Exploitation of Langerhans cells for in vivo DNA vaccine delivery into the lymph nodes.

There is no clinically available cancer immunotherapy that exploits Langerhans cells (LCs), the epidermal precursors of dendritic cells (DCs) that are the natural agent of antigen delivery. We developed a DNA formulation with a polymer and obtained synthetic 'pathogen-like' nanoparticles that preferentially targeted LCs in epidermal cultures. These nanoparticles applied topically under a patch-elicited robust immune responses in human subjects. To demonstrate the mechanism of action of this novel vaccination strategy in live animals, we assembled a high-resolution two-photon laser scanning-microscope. Nanoparticles applied on the native skin poorly penetrated and poorly induced LC motility. The combination of nanoparticle administration and skin treatment was essential both for efficient loading the vaccine into the epidermis and for potent activation of the LCs to migrate into the lymph nodes. LCs in the epidermis picked up nanoparticles and accumulated them in the nuclear region demonstrating an effective nuclear DNA delivery in vivo. Tissue distribution studies revealed that the majority of the DNA was targeted to the lymph nodes. Preclinical toxicity of the LC-targeting DNA vaccine was limited to mild and transient local erythema caused by the skin treatment. This novel, clinically proven LC-targeting DNA vaccine platform technology broadens the options on DC-targeting vaccines to generate therapeutic immunity against cancer.

Mapping average axon diameters in porcine spinal cord white matter and rat corpus callosum using d-PFG MRI.

Knowledge of microstructural features of nerve fascicles, such as their axon diameter, is crucial for understanding normal function in the central and peripheral nervous systems as well as assessing changes due to pathologies. In this study double-pulsed field gradient (d-PFG) filtered MRI was used to map the average axon diameter (AAD) in porcine spinal cord, which was then compared to AADs measured with optical microscopy of the same specimen, as a way to further validate this new MRI method. A novel 3D d-PFG acquisition scheme was used to obtain AADs in each voxel of a coronal slice of rat brain corpus callosum. AAD measurements were also acquired using optical microscopy performed on histological sections and validated using a glass capillary array phantom.

Thermodynamic Analysis of the Selectivity Enhancement Obtained by Using Smart Hydrogels That Are Zwitterionic When Detecting Glucose With Boronic Acid Moieties.

Because the boronic acid moiety reversibly binds to sugar molecules and has low cytotoxicity, boronic acid-containing hydrogels are being used in a variety of implantable glucose sensors under development, including sensors based on optical, fluorescence, and swelling pressure measurements. However, some method of glucose selectivity enhancement is often necessary, because isolated boronic acid molecules have a binding constant with glucose that is some forty times smaller than their binding constant with fructose, the second most abundant sugar in the human body. In many cases, glucose selectivity enhancement is obtained by incorporating pendant tertiary amines into the hydrogel network, thereby giving rise to a hydrogel that is zwitterionic at physiological pH. However, the mechanism by which incorporation of tertiary amines confers selectivity enhancement is poorly understood. In order to clarify this mechanism, we use the osmotic deswelling technique to compare the thermodynamic interactions of glucose and fructose with a zwitterionic smart hydrogel containing boronic acid moieties. We also investigate the change in the structure of the hydrogel that occurs when it binds to glucose or to fructose using the technique of small angle neutron scattering.

Double pulsed field gradient (double-PFG) MR imaging (MRI) as a means to measure the size of plant cells.

Measurement of diffusion in porous materials and biological tissues with the pulsed field gradient (PFG) MR techniques has proven useful in characterizing the microstructure of such specimens noninvasively. A natural extension of the traditional PFG technique comprises multiple pairs of diffusion gradients. This approach has been shown to provide the ability to characterize anisotropy at different length scales without the need to employ very strong gradients. In this work, the double-PFG imaging technique was used on a specimen involving a series of glass capillary arrays with different diameters. The experiments on the phantom demonstrated the ability to create a quantitative and accurate map of pore sizes. The same technique was subsequently employed to image a celery stalk. A diffusion tensor image (DTI) of the same specimen was instrumental in accurately delineating the regions of vascular tissue and determining the local orientation of cells. This orientation information was incorporated into a theoretical double-PFG framework and the technique was employed to estimate the cell size in the vascular bundles of the celery stalk. The findings suggest that the double-PFG MRI framework could provide important new information regarding the microstructure of many plants and other food products.

Strong relationship between NT-proXNP levels and cardiac output following cardiac surgery in neonates and infants.

BACKGROUND: NT-proXNP, a new natriuretic peptide analyte, incorporates information about the concentrations of both N-terminal pro-atrial and pro-brain natriuretic peptides (NT-proANP, NT-proBNP). We aimed to investigate whether NT-proXNP is a reliable indicator of the cardiac index (CI) and the hemodynamic state in neonates and infants undergoing an open heart surgery. METHODS: We enrolled 26 children under the age of 1 year into this prospective study. All patients underwent an elective cardiac operation with cardiopulmonary bypass (CPB) to achieve complete biventricular repair. Peri-operative hemodynamic parameters were assessed by transpulmonary thermodilution and natriuretic peptide levels were recorded. RESULTS: The NT-proXNP level correlated significantly with the simultaneously measured NT-proANP level (r=0.60, P<0.001), but more strongly with the NT-proBNP level (r=0.89, P<0.001) and the arithmetic sum of both (r=0.88, P<0.001). NT-proXNP had a strong correlation with CI (r=-0.85, P<0.001), the stroke volume index (r=-0.80, P<0.001) and the global ejection fraction (r=-0.67, P<0.009) throughout the post-operative period. Conventionally measured parameters such as heart rate, mean arterial pressure and pulse-pressure product exhibited weaker correlations with CI than NT-proXNP. Among laboratory values, creatinine levels correlated significantly with CI (r=-0.77, P<0.001) and NT-proXNP (r=0.76, P<0.001) during the post-operative period. A post-operative NT-proXNP level of 3079 pmol/l was diagnostic for CI <3 l/min/m(2) with 89% sensitivity and 90% specificity (area under the curve: 0.91 +/- 0.05). CONCLUSION: NT-proXNP is a good marker of cardiac output following pediatric cardiac surgery and might be a useful tool in the recognition of a low output state.

A novel MRI phantom to study interstitial fluid transport in the glymphatic system.

The glymphatic system is a recently discovered transport system, mediated by cerebral spinal fluid (CSF), that clears metabolic and cellular waste products in the brain. This system's function in the brain is analogous to that of the lymphatic system in the rest of the mammalian body. It is hypothesized that CSF clears harmful chemicals from the brain by flowing through interstitial spaces in the brain during sleep. While there is growing recognition of the critical role the glymphatic system plays in maintaining normal brain health and in explaining pathology, there are few noninvasive imaging methods that measure and characterize the efficacy of glymphatic transport in vivo. In this study we designed, constructed, and tested a glymphatic transport magnetic resonance imaging (MRI) flow phantom, which combines regions that mimic CSF-filled ventricles and brain interstitial space. We tested high- and low-q space diffusion MRI and diffusion tensor imaging (DTI) acquisitions to determine if they could detect, measure, and map interstitial glymphatic flows. The results suggest that, under certain flow conditions, diffusion-weighted MRI can detect the enhanced mixing that occurs during glymphatic clearance.

Detection of microscopic anisotropy in gray matter and in a novel tissue phantom using double Pulsed Gradient Spin Echo MR.

A double Pulsed Gradient Spin Echo (d-PGSE) MR experiment was used to measure and assess the degree of local diffusion anisotropy in brain gray matter, and in a novel "gray matter" phantom that consists of randomly oriented tubes filled with water. In both samples, isotropic diffusion was observed at a macroscopic scale while anisotropic diffusion was observed at a microscopic scale, however, the nature of the resulting echo attenuation profiles were qualitatively different. Gray matter, which contains multiple cell types and fibers, exhibits a more complicated echo attenuation profile than the phantom. Since microscopic anisotropy was observed in both samples in the low q regime comparable to that achievable in clinical scanner, it may offer a new potential contrast mechanism for characterizing gray matter microstructure in medical and biological applications.

Influence of free chains on the swelling pressure of PEG-HEMA and dex-HEMA hydrogels.

Insight in the osmotic behavior of degrading hydrogels is of great importance in the design of biodegradable hydrogels for biomedical applications. This study compares the degradation behavior of PEG-HEMA (hydroxyethylmethacrylated polyethylene glycol) and dex-HEMA (hydroxyethylmethacrylated dextran) hydrogels. The degradation of PEG-HEMA gels takes several months to over a year, while that of dex-HEMA gels takes only days or weeks. The faster degradation kinetics of dex-HEMA networks can be attributed to stabilization of the keto-enol form by hydroxyl groups. Upon degradation of PEG-HEMA and dex-HEMA hydrogels, respectively, free PEG and free dextran chains are produced. We investigated the effect of unattached PEG and dextran chains on the swelling pressure of the degrading gels. It is found that low molecular weight free chains significantly increase the swelling pressure. However, the contribution of higher molecular weight free chains (M(w)>10 kDa) is similar to that of the network chains.

Anisotropic phantom to calibrate high-q diffusion MRI methods.

A silicon oil-filled glass capillary array is proposed as an anisotropic diffusion MRI phantom. Together with a computational/theoretical pipeline these provide a gold standard for calibrating and validating high-q diffusion MRI experiments. The phantom was used to test high angular resolution diffusion imaging (HARDI) and double pulsed-field gradient (d-PFG) MRI acquisition schemes. MRI-based predictions of microcapillary diameter using both acquisition schemes were compared with results from optical microscopy. This phantom design can be used for quality control and quality assurance purposes and for testing and validating proposed microstructure imaging experiments and the processing pipelines used to analyze them.

Intrapericardial infusion of endothelin-1 induces ventricular arrhythmias in dogs.

OBJECTIVES: Recently, extremely high levels of endothelin-1 (ET-1) were detected in the pericardial fluid of patients with heart disease; however, the pathophysiological importance of this finding is not known. The present study was designed to characterize ET-1 levels in canine pericardial fluid and to investigate the effects of local high concentrations of exogenous ET-1 in vivo. METHODS: In anesthetized, open-chest dogs ET-1 (Groups 1 and 2: 11 and 33 pmol.kg-1.min-1; n = 6 and 6, respectively) or physiological saline (Group 3, n = 5) were infused into the closed pericardial sac for 40 min. In serial pericardial fluid and aortic blood plasma samples, ET-1 levels were measured by radioimmunoassay, and analysed by high-performance liquid chromatography (HPLC). Systemic arterial blood pressure, heart rate, cardiac output (CO), standard ECG and right ventricular endocardial monophasic action potentials (MAPs) were recorded. RESULTS: Basal pericardial fluid ET-1 levels were significantly higher than respective plasma levels (342 +/- 210 vs. 8.0 +/- 5.2 pmol.l-1, n = 14, P < 0.001. In HPLC analysis pericardial fluid ET-1 was indistinguishable from ET-1(1-21). Infusion of exogenous ET-1 into the pericardial space induced ventricular arrhythmias in all instances, which were associated with 9.7-fold increase in pericardial fluid ET-1 levels. Ventricular tachycardias developed in 9 of 12 animals. The arrhythmogenic effect of ET-1 was more apparent in dogs with the larger dose. Before the onset of arrhythmias, intrapericardial infusion of ET-1 increased QT time (Group 1: 207 +/- 18 to 230 +/- 23 ms, P < 0.01; Group 2: 220 +/- 12 to 277 +/- 17 ms, P < 0.01) and MAP duration at 90% repolarization (at 300 ms cycle length) (Group 1: 192 +/- 9 to 216 +/- 9 ms, P < 0.01; Group 2: 205 +/- 9 to 255 +/- 9 ms, P < 0.001). Hemodynamic variables did not change significantly prior to the onset of ventricular tachyarrhythmias. In Group 3, arrhythmias were not observed and all electrophysiological and hemodynamic parameters remained unchanged. CONCLUSIONS: Administration of exogenous ET-1 into the pericardial space induces ventricular arrhythmias associated with prolongation of QT time and MAP duration. Whether pericardial fluid ET-1 under pathophysiological conditions can ever reach sufficiently high levels to induce ventricular arrhythmias remains to be elucidated.

Response to heparinization in adults and children undergoing cardiac operations.

The activated clotting time is an unreliable index of anticoagulation status during cardiopulmonary bypass procedures. However, modern instrumentation (Hemotec Hepcon HMS) now allows the monitoring of free heparin levels via automated protamine titration. In the present study, the standard procedure of anticoagulation at Killingbeck Hospital, Leeds, was investigated. Twenty-two pediatric patients and 20 adult patients undergoing open heart procedures involving cardiopulmonary bypass were given empirical doses of heparin (3 mg/kg body weight bolus), and activated clotting time was maintained at a level greater than 450 seconds using the Hemochron Timer. Heparin neutralization was performed at the termination of the bypass period using an empirical equivalent (3 mg/kg) of protamine sulfate. Mean free heparin concentration (+/- standard deviation) fell from 2.26 (+/- 0.45) mg/kg to 1.39 (+/- 0.34) mg/kg over the period 10 to 40 minutes on bypass in children. In adults, free heparin level declined from 2.56 (+/- 0.58) mg/kg to 1.81 (+/- 0.58) mg/kg over the same period. The biological half-life for heparin was 60 minutes in adults and 35 minutes in pediatric patients. Empirical protamine dosing resulted in excess protamine administration when compared with Hepcon titrated dose requirements: for children: median (range), 80 (12 to 350) versus 33 (12 to 97) mg, p less than 0.001; and for adults: 350 (200 to 500) versus 130 (61 to 237) mg, p less than 0.001. In conclusion, empirical heparin administration (3 mg/kg) does not result in "steady-state" anticoagulation during cardiopulmonary bypass, and empirical administration of protamine takes no account of interindividual differences in heparin sensitivity and biological half-life, which may be assessed using the Hepcon HMS.

Characterization and stimuli for production of pericardial fluid atrial natriuretic peptide in dogs.

Recently high immunoreactive atrial natriuretic peptide (ir-ANP) levels have been found in the pericardial fluid of patients undergoing cardiac surgery. The present study was designed to characterize pericardial fluid ANP in anesthetized dogs. Pericardial fluid ir-ANP levels were 3.4-fold higher than plasma levels and the molecular form, revealed by high performance liquid chromatography, was indistinguishable from ANP[99-126]. Elimination of [125I]ANP was 5-fold slower in the pericardial space than in plasma. Activity of the major ANP degrading enzyme, neutral endopeptidase (NEP, EC 3.4.24.11), was 15-times higher in the pericardial fluid than in plasma. Right atrial balloon distension and rapid right ventricular pacing induced maximally 2.3-fold and 1.5-fold increases of pericardial fluid ir-ANP, respectively. Pericardial fluid ir-ANP concentrations and right atrial pressure values showed significant correlation during the stimuli. Our present results show that high concentrations of ir-ANP can be found in the dog pericardial fluid even under unstimulated conditions. Slow elimination of ANP from the pericardial fluid compartment may contribute to the high peptide levels. However this slow elimination cannot be attributed to a lower NEP activity. High basal levels of ANP in the pericardial fluid could be further increased by atrial balloon stretch and rapid ventricular pacing. The increase of pericardial fluid ir-ANP appeared to be a stretch-dependent response. ANP released into the pericardial fluid may be involved in the regulation of cardiac function and coronary vascular tone.

Presence of immunoreactive endothelin-1 and atrial natriuretic peptide in human pericardial fluid.

This study was undertaken to characterize endothelin-1 (ET-1) and atrial natriuretic peptide (ANP) concentrations in human pericardial fluid, blood plasma, right atrial appendage and papillary muscle by use of specific radioimmunoassays. In patients undergoing cardiac surgery (n=16) pericardial fluid mean immunoreactive (ir-) ET-1 and ir-ANP levels were 36-fold and 4-fold higher than corresponding plasma levels, respectively. In high performance liquid chromatography (HPLC) pericardial fluid ir-ET-1 was indistinguishable from human ET-1[1-21] and the majority of pericardial fluid ir-ANP coeluted with human ANP[99-126]. Atrial tissue ir-ET-1 and ir-ANP concentrations were 17-fold and 870-fold higher than in ventricular tissue. Our present study demonstrated for the first time the presence of ir-ET-1 in the pericardial fluid in humans. Human pericardial fluid contained far the highest concentrations of ET-1 among all biological fluids tested thus far. The functions of pericardial fluid ET-1 and ANP on cardiac performance and coronary vascular tone require further investigations.

Enhanced accumulation of pericardial fluid adenosine and inosine in patients with coronary artery disease.

Adenosine and inosine are believed to have cardioprotective effects. However, little is known about their possible role in the metabolic autoregulation of human coronaries and in pathologic conditions with supply/demand imbalance of the heart such as coronary artery disease. Since these low molecular weight nucleosides freely diffuse through the monolayer of the visceral pericardium, adenosine and inosine concentrations in pericardial fluid may well reflect the conditions in cardiac interstitium. The pericardial fluid and systemic venous blood adenosine and inosine concentrations were measured in 98 human subjects undergoing heart surgery for coronary artery disease or valvular heart disease. Adenosine and inosine concentrations were measured by HPLC with UV detection. In subjects with coronary artery disease pericardial fluid nucleoside concentrations were significantly higher than in patients with valvular heart disease (adenosine: 1545 (996-3146) nmol/L [median (25th-75th quartiles)] vs. 738 (390-2527) nmol/L, P<0.01; inosine: 658 (321-1331) nmol/L vs. 347 (159-1037) nmol/L, P<0.05), while in both patient groups pericardial fluid nucleoside concentrations were higher by an order of magnitude than in venous plasma. Our results show the enhanced release of adenosine and inosine by the ischemic myocardium as a marker of supply/demand imbalance and support the hypothesis that these cardiac nucleosides may have an important role in the adaptation of coronary blood flow in human coronary artery disease.

Enhanced accumulation of pericardial fluid ferritin in patients with coronary artery disease.

BACKGROUND: Ferritin is a storage protein for iron that can either represent a source of iron or perform a cytoprotective action as an iron sequestrant. OBJECTIVE: To compare the concentrations of ferritin in pericardial fluid of patients with valvular heart disease, serving as controls, and in patients with coronary artery disease. DESIGN: We studied a total of 59 consecutive male patients undergoing elective heart valve replacement (group 1: n = 22, mean +/- SD age 55 +/- 11 years) or elective coronary artery bypass grafting (group 2: n = 37, mean +/- SD age 59 +/- 9 years). METHODS: Iron status indicators, total protein and albumin concentrations, and lactate dehydrogenase activities were determined in pericardial fluid and serum samples obtained from patients during surgery. RESULTS: Pericardial fluid concentrations of ferritin in both patient populations were significantly (P < 0.001) greater than the concentrations in sera: group 1, 375 (107-2030) micrograms/l compared with 146.5 (21-407) micrograms/l; group 2, 1115 (226-2500) micrograms/l compared with 152.0 (16-398) micrograms/l (median (range)), respectively. Moreover, pericardial fluid ferritin concentration was significantly (P < 0.01) greater in patients undergoing coronary artery bypass grafting than in those undergoing heart valve replacement, whereas serum ferritin concentrations did not differ between the two patient populations. CONCLUSIONS: As pericardial fluid reflects the composition of the myocardial interstitium, we suggest that ferritin released can serve as a potential source of iron in the cardiac interstitium that may promote the generation of oxygen free radicals. Conversely, we presume that induction of ferritin synthesis, representing an important mechanism by which tissue adapts to hypoxic damage, can afford myocardial cytoprotection.

Heparin rebound phenomenon--much ado about nothing?

Significant postoperative bleeding following open-heart surgery is often ascribed to the so-called heparin 'rebound' phenomenon and as such is treated with additional empiric doses of protamine sulphate. However, inappropriate protamine administration has been reported to be associated with acute pulmonary hypertension. The efficacy of heparin reversal was investigated in 42 patients undergoing open-heart surgery. The standard heparin bolus of 3 mg/kg body weight (4.1 IU/ml blood) administered before cardiopulmonary bypass was countered at the end of bypass using an empirical equivalent (3 mg/kg) of protamine. This regimen resulted in complete heparin neutralization (measured by the Hepcon HMS [Hemotec Inc., Englewood, CO, USA]) 15 min after protamine administration in all 42 patients, but heparin levels (0.4 IU/ml) were transiently detectable (duration less than 1 h) in six (14%) of the 42 cases 2 h later. Twenty-four hour postoperative bleeding in these patients did not differ significantly from that seen in patients who did not exhibit heparin rebound. Similarly, the thrombelastographic profiles (at 15 min and 2 h post-operation) and coagulation screen (prothrombin time, activated partial thromboplastin time, activated clotting time and platelets) did not differ significantly from those of non-rebound patients. The significance, if any, of the phenomenon of heparin rebound following cardiac surgery remains to be elucidated, and, until such time, conservative administration of protamine in response to 'rebound' is recommended.

Structure of polymer solutions containing fumed silica.

Small angle neutron scattering is employed to estimate the interactions in complex multicomponent systems. The method is applied to describe a ternary system, poly(dimethyl siloxane) toluene with silica filler particles. Contrast variation by solvent deuteration is used to distinguish the three different partial structure factors of filled polymer samples, S(pp)(q), S(pf)(q), and S(ff)(q), where the subscripts p and f refer to polymer and filler. This procedure allows changes to be detected in the distribution of the polymer close to the solid surface. Independent dynamic light scattering measurements are used to validate the method. A comparison is made between the behavior of different filled poly(dimethyl siloxane) PDMS samples. It is found that the polymer distribution is perturbed in the vicinity of the solid surfaces. In the case of weak interactions, the solvent removes the polymer almost completely from the filler particles, while, when the interaction is strong, the surface remains covered.

[Experience with the surgical management of constrictive pericarditis]. / Tapasztalataink a constrictiv pericarditis sebészi kezelésében.

61 patients were operated on for constrictive pericarditis at the Cardiovascular Surgical Clinic of Semmelweis Medical University in the last 33 years. The average hospital mortality of the surgical pericardiectomy was 4.9%. The final conclusions of this retrospective and follow up study are as follows: pericardiectomy is the method of choice in the treatment of constrictive pericarditis, since it does not has any therapeutic alternative, its hospital mortality is low and it results excellent early and late postoperative functional effects.

[Development and results of valve replacement surgery at the Városmajor Clinic 1976-1990 (retorospective clinical analysis)]. / A szívbillentyü-sebészet fejlödése és eredményei a Városmajori Klinikán 1976-1990 (retrospektív klinikai analízis).

In Hungary valve replacement is still a major indication for heart surgery in adults. In the Cardiovascular Surgical Clinic of Semmelweis Medical University of Budapest from 1976 to 1990 2435 patients were operated for valve disease. Majority of the cases had single (aortic n = 856, mitral n = 912) or double (aortic + mitral n = 513) valve replacement. Over this 15 years period there have been many alterations in patients characteristics and surgical technique as well. In spite of the increasing mean age of patients the operative mortality has decreased (in the last 5 years period it was 2.7%, 5.5% and 7.9% in the three groups respectively). At the same time the number of patients requiring valve re-replacement or combined valve + coronary procedure has increased. The use of bioprosthetic valves has fallen below 10 percent from the 60--80 percent observed between 1976--1980. The analysis showes excellent surgical results in the field of valve replacement in Hungary.

[Coronary surgery at the Cardiovascular Surgical Clinic (1976-1990)]. / Coronariasebészet az Er- és Szívsebészeti Klinikán (1976-1990).

In the Cardiovascular Surgical Clinic of the Semmelweis Medical University of Budapest the first coronary artery bypass grafting (CABG) procedure was performed in 1975. Since that time coronary artery surgery has become a routine everyday practice representing more than half of the total workload of adult cardiac surgery. The analysis of 1347 operations performed between 1976-1990 on patients with coronary heart disease showed the followings: the first few years--so called learning curve of CABG operations is characterised by high mortality. With passing time the number of cases performed each year increased rapidly and the surgical technique has improved too. At the same time the operative mortality figures showed decreasing tendency--it was 2.1% for the last 609 cases. All observed parameters showed some progress: in 1990 the average number of grafts per patient was 3.09, internal mammary artery usage 15 percent and the mean aortic cross clamp time per anastomosis 24.5 minutes. Complete myocardial revascularisation is the key point of coronary artery surgery. In order to achieve this target in all operated cases further technical improvement is necessary.