Implementation of an epicardial implantable MEMS sensor for continuous and real-time postoperative assessment of left ventricular activity in adult minipigs over a short- and long-term period.

The sensing of left ventricular (LV) activity is fundamental in the diagnosis and monitoring of cardiovascular health in high-risk patients after cardiac surgery to achieve better short- and long-term outcome. Conventional approaches rely on noninvasive measurements even if, in the latest years, invasive microelectromechanical systems (MEMS) sensors have emerged as a valuable approach for precise and continuous monitoring of cardiac activity. The main challenges in designing cardiac MEMS sensors are represented by miniaturization, biocompatibility, and long-term stability. Here, we present a MEMS piezoresistive cardiac sensor capable of continuous monitoring of LV activity over time following epicardial implantation with a pericardial patch graft in adult minipigs. In acute and chronic scenarios, the sensor was able to compute heart rate with a root mean square error lower than 2 BPM. Early after up to 1 month of implantation, the device was able to record the heart activity during the most important phases of the cardiac cycle (systole and diastole peaks). The sensor signal waveform, in addition, closely reflected the typical waveforms of pressure signal obtained via intraventricular catheters, offering a safer alternative to heart catheterization. Furthermore, histological analysis of the LV implantation site following sensor retrieval revealed no evidence of myocardial fibrosis. Our results suggest that the epicardial LV implantation of an MEMS sensor is a suitable and reliable approach for direct continuous monitoring of cardiac activity. This work envisions the use of this sensor as a cardiac sensing device in closed-loop applications for patients undergoing heart surgery.

Cardiovascular response to closed-loop intraneural stimulation of the right vagus nerve: a proof-of-concept study.

Vagus nerve stimulation (VNS) is an FDA-approved technique for the neuromodulation of the autonomic nervous system. There are many therapeutic applications where VNS could be used as a therapy, such as cardiovascular diseases, epilepsy, depression, and inflammatory conditions. Cardiovascular applications are particularly relevant, since cardiovascular diseases are the top causes of death worldwide. VNS clinical trials have been performed in the last 15 years for the treatment of heart failure (HF), achieving controversial results. Typically VNS is applied with a cuff electrode placed around the nerve, in an open-loop or cardiac synchronized design. The effectiveness of this approach is hindered by the multifunctional nature of the VN, which is involved in a variety of homeostatic controls. When a high current is applied, adverse effects arise from the stimulation of undesired fibers. An alternative strategy is represented by intraneural stimulation, which can guarantee higher selectivity. Moreover, closed-loop modalities allow the delivery of electrical current inside the nerves only if needed, with a reduced risk of untargeted nerve activation and lower energy consumption. Here we propose a closed-loop intraneural stimulation of the right cervical VN in a clinically relevant animal model. The intraneural was designed according to the internal structure of the VN. A threshold-based closed-loop algorithm was developed using HR as a control variable to produce a chronotropic effect.Clinical Relevance-This work analyzes the closed-loop intraneural VNS for the treatment of cardiovascular disorders, and supports the possibility of developing fully implantable devices with a high degree of selectivity in stimulation and prolonged lifespan.

Neural Stimulation Hardware for the Selective Intrafascicular Modulation of the Vagus Nerve.

The neural stimulation of the vagus nerve is able to modulate various functions of the parasympathetic response in different organs. The stimulation of the vagus nerve is a promising approach to treating inflammatory diseases, obesity, diabetes, heart failure, and hypertension. The complexity of the vagus nerve requires highly selective stimulation, allowing the modulation of target-specific organs without side effects. Here, we address this issue by adapting a neural stimulator and developing an intraneural electrode for the particular modulation of the vagus nerve. The neurostimulator parameters such as amplitude, pulse width, and pulse shape were modulated. Single-, and multi-channel stimulation was performed at different amplitudes. For the first time, a polyimide thin-film neural electrode was designed for the specific stimulation of the vagus nerve. In vivo experiments were performed in the adult minipig to validate to elicit electrically evoked action potentials and to modulate physiological functions, validating the spatial selectivity of intraneural stimulation. Electrochemical tests of the electrode and the neurostimulator showed that the stimulation hardware was working correctly. Stimulating the porcine vagus nerve resulted in spatially selective modulation of the vagus nerve. ECAP belonging to alpha and beta fibers could be distinguished during single- and multi-channel stimulation. We have shown that the here presented system is able to activate the vagus nerve and can therefore modulate the heart rate, diastolic pressure, and systolic pressure. The here presented system may be used to restore the cardiac loop after denervation by implementing biomimetic stimulation patterns. Presented methods may be used to develop intraneural electrodes adapted for various applications.

Decoding bladder state from pudendal intraneural signals in pigs.

Neuroprosthetic devices used for the treatment of lower urinary tract dysfunction, such as incontinence or urinary retention, apply a pre-set continuous, open-loop stimulation paradigm, which can cause voiding dysfunctions due to neural adaptation. In the literature, conditional, closed-loop stimulation paradigms have been shown to increase bladder capacity and voiding efficacy compared to continuous stimulation. Current limitations to the implementation of the closed-loop stimulation paradigm include the lack of robust and real-time decoding strategies for the bladder fullness state. We recorded intraneural pudendal nerve signals in five anesthetized pigs. Three bladder-filling states, corresponding to empty, full, and micturition, were decoded using the Random Forest classifier. The decoding algorithm showed a mean balanced accuracy above 86.67% among the three classes for all five animals. Our approach could represent an important step toward the implementation of an adaptive real-time closed-loop stimulation protocol for pudendal nerve modulation, paving the way for the design of an assisted-as-needed neuroprosthesis.

Vitamin D replacement ameliorates serum lipoprotein functions, adipokine profile and subclinical atherosclerosis in pre-menopausal women.

BACKGROUND AND AIMS: Low vitamin D (vitD) has been linked to increased cardiovascular (CV) risk, but the effects of vitD supplementation are not clarified. We evaluated the impact of vitD normalization on HDL cholesterol efflux capacity (CEC), which inversely correlates with CV risk, the proatherogenic serum cholesterol loading capacity (CLC), adipokine profile and subclinical atherosclerosis. METHODS AND RESULTS: Healthy premenopausal women with vitD deficiency (n = 31) underwent supplementation. Subclinical atherosclerosis was evaluated by flow-mediated dilation (FMD), pulse wave velocity (PWV) and augmentation index (AIx), measured with standard techniques. HDL CEC and serum CLC were measured by a radioisotopic and fluorimetric assay, respectively. Malondialdehyde (MDA) in HDL was quantified by the TBARS assay. Pre-ß HDL was assessed by 2D-electrophoresis. Serum adipokines were measured by ELISA. VitD replacement restored normal levels of serum 25-hydroxyvitamin D (25OHD) and significantly improved FMD (+4%; p < 0.001), PWV (-4.1%: p < 0.001) and AIx (-16.1%; p < 0.001). Total CEC was significantly improved (+19.5%; p = 0.003), with a specific increase in the ABCA1-mediated CEC (+70.8%; p < 0.001). HDL-MDA slightly but significantly decreased (-9.6%; p = 0.027), while no difference was detected in pre-ß HDL. No change was observed in aqueous diffusion nor in the ABCG1-mediated CEC. Serum CLC was significantly reduced (-13.3%; p = 0.026). Levels of adiponectin were increased (+50.6%; p < 0.0001) and resistin levels were decreased (-24.3%; p < 0.0001). After vitD replacement, an inverse relationship was found linking the ABCA1-mediated CEC with pre-ß HDL (r2 = 0.346; p < 0.001) and resistin (r2 = 0.220; p = 0.009). CONCLUSION: Our data support vitD supplementation for CV risk prevention.

Joint position statement on "Nutraceuticals for the treatment of hypercholesterolemia" of the Italian Society of Diabetology (SID) and of the Italian Society for the Study of Arteriosclerosis (SISA).

AIM: Evidence showed that LDL-cholesterol lowering is associated with a significant cardiovascular risk reduction. The initial therapeutic approach to hypercholesterolemia includes dietary modifications but the compliance to recommendations is often inadequate. Some dietary components with potential cholesterol-lowering activity are present in small amounts in food. Therefore, in recent years the use of "nutraceuticals" (i.e., nutrients and/or bioactive compounds with potential beneficial effects on human health) has become widespread. Such substances may be added to foods and beverages, or taken as dietary supplements (liquid preparations, tablets, capsules). In the present manuscript, the cholesterol-lowering activity of some nutraceuticals (i.e. fiber, phytosterols, soy, policosanol, red yeast rice and berberine) will be discussed along with: 1) the level of evidence on the cholesterol-lowering efficacy emerging from clinical trial; 2) the possible side effects associated with their use; 3) the categories of patients who could benefit from their use. DATA SYNTHESIS: Based on the current literature, the cholesterol-lowering effect of fiber, phytosterols and red yeast rice is consistent and supported by a good level of evidence. Over berberine, there is sufficient evidence showing significant cholesterol-lowering effects, although the results come from studies carried out almost exclusively in Asian populations. Data on the effects of soy are conflicting and, therefore, the strength of recommendation is quite low. The evidence on policosanol is inconclusive. CONCLUSION: Although health benefits may arise from the use of nutraceuticals with cholesterol-lowering activity, their use might be also associated with possible risks and pitfalls, some of which are common to all nutraceuticals whereas others are related to specific nutraceuticals.

Lomitapide affects HDL composition and function.

BACKGROUND: Lomitapide reduces low-density lipoprotein-cholesterol (LDL-C) but also high-density lipoprotein-cholesterol (HDL-C) levels. The latter may reduce the clinical efficacy of lomitapide. We investigated the effect of lomitapide on HDL-C levels and on cholesterol efflux capacity (CEC) of HDL in patients with homozygous familial hypercholesterolemia (HoFH). METHODS AND RESULTS: Four HoFH patients were treated with increasing dosages of lomitapide. Lomitapide decreased LDL-C (range -34 to -89%). Total HDL-C levels decreased (range -16 to -34%) with a shift to buoyant HDL. ABCA1-mediated CEC decreased in all patients (range -39 to -99%). The changes of total, ABCG1- and SR-BI-mediated CEC were less consistent. CONCLUSION: Lomitapide decreased LDL-C and HDL-C levels. Our report raises the hypothesis that the anti-atherogenic potential of HDL seems to be unaffected as total CEC did not seem to change consistently. Combined with the reduction of atherogenic lipoproteins, the net effect of lomitapide appears to be beneficial in HoFH patients.

The natural compound berberine positively affects macrophage functions involved in atherogenesis.

BACKGROUND AND AIMS: We investigated the effect of berberine (BBR), an alkaloid showing antiatherogenic properties beyond the cholesterol lowering capacity, on macrophage cholesterol handling upon exposure to human serum and on macrophage responses to excess free cholesterol (FC) loading. METHODS AND RESULTS: Mouse and human macrophages were utilized as cellular models. Cholesterol content was measured by a fluorimetric assay; cholesterol efflux, cytotoxicity and membrane FC distribution were evaluated by radioisotopic assays. Monocyte chemotactic protein-1 (MCP-1) secretion was measured by ELISA; membrane ruffling and macropinocytosis were visualized by confocal microscopy. Exposure of cholesterol-enriched MPM to serum in the presence of 1 µM BBR resulted in a reduction of intracellular cholesterol content twice greater than exposure to serum alone (-52%; p < 0.01 and -21%; p < 0.05), an effect not mediated by an increase of cholesterol efflux, but rather by the inhibition of cholesterol uptake from serum. Consistently, BBR inhibited in a dose-dependent manner cholesterol accumulation in human macrophages exposed to hypercholesterolemic serum. Confocal microscope analysis revealed that BBR inhibited macropinocytosis, an independent-receptor process involved in LDL internalization. Macrophage FC-enrichment increased MCP-1 release by 1.5 folds, increased cytotoxicity by 2 fold, and induced membrane ruffling; all these responses were markedly inhibited by BBR. FC-enrichment led to an increase in plasma membrane cholesterol by 4.5 folds, an effect counteracted by BBR. CONCLUSION: We showed novel potentially atheroprotective activities of BBR in macrophages, consisting in the inhibition of serum-induced cholesterol accumulation, occurring at least in part through an impairment of macropinocytosis, and of FC-induced deleterious effects.

Flow-mediated dilation, carotid wall thickness and HDL function in subjects with hyperalphalipoproteinemia.

BACKGROUND AND AIMS: The relationships between very high plasma HDLc and subclinical atherosclerosis are still a matter of debate. METHODS AND RESULTS: Twenty subjects with primary hyperalphalipoproteinemia (HAL, with HDLc in the highest 10th percentile and absence of overt secondary causes of this condition), aged 30-65 years, were compared with 20 age and sex-matched controls. Lipid determination, lipoprotein particle distribution (Lipoprint(®)), Cholesterol Efflux Capacity (CEC), plasma adhesion molecule, analyses of CETP, SRB1 and LIPG genes and of different markers of subclinical vascular disease (ankle-brachial index, ABI; carotid intima-media thickness, cIMT; brachial-artery flow mediated dilation, FMD) were performed. Fasting HDLc levels were 40 mg/dl higher in HAL subjects while LDLc concentration was comparable to control group. CETP gene analysis in HAL subjects identified one novel rare Single Nucleotide Polymorphism (SNP, Asp131Asn), possibly damaging, while the common SNP p.Val422Ile was highly prevalent (50% vs. 27.4% in a control population). No rare mutations associated with HAL were found in SR-B1 and LIPG genes. Polyacrylamide gel electrophoresis in HAL subjects disclosed larger and more buoyant HDL particles than in controls, while LDL profile was much more similar. ABI, cIMT and arterial plaques did not differ in cases and controls and the two groups showed comparable FMD at brachial artery examination. Similarly, ABCA1 and ABCG1 HDL-mediated CEC, the most relevant for atheroprotection, did not discriminate between the groups and only ABCG1 pathway seemed somewhat related to arterial reactivity. CONCLUSIONS: HDL dimension, function and genetics seem scarcely related to subclinical atherosclerosis and vascular reactivity in middle-aged HAL subjects.

Cellular cholesterol efflux and cholesterol loading capacity of serum: effects of LDL-apheresis.

High LDL-cholesterol (LDL-C) characterizes familial hypercholesterolemia (FH) and familial combined hyperlipidemia (FCH). LDL-apheresis, used in these patients to reduce LDL-C levels, has been shown to also affect HDL levels and composition. We studied LDL-apheresis effects on six FH and nine FCH subjects' serum capacity to modulate cellular cholesterol efflux, an index of HDL functionality, and to load macrophages with cholesterol. Serum cholesterol efflux capacity (CEC) and macrophage cholesterol loading capacity (CLC) were measured before, immediately after, and two days after LDL-apheresis. The procedure reduced total cholesterol (TC), LDL-C, and apoB plasma levels (-69%, -80% and -74%, respectively), parameters only partially restored two days later. HDL-C and apoA-I plasma levels, reduced after LDL-apheresis (-27% and -16%, respectively), were restored to almost normal levels two days later. LDL-apheresis reduced serum aqueous diffusion (AD) CEC, SR-BI-CEC, and ABCA1-CEC. AD and SR-BI were fully restored whereas ABCA1-CEC remained low two days later. Sera immediately and two days after LDL-apheresis had a lower CLC than pre-LDL-apheresis sera. In conclusion, LDL-apheresis transiently reduces HDL-C levels and serum CEC, but it also reduces also serum capacity to deliver cholesterol to macrophages. Despite a potentially negative effect on HDL levels and composition, LDL-apheresis may counteract foam cells formation.

The thienotriazolodiazepine Ro 11-1464 increases plasma apoA-I and promotes reverse cholesterol transport in human apoA-I transgenic mice.

BACKGROUND AND PURPOSE: Ro 11-1464 is a thienotriazolodiazepine previously described to selectively stimulate apolipoprotein A-I (apoA-I) production and mRNA level in human liver cells. Here, we studied its effects upon oral administration to human apoA-I transgenic (hapoA-I) mice. EXPERIMENTAL APPROACH: HapoA-I mice were treated for 5 days with increasing doses of Ro 11-1464. Macrophage reverse cholesterol transport (mph-RCT) was assessed by following [(3) H]-cholesterol mobilization from pre-labelled i.p. injected J774 macrophages to plasma, liver and faeces. Effects on plasma lipids, apoproteins, lecithin-cholesterol : acyltransferase (LCAT) and liver enzymes, as well as on faecal excretion of cholesterol and bile salts, and on liver lipids and mRNA contents were determined. KEY RESULTS: Treatment with Ro 11-1464 300 mg·kg(-1) ·day(-1) resulted in a nearly 2-fold increase in plasma apoA-I, a 2- to 3-fold increase in the level of large sized-pre-ß high-density lipoprotein and a 3-fold selective up-regulation of hepatic apoA-I mRNA, but a marked decrease in all plasma lipids and LCAT activity. Mpm-RCT was decreased in blood but markedly increased in faecal sterols (4-fold) and bile acids (1.7-fold). However, liver weight and liver enzymes in plasma were also increased, in parallel with an increase in liver cholesterol ester content (all these effect being significant). CONCLUSION AND IMPLICATIONS: In this model Ro 11-1464 causes increased hepatic expression and plasma levels of apoA-I and a suppression of LCAT, and a marked enhancement of reverse cholesterol transport, but also some symptoms of liver toxicity. The compound may therefore be a prototype for a next generation of anti-atherosclerotic medicines.

Computer simulation of coronary flow waveforms during caval occlusion.

OBJECTIVES: Mathematical modeling of the cardiovascular system is a powerful tool to extract physiologically relevant information from multi-parametric experiments. The purpose of the present work was to reproduce by means of a computer simulator, systemic and coronary measurements obtained by in vivo experiments in the pig. METHODS: We monitored in anesthetized open-chest pig the phasic blood flow of the left descending coronary artery, aortic pressure, left ventricular pressure and volume. Data were acquired before, during, and after caval occlusion. Inside the software simulator (CARDIOSIM) of the cardiovascular system, coronary circulation was modeled in three parallel branching sections. Both systemic and pulmonary circulations were simulated using a lumped parameter mathematical model. Variable elastance model reproduced Starling's law of the heart. RESULTS: Different left ventricular pressure-volume loops during experimental caval occlusion and simulated cardiac loops are presented. The sequence of coronary flow-aortic pressure loops obtained in vivo during caval occlusion together with the simulated loops reproduced by the software simulator are reported. Finally experimental and simulated instantaneous coronary blood flow waveforms are shown. CONCLUSIONS: The lumped parameter model of the coronary circulation, together with the cardiovascular system model, is capable of reproducing the changes during caval occlusion, with the profound shape deformation of the flow signal observed during the in vivo experiment. In perspectives, the results of the present model could offer new tool for studying the role of the different determinants of myocardial perfusion, by using the coronary loop shape as a "sensor" of ventricular mechanics in various physiological and pathophysiological conditions.

Severe HDL deficiency due to novel defects in the ABCA1 transporter.

OBJECTIVES: The objective was the identification and functional characterization of mutations in the ABCA1 gene in four patients with severe HDL deficiency. SUBJECTS: Patients were referred to the clinic because of almost complete HDL deficiency. METHODS: The ABCA1 gene was sequenced directly. The analysis of the ABCA1 protein, ABCA1 mRNA and ABCA1-mediated cholesterol efflux was performed in cultured fibroblasts. Intracellular localization of ABCA1 mutants was investigated in transfected HEK293 cells. RESULTS: Two patients were homozygous for mutations in the coding region of the ABCA1 gene, resulting in an amino acid substitution (p.A1046D) and a truncated protein (p.I74YFsX76). The third patient was homozygous for a splice site mutation in intron 35 (c.4773 + 1g>a), resulting in an in-frame deletion of 25 amino acids (del p.D1567_K1591) in ABCA1. These patients had clinical manifestations of accumulation of cholesterol in the reticulo-endothelial system. The fourth patient, with preclinical atherosclerosis, was a compound heterozygote for two missense mutations (p.R587W/p.W1699C). ABCA1-mediated cholesterol efflux was abolished in fibroblasts from patients with p.A1046D and del p.D1567_K1591 mutants and in fibroblasts homozygous for p.R587W. A reduced ABCA1 protein content was observed in these cells, suggesting an increased intracellular degradation. The mutant p.W1699C was largely retained in the endoplasmic reticulum, when expressed in HEK293 cells. CONCLUSIONS: The homozygotes for mutations which abolish ABCA1 function showed overt signs of involvement of the reticulo-endothelial system. This was not the case in the compound heterozygote for missense mutations, suggesting that this patient retains some residual ABCA1 function that reduces cholesterol accumulation in the reticulo-endothelial system.

Localisation of heavy metals in the midgut epithelial cells of Xenillus tegeocranus (Hermann, 1804) (Acari: Oribatida).

Sites of intracellular metal deposition in the midgut ventriculus and in the proventricular glands of Xenillus tegeocranus (Hermann, 1804) (Acari: Oribatida) were studied by TEM. The study aimed to obtain new information on the ultrastructural features of heavy metal compartmentalisation and elimination mechanisms in oribatid mites. Specimens of X. tegeocranus were collected from an abandoned mining and smelting area and from an unpolluted site. A large number of electron-dense granules (EDGs) were detected: concentric spherocrystals were observed mainly in the epithelium of the midgut ventriculus, while homogeneous dark granules were found exclusively in proventricular gland cells. The elemental composition of EDGs, studied by X-ray microanalysis, showed that midgut cells of X. tegeocranus can store metals (Fe, Mn, Zn, Ni, and Cu) in granules. The chemical composition of EDGs seems to be influenced by the presence and bioavailability of heavy metals in soil, with different kinds of metals accumulating in different types of granules.

Tethered cord syndrome.

AIM: Tethered cord syndrome (TCS) is a stretch-induced disorder of the spinal cord. Tethering is due to an inelastic structure anchoring the caudal end of the spinal cord as a short and thick filum terminale. Spinal dysraphism is occasionally associated,but the etiological relationship between these disorders remain unclear. Other anomalies may be concurrently found as hydromielia and Arnold-Chiari malformation. METHODS: The authors analysed neuroradiological findings in 5 children and 9 patients of 20-24 years of age; there were four male and ten female. The criteria for inclusion were neurological disturbances (disorders!) localizable to the level of the conus and evidence for spinal dysraphism. The purpose of this study was to make the precise diagnosis and make also precise planning for therapy, conservative or surgical treatment. RESULTS: The age of diagnosis of TCS varies from under 1 year to 14 years and is very rarely as late as adulthood. TCS can present late and insidiously with progressive gait disturbances, atrophy of various muscle groups or the entire limb, loss of reflexes,loss of sensation in the sacral dermatomes, sphincter disturbances, gait abnormality and pain in the gluteal, perianal and other pelvic areas. The diagnosis involves standard X-RAY examination, CT and CT-Mielography but MRI is now a diagnostic method of choice. Surgical untethering of the cord is recommended. The associated pain responds best to surgical treatment; ambulation and bladder function may improve as well. CONCLUSIONS: However sphincter dysfunction often remains a permanent problem. Given the potential for rapid deterioration with incomplete neurological recovery, even prophilactic surgery seems advisable. The patient need to be followed up, if not operated upon.

Fine structure of the midgut and Malpighian papillae in Campodea (Monocampa) quilisi Silvestri, 1932 (Hexapoda, Diplura) with special reference to the metal composition and physiological significance of midgut intracellular electron-dense granules.

The fine structure of the midgut and the Malpighian papillae in Campodea (Monocampa) quilisi Silvestri, 1932 (Hexapoda, Diplura) specimens was described. We observed the presence of electron-dense granules (EDGs) in the midgut epithelial cells, similar in genesis, structure and aspect to the type A spherocrystals described in the midgut epithelium of Collembola and Diplopoda. Energy-dispersive X-ray microanalysis was used to detect the chemical composition of the granules and to relate it to the concentrations of some potential toxic heavy metals (Pb, Cu, Zn) in soil and litter. Chemical composition of the granules seems strongly influenced by the presence and bioavailability of heavy metals in the external environment. Specimens from a contaminated abandoned mining and smelting area (Colline Metallifere, southern Tuscany) were able to accumulate Fe, Mn, Zn, Pb and Cu in their midgut EDGs. In addition, we observed that C. (M.) quilisi was able to excrete the metal-containing granules into the external medium by the moulting of the intestinal epithelium. This confirms that the process of ionic retention of midgut cells is particularly significant in animals lacking Malpighian tubules.

Modulation of macrophage function and metabolism.

Several drugs or pharmacologically active molecules such as statins, calcium antagonists, and PPAR agonists have been shown to affect macrophage functions that contribute to atherosclerosis and modulate plaque stability. For example, the modulation of matrix metalloproteinase secretion and cholesterol metabolism in macrophages may help to prevent cardiovascular disease independently of the correction of risk factors.

Inhibition of metalloproteinase-9 activity and gene expression by polyphenolic compounds isolated from the bark of Tristaniopsis calobuxus (Myrtaceae).

Excessive breakdown of extracellular matrix by metalloproteinases (MMPs) occurs in many pathological conditions, and thus inhibition of MMP activity might have therapeutic potential. The methanolic extract and the identified compounds from the bark of Tristaniopsis calobuxus Brongniart & Gris (Myrtaceae) were tested on the activity, production, and gene expression of MMP-9. The extract produced a concentration-dependent inhibition (50-95% at 10-50 microg/ml) of MMP-9 activity. The inhibitory activity was retained in the ethyl acetate-soluble fraction (50-95% inhibition at 10-50 microg/ml) which also reduced the release of MMP-9 by mouse peritoneal macrophages up to 80%. In the ethyl acetate-soluble fraction, two active fractions, 5A and 5B were identified. HPLC-MS and NMR analyses of these fractions indicated the presence of gallocatechin, ellagic acid, and its glycoside derivatives. Since the absolute configuration of gallocatechin was not determined, in the next experiments both (+)-gallocatechin (2R,3S) and (-)-gallocatechin (2S,3R) were tested, and (-)-epigallocatechin (2R,3R) was included for comparison. 5A and 5B inhibited MMP-9 secretion, an observation which correlated with the decrease of MMP-9 promoter activity and the downregulation of mRNA levels. All compounds decreased MMP-9 mRNA levels and secretion. Ellagic acid, (+)-gallocatechin and (-)-epigallocatechin, but not (-)gallocatechin inhibited promoter-driven transcription. Thus configuration at C2 (R) of the flavanol seem to be critical for the interaction with the promoter.

Normal human IgG prevents endothelial cell activation induced by TNFalpha and oxidized low-density lipoprotein atherogenic stimuli.

Normal human immunoglobulin G (IgG) has anti-inflammatory and immuno-regulatory properties, which are exploited in the therapy of selected diseases. A putative mechanisms of action is the direct regulation of endothelial cell function by natural antiendothelial cell antibodies. Endothelium activation is a critical event in atherosclerosis. We have verified the ability of normal human IgG to modulate endothelial responses to the atherogenic stimuli tumour necrosis factor-alpha (TNFalpha) and oxidized low-density lipoproteins (oxLDL) in vitro. Confocal microscopy was used to visualize vascular cell adhesion molecule-1 (CD106) expression on endothelial cells, cytoplasmic free calcium ([Ca++]i) modifications and fluorescein-coupled oxLDL internalization. Cytokine secretion was measured by ELISA on cell supernatants. IgG prevented TNFalpha induced CD106 membrane expression and an increase in [Ca++]i, and inhibited the secretion of interleukin-6 (IL-6) and macrophage-colony-stimulating factor (M-CSF). IgG also inhibited CD106 expression induced by oxLDL and one pathway of their internalization, but were ineffective on oxLDL induced [Ca++]i rise and apoptosis. F(ab)'2 fragments from IgG, but not monoclonal IgG, reproduce IgG effects. These findings point to a regulatory role for specific antibodies included in circulating normal IgG towards proinflammatory responses of endothelial cells in atherogenesis and suggest possible development of new therapeutic strategies.