Biomedical subjects

Douglas J Taatjes

Publications and source records attributed to Douglas J Taatjes.

At least 19 recordsLinked to original sources

PubMed2007

Group v secretory phospholipase A2 promotes atherosclerosis: evidence from genetically altered mice.

OBJECTIVE: Group V secretory phospholipase A2 (GV sPLA2) has been detected in both human and mouse atherosclerotic lesions. This enzyme has potent hydrolytic activity towards phosphatidylcholine-containing substrates, including lipoprotein particles. Numerous studies in vitro indicate that hydrolysis of high density lipoproteins (HDL) and low density lipoproteins (LDL) by GV sPLA2 leads to the formation of atherogenic particles and potentially proinflammatory lipid mediators. However, there is no direct evidence that this enzyme promotes atherogenic processes in vivo. METHODS AND RESULTS: We performed gain-of-function and loss-of-function studies to investigate the role of GV sPLA2 in atherogenesis in LDL receptor-deficient mice. Compared with control mice, animals overexpressing GV sPLA2 by retrovirus-mediated gene transfer had a 2.7 fold increase in lesion area in the ascending region of the aortic root. Increased atherosclerosis was associated with an increase in lesional collagen deposition in the same region. Mice deficient in bone marrow-derived GV sPLA2 had a 36% reduction in atherosclerosis in the aortic arch/thoracic aorta. CONCLUSIONS: Our data in mouse models provide the first in vivo evidence that GV sPLA2 contributes to atherosclerotic processes, and draw attention to this enzyme as an attractive target for the treatment of atherosclerotic disease.

Animals↗

PubMed2007

Asbestos-induced peribronchiolar cell proliferation and cytokine production are attenuated in lungs of protein kinase C-delta knockout mice.

The signaling pathways leading to the development of asbestos-associated diseases are poorly understood. Here we used normal and protein kinase C (PKC)-delta knockout (PKCdelta-/-) mice to demonstrate multiple roles of PKC-delta in the development of cell proliferation and inflammation after inhalation of chrysotile asbestos. At 3 days, asbestos-induced peribronchiolar cell proliferation in wild-type mice was attenuated in PKCdelta-/- mice. Cytokine profiles in bronchoalveolar lavage fluids showed increases in interleukin (IL)-1beta, IL-4, IL-6, and IL-13 that were decreased in PKCdelta-/- mice. At 9 days, microarray and quantitative reverse transcriptase-polymerase chain reaction analysis of lung tissues revealed increased mRNA levels of the profibrotic cytokine, IL-4, in asbestos-exposed wild-type mice but not PKCdelta-/- mice. PKCdelta-/- mice also exhibited decreased lung infiltration of polymorphonuclear cells, natural killer cells, and macrophages in bronchoalveolar lavage fluid and lung, as well as increased numbers of B lymphocytes and plasma cells. These changes were accompanied by elevated mRNA levels of immunoglobulin chains. These data show that modulation of PKC-delta has multiple effects on peribronchiolar cell proliferation, proinflammatory and profibrotic cytokine expression, and immune cell profiles in lung. These results also implicate targeted interruption of PKC-delta as a potential therapeutic option in asbestos-induced lung diseases.

Animals↗

PubMed2006

The histochemistry and cell biology vade mecum: a review of 2005-2006.

The procurement of new knowledge and understanding in the ever expanding discipline of cell biology continues to advance at a breakneck pace. The progress in discerning the physiology of cells and tissues in health and disease has been driven to a large extent by the continued development of new probes and imaging techniques. The recent introduction of semi-conductor quantum dots as stable, specific markers for both fluorescence light microscopy and electron microscopy, as well as a virtual treasure-trove of new fluorescent proteins, has in conjunction with newly introduced spectral imaging systems, opened vistas into the seemingly unlimited possibilities for experimental design. Although it oftentimes proves difficult to predict what the future will hold with respect to advances in disciplines such as cell biology and histochemistry, it is facile to look back on what has already occurred. In this spirit, this review will highlight some advancements made in these areas in the past 2 years.

Animals↗

PubMed2006

Unique uptake of acid-prepared mesoporous spheres by lung epithelial and mesothelioma cells.

Lung cancers, malignant mesotheliomas (MM), and fibrosis are devastating diseases with limited treatment strategies, in part due to poorly-effective drug delivery to affected areas of lung. We hypothesized that acid-prepared mesoporous spheres (APMS) (1-2 microm diameter, 40 A pore size) might be effective vehicles for pulmonary chemotherapeutic drug delivery. To assess this, APMS, chemically modified with different surface molecules (lipid, a linker having a terminal amine group, a thiol group, or tetraethylene glycol [TEG]), were evaluated for uptake and possible cytotoxic effects after in vitro administration to murine alveolar epithelial Type II (C10) and human mesothelioma (MM) cells and after intrapleural or intranasal administration to C57Bl/6 mice. APMS coated with TEG (APMS-TEG) were most efficiently taken up by C10 and MM cells. The mechanism of cell uptake was rapid, actin-dependent, and did not involve clathrin- or caveolae-mediated mechanisms nor fusion of membrane-bound APMS with lysosomes. When injected intrapleurally in mice, APMS-TEG were taken up by both CD45-positive and -negative cells of the diaphragm, lung, and spleen, whereas APMS administered by the intranasal route were predominantly in lung epithelial cells and alveolar macrophages. After intrapleural or intranasal administration, APMS were nonimmunogenic and nontoxic as evaluated by differential cell counts and lactate dehydrogenase levels in bronchoalveolar and pleural lavage fluids. In the treatment of lung and pleural diseases, APMS-TEG may be useful tools to deliver chemotherapeutic drugs or molecular constructs.

Administration, Intranasal↗

PubMed2006

Cloning and functional characterization of allelic variation in the porcine prolactin receptor.

Prolactin (PRL) regulates various functions in pigs including reproduction, mammary development and lactation. We used 5'-rapid amplification of cDNA ends (5'-RACE) to clone three full-length alleles of the porcine PRL receptor (pPRLR) from Landrace (alleles LR2 and LR4) and Yucatan miniature (MP) pigs, corresponding to the A and B alleles previously reported to be associated with reproductive traits. When expressed in Chinese hamster ovary (CHO-K1) cells, all three pPRLRs transduced differentiation signals to a beta-casein promoter with the same effectiveness, where human growth hormone (hGH) and porcine PRL (pPRL) were more effective ligands than ovine PRL (oPRL). The pPRLR had a lower binding affinity for oPRL than pPRL while binding affinity for hGH was not different between the three pPRLR variants. The pPRLRs primarily localized to the cytoplasm with perinuclear concentration. In conclusion, we have cloned three allelic variants of the pPRLR and have functionally characterized these as different from the hPRLR. However, our data do not support the proposal that allelic variation of the pPRLR confers functional differences in vivo.

Alleles↗

PubMed2006

Fibroblast spreading induced by connective tissue stretch involves intracellular redistribution of alpha- and beta-actin.

Mechanical stretching of connective tissue occurs with normal movement and postural changes, as well as treatments including physical therapy, massage and acupuncture. Connective tissue fibroblasts were recently shown to respond actively to short-term mechanical stretch (minutes to hours) with reversible cytoskeletal remodeling, characterized by extensive cell spreading and lamellipodia formation. In this study, we have examined the effect of tissue stretch on the distribution of alpha- and beta-actin in subcutaneous tissue fibroblasts ex vivo. Normal fibroblasts uniformly exhibited alpha-smooth muscle actin (alpha-SMA) immunoreactivity. Unlike cultured fibroblasts and smooth muscle cells, alpha-SMA in these fibroblasts was not in F-actin form (indicated by lack of phalloidin co-localization) nor was it organized into distinct stress fibers. The lack of stress fibers and fibronexus was confirmed by electron microscopy, indicating that these cells were not myofibroblasts. In unstretched tissue, the pattern of alpha-actin was diffuse and granular. With tissue stretch (30 min), alpha-actin formed a star-shaped pattern centered on the nucleus, while beta-actin extended throughout the cytoplasm including lamellipodia and cell cortex. This dual response pattern of alpha- and beta-actin may be an important component of cellular mechanotransduction mechanisms relevant to physiologic and therapeutic mechanical forces applied to connective tissue.

Actins↗

PubMed2006

Energy-dependent disassembly of self-assembled SNARE complex: observation at nanometer resolution using atomic force microscopy.

Full-length v-SNARE protein reconstituted in lipid vesicles, when exposed to t-SNARE-reconstituted lipid membrane, results in the self-assembly of a t-/v-SNARE complex in a ring pattern, forming pores and the establishment of continuity between the opposing bilayers. In contrast, when v-SNARE protein alone (without liposomes) is exposed to t-SNARE-reconstituted lipid membrane, they also self-assemble to form t-/v-SNARE complexes, although such complexes fail to possess the characteristic ring pattern, nor do they help in the establishment of continuity between the opposing bilayers. Hence, t-SNAREs and v-SNARE need to be membrane-associated to interact in a circular array to form conducting pores in the presence of calcium. This study demonstrates that, irrespective of their arrangement, both forms of the SNARE complex can be disassembled in the presence of NSF-ATP.

Adenosine Triphosphate↗

PubMed2006

Laser scanning cytometer-based assays for measuring host cell attachment and invasion by the human pathogen Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii is among the most common protozoan parasites of humans. Both attachment to and invasion of host cells by T. gondii are necessary for infection, yet little is known about the molecular mechanisms underlying these processes. T. gondii's etiological importance and its role as a model organism for studying invasion in related parasites necessitate a means to quantitatively assay host cell attachment and invasion. METHODS: We present here Laser Scanning Cytometer (LSC)-based assays of T. gondii invasion and attachment. The invasion assay involves automated counting of invaded and non-invaded parasites, differentially labeled with distinct fluorochromes. The attachment assay compares the relative binding of differentially labeled parasites. The assays were evaluated using treatments known to decrease invasion or attachment. RESULTS: The LSC-based assays are robust and reproducible, remove operator bias, and significantly increase the sample size that can be feasibly counted compared to other currently available microscope-based methods. In the first application of the new assays, we have shown that parasites attach to fixed and unfixed host cells using different mechanisms. CONCLUSIONS: The LSC-based assays represent useful new methods for quantitatively measuring attachment and invasion by T. gondii, and can be readily adapted to study similar processes in other host-pathogen systems.

Animals↗

PubMed2006

Analysis of asbestos-induced gene expression changes in bronchiolar epithelial cells using laser capture microdissection and quantitative reverse transcriptase-polymerase chain reaction.

Laser capture microdissection (LCM) enables the removal of discrete microstructures or cell types from properly prepared histological sections. Extraction of RNA from microdissected tissue followed by quantitative reverse transcriptase-polymerase chain (QRT-PCR) reaction permits the analysis of cell-type or microstructure-specific gene expression changes that occur in response to various stimuli in the environment. In our lab, the combination of LCM and QRT-PCR has proven very useful in the determination of the in vivo gene expression changes that occur in bronchiolar epithelium in response to inhalation of crocidolite asbestos. A detailed description of the preparation of cDNA from bronchiolar epithelial cells obtained by LCM is described in this work.

Asbestos, Crocidolite↗

PubMed2006

Multifluorescence labeling techniques and confocal laser scanning microscopy on lung tissue.

Lung tissue consists of more than 40 individual cell types that might interact to produce adverse pathologies. After injury, a number of signaling proteins expressed in various epithelial and other cell types have been linked to the advent of apoptosis, compensatory proliferation, and adaptation to stress. We describe here the use of immunochemistry and multifluorescence approaches using confocal laser scanning microscopy to define the signaling pathways (protein kinases C and mitogen-activated protein kinases) activated by asbestos fibers after inhalation. Using these approaches, we are able to localize signaling events in distinct cell types of the lung and determine their status in the cell cycle (resting or nonresting). Moreover, we are able to determine whether various signaling proteins colocalize in cells and the sites affected by asbestos fibers.

Animals↗

PubMed2006

Quantitative analysis of atherosclerotic lesion composition in mice.

Comparative quantitation has become an increasingly desirable tool in determining compositional differences of aortic plaque lesion in transgenically altered mice. To this end, methodology has been developed to identify lipid, cellularity, collagen, and elastin components using traditional bright-field microscopy, fluorescence, and polarized light microscopy, employing both confocal and wide-field imaging systems. Subsequent imaging processing and analysis on the digitally captured images reveals differences in compositional components as influenced by diet, age and gender. This method can be expanded to employ a rich variety of histochemical and immunohistochemical staining protocols.

Animals↗

PubMed2005

Recent progress in histochemistry and cell biology: the state of the art 2005.

Advances in the field of histochemistry, a multidisciplinary area including the detection, localization and functional characterization of molecules in single cells and complex tissues, often drives the attainment of new knowledge in the broadly defined discipline of cell biology. These two disciplines, histochemistry and cell biology, have been joined in this journal to facilitate the flow of information with celerity from technical advancement in histochemical procedures, to their utilization in experimental models. This review summarizes advancements in these fields during the past year.

Animals↗

PubMed2005

Nitrogen dioxide enhances allergic airway inflammation and hyperresponsiveness in the mouse.

In addition to being an air pollutant, NO2 is a potent inflammatory oxidant generated endogenously by myeloperoxidase and eosinophil peroxidase. In these studies, we sought to determine the effects of NO2 exposure on mice with ongoing allergic airway disease pathology. Mice were sensitized and challenged with the antigen ovalbumin (OVA) to generate airway inflammation and subsequently exposed to 5 or 25 ppm NO2 for 3 days or 5 days followed by a 20-day recovery period. Whereas 5 ppm NO2 elicited no pathological changes, inhalation of 25 ppm NO2 alone induced acute lung injury, which peaked after 3 days and was characterized by increases in protein, LDH, and neutrophils recovered by BAL, as well as lesions within terminal bronchioles. Importantly, 25 ppm NO2 was also sufficient to cause AHR in mice, a cardinal feature of asthma. The inflammatory changes were ameliorated after 5 days of inhalation and completely resolved after 20 days of recovery after the 5-day inhalation. In contrast, in mice immunized and challenged with OVA, inhalation of 25 ppm NO2 caused a marked augmentation of eosinophilic inflammation and terminal bronchiolar lesions, which extended significantly into the alveoli. Moreover, 20 days postcessation of the 5-day 25 ppm NO2 inhalation regimen, eosinophilic and neutrophilic inflammation, pulmonary lesions, and AHR were still present in mice immunized and challenged with OVA. Collectively, these observations suggest an important role for NO2 in airway pathologies associated with asthma, both in modulation of degree and duration of inflammatory response, as well as in induction of AHR.

Animals↗

PubMed2005

Gene expression profiles reveal increased mClca3 (Gob5) expression and mucin production in a murine model of asbestos-induced fibrogenesis.

To elucidate genes important in development or repair of asbestos-induced lung diseases, gene expression was examined in mice after inhalation of chrysotile asbestos for 3, 9, and 40 days. We identified changes in the expression of genes linked to proliferation (cyclin B2, CDC20, and CDC28 protein kinase regulatory subunit 2), inflammation (CCL9, CCL6, complement component 1, chitinase3-like 3, TNF superfamily member 10, and IL-1B), and matrix remodeling (MMP12, MMP3, integrin alphaX, and cathepsins K, Z, B, and S). The most highly induced gene at all time points was mclca3 (gob5), a putative calcium-activated chloride channel involved in the regulation of mucus production and/or secretion. Using histochemistry, we demonstrated accumulation of mucus and increased mClca3 protein in the bronchiolar epithelium of asbestos-exposed mice at all time points but peaking at 9 days. Cytokine levels (interleukin-1beta, interleukin-4, interleukin-6) in bronchoalveolar lavage fluid also increased at 9 days, suggesting Th2-mediated immunity may play a role in asbestos-induced mucus production. In contrast, levels of cathepsin K, a potent elastase, increased between 3 and 40 days at both the mRNA and protein levels, localizing primarily in CD45-positive leukocytes and interstitial cells. Identification of genes involved in lung injury and remodeling after asbestos exposure could aid in defining mechanisms of airborne particulate-induced disease and in developing therapeutic strategies.

Animals↗

PubMed2005

Attenuation of accumulation of neointimal lipid by pioglitazone in mice genetically deficient in insulin receptor substrate-2 and apolipoprotein E.

Rupture of vulnerable atherosclerotic plaques that are characterized by extensive neointimal accumulation of lipid is a cause of acute coronary syndromes. To identify whether insulin resistance alters atherogenesis, we characterized the composition of atherosclerotic lesions in the proximal aortas in mice deficient in apolipoprotein E (ApoE(-/-)) and in ApoE(-/-) mice in which insulin resistance was intensified by a concomitant heterozygous deficiency in insulin receptor substrate type 2 (IRS2(+/-) ApoE(-/-) mice). In addition, we characterized the effect of an insulin sensitizer, pioglitazone, on the atherogenesis in IRS2(+/-) ApoE(-/-) mice. The extent of the aortic intima occupied by lesion was increased in the IRS2(+/-) ApoE(-/-) compared with ApoE(-/-) mice (79 +/- 3% compared with 68 +/- 8%, p<0.05). Treatment with pioglitazone decreased the neointimal content of lipid in 20-week-old mice from 50 +/- 6% to 30 +/- 7%, p=0.005 and decreased the cellularity reflected by the multisection cross-sectional areas of lesions comprising cells in atheroma from 24 +/- 1% to 19 +/- 3%, p=0.018. Accordingly, genetically induced intensification of insulin resistance increases atheroma formation. Furthermore, attenuation of insulin resistance by treatment with pioglitazone decreases accumulation of lipid in the neointima.

Administration, Oral↗

PubMed2004

NF-kappa B activation in airways modulates allergic inflammation but not hyperresponsiveness.

Airways display robust NF-kappaB activation and represent targets for anti-inflammatory asthma therapies, but the functional importance of NF-kappaB activation in airway epithelium remains enigmatic. Therefore, transgenic mice were created in which NF-kappaB activation is repressed specifically in airways (CC10-IkappaBalpha(SR) mice). In response to inhaled Ag, transgenic mice demonstrated significantly ameliorated inflammation, reduced levels of chemokines, T cell cytokines, mucus cell metaplasia, and circulating IgE compared with littermate controls. Despite these findings, Ag-driven airways hyperresponsiveness was not attenuated in CC10-IkappaBalpha(SR) mice. This study clearly demonstrates that airway epithelial NF-kappaB activation orchestrates Ag-induced inflammation and subsequent adaptive immune responses, but does not contribute to airways hyperresponsiveness, the cardinal feature that underlies asthma.

Allergens↗

PubMed2004

The duration of nuclear extracellular signal-regulated kinase 1 and 2 signaling during cell cycle reentry distinguishes proliferation from apoptosis in response to asbestos.

Asbestos exposure causes activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in lung epithelial cells, the targets of asbestos-associated lung carcinomas. The functional significance of ERK1/2 activation in pulmonary epithelial and mesothelial cells is unclear. Using serum-stimulated mouse alveolar type II epithelial cells as a model for cell cycle reentry, we show that the duration of phospho-ERK1/2 in the nucleus determines cell fate in response to crocidolite asbestos. In response to 10% serum, a proliferative stimulus, phosphorylated ERK1/2 initially accumulated in the nucleus, and reduction of nuclear phospho-ERK1/2 after 2 to 4 hours was followed by expression of cyclin D1 and S-phase entry. Low levels of asbestos (<0.5 microg/cm2) promoted S-phase entry in low (2%) serum through an epidermal growth factor receptor-dependent pathway but did not promote cell cycle progression or induce apoptosis in the presence of high (10%) serum-containing medium. Higher levels of asbestos (1.0 to 5.0 microg/cm2) prolonged the localization of phospho-ERK1/2 in the nucleus in the presence of high serum, impeded S-phase entry, and induced apoptosis in a dose-dependent manner. Immunofluorescence microscopy indicated that the duration of signaling by phospho-ERK1/2 in the nucleus was predictive of cell fate at any concentration of asbestos. After 8 hours of exposure, cells with nuclear phospho-ERK1/2 also were positive for nuclear localization of apoptosis-inducing factor (AIF), an early event in apoptosis. In contrast, asbestos-exposed cells that displayed cytoplasmic phospho-ERK1/2 at 8 hours expressed cyclin D1 and proceeded to S phase. Our studies show that prolonged localization of phospho-ERK1/2 in the nucleus is incompatible with expression of cyclin D1 and is predictive of asbestos-associated cell death by AIF, thereby providing an approach for determining cell fate in asbestos-induced tumorigenesis.

Animals↗

PubMed2004

Fibroblasts form a body-wide cellular network.

"Loose" connective tissue forms a network extending throughout the body including subcutaneous and interstitial connective tissues. The existence of a cellular network of fibroblasts within loose connective tissue may have considerable significance as it may support yet unknown body-wide cellular signaling systems. We used a combination of histochemistry, immunohistochemistry, confocal scanning laser microscopy (confocal microscopy), and electron microscopy to investigate the extent and nature of cell-to-cell connections within mouse subcutaneous connective tissue. We found that fibroblasts formed a reticular web throughout the tissue. With confocal microscopy, 30% of fibroblasts' processes could be followed continuously from one cell to another. Connexin 43 immunoreactivity was present at apparent points of cell-to-cell contact. Electron microscopy revealed that processes from adjacent cells were in close apposition to one another, but gap junctions were not observed. Our findings indicate that soft tissue fibroblasts form an extensively interconnected cellular network, suggesting they may have important and so far unsuspected integrative functions at the level of the whole body.

Animals↗