Catherine Pugliese-Sivo

SCH-C (SCH 351125), an orally bioavailable, small molecule antagonist of the chemokine receptor CCR5, is a potent inhibitor of HIV-1 infection in vitro and in vivo

We describe here the identification and properties of SCH-C (SCH 351125), a small molecule inhibitor of HIV-1 entry via the CCR5 coreceptor. SCH-C, an oxime–piperidine compound, is a specific CCR5 antagonist as determined in multiple receptor binding and signal transduction assays. This compound specifically inhibits HIV-1 infection mediated by CCR5 in U-87 astroglioma cells but has no effect on infection of CXCR4-expressing cells. SCH-C has broad and potent antiviral activity in vitro against primary HIV-1 isolates that use CCR5 as their entry coreceptor, with mean 50% inhibitory concentrations ranging between 0.4 and 9 nM. Moreover, SCH-C strongly inhibits the replication of an R5-using HIV-1 isolate in SCID-hu Thy/Liv mice. SCH-C has a favorable pharmacokinetic profile in rodents and primates with an oral bioavailability of 50–60% and a serum half-life of 5–6 h. On the basis of its novel mechanism of action, potent antiviral activity, and in vivo pharmacokinetic profile, SCH-C is a promising new candidate for therapeutic intervention of HIV infection.

Discovery and characterization of vicriviroc (SCH 417690), a CCR5 antagonist with potent activity against human immunodeficiency virus type 1.

ABSTRACT Inhibiting human immunodeficiency virus type 1 (HIV-1) infection by blocking the host cell coreceptors CCR5 and CXCR4 is an emerging strategy for antiretroviral therapy. Currently, several novel coreceptor inhibitors are being developed in the clinic, and early results have proven promising. In this report, we describe a novel CCR5 antagonist, vicriviroc (formerly SCH-D or SCH 417690), with improved antiviral activity and pharmacokinetic properties compared to those of SCH-C, a previously described CCR5 antagonist. Like SCH-C, vicriviroc binds specifically to the CCR5 receptor and prevents infection of target cells by CCR5-tropic HIV-1 isolates. In antiviral assays, vicriviroc showed potent, broad-spectrum activity against genetically diverse and drug-resistant HIV-1 isolates and was consistently more active than SCH-C in inhibiting viral replication. This compound demonstrated synergistic anti-HIV activity in combination with drugs from all other classes of approved antiretrovirals. Competition binding assays revealed that vicriviroc binds with higher affinity to CCR5 than SCH-C. Functional assays, including inhibition of calcium flux, guanosine 5′-[35S]triphosphate exchange, and chemotaxis, confirmed that vicriviroc acts as a receptor antagonist by inhibiting signaling of CCR5 by chemokines. Finally, vicriviroc demonstrated diminished affinity for the human ether a-go-go related gene transcript ion channel compared to SCH-C, suggesting a reduced potential for cardiac effects. Vicriviroc represents a promising new candidate for the treatment of HIV-1 infection.

Blocking ion channel KCNN4 alleviates the symptoms of experimental autoimmune encephalomyelitis in mice

The KCNN4 potassium‐ion channel has been reported to play an important role in regulating antigen‐induced T cell effector functions in vitro. This study presents the first evidence that a selective KCNN4 blocker, TRAM‐34, confers protection against experimental autoimmune encephalomyelitis (EAE) in the mouse model. Treatment with the KCNN4 blocker did not prevent infiltration of T cells in the spinal cord, but resulted in the reduction of both the protein and the message levels of TNF‐α and IFN‐γ as well as the message levels of several other pro‐inflammatory molecules in the spinal cord. Plasma concentrations of TRAM‐34 within a 24‐h period were between the in vitro IC50 and IC90 values for the KCNN4 channel. The effect of TRAM‐34 was reversible, as indicated by the development of clinical EAE symptoms within 48 h after withdrawal of treatment. In summary, our data support the idea that KCNN4 channels play a critical role in the immune response during the development of MOG‐induced EAE in C57BL/6 mice.

The effect of GM-CSF and G-CSF on human neutrophil function

A direct comparison of GM-CSF and G-CSF in a panel of in vitro neutrophil-function assays was performed to investigate any differences in activity profiles. In our modified chemotactic assay, GM-CSF rapidly increased the migratory capacity of polymorphonuclear cells (PMNs) to move toward fMLP and LTB4. In contrast, G-CSF only stimulated PMN migration towards fMLP. GM-CSF, but not G-CSF, increased PMN cytotoxic killing of C. albicans blastospores. The expression of PMN surface antigens associated with Fc- and complement-mediated cell-binding (Fc gamma R1, CR-1 and CR-3), and adhesion signalling (ICAM-1), was increased after the exposure of GM-CSF, but not to G-CSF. In contrast these CSFs demonstrated relative equipotency in their ability to induce PMN anti-bacterial phagocytosis, and to restore the Staphylococcus aureus killing capacity of dexamethasone-suppressed neutrophils. The phagocytic activity of PMNs for opsonized yeast, as well as hexose-monophosphate shunt activity, was equivalent following GM-CSF or G-CSF treatment. We discuss the significance of the difference in activity profiles in this article.

IL-4 induces neutrophilic maturation of HL-60 cells and activation of human peripheral blood neutrophils

IL‐4 is a T‐helper cell derived cytokine that has effects on myelomonocytic cell maturation and activation. We have studied the effect of IL‐4 on neutrophilic maturation using the cell line HL‐60 and found that it has a profound effect on the maturation and activation of the cell line. The treatment of HL‐60 cells with recombinant hu IL‐4 (0.15 to 15.0 ng/ml) induced a shift in the percentage of HL‐60 cells staining positive for chloroacetate esterase enzyme activity (indicating commitment to the neutrophilic lineage). IL‐4 increased surface expression of the neutrophil‐lineage antigen WEM G11, the complement receptors CR3 (CD11b) and CR1 (CD35), but not for the monocyte differentiation antigen CD14. IL‐4 treated HL‐60 cells demonstrated enhanced Fc‐ and complement‐mediated phagocytic capacity and increased hexose‐monophosphate shunt activity. In addition, IL‐4 was capable of sustaining the neutrophil maturation of HL‐60 cells that had been pre‐treated for 24h with DMSO. To investigate the effect of IL‐4 on the mature neutrophil, we studied freshly isolated and rested human peripheral blood neutrophils. In the absence of other stimuli, neutrophils were induced by IL‐4 to have significantly elevated phagocytic responses. The response was specific since treatment with anti‐human IL‐4 abolished phagocytic stimulation. Finally, IL‐4 treatment also stimulated resting neutrophils to migrate toward zymosan‐activated serum (ZAS) and human IL‐5. The results demonstrate that IL‐4 is a potent maturation factor for myelocytes to become neutrophils and that IL‐4 can stimulate resting mature neutrophils.

Pharmacological characterization of the chemokine receptor, hCCR1 in a stable transfectant and differentiated HL-60 cells: antagonism of hCCR1 activation by MIP-1β

C‐C chemokine receptor‐1 (CCR1) has been implicated in mediating a variety of inflammatory conditions including multiple sclerosis and organ rejection. Although originally referred to as the MIP‐1α/RANTES receptor, CCR1 is quite promiscuous and can be activated by numerous chemokines. We used radioligand binding and [35S]‐GTPγS exchange assays in membranes from a cell line transfected to express CCR1 (Ba/F3‐hCCR1) to characterize a panel of chemokines (HCC‐1, MIP‐1α, MIP‐1β, MIP‐1δ, MPIF‐1, MCP‐2, MCP‐3, and RANTES) as CCR1 ligands. In this recombinant model, these chemokines displaced 125I‐MIP‐1α with a wide range of potencies and, with the exception of MCP‐2, acted as full agonists in stimulating [35S]‐GTPγS exchange. We then assessed the utility of HL‐60 cells cultured with known differentiating agents (PMA, DMSO, dibutyryl‐cAMP or retinoic acid) for investigating CCR1 pharmacology. In [35S]‐GTPγS exchange assays, membranes from cells cultured with retinoic acid (4–6 days) were the most responsive to activation by MIP‐1α and MPIF‐1. FACS analysis and comparative pharmacology confirmed that these activities were mediated by CCR1. Using [35S]‐GTPγS exchange assays, intracellular calcium flux and/or whole cell chemotaxis assays in HL‐60(Rx) cells, we validated that MIP‐1α was the most potent CCR1 ligand (MIP‐1α>MPIF‐1>RANTESMIP‐1β) although the ligands differed in their efficacy as agonists. MPIF‐1 was the more efficacious (MPIF‐1>RANTES=MIP‐1α>>MIP‐1β). 125I‐MIP‐1β binding in Ba/F3‐hCCR1 and HL‐60(Rx) membranes was competitively displaced by MIP‐1α, MPIF‐1 and MIP‐1β. The binding Ki for these chemokines with 125I‐MIP‐1β were essentially identical in the two membrane systems. Lastly, MIP‐1β antagonized [35S]‐GTPγS exchange, Ca2+ flux and chemotaxis in HL‐60(Rx) cells in response to robust agonists such as MIP‐1α, RANTES and MPIF‐1. Based on our results, we propose that MIP‐1β could function as an endogenous inhibitor of CCR1 function.

The effects of colony stimulating factors on human monocyte cell function.

We used a panel of functional assays to compare directly the pattern and potency of GM-CSF and M-CSF on monocyte activity associated with cell-mediated immune defense. GM-CSF and M-CSF were found to be equivalent both in their capacity to stimulate human monocyte functions in vitro and in their pattern of monocyte activation. The two CSFs were effective in inducing monocyte chemotaxis towards either fMLP or LTB4 at equivalent concentrations across a panel of donors. GM-CSF and M-CSF demonstrated equipotency in the induction of monocyte phagocytosis of heat-killed bakers yeast and in the regulation of the hexose-monophosphate shunt (NBT reduction). Both were also found to be equivalent in preventing steroid (dexamethasone)-induced suppression of monocyte anti-bacterial (Candida albicans) and anti-fungal (Staphylococcus aureus) phagocytic capacities. GM-CSF was somewhat more effective than M-CSF in stimulating monocyte C. albicans killing at a lower E:T ratio.

Human IL-10 reduces the number of IL-4-induced IgE B cells in cultures of atopic mononuclear cells

We investigated the effect of recombinant human IL-10 on IL-4- and antigen-driven human peripheral blood mononuclear cell cultures derived from atopic donors. These cultures were phenotyped for the percentage of B cell (CD20 HLA-DR) population by flow cytometry and for intracellular IgE using epifluorescence microscopy. The addition of IL-4 (100 U/ml) to these cultures resulted in an increase in the percentage of IgE B cells. However, the percentage of IgE B cells in cultures coincubated with IL-10 (2 or 20 ng/ml) and IL-4 was reduced to the level of medium control. Peripheral-blood mononuclear (PBMN) cultures driven with dust mite allergen demonstrated a significant increase in cellular proliferation, as measured by 3[H] thymidine uptake, and in the percentage of IgE B cells. The coaddition of IL-10 (2 or 20 ng/ml) to these cultures significantly inhibited both proliferation and the mean percentage of IgE B cells. The inhibitory effect of IL-10 on IgE B cell percentages in both the IL-4- and the allergen-driven cultures, and on allergen-driven proliferation, was dependent upon the presence of monocytes. Interestingly, the inhibitory effect of IL-10 on allergen-driven proliferation in the atopic PBMN cultures was reversible by the coaddition of exogenous IFN gamma (1 ng/ml) and IL-2 (2 U/ml). The addition of IL-2 (2 U/ml) partially reversed IL-10-inhibited allergen-driven proliferation while alone IFN gamma had no effect (1 ng/ml). In fact, the addition of IFN gamma (1 ng/ml) in the absence of either IL-10 or IL-2 (2 U/ml) partially inhibited allergen-driven proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Potential Interaction of lnterleukin-4 with Endogenous Cytokines in vivo

We employed alginate-entrapped cells secreting recombinant murine interleukin-4 (IL-4) to study the effect of IL-4 on hematopoietic cells of normal mice. The most dramatic effect was registered in an increase in the neutrophils and to a lesser extent the monocytes. A small increase in the CD4+, CD8+ and B220+ populations was also observed. Serum IgE levels also increased dramatically. All of these increases could be specifically inhibited with anti-IL-4. Antibodies to granulocyte-macrophage colony-stimulating factor, IL-3 and IL-5 could also inhibit some IL-4-mediated actions suggesting an interplay between these cytokines in vivo.