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Biomedical subjects

James P Fabisiak

Publications and source records attributed to James P Fabisiak.

10 recordsLinked to original sources
PubMed2025

Epigenome-Wide Analysis Identifies Pollution-Sensitive Loci in Fibrotic Interstitial Lung Disease.

Rationale: Particulate matter &#x2a7d;2.5 &#x3bc;m (PM2.5) adversely impacts patients with fibrotic interstitial lung disease (fILD). Objectives: We sought to determine whether PM2.5-associated epigenetic alterations contribute to the environmental pathogenesis of fILD. Methods: A retrospective two-cohort study applied satellite-derived PM2.5 and constituent exposure matching to the residential location of patients with fILD. Robust linear regressions were used to evaluate cohort-specific, epigenome-wide differential blood DNA methylation with increasing pollutant exposures (Illumina MethylationEPIC BeadChip). Cox and linear regressions were used to evaluate associations of cytosine-phosphate-guanine (CpG) loci with transplant-free survival and lung function. A Wilcoxon test was used to evaluate cartilage-associated protein (CRTAP) levels in fILD and control lungs. Measurements and Main Results: The University of Pittsburgh cohort (n&#x2009;=&#x2009;306) had 5-year median PM2.5 exposures of 12.1 &#x3bc;g/m3 compared with 5.1 &#x3bc;g/m3 in the University of British Columbia cohort (n&#x2009;=&#x2009;170). Higher pollutant exposures in the University of Pittsburgh cohort were associated with lower methylation at cg25354716, annotated to CRTAP, a critical extracellular matrix remodeling enzyme. Higher exposures in the University of British Columbia cohort were associated with higher methylation at cg01019301, annotated to TLN2 (talin-2), a cytoskeletal protein involved in fibroblast migration. A 10% increase in cg25354716 methylation was associated with a hazard ratio of 0.81 for death or lung transplantation in the meta-analyzed cohorts (95% confidence interval&#x2009;=&#x2009;0.69-0.96; P&#x2009;=&#x2009;0.01), whereas the same change in cg01019301 was associated with a hazard ratio of 1.36 (95% confidence interval&#x2009;= 1.07-1.74; P&#x2009;=&#x2009;0.01). CRTAP protein was more abundant in lungs from patients with fILD compared with those from donor controls (P&#x2009;<&#x2009;0.001). Conclusions: PM2.5 is associated with altered blood DNA methylation in fILD. This work identifies novel pollution-sensitive targets that hold potential for therapeutic modulation in fILD.

Humans↗
PubMed2006

Mycoplasma fermentans and TNF-beta interact to amplify immune-modulating cytokines in human lung fibroblasts.

Mycoplasma can establish latent infections and are associated with arthritis, leukemia, and chronic lung disease. We developed an experimental model in which lung cells are deliberately infected with Mycoplasma fermentans. Human lung fibroblasts (HLF) were exposed to live M. fermentans and immune-modulating cytokine release was assessed with and without known inducers of cytokine production. M. fermentans increased IL-6, IL-8/CXCL8, MCP-1/CCL2, and Gro-alpha/CXCL1 production. M. fermentans interacted with TNF-beta to release more IL-6, CXCL8, and CXCL1 than predicted by the responses to either stimulus alone. The effects of live infection were recapitulated by exposure to M. fermentans-derived macrophage-activating lipopeptide-2 (MALP-2), a Toll-like receptor-2- and receptor-6-specific ligand. The synergistic effect of combined stimuli was more pronounced with prolonged incubations. Preexposure to TNF-beta sensitized the cells to subsequent MALP-2 challenge, but preexposure to MALP-2 did not alter the IL-6 response to TNF-beta. Exposure to M. fermentans or MALP-2 did not enhance nuclear localization, DNA binding, or transcriptional activity of NF-kappaB and did not modulate early NF-kappaB activation in response to TNF-beta. Application of specific inhibitors of various MAPKs suggested that p38 and JNK/stress-activated protein kinase were involved in early IL-6 release after exposure to TNF-beta and M. fermentans, respectively. The combined response to M. fermentans and TNF-beta, however, was uniquely sensitive to delayed application of SP-600125, suggesting that JNK/stress-activated protein kinase contributes to the amplification of IL-6 release. Thus M. fermentans interacts with stimuli such as TNF-beta to amplify lung cell production of immune-modulating cytokines. The mechanisms accounting for this interaction can now be dissected with the use of this in vitro model.

Cells, Cultured↗
PubMed2005

Quantification of selective phosphatidylserine oxidation during apoptosis.

Membrane phospholipids are gaining increasing attention as important mediators in a variety of signal transduction processes. Oxidation and changes in membrane topography of lipids are probably important elements in the regulation of phospholipid-dependent signaling. Phosphatidylserine (PS), in particular, is implicated in the regulation of macrophage-dependent clearance of apoptotic cell "corpses" in a pathway probably mediated by selective oxidation and translocation of PS in the plasma membrane. Here we describe our highly sensitive and specific assay to measure differential lipid peroxidation in individual phospholipid classes in live cells using metabolic integration of the fluorescent oxidation-sensitive fatty acid analog cis-parinaric acid (cis-PnA) and resolution of specific phospholipids by high-performance liquid chromatography (HPLC). These experimental approaches can provide insight into the roles and mechanisms of PS oxidation in the identification and clearance of apoptotic cells.

Apoptosis↗
PubMed2005

Quantitative method of measuring phosphatidylserine externalization during apoptosis using electron paramagnetic resonance spectroscopy and annexin-conjugated iron.

We present here the application of a novel assay that measures the absolute amount of phosphatidylserine (PS) externalized on the surface of cells. Although the assay is based on the same annexin binding principle as the fluorescent flow cytometry assay, we use paramagnetic iron as the ultimate reporter molecule, establishing a linear relationship between signal amplitude and amount of PS on the cell surface, allowing a quantitative assay of PS externalization over a wide dynamic range. The application of this technique, alone and in concert with the PS oxidation method presented in the previous chapter, will greatly aid in studying the mechanistic connection between lipid peroxidation and translocation events during apoptosis.

Annexins↗
PubMed2004

Microbial stimulation by Mycoplasma fermentans synergistically amplifies IL-6 release by human lung fibroblasts in response to residual oil fly ash (ROFA) and nickel.

Mycoplasma (MP), such as the species M. fermentans, possess remarkable immunoregulatory properties and can potentially establish chronic latent infections with little signs of disease. Atmospheric particulate matter (PM) is a complex and diverse component of air pollution associated with adverse health effects. We hypothesized that MP modulate the cellular responses induced by chemical stresses such as residual oil fly ash (ROFA), a type of PM rich in transition metals. We assessed the release of interleukin-6 (IL-6), a prototypic immune-modulating cytokine, in response to PM from different sources in human lung fibroblasts (HLF) deliberately infected with M. fermentans. We found that M. fermentans and ROFA together synergistically stimulated production of IL-6 compared to either stimuli alone. Compared to several other PM, ROFA appeared most able to potentiate IL-6 release. The potentiating effect of live MP infection could be mimicked by M. fermentans-derived macrophage-activating lipopeptide-2 (MALP-2), a known Toll-like receptor-2 agonist. The aqueous fraction of ROFA also contained potent IL-6 inducing activity in concert with MALP-2, and exposure to several defined metal salts indicated that Ni and, to a lesser extent V, (but not Cu) could synergistically act with MALP-2 to induce IL-6. These data indicate that microorganisms like MP can interact with environmental stimuli such as PM-derived metals to synergistically activate signaling pathways that control lung cell cytokine production and, thus, can potentially modulate adverse health effects of PM exposure.

Air Pollutants, Occupational↗
PubMed2004

The plasma membrane is the site of selective phosphatidylserine oxidation during apoptosis: role of cytochrome C.

Phosphatidylserine (PS) externalization, a functional end point of apoptosis that triggers phagocytic recognition of dying cells, may be modulated by oxidative stress in biological membranes. We previously observed selective oxidation of PS during apoptosis, but the intracellular location and molecular mechanisms responsible for PS oxidation remain to be described. Peroxidation in individual classes of cellular phospholipids was monitored in whole cells and various subcellular fractions obtained from HL-60 cells undergoing apoptosis in response to tert-butyl hydroperoxide (t-BuOOH) after metabolic acylation of phospholipids with the oxidation-sensitive fluorescent fatty acid, cis-parinaric acid. Nonrandom selective oxidation of PS was observed in whole cells, as well as in plasma membrane. PS in mitochondria appeared selectively resistant to oxidation during apoptosis. All phospholipids in nuclear membranes appeared resistant to oxidation after t-BuOOH treatment. Selective PS oxidation was accompanied by cytochrome c release and PS externalization. PS oxidation and externalization were followed by caspase activation and other end points of apoptosis. HL-60 cells "loaded" with exogenous cytochrome c by mild sonication showed selective oxidation of PS in both the absence and presence of t-BuOOH. Cytochrome c/hydrogen peroxide could effectively oxidize purified PS but not phosphatidylcholine in a cell-free model system. Selective plasma membrane-based PS oxidation and subsequent externalization during oxidant-induced apoptosis may be mediated through the redox activity of cytochrome c.

Apoptosis↗
PubMed2002

Redox sensor function of metallothioneins.

In summary, the redox conversions of MT cysteines are likely to be the principal mechanisms for regulation of metal binding and release by this protein. Oxidative and/or nitrosative challenges can serve to promote metal ion release from MT to render their delivery to specific target proteins. It is tempting to consider the potential roles of MTs as redox sensors because of their high sensitivity to cysteine modification, as well as their potential to amplify signals by releasing multiple metal ions. In other words, MTs may act early in a biological signaling cascade that triggers metal-dependent biochemical and cellular responses. Alternatively, uncontrolled release of metals by excessive oxidative stress may contribute to metal toxicity. Because oxidative and nitrosative signaling is ubiquitous within cells, the physiological function of MT demands that efficient recycling of modified cysteines be operative. Little is known regarding the potential mechanisms for the regeneration of MT after oxidative/nitrosative modification, but they may involve endogenous dithiols, such as thioredoxin, and pharmacologically relevant dithiols, such as dihydrolipoate.

Animals↗
PubMed2002

Selective peroxidation and externalization of phosphatidylserine in normal human epidermal keratinocytes during oxidative stress induced by cumene hydroperoxide.

Reactive oxygen species not only modulate important signal transduction pathways, but also induce DNA damage and cytotoxicity in keratinocytes. Hydrogen peroxide and organic peroxides are particularly important as these chemicals are widely used in dermally applied cosmetics and pharmaceuticals, and also represent endogenous metabolic intermediates. Lipid peroxidation is of fundamental interest in the cellular response to peroxides, as lipids are extremely sensitive to oxidation and lipid-based signaling systems have been implicated in a number of cellular processes, including apoptosis. Oxidation of specific phospholipid classes was measured in normal human epidermal keratinocytes exposed to cumene hydroperoxide after metabolic incorporation of the fluorescent oxidation-sensitive fatty acid, cis-parinaric acid, using a fluorescence high-performance liquid chromatography assay. In addition, lipid oxidation was correlated with changes in membrane phospholipid asymmetry and other markers of apoptosis. Although cumene hydroperoxide produced significant oxidation of cis-parinaric acid in all phospholipid classes, one phospholipid, phosphatidylserine, appeared to be preferentially oxidized above all other species. Using fluorescamine derivatization and annexin V binding it was observed that specific oxidation of phosphatidylserine was accompanied by phosphatidylserine translocation from the inner to the outer plasma membrane surface where it may serve as a recognition signal for interaction with phagocytic macrophages. These effects occurred much earlier than any detectable changes in other apoptotic markers such as caspase-3 activation, DNA fragmentation, or changes in nuclear morphology. Thus, normal human epidermal keratinocytes undergo profound lipid oxidation with preference for phosphatidylserine followed by phosphatidylserine externalization upon exposure to cumene hydroperoxide. It is therefore likely that normal human epidermal keratinocytes exposed to similar oxidative stress in vivo would under go phosphatidylserine oxidation/translocation. This would make them targets for macrophage recognition and phagocytosis, and thus limit their potential to invoke inflammation or give rise to neoplastic transformations.

Adult↗
PubMed2002

Quantification of oxidative/nitrosative modification of CYS(34) in human serum albumin using a fluorescence-based SDS-PAGE assay.

The SH group represented by cysteine in proteins is fundamental to the redox regulation of protein structure and function. Albumin is the most abundant serum protein whose redox modification modulates its physiologic function, as well as serves as a biomarker of oxidative stress. Measurement of selective Cys modification (S-oxidation/nitrosation, electrophilic substitution) on specific proteins, however, is problematic within complex biological mixtures such as plasma. We have utilized a maleimide fluorogenic SH reagent, ThioGlo-1, to develop a fluorescence-based quantitative assay of SH modification of human serum albumin (hSA) using SDS-PAGE. Fully reduced native albumin containing one free SH (Cys(34)) per molecule was utilized as a model protein to characterize the kinetics of ThioGlo-1 reaction using a solution-based spectrofluorometric assay. Optimum labeling of hSA Cys(34) was achieved within 10 min at 60 degrees C using a threefold molar excess of ThioGlo-1 relative to hSA and required SDS. Comparison of the solution spectrofluorometric assay to fluorescent image analysis of hSA bands localized by SDS-PAGE revealed that SH groups in hSA could be quantified after gel electrophoresis. The solution- and gel-based methods were in excellent concordance in their ability to quantify SH modification of hSA following exposure to phenoxyl radicals and nitric oxide. The application of ThioGlo-1 staining and SDS-PAGE quantified the degree of hSA modification in complex human plasma exposed to oxidative or nitrosative stress and revealed that hSA is more sensitive to S modification than other SH-containing plasma proteins.

Binding Sites↗