BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major components of plaque, beta-amyloid peptides (Abetas), are produced from amyloid precursor protein (APP) by the activity of beta- and gamma-secretases. beta-secretase activity cleaves APP to define the N-terminus of the Abeta1-x peptides and, therefore, has been a long- sought therapeutic target for treatment of AD. The gene encoding a beta-secretase for beta-site APP cleaving enzyme (BACE) was identified recently. However, it was not known whether BACE was the primary beta-secretase in mammalian brain nor whether inhibition of beta-secretase might have effects in mammals that would preclude its utility as a therapeutic target. In the work described herein, we generated two lines of BACE knockout mice and characterized them for pathology, beta-secretase activity and Abeta production. These mice appeared to develop normally and showed no consistent phenotypic differences from their wild-type littermates, including overall normal tissue morphology and brain histochemistry, normal blood and urine chemistries, normal blood-cell composition, and no overt behavioral and neuromuscular effects. Brain and primary cortical cultures from BACE knockout mice showed no detectable beta-secretase activity, and primary cortical cultures from BACE knockout mice produced much less Abeta from APP. The findings that BACE is the primary beta-secretase activity in brain and that loss of beta-secretase activity produces no profound phenotypic defects with a concomitant reduction in beta-amyloid peptide clearly indicate that BACE is an excellent therapeutic target for treatment of AD.

Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain.

Converging lines of evidence implicate the beta-amyloid peptide (Ass) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce A beta production by functionally inhibiting gamma-secretase, the activity responsible for the carboxy-terminal cleavage required for A beta production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon A beta production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP(V717F) reduces brain levels of Ass in a dose-dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain A beta in vivo. Development of such novel functional gamma-secretase inhibitors will enable a clinical examination of the A beta hypothesis that Ass peptide drives the neuropathology observed in Alzheimer's disease.

TNF-alpha blockade by a dimeric TNF type I receptor molecule selectively inhibits adaptive immune responses.

Tumor necrosis factor-alpha (TNF-alpha) is a mediator of severe inflammatory processes, including rheumatoid arthritis. Suppression of TNF with a soluble type I or type II receptor molecule (TNF-RI or TNF-RII) has the potential to decrease cytokine levels and modulate inflammatory diseases in humans. However, it has recently been reported that treatment of mice with a TNF-RI:Fc immunoadhesin protein augmented Gram positive infections and subsequent mortality. To determine if TNF-alpha blockade with soluble TNF-alpha receptors might alter immune system function, assays were assessed in rodents treated with a dimeric form of the p55 TNF-RI, Tumor Necrosis Factor-binding protein (TNFbp). Administration of TNFbp resulted in suppression of primary and secondary IgG antibody responses and cell-mediated immune function. No treatment-related differences were detected in immune-enhancing assays or non-specific immune function parameters. Bacterial host resistance assays with Listeria monocytogenes, Staphylococcus aureus or Escherichia coli showed an increase in tissue colony counts only with L. monocytogenes challenged animals following TNFbp administration. These results suggest that TNFbp has the capacity to inhibit adaptive immune function in experimental animal models. Studies suggest that while reducing TNF-alpha is important in controlling cytokine-dependent disease states, maintenance of a threshold level may be critical for normal immune function.

Preclinical safety evaluation of rhuMAbVEGF, an antiangiogenic humanized monoclonal antibody.

Recombinant humanized antivascular endothelial growth factor (rhuMAbVEGF) is a monoclonal IgG1 antibody that is being developed as an antiangiogenic agent for use in treating a variety of solid tumors. Preclinical safety studies included an immunohistochemical tissue cross-reactivity study, in vitro hemolytic potential and blood compatibility studies, and multiple dose toxicity studies. Toxicity studies were conducted in cynomolgus monkey because rhuMAbVEGF is pharmacologically active in this species and does not bind rat or mouse vascular endothelial growth factor (VEGF). Following twice weekly administration of rhuMAbVEGF for 4 or 13 wk, young adult cynomolgus monkeys exhibited physeal dysplasia characterized by a dose-related increase in hypertrophied chondrocytes, subchondral bony plate formation, and inhibition of vascular invasion of the growth plate. In addition, decreased ovarian and uterine weights and an absence of corpora lutea were observed in females receiving 10 and 50 mg/kg/dose in the 13-wk study. Both the physeal and ovarian changes were reversible with cessation of treatment. No other treatment-related effects were observed following rhuMAbVEGF administration at doses up to 50 mg/kg. These findings indicate that VEGF is required for longitudinal bone growth and corpora lutea formation and that rhuMAbVEGF can reversibly inhibit physiologic neovascularization at these sites.

Evaluation of interleukin-1 receptor antagonist (IL-1RA) and tumor necrosis factor binding protein (TNF-BP) in a rodent abscess model of host resistance.

Modulation of pro-inflammatory cytokine responses can alter the normal protective mechanisms against invading pathogens. The cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) are crucial in the inflammatory cascade for upregulation of adhesion molecule expression, neutrophil recruitment, and additional cytokine induction. To determine if the cytokine antagonists interleukin-1 receptor antagonist (IL-1ra) and tumor necrosis factor-binding protein (TNF-bp) alter host resistance mechanisms they were evaluated in a rodent abscess model. It has previously been shown that subcutaneous Staphylococcus aureus injections induce abscess formation in rats. These abscesses can be examined over a pre-determined time course for evaluation of size, severity and time to resolution. Treatment with immunosuppressive drug therapy can modify the normal course of abscess formation and/or resolution. IL-1ra and TNF-bp were administered either alone or in combination. Also, the effects of these cytokine antagonists in combination with dexamethasone were tested. Results indicated TNF-bp at any dose examined did not adversely alter any parameter of abscess formation or resolution. In contrast, high doses of IL-1ra increased abscess severity, while more clinically relevant doses did not. Combination treatment with IL-1ra and TNF-bp did not alter abscess parameters above individual findings. Dexamethasone, given in combination with either cytokine antagonist, significantly increased severity grading scores above dexamethasone given alone. Overall the data indicated high dosing regimens of IL-1ra or TNF-bp only caused transient impacts on this host resistance model, while more clinically relevant doses did not impact any aspect of the abscess. These findings demonstrate that these anti-cytokine therapies do not alter general host resistance.

In vivo exposure to ultraviolet radiation enhances pathogenic effects of murine leukemia virus, LP-BM5, in murine acquired immunodeficiency syndrome.

LP-BM5 murine leukemia virus (MuLV) induces an immunodeficiency syndrome (MAIDS) in C57BL/6 mice which resembles immunological abnormalities observed in early stages of human AIDS. In our study, MAIDS virus-infected mice were exposed to low doses of ultraviolet radiation (UVR) before and after virus inoculation and compared with MAIDS-infected but not UVR-exposed mice. In all tested parameters (blood IgM levels; mitogenic responses to PHA, ConA, LPS and anti-mu; MLR; antigenic response to SRBC; enlargement and histopathologic changes of the spleen) we observed the same trend: changes due to MAIDS infection were more pronounced in the UVR-exposed group than in the unexposed group. Statistically significant differences between these two groups were seen for mitogenic responses at two different time points after virus inoculation. These results demonstrate that in vivo UVR exposure enhances the immunosuppressive effects of a retroviral infection. UVR exposure may affect the progression of AIDS in a similar manner.

Primary immunization in the canine lung. Soluble antigen induces a localized response.

Primary immunization of the dog by intralobar instillation of particulate antigen induces an intense, localized pulmonary antibody response. In contrast, although soluble antigen can also induce local antibody responses after repeated deposition in the canine respiratory tract, its ability to induce local responses after primary immunization has not been well characterized. To document such responses, we immunized five beagle dogs using a bronchoscope to instill 10 mg keyhole limpet hemocyanin (KLH) into a single lung lobe (immunized) and saline into a contralateral lung lobe (control). Over the next 3 wk, we monitored specific immune responses in blood and bronchoalveolar lavage (BAL) fluids obtained from the immunized and control lung lobes. Primary intrapulmonary immunization of dogs with KLH resulted in anti-KLH antibody responses both in blood and in immunized and control BAL fluids. However, immunoglobulin class-specific expression of response differed between the immunized and control lung lobes. Specific IgM and IgA responses were significantly greater in the immunized lobes. In contrast, specific IgG, and cells producing specific IgG, were quantitatively similar in lavage fluids derived from immunized and control lung lobes. These studies demonstrate that primary immunization of the dog by intralobar instillation of soluble antigen stimulates a local IgM and IgA response and an IgG response that distributes to both immunized and unimmunized lung. This pattern of immunoglobulin class-specific pulmonary antibody response has the potential to importantly influence regional responses to intrapulmonary antigen.

The immunotoxicity of three nickel compounds following 13-week inhalation exposure in the mouse.

Groups of B6C3F1 mice were exposed to aerosols of nickel subsulfide (Ni3S2), nickel oxide (NiO), or nickel sulfate hexahydrate (NiSO4.6H2O) 6 hr/day, 5 days per week for 65 days to determine the immunotoxicity of these compounds. Exposure concentrations were 0.11, 0.45, and 1.8 mg Ni/m3 for Ni3S2, 0.47, 2.0, and 7.9 mg Ni/m3 for NiO; and 0.027, 0.11, and 0.45 mg Ni/m3 for NiSO4. Thymic weights were decreased only in mice exposed to 1.8 mg Ni/m3 Ni3S2. Increased numbers of lung-associated lymph nodes (LALN), but not spleen nucleated cells, were seen with all compounds. Nucleated cells in lavage samples were increased in mice exposed to the highest concentrations of NiSO4 and NiO and to 0.45 and 1.8 mg Ni/m3 Ni3S2. Increased antibody-forming cells (AFC) were seen in LALN of mice exposed to 2.0 and 7.9 mg Ni/m3 NiO and 1.8 mg Ni/m3 Ni3S2. Decreased AFC/10(6) spleen cells were observed in mice exposed to NiO, and decreased AFC/spleen were seen for mice exposed to 1.8 mg Ni/m3 Ni3S2. Only mice exposed to 1.8 mg Ni/m3 Ni3S2 had a decrease in mixed lymphocyte response. All concentrations of NiO resulted in decreases in alveolar macrophage phagocytic activity, as did 0.45 and 1.8 mg Ni/m3 Ni3S2. None of the nickel compounds affected the phagocytic activity of peritoneal macrophages. Only 1.8 mg Ni/m3 Ni3S2 caused a decrease in spleen natural killer cell activity. Results indicate that inhalation exposure of mice to nickel can result in varying effects on the immune system, depending on dose and physicochemical form of the nickel compound. These nickel-induced changes may contribute to significant immunodysfunction.

Separation and characterization of basal and secretory cells from the rat trachea by flow cytometry.

Basal and secretory cells have been separated as highly enriched viable populations from single-cell suspensions of rat tracheal epithelial cells. Isolation of the populations was achieved by preparation of a cell suspension and separation by flow cytometry using contour maps generated from 2 degrees and 90 degrees light scatter signals. Flow cytometric analysis of cells showed 10% of the whole preparation were cells in SG2M phase of the cell cycle. The secretory cells accounted for 86% of these cycling cells; the remainder were accounted for by the basal cells. Culture of sorted populations of basal and secretory cells in serum free defined medium showed that basal cells had a lower (0.6%) colony-forming efficiency than secretory cells (3.4%). Significant differences in blue auto-fluorescence, Hoechst 33342 uptake, and lectin staining were apparent between basal and secretory cells. These results suggest that the secretory cell rather than the basal cell is primarily the cell type involved in maintenance of the normal tracheal epithelium. Secretory cells are greater in number, have a higher proliferative potential, and greater metabolic capability. Because of these traits they may be a critical cell at risk from damage by environmental agents.

Bronchoalveolar lavage cytology in cynomolgus monkeys and identification of cytologic alterations following sequential saline lavage.

Total and differential cell counts were determined on cytolytic specimens obtained by fiberoptic bronchoscopy and bronchoalveolar lavage (BAL) of five normal cynomolgus monkeys. Total nucleated cell counts ranged from 100 to 430 cells/microliters. Macrophages were approximately 91% of total nucleated cells, while lymphocytes were 3%, neutrophils 4%, and eosinophils 2% of the initial BAL from each monkey. Less than 1% of the cells were mast cells and ciliated or nonciliated epithelial cells. The effects of repeated saline BAL on pulmonary cell populations were evaluated. Saline lavage of individual lung lobes resulted in a marked rise in circulating blood neutrophils at 4 hr after BAL; there was a similar rise in neutrophils in lavage fluids 24 hr after the initial lavage. Differential and total cell counts of both blood and lavage fluid returned to normal if subsequent lavages were spaced at 48-hr intervals. Lymphocytes were not present in saline-lavaged lung lobes, and protein levels of lavage fluids did not rise significantly. BAL produced a transient, reversible, intra-alveolar influx of neutrophils which was preceded by mobilization of bone marrow-stored neutrophils. Neutrophilia in the lavage fluid and blood was not detectable if lavage and blood sampling procedures were done at 48-hr intervals (which did not alter Ia antigen expression among BAL cells). These observations indicate that BAL is a valid method for sampling and assessing pulmonary cellular and fluid constituents if the procedures are done at intervals of at least 48 hr.

Lymphocyte subset analysis by flow cytometry. Comparison of three different staining techniques and effects of blood storage.

Flow cytometric analysis enables the researcher and clinician to enumerate lymphocyte subsets in peripheral blood mononuclear cells (PBMC). Often blood samples are collected at one site and then shipped to another site for analysis. Many options in the storage, preparation, and staining of PBMC for flow cytometric analysis exist. Preparation techniques include the conventional Ficoll-Paque (FP) density centrifugation versus the whole blood technique with red blood cells (RBC) lysed using a lysing reagent. In comparing three methods of PBMC preparation, and comparing the staining of fresh blood cells with staining of cells after storage for 24 h at 4 degrees C, analyses show how these different techniques affect the results.

Cigarette smoke causes inhibition of the immune response to intratracheally administered antigens.

Chronic inhalation of cigarette smoke in rats preferentially inhibited the plaque-forming cell (PFC) response of lung-associated lymph nodes (LALN) to sheep red blood cells (SRBC), compared to anatomically distant lymph nodes. Inhibition of the antibody response in LALN of smoke-exposed animals was first detected at 21 weeks of smoke inhalation and was well established by the 27th week of smoke exposure. After prolonged exposure (greater than 34 weeks) to cigarette smoke, similar smoke-induced changes in PFC response took place in other lymphoid tissues as well. Cigarette smoke affected the response of LALN cells to a T cell-dependent antigen (SRBC). Exposure to cigarette smoke, however, did not alter the relative percentages of W3/13-positive (T cells) or Ig-positive (B cells) cells, nor did it alter the relative percentages of T cell subsets as scored by their surface phenotypes, i.e., T helper (W3/25+) or T suppressor/cytotoxic (OX-8+) cells. The percentage of phagocytic cells and the accessory cell functions of macrophages remained comparable between sham and smoke-exposed animals. Exposure to cigarette smoke did not significantly alter the response of LALN cells to T cell mitogens (concanavalin A and phytohemagglutinin). However, response to a T cell-independent antigen trinitrophenyl Brucella abortus was also significantly reduced. These results show that cigarette exposure in the rat results in a decreased antibody response and this exposure to cigarette smoke may primarily affect the B cell function.

Efficient use of monoclonal antibodies for immunofluorescence.

We describe here a simple and rapid small volume microplate-based immunofluorescence staining method in which fluorochrome-conjugated monoclonal antibodies (MAb) from three different manufacturers, used at a single standardized quantity (50 ng per test), resulted in optimal staining of human lymphocyte subsets. Staining reactions were robust, in that the number of lymphocytes used could be varied over a wide range (3 x 10(4)-1 x 10(6) cells per microplate well) without significant effects on the fluorescence intensity of staining or nonspecific binding by MAb. A measure of the efficiency of MAb use was the number of tests theoretically possible to perform with nominal 100 test kits; this figure ranged from 400 to 20,000 tests, depending on the MAb in question. This method was readily adaptable to both single- and two-color immunofluorescence analysis.

The determination of biurea in the presence of azodicarbonamide by HPLC.

Azodicarbonamide (ADA), a solid blowing agent used in the manufacture of plastics, has been selected for inhalation toxicity testing by the National Toxicology Program. To test for decomposition of ADA during aerosolization, an HPLC method was developed to quantitate the relative amounts of one possible degradation product, biurea, in bulk samples and filter samples collected after aerosolization. The method uses a C18 column with 10-micron particles, UV monitoring at 190 nm for biurea and 425 nm for ADA, and a mobile phase of 100% water. Quantitation is with 14C-labeled biurea and ADA as external standards. The assay was validated by spiking bulk ADA with projected levels of 1, 2, and 3% biurea. Levels of biurea bound in both bulk and filter-collected aerosol samples of ADA were both 0.50%, with relative standard deviations of 13 and 26%, respectively.

Acute inhalation exposure of azodicarbonamide in the guinea pig.

Humans have been exposed to azodicarbonamide (ADA) by inhalation where bulk quantities of ADA are handled in the workplace. Responses of some workers have led to concern for the potential irritant and sensitizing properties of inhaled ADA. This study examined the effects of inhaling ADA on lung structure and function of guinea pigs during and after an acute exposure. Groups of 20 guinea pigs were exposed to each of 3 concentrations of ADA (19, 58, and 97 mg/m3), plus air as a control, for 1 hr. Pulmonary function was measured before exposure (baseline), during exposure, immediately after exposure and 24 hr after exposure. Dynamic compliance (Cdyn), total pulmonary resistance (RL), tidal volume (VT), respiratory frequency and minute volume were measured. In addition, gross necropsies and histological examinations of respiratory tract tissues were done either immediately following the exposure or 24 hr after exposure. There were no effects of ADA exposure on gross necropsy, histology, Cdyn, or RL. Some significant, concentration-related decreases in VT, respiratory frequency and minute volume were seen. The magnitudes of these changes were small: the largest change was seen in minute volume, amounting to a 24% decrease in the high concentration group. Inhalation exposure of guinea pigs to ADA at concentrations of up to 97 mg/m3 resulted in minor changes in pulmonary function without any changes in lung histology.

The pulmonary antibody-forming cell response in the guinea pig after intratracheal immunization.

Guinea pigs were immunized by an intratracheal instillation of 5 X 10(9) sheep red blood cells (sRBCs) with or without the immunoadjuvant maleic vinyl ether-2 (MVE). At 6 days post immunization, a peak IgM antibody-forming cell (AFC) response was detected in lung-associated lymph nodes (LALNs), lung tissue, lavage fluid, blood, and spleen. The time course of the response in the LALNs was similar to that of the response of the popliteal lymph node after footpad immunization. The use of MVE significantly enhanced this response. In addition, immunization with the sRBCs enhanced the phagocytic activity of the lung macrophages. The magnitude and kinetics of the AFC response seen in the guinea pig lung is compared to the response seen in other animals.

Suppression of the antibody response by phorbol esters in the mouse is due to an effect on the nylon wool adherent cell population.

Phorbol esters, in particular 12-O-tetradecanoyl-phorbol-13-acetate (TPA), have been shown to have profound effects on most biological systems including tumor promotion. Presented here are studies on the acute toxic effects of TPA, and the effects of phorbol esters on the in vivo and in vitro, T cell-dependent, antigen-specific antibody response in the mouse. The LD50 of a single i.v. dose of TPA in the mouse was 309 micrograms/kg. Acute toxic effects included lethargy, hypothermia and enlarged, hemorrhagic spleens at the higher doses. TPA was shown to be a potent inhibitor of the in vivo primary antibody response as measured by the IgM antibody-forming cell (AFC) response to sheep red blood cells (sRBC). The ED50 of a cumulative i.v. dose was 145 micrograms/kg administered the day before and the day of immunization (72.5 micrograms/kg/day). A cumulative dose of 500 micrograms/kg (250 micrograms/kg/day) resulted in a 100% suppression of the response. This in vivo exposure to TPA did not alter B cell/T cell ratio in the spleen. Phorbol ester analogs inactive in other biological systems were also inactive in the in vivo AFC response. The in vitro AFC assay was used to determine what cell type was being affected by TPA. Separation of the adherent spleen cells into B and T cell populations was done using nylon wool columns and anti-theta plus complement treatment. Experiments with these cell populations indicated that TPA produced suppression of the response due to an effect on the nylon wool adherent cell population.