Adjuvant activities of immune response modifier R-848: comparison with CpG ODN.

R-848 and imiquimod belong to a class of immune response modifiers that are potent inducers of cytokines, including IFN-alpha, TNF-alpha, IL-12, and IFN-gamma. Many of these cytokines can affect the acquired immune response. This study examines the effects of R-848 on aspects of acquired immunity, including immunoglobulin secretion, in vivo cytokine production, and Ag-specific T cell cytokine production. Results are compared with those of Th1 CpG ODN. R-848 and CpG ODN are effective at skewing immunity in the presence of Alum toward a Th1 Ab response (IgG2a) and away from a Th2 Ab response (IgE). R-848 and CpG ODN are also capable of initiating an immune response in the absence of additional adjuvant by specifically enhancing IgG2a levels. Both R-848 and imiquimod showed activity when given subcutaneously or orally, indicating that the compound mechanism was not through generation of a depot effect. Although CpG ODN behaves similarly to R-848, CpG ODN has a distinct cytokine profile, is more effective than R-848 when given with Alum in the priming dose, and is active only when given by the same route as the Ag. The mechanism of R-848's adjuvant activity is linked to cytokine production, where increases in IgG2a levels are associated with IFN-alpha, TNF-alpha, IL-12, and IFN-gamma induction, and decreases in IgE levels are associated with IFN-alpha and TNF-alpha. Imiquimod also enhances IgG2a production when given with Ag. The above results suggest that the imidazoquinolines R-848 and imiquimod may be attractive compounds for use as vaccine adjuvants and in inhibiting pathological responses mediated by Th2 cytokines.

Dendritic cell maturation and subsequent enhanced T-cell stimulation induced with the novel synthetic immune response modifier R-848.

Agents that enhance dendritic cell maturation can enhance T-cell activation and therefore may improve the efficiency of vaccines or improve cellular immunotherapy. Previously, we demonstrated that a novel low-molecular-weight synthetic immune response modifier, R-848, induces IL-12 and IFN-alpha secretion from monocytes and macrophages. Here we report that R-848 induces the maturation of human monocyte-derived dendritic cells. Characteristic of dendritic cell maturation, R-848 treatment induces cell surface expression of CD83 and increases cell surface expression of CD80, CD86, CD40, and HLA-DR. Additionally, R-848 induces cytokine (IL-6, IL-12, TNF-alpha, IFN-alpha) and chemokine (IL-8, MIP-1alpha, MCP-1) secretion from dendritic cells. Most significantly, R-848 enhances dendritic cell antigen presenting function, as measured by increased T-cell proliferation and T-cell cytokine secretion in both allogeneic and autologous T-cell systems. Consequently, low-molecular-weight synthetic molecules such as R-848 and its derivatives may be useful as vaccine adjuvants or as ex vivo stimulators of dendritic cells for cellular immunotherapy.

Modulation of TH1 and TH2 cytokine production with the immune response modifiers, R-848 and imiquimod.

Cytokines produced by antigen-presenting cells are known to affect the development and cytokine profile of T cells. The immune response modifiers imiquimod and R-848 were previously shown to stimulate human and mouse cultures to secrete interferon-alpha. Results from the present study demonstrate that R-848 and imiquimod are capable of inducing interleukin-12 and interferon-gamma in mouse and human cell cultures. Both CD4(+) and CD8(+) T lymphocytes were responsible for producing IFN-gamma following stimulation with R-848. Macrophages were required for induction of interferon-gamma by R-848 and the cytokines IFN-alpha and IL-12 mediated this response. R-848 and imiquimod were also found to inhibit IL-4 and IL-5 production in mouse and human culture systems. The inhibition of IL-5 in response to R-848 is seen in cultures containing CD4(+) lymphocytes and macrophages and is mediated in part by IFN-alpha. These data suggest that imiquimod and R-848 may have clinical utility in diseases where cell-mediated immune responses are important and in diseases associated with overexpression of IL-4 or IL-5 such as atopic disease.

Contribution of the ICE family to neurodegeneration.

The ICE family of cysteine proteases mediates necrotic or apoptotic events in the nervous system as well as in other tissues. This suggests that inhibitors may be of therapeutic value in acute and, perhaps, chronic neurodegenerative disease. In addition, some members of this family may respond to intercellular signals controlling proliferation or differentiation. This possibility should be kept in mind as therapeutics are pursued.

Inhibition of the interleukin-1 beta converting enzyme family rescues neurons from apoptotic death.

The interleukin-1 beta converting enzyme (ICE) family of cysteine proteases has been implicated in apoptosis. This study tested the effects of a novel pan-ICE family inhibitor, bocaspartyl(OMe)-fluoromethylketone (boc-Asp-CH2F), against low potassium-induced apoptosis of cultured rat cerebellar granule neurons (CGN). A single application of this cell-permeant compound (20 microM) inhibited apoptotic cell death up to 48 h. Classical apoptotic changes were monitored by fluorescence microscopy, DNA fragmentation and scanning electron microscopy (SEM). A control peptidic fluoromethylketone (boc-Thr-CH2F), and inhibitors to calpain (Ac-Leu-Leu-norleucinal), cathepsin B (Z-Phe-Ala-CH2F), and CPP32-like proteases (Z-DEVD-CH2F), failed to prevent apoptotic death. An 35S-methionine incorporation assay verified that, unlike cycloheximide, boc-Asp-CH2F did not inhibit protein synthesis, hence excluding this as a rescuing mechanism. Although ICE was not detected by northern blot analysis, both CPP32 and Nedd2 expression were found to increase during apoptosis. Kinetic assays with cell extracts from boc-Asp-CH2F-treated neurons measured reduced rates of cleavage for DEVD-pNA and LEVD-pNA. At present, ICE-like proteases remain viable candidates for mediating neuronal death.

Caspase-3/CPP32-like activity is not sufficient to mediate apoptosis in an IL-2 dependent T cell line.

CTLL cells undergo apoptosis when cultured in the absence of IL-2. The IL-1beta-converting-enzyme (ICE)/ caspase family has been implicated as an integral component of some forms of apoptosis. Numerous members of the caspase family have been identified, and it appears as if caspase-3/CPP32 plays a critical role. Previously we demonstrated that ICE/caspase-1 expression increases in CTLL cells during apoptosis; however, inhibition of ICE activity did not abrogate apoptotic death. The purpose of this report is to determine if other members of the caspase family are involved in T cell apoptosis induced by growth factor starvation. We show that cytosolic CPP32-like activity, as measured by the cleavage of DEVD-pNA and poly(ADP-ribose) polymerase (PARP), increases during apoptosis following growth factor deprivation. Cytosolic CPP32-like activity is inhibited in cells treated with the broad spectrum ICE family inhibitor boc-aspartyl(OMe)-fluoromethylketone (D-FMK) and by VAD-FMK and DEVD-FMK which have greater specificity for CPP32-like ICE homologs; however, only the broad spectrum ICE inhibitor D-FMK inhibited apoptosis. Our results suggest that apoptosis induced by growth factor deprivation involves the caspase family, but increased CPP32-like activity is not sufficient to mediate apoptosis induced by IL-2 starvation.

Genetic and metabolic status of NGF-deprived sympathetic neurons saved by an inhibitor of ICE family proteases.

Sympathetic neurons undergo programmed cell death (PCD) when deprived of NGF. We used an inhibitor to examine the function of interleukin-1 beta-converting enzyme (ICE) family proteases during sympathetic neuronal death and to assess the metabolic and genetic status of neurons saved by such inhibition. Bocaspartyl(OMe)-fluoromethylketone (BAF), a cell-permeable inhibitor of the ICE family of cysteine proteases, inhibited ICE and CPP32 (IC50 approximately 4 microM) in vitro and blocked Fas-mediated apoptosis in thymocytes (EC50 approximately 10 microM). At similar concentrations, BAF also blocked the NGF deprivation-induced death of rat sympathetic neurons in culture. Compared to NGF-maintained neurons, BAF-saved neurons had markedly smaller somas and maintained only basal levels of protein synthesis; readdition of NGF restored growth and metabolism. Although BAF blocked apoptosis in sympathetic neurons, it did not prevent the fall in protein synthesis or the increase in the expression of c-jun, c-fos, and other mRNAs that occur during neuronal PCD, implying that the ICE-family proteases function downstream of these events during PCD.NGF and BAF rescued sympathetic neurons with an identical time course, suggesting that NGF, in addition to inhibiting metabolic and genetic events associated with neuronal PCD, can act posttranslationally to abort apoptosis at a time point indistinguishable from the activation of cysteine proteases. Both poly-(ADP ribose) polymerase and pro-ICE and Ced-3 homolog-1 (ICH-1) appear to be cleaved in a BAF-inhibitable manner, although the majority of pro-CPP32 appears unchanged, suggesting that ICH-1 is activated during neuronal PCD. Potential implications of these findings for anti-apoptotic therapies are discussed.

Watch for ICE in neurodegeneration.

Neuronal death occurs naturally during brain development and is a common response to an external insult. Cell death, whose mechanisms are currently being elucidated, appears in three forms: necrosis, apoptosis and programmed cell death. Recently, attention has focused on a family of cysteine proteases whose prototype is interleukin-1 beta converting enzyme (ICE). ICE, essential for IL-1 beta production and, thus, critical to necrotic mechanisms, also plays a role in apoptosis mediated through the stimulation of the lymphocyte fas antigen. The absence of ICE expression in neurons makes ICE an unlikely direct participant in neuronal death. However, the existence of ICE family members in neurons combined with the pharmacological inhibition of both apoptosis in vitro and programmed cell death during development make ICE homologs candidates for mediating these two forms of cell death. Since several neurodegenerative diseases as well as at least one neurological disorder may have an apoptotic component, antagonists of this protease family may be neuroprotective.

IL-1 beta converting enzyme (ICE) is not required for apoptosis induced by lymphokine deprivation in an IL-2-dependent T cell line.

Clonal T cells undergo programmed cell death (PCD) or apoptosis when cultured without the appropriate cytokines. The cysteine protease, IL-1 beta converting enzyme (ICE), is implicated in apoptosis based on its structural similarity to the PCD gene, ced-3, in Caenorhabditis elegans and the induction of PCD in fibroblasts transfected with recombinant ICE. We show that the murine IL-2-dependent CTLL T cell line expresses ICE but not IL-1 beta. Interestingly, ICE mRNA and protein levels increase during apoptosis. Yet inhibition of ICE enzymatic activity (> 90%) with either of two cell-permeable ICE inhibitors does not abrogate or delay apoptosis following IL-2 deprivation, as measured by DNA fragmentation and viability. Our results suggest that ICE is not required for apoptosis in lymphokine-deprived T cells.

Interleukin-1 beta dissociates beta-amyloid precursor protein and beta-amyloid peptide secretion.

A heightened production of interleukin 1 beta (IL-1 beta) has been reported in microglial-associated amyloid deposits in Alzheimer's disease (AD) brains. These plaques are composed predominantly of beta/A4 peptide derived from beta-amyloid precursor protein (beta APP). We demonstrate that short-term (1 h) IL-1 beta-treatment increases beta APPs secretion and concomitantly decreases cell-associated beta APP in human H4 neuroglioma cells. Long-term (5 h) IL-1 beta treatment did not alter secreted or cell-associated beta APP content. In contrast, the secretion of beta/A4-containing epitope was not affected by short-term IL-1 beta stimulation; however, long-term IL-1 beta treatment decreased the amount of beta/A4-containing epitope secreted from the cells. These results show that IL-1 beta modifies the processing and secretion of beta APP to exacerbate perhaps the neuropathology of AD.

Enhancement of CTL response induced by a viral peptide using cationized BSA, a Th1-stimulating adjuvant.

We have recently shown that it is possible to activate cytotoxic T cells (CTL) in vivo with HSV-1 glycoprotein B H-2Kb-restricted peptide (gB peptide) independent of CD4+ T cell help. Here we report that the gB peptide-specific CTL response is significantly enhanced when mice are immunized with a mixture of gB peptide and cationized BSA (cBSA). The latter molecule is a positively charged form of the native BSA molecule that stimulates CD4+ T cells to produce cytokines characteristic of Th1 cells. The cBSA-enhanced CTL response required the presence of CD4+ T cells, but it did not require stimulation in vitro by antigen or exogenous cytokines. gB peptide/cBSA-activated LN cells transcribed IL-2 and IFN-gamma, but only IL-2 was essential for CTL development. Our data demonstrate that while activation of CTL may occur in the absence of CD4+ cells, cytokines produced by CD4+ Th1 cells provide stimulatory signals during CTL maturation. Thus, cotreatment with a substance that activates Th1 CD4+ cells may be useful for achieving maximal CTL responsiveness.

Herpes simplex virus class I-restricted peptide induces cytotoxic T lymphocytes in vivo independent of CD4+ T cells.

Cytotoxic T cells were induced in vivo by immunizing C57BL/6 mice with a single dose of a short synthetic peptide representing amino acid residues 497-507 from HSV-1 glycoprotein B. Primed lymph node cells did not require in vitro boosting with peptide and APC for CTL detection. The CTL were CD8+ and H-2b restricted. They were capable of lysing target cells exogenously sensitized with peptide or endogenously processed glycoprotein B. Virus-primed CTL produced an anamnestic response in vivo upon peptide challenge, indicating that peptide-specific CTL may be relevant to infection. The requirement of CD4+ T cells for CD8+ CTL activation was investigated by depleting CD4+ cells in vivo with GK1.5 mAb. CD4+ T cell depletion did not abrogate CTL generation. These results suggest that glycoprotein B peptide 497-507 activates CD8+ CTL in vivo in a manner independent of CD4+ T cells. This is the first study in which a class I-restricted peptide derived from a HSV protein was used to activate CTL in vivo, and in which a synthetic peptide was shown to activate CTL independent of CD4+ T cell help. Our data are relevant to viral vaccine development and to processing and presentation of viral epitopes.

Mitral valve prolapse: a review of the syndrome with emphasis on current antibiotic prophylaxis.

Mitral valve prolapse syndrome (MVPS) is the name given to the heart valve abnormality described by Barlow over two decades ago. This condition is of particular importance to the dentist as these patients are thought to be at risk of developing infective endocarditis with routine dental procedures which may cause gingival bleeding. This paper is an updated version of an article that originally appeared in the University of Toronto Dental Journal in 1990. In 1984, guidelines for antibiotic prophylaxis were provided by the American Heart Association (AHA). More current prophylactic regimens are also presented, with emphasis on the current recommendations of the AHA, recently published in December of 1990.

Effect of in vivo T-cell depletion on the effector T-cell function of immunity to Eimeria falciformis.

BALB/c mice were exposed to the enteric parasite Eimeria falciformis to produce a natural acquired immunity. The mice were then depleted of their effector T-cell function by in vivo administration of a cytotoxic Thy-1.2 mouse monoclonal antibody. T-cell depletion was demonstrated by a reduction in concanavalin A-induced proliferation of splenic lymphocytes in treated mice compared with that in controls. Twenty-four hours following T-cell depletion, the mice were challenged with 5,000 oocysts of E. falciformis. Daily total oocyst counts were done for each mouse from days 6 to 21 following challenge. Our studies demonstrated that depleting mice of their effector T-cell function following establishment of immunity caused an abrogation of protective immunity to this parasite.