Antiretroviral agents as inhibitors of both human immunodeficiency virus type 1 integrase and protease.

The human immunodeficiency virus type one integrase (HIV-1 integrase) is required for integration of a double-stranded DNA copy of the viral RNA genome into a host chromosome and for HIV replication. We have previously reported that phenolic moieties in compounds such as flavones, caffeic acid phenethyl ester (CAPE), tyrphostins, and curcumin confer inhibitory activity against HIV-1 integrase. We have investigated the actions of several recently described protease inhibitors, possessing novel structural features, on HIV-1 integrase.

Towards endothelial-cell-directed cancer immunotherapy: in vitro expression of human recombinant cytokine genes by human and mouse primary endothelial cells.

Recent studies have demonstrated the feasibility of cytokine gene transfer to enhance the antitumor activities of host immune cells. Endothelial cells forming the vascular supply of tumors may be useful vehicles for the delivery of cytokine molecules in order to effect tumor immunotherapy. In order to determine whether primary endothelial cells can express cytokine transgenes efficiently, we constructed two retroviral vectors containing a cDNA encoding either recombinant human interleukin-1 alpha (rhIL-1 alpha) or recombinant human interleukin-2 (rhIL-2), called LNCIL-1 alpha and LNCIL-2 respectively, and studied the expression of the two cytokines in vitro in non-immortalized endothelial cells.

The effect of hydroxychloroquine on alloreactivity and its potential use for graft-versus-host disease.

Hydroxychloroquine (HCQ) interferes with antigen processing and with receptor loading and recycling by raising the pH of lysosomes and endosomes. HCQ thus inhibits MHC class II-restricted antigen presentation by blocking the binding of peptides to MHC molecules. Additionally, HCQ has been shown to diminish the release of several cytokines.

Modulation of B16 melanoma growth and metastasis by anti-transforming growth factor beta antibody and interleukin-2.

Earlier evidence suggests that transforming growth factor beta (TGF beta) plays a significant role in tumor progression and metastasis. The most likely mechanism of the action of TGF beta is induction of immunosuppression in the host, allowing for unchecked tumor growth and metastasis. We attempted to test that hypothesis and to compare antitumor effects of anti-TGF beta antibody alone and in combination with interleukin-2 (IL-2).

Inhibitors of Escherichia coli RNA polymerase specific for the single-stranded DNA of transcription intermediates. Tetrahedral cuprous chelates of 1,10-phenanthrolines.

Single-stranded DNA of the lacUV-5 promoter formed at the active site of Escherichia coli RNA polymerase during transcription is specifically cleaved by the redox active tetrahedral cuprous chelates of 1,10-phenanthroline and its derivatives. The cleavage sites are observed in the open, initiating, and elongating complexes. Redox-inert, tetrahedral cuprous chelates of neocuproine (2,9-dimethyl-1,10- phenanthroline) and its 5-phenyl and 4-phenyl derivatives protect the template strand of DNA from scission within these steady state intermediates and inhibit transcription.

Effects of nucleotide analogues on human immunodeficiency virus type 1 integrase.

We extended our previous study with 3'-azido-3'-deoxythymidine nucleotides [Proc. Natl. Acad.

Differential induction of programmed cell death in CD8+ and CD4+ T cells by the B subunit of cholera toxin.

We have recently demonstrated that a short-term treatment of parental splenocytes with the B subunit of cholera toxin (CT-B) abrogates the development of acute GVHD in F1 hybrid mice transplanted with these cells. In order to obtain better insight into the mechanism of the action of CT-B, we studied the effect of CT-B on survival of purified murine T cells and their subsets. We show that treatment with B subunit stimulates apoptosis in T cells, detectable following incubation in vitro.

Probing the active site residues in aromatic donor oxidation in horseradish peroxidase: involvement of an arginine and a tyrosine residue in aromatic donor binding.

The plausible role of arginine and tyrosine residues at the active side of horseradish peroxidase (HRP) in aromatic donor (guaiacol) oxidation was probed by chemical modification followed by characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), 2,3-butanedione and 1,2-cyclohexanedione all inactivated the enzyme, following pseudo-first-order kinetics with second-order rate contents of 24M(-1.)min(-1), 0.

Allogeneic bone marrow transplantation in a patient with hypereosinophilic syndrome.

We describe a 32-year-old man with idiopathic hypereosinophilic syndrome (HES) who presented with pulmonary dysfunction, thrombocytopenia, lymphadenopathy, and hepatosplenomegaly. The patient developed progressive disease on prednisone and hydroxyurea therapy, and he underwent a successful allogeneic bone marrow transplantation (BMT). The patient is asymptomatic with no evidence of eosinophilia 30 months after transplantation.

(-)-Arctigenin as a lead structure for inhibitors of human immunodeficiency virus type-1 integrase.

The natural dibenzylbutyrolactone type lignanolide (-)-arctigenin (2), an inhibitor of human immunodeficiency virus type-1 (HIV-1) replication in infected human cell systems, was found to suppress the integration of proviral DNA into the cellular DNA genome. In the present study 2 was tested with purified HIV-1 integrase and found to be inactive in the cleavage (3'-processing) and integration (strand transfer) assays. However, the semisynthetic 3-O-demethylated congener 9 characterized by a catechol substructure exhibited remarkable activities in both assays.

Immunomodulation following chemotherapy.

In the last decade, immunomodulation has emerged as a mode of therapy capable of mediating the regression of cancer in some patients. This article reviews our experience with immunomodulation following transplant and non-transplant chemotherapy. We used interferon and cyclosporine A following conventional chemotherapy in a non-transplant setting for a B16 melanoma in a murine model.

Stem cell transplantation with chemoradiotherapy myeloablation and interleukin-2.

Interleukin 2 (IL-2) stimulates the proliferation of T-cells both in vitro and in vivo. When murine or human peripheral blood (PB) or bone marrow (BM) mononuclear cells are incubated with IL-2 in vitro for 24 hours, cytotoxic T-cells are generated. If these activated cells are infused into mice, the enhanced cytotoxicity continues if low dose IL-2 is administered.

Effects of tyrphostins, protein kinase inhibitors, on human immunodeficiency virus type 1 integrase.

Efficient replication of HIV-1 requires establishment of the proviral state, i.e., the integration of a DNA copy of the viral genome, synthesized by reverse transcriptase, into a chromosome of the host cell.

T30177, an oligonucleotide stabilized by an intramolecular guanosine octet, is a potent inhibitor of laboratory strains and clinical isolates of human immunodeficiency virus type 1.

T30177, an oligonucleotide composed of only deoxyguanosine and thymidine, is 17 nucleotides in length and contains single phosphorothioate internucleoside linkages at its 5' and 3' ends for stability. This oligonucleotide does not share significant primary sequence homology with or possess any complementary (antisense) sequence motifs to the human immunodeficiency virus type 1 (HIV-1) genome. T30177 inhibited replication of multiple laboratory strains of HIV-1 in human T-cell lines, peripheral blood lymphocytes, and macrophages.

Hydroxylated aromatic inhibitors of HIV-1 integrase.

Efficient replication of HIV-1 requires integration of a DNA copy of the viral genome into a chromosome of the host cell. Integration is catalyzed by the viral integrase, and we have previously reported that phenolic moieties in compounds such as flavones, caffeic acid phenethyl ester (CAPE, 2), and curcumin confer inhibitory activity against HIV-1 integrase. We now extend these findings by performing a comprehensive structure-activity relationship using CAPE analogues.

Concurrent reduction of iodine and oxidation of EDTA at the active site of horseradish peroxidase: probing the iodine binding site by optical difference spectroscopy and steady state kinetic analysis for the formation of active enzyme-I(+)-EDTA ternary complex for iodine reductase activity.

Horseradish peroxidase (HRP) catalyzes the reduction of iodine to iodide by EDTA with pseudocatalatic degradation of H2O2 to O2 (Banerjee et al., (1986) J. Biol.

Augmentation of murine hematopoiesis by interleukin 2-activated irradiated T cells.

We have examined the role of T cells activated with interleukin-2 (IL-2) in vitro and subsequently irradiated (2500 rads), in stimulating murine hematopoiesis in a syngeneic system. Our data suggest that activated, irradiated T (AIT) cells significantly increased the progenitor cell activity of T cell-depleted bone marrow (BM) both in vitro and in vivo as compared with controls (P < 0.001).

Interleukin-12 (IL-12) alone or in synergistic combination with IL-2 for in vitro activation of human bone marrow: differential effects at different time points.

IL-12 augments many effector functions associated with T and NK cells including induction of cytotoxic activity and secretion of various cytokines. In addition, synergistic responses with IL-2 in most of the functions attributed to that cytokine make IL-12 an attractive candidate to have a potential role as an immunotherapeutic agent. We have evaluated the effect of rhIL-12 either alone or in combination with IL-2 on the generation of cytotoxic effectors from normal human bone marrow.

Incorporation of a fluorescent guanosine analog into oligonucleotides and its application to a real time assay for the HIV-1 integrase 3'-processing reaction.

We have synthesized a highly fluorescent (quantum yield 0.88) guanosine analog, (3-methyl-8-(2-deoxy-beta-D-ribofuranosyl) isoxanthopterin (3-Mi) in a dimethoxytrityl, phosphoramidite protected form, which can be site-specifically inserted into oligonucleotides through a 3',5'-phosphodiester linkage using an automated DNA synthesizer. Fluorescence is partially quenched within an oligonucleotide and the degree of quench is a function of the fluorophore's proximity to purines and its position in the oligonucleotide.

Processing of deoxyuridine mismatches and abasic sites by human immunodeficiency virus type-1 integrase.

We have examined the activities of HIV-1 integrase on substrates containing mismatches, composed of deoxyuridine at different positions in either the processed or nonprocessed strand of viral DNA, within and near the conserved CA dinucleotide of the U5 end of the HIV-1 LTR. Substitution in the processed strand of either the C or A of the CA dinucleotide or of the G 5' to the CA reduced strand transfer six-, three- and seven-fold respectively. 3'-processing was also reduced by substitution at the GC but not at the A.

Role of granulocyte colony-stimulating factor in preventing ceftazidime-induced myelosuppression in vitro.

Ceftazidime has been reported to cause myelosuppression both in vitro and in vivo. Granulocyte colony-stimulating factor (G-CSF) is known to enhance the proliferation and differentiation of myeloid cells. The present study was carried out to define the role of G-CSF in preventing the ceftazidime-induced suppression of BM progenitor cells in vitro, and to define the mechanisms involved in ceftazidime-induced myelosuppression.

Inhibition of human immunodeficiency virus type-1 integrase by curcumin.

Curcumin (diferuloylmethane) is the yellow pigment in turmeric (Curcuma longa L.) that is widely used as a spice, food coloring (curry) and preservative. Curcumin exhibits a variety of pharmacological effects including antitumor, anti-inflammatory, and anti-infectious activities and is currently in clinical trials for AIDS patients.

Prevention of acute graft-versus-host disease by treatment with a novel immunosuppressant. Cholera toxin B subunit.

Transplantation of allogeneic bone marrow (BM) is often complicated by the development of acute graft-vs-host disease (GVHD) caused by contaminating T cells in BM inocula because of activation of the host reactive T effector cells. Using a murine model for acute GVHD caused by injection of parental C57Bl/6 splenocytes into unirradiated (C57Bl/6 x DBA/2) F1 hybrids, we demonstrated that pretreatment of the inocula with a novel immunosuppressant, B subunit of cholera toxin (CT-B) impaired the ability of C57Bl/6 T cells to induce acute GVHD in F1 recipients. F1 mice injected with CT-B-treated C57Bl/6 splenocytes did not develop significant splenomegaly, and no antihost CTLs were found in their spleens.

Phase I-II trial of high-dose cyclophosphamide, carboplatin and autologous bone marrow or peripheral blood stem cell rescue.

In an effort to evaluate the toxicities and anti-tumor efficacy of the combination of high-dose cyclophosphamide (CY) and carboplatin, we undertook a phase I-II trial with autologous bone marrow (BM) or peripheral blood stem cell (PBSC) rescue for patients with solid tumors. Forty three patients, 39 of whom had either high risk stage II or III or metastatic breast cancer were treated with escalating doses of carboplatin 1200-1800 mg/m2 and cyclophosphamide 4800-6000 mg/m2 over 3 days followed by autologous BM or PBSC infusion. No life-threatening or fatal toxicities were observed.