CK 8 is associated with the external cell surface where it may express important proteinase receptor function.
Thesis (Ph. D.)--University of Virginia, 2003.
Includes bibliographical references (leaves 501-511). Also available online through Digital Dissertations.
Following HIV-1 infection in a new host and during the asymptomatic phase of infection, the viral species that predominate use CCR5 as a coreceptor and are called R5 HIV-1. During progression to ...disease, the virus switches to using CXCR4 in addition to, or rarely, instead of CCR5 and is called R5X4 (dual tropic) or X4 tropic. We used an HIV-1 luciferase vector pseudotyped with X4 tropic HXB-2 or an R5X4 patient isolate envelope protein, to transduce COS-CD4 cells transfected with chimp, rhesus macaque, African green monkey, rat and human CXCR4. HXB-2 envelope protein allowed the vector to effectively utilize CXCR4 from all species. The virus pseudotyped with the dual tropic envelope clones showed greater sensitivity to variability in CXCR4 and did not utilize rat CXCR4 or a chimeric human-rat CXCR4 molecule as effectively as virus pesudotyped with HXB-2 envelope protein. This suggests that the regions that most affect the binding of this dual tropic HIV-1 envelope, lie in the carboxy-terminal portion of CXCR4. We hypothesize that a charge-mediated interaction may play a role in overcoming the loss of specific contacts between envelope protein and the receptor that arise from sequence dissimilarities between human and rat CXCR4. This would allow the X4 tropic, HXB-2 virus to broaden its coreceptor range. We also studied the role of signaling through CCR5 in the establishment of HIV-1 infection. In resting memory CCR5 Δ32/Δ32 CD4 positive T cells, only a transduced, signaling proficient, CCR5 was able to support detectable replication by R5 HIV-1. Infection of normal resting memory CD4 positive T cells with R5 HIV-1 was inhibited in the presence of the PI3K inhibitor, wortmannin, and the Src inhibitor PP2. The Gα i inhibitor pertussis toxin aad the Gαs inhibitor NF449 did not prevent viral replication. These data suggest that the inability of HIV-1 to infect resting memory T cells can be overcome by signaling through CCR5 by R5 HIV-1. This may be a crucial step in establishing HIV-1 infection in a healthy host where most of the CD4 positive cells are resting. Moreover, it may explain the selection and exclusive maintenance of R5 HIV-1, following transmission and during the asymptomatic period.
Nerve growth factor-gamma (NGF-gamma) is a serine proteinase which reversibly associates with the well characterized neurotrophin
NGF-beta. In this study, we demonstrated that NGF-gamma cleaves ...recombinant single chain urokinase-type plasminogen activator
(scu-PA), converting the zymogen into a two-chain form (tcu-PA). The apparent masses of the two u-PA chains were 33 and 22
kDa, as determined by SDS-polyacrylamide gel electrophoresis (PAGE). There was no evidence for secondary cleavage sites or
further digestion of tcu-PA by NGF-gamma, even when conversion of scu-PA was complete. The NH2-terminal sequence of the 33-kDa
band was Ile-Ile-Gly-Gly-Glu, indicating that NGF-gamma cleaved scu-PA at Lys158-Ile159, the plasmin cleavage site. Cleavage
of scu-PA by NGF-gamma resulted in scu-PA activation. The kcat and Km for this reaction, as determined in a continuous assay
with the tcu-PA-specific substrate L-pyroglutamyl-glycyl-arginine-p-nitroanilide hydrochloride (S-2444), were (4.1 +/- 0.6)
x 10(-2) s-1 and 2.3 +/- 0.4 microM, respectively. The catalytic efficiency (kcat/Km) for scu-PA activation by NGF-gamma was
1.3 x 10(4) M-1 s-1, compared with 6.2 x 10(5) M-1 s-1 for the activation of scu-PA by plasmin. NGF-gamma-cleaved scu-PA which
was bound to receptors on U937 monocytoid cells. The apparent masses of the resulting u-PA cleavage products were identical
to those generated in solution as determined by SDS-PAGE. Cell-associated scu-PA was activated by NGF-gamma, as determined
by the generation of activity against the tcu-PA-specific fluorogenic substrate, glutamyl-glycyl-arginine-7-amino-4-methyl
coumarin. By activating scu-PA, NGF-gamma may initiate the u-PA-dependent cell-surface proteinase cascade and support NGF-beta
activities which involve cellular migration and/or extracellular matrix remodeling.
Summary
Anisoylated Lys-plasminogen streptokinase activator complex (APSAC) was purified from Eminase
®
by chromatography on Superose-12. Purified APSAC did not significantly deacylate within 4 h at ...4° C in solution as determined by hydrolysis of D-Val-L-leu-L-lys-p-nitroanilide HCl (S-2251). At 37° C, maximum amidase activity developed in 120 min; ε-amino-n-caproic acid (EACA) did not affect the apparent rate of APSAC deacylation but stabilized the streptokinase-plasmin(ogen) complex (SkPl) which formed. APSAC bound to C6 glioma cells and human umbilical vein endothelial cells (HUVECs) in culture. Binding was completely inhibited by EACA suggesting an essential role for the plasminogen kringle domains. Cell-associated APSAC deacylated to form active SkPl which hydrolyzed S-2251 and D-Val-Leu-Lys-7-amino-4-methyl coumarin. The rate of APSAC deacylation was increased when the APSAC was cell-associated. APSAC that was initially bound to C6 cells or HUVECs also activated
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I-plasminogen. This activity may have reflected cell-associated APSAC or APSAC that dissociated into solution. Plasmin was recovered bound to cells and in solution. These studies demonstrate that APSAC associates with cell-surfaces and retains activity. In the circulation, cell-surfaces may provide a significant pharmacologic compartment for intravenously administered APSAC.
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