Inhibition of Premature Death by Isothiocyanates through Immune Restoration in LP-BM5 Leukemia Retrovirus-Infected C57BL/6 Mice

The purpose of this study was to determine the effect of isothiocyanates (ITCs) in delaying the progression of the murine immunodeficiency virus to murine AIDS, resulting in increased life span. Furthermore, we investigated the role of ITCs in modulating immune dysfunction caused by LP-BM5 retrovirus infection. Among the tested ITCs, oral administration of sulforaphane (SUL), benzyl isothiocyante (BITC), and phenethyl isothiocyanate (PEITC) showed the inhibition of premature death caused by LP-BM5 retrovirus infection, while indolo[3,2-b] carbazole (ICZ) and indole-3-carbinol (I3C) did not delay the progress of the LP-BM5 retrovirus to murine AIDS. Inhibition of premature death by BITC, PEITC, and SUL could be explained by restoration of the immune system and down regulation of free radicals. Dysfunction of T and B cell mitogenesis caused by retrovirus infection in primary cultured splenocytes has been partially recovered with administration of BITC, PEITC, and SUL. There was a shift from imbalanced cytokine production (increased Th2 and decreased Th1 cell cytokine production) into balanced Th1/Th2 cell secretion of cytokines under administration of these ITCs during the development of murine AIDS. Hepatic vitamin E level was significantly restored by administration of these ITCs, in accordance with reduced hepatic lipid peroxidation levels. This study suggests that certain types of ITCs have beneficial effects in preventing premature death during progression to murine AIDS by restoration of immune dysfunction and removal of excessive free radicals, implying that selective usage of ITCs would be helpful in retarding the progression from HIV infection to AIDS.     

Development and application of a method for identification of isothiocyanate-targeted molecules in colon cancer cells

In this study, we have developed a novel method to identify isothiocyanate (ITC)-targeted molecules using two well-studied ITCs: benzyl ITC (BITC) and phenethyl ITC (PEITC). The principle of this method is based on identifying a pattern of differences between BITC and PEITC given that they show similar chemical and biological behaviors. For method validation, dithiothreitol-reduced bovine insulin as a model molecule was incubated with either BITC or PEITC, and digested peptides were analyzed by ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) and liquid chromatography quadrupole TOF-MS (LC-Q-TOF-MS). Three peptides-NYCN, FVNQHLCGSHLVE, and ALYLVCGE-were identified as being adducted with BITC or PEITC on their cysteine residues. Each set of peptides adducted with either BITC or PEITC showed retention times (RT(BITC)相似文献   

Changes in hepatic lipid composition after infection by LP-BM5 murine leukemia virus causing murine AIDS.

The severe hepatic disorders in patients with acquired immune deficiency syndrome (AIDS) is often attributed to a variety of other factors which could affect hepatic function. To evaluate the mechanism of liver damage in murine AIDS-induced immune-suppressed animal, a murine model of AIDS (MAIDS), caused by infection with LP-BM5 murine leukemia virus was used at a late stage of the disease. Retroviral infection significantly increased hepatic cholesterol, triacylgycerol and the cholesterol/phospholipid ratio. Similarly, the proportions of palmitic, palmitoleic, linoleic, ratios of linoleic to arachidonic and saturated to unsaturated fatty acids were significantly lower while the proportion of oleic, docosatetraenoic and docosahexenoic fatty acids were significantly increased in the retrovirus infected mice. Hepatic dysfunction as evidence by increased serum transaminase levels were also observed in the retrovirus infected animals. The data suggest that the liver damage in murine AIDS is induced by retroviral infection and the desaturase enzymes system necessary to maintain regular balance of the fatty acids in the cells may be affected during retroviral infection.     

Covalent binding to tubulin by isothiocyanates. A mechanism of cell growth arrest and apoptosis

Isothiocyanates (ITCs) found in cruciferous vegetables, including benzyl-ITC (BITC), phenethyl-ITC (PEITC), and sulforaphane (SFN), inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in various cell types. The biochemical mechanisms of cell growth inhibition by ITCs are not fully understood. Our recent study showed that ITC binding to intracellular proteins may be an important initiating event for the induction of apoptosis. However, the specific protein target(s) and molecular mechanisms were not identified. In this study, two-dimensional gel electrophoresis of human lung cancer A549 cells treated with radiolabeled PEITC and SFN revealed that tubulin may be a major in vivo binding target for ITC. We examined whether binding to tubulin by ITCs could lead to cell growth arrest. The proliferation of A549 cells was significantly reduced by ITCs, with relative activities of BITC > PEITC > SFN. All three ITCs also induced mitotic arrest and apoptosis with the same order of activity. We found that ITCs disrupted microtubule polymerization in vitro and in vivo with the same order of potency. Mass spectrometry demonstrated that cysteines in tubulin were covalently modified by ITCs. Ellman assay results indicated that the modification levels follow the same order, BITC > PEITC > SFN. Together, these results support the notion that tubulin is a target of ITCs and that ITC-tubulin interaction can lead to downstream growth inhibition. This is the first study directly linking tubulin-ITC adduct formation to cell growth inhibition.     

Minireview Murine models for Acquired Immune Deficiency Syndrome

The mouse has been suggested as a host for comparative studies of several aspects of Human Acquired Immune Deficiency Syndrome (AIDS). Models include studies where part or all of the genome of Human Immunodeficiency Virus (HIV) has been incorporated into murine DNA in living mice. However, the most promising oppurtunities for study of immunological changes, vaccine development, cofactor involvement in disease, and anti-retroviral and immunostimulatory drug testing involve infection with murine retroviruses which cause many functional changes similar to AIDS. The viruses' effects on immune systems are reviewed with special emphasis on LP-BM5 murine leukemia virus which infects T and B cells, and macrophages. LP-BM5 infection suppresses cell functions while causing polyclonal lymphocyte activation. Murine immunological characterization, availability of inbred mouse strains, economy of using mice versus primates or humans models, and similarity of immune change caused by murine retroviruses compared to those seen in AIDS caused by HIV encourage rapid development of the LP-BM5 murine leukemia model.     

Murine models for acquired immune deficiency syndrome

The mouse has been suggested as a host for comparative studies of several aspects of Human Acquired Immune Deficiency Syndrome (AIDS). Models include studies where part or all of the genome of Human Immunodeficiency Virus (HIV) has been incorporated into murine DNA in living mice. However, the most promising opportunities for study of immunological changes, vaccine development, cofactor involvement in disease, and anti-retroviral and immunostimulatory drug testing involve infection with murine retroviruses which cause many functional changes similar to AIDS. The viruses' effects on immune systems are reviewed with special emphasis. LP-BM5 murine leukemia virus which infects T and B cells, and macrophages. LP-BM5 infection suppresses cell functions while causing polyclonal lymphocyte activation. Murine immunological characterization, availability of inbred mouse strains, economy of using mice versus primates or humans models, and similarity of immune change caused by murine retroviruses compared to those seen in AIDS caused by HIV encourage rapid development of the LP-BM5 murine leukemia model.     

Effect of cocaine and murine aids on lamina propria T and B cells in normal mice

We developed an experimental model to study the effect of daily cocaine administration on the mucosal immune system during murine acquired immune deficiency syndrome (MAIDS). Mice were infected with LP-BM5 murine leukemia virus, a retrovirus which causes immunosuppression with development of functional murine AIDS. Mice were given cocaine by daily intraperitoneal injection for 11 weeks. Our objective was to investigate if cocaine treatment could alter the mucosal immune system at the level of the intestinal lamina propria (ILP) and if it could further modify the already altered mucosal immunity when it was administered to MAIDS-mice. Daily cocaine administration induced a significant decrease in the number of IgA⁺ cells with a concomitant increase in the number of CD8⁺ cells per villi in the ILP. Murine retrovirus infection alone decreased the number of IgA⁺ and CD4⁺ cells in the ILP, and this decreased was even more marked when MAIDS mice also received cocaine. These data indicate that cocaine administration could potentiate the dramatic effect that MAIDS infection has in the mucosal-associated lymphoid tissues.     

Dietary organic isothiocyanates are cytotoxic in human breast cancer MCF-7 and mammary epithelial MCF-12A cell lines

Organic isothiocyanates (ITCs) are dietary components present in cruciferous vegetables. The purpose of this investigation was to examine the cytotoxicity of 1-naphthyl isothiocyanate (NITC), benzyl isothiocyanate (BITC), beta-phenethyl isothiocyanate (PEITC), and sulforaphane in human breast cancer MCF-7 and human mammary epithelium MCF-12A cell lines, as well as in a second human epithelial cell line, human kidney HK-2 cells. The cytotoxicity of NITC, BITC, PEITC, and sulforaphane, as well as the cytotoxicity of the chemotherapeutic agents daunomycin (DNM) and vinblastine (VBL), were examined in MCF-7/sensitive (wt), MCF-7/Adr (which overexpresses P-glycoprotein), MCF-12A, and HK-2 cells. Cell growth was determined by a sulforhodamine B assay. The IC50 values for DNM and VBL in MCF-7/Adr cells were 7.12 +/- 0.42 microM and 0.106 +/- 0.004 microM (mean +/- SE) following a 48-hr exposure; IC50 values for BITC, PEITC, NITC, and sulforaphane were 5.95 +/- 0.10, 7.32 +/- 0.25, 77.9 +/- 8.03, and 13.7 +/- 0.82 microM, respectively, with similar values obtained in MCF-7/wt cells. Corresponding values for BITC, PEITC, NITC, and sulforaphane in MCF-12A cells were 8.07 +/- 0.29, 7.71 +/- 0.07, 33.6 +/- 1.69, and 40.5 +/- 1.25 microM, respectively. BITC and PEITC can inhibit the growth of human breast cancer cells as well as human mammary epithelium cells at concentrations similar to those of the chemotherapeutic drug DNM. Sulforaphane and NITC exhibited higher IC50 values. The effect of these ITCs on cell growth may contribute to the cancer chemopreventive properties of ITCs by suppressing the growth of preclinical tumors, and may indicate a potential use of these compounds as chemotherapeutic agents in cancer treatment.     

Immunotherapy of murine retrovirus-induced acquired immunodeficiency by CD4 T regulatory cell depletion and PD-1 blockade

LP-BM5 retrovirus induces a complex disease featuring an acquired immunodeficiency syndrome termed murine AIDS (MAIDS) in susceptible strains of mice, such as C57BL/6 (B6). CD4 T helper effector cells are required for MAIDS induction and progression of viral pathogenesis. CD8 T cells are not needed for viral pathogenesis, but rather, are essential for protection from disease in resistant strains, such as BALB/c. We have discovered an immunodominant cytolytic T lymphocyte (CTL) epitope encoded in a previously unrecognized LP-BM5 retroviral alternative (+1 nucleotide [nt]) gag translational open reading frame. CTLs specific for this cryptic gag epitope are the basis of protection from LP-BM5-induced immunodeficiency in BALB/c mice, and the inability of B6 mice to mount an anti-gag CTL response appears critical to the initiation and progression of LP-BM5-induced MAIDS. However, uninfected B6 mice primed by LP-BM5-induced tumors can generate CTL responses to an LP-BM5 retrovirus infection-associated epitope(s) that is especially prevalent on such MAIDS tumor cells, indicating the potential to mount a protective CD8 T-cell response. Here, we utilized this LP-BM5 retrovirus-induced disease system to test whether modulation of normal immune down-regulatory mechanisms can alter retroviral pathogenesis. Thus, following in vivo depletion of CD4 T regulatory (Treg) cells and/or selective interruption of PD-1 negative signaling in the CD8 T-cell compartment, retroviral pathogenesis was significantly decreased, with the combined treatment of CD4 Treg cell depletion and PD-1 blockade working in a synergistic fashion to substantially reduce the induction of MAIDS.     

Characterization of the CD154-positive and CD40-positive cellular subsets required for pathogenesis in retrovirus-induced murine immunodeficiency

Genetically susceptible C57BL/6 (B6) mice that are infected with the LP-BM5 isolate of murine retroviruses develop profound splenomegaly, lymphadenopathy, hypergammaglobulinemia, terminal B-cell lymphomas, and an immunodeficiency state bearing many similarities to the pathologies seen in AIDS. Because of these similarities, this syndrome has been called murine AIDS (MAIDS). We have previously shown that CD154 (CD40 ligand)-CD40 molecular interactions are required both for the initiation and progression of MAIDS. Thus, in vivo anti-CD154 monoclonal antibody (MAb) treatment inhibited MAIDS symptoms in LP-BM5-infected wild-type mice when either a short course of anti-CD154 MAb treatment was started on the day of infection or a course was initiated 3 to 4 weeks after LP-BM5 administration, after disease was established. Here, we further characterize this required CD154-CD40 interaction by a series of adoptive transfer experiments designed to elucidate which cellular subsets must express CD154 or CD40 for LP-BM5 to induce MAIDS. Specifically with regard to CD154 expression, MAIDS-insusceptible B6 nude mice reconstituted with highly purified CD4+ T cells from wild-type, but not from CD154 knockout, B6 donors displayed clear MAIDS after LP-BM5 infection. In contrast, nude B6 recipients that received CD8+ T cells from wild-type B6 donors did not develop MAIDS after LP-BM5 infection. B6 CD40 knockout mice, which are also relatively resistant to LP-BM5-induced MAIDS, became susceptible to LP-BM5-induced disease after reconstitution with highly purified wild-type B cells but not after receiving purified wild-type dendritic cells (DC) or a combined CD40+ population composed of DC and macrophages obtained from B6 SCID mouse donors. Based on these and other experiments, we thus conclude that the cellular basis for the requirement for CD154-CD40 interactions for MAIDS induction and progression can be accounted for by CD154 expression on CD4+ T cells and CD40 expression on B cells.     

Cell death induction by isothiocyanates and their underlying molecular mechanisms

An important and promising group of compounds that have a chemopreventive property are organosulfur compounds, such as isothiocyanates (ITCs). In recent years, it has been shown that ITCs induce apoptosis in various cancer cell lines and experimental rodents. During the course of apoptosis induction by ITC, multiple signal-transduction pathways and apoptosis intermediates are modulated. We have also clarified the molecular mechanism underlying the relationship between cell cycle arrest and apoptosis induced by benzyl isothiocyanate (BITC), a major ITC compound isolated from papaya. The exposure of cells to BITC resulted in the inhibition of the G2/M progression that coincided with not only the up-regulated expression of the G2/M cell cycle arrest-regulating genes but also the apoptosis induction. The experiment using the phase-specific synchronized cells demonstrated that the G2/M phase-arrested cells are more sensitive to undergoing apoptotic stimulation by BITC than the cells in other phases. We identified the phosphorylated Bcl-2 as a key molecule linking the p38 MAPK-dependent cell cycle arrest with the JNK activation by BITC. We also found that BITC induced the cytotoxic effect more preferentially in the proliferating normal human colon epithelial cells than in the quiescent cells. Conversely, treatment with an excessive concentration of BITC resulted in necrotic cell death without DNA ladder formation. This review addresses the biological impact of cell death induction by BITC as well as other ITCs and the involved signal transduction pathways.     

Cytolytic T lymphocytes specific for tumors and infected cells from mice with a retrovirus-induced immunodeficiency syndrome.

LP-BM5 retrovirus complex-infected C57BL/6 mice develop immunodeficiency, somewhat analogous to AIDS, termed murine AIDS (MAIDS). After secondary stimulation with syngeneic B-cell lymphomas from LP-BM5-infected mice, C57BL/6 mice produced vigorous CD8+ cytotoxic T lymphocytes specific for MAIDS-associated tumors. An anti-LP-BM5 specificity was suggested because spleen and lymph node cells from LP-BM5-infected mice served as target cells in competition assays, and cells from LP-BM5, but not ecotropic, virus-infected mice functioned as secondary in vitro stimulators to generate cytotoxic T lymphocytes to MAIDS tumors.     

T helper cell activation and human retroviral pathogenesis.

T helper (Th) cells are of central importance in regulating many critical immune effector mechanisms. The profile of cytokines produced by Th cells correlates with the type of effector cells induced during the immune response to foreign antigen. Th1 cells induce the cell-mediated immune response, while Th2 cells drive antibody production. Th cells are the preferential targets of human retroviruses. Infections with human T-cell leukemia virus (HTLV) or human immunodeficiency virus (HIV) result in the expansion of Th cells by the action of HTLV (adult T-cell leukemia) or the progressive loss of T cells by the action of HIV (AIDS). Both retrovirus infections impart a high-level activation state in the host immune cells as well as systemically. However, diverging responses to this activation state have contrasting effects on the Th-cell population. In HIV infection, Th-cell loss has been attributed to several mechanisms, including a selective elimination of cells by apoptosis. The induction of apoptosis in HIV infection is complex, with many different pathways able to induce cell death. In contrast, infection of Th cells with HTLV-1 affords the cell a protective advantage against apoptosis. This advantage may allow the cell to escape immune surveillance, providing the opportunity for the development of Th-cell cancer. In this review, we will discuss the impact of Th-cell activation and general immune activation on human retrovirus expression with a focus upon Th-cell function and the progression to disease.     

Mice with disrupted type I protein kinase A anchoring in T cells resist retrovirus-induced immunodeficiency

Type I protein kinase A (PKA) is targeted to the TCR-proximal signaling machinery by the A-kinase anchoring protein ezrin and negatively regulates T cell immune function through activation of the C-terminal Src kinase. RI anchoring disruptor (RIAD) is a high-affinity competitor peptide that specifically displaces type I PKA from A-kinase anchoring proteins. In this study, we disrupted type I PKA anchoring in peripheral T cells by expressing a soluble ezrin fragment with RIAD inserted in place of the endogenous A-kinase binding domain under the lck distal promoter in mice. Peripheral T cells from mice expressing the RIAD fusion protein (RIAD-transgenic mice) displayed augmented basal and TCR-activated signaling, enhanced T cell responsiveness assessed as IL-2 secretion, and reduced sensitivity to PGE(2)- and cAMP-mediated inhibition of T cell function. Hyperactivation of the cAMP-type I PKA pathway is involved in the T cell dysfunction of HIV infection, as well as murine AIDS, a disease model induced by infection of C57BL/6 mice with LP-BM5, a mixture of attenuated murine leukemia viruses. LP-BM5-infected RIAD-transgenic mice resist progression of murine AIDS and have improved viral control. This underscores the cAMP-type I PKA pathway in T cells as a putative target for therapeutic intervention in immunodeficiency diseases.     

Clonal expansion of superantigen-reactive T cells is resistant to FK506 in mice with AIDS.

Subcutaneous injection of FK506 (10 mg/kg of body weight) completely blocked the clonal expansion of staphylococcal enterotoxin A (SEA)-reactive T cells in healthy (control) mice after SEA injection but did not disturb it in mice with murine AIDS (MAIDS) caused by infection with LP-BM5 murine leukemia virus. MAIDS mice are characterized by utilization of a FK506-insensitive pathway for clonal expansion of superantigen-reactive T cells.     

Determination of new biomarkers to monitor the dietary consumption of isothiocyanates

Isothiocyanates (ITCs) found in cruciferous vegetables have been associated with a reduced cancer risk in humans. We determined serum albumin adducts of allyl isothiocyanate (AITC), benzylisothiocyanate (BITC), phenylethylisothiocyanate (PEITC) and sulforaphane (SFN) in 85 healthy men from a dietary, randomized, controlled trial. After enzymatic digestion of albumin we determined the adducts of the ITCs with lysine (Lys) using liquid chromatography–tandem mass spectrometry. At the beginning of the study (and after 4 weeks) 4.7% (2.4%), 48.2% (35.3%), 5.9% (10.6%), and 24.7% (23.5%) of the samples were found positive for AITC-Lys, BITC-Lys, PEITC-Lys and SFN-Lys, respectively. This method enables the quantification of ITC adducts in albumin from large, prospective studies on diet and cancer.     

Modulation of murine AIDS-related pathology by concurrent antibody treatment and coinfection with Leishmania major.

Infection of C57BL/6 mice with a mixture of murine leukemia viruses (MuLVs) designated LP-BM5 MuLV leads to a disease characterized by progressive immunodeficiency and lymphoproliferation, known as murine AIDS (MAIDS). The development of MAIDS is associated with increased B-cell lymphoblast proliferation, but there is reason to believe that T-cell function and, particularly, T-cell-derived cytokines may also play a role. We have previously shown that concurrent infection with Leishmania major (which induces a strongly polarized Th1 response in C57BL/6 mice) and LP-BM5 MuLV modulates the disease induced by both infections. Here we show by treatment of mice with anticytokine antibodies that this modulation is largely exerted through the balance of Th1 and Th2 cytokines. Infected mice treated with antibodies to interleukin-4 and interleukin-10 exhibited a delayed development of MAIDS-related pathology and maintained T-cell responsiveness longer than mice treated with control antibody. Gamma interferon induced by coinfection with L. major synergized with anti-IL-4 treatment to inhibit the development of MAIDS pathology. Conversely, treatment with anti-gamma interferon led to a significant increase in splenomegaly and lymphadenopathy and slightly exacerbated loss of T-cell function. These data suggest that the production of Th2-associated cytokines may promote MAIDS pathology, while Th1-associated cytokines may help control the disease.     

Quinolinic Acid Levels in a Murine Retrovirus-Induced Immunodeficiency Syndrome

Abstract: Mice infected with the retrovirus mixture designated LP-BM5 murine leukemia virus (MuLV) develop an immunosuppressive disease. Quinolinic acid (QUIN) is an endogenous neurotoxic N -methyl- d -aspartate agonist that may contribute to the pathogenesis of HIV-associated neurologic disease. In the present study, the levels of QUIN in brain and blood were measured in mice infected with LP-BM5 MuLV and compared with those in uninfected mice and mice infected with the nonpathogenic strain of ecotropic MuLV (helper component of LP-BM5 MuLV). Infection with LP-BM5 MuLV resulted in progressive increases in blood QUIN levels beginning 2 weeks after inoculation that peaked by 16 weeks postinfection. QUIN levels were also increased in cerebral cortex, hippocampus, and striatum. In systemic tissues, QUIN levels were increased in lung, liver, and spleen. In contrast, infection with the ecotropic viral component of the LP-BM5 MuLV mixture was not associated with any changes in brain, blood, or systemic tissue QUIN levels, even though helper virus burdens were comparable to those in mice infected with LP-BM5 MuLV. Treatment of LP-BM5 MuLV-infected mice with the antiretroviral agent zidovudine (azidothymidine) significantly reduced blood and brain QUIN levels in association with reductions in viral load in brain and spleen. These observations suggest that elevated QUIN production is not attributable to productive infection with retrovirus per se but occurs in response to an agent or agents, such as cytokines, that are produced by the host in response to virus infection.     

Immunomodulation by pycnogenol® in retrovirus-infected or ethanol-fed mice

Pycnogenol^® is a commercial mixture of bioflavonoids that exhibits antioxidative activity. The effects of dietary pycnogenol on immune dysfunction in normal mice as well as those fed ethanol or infected with the LP-BM5 murine retrovirus were determined. The ethanol consumption and retrovirus infection cause abnormalities in the function and/or structure of a broad array of cells involved in humoral and cellular immunity. Pycnogenol enhanced in vitro IL-2 production by mitogen-stimulated splenocytes if its production was suppressed in ethanolfed or retrovirus-infected mice. Mitogenesis of splenocytes did not show a significant change in mice treated with pycnogenol. It reduced the elevated levels of interleukin-6 produced in vitro by cells from retrovirus infected mice and IL-10 secreted by spleen cells from mice consuming ethanol. Natural killer cell cytotoxicity was increased with pycnogenol treatment.