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D A Pettit

Publications and source records attributed to D A Pettit.

10 recordsLinked to original sources
PubMed2001

The determination of draining lymph node cell cytokine mRNA levels in BALB/c mice following dermal sodium lauryl sulfate, dinitrofluorobenzene, and toluene diisocyanate exposure.

Differential modulation has been demonstrated in interleukin-4 (IL-4), IL-10, and interferon gamma (IFN-gamma) mRNA and protein secretion patterns of cells isolated from the draining lymph nodes of mice following exposure to T cell and respiratory sensitizers. Using a multiprobe ribonuclease protection assay, the following investigation examined the mRNA expression patterns of multiple cytokines associated with respiratory sensitization for modulation following exposure to chemicals known primarily to induce irritation (sodium lauryl sulfate), respiratory sensitization (toluene diisocyanate), or T cell-mediated hypersensitivity (dinitrofluorobenzene) responses. On days 0 and +5 female BALB/c mice were exposed to either test article or vehicle on the shaven dorsal lumbar region; on days +10 through +12 the mice received test article on the dorsal aspect of each ear. On day +13 animals were euthanized, draining lymph nodes were excised, and mRNA was isolated immediately or following 24 or 48 h of culture in the presence or absence of concanavalin (Con) A. Differential expression of cytokine mRNA was most notable following 24 h incubation with Con A. Modulation of IL-4, -10, and IFN-gamma following chemical exposure was consistent with previous studies. In addition, IL-9, -13, and -15 were significantly elevated only following toluene diisocyanate exposure. Further investigations of these cytokines may provide additional insight into the mechanisms of chemically induced respiratory sensitization and provide endpoints for the detection of a chemical's ability to elicit IgE-mediated hypersensitivity responses.

Administration, Topical↗
PubMed2000

Thalidomide stimulates splenic IgM antibody response and cytotoxic T lymphocyte activity and alters leukocyte subpopulation numbers in female B6C3F1 mice.

Thalidomide has been shown to have antiinflammatory and, more recently, immunomodulating properties, which are beneficial for the treatment of an ever-increasing list of immune related diseases. Although considerable knowledge regarding thalidomide s antiinflammatory properties has been acquired, relatively little is known about its immunomodulating properties in vivo. In this paper, a panel of immune assays was used to evaluate immunomodulation in female B6C3F1 mice treated intraperitoneally for 28 days with thalidomide (30, 100, or 150 mg/kg/day). Spleen antibody forming cell response was significantly enhanced by 37% in mice treated with 150 mg/kg/day, despite an 8% decrease in the percentage of Ig+ B cells. A significant stimulatory trend was observed for the cytotoxic T cell response across thalidomide treatment groups. An evaluation of the spleen leukocyte subpopulations revealed a 23% increase in the absolute number of CD8+ T cells in the 150 mg/kg treatment group and a 9 and 11% decrease in the absolute number of NK cells in both the 100 and 150 mg/kg thalidomide treatment groups, respectively. These findings demonstrate that, in addition to modulating spleen leukocyte numbers, thalidomide also stimulates murine humoral and cellular immune responses in vivo.

Animals↗
PubMed1998

High-level expression of the human CB2 cannabinoid receptor using a baculovirus system.

A human CB2 recombinant baculovirus (AcNPV-hCB2) was generated by site-specific transposition and employed to express the human CB2 cannabinoid receptor. Northern analysis of total RNA from Spodoptera frugiperda (Sf9) insect cells infected with AcNPV-hCB2 revealed novel expression of a unique 2.3 kb transcript when probed with hCB2 cDNA. This transcript corresponded to the size expected for hCB2 generated from the recombinant virus construct. Western immunoblot analysis of whole cell homogenates of recombinant baculovirus-infected Sf9 cells, using affinity-purified antibody to a human CB2 carboxy terminal domain (anti-hCB2.CV), revealed the presence of novel immunoreactive protein. In addition, when anti-hCB2.CV was employed in immunofluorescence staining, an intense signal was observed within AcNPV-hCB2-infected cells but not within uninfected cells or cells infected with a control beta-galactosidase recombinant baculovirus. The pattern of immunofluorescence at early periods post-infection was in a perinuclear arrangement with a "signet-ring" appearance, suggestive of glycosylation of the expressed recombinant protein. Transmission electron microscopy revealed regions of intranuclear recombinant virus assembly and the presence of numerous intracytoplasmic proteinaceous vesicular inclusions consistent with hyperproduction of hCB2. Scatchard-Rosenthal analysis of [3H]-(-)3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxypro pyl]cyclohexan-1-ol ([3H]CP 55,940) receptor binding indicated a Kd of 2.24 nM and a Bmax equal to 5.24 pmol/mg of protein. The lack of [3H]CP 55,940 displacement with N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-met hyl-1H-pyrazole-3-carboxamidehydrochloride (SR 141716A), the CB1-selective antagonist, confirmed the identity of the receptor as CB2. These data indicate that AcNPV-hCB2 expresses high levels of the human CB2, which retains properties of the native receptor. Thus, this recombinant virus may prove suitable for hyperproduction of receptor for basic biochemical and biophysical characterization studies.

Animals↗
PubMed1998

Immunohistochemical localization of the neural cannabinoid receptor in rat brain.

The cannabinoid receptor family consists of two inhibitory G-protein-coupled receptors, CB1 and CB2. CB1 is distributed primarily in neural tissue, whereas CB2 is distributed predominantly in immune cells. The distribution of cannabinoid receptors in neural tissue has been demonstrated by using ligand binding autoradiography with CP55,940, a high-affinity cannabinoid receptor ligand, and in situ hybridization. However, the localization of CB1 within individual cells in the brain remains to be defined. In the present study, domain-specific polyclonal antibody to amino acids 83-98 of CB1 was used to define the expression of the neural cannabinoid receptor at the histochemical level. The use of CB1-specific antiserum is advantageous in view of recent reports that CB2 also is expressed in the brain and binds CP55,940. Thus, utilization of anti-CB1 antiserum would allow for the specific detection of CB1 protein expression. The regional staining pattern for CB1 in rat brain was consistent with that reported for CB1 using ligand binding autoradiography and in situ hybridization. Intense immunoreactivity was present in the hippocampal formation, the basal ganglia, and the molecular layer of the cerebellum. Moderate immunohistochemical staining was observed in the olfactory bulb, piriform cortex, cerebral cortex, and the granular layer of the cerebellum. In addition, immunoreactive staining was concentrated on afferent projections and dendritic processes of neuronal cells and was present within cell bodies and on cell surfaces. These data indicate that the anti-CB1 antibody is a sensitive probe for the unequivocal histological discrimination of CB1 protein expression.

Animals↗
PubMed1996

Expression and purification of recombinant human tryptase in a baculovirus system.

B2 is a mAb that recognizes a conformational determinant on the active form of native tryptase, but does not recognize native tryptase that spontaneously loses activity in physiologic buffer. Precursor forms of recombinant human (rh) alpha- and rh beta-tryptase have been expressed in a baculovirus system. In each case, multiple electrophoretic forms were detected in both culture media and cell lysates of infected insect cells by Western blots developed with the G3 mAb made against native human tryptase. Although only 4 of 30 amino acids in the leader sequences of alpha- and beta-tryptase differ, rh alpha-tryptase appeared predominantly in the cell lysates, rh beta-tryptase predominantly in the culture media. B2 recognized rh alpha-tryptase and rh beta-tryptase found in the culture media of infected Sf-9 cells, but not that in cell lysates. Secreted forms of tryptase were purified to homogeneity by B2-immunoaffinity chromatography. From 1 liter of culture fluid 1.5 to 3 mg of rh-tryptase could be purified. Each rh-tryptase precursor was enzymatically inactive with synthetic substrates. Analysis of the N-terminal amino acid sequences of purified rh alpha- and rh beta-tryptase precursors (APAPVQA and APAPGQA, respectively) indicated that the initial 18 amino acids of the 30-amino-acid leader sequence had been removed. Differential N-glycosylation was found in both rh alpha-tryptase (one or two carbohydrate groups per molecule) and rh beta-tryptase (zero or one carbohydrate group per molecule). Thus, the baculovirus expression system is a useful tool for generation of rh alpha- and rh beta-tryptase precursors that exhibit a conformational epitope also present on natural tryptase and that are preferentially secreted into the culture media of infected cells.

Amidohydrolases↗
PubMed1996

In vitro destruction of nerve cell cultures by Acanthamoeba spp.: a transmission and scanning electron microscopy study.

Trophozoites of 4 species of Acanthamoeba were cytopathic for cultured rat B103 neuroblastoma cells. Cytopathogenicity was evaluated by a chromium release assay and by transmission and scanning electron microscopy. Acanthamoeba culbertsoni, Acanthamoeba castellanii, and Acanthamoeba polyphaga destroyed B103 target cells at 37 C as evidenced by the release of radiolabel. Acanthamoeba astronyxis did not produce cytopathology at 37 C but destroyed nerve cells at 25 C. Transmission and scanning electron microscopy of cocultures maintained at different time periods revealed that all species of Acanthamoeba exhibited long cylindrical structures, termed digipodia, which made contact with target cells. Following this effector cell-target cell contact, membrane blebbing on the nerve cells was observed. These events were followed either by lysis of target nerve cells or ingestion of the target cells via food-cups and their subsequent channeling into intracytoplasmic food vacuoles. Use of the TUNEL (TdT-mediated dUTP nick end labeling) technique indicated that approximately 40% of B103 cells incubated with A. culbertsoni, 20% of B103 cells cocultured with A. castellanii or with A. polyphaga, and less than 1% of B103 cells incubated with A. astronyxis at 37 C were apoptotic after 24 hr of coculture. Studies using electron microscopy indicated that Acanthamoeba trophozoites destroyed nerve cells both by cytolysis and by ingestion of whole nerve cells via food-cups.

Acanthamoeba↗
PubMed1994

Expression of a cannabinoid receptor in baculovirus-infected insect cells.

A cannabinoid receptor recombinant baculovirus (AcNPV-THCR) has been constructed and employed to express rat neural cannabinoid receptors. Northern analysis of total RNA from Spodoptera frugiperda (Sf9) insect cells infected with AcNPV-THCR revealed novel hyper-production of a 3.3 kb transcript when probed with nick-translated rat cannabinoid receptor cDNA. Optimal viral protein expression was observed in 35S-metabolically labeled AcNPV-THCR-infected Sf9 cells at a multiplicity of infection of 2.5. Transmission electron microscopy of AcNPV-THCR-infected Sf9 cells showed extensive membrane perturbation and electron-dense cytoplasmic perinuclear accumulation, indicative of receptor glycoprotein expression. Immunofluorescence staining using antiserum produced to a fusion protein consisting of the external domain of the cannabinoid receptor and hepatitis B core antigen revealed cannabinoid receptor expression in AcNPV-THCR-infected Sf9 cells. Scatchard-Rosenthal analysis of [3H]CP55,940 receptor binding indicated a Kd of 3.4 nM and a Bmax equal to 3.17 pmol/mg protein. Western immunoblotting performed on AcNPV-THCR-infected Sf9 cell lysates revealed immunoreactive bands with relative molecular weights ranging from 45 to 79 kDa. The predominant species (55 kDa) exhibited a relative molecular weight consistent with that predicted for the translational product obtained from the cannabinoid receptor cDNA coding sequence. In vitro translation using AcNPV-THCR mRNA also yielded a 55 kDa immunoreactive species. These data indicate that the baculovirus expression system is a viable means of expressing relatively large quantities of cannabinoid receptor recombinant protein.

Animals↗
PubMed1989

The quantitation of biotinidase activity in dried blood spots using microtiter transfer plates: identification of biotinidase-deficient and heterozygous individuals.

A simple and rapid method for the quantitation of biotinidase activity in blood-soaked filter paper spots was developed. The assay measures the release of p-aminobenzoate from N-biotinyl-p-aminobenzoate. A microtiter transfer plate is used to rapidly separate the reaction solution from the filter paper spots. Color is developed and the absorbance is determined using a microplate reader. The biotinidase activity in frozen filter spots correlates well with the activity in serum (r = 0.94). The enzyme activities of obligate heterozygotes for biotinidase deficiency were significantly different from those with normal activity (P = 0.03). This rapid screening procedure can be used to quantitate biotinidase activity in newborn screening samples, identify heterozygotes, and estimate the gene frequency and incidence of biotinidase deficiency in large populations. In addition, the use of microtiter transfer plates can be applied to other assays in which the separation of the incubation solution from a filter paper spot is required.

Amidohydrolases↗