Human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs

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.

methyl ester (NALME) is a lysine analogue that reportedly binds to low-affinity lysine binding sites in plasmin(ogen) and miniplasmin(ogen). In the studies presented here, we show that NALME has antifibrinolytic activity; however, unlike the therapeutic agents e-amino-n-caproic acid (eACA) and tranexamic acid (TEA), the activity of NALME is based on inhibition of the plasmin active site. NALME (0.1-10 mM) significantly inhibited the amidase activity of plasmin, miniplasmin, and streptokinase-plasmin complex without affecting er-thrombin or tissue plasminogen activator. eACA and TEA (0.1-10 mM) did not affect the amidase activity of plasmin or miniplasmin. A kinetic analysis showed that NALME is a competitive inhibitor of D-Val-L-Leu-L-Lys-p-nitroanilide HC1 (S-2251) hydrolysis by plasmin; NALME binding to plasmin completely prevented S-2251 binding. The K, for the plasmin-NALME interaction was 0.4 mM. eACA and TEA inhibited fibrin monomer digestion by plasmin and miniplasmin without binding to the active site of either enzyme. This result suggests that eACA and TEA function as antifibrinolytics by disrupting the noncovalent association of fibrin monomer with a domain common to both plasmin and miniplasmin (probably kringle 5). NALME inhibited fibrin monomer digestion principally by decreasing amidase activity. NALME was the only lysine analogue that prevented fragment X formation; TEA and eACA primarily inhibited the formation of fragments Y and D. When plasmin was incubated simultaneously with o^-antiplasmin and a 2 -macroglobulin, eACA increased the fraction of plasmin reacting with o^-macroglobulin; NALME had no effect on the plasmin distribution. eACA, TEA, and NALME increased the euglobulin clot lysis time of normal plasma. NALME did not prolong the prothrombin time or activated partial thromboplastin time. These studies demonstrate that the antifibrinolytic activity of NALME is based on inhibition of the plasmin active site, whereas eACA and TEA are active due to kringle domain interactions. ( I n the circulation, fibrinolysis and fibrinogenolysis are mediated primarily by plasmin, the active serine proteinase counterpart of the zymogen plasminogen. 1 ' 2 The intact structure of plasminogen includes 791 amino acids with an Af-terminal glutamic acid 2 " 5 and either one or two oligosaccharide chains. 6 -8 Plasminogen activation results from the cleavage of the Arg^-Val 56 ' peptide bond. 9 Low concentrations of plasmin cleave [Glu'J-plasminogen to form [Lys 78 ]-plasminogen, a second more readily activatable form of the zymogen. 10 Plasminogen activation and the subsequent function of plasmin are modulated by noncovalent binding inter-

Plasmin regulates the activity and distribution of transforming growth factor beta (TGF-beta) and other growth factors. The purpose of the present investigation was to determine the effects of plasmin on cellular receptors for TGF-beta. AKR-2B fibroblasts were affinity-labelled with 125I-TGF-beta 1 and 125I-TGF-beta 2, demonstrating betaglycan, the type-I TGF-beta receptor and the type-II TGF-beta receptor. Treatment of TGF-beta-affinity-labelled cells with plasmin (10-100 nM) for 1 h profoundly and selectively decreased recovery of TGF-beta-betaglycan complex. The type-I and type-II receptors were not plasmin substrates. A radiolabelled complex with an apparent mass of 60 kDa was detected by SDS/PAGE in both the medium and cell extracts of plasmin-treated affinity-labelled cells. In order to demonstrate that plasmin cleavage of betaglycan did not require prior exposure of the betaglycan to cross-linking agent, AKR-2B cells were treated with plasmin first and then affinity-labelled. Markedly decreased TGF-beta binding to cellular betaglycan was observed. Although plasmin treatment of AKR-2B cells decreased overall binding of 125I-TGF-beta 1 and 125I-TGF-beta 2, the rate at which the cells degraded bound 125I-TGF-beta at 37 degrees C was not changed. AKR-2B cells treated with plasmin demonstrated slightly increased [3H]thymidine incorporation; the plasmin-treated cells retained their ability to respond to TGF-beta. Conditioned medium from plasmin-treated AKR-2B cells contained increased amounts of active TGF-beta as determined in Mv 1 Lu epithelial-cell-proliferation assays. Specific cleavage of betaglycan represents a novel mechanism whereby plasmin may regulate the assortment of receptors available for TGF-beta. In addition, plasmin may facilitate transfer of active TGF-beta between neighbouring cells by releasing the active growth factor from the cell surface.