The functional gamma-secretase inhibitor prevents production of amyloid beta 1-34 in human and murine cell lines.

The amyloid precursor protein (APP) undergoes two consecutive cleavages by different proteases, beta-secretase and gamma-secretase, leading to the release of an amyloidogenic 4 kDa fragment called amyloid beta (Abeta). Combining immunoprecipitation and mass spectrometry, we characterized soluble Abeta in cultured cell media of mouse neuroblastoma N2a cells and double hAPP/hBACE-1 transfected HEK293. The major Abeta isoforms detected were Abeta11-34, Abeta1-34, Abeta11-40 and Abeta1-40. In this study, we demonstrate that overexpression of human beta-secretase (BACE-1) in HEK293 cells resulted in predominant Abeta cleavage at position Glu(11) rather than Asp(1), as well as increased production of Abeta(x)-34, but not Abeta(x)-40. Incubation of cells with a specific gamma-secretase inhibitor suggests that cleavage of APP at Leu(34) could be mediated by gamma-secretase itself or by a gamma-secretase dependent process.

Development and application of monoclonal antibodies against SKALP/elafin and other trappin family members.

SKALP/elafin is an epithelial proteinase inhibitor with antimicrobial properties that is not normally expressed in human epidermis, but is induced under inflammatory conditions and in some types of skin cancer. SKALP is a member of the recently described trappin gene family, which encodes a new class of proteins, characterized by a four-disulphide core and a transglutaminase substrate domain. Polyclonal antisera against SKALP have been shown to be useful for monitoring disease activity in psoriasis and tumour differentiation in squamous cell carcinoma. We developed ten different mouse monoclonal antibodies (mAbs) against synthetic peptides corresponding to a hexapeptide epitope in the transglutaminase substrate domain and three mAbs recognizing an epitope in the proteinase-inhibiting domain. The antibodies could be used with high specificity by immunohistochemistry on formalin-fixed tissue, by affinity chromatography, by Western blotting, and by enzyme-linked immunoadsorbent assay (ELISA) for the detection of SKALP/elafin. These antibodies have several advantages over existing polyclonal antisera, such as a defined epitope, the detection of full-length SKALP/elafin and unlimited supply. An antibody against the hexapeptide epitope, which is common to all known human, simian, bovine and swine trappin family members, was used to immunolocalize bovine trappins expressed in trachea, that have recently been discovered. These mAbs will serve as important new tools to measure SKALP/elafin and trappin family members in research and diagnostics.

Dimethylfumarate is an inhibitor of cytokine-induced nuclear translocation of NF-kappa B1, but not RelA in normal human dermal fibroblast cells.

We previously demonstrated that the oral antipsoriatic dimethylfumarate is an inhibitor of cytokine-induced adhesion molecule expression in endothelial HUVEC cells. We now report the inhibitory effect of dimethylfumarate on tumor-necrosis-factor-alpha- or interleukin-1 alpha-induced intercellular adhesion molecule 1 expression in normal human dermal fibroblasts. Western blots of normal human dermal fibroblast cytoplasmic extracts showed that dimethylfumarate has minor effects on the I kappa B alpha, beta and epsilon proteins: their cytokine-induced degradation and resynthesis is only slowed down, an effect most prominently observed for I kappa B beta. No inhibitory effect of dimethylfumarate was observed on cytokine-induced RelA/p65 or c-Rel accumulation in nuclear extracts of cytokine-treated normal human dermal fibroblast cells. In contrast, cytokine-induced nuclear factor kappa B1/p50 nuclear accumulation was specifically inhibited by dimethylfumarate. This inhibitory effect on nuclear factor kappa B1 nuclear localization in normal human dermal fibroblasts proved sufficient to inhibit nuclear factor kappa B1-RelA binding to nuclear factor kappa B consensus oligonucleotides in DNA binding assays. Likewise, cytokine-induced activation of a pNF kappa B::luciferase reporter construct in transiently transfected normal human dermal fibroblasts was inhibited by dimethylfumarate. The observations support a mechanistic model for the oral antipsoriatic dimethylfumarate in which lowering of nuclear factor kappa B1 leads to changes in the nuclear factor kappa B1-RelA nuclear balance and inhibition of cytokine-induced adhesion molecule expression in normal human dermal fibroblasts.

Biological activity of hexitol nucleic acids targeted at Ha-ras and intracellular adhesion molecule-1 mRNA.

Hexitol nucleic acid (HNA) is a new steric blocking oligonucleotide, hybridizing sequence selectively with RNA. The biological activity of HNA was evaluated in an in vitro translation arrest system targeting Ha-ras mRNA and in a cellular system targeting intracellular adhesion molecule-1 (ICAM-1) expression. HNA very efficiently and selectively inhibited Ha-ras mRNA translation (IC(50) of 50 nM) when targeted at the translation initiation region. When targeting at the 12th codon region, a gap-mer approach was needed to inhibit mRNA translation. Similarly, HNA inhibited ICAM-1 expression in keratinocytes when targeting at codon sequences. In this test system, HNA is less active but more selective than phosphorothioates, but needs lipofection to become active in keratinocytes. This new steric blocker may be an efficient antisense agent providing that enough material can be brought into cells.

Retinoic acid potentiates TNF-alpha-induced ICAM-1 expression in normal human epidermal keratinocytes.

ICAM-1 protein in keratinocytes is thought to contribute to cutaneous inflammatory reactions. Its induction depends-among others-on cytokines such as TNF-alpha, IFN-gamma, IL-1 or on retinoic acid (RA), a key regulator of epidermal homeostasis. We investigated the effect of treatments with TNF-alpha, RA or their combination on ICAM-1 expression on proliferative or differentiating keratinocytes over an 8 day culture period. Basal ICAM-1 levels were undetectable at low (30 microM) and standard (88 microM) Ca2+ and RA alone did not induce ICAM-1. However, at high Ca2+ (1500 microM), ICAM-1 levels were augmented in response to RA-treatment. TNF-alpha induced a transient ICAM-1 increase in NHK, which reached peak-levels 2-4 days post cytokine stimulus. RA potentiated the TNF-alpha-induced ICAM-1 response in all Ca2+-concentrations. This potentiating effect of RA was confirmed at the mRNA level. In summary, our results establish retinoic acid as an enhancer of TNF-alpha-induced ICAM-1 levels in NHK.

Dimethylfumarate is an inhibitor of cytokine-induced E-selectin, VCAM-1, and ICAM-1 expression in human endothelial cells.

Most studies on the antipsoriatic mode of action of dimethylfumarate focused on its antiproliferative effects in keratinocytes. Because inflammatory skin diseases are associated with an upregulation of endothelial cell adhesion molecules and because the presence of inflammatory cells in dermis and epidermis is considered an important feature in psoriasis, we tested the effect of DMF on cytokine-induced adhesion molecule expression in HUVEC, using in situ ELISA and Northern blotting. Dimethylfumarate inhibited ICAM-1, VCAM-1, and E-selectin expression and reduced adhesion of U937 cells to stimulated HUVEC. Monoethylfumarate and fumaric acid had no effect. Similar inhibitory effects for DMF on VCAM-1 expression were observed after stimulation of HUVEC with LPS, PMA, IL-4, and IL-1 alpha or in combinations with TNF alpha. These data are in agreement with previously reported effects of DMF on intracellular thiol levels and inhibition of NF-kappa B activation. The inhibitory effect on cytokine-induced endothelial adhesion molecule expression may represent another target of dimethylfumarate in psoriasis.

Microtubule-associated protein tau epitopes are present in fiber lesions in diverse muscle disorders.

The microtubule-associated protein tau is a major cytoskeletal protein involved in the neurofibrillary tangles of Alzheimer's disease. Although tau is predominantly a neuronal protein, it has been demonstrated in glia and other nonneuronal cells. We describe the presence of microtubule-associated protein tau epitopes in various muscle fiber lesions in oculopharyngeal and Becker muscular dystrophy, dermatomyositis, central core disease, neurogenic atrophy, and in the recovery phase of an attack of malignant hyperthermia. Western blot demonstrated a 100- to 110-kd tau-immunoreactive protein probably corresponding to 'big tau' as described in peripheral nerves. Tau immunoreactivity in muscle fiber lesions usually co-localized with tubulin, although electron microscopy failed to show an increase in microtubules. Tau and tubulin reactivity also correlated with the presence of desmin and vimentin epitopes. Possible explanations for the presence of tau are briefly discussed.

The phosphatase inhibitor okadaic acid induces a phosphorylated paired helical filament tau epitope in human LA-N-5 neuroblastoma cells.

Recently, a mitogen activated protein kinase has been implicated in the generation of a phosphorylated paired helical filament (PHF) epitope recognized by the monoclonal antibody AT8. This epitope consists of phosphorylated serines 199 and/or 202 of the human microtubule associated protein tau. Theoretically, aside from abnormal kinase activity, inhibition of phosphatase activity could also be involved in the abnormal phosphorylation status of the microtubule associated protein tau. To investigate this, we incubated LA-N-5 neuroblastoma cells with okadaic acid, a specific inhibitor of phosphatase 2A. We found that incubating neuroblastoma cells with okadaic acid induces the abnormally phosphorylated AT8 epitope. The effect of okadaic acid is time and dose dependent and is reversible. Our findings suggest that phosphatase activity is important in the regulation of the phosphorylation state of tau. Phosphatases may act directly on tau or may influence the activity of mitogen activated protein kinase. Incubation of LA-N-5 neuroblastoma cells with okadaic acid provides a cellular model in which the generation of a well-defined PHF-tau epitope can be investigated.

Isolation of IgG1-secreting switch variants from IgM hybridomas produced after in vitro immunization.

IgG1-secreting variants have been isolated from three different IgM-secreting hybridomas, in two instances following in vitro immunization. The method used was based on sequential sublining in combination with selection by an IgG1-specific two-site ELISA system employing two different IgG1-specific polyclonal antisera. Idiotypic identity between the IgG1 variants and their respective IgM parent was demonstrated using syngeneic anti-idiotypic antisera. The antigen binding specificity in the IgG1 variants was also conserved. Isolation of naturally occurring IgG1 switch variants from IgM-secreting hybridomas that are produced after in vivo immunization offers a solution to the major disadvantages associated with the generation of IgM hybridomas.

A monoclonal antibody raised against Alzheimer cortex that specifically recognizes a subpopulation of microglial cells.

A monoclonal antibody (MAb), termed AMC30, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer's brain. The antigen to which AMC30 is directed was expressed by microglial cells in senile plaques of Alzheimer's disease (AD). Microglia in the parenchyma surrounding brain tumors or infarctions, multinuclear giant cells, perivascular and parenchymal macrophages throughout the brain of AIDS patients were also labeled. Different non-nervous system lesions in which macrophages participate were also stained. Microglial cells in normal areas of the cortex or white matter were not labeled with MAb AMC30. The antigen to which AMC30 is directed was not detected in normal bone marrow, lymph nodes, lung, or spleen monocytes or macrophages. The epitope recognized by MAb AMC30 was present after formalin fixation and paraffin embedding. Our findings suggest that this MAb is directed against an antigen that is specifically expressed in a subpopulation of microglial cells and macrophages reactive to various pathological conditions.

Human myelin basic protein-specific cytolytic T lymphocyte clones are functionally restricted by HLA class II gene products.

Cellular immune reactions against the autoantigen myelin basic protein (MBP) are strongly implicated in the occurrence of postinfectious and postvaccination encephalomyelitis. Clinical autoimmune encephalomyelitis in experimental animals can be transferred with cloned MBP-specific cytolytic major histocompatibility complex Class II-restricted T lymphocytes. The HLA restriction pattern of specific proliferative and cytolytic functions of two human MBP-specific cytotoxic T lymphocyte clones, derived from two different multiple sclerosis patients, was analyzed in detail. Using monoclonal antibodies against various HLA gene products and allogeneic Epstein-Barr virus-transformed B cells as antigen-presenting cells and as targets for cytolysis, it was found that MBP-specific functions of the T cell clones was restricted by HLA class II antigens, and, more specifically, by molecules encoded for by DR locus genes.

Activation through CD3 molecule leads to clonal expansion of all human peripheral blood T lymphocytes: functional analysis of clonally expanded cells.

Monoclonal antibodies directed against CD3, a T cell-specific surface molecule essentially required for activation of these cells, are highly mitogenic for resting human peripheral blood T lymphocytes. A predetermined optimal concentration of anti-CD3 monoclonal antibody WT32 was employed to activate T cells cultured in limiting-dilution microcultures containing irradiated feeder cells and exogeneous interleukin 2. Frequencies of cells triggered into clonal expansion by WT32 under these culture conditions were 0.57 to 0.72 and 0.90 to 1.10 in peripheral blood mononuclear cells and E rosette-positive cells, respectively. It appeared that WT32 could induce virtually every human peripheral blood T lymphocyte to expand into a clonal progeny of 5 to 40 X 10(4) cells in 14 to 18 days of culture. This progeny was tested for cytolytic effector function with 51Cr-labeled murine P815 targets in the presence of PHA to detect all cytotoxic T lymphocytes (CTL) regardless of specificity, and was also assayed for natural killer like activity against K562 target cells. Frequencies of cells in the human peripheral blood T cell compartment giving rise to a clonal progeny expressing CTL function was 1/3, whereas 1/6 to 1/5 expanded into effector cell populations possessing NK activity. Frequency analysis of CD4-positive and CD8-positive populations, activated by WT32 in limiting dilution microcultures, demonstrated that 1 to 6% of the CD4-positive and 100% of the CD8-positive peripheral blood T lymphocytes expanded into CTL.