Myocardial edema in acute myocarditis: relationship of T2 relaxometry and late enhancement burden by using dual-contrast turbo spin-echo MRI.

To quantify myocardial edema by using a T2 relaxometry approach with a dual-contrast turbo spin-echo (dcTSE) sequence in patients with acute myocarditis regarding focal late gadolinium enhancement (LGE) burden. CMR T2 relaxometry was performed in 39 patients (age 41 ± 19 years; 36% women) with LGE in a typical myocarditis pattern and in ten healthy volunteers (age 46 ± 12; 60% woman). dcTSE sequence (echo time 29 and 75 ms, respectively) was used for T2 mapping, analysis were performed on the basis of region of interest (ROI). Myocardial T2 relaxation times (T2 RT) in patients-ROI with focal LGE were significantly (p < 0.001) higher than T2 RT in patients-ROI without apparent LGE pattern (65 ms (IQR 36-95) vs. 60 ms (IQR 26-88), respectively). T2 RT in healthy volunteers [55 ms (IQR 35-71)] were significantly lower than in patients ROI with or without focal LGE-pattern (p < 0.001, respectively). T2 RT assessed by dcTSE are significantly higher in patients segments with and without focal LGE compared to normal controls, supporting a global myocardial inflammatory process in acute myocarditis. Furthermore, this quantitative T2-mapping approach highlights the potential to identify patients with diffuse myocarditis.

Association of aortic stiffness with biomarkers of myocardial wall stress after myocardial infarction.

BACKGROUND: Aortic pulse wave velocity (PWV) was linked to LV-geometry and -function in patients with kidney disease and non-ischemic cardiomyopathy. The role of aortic compliance after acute STEMI is so far unknown. In the present study, we prospectively investigated the relationship of increased aortic stiffness with biomarkers of myocardial wall stress 4 months after STEMI. METHODS: 48 STEMI patients who were reperfused by primary coronary angioplasty underwent cardiovascular magnetic resonance (CMR) at baseline and at 4-month follow-up. The CMR protocol comprised cine-CMR as well as gadolinium contrast-enhanced CMR. Aortic PWV was determined by velocity-encoded, phase-contrast CMR. Blood samples were routinely drawn at baseline and follow-up to determine N-terminal pro-B-type natriuretic peptide (NT-proBNP). In a subgroup of patients, mid-regional pro-adrenomedullin (MR-proADM) and mid-regional pro-A-type natriuretic peptide (MR-proANP) levels were determined. RESULTS: Patients with a PWV above median (>7.0m/s) had significantly higher NT-proBNP, MR-proADM and MR-proANP concentrations at 4-month follow-up than patients with a PWV below median (all p<0.02). PWV showed moderate to good correlation with NT-proBNP, MR-proAMD and MR-proANP levels 4 months after STEMI (all p<0.05). Multivariate analysis revealed PWV, beside myocardial infarct size, as an independent predictor of 4-month NT-proBNP levels after correction for age, creatinine and LV ejection fraction (model r: 0.781, p<0.001). CONCLUSION: Aortic stiffness is directly associated with biomarkers of myocardial wall stress 4 months after reperfused STEMI, suggesting a role for aortic stiffness in chronic LV-remodelling.

Relation of plasma adiponectin levels and aortic stiffness after acute ST-segment elevation myocardial infarction.

BACKGROUND: Pulse wave velocity is a measure of aortic stiffness and an independent predictor of cardiovascular morbidity and mortality. Adiponectin is involved in atherosclerosis and inflammation. In the present study we aimed to explore the association between plasma adiponectin concentrations and pulse wave velocity in the acute phase after ST-segment elevation myocardial infarction (STEMI). METHODS: Forty-six consecutive STEMI patients (mean age 57 ± 11 years) treated with primary percutaneous coronary intervention (PCI) were enrolled in this cross-sectional study. Plasma adiponectin was measured 2 days after index event by enzyme-linked immunosorbent assay. Aortic pulse wave velocity (PWV) was calculated by the transit-time method with the use of a velocity-encoded, phase-contrast cardiac magnetic resonance protocol. RESULTS: Median plasma adiponectin concentration was 2385 ng/ml (interquartile range 1735-5403). Males had lower plasma adiponectin values than females and current smokers had lower values than non-smokers (all p<0.02). Adiponectin was significantly associated with PWV (r=0.505, p<0.001), age (r=0.437, p=0.002), and total cholesterol (r=0.468, p=0.001). Multiple linear regression analysis revealed adiponectin as a predictor of PWV independently of age, sex, smoking status, total cholesterol, and N-terminal pro-B-type natriuretic peptide (p=0.027). CONCLUSIONS: Plasma adiponectin concentrations are strongly associated with aortic stiffness in patients after acute STEMI treated with primary PCI. Our data support a possible role for adiponectin as an independent risk marker for increased aortic stiffness in STEMI patients.

Occurrence of acute myocardial infarction in winter tourists: data from a retrospective questionnaire.

BACKGROUND: Every year millions of tourists spend their vacation in Tyrol, Austria during the winter season. They often perform sports at high altitudes and at low temperatures, factors that might cause acute myocardial infarction (AMI). This study aimed to evaluate the relationship of first physical activity and the onset of AMI in winter tourists. METHODS: We carried out a retrospective analysis of consecutive patients admitted to the Department of Internal Medicine III at the Medical University of Innsbruck with the diagnosis of an AMI between 2006 and 2010. We identified 172 patients as potential candidates for the questionnaire. We successfully contacted 110 patients (mean age: 60 ± 10 years). The location of visit, duration of stay, time of arrival, first sportive activity and onset of symptoms were assessed. RESULTS: During the first 2 days of physical activity , 56% of AMIs occurred. In tourists who suffered AMI during, or within 1 h after cessation of activity (52%), the mean time from the start of the activity to the onset of symptoms was 2.0 ± 1.7 h. 56% of patients performed less than 2.5 h of sport per week before their vacation and 70% had ≥2 cardiovascular risk factors. Although the mean planned vacation time was 8.3 ± 3.7 days, 39% of the patients suffered from AMI on the day of arrival or the day after. CONCLUSION: The majority of AMIs in winter tourists happens within the first 2 days after arrival and within the first 2 days of physical activity.

Effects of fermentable carbohydrates and low dietary phosphorus supply on the chemical composition of faecal bacteria and microbial metabolites in the gastrointestinal tract of pigs.

The objective was to determine the effects of fermentable carbohydrates on phosphorus (P) metabolism, the chemical composition of the faecal mixed bacterial mass (MBM) and the microbial activity in the large intestine (LI) of pigs. Eight barrows (mean BW 35.9 +/- 0.9 kg), fitted with simple T-cannulas at the terminal ileum, were either fed a low-P corn-soybean meal-based control diet or 75% of the control diet supplemented with 25% cellulose, starch or pectin according to a 4 x 3 Latin Square design. Both pectin and cellulose caused higher faecal than ileal P recoveries. Ileal volatile fatty acids (VFA) levels were more pronounced for the starch (p < 0.05) rather than the cellulose and pectin treatments, whereas pectin resulted in a higher faecal VFA concentration in comparison to starch and cellulose (p < 0.05). The differences in faecal VFA concentrations corresponded to the pH values obtained in faeces. The N content of MBM was higher (p < 0.05) when cellulose was supplemented. Pectin caused a decrease in the P content of the MBM compared to the control (p < 0.05). As a result, the N:P ratio was significantly higher for the pectin (N:P = 4.33) than for the control treatment (N:P = 2.63), while the Ca:P ratio remained constant for all treatments, suggesting changes in the accumulation of N, P and Ca in MBM, probably due to changes in the species composition and activity of the microflora.

The effect of dietary phosphorus and calcium level, phytase supplementation, and ileal infusion of pectin on the chemical composition and carbohydrase activity of fecal bacteria and the level of microbial metabolites in the gastrointestinal tract of pigs.

Two experiments with growing pigs were conducted to determine the effects of dietary P and Ca level, phytase supplementation, and ileal pectin infusion on ileal and fecal P and Ca balance, chemical composition of fecal mixed bacterial mass (MBM), and bacterial metabolic activity. Pigs (initial BW = 30 kg) were fitted with simple T-cannulas at the distal ileum. They were fed a low-P corn-soybean meal control diet (3 g of P/kg) or the control diet supplemented with monocalcium phosphate (MCP; 7 g of P/kg; Exp. 1) or 1,000 FTU phytase/kg (Exp. 2). The daily infusion treatments consisted of 60 g of pectin dissolved in 1.8 L of demineralized water or 1.8 L of demineralized water as the control infusion, infused via the ileal cannula. In each experiment, 8 barrows were assigned to 4 dietary treatments according to a double, incomplete 4 x 2 Latin square. The dietary treatments in Exp. 1 were the control (Con-) diet with water infusion; the control (Con+) diet with pectin infusion; the MCP diet with water infusion; and the MCP diet with pectin infusion. In Exp. 2, the pigs received the same Con- and Con+ treatments as in Exp. 1 and, in addition, the phytase-supplemented diet in combination with water or pectin infusion. After a 15-d adaptation period, feces were collected for 5 d followed by ileal digesta collection for 24 h. In Exp. 1, supplemental MCP increased (P

Impact of exercise capacity on myocardial high-energy phosphate metabolism.

31-Phosphorous magnetic resonance spectroscopy (31P MRS) is a unique tool to investigate IN VIVO high-energy phosphates (HEP) in the human heart. We hypothesized that physical capacity may be associated with myocardial HEP status. Healthy, male volunteers (n = 105, mean age 51 +/- 7 years) underwent bicycle ergometry with a stepwise increasing workload to determine maximal working capacity (MWC). Heart rate (HR) and blood pressure (BP) were measured continuously during exercise and 4 minutes of recovery. Further 31-Phosphorous 2-dimensional chemical shift imaging (31P 2D CSI) MRS was performed to assess myocardial HEP metabolism by determining phosphocreatinine to beta-ATP ratios (PCr/b-ATP) using a 1.5 tesla scanner. Volunteers with MWC > 230 Watt had significantly higher PCr/b-ATP ratios than those with MWC < 200 Watt (1.93 +/- 0.36 vs. 1.59 +/- 0.35; p < 0.001). Additionally, those with a recovery systolic (S)BP < 195 mmHg had significantly higher ratios than those with a recovery SBP > 195 mmHg (1.74 +/- 0.3 vs. 1.51 +/- 0.2; p < 0.05). We observed a linear correlation between the PCr/b-ATP ratio and MWC (r = 0.411; p < 0.001) and recovery SBP (r = - 0.290; p < 0.01). After statistical correction for age, these correlations remained significant. In this study, we observed a correlation of parameters of physical fitness determined by bicycle exercise testing and cardiac PCr/b-ATP ratios.

Evaluation of transient apical ballooning with cardiac magnetic resonance imaging and 31-phosphorous magnetic resonance spectroscopy.

Apical ballooning is an increasingly reported transient cardiomyopathy with yet unknown origin. In this study 2 cases of apical ballooning are described in whom we used a combined approach of cardiac magnetic resonance imaging (CMR) and 31-Phosphorous magnetic resonance spectroscopy (31P MRS). Electrocardiogram showed ST abnormalities and cardiac serum markers were mildly elevated, but CAG demonstrated smooth coronary arteries. Cine-CMR revealed severe apical akinesia and significantly decreased ejection fraction. Furthermore we detected reduced myocardial phosphocreatine to beta-ATP (PCr/b-ATP) ratios during the first week of acute disease. After 1 week we observed an improvement of PCr/b-ATP ratios by 68% and 34%, which was associated with an increase in left ventricular function. Our data suggest that 31P MRS might be a valuable tool in the evaluation of apical ballooning, but larger cohorts are needed to improve the understanding of metabolic changes during transient apical ballooning.

Effects of physically effective fiber on digestive processes and milk fat content in early lactating dairy cows fed total mixed rations.

Data from recent research studies were analyzed quantitatively, and the random effect of experiment was assessed to define the physiological responses of dairy cows in early lactation to intake of physically effective neutral detergent fiber (peNDF). All studies were conducted with lactating Holstein cows (84.8 +/- 3.54 days in milk) in Latin square designs, and feeds were offered ad libitum as total mixed rations (TMR). The peNDF was estimated by 2 measurement techniques, the NDF content of TMR multiplied by amount of dry matter (DM) retained on a 1.18-mm screen (peNDF(> 1.18)) and NDF content of TMR multiplied by the proportion of DM retained by 19- and 8-mm Penn State Particle Separator screens (peNDF(> 8)). Other factors, including concentrations of NDF, forage NDF, non-fiber carbohydrates, the amount of digestible organic matter of forages (FDOM), and the intake of ruminally degradable starch (RDSI) from grain in the diet were also investigated. The studied animal response variables included feed intake, ruminal fermentation, chewing activity, fiber digestibility, and milk production and composition. The ruminal pH (day mean) in this study ranged from 5.30 to 6.59. Using peNDF(> 1.18) approach, the requirements for physically effective fiber in high-yielding dairy cows fed TMR in an ad libitum intake were estimated to be about 19% of ration DM or 4.1 kg/d or 0.6 kg/100 kg of body weight to maintain a ruminal pH of about 6.0. When peNDF was measured as peNDF(> 8), ruminal pH responded in a quadratic fashion but the confidence of estimation was lower (R(2) = 0.27) compared with the peNDF(> 1.18) approach (R(2) = 0.67). Results of these data analyses showed that peNDF(> 1.18) provided a satisfactory estimation of the mean ruminal pH (R(2) = 0.67) and NDF digestibility (R(2) = 0.56). Furthermore, peNDF(> 1.18) was poorly, although positively, correlated to daily chewing (R(2) = 0.17), and rumination (R(2) = 0.24) activity. On the other hand, results from these analyses showed that milk parameters are less sensitive to the effects of dietary peNDF than other variables, such as ruminal pH, chewing activity, and fiber digestibility. Dietary FDOM correlated positively (moderately) to ruminal pH (R(2) = 0.24), daily chewing (R(2) = 0.23), and rumination (R(2) = 0.29) activity, whereas the daily RDSI from grain correlated negatively to ruminal pH (R(2) = 0.55) and positively to total volatile fatty acids (R(2) = 0.27). Inclusion of FDOM and RDSI from grain along with peNDF(> 1.18) in the models that predict rumen pH further improved the accuracy of prediction. This approach appeared to further complement the concept of peNDF that does not account for differences in ruminal fermentability of feeds.

Mediastinoscopic ultrasonography (MUS).

Correct pre-therapeutic T4 staging is mandatory for neo-adjuvant studies and for the decision on surgical therapy of high-risk patients. T4-staging of centrally located lung-cancer by means of non-invasive imaging techniques is either of low accuracy (CT and NMR) or important regions are not accessible due to air interference with the tracheo-bronchial tree (trans-esophageal-endosonography, TEE). We here describe for the first time the new technique of mediastinoscopic ultrasonography (MUS). A fingertip ultrasound probe is introduced through the video-mediastinoscope. The probe lies in front of the tracheo-bronchial tree and in direct contact with the vena cava and pulmonary artery. This position allows examining those regions that are not accessible with TEE. In a pilot study with 12 patients, visualization of central vessels and their relation to the tumor was excellent and without artifacts. In 3 patients, MUS did not confirm the T4 stage predicted by CT Scan. Those three patients underwent successful pneumonectomy (R0-resection) while the other nine patients received induction treatment. MUS is a promising addition to CT scanning, NMR, and transesophageal ultrasound in staging of centrally located tumors.

[Persistent left superior vena cava with right-left shunt into the left atrium]. / Persistierende linke Vena cava superior mit Rechts-Links-Shunt zum linken Atrium.

UNLABELLED: Persistent left superior vena cava with right-left shunt into the left atrium. HISTORY AND CLINICAL FINDINGS: A 72-year-old patient was admitted to the hospital following bleeding into the basal ganglia secondary to a hypertensive crisis. INVESTIGATIONS: The patient was found to suffer from marked hypoxaemia (pO2 49 mmHg) and erythrocytosis (Hb 18,5 g/dl). Subsequent investigations raised suspicion of a right-left shunt. This was verified by a contrast echocardiogram which was performed transthoracically by injection of echo-contrast material from the left. To improve imaging of the shunt a transoesophageal contrast-echocardiogram was carried out. This showed that the persistent left superior vena cava did not, as previously expected, lead directly into the left atrium, but had a connection to the left superior pulmonary vein. This anatomical variant, which so far to our knowledge has not been reported in the literature, could be confirmed by spiral computed tomography. Apart from an atrial septal aneurysm no other cardiac anomaly could be identified. TREATMENT AND COURSE: Ligation of the left superior vena cava could have been a therapeutic option, but the patient declined operative intervention. CONCLUSION: In cases of profound hypoxemia and erythrocytosis the differential diagnosis must include a persistent left superior vena cava with anomalous connection to the left atrium. Trans-thoracic and transoesophageal contrast-echocardiography is a simple and reliable method to diagnose persistent left superior vena cava as well as concomitant cardiac anomalies.

Lack of a role for transforming growth factor-beta in cytotoxic T lymphocyte antigen-4-mediated inhibition of T cell activation.

Similarities in the phenotypes of mice deficient for cytotoxic T lymphocyte antigen-4 (CTLA-4) or transforming growth factor-beta1 (TGF-beta1) and other observations have led to speculation that CTLA-4 mediates its inhibitory effect on T cell activation via costimulation of TGF-beta production. Here, we examine the role of TGF-beta in CTLA-4-mediated inhibition of T cell activation and of CTLA-4 in the regulation of TGF-beta production. Activation of AND TCR transgenic mouse T cells with costimulatory receptor-specific antigen presenting cells results in efficient costimulation of proliferation by CD28 ligation and inhibition by CTLA-4 ligation. Neutralizing antibody to TGF-beta does not reverse CTLA-4-mediated inhibition. Also, CTLA-4 ligation equally inhibits proliferation of wild-type, TGF-beta1(-/-), and Smad3(-/-) T cells. Further, CTLA-4 engagement does not result in the increased production of either latent or active TGF-beta by CD4(+) T cells. These results indicate that CTLA-4 ligation does not regulate TGF-beta production and that CTLA-4-mediated inhibition can occur independently of TGF-beta. Collectively, these data demonstrate that CTLA-4 and TGF-beta represent distinct mechanisms for regulation of T cell responses.

Mouse model of myocardial remodelling after ischemia: role of intercellular adhesion molecule-1.

OBJECTIVE: We studied the effects of temporary myocardial ischemia and reperfusion on myocyte injury and ventricular remodelling in wildtype and intercellular adhesion molecule-1- (ICAM-1) deficient mice. METHODS: ICAM-1-/- and ICAM-1+/+ mice were subjected to 30 min of myocardial ischemia and subsequent reperfusion for 2 h, 1 week and 3 weeks, respectively. The evaluation of tissue damage and scar size was performed with histological sections stained with hematoxilin and eosin. Serum levels of troponin T, creatine kinase and lactate dehydrogenase isoenzyme 1 were evaluated as an index of cardiac cellular damage. Immunohistological analysis was employed to determine cell compositions in ischemic regions. RESULTS: After myocardial ischemia (30 min) and 2 h reperfusion, elevation in serum troponin T, creatine kinase and lactate dehydrogenase isoenzyme 1 were found in both groups, but significantly reduced in ICAM-1-/- mice compared with wildtype mice (P<0.05). Absence of a functional ICAM-1 gene in ICAM-1-/- mice resulted in a marked reduction of ischemia-reperfusion injury at the early stage. The damage score and size of the infarct area were lower in ICAM-1 -/- mice by 30 min of ischemia and 2 h of reperfusion (1.4+/-0.54 vs. 2.4+/-0.47, P<0.05). The percentage of MAC-1-positive cells in the ischemic region and the border zone was also significantly diminished in groups of ICAM-1-/- mice. Surprisingly, the scar size in ventricles in animals 1 or 3 weeks after ischemia was similar between ICAM-1-/- and ICAM-1+/+ mice, although the number of infiltrated MAC-1 positive cells in the scar in wildtype mice was higher. CONCLUSION: Our results demonstrate that the absence of ICAM-1 expression results in less myocardial damage induced by ischemia-reperfusion at the early stage, but does not influence the size of myocardial infarction and scar formation.

Rapid development of vein graft atheroma in ApoE-deficient mice.

Several animal models manifesting lesions resembling neointimal hyperplasia of human vein grafts have been developed, but no spontaneous atheromatous lesions in their vein grafts have been observed. We developed and here characterize a new animal model of vein graft atheroma, a maturated atherosclerotic plaque, in apoE-deficient mice. The lesion displayed classical complex morphological features and heterogeneous cellular compositions and consisted of a fibrous cap, infiltrated mononuclear cells, foam cells, cholesterol crystal structure, necrotic core with calcification, and neovasculature. Cell component analysis revealed smooth muscle cells (SMCs) localized in the cap region, macrophages which made up a large portion of the lesions, and CD4+ T cells scattered under the cap. Importantly, apoptotic/necrotic cells determined by TUNEL assay in vein grafts into apoE-/- mice were significantly higher than wild-type mice, although a similar number of proliferating cell nuclear antigen-positive cells in both types of lesions was found. Interestingly, vascular SMCs cultivated from aortas of apoE-deficient mice showed a high rate of spontaneous apoptosis/necrosis and a higher rate of cell death stimulated by a nitric oxide donor, sodium nitroprusside, H(2)O(2), and oxidized low density lipoprotein (LDL), although no difference in proliferation of both SMCs incubated with platelet-derived growth factor, angiotensin II, LDL, and oxidized LDL was seen. Thus, the pathogenic mechanisms of vein graft atheroma involve increased intimal cell death initiated by biomechanical stress and amplified by hypercholesterolemia, which leads to continuous recruitment of blood mononuclear cells to constitute atheromatous lesions. This mouse model resembling human vein graft disease has many advantages over other animal models.

Increased expression and activation of stress-activated protein kinases/c-Jun NH(2)-terminal protein kinases in atherosclerotic lesions coincide with p53.

Hyperlipidemia alters gene expression of arterial endothelial and smooth muscle cells (SMCs) and induces atherosclerotic lesions, in which cell proliferation and apoptosis co-exist. The signal transduction pathways that mediate these responses in the vessel wall in vivo have yet to be identified. Stress-activated protein kinases (SAPKs) or c-Jun NH(2)-terminal protein kinases (JNKs) are thought to be crucial in transmitting transmembrane signals required for cell differentiation and apoptosis in vitro. In the present study, we investigated the localization and activity of SAPK/JNK in atherosclerotic lesions of cholesterol-fed rabbits. Immunofluorescence analysis revealed abundant and heterogeneous distribution of pan-SAPK/JNK and phosphorylated SAPK/JNK, which were mainly localized in cell nuclei of the lesional cap and basal regions. Double staining of the lesions demonstrated that a portion of alpha-actin(+) SMCs and RAM11(+) macrophages contained abundant phosphorylated SAPK/JNK proteins. SAPK/JNK protein levels in protein extracts from atherosclerotic lesions were two- to threefold higher than the vessels of chow-fed rabbits. SAPK/JNK activities were elevated three- to fivefold higher than the normal vessels. Interestingly, increased SAPK/JNK in lesions was co-localized or coincided with high levels of transcription factor p53 as identified by double labeling and immunoprecipitation. Abundant pro-apoptotic protein BAX and BCL-X(S) were also observed. Furthermore, low-density lipoprotein (LDL) and oxidized LDL stimulated SAPK/JNK activation in cultured SMCs in a time- and dose-dependent manner. LDL also induced SAPK/JNK activation in vascular SMCs derived from LDL-receptor-deficient Watanabe rabbits, indicating a LDL-receptor-independent process. Thus, SAPK/JNK persistently hyperexpressed and activated in lesions may play a key role in mediating cell differentiation and apoptosis during the development of atherosclerosis via activation of transcription factor p53.

Kinetics of peptide uptake and tissue distribution following a single intranasal dose of peptide.

We have previously used intranasal (i.n.) peptide application to induce mucosal tolerance in experimental autoimmune encephalomyelitis (EAE). This strategy, however, appeared to give rise to similar phenomena of tolerance observed as a result of systemic administration of soluble antigenic peptide. We were interested, therefore, in the uptake and tissue distribution of peptide following i.n. treatment. In the H-2u mouse model of EAE, the highly tolerogenic peptide analogue Ac1-9[4Y] of myelin basic protein (MBP) displays high affinity binding to Au MHC class II. For the purpose of the present study this peptide was synthesised to contain a tritiated acetyl group and a protocol was developed to recover radioactivity in solubilised tissues taken at various times after [3H]Ac1-9[4Y] i.n.. Radiolabel loads of the lung and gastro-intestinal tract were initially high but declined rapidly. Radiolabel uptake by blood and lymphoid tissues followed similar kinetics with peak levels around 2.5-4 hours after i.n. administration. Concentrations were high in the draining cervical lymph nodes (CLN) but also reached significant levels in the spleen and 'nondraining' inguinal lymph nodes. The presence of intact antigenic peptide was demonstrated in spleens and CLN from Ac1-9[4Y] i.n. treated mice. Cell suspensions prepared from these tissues at selected time points after peptide i.n. were able to stimulate peptide-specific T cell lines up to at least one day after peptide i.n., suggesting long lasting formation of stable Au-Ac1-9[4Y] complexes in vivo.

Hyperexpression and activation of extracellular signal-regulated kinases (ERK1/2) in atherosclerotic lesions of cholesterol-fed rabbits.

A hallmark of hyperlipidemia-induced atherosclerosis is altered gene expression that initiates cell proliferation and (de)differentiation in the intima of the arterial wall. The molecular signaling that mediates this process in vivo has yet to be identified. Extracellular signal-regulated kinases (ERKs) are thought to play a pivotal role in transmitting transmembrane signals required for cell proliferation in vitro. The present studies were designed to investigate the activity, abundance, and localization of ERK1/2 in atherosclerotic lesions of cholesterol-fed rabbits. Immunofluorescence analysis revealed abundant and heterogeneous distribution of ERK1/2, mainly localized in the cap and basal regions of atheromas. A population of ERK-enriched cells was identified as alpha-actin-positive smooth muscle cells (SMCs). ERK1 and 2 were heavily phosphorylated on tyrosyl residues and coexpressed with proliferating cell nuclear antigen in atherosclerotic lesions. ERK1/2 protein levels in protein extracts from atherosclerotic lesions were 2- to 3-fold higher than the vessels of chow-fed rabbits, and their activities were elevated 3- to 5-fold over those of the normal vessel. SMCs derived from atherosclerotic lesions had increased migratory/proliferative ability and higher ERK activity in response to LDL stimulation compared with cells from the normal vessel. Inhibition of ERK activation by PD98059, a specific inhibitor of mitogen-activated protein kinase kinases (MEK1/2), abrogated LDL-induced SMC proliferation in vitro. Taken together, our findings support the proposition that persistent activation and hyperexpression of ERK1/2 may be a critical element to initiate and perpetuate cell proliferation during the development of atherosclerosis.

Peptide-induced T cell regulation of experimental autoimmune encephalomyelitis: a role for IL-10.

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-mediated, inflammatory disease with similarities to multiple sclerosis in humans. Intranasal (i.n.) administration of a myelin basic protein (MBP)-derived peptide can protect susceptible mice from EAE. The mechanisms underlying this phenomenon, however, remain unclear. To analyze the phenotypic and functional changes taking place during the induction of tolerance by peptide inhalation, we have studied the fate of CD4(+) T cells after i.n. peptide application using transgenic mice expressing a TCR specific for the N-terminal peptide (Ac1-9) of MBP. Peripheral T cell death was variably observed in TCR transgenic mice after a single i.n. administration of antigenic peptide but was transient and incomplete. Transgenic spleen cells and cervical lymph node cells responded with a cytokine burst to peptide inhalation and hyperproliferation when re-stimulated in vitro. Transfer experiments demonstrated that the duration of peptide administration required to induce tolerance depended on the precursor frequency of T cells in recipient animals. The stringency of i.n. peptide treatment was increased so as to test the efficacy of tolerance induction both in vitro and in vivo in the presence of high precursor frequencies of antigen-specific T cells. Multiple i.n. doses of peptide completely protected TCR transgenic mice from EAE induced with myelin. Such repeated peptide administration resulted in down-regulation of the capacity of antigen-specific CD4(+) T cells to proliferate or to produce IL-2, IFN-gamma and IL-4 but increased the production of IL-10. The role of IL-10 in suppression of EAE in vivo was demonstrated by neutralization of IL-10. This completely restored susceptibility to EAE in mice previously protected by i.n. peptide. Considering the immunosuppressive properties of IL-10, T cells which are resistant to apoptosis might act as regulatory cells and mediate bystander suppression.