Immune interferon and leukocyte-conditioned medium induce normal and leukemic myeloid cells to differentiate along the monocytic pathway.

Conditioned medium from phytohemagglutinin-stimulated human leukocytes contains a factor that can induce promyelocytic cell lines and certain acute myelogenous leukemia cells to differentiate along the monocytic pathway. In this report, we show that immature myeloid cells from normal bone marrow or the peripheral blood of patients with chronic myelogenous leukemia can be induced to differentiate to monocyte-like cells by immune gamma interferon (IFN gamma). We have identified IFN gamma as the predominant differentiation factor contained in the conditioned medium. Purified or recombinant IFN gamma, but not various preparations of IFN alpha or beta, can induce monocytic differentiation in myeloid cells. In cultures containing conditioned medium, the cells fail to continue myeloid maturation, and are induced to express monocyte markers and functions, such as monocyte-specific surface antigens, HLA-DR antigens, Fc receptors for monomeric immunoglobulins, nonspecific esterase, and the ability to mediate antibody-dependent, cell-mediated cytotoxicity. Even myeloid cells as mature as metamyelocytes or band cells can be induced by IFN gamma to undergo monocyte differentiation, but monocyte-specific or HLA-DR antigens are not induced in mature neutrophils. These findings reveal a previously unknown, specific function of human IFN gamma and offer new insights to the regulation of monocyte recruitment and differentiation during a virus infection or immune response.

Transendothelial migration of megakaryocytes in response to stromal cell-derived factor 1 (SDF-1) enhances platelet formation.

Although thrombopoietin has been shown to promote megakaryocyte (MK) proliferation and maturation, the exact mechanism and site of platelet formation are not well defined. Studies have shown that MKs may transmigrate through bone marrow endothelial cells (BMEC), and release platelets within the sinusoidal space or lung capillaries. In search for chemotactic factor(s) that may mediate transmigration of MKs, we have discovered that mature polyploid MKs express the G protein-coupled chemokine receptor CXCR4 (Fusin, LESTR). Therefore, we explored the possibility that stromal cell-derived factor 1 (SDF-1), the ligand for CXCR4, may also induce transendothelial migration of mature MKs. SDF-1, but not other CXC or CC chemokines, was able to mediate MK migration (ED50 = 125 pmol/liter). The MK chemotaxis induced by SDF-1 was inhibited by the CXCR4-specific mAb (12G5) and by pertussis toxin, demonstrating that signaling via the G protein-coupled receptor CXCR4 was necessary for migration. SDF-1 also induced MKs to migrate through confluent monolayers of BMEC by increasing the affinity of MKs for BMEC. Activation of BMEC with interleukin 1beta resulted in a threefold increase in the migration of MKs in response to SDF-1. Neutralizing mAb to the endothelial-specific adhesion molecule E-selectin blocked the migration of MKs by 50%, suggesting that cellular interaction of MKs with BMEC is critical for the migration of MKs. Light microscopy and ploidy determination of transmigrated MKs demonstrated predominance of polyploid MKs. Virtually all platelets generated in the lower chamber also expressed CXCR4. Platelets formed in the lower chamber were functional and expressed P-selectin (CD62P) in response to thrombin stimulation. Electron microscopy of the cells that transmigrated through the BMEC monolayers in response to SDF-1 demonstrated the presence of intact polyploid MKs as well as MKs in the process of platelet formation. These results suggest that SDF-1 is a potent chemotactic factor for mature MKs. Expression of CXCR4 may be the critical cellular signal for transmigration of MKs and platelet formation.

Productive infection of neonatal CD8+ T lymphocytes by HIV-1.

CD8+ T lymphocytes confer significant but ultimately insufficient protection against HIV infection. Here we report that activated neonatal CD8+ T cells can be productively infected in vitro by macrophage-tropic (M-tropic) HIV-1 isolates, which are responsible for disease transmission, whereas they are resistant to T cell-tropic (T-tropic) HIV strains. Physiological activation of CD8-alpha/beta+ CD4- T cell receptor-alpha/beta+ neonatal T cells, including activation by allogeneic dendritic cells, induces the accumulation of CD4 messenger RNA and the expression of CD4 Ag on the cell surface. The large majority of anti-CD3/B7.1-activated cord blood CD8+ T cells coexpress CD4, the primary HIV receptor, as well as CCR5 and CXCR4, the coreceptors used by M- and T-tropic HIV-1 strains, respectively, to enter target cells. These findings are relevant to the rapid progression of neonatal HIV infection. Infection of primary HIV-specific CD8+ T cells may compromise their survival and thus significantly contribute to the failure of the immune system to control the infection. Furthermore, these results indicate a previously unsuspected level of plasticity in the neonatal immune system in the regulation of CD4 expression by costimulation.

Roles for endocytosis in lentiviral replication.

Endocytosis is essential for the entry of many viruses into cells. The primate lentiviruses [human immunodeficiency virus (HIV) 1 and 2, and the simian immunodeficiency viruses (SIVs)], however, use endocytosis in other aspects of their life cycles. Here, the authors describe the ways in which the endocytic pathway is used by HIV and SIV and discuss the mechanisms through which endocytosis may contribute to the pathogenic properties of these viruses.

An internalization signal in the simian immunodeficiency virus transmembrane protein cytoplasmic domain modulates expression of envelope glycoproteins on the cell surface.

A Tyr to Cys mutation at amino acid position 723 in the cytoplasmic domain of the simian immunodeficiency virus (SIV) transmembrane (TM) molecule has been shown to increase expression of envelope glycoproteins on the surface of infected cells. Here we show that Tyr-723 contributes to a sorting signal that directs the rapid endocytosis of viral glycoproteins from the plasma membrane via coated pits. On cells infected by SIVs with a Tyr at position 723, envelope glycoproteins were transiently expressed on the cell surface and then rapidly endocytosed. Similar findings were noted for envelope molecules expressed in the absence of other viral proteins. Immunoelectron microscopy demonstrated that these molecules were localized in patches on the cell surface and were frequently associated with coated pits. In contrast, envelope glycoproteins containing a Y723C mutation were diffusely distributed over the entire plasma membrane. To determine if an internalization signal was present in the SIV TM, chimeric molecules were constructed that contained the CD4 external and membrane spanning domains and a SIV TM cytoplasmic tail with a Tyr or other amino acids at SIV position 723. In Hela cells stably expressing these molecules, chimeras with a Tyr-723 were rapidly endocytosed, while chimeras containing other amino acids at position 723, including a Phe, were internalized at rates only slightly faster than a CD4 molecule that lacked a cytoplasmic domain. In addition, the biological effects of the internalization signal were evaluated in infectious viruses. A mutation that disrupted the signal and as a result, increased the level of viral envelope glycoprotein on infected cells, was associated with accelerated infection kinetics and increased cell fusion during viral replication. These results demonstrate that a Tyr-dependent motif in the SIV TM cytoplasmic domain can function as an internalization signal that can modulate expression of the viral envelope molecules on the cell surface and affect the biological properties of infectious viruses. The conservation of an analogous Tyr in all human and simian immunodeficiency viruses suggests that this signal may be present in other primate lentiviruses and could be important in the pathogenesis of these viruses in vivo.

Phorbol esters and SDF-1 induce rapid endocytosis and down modulation of the chemokine receptor CXCR4.

The chemokine receptor CXCR4 is required, together with CD4, for entry by some isolates of HIV-1, particularly those that emerge late in infection. The use of CXCR4 by these viruses likely has profound effects on viral host range and correlates with the evolution of immunodeficiency. Stromal cell-derived factor-1 (SDF-1), the ligand for CXCR4, can inhibit infection by CXCR4-dependent viruses. To understand the mechanism of this inhibition, we used a monoclonal antibody that is specific for CXCR4 to analyze the effects of phorbol esters and SDF-1 on surface expression of CXCR4. On human T cell lines SupT1 and BC7, CXCR4 undergoes slow constitutive internalization (1.0% of the cell surface pool/min). Addition of phorbol esters increased this endocytosis rate >6-fold and reduced cell surface CXCR4 expression by 60 to 90% over 120 min. CXCR4 was internalized through coated pits and coated vesicles and subsequently localized in endosomal compartments from where it could recycle to the cell surface after removal of the phorbol ester. SDF-1 also induced the rapid down modulation (half time approximately 5 min) of CXCR4. Using mink lung epithelial cells expressing CXCR4 and a COOH-terminal deletion mutant of CXCR4, we found that an intact cytoplasmic COOH-terminal domain was required for both PMA and ligand-induced CXCR4 endocytosis. However, experiments using inhibitors of protein kinase C indicated that SDF-1 and phorbol esters trigger down modulation through different cellular mechanisms. SDF-1 inhibited HIV-1 infection of mink cells expressing CD4 and CXCR4. The inhibition of infection was less efficient for CXCR4 lacking the COOH-terminal domain, suggesting at least in part that SDF-1 inhibition of virus infection was mediated through ligand-induced internalization of CXCR4. Significantly, ligand induced internalization of CXCR4 but not CD4, suggesting that CXCR4 and CD4 do not normally physically interact on the cell surface. Together these studies indicate that endocytosis can regulate the cell-surface expression of CXCR4 and that SDF-1-mediated down regulation of cell-surface coreceptor expression contributes to chemokine-mediated inhibition of HIV infection.

Persistent noncytopathic infection of normal human T lymphocytes with AIDS-associated retrovirus.

Infection of normal peripheral blood T cells by the acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV) was evaluated in long-term cultures of helper-inducer T cells (T4 cells). Cells that were inoculated with ARV and maintained in medium supplemented with interleukin-2 remained productively infected with this virus for more than 4 months in culture, although they showed no cytopathic effects characteristic of acute ARV infection. The presence of replicating virus was demonstrated by reverse transcriptase activity of culture fluids and by viral antigens and budding particles detected on cells by immunofluorescence and electron microscopy. Virus produced in these cultures remained infectious and could induce cytopathic effects and viral antigens in uninfected lymphoid cells. The finding that normal lymphocytes may be productively infected by an AIDS retrovirus in the absence of cell death suggests that a range of biologic effects may occur after infection in vivo.

Alterations in T4 (CD4) protein and mRNA synthesis in cells infected with HIV.

Cells infected with the human immunodeficiency virus (HIV) show decreased expression of the 58-kilodalton T4 (CD4) antigen on their surface. In this study, the effect of HIV infection on the synthesis of T4 messenger RNA (mRNA) and protein products was evaluated in T-cell lines. Metabolically labeled lysates from the T4+ cell line Sup-T1 were immunoprecipitated with monoclonal antibodies to T4. Compared with uninfected cells, HIV-infected Sup-T1 cells showed decreased amounts of T4 that coprecipitated with both the 120-kilodalton viral envelope and the 150-kilodalton envelope precursor molecules. In four of five HIV-producing T-cell lines studied, the steady-state levels of T4 mRNA were also reduced. Thus, the decreased T4 antigen on HIV-infected cells is due to at least three factors: reduced steady-state levels of T4-specific mRNA, reduced amounts of immunoprecipitable T4 antigen, and the complexing of available T4 antigen with viral envelope gene products. The data suggested that the T4 protein produced after infection may be complexed with viral envelope gene products within infected cells. Retroviral envelope-receptor complexes may thus participate in a general mechanism by which receptors for retroviruses are down-modulated and alterations in cellular function develop after infection.

Targeting of HIV- and SIV-infected cells by CD4-chemokine receptor pseudotypes.

Retroviral vectors containing CD4 and an appropriate chemokine receptor were evaluated for the ability to transduce cells infected with human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). These CD4-chemokine receptor pseudotypes were able to target HIV- and SIV-infected cell lines and monocyte-derived macrophages in a manner that corresponded to the specificity of the viral envelope glycoprotein for its CD4-chemokine receptor complex. This approach could offer a way to deliver antiviral genes directly to HIV-infected cells in vivo and could provide an additional treatment strategy in conjunction with existing antiviral therapies.

West African HIV-2-related human retrovirus with attenuated cytopathicity.

Clinical and seroepidemiological studies in West Africa indicate that human immunodeficiency virus type 2 (HIV-2) is widespread and associated with immunodeficiency states of variable degree. In this study, an isolate of HIV-2 from a patient in Senegal was molecularly cloned and characterized. This isolate (HIV-2ST) was shown by hybridization and restriction enzyme analysis to be more related to the prototype HIV-2ROD than to other human or primate retroviruses. Cultures of HIV-2ST showed genotypic polymorphism, and clones of the virus had transmembrane envelope glycoproteins of 30 and 42 kilodaltons. Unlike other immunodeficiency viruses, HIV-2ST did not cause cell death or induce cell fusion in peripheral blood lymphocytes or in any of four CD4+ cell lines tested. Although HIV-2ST entered cells by a CD4-dependent mechanism and replicated actively, cell-free transmission of the virus was retarded at the level of cell entry. These findings suggest that immunodeficiency viruses prevalent in West African populations are members of the HIV-2 virus group and that certain strains of this virus have attenuated virulence.

Neuronal apoptosis induced by HIV-1 gp120 and the chemokine SDF-1 alpha is mediated by the chemokine receptor CXCR4.

CXCR4, a seven transmembrane domain G-protein-coupled receptor for the Cys-X-Cys class of chemokines, is one of several chemokine receptors that can act as a co-receptor with CD4 for the human immunodeficiency virus (HIV-1) glycoprotein gp120 [1-3]. CXCR4 can mediate the entry of HIV-1 strains that specifically infect T cells, such as the IIB strain (see [4] for review). Recent reports indicate that gp120 can signal through CXCR4 [5] and it has been suggested that signal transduction, mediated by the viral envelope, might influence viral-associated cytopathicity or apoptosis [6]. Neuronal apoptosis is a feature of HIV-1 infection in the brain [7,8], although the exact mechanism is unknown. Here, we address the possible role of CXCR4 in inducing apoptosis using cells of the hNT human neuronal cell line; these cells resemble immature post-mitotic cholinergic neurons and have a number of neuronal characteristics [9-15]. We have previously shown that gp120 from the HIV-1 IIIB strain binds with high affinity to CXCR4 expressed on hNT neurons [15]. We now find that both IIIB gp120 and the Cys-X-Cys chemokine SDF-1 alpha can directly induce apoptosis in hNT neurons in the absence of CD4 and in a dose-dependent manner. To our knowledge, this is the first report of a chemokine and an HIV-1 envelope glycoprotein eliciting apoptotic responses through a chemokine receptor.

CD4-independent association between HIV-1 gp120 and CXCR4: functional chemokine receptors are expressed in human neurons.

BACKGROUND: Chemokines are a family of proteins that chemoattract and activate immune cells by interacting with specific receptors on the surface of their targets. We have shown previously that chemokine receptors including the interleukin-8 receptor B (CXCR2) and the Duffy blood group antigen are expressed on subsets of neurons in various regions of the adult nervous system. RESULTS: Using a combination of immunohistochemical staining and receptor binding studies, we show that hNT cells, which are differentiated human neurons derived from the cell line NTera2, express functional chemokine receptors of the C-X-X and C-C types. These chemokine receptors include CXCR2, CXCR4, CCR1 and CCR5. We demonstrate high-affinity binding of both types of chemokines to hNT neurons and dose-dependent chemotactic responses to these chemokines in differentiated, but no t undifferentiated, NTera 2 cells. In addition, we show that the envelop glycoprotein from the T-cell-tropic human immunodeficiency virus 1 (HIV-1) strain IIIB is a CD4-independent, dose-dependent inhibitor of the binding of stromal cell-derived factor 1 to its receptor, CXCR4. CONCLUSIONS: These data support recent findings that members of the chemokine family, including CCR5 and LESTR/Fusin (CXCR4), function as coreceptors in combination with CD4 for HIV-1 invasion. This is the first report of functional expression of chemokine receptors on human neurons. Furthermore, our studies provide for direct CD4-independent association of the viral envelope protein of the HIV-1 strain III with the chemokine receptor CXCR4.

Phorbol ester modulation of T-cell antigens in the Jurkat lymphoblastic leukemia cell line.

The effect of phorbol dibutyrate (PDB) on the cell surface antigens of the human T-cell acute lymphoblastic leukemia cell line, Jurkat, was studied with OKT monoclonal antibodies by indirect immunofluorescence assay. Cells were analyzed in an Ortho Spectrum III fluorescence-activated flow cytometer. The surface antigen profile of untreated Jurkat cells resembled that of thymocytes; high levels of T3, T4, T6, T8, T9, T10, and T11 antigens were detected. Although 89% of cells were positive for T11, the putative sheep erythrocyte receptor, only 12% were able to form erythrocyte (sheep) rosettes. Exposure of the cells to 1.0 microM PDB for up to 7 days resulted in a rapid loss in T4 expression and a slower decrease in T6 reactivity, while the percentage of cells positive for T3, T8, T10, and T11 remained high. T4 reappeared on the cell surface when PDB was removed by washing. T11 antigen density increased 70%, and this was accompanied by an increase in the percentage of erythrocyte-rosetting cells from 12 to 55%. These changes in cell surface antigens induced by PDB suggested differentiation to a more mature state (i.e., a precursor cytotoxic-suppressor T-lymphocyte, T3+T8+T10+T11+). However, the reversibility of the change in T4 expression indicated that T4 loss was not a manifestation of terminal differentiation but rather was consistent with a phorbol ester-induced modulation of the cell surface T4 antigen.

Differential effects of phorbol esters on normal myeloid precursors and leukemic cells: basis for autologous bone marrow reconstitution in acute nonlymphocytic leukemia using phorbol ester-treated bone marrow from patients in remission.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces macrophage-like differentiation of HL60 cells and cells from patients with acute nonlymphocytic leukemia (ANLL). We assessed the use of TPA as a means of eradicating residual leukemia from remission bone marrow prior to autologous bone marrow reconstitution. A 30-min incubation with TPA led to marked growth arrest in HL60 cells and in cells from most patients with acute myelogenous leukemia and acute myelomonocytic leukemia, whereas cells from most patients with acute promyelocytic leukemia and acute undifferentiated leukemia demonstrated a lesser degree of growth arrest. Freezing and thawing, a necessary step in autologous reconstitution, had no effect on the cessation of proliferation induced in HL60 or ANLL cells preincubated with TPA for 30 min. Virtually normal myeloid precursor growth occurred in normal or remission bone marrow cells preincubated with TPA and then frozen and thawed. Based on these observations, two patients with advanced ANLL in remission underwent marrow ablative therapy followed by autologous reconstitution using TPA-treated bone marrow. Limited normal hematopoiesis was reestablished in both patients, although they subsequently experienced leukemic relapse. These studies demonstrate that in ANLL cells, TPA stimulates growth arrest; in contrast, hematopoiesis is able to proceed both in vitro and in vivo.

Pre-B-cell leukemia with a t(8; 14) and a t(14; 18) translocation is preceded by follicular lymphoma.

We have performed gene rearrangement studies on the leukemic blasts of a patient with acute pre-B-cell leukemia. The patient had a 5 year history of follicular lymphoma, which developed into acute pre-B-cell leukemia. The leukemic blasts revealed a karyotype with two translocations, t(8; 14) and t(14; 18), characteristic for Burkitt's lymphoma and follicular lymphoma. The cells are TdT positive, do not possess surface immunoglobulin, and they show immunoglobulin gene rearrangement. The mu heavy chain and kappa light chain constant (C mu and C kappa) loci are deleted, while the gamma and lambda light chain constant (C gamma and C lambda) region genes are rearranged. Both alleles of the heavy chain joining segment (JH) are rearranged on chromosome 14q+, one of them with the bcl-2 oncogene from chromosome 18. The breakpoint of the t(14; 18) translocation occurs in the major breakpoint cluster region in the 3' untranslated region of bcl-2. On chromosome 8 a c-myc rearrangement was mapped immediately 5' to the c-myc first exon in a region involved in sporadic Burkitt lymphoma. The data are consistent with our previous hypothesis on the evolution of B-cell malignancies: a rare pre-B cell develops a t(14; 18) translocation during immunoglobulin VDJ joining that results in an expansion of a follicular lymphoma clone carrying an activated bcl-2 gene. Within the clone of pre-B cells a second translocation, t(8; 14), occurs during heavy chain isotype switching that results in the deregulation of the c-myc involved in the translocation.

The severe combined immunodeficient (SCID) mouse as a model for human myeloid leukemias.

Recent work has demonstrated the ability of lymphoblastic leukemias of pre-B- and T-cell origin to grow in severe combined immunodeficient (SCID) mice with a pattern reminiscent of the human clinical disease. Here, we investigated the possibility of engrafting human myeloid leukemias using both established cell lines and primary patient material. Whereas the two growth factor-independent cell lines K562 and U937 grew aggressively and induced leukemia in these animals, three other myeloid cell lines which require interleukin 3 or granulocyte-macrophage colony-stimulating factor for continuous growth in vitro failed to induce disease. Primary bone marrow and peripheral blood cells from five out of seven patients with different types of myeloid leukemias (undifferentiated, megakaryoblastic, monoblastic and chronic myelogenous leukemia in blast crisis) induced patterns of leukemic infiltration that were distinct for each leukemia subtype. The diagnosis of leukemia in SCID mice was established by microscopic detection of myeloblasts in the bone marrow, peripheral blood and, in some instances, in extramedullary sites, including the central nervous system and gonads. The karyotype and phenotype of the blasts recovered from mouse tissues were identical to those of the original patient cells. Moreover, human specific ALU sequences were amplified from the bone marrow DNA by polymerase chain reaction. Despite their ability to grow in vivo by serial transfers in SCID mice, the leukemic cells recovered from mouse tissues could not be maintained in vitro, even in the presence of recombinant cytokines. Overall, these data indicate that the SCID mouse may represent a useful animal model for human myeloid leukemias and for the development of new pharmacological and molecular approaches to therapy.

CXCR4 on human endothelial cells can serve as both a mediator of biological responses and as a receptor for HIV-2.

It has been shown that deletion of the chemokine receptor, CXCR4, causes disordered angiogenesis in mouse models. In the present studies, we examined the distribution and trafficking of CXCR4 in human endothelial cells, tested their responses to the CXCR4 ligand, SDF-1, and asked whether endothelial cell CXCR4 can serve as a cell surface receptor for the binding of viruses. The results show that CXCR4 is present on endothelial cells from coronary arteries, iliac arteries and umbilical veins (HUVEC), but expression was heterogeneous, with some cells expressing CXCR4 on their surface, while others did not. Addition of SDF-1 caused a rapid decrease in CXCR4 surface expression. It also caused CXCR4-mediated activation of MAPK, release of PGI(2), endothelial migration, and the formation of capillary-like structures by endothelial cells in culture. Co-culture of HUVEC with lymphoid cells that were chronically infected with a CD4-independent/CXCR4-tropic variant of HIV-2 resulted in the formation of multinucleated syncytia. Formation of the syncytia was inhibited by each of several different CXCR4 antibodies. Thus, our findings indicate: (1) that CXCR4 is widely expressed on human endothelial cells; (2) the CXCR4 ligand, SDF-1, can evoke a wide variety of responses from human endothelial cells; and (3) CXCR4 on endothelial cells can serve as a receptor for isolates of HIV that can utilize chemokine receptors in the absence of CD4.

Trafficking thrombin receptors Biodiversity on the receptor superhighway.

In the past several years, the identification of the human thrombin receptor has permitted considerable progress to be made in the understanding of the ways in which thrombin activates cells. To date, only a single receptor for thrombin has been identified: a member of the G protein-coupled family of receptors that has proved to be a proteolytic substrate for thrombin. Cleavage of the receptor enables it to activate, but also leaves it in a state in which it is unable to respond to thrombin a second time. This review examines the variety of cellular response mechanisms to thrombin, and the growing evidence for diversity among cells in the processes that remove and replace cleaved thrombin receptors, issues that are central to the development of therapeutically useful thrombin receptor antagonists.

Monoclonal anti-idiotypic antibodies that mimic the epitope on gp120 defined by anti-HIV-1 monoclonal antibody 0.5 beta.

OBJECTIVE: To develop effective, specific and safe anti-idiotypic antibody (Ab2) vaccines against HIV-1. DESIGN: Murine monoclonal Ab2 were generated against anti-HIV-1 antibody 0.5 beta (Ab1), which binds to gp120, neutralizes HIV-1 and inhibits virus-induced syncytia formation. METHODS: Mice were immunized with Ab1, and Ab2 were produced from immunized mice by the hybridoma technique. The Ab2 were characterized in vitro, injected into rabbits, and the anti-anti-idiotypes (Ab3) induced in the rabbits were analyzed for binding and antiviral reactivities by enzyme-linked immunosorbent assay, p24gag release and syncytia formation assays. RESULTS: Seven Ab2 bound to the antigen-combining site of Ab1, one of which (UD7) induced Ab3 in rabbits that were Ab1-like in their binding reactivities to PB1 (recombinant gp120 fragment) or peptides of gp120, and shared idiotypes with the Ab1. Crude Ab3-containing sera specifically and effectively neutralized the virus. CONCLUSION: Monoclonal Ab2 UD7 has potential as a vaccine against HIV-1.

Thrombin receptors: turning them off after turning them on.

Recent studies have helped to define the mechanisms by which thrombin activates platelets and other cells. Those studies show that the human thrombin receptor has a structure similar to other G protein-coupled receptors, but is activated by a novel mechanism in which thrombin cleaves its receptor, creating a new N-terminus that can serve as a tethered ligand. Shortly after activation, thrombin receptors become temporarily resistant to re-activation. Present evidence suggests that this loss of function is due to a combination of receptor desensitization, phosphorylation and internalization, and that recovery may involve dephosphorylation, as well as receptor recycling and the expression of newly-synthesized receptors. Together these processes provide a potent mechanism for limiting the duration of thrombin-initiated events in platelets and other thrombin-responsive vascular cells.