Lung surfactant gelation induced by epithelial cells exposed to air pollution or oxidative stress

Lung surfactant lowers surface tension and adjusts interfacial rheology to facilitate breathing. A novel instrument, the interfacial stress rheometer (ISR), uses an oscillating magnetic needle to measure the shear viscosity and elasticity of a surfactant monolayer at the air-water interface. The ISR reveals that calf lung surfactant, Infasurf, exhibits remarkable fluidity, even when exposed to air pollution residual oil fly ash (ROFA), hydrogen peroxide (H2O2), or conditioned media from resting A549 alveolar epithelial cells (AEC). However, when Infasurf is exposed to a subphase of the soluble fraction of ROFA- or H2O2-treated AEC conditioned media, there is a prominent increase in surfactant elasticity and viscosity, representing two-dimensional gelation. Surfactant gelation is decreased when ROFA-AEC are pretreated with inhibitors of cellular reactive oxygen species (ROS), or with a mitochondrial anion channel inhibitor, as well as when A549-rho0 cells that lack mitochondrial DNA and functional electron transport are investigated. These results implicate both mitochondrial and nonmitochondrial ROS generation in ROFA-AEC-induced surfactant gelation. A549 cells treated with H2O2 demonstrate a dose-dependent increase in lung surfactant gelation. The ISR is a unique and sensitive instrument to characterize surfactant gelation induced by oxidatively stressed AEC.     

Cyclin alterations in giant cell tumor of bone.

Cyclins play an important role in regulating the passage of dividing cells through critical checkpoints in the cell cycle. Because alterations of several cyclins, especially cyclin D1, have been implicated in the development of many human neoplasms, we examined 32 cases of giant cell tumor of long bones for cyclin D1 gene amplification and protein overexpression using differential polymerase chain reaction and immunohistochemistry, respectively. In addition, the expression of cyclin D3, cyclin B1, and the proliferation-associated antigen Ki-67 (MIB-1) was assessed immunohistochemically. Low-level cyclin D1 gene amplification was detected in 61% of giant cell tumor cases. All tumors showed cyclin D1, cyclin D3, cyclin B1, and Ki-67 (MIB-1) staining; however, the distribution was very characteristic. Cyclin D1 protein expression was seen predominantly in the nuclei of the giant cells, with occasional mononuclear cells staining. There was no correlation between cyclin D1 gene amplification and protein overexpression. Cyclin D3 staining showed a similar distribution, with 88% of cases showing protein overexpression. Cyclin D1 and/or D3 staining in the giant cells was never associated with staining for either cyclin B1 or Ki-67 (MIB-1), as the expression of the latter two proteins was restricted to the mononuclear cells. Cyclin B1 overexpression was seen in 44% of cases. Ki-67 (MIB-1) staining was present in all cases, and between 10 to 50% of the mononuclear cells were positive. These results suggest that alterations in cyclin D1 and/or D3 might play a role in the pathogenesis of giant cell tumor of bone.     

A screening assay for detecting CD8+ cell non-cytotoxic anti-HIV responses

The rate of HIV-1 disease progression is influenced by several factors that include pathogen and host genetic variations and the quality of antiviral immune responses. The CD8+ cell non-cytotoxic antiviral response (CNAR) substantially suppresses HIV replication in CD4+ cells and is positively associated with an asymptomatic clinical state. Traditionally, the measurement of CNAR has required several culture procedures and costly reagents. Here we report the development and validation of a screening assay for detection of CNAR that accurately identifies individuals benefiting from this response. Use of the CNAR screening assay should facilitate the evaluation of this important immune parameter in studies of HIV pathogenesis, resistance to infection, and vaccine development.     

New rat model of Pneumocystis carinii infection.

Rats free of latent Pneumocystis carinii organisms were immunosuppressed with adrenal corticosteroids and transtracheally injected with P. carinii. These animals subsequently developed P. carinii pneumonia. Infection was accomplished by using organisms from infected rat lung or from culture. Diffuse infection was produced with no significant differences in the numbers of organisms found in various lobes of the lungs. Infections progressed over time so that by 6 weeks postinoculation all animals were heavily infected. Infection by transtracheal injection has three advantages over current models. First, transtracheal injection provides a reliable model which is not dependent on naturally occurring latent Pneumocystis infection. Second, transtracheal injection allows the perpetuation of specific Pneumocystis strains. Third, transtracheal injection is a more rapid and economical means of producing severe Pneumocystis pneumonia.     

In vitro effector activity of Pneumocystis murina-specific T-cytotoxic-1 CD8+ T cells: role of granulocyte-macrophage colony-stimulating factor

Human immunodeficiency virus (HIV)-related opportunistic infections continue to occur in patients who are newly diagnosed with HIV infection, those in the early course of highly active antiretroviral therapy or nonadherent to HIV care, and other immunosuppressed individuals. One of the most common opportunistic infections in these patients is Pneumocystis pneumonia. CD8+ T cells are recruited to the lung after P. carinii infection and have been associated with both lung injury and host defense. This variability may be due to subpopulations of CD8+ T cells recruited to the lung. We have previously shown using adoptive transfer studies that in vivo-generated T-cytotoxic-1 (Tc1) CD8+ T cells, defined by the secretion of gamma interferon (IFN-gamma), have effector activity against Pneumocystis spp. in vitro as well as in vivo. To better understand the mechanisms of these effects, we generated, expanded, and tested Tc1 and Tc2 CD8+ T cells specific for P. murina ex vivo. Tc1-polarized CD8+ T cells secreted higher levels of IFN-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) and lower levels of interleukin-4 (IL-4), IL-5, IL-10, and IL-13 than Tc2 CD8+ T cells when stimulated with P. murina antigen. Moreover, Tc1 CD8+ T cells demonstrated enhanced effector activity in a macrophage-mediated killing assay which was independent of cell contact. The augmentation in macrophage-mediated P. murina killing was significantly abrogated when GM-CSF was neutralized in the Tc1 CD8+ T cells. These data support the possibility that antigen-specific GM-CSF secretion is critical for effector activity of P. murina-specific Tc1 CD8+ T cells in vitro.     

Is Irregular Regression of Corpora Lutea in Climacteric Women Caused by Age-Induced Alterations in the “Tissue Control System”?

PROBLEM: We have recently observed that the regression of corpora lutea (CL) in women during the reproductive period of life is accompanied by a diminution of Thy-1 differentiation protein release from vascular pericytes and an accumulation of T lymphocytes and activated macrophages among both degenerating granulosa lutein cells (GLC) and theca lutein cells. These data suggest that the immune system and other stromal factors, representing components of the “tissue control system,” may play a role in regression of the CL. We investigated degenerating CL from climacteric women to address the possibility that the decline of immune functions with advancing age may result in incomplete regression of luteal tissue. This could contribute to the altered hormonal profiles and abnormal uterine bleeding that frequently occur during the climacteric. METHOD: Immunoperoxidase staining and image analysis were used to localize Thy-1 differentiation protein of vascular pericytes, cytokeratin staining of GLC, neural cell adhesion molecule expression by theca lutein cells, CD15 of neutrophils, CD4, CD14, CD68, and leukocyte common antigens of macrophages, and CD3 and CD8 determinants of T lymphocytes. We also investigated the expression of luteinizing hormone receptor (LH receptor) and mitogen activated protein kinases (MAP kinases) in luteal cells. Samples of regressing luteal tissue were obtained during the follicular phase from perimenopausal women (age 45–50) who exhibited prolonged or irregular cycles. For comparison, luteal tissues from women with regular cycles (age 29–45) and CL of pregnancy were also investigated. RESULTS: Corpora lutea of the climacteric women exhibited irregular regression of luteal tissue characterized by a lack of cytoplasmic vacuolization and nuclear pyknosis in GLC, and by a persistence of theca lutein cells exhibiting hyperplasia and adjacent theca externa layers. This was accompanied by a continuing release of Thy-1 differentiation protein from vascular pericytes. Persisting GLC lacked surface expression of macrophage markers (CD4, CD14, CD68 and leukocyte common antigen) as well as nuclear granules exhibiting CD15 of neutrophils, detected in regularly regressing GLC. In addition, such persisting GLC showed weak or no LH receptor expression, and retained the expression of cytokeratin. They also exhibited enhanced staining for MAP kinases. Strong cytoplasmic MAP kinase expression with occasional nuclear translocation was also detected in persisting theca lutein cells, indicating high metabolic activity of these cells. T lymphocytes, although occasionally present in luteal stroma within luteal convolutions, did not invade among persisting GLC and were virtually absent from layers of theca externa and theca lutein cells. CONCLUSIONS: These data indicate that the regressing CL in climacteric women may exhibit persistence of luteal cells, perhaps because of age-induced alterations of the immune system and other local stromal homeostatic mechanisms involved in the elimination of luteal cells. Persisting GLC and/or theca lutein cells may exhibit abnormal hormonal secretion that contributes to the alteration of target tissues, such as the endometrium, resulting in abnormal uterine bleeding, hyperplasia, and neoplasia.     

Vitamin C-induced loss of redox-dependent viability in lung microvascular endothelial cells

Recent clinical trials have shown that vitamin C, at pharmacological concentrations (milligram to approximately gram), upon infusion into circulation, modulates vasodilation and vascular tone in humans. This also results in the elevated concentrations of vitamin C in circulation in the millimolar range. Here, it was hypothesized that vitamin C at pharmacological concentrations (millimolar) would induce oxidative stress and cause loss of redox-dependent cell viability in vascular endothelial cells (ECs). To test the hypothesis, bovine lung microvascular ECs (BLMVECs) in monolayer cultures were exposed to vitamin C (0-10 mM) for different time periods (0-2 h). Electron paramagnetic resonance spectroscopy revealed the intracellular formation of ascorbate free radical in a dose- and time-dependent fashion. Vitamin C also induced formation of intracellular reactive oxygen species in a dose-dependent fashion. It was observed that vitamin C induced morphological alterations and loss of cell viability in a dose- and time-dependent fashion, as measured by light microscopy and Alamar Blue redox cell viability assay, respectively. Vitamin C analogues failed to induce such changes. Vitamin C depleted cellular GSH levels in a dose-dependent fashion, suggesting that vitamin C altered thiol-redox status in BLMVECs. Antioxidants, intracellular iron chelator, and catalase protected cells against vitamin C-induced loss of redox-dependent cell viability, confirming the role of hydrogen peroxide and iron during redox cycling of vitamin C. These results, for the first time in detail, established that vitamin C at pharmacological doses induced oxidative stress and loss of redox-dependent cell viability in microvascular ECs.