In vitro priming with adenovirus/gp100 antigen-transduced dendritic cells reveals the epitope specificity of HLA-A*0201-restricted CD8+ T cells in patients with melanoma

Replication-deficient recombinant adenovirus (Ad) encoding human gp100 or MART-1 melanoma Ag was used to transduce human dendritic cells (DC) ex vivo as a model system for cancer vaccine therapy. A second generation E1/E4 region deleted Ad which harbors the CMV immediate-early promoter/enhancer and a unique E4-ORF6/pIX chimeric gene was employed as the backbone vector. We demonstrate that human monocyte-derived DC are permissive to Ad infection at multiplicity of infection between 100 and 500 and occurs independent of the coxsackie Ad receptor. Fluorescent-labeled Ad was used to assess the kinetics and distribution of viral vector within DC. Ad-transduced DC show peak transgene expression at 24-48 h and expression remains detectable for at least 7 days. DC transduced with replication-deficient Ad do not exhibit any unusual phenotypic characteristics or cytopathic effects. DC transduced with Ad2/gp100v2 can elicit tumor-specific CTL in vitro from patients bearing gp100+ metastatic melanoma. Using a panel of gp100-derived synthetic peptides, we show that Ad2/gp100v2-transduced DC elicit Ag-specific CTL that recognize only the G209 and G280 epitopes, both of which display relatively short half-lives ( approximately 7-8 h) on the surface of HLA-A*0201+ cells. Thus, patients with metastatic melanoma are not tolerant to gp100 Ag based on the detection of CD8+ T cells specific for multiple HLA-A*0201-restricted, gp100-derived epitopes.     

HLA-A2.1/K(b) transgenic murine dendritic cells transduced with an adenovirus encoding human gp100 process the same A2.1-restricted peptide epitopes as human antigen-presenting cells and elicit A2.1-restricted peptide-specific CTL

HLA-A2.1/K(b) transgenic mice (A2.1/K(b) mice) were used to investigate the processing of human gp100 melanoma antigen by murine antigen presenting cells (APC). Bone marrow-derived dendritic cells (DC) from A2.1/K(b) mice were transduced with adenovirus encoding human gp100 (Ad2/hugp100v2). The Ad2/hugp100v2-transduced DC express human gp100, as documented by immunoperoxidase staining. Flow cytometric analysis demonstrates that Ad vector transduction does not downregulate expression of several markers, including MHC class I. We show that Ad2/hugp100v2-transduced DC are recognized by peptide-specific, A2.1-restricted CTL, suggesting correct processing and presentation of the hugp100 antigen by murine DC. To assess dominance among the various A2.1-restricted epitopes encoded by hugp100, A2.1/K(b) transgenic mice were immunized with Ad2/hugp100v2-transduced DC. Resulting effector cytotoxic T lymphocytes (CTL) were assayed for peptide specificity using a panel of six synthetic peptides known to encode A2.1-restricted epitopes of human gp100 (denoted G154, G177, G209, G280, G457, G476). CTL obtained from Ad2/hugp100v2-transduced DC immunized A2.1/K(b) mouse lysed target cells presenting five of the six epitopes, supporting the observation that murine cells correctly process the hugp100 antigen. The immunogenicity of individual gp100 epitopes correlates with their binding affinity to A2.1. CTL generated from A2.1/K(b) mice immunized with Ad2/hugp100v2-transduced DC also specifically recognize A2.1(+)/gp100(+) human melanoma cells. These data suggest that murine APC process and present the same set of HLA-restricted peptides, similar to human APC. HLA transgenic mice serve as a useful model system to study class I-restricted epitopes of human tumor-associated antigens.     

Combining fluorescence lifetime and polarization microscopy to discriminate phase separated domains in giant unilamellar vesicles

Using fluorescence lifetime microscopy we study the structure of lipid domains in giant unilamellar vesicles made from sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, and cholesterol. Lifetimes and orientation of a derivative of the fluorescent probe DPH embedded in the membrane were measured for binary and ternary lipid mixtures incorporating up to 42 mol % of cholesterol. The results show that adding cholesterol always increases the lifetime of the probe studied. In addition, the analysis of the probe orientation indicates that cholesterol has little influence on the ordering of the sphingomyelin alkyl chains whereas it has a noticeable effect on the structure of the 1,2-dioleoyl-sn-glycero-3-phosphocholine chains. The measurements made on the orientation and lifetime of the probe show the structure of the membrane in its liquid ordered and liquid disordered domains.     

A new technique for assessing arterial pressure wave forms and central pressure with tissue Doppler

Background

Arterial diameters enlarge in response to wall thickening, plaques, and many atherosclerotic risk factors. We hypothesized that right common carotid artery (RCCA) diameter would be independently associated with cardiac disease and improve risk discrimination.

Methods

In a middle-aged, biracial population (baseline n = 11225), we examined associations between 1 standard deviation increments of baseline RCCA diameter with prevalent myocardial infarction (MI) and incident cardiac events (MI or cardiac death) using logistic regression and Cox proportional hazards models, respectively. Areas under the receiver operator characteristic curve (AUC) were used to estimate model discrimination.

Results

MI was present in 451 (4%) participants at baseline (1987–89), and incident cardiac events occurred among 646 (6%) others through 1999. Adjusting for IMT, RCCA diameter was associated with prevalent MI (female OR = 2.0, 95%CI = 1.61–2.49; male OR = 1.16, 95% CI = 1.04–1.30) and incident cardiac events (female HR = 1.75, 95% CI = 1.51–2.02; male HR = 1.27, 95% CI = 1.15–1.40). Associations were attenuated but persisted after adjustment for risk factors (not including IMT) (prevalent MI: female OR = 1.73, 95% CI = 1.40–2.14; male OR = 1.14, 95% CI = 1.02–1.28, and incident cardiac events: female HR = 1.26, 95% CI = 1.08–1.48; male HR = 1.19, 95% CI = 1.08–1.32). After additional adjustment for IMT, diameter was associated with incident cardiac events in women (HR = 1.18, 95% CI = 1.00–1.40) and men (HR = 1.17, 95% CI = 1.06–1.29), and with prevalent MI only in women (OR = 1.73; 95% CI = 1.37–2.17). In women, when adjustment was limited, diameter models had larger AUC than other models.

Conclusion

RCCA diameter is an important correlate of cardiac events, independent of IMT, but adds little to overall risk discrimination after risk factor adjustment.     

Acute elevation of triglycerides increases left ventricular contractility and alters ventricular-vascular interaction

Acute elevation of circulating lipids, such as the postprandial state, contributes to increased cardiovascular risk. However, the effect of acutely elevated triglycerides on arterial and left ventricular function is not completely understood. We aimed to assess whether an acute increase in triglycerides affects ventricular-vascular interaction. Fifteen healthy men (age, 49 ± 8 yr) underwent blinded, randomized infusion of saline and intravenous fat emulsion to acutely raise plasma triglycerides. All subjects underwent both randomization trials, in random order on two separate days. Ventricular-vascular interaction measures were recorded by tonometry (central blood pressure) and echocardiography (left ventricular volumes, strain, and strain rate) at baseline and after 1 h infusion. Net ventricular-vascular interaction was defined by the effective arterial elastance (E(A))-to-left ventricular end-systolic elastance (E(LV)) ratio (E(A)/E(LV)). When compared with saline, the infusion of intravenous fat emulsion increased triglycerides and free fatty acids (ΔP < 0.001 for both) and improved left ventricular contractility (ΔE(LV), end-systolic volume and strain rate; P < 0.05 for all). However, arterial function was unchanged (ΔE(A), brachial and central blood pressure; P > 0.05 for all). Overall, E(A)/E(LV) was decreased by an infusion of intravenous fat emulsion (P = 0.004) but not saline (P > 0.05, P = 0.001 for Δ between trials). We conclude that intravenous fat emulsion and acute elevation of blood lipids (including triglycerides and free fatty acids) alter ventricular-vascular interaction by increasing left ventricular contractility without affecting arterial load. These findings may have implications for cardiovascular responses to parenteral nutrition.     

Amniotic fluid lipoxygenase metabolites during spontaneous labor and after RU486 treatment during late pregnancy in rhesus macaques

To determine changes in amniotic fluid (AF) lipoxygenase metabolites prior to spontaneous labor and after RU486 administration, we implanted AF and vascular catheters and myometrial electromyographic (EMG) electrodes in 8 rhesus macaques at 120-130 days of pregnancy (term = 167 days). Four animals had AF samples taken serially until they delivered their infants normally at term. The other four animals received RU486 (20 mg/kg/day) for 3 days. AF samples were collected every 2-3 days and at 12 hour intervals for 72 hours before and after treatment with RU486. Uterine activity was monitored continuously. LTB4, 5-HETE and 15-HETE were measured by radioimmunoassay. In untreated animals, LTB4 and 5-HETE concentrations in AF increased significantly (P less than 0.05) 4 days before delivery with no change in 15-HETE. After RU486, mean levels of LTB4 and 5-HETE were increased although the difference was not statistically significant. No change in 15-HETE levels was observed. In conclusion, LTB4 and 5-HETE increase in AF before the onset of spontaneous labor. Progesterone receptor blockade by RU486 does not reproduce the changes in AF lipoxygenase metabolites observed during normal parturition.     

Ferric iron and extracellular electron shuttling increase xylose utilization and butanol production during fermentation with multiple solventogenic bacteria

Xylose is the second most abundant sugar derived from lignocellulose; it is considered less desirable than glucose for fermentation, and strategies that specifically increase xylose utilization in wild-type cells are goals for biofuel production. Xylose consumption, butanol production, and hydrogen production increased in both Clostridium beijerinckii and a novel solventogenic bacterium (strain DC-1) when anthraquinone-2,6,-disulfonate (AQDS) or riboflavin were used as redox mediators to transfer electrons to poorly crystalline Fe(OH)₃ as an extracellular electron sink. Strain DC-1 was most closely related to Rhizobiales bacterium Mfc52 based on 95% 16S rRNA gene sequence similarity, which demonstrates that this response is not limited to a single genus of xylose-fermenting bacteria. Xylose utilization and butanol production were negligible in control incubations containing cells plus 3% (w/v) xylose alone during a 10-day batch fermentation, for both strains tested (n-butanol titers of 0.05 g L⁻¹). Micromolar concentrations of AQDS and riboflavin were added as electron shuttling compounds with poorly crystalline Fe(OH)₃ as an insoluble electron acceptor, and respective n-butanol titers increased to 6.35 and 7.46 g L⁻¹. Increases in xylose consumption for the iron treatments were relatively high, from less than 0.49 g L⁻¹ (xylose alone, no iron or electron shuttling molecules) to 25.98 and 29.15 g L⁻¹ for the AQDS and riboflavin treatments, respectively. Hydrogen production was also 3.68 times greater for the AQDS treatment and 5.27 greater for the riboflavin treatment relative to controls. Strain DC-1 data were similar, again indicating that the effects are not specific to the genus Clostridium.

     

Treatment of melanoma with 5-fluorouracil or dacarbazine in vitro sensitizes cells to antigen-specific CTL lysis through perforin/granzyme- and Fas-mediated pathways

Several factors may influence sensitivity of melanoma cells to CTL lysis. One is the avidity of the CTL TCR. A second is that certain cytotoxic drugs have been reported to sensitize cancer cells to CTL lysis through Fas-mediated apoptosis. In this study, we examined whether antineoplastic agents 5-fluorouracil (5-FU) and dacarbazine (DTIC) sensitize melanoma cells to lysis of G209 peptide-specific CTL. Our results show that CTL generated from PBMC are HLA-A2 restricted and gp100 specific. Treatment with 5-FU or DTIC sensitized melanoma cells to lysis of G209-specific CTL. Most importantly, 5-FU- or DTIC-treated melanoma cells also became sensitive to low-avidity CTL, which per se are less cytolytic to melanomas. We sought to identify apoptotic pathways mediating this effect. The enhanced cytolysis was mediated through the perforin/granzyme pathway. Although 5-FU up-regulated FasR expression on melanoma cells, sensitization was not blocked by anti-Fas Ab, and the G209-specific CTL was Fas ligand (FasL) negative. However, when G209-specific CTL were stimulated to express FasL, FasL signaling also contributed to enhanced cytolysis. DTIC treatment, which did not increase FasR expression, also sensitized FasL-mediated killing induced by neutralizing anti-Fas Ab. For CD95L-positive G209-specific CTL, the sensitization was primarily mediated through the perforin/granzyme pathway regardless of up-regulation of FasR. The findings demonstrate that cytotoxic drug-mediated sensitization primes both perforin/granzyme and Fas-mediated killing by melanoma-specific CTL. Considering that most of autoreactive antitumor CTL are low avidity, the findings provide experimental basis for understanding cytotoxic and immunologic therapeutic synergy in melanoma.     

Atherosclerotic disease is increased in recent-onset rheumatoid arthritis: a critical role for inflammation

Rheumatoid arthritis (RA) patients have increased mortality and morbidity as a result of cardiovascular and cerebrovascular disease. What is not clear, however, is either how early accelerated atherosclerosis begins in RA or how soon risk factors must be rigorously controlled. Furthermore, given the strong relationship of vascular disease to RA mortality and of inflammation to the accelerated atherosclerosis associated with RA, it is important to evaluate indices that could serially and noninvasively quantify atherosclerotic disease in RA patients. The carotid intima-media thickness (cIMT) and plaque, measured by ultrasound, correlate closely with direct measurement of the local and systemic atherosclerotic burden. To investigate the presence of subclinical atherosclerosis in the early stages of RA, the cIMT and plaque were measured using carotid duplex scanning in 40 RA patients with disease duration < 12 months and in 40 control subjects matched for age, sex and established cardiovascular risk factors. Patients with RA had significantly higher average cIMT values and more plaque than the control group (cIMT 0.64 ± 0.13 mm versus 0.58 ± 0.09 mm, respectively; P = 0.03). In RA patients, the cIMT was predicted by age and C-reactive protein level at first presentation to the clinic (R ² = 0.64). C-reactive protein was associated with age of disease onset and history of smoking. Since inflammation has been shown to predate onset of clinical RA, the accelerated atherogenic process related to inflammation may precede RA symptom onset.     

Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast

DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and protein phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2A^Cdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We show that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1, which encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK (cdc28F19), also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.