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.     

Loss of LFA-1, but not Mac-1, protects MRL/MpJ-Fas(lpr) mice from autoimmune disease

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by immune complex-mediated tissue injury. Many different adhesion molecules are thought to participate in the development of SLE; however, few studies have directly examined the contributions of these proteins. Here we demonstrate that LFA-1 plays an essential role in the development of lupus in MRL/MpJ-Fas(lpr) mice. Mice deficient in LFA-1, but not Mac-1, showed significantly increased survival, decreased anti-DNA autoantibody formation, and reduced glomerulonephritis. The phenotype of the LFA-1-deficient mice was similar to that observed in beta(2) integrin-deficient (CD18-null) MRL/MpJ-Fas(lpr) mice, suggesting a lack of redundancy among the beta(2) integrin family members and other adhesion molecules. These studies identify LFA-1 as a key contributor in the pathogenesis of autoimmune disease in this model, and further suggest that therapeutic strategies targeting this adhesion molecule may be beneficial for the treatment of SLE.     

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.     

Neuroprotective effects of IGF-I against TNFalpha-induced neuronal damage in HIV-associated dementia

Human immunodeficiency virus type 1 (HIV-1) infection often results in disorders of the central nervous system, including HIV-associated dementia (HAD). It is suspected that tumor necrosis factor-alpha (TNFalpha) released by activated and/or infected macrophages/microglia plays a role in the process of neuronal damage seen in AIDS patients. In light of earlier studies showing that the activation of the insulin-like growth factor I receptor (IGF-IR) exerts a strong neuroprotective effect, we investigated the ability of IGF-I to protect neuronal cells from HIV-infected macrophages. Our results demonstrate that the conditioned medium from HIV-1-infected macrophages, HIV/CM, causes loss of neuronal processes in differentiated PC12 and P19 neurons and that these neurodegenerative effects are associated with the presence of TNFalpha. Furthermore, we demonstrate that IGF-I rescues differentiated neurons from both HIV/CM and TNFalpha-induced damage and that IGF-I-mediated neuroprotection is strongly enhanced by overexpression of the wt IGF-IR cDNA and attenuated by the antisense IGF-IR cDNA. Finally, IGF-I-mediated antiapoptotic pathways are continuously functional in differentiated neurons exposed to HIV/CM and are likely supported by TNFalpha-mediated phosphorylation of I(kappa)B. All together these results suggest that the balance between TNFalpha and IGF-IR signaling pathways may control the extent of neuronal injury in this HIV-related experimental setting.     

Use of 16S rRNA gene sequencing for rapid identification and differentiation of Burkholderia pseudomallei and B. mallei

Burkholderia pseudomallei and B. mallei, the causative agents of melioidosis and glanders, respectively, are designated category B biothreat agents. Current methods for identifying these organisms rely on their phenotypic characteristics and an extensive set of biochemical reactions. We evaluated the use of 16S rRNA gene sequencing to rapidly identify these two species and differentiate them from each other as well as from closely related species and genera such as Pandoraea spp., Ralstonia spp., Burkholderia gladioli, Burkholderia cepacia, Burkholderia thailandensis, and Pseudomonas aeruginosa. We sequenced the 1.5-kb 16S rRNA gene of 56 B. pseudomallei and 23 B. mallei isolates selected to represent a wide range of temporal, geographic, and origin diversity. Among all 79 isolates, a total of 11 16S types were found based on eight positions of difference. Nine 16S types were identified in B. pseudomallei isolates based on six positions of difference, with differences ranging from 0.5 to 1.5 bp. Twenty-two of 23 B. mallei isolates showed 16S rRNA gene sequence identity and were designated 16S type 10, whereas the remaining isolate was designated type 11. This report provides a basis for rapidly identifying and differentiating B. pseudomallei and B. mallei by molecular methods.     

Comparative genomic indexing reveals the phylogenomics of Escherichia coli pathogens

The Escherichia coli O26 serogroup includes important food-borne pathogens associated with human and animal diarrheal disease. Current typing methods have revealed great genetic heterogeneity within the O26 group; the data are often inconsistent and focus only on verotoxin (VT)-positive O26 isolates. To improve current understanding of diversity within this serogroup, the genomic relatedness of VT-positive and -negative O26 strains was assessed by comparative genomic indexing. Our results clearly demonstrate that irrespective of virulence characteristics and pathotype designation, the O26 strains show greater genomic similarity to each other than to any other strain included in this study. Our data suggest that enteropathogenic and VT-expressing E. coli O26 strains represent the same clonal lineage and that VT-expressing E. coli O26 strains have gained additional virulence characteristics. Using this approach, we established the core genes which are central to the E. coli species and identified regions of variation from the E. coli K-12 chromosomal backbone.     

Extravasation and transcytosis of liposomes in Kaposi's sarcoma-like dermal lesions of transgenic mice bearing the HIV tat gene.

Transgenic mice bearing the HIV tat gene develop dermal lesions resembling a common malignant tumor in AIDS, Kaposi's sarcoma (KS). To evaluate the permeability characteristics of these lesions and the therapeutic potential of drug-carrying liposomes, we have studied the localization of sterically stabilized liposomes, which show long circulation time in blood and increased accumulation in tumors. Liposomes encapsulating colloidal gold were injected intravenously into transgenic mice bearing KS lesions, and tissues were processed 24 hours later for both electron microscopy and for light microscopy with silver enhancement. Liposomes and silver marker were detected predominantly in the dermis surrounding the early and mature KS lesions, which were characterized by a proliferation of fibroblast-like spindle cells and abnormal blood vessels close to the epidermis. The silver-enhanced gold marker often surrounded vascular channels and scattered erythrocytes. As determined by electron microscopy, some spindle cells and macrophages had ingested intact liposomes. Transendothelial transport of liposomes was observed both through open channels between endothelial cells and also through endothelial cells lining intact vessels. Both extravasation and transcytosis of liposomes through irregular endothelium were much higher in KS lesions than in the adjacent normal skin. The high accumulation of sterically stabilized liposomes in KS lesions and their intracellular uptake by some spindle cells enhances their potential as carriers of chemotherapeutic agents against this neoplasm.     

Serial Testing of Postural Control After Acute Lateral Ankle Sprain

OBJECTIVE: To identify subjects' changes in postural control during single-leg stance in the 4 weeks after acute lateral ankle sprain. DESIGN AND SETTING: We used a 2 x 2 x 3 (side-by-plane-by-session) within-subjects design with repeated measures on all 3 factors. All tests were performed in a university laboratory. SUBJECTS: Seventeen young adults (9 men, 8 women; age, 21.8 +/- 5.9 years; mass, 74.9 +/- 10.5 kg; height, 176.9 +/- 7.1 cm) who had sustained unilateral acute mild or moderate lateral ankle sprains. MEASUREMENTS: Measures of center-of-pressure excursion length, root mean square velocity of center-of-pressure excursions (VEL), and range of center-of-pressure excursions (RANGE) were calculated separately in the frontal and sagittal planes during 5-second trials of static single-leg stance. RESULTS: We noted significant side-by-plane-by-session interactions for magnitude of center-of-pressure excursions in a given trial (PSL) (P =.004), VEL (P =.011), and RANGE (P =.009). Both PSL and VEL in the frontal plane were greater in the injured limbs compared with the uninjured limbs on day 1 and during week 2 but not during week 4, whereas sagittal-plane differences existed during all 3 testing sessions. Injured-limb, frontal-plane RANGE scores were greater than uninjured values at day 1 but not during weeks 2 or 4. No significant differences in sagittal-plane RANGE scores were seen. CONCLUSIONS: Postural control was significantly impaired in the injured limbs at day 1 and during week 2 after lateral ankle sprain but not during week 4. Consistent improvement in postural control measures on both injured and uninjured limbs was seen throughout the 4 weeks after ankle sprain.