Increased CD8(+) T cells associated with Chlamydia pneumoniae in symptomatic carotid plaque.

BACKGROUND AND PURPOSE: The presence of Chlamydia pneumoniae has been reported in carotid atheroma, but its causative effect in the activation of an atherosclerotic plaque to a prothrombotic state remains unproved. Antigen- mediated activation of T lymphocytes within plaque may represent a mechanism by which infection can result in plaque conversion. The goal of the present study was to characterize the T-cell subtype profile related to the presence of C pneumoniae in patients with symptomatic versus asymptomatic carotid atherosclerosis. METHODS: We studied 14 plaques (5 symptomatic and 9 asymptomatic) positive for C pneumoniae confirmed by polymerase chain reaction and 14 plaques (6 symptomatic and 8 asymptomatic) from age- and stenosis-matched patients negative for C pneumoniae by polymerase chain reaction. T-cell subpopulations of T-helper, T-cytotoxic, and T-memory lymphocytes were identified through indirect enzyme immunohistochemistry with anti-CD3+, anti-CD4+, anti-CD8+, and anti-CD45RO+ monoclonal antibodies, respectively. Results are expressed as the number of positive cells per millimeter squared. RESULTS: In the absence of C pneumoniae, symptomatic plaques had a modest but significant increase of CD3+ (89.6 versus 55.3, P=0.013), CD4+ (57.3 versus 32.7, P=0.01), and CD45RO+ (82.8 versus 43.7, P=0.007), but not CD8+ T cells (28.5 versus 25.5, P=0.245) compared with asymptomatic. However, in the presence of C pneumoniae, there was significant increase of all T-lymphocyte subtypes in symptomatic plaques, including CD8+ (76.8 versus 30.3, P=0.03), CD3+ (192.1 versus 80.4, P=0.004), CD4+ (111.9 versus 37.9, P=0.003), and CD45RO+ (120.2 versus 72.9, P=0.003) cells compared with asymptomatic plaques. With use of 2-way ANOVA, both the presence of chlamydia and symptoms were associated with significantly higher T-cell counts (P<0.005 for all subtypes). CONCLUSIONS: Although all patients with symptomatic disease show a modest elevation in the concentration of intraplaque lymphocytes, a preferential increase in CD8+ class I-restricted T cells is observed in symptomatic carotid plaque positive for C pneumoniae. These data provide incentive to further explore the role of Chlamydia in the modification of immune-mediated mechanisms in active atherosclerotic plaque.

Quantitative expression profile of androgen-regulated genes in prostate cancer cells and identification of prostate-specific genes.

Quantitative expression profile of androgen-regulated genes (ARGs) was evaluated in the hormone-responsive prostate cancer cell line LNCaP by serial analysis of gene expression (SAGE). A total of 83,489 SAGE tags representing 23,448 known genes or expressed sequence tags (ESTs) and 1,655 potentially novel sequences have unraveled the transcriptome of LNCaP cells, the most common cell line used in prostate cancer research. Comparison of transcripts between control and R1881-treated LNCaP cells revealed the induction of 136 genes and repression of 215 genes in response to androgen (p < 0.05). Strikingly, a high fraction ( approximately 90%) of ARGs identified in our study has not been described as ARGs previously. A number of prostate-specific transcription factors were among the ARGs identified here. Classification of the ARGs on the basis of biochemical functions revealed that a great majority of ARGs identified in our experimental system appear to be involved in regulation of transcription, splicing, ribosomal biogenesis, mitogenesis, bioenergetics and redox processes. One of the novel aspects of androgen signaling included androgen regulation of genes involved in DNA repair/recombination process. By comparing our LNCaP-C and LNCaP-T SAGE libraries with SAGE tag libraries available at the NCBI-SAGE website, we have identified >200 potential prostate specific/abundant transcripts. The discovery of new prostate-specific genes and ARGs provides a unique opportunity to determine the role of these genes in prostate cell growth, differentiation and tumorigenesis.

Presence of Chlamydia pneumoniae in human symptomatic and asymptomatic carotid atherosclerotic plaque.

BACKGROUND: Chlamydia pneumoniae has been identified in atherosclerotic plaques of patients with cerebrovascular and cardiovascular disease. However, the direct causative effect of C pneumoniae infection in the activation of atherosclerotic plaque to a prothrombotic state remains to be established. The aim of the present study is to examine the correlation between intraplaque presence of chlamydiae and symptomatic carotid disease in humans. METHODS: Plaques from 37 symptomatic and 57 asymptomatic consenting patients undergoing carotid endarterectomy were snap-frozen, and the tissue was prepared for polymerase chain reaction analysis for Chlamydia pneumoniae per Institutional Review Board-approved protocol. Blood was drawn from each patient at the time of surgery for serological analysis. RESULTS: The overall rate of plaques positive for C pneumoniae was 14.82%, with 5 of 37 (13.5%) plaques from symptomatic patients and 9 of 57 (15.8%) from asymptomatic patients, which revealed a definitive presence of the organism. No association existed between C pneumoniae presence and symptomatic disease (P:=1.0). Also, no association existed between presence of C pneumoniae and severity of stenosis. Finally, seropositivity for anti-chlamydial IgG, IgA, and IgM anti-chlamydial antibodies did not correlate with identification of C pneumoniae in the plaques. However, high-serum anti-chlamydial IgA levels (>/=1:128) were associated with occurrence of symptomatic disease (P=0.03; odds ratio, 2.86; 95% CI, 1.12 to 7.28). CONCLUSIONS: Presence of C pneumoniae as a single factor does not appear to be sufficient to explain the occurrence of cerebrovascular symptoms. Low sensitivity of seropositivity for IgG, IgA, or IgM associated with PCR-identified C pneumoniae presence in the plaque makes it unlikely to be valuable as the single determining factor for actively infected plaque. Association of high-level anti-chlamydial IgA with symptomatic disease suggests that chronic or acute chlamydial infection anywhere in the body could play a role in atherosclerotic plaque activation and be used as a marker to target populations in future stroke prevention trials.

Selection for enhanced adhesion to microvessel endothelial cells or resistance to interferon-gamma modulates the metastatic potential of murine RAW117 large-cell lymphoma cells.

Poorly liver metastatic large-cell lymphoma RAW117-P cells were sequentially selected in vitro for increased adhesion to murine hepatic sinusoidal endothelial cells. After three or four sequential selections, the selected sublines showed increased rates of adhesion to target hepatic microvessel endothelial cells and increased formation of experimental metastases in the liver. However, the endothelial cell adhesion-selected RAW117 sublines were generally unstable and gradually lost their enhanced adhesive and metastatic properties during passage in culture. Highly metastatic, liver-selected RAW117-H10 large-cell lymphoma cells were more resistant to the cytostatic effects of interferon-gamma (IFN-gamma) than poorly metastatic unselected parental RAW117-P cells. When tested for their sensitivity to IFN-gamma, the endothelial cell adhesion variants were significantly more resistant than the unselected RAW117-P cells, but after a 72-h treatment with IFN-gamma, the in vitro-selected cells lost their enhanced endothelial cell adhesion characteristics, their potential to colonize the liver, and their ability to grow when injected at subcutaneous or intramuscular sites. In contrast, the metastatic potential of similarly treated RAW117-P cells was unaffected by IFN-gamma during a 72-h treatment. Sequential selection of RAW117-P cells for increased resistance to IFN-gamma in vitro resulted in variant lines that were refractory to the growth-inhibiting effects of IFN-gamma, and these IFN-gamma-selected variants were also less adhesive to liver microvessel endothelial cells. The IFN-gamma-selected variants also lost their experimental metastatic potentials completely and their tumorigenicities at sites of subcutaneous or intramuscular injection. Cytofluorographic analysis indicated reduced cell surface expression of H-2Kd antigen and fibronectin receptor on the selected variant cells but no change in cell surface mu heavy chain immunoglobulin. The unselected and selected RAW117 lines had similar sensitivities to natural killer (NK) cell-mediated cytolysis, indicating that the in vivo differences were probably not due to differences in NK cell-mediated cytolysis. The results suggest that selection for adhesion to organ microvessel endothelial cells or sequential exposure to certain cytokines can affect the adhesive, growth and metastatic properties of RAW117 cells without modifying their responses to NK cells.

Modulating the metastatic potential of murine RAW117 large-cell lymphoma cells by selection for resistance to interferon-gamma.

Highly metastatic, in vivo-selected cells of RAW117-H10 large-cell lymphoma have been shown to be more resistant than poorly metastatic parental RAW117-P cells to the cytolytic and cytostatic activities of activated macrophages in co-culture experiments. Activated macrophages are known to produce soluble, cytostatic respiration-inhibiting factors, and such activities can be duplicated by interferon-gamma (IFN-gamma) or by combinations of IFN-gamma and Escherichia coli lipopolysaccharide (LPS). Highly metastatic RAW117-H10 cells are more resistant to the cytostatic effects of IFN-gamma and LPS than poorly metastatic RAW117-P cells, and short-term (up to 72 hr) treatment with IFN-gamma and LPS does not change the metastatic potentials of RAW117 cells. We have studied the effects of sequential selection of RAW117-P cells for increased resistance to IFN-gamma and LPS. After 7 to 13 sequential selections, the resulting variant lines were completely refractory to the growth-inhibitory effects of IFN-gamma and LPS and cross-tolerant to macrophage-conditioned medium. The selected variants also completely lost their experimental metastatic potentials and their tumorigenicities after s.c. or i.m. injection. Cytofluorographic analysis indicated reduced cell-surface expression of H-2Kd antigen and fibronectin receptor on the variant cells but no change in surface mu heavy-chain immunoglobulin. The IFN-gamma-selected lines were less adhesive to liver microvascular endothelial cells than the unselected RAW117 cell lines, but were equally sensitive to NK cytolysis by spleen cells. Our results suggest that exposure to certain cytokines can affect the growth and metastatic potential of RAW117 cells and result in the selection of resistant variants with altered biologic properties.

Transcripts of the mitochondrial gene ND5 are overexpressed in highly metastatic murine large cell lymphoma cells.

The relative levels of mitochondrial specific gene transcripts were compared in two murine large cell lymphoma cell lines that differ in their propensities to form liver metastases and in their sensitivity to macrophage mediated antitumor cytostasis and cytolysis. Full-length transcripts of the mitochondrial genes were hybridized on electroblots from citrate/urea gels with specific gene prodes. The mitochondrially encoded gene NADH dehydrogenase subunit 5 (ND5), that encodes a component of NADH dehydrogenase (complex I) of the electron transport chain, was significantly overexpressed in the highly metastatic RAW117-H10 compared to low metastatic RAW117-P cells. Results from analysis of RNA blots were confirmed in an S1 nuclease protection assay. Since RAW117-H10 cells are significantly more resistant to cytostasis by activated macrophages in coculture and such macrophage activity can inhibit RAW117 tumor cell respiration and growth, a mechanism was suggested that allows RAW117 cell escape from certain host effector mechanisms that block cellular respiration by an increase in the in vivo concentrations of translatable messenger RNA (mRNA) that codes for key components of the electron transport chain.

Gene expression and tumor cell escape from host effector mechanisms in murine large cell lymphoma.

Using in vivo selection methods, we obtained metastatic sublines of the murine RAW117 large cell lymphoma that form multiple liver metastases. The highly metastatic subline RAW117-H10 has a low number of gp70 molecules expressed at the cell surface and low cytostatic sensitivity to activated syngeneic macrophages. This subline was infected with endogenous RNA tumor virus isolated from a high virus-expressing RAW117-P subline of low metastatic potential. After superinfection the H10 subline gradually increased its expression of cell surface gp70 and showed enhanced sensitivity to macrophage-mediated cytostasis, suggesting that gp70 might be involved in host macrophage-mediated surveillance. Culture of RAW117-P and H10 cells in media conditioned by activated macrophages indicated that parental cells are severely growth inhibited in a dose dependent fashion while H10 cells showed almost no effect. Examination of differentially expressed genes in the highly metastatic RAW117-H10 cells by analysis of RNA blots indicated that a mitochondrial gene was expressed at a level that was approximately 10 times higher in H10 cells than in parental cells. This gene was identified as ND5, which codes for a subunit of NADH dehydrogenase (complex I of the mitochondrial electron transport chain); this complex is the target for an activated macrophage-released cytostatic factor. Among other possibilities, the results are consistent with the suggestion that highly metastatic RAW117 cells may escape macrophage surveillance by decreasing the synthesis of specific cell-surface receptors for cytostatic molecules and increasing the synthesis of specific cellular targets for such molecules.

Differential expression of metastasis-associated cell surface glycoproteins and mRNA in a murine large cell lymphoma.

A metastatic variant cell subline of the Abelson virus-transformed murine large lymphoma/lymphosarcoma RAW117 has been selected in vivo ten times for liver colonization. Highly metastatic subline RAW117-H10 forms greater than 200 times as many gross surface liver tumor nodules as the parental line RAW117-P. Analysis of cellular proteins and glycoproteins indicates reduced expression of murine Moloney leukemia virus-associated p15, p30, and gp70, and increased expression of a sialoglycoprotein, gp150, in the highly metastatic H10 cells. Northern analyses of oncogene expression suggested that mRNA of various oncogenes was expressed equally or not expressed in the RAW117 cells of differing metastatic potential. Differential gene expression was examined using a cDNA library of 17,600 clones established from poly A+ mRNA isolated from H10 cells. The cDNA library was screened by the colony hybridization technique using probes made from both RAW117-P and -H10 cells. Approximately 99.5% of these cDNA clones were expressed identically in P and H10 cells. Of the few differentially expressed cDNA clones (approx. 150/17,600), one-half of these were identified as Moloney leukemia virus sequences in a separate probing with a radiolabeled Moloney leukemia virus probe. The remainder of the differentially expressed mRNA detected by colony hybridization of the cDNA library were expressed at higher levels (approx. 1/6) or lower levels (approx. 1/3) in the highly metastatic H10 cells.

Nuclear-hormone mediated changes in chromatin solubility.

Rat liver nuclei were incubated with either thyroid hormone or angiotensin (AII) at varying concentrations or with buffer (control) prior to digestion with micrococcal nuclease. Concentrations of hormones greater than 10(-10)M were effective in increasing the solubilization of chromatin with physiological levels (10(-9)M) of AII showing an approximate 2.4 fold increase over control. Nuclei were also isolated from animals treated in-vivo with either AII or buffer (control) and chromatin solubility was increased in the AII treated nuclei even prior to the addition of exogenous nuclease, presumably from the action of endogenous nucleases. The data suggest that hormone-induced increases in solubility are a reflection of structural changes in chromatin which enhance the accessibility of DNA to endonuclease attack.

Studies on two novel angiotensin II actions.

Animal data suggest that angiotensin II may directly affect renal sodium retention independent of aldosterone (4,6). Additionally there is evidence to suggest that the hormone can stimulate protein synthesis in a variety of tissues and that indeed it may be a vasculotoxin (5,7). We describe here experiments designed to elucidate the role of angiotensin II in renal sodium retention in normal man. Additionally we present preliminary evidence suggesting that unlike the rapid sodium retaining effect, some delayed (trophic) actions of the hormone may be generated by a hitherto unappreciated mechanism.

Partitioning of zinc and copper within subnuclear nucleoprotein particles.

Nuclei from frozen calf thymus suspended in buffer were analyzed for metal content prior to and after repeated washing. After three such extractions about 0.1 micrograms Zn/mg DNA and 0.025 micrograms Cu/mg DNA remained tightly associated with chromatin. This level of metal was essentially unchanged with subsequent washings. Digestion of extracted nuclei with micrococcal nuclease yielded soluble nucleoprotein containing zinc and copper. Metal enriched regions of chromatin appeared to be preferentially solubilized by digestion, and the solubilized metal was only partially dializable either with or without EDTA. Metal profiles generated from gel (A-5m) chromatography analysis of chelated and non-chelated solubilized chromatin were distinctive in that copper was undetectable (by flame AA) while zinc was associated only with low molecular weight products when EDTA was used. In contrast, both metals were detected with higher molecular weight oligonucleosomes in the absence of chelating agents. Additionally, the two metals localized within nucleoprotein peaks and these metal-containing regions were only resolved by gel chromatography when EDTA was omitted throughout the procedure. A discrete Cu-rich species in a region of the profile suggests a subset of Cu-rich nucleoprotein complexes.