Commercial α1-antitrypsin preparations markedly differ in their potential to inhibit the ATP-induced release of monocytic interleukin-1ß.

The acute phase protein α1-antitrypsin (AAT) inhibits numerous proteases, specifically neutrophil elastase. Patients with an AAT deficiency due to mutations frequently develop early onset emphysema. The commercial preparations of human plasma AAT are clinically used as biopharmaceuticals to protect the lung tissue of AAT-deficient patients from damage caused by neutrophil elastase. Accordingly, preparations of AAT are validated for their anti-elastase activity. However, several anti-inflammatory effects of AAT were described, some of them being independent from its anti-protease function. We recently demonstrated that AAT isolated from the blood of healthy persons efficiently inhibits the ATP-induced release of interleukin-1ß by human monocytes. This finding is of therapeutic relevance, because IL-1ß plays an important role in numerous debilitating and life-threatening inflammatory diseases. As anti-inflammatory functions of AAT are of increasing clinical interest, we compared the potential of two widely used AAT preparations, Prolastin® and Respreeza®, to inhibit the ATP-induced release of IL-1ß using human monocytic U937 cells. We detected marked functional differences between both medicaments. The AAT preparation Respreeza® is less active compared to Prolastin® regarding the inhibition of the ATP-induced release of monocytic IL-1ß. Chemical oxidation of Respreeza® restored this anti-inflammatory activity, while destroying its anti-protease function. Our data suggest that the anti-inflammatory potential and the anti-protease function of AAT can be fully uncoupled. In the light of the increasing clinical interest in anti-inflammatory functions of AAT, commercial AAT preparations should be carefully reinvestigated and optimized to preserve the dual anti-protease and anti-inflammatory activity of native AAT.

Neutrophilic panniculitis associated with alpha-1-antitrypsin deficiency: an update.

Neutrophilic panniculitis associated with alpha-1-antitrypsin deficiency (AATD) is a very rare disease. Its estimated prevalence is 1 in 1000 subjects with severe AATD (usually white individuals with a Pi*ZZ genotype). It is manifested clinically by painful recurrent ulcerating subcutaneous nodules, and characterized histologically by dense infiltrates of neutrophils in the deep dermis and connective-tissue septae, with secondary lobular panniculitis. It may be the only clinical manifestation of AATD, although it can also occur together with the classical pulmonary or hepatic manifestations of the disease. AATD-associated panniculitis is not only very rare but may also be significantly underdiagnosed. The physician managing a case of panniculitis with a clinical presentation suggestive of AATD and a compatible skin biopsy should measure serum AAT concentration and, if low, determine the AAT phenotype by isoelectric focusing. If uncertainty remains, the SERPINA1 gene should be sequenced to identify the genotype. If AATD is diagnosed, AATD testing of first-degree family members should be performed in order to take appropriate preventive and therapeutic measures, including genetic counselling, education on inheritance, risk arising from tobacco smoke, occupational exposure to pollutants and hepatotoxic substances, and the provision of information on clinical management. Cases of panniculitis in which conventional therapy with dapsone has failed may be managed with intravenous augmentative therapy using human AAT. The current manuscript addresses the fundamental concepts of the pathogenesis of AATD-associated panniculitis and describes the clinical presentation and management of cases in order to reduce underdiagnosis and improve outcomes.

α-Methylacyl-coenzyme A racemase (AMACR, p504s) is a marker to distinguish malignant melanomas from dysplastic nevi and melanocytic nevi.

Routinely processed skin biopsies are still the mainstay for the diagnosis of melanocytic skin neoplasms (MSNs) and are considered the "gold standard" for individual patient management and clinical trials. The diagnostic challenge of melanocytic lesions of the skin prompts histopathologists to consider new diagnostic tools; among these, immunohistochemistry. We aimed to find putative new immunohistochemical markers, which can supplement the histological criteria used to detect dysplasia. In this immunohistochemical study, we chose a panel of promising biomarkers which could potentially differentiate between different MSN entities. These included α-methylacyl-coenzyme A racemase (AMACR; p504s), which is involved in the degradation of branched chained fatty acid derivates. We analysed a cohort of benign nevi and malignant melanomas. The design of the study included 78 melanocytic skin neoplasms (26 malignant melanomas and 52 benign nevi) in a tissue microarray. Immunohistochemistry of cyclin-dependent kinase inhibitor 2A (p16Ink4a), methylacyl-coenzyme A racemase (AMACR), cyclin D1, and E-cadherin was performed and assessed. We have observed that the p16Ink4a, AMACR, cyclin D1, and E-cadherin showed no exclusive staining for nevi or melanomas. However, a significant overexpression of AMACR was found in malignant melanomas compared to benign nevi. AMACR overexpression was also associated with an increased p16Ink4a staining. Our results suggest AMACR as an immunohistochemical marker for distinguishing malignant melanomas and dysplastic nevi from conventional melanocytic nevi.

Heterogeneity among septic shock patients in a set of immunoregulatory markers.

Immune activation is a regular feature of sepsis, but the incidence and nature of the ensuing inflammation-resolving and immunosuppressive component is less well understood. In this study, we compared immunoregulatory markers on blood leukocytes from patients with Gram-negative or Gram-positive sepsis or septic shock, and compared this to blood from patients with severe virosis or healthy controls. To this end, blood from 32 patients with sepsis, including ten cases with shock, and 12 patients with severe virosis were analysed by flow cytometry for the expression levels of monocyte HLA-DR, CD11c, CD14 and CD40, and for frequencies of CD163(+)-suppressive monocytes, HLA-DR(+) or CD40(+)-activated T cells and Tregs. Plasma cytokine levels were analysed as a functional measurement. Signs of immunosuppression dominated in the septic shock and Gram-positive sepsis groups, whereas monocyte activation was common in Gram-negative sepsis patients without shock. However, the main finding was the large inter-individual variation of immune activation and immunosuppression, with no correlation to prognosis among the shock patients. The pronounced inter-individual variation in the analysed monocyte and lymphocyte markers forms a strong argument that, when immunomodulatory treatment is considered in a sepsis patient, it should be personalised and guided by a detailed immune status assessment.

Does heart surgery change the capacity of α1-antitrypsin to inhibit the ATP-induced release of monocytic interleukin-1ß? A preliminary study.

Heart surgery involving cardiopulmonary bypass induces systemic inflammation that is, at least in part, caused by extracellular ATP originating from damaged cells and by proteases secreted by activated neutrophils. The anti-protease α1-antitrypsin (AAT) forms complexes with several proteases including neutrophil elastase, resulting in a mutual loss of activity. We demonstrated recently that AAT inhibits the ATP-induced release of the pro-inflammatory cytokine interleukin-1ß by human monocytes by a mechanism involving activation of metabotropic functions at nicotinic acetylcholine receptors. Interleukin-1ß importantly contributes to the pathogenesis of sterile inflammatory response syndrome. Thus, AAT might function as an endogenous safeguard against life-threatening systemic inflammation. In this preliminary study, we test the hypothesis that during cardiopulmonary bypass, AAT is inactivated as an anti- protease and as an inhibitor of ATP-induced interleukin-1ß release. AAT was affinity-purified from the blood plasma of patients before, during and after surgery. Lipopolysaccharide-primed human monocytic U937 cells were stimulated with ATP in the presence or absence of patient AAT to test for its inhibitory effect on interleukin-1ß release. Anti-protease activity was investigated via complex formation with neutrophil elastase. The capacity of patient AAT to inhibit the ATP-induced release of interleukin-1ß might be slightly reduced in response to heart surgery and complex formation of patient AAT with neutrophil elastase was unimpaired. We conclude that surgery involving cardiopulmonary bypass does not markedly reduce the anti-inflammatory and the anti-protease activity of AAT. The question if AAT augmentation therapy during heart surgery is suited to attenuate postoperative inflammation warrants further studies in vivo.

New Findings in PiZZ alpha1-antitrypsin deficiency-related panniculitis. Demonstration of skin polymers and high dosing requirements of intravenous augmentation therapy.

Panniculitis is a recognized but unusual complication of a severe deficiency of alpha1-antitrypsin (AAT), with fewer than 100 cases described to date. Like the pathogenesis of emphysema in severe PiZZ deficiency of AAT, panniculitis has been hypothesized to be an inflammatory process, possibly related to Z AAT polymer formation and to an unopposed anti-inflammatory screen in the context of deficient serum levels of AAT. The current report presents a 31-year-old woman with PiZZ AAT deficiency-associated panniculitis. Our case extends current knowledge of AAT-associated panniculitis in 2 ways: (1) we demonstrate Z-type AAT polymers in the skin, which supports the inflammatory pathogenesis of panniculitis and the potential pro-inflammatory role of polymers; (2) we show that a high dose and long-term use of intravenous augmentation therapy (90 mg/kg body weight once weekly during 3 years) can ameliorate the frequency and severity of panniculitis associated with AAT deficiency.

Blood monocyte profiles in COPD patients with PiMM and PiZZ α1-antitrypsin.

Human blood monocytes are divided into populations based on the differential expression of CD14 and CD16 receptors: CD14 + CD16(classical), CD14 + CD16 + (intermediate), and CD14-CD16+ (non-classical). Given their functional differences and their role in pathogenesis of chronic obstructive pulmonary disease (COPD), monocyte profiling is of clinical interest. Here we investigated blood monocyte subsets in clinically stable COPD patients with alpha1-antitrypsin (AAT) deficiency (PiZZ, n = 7) and with normal AAT variant (PiMM, n = 7). Peripheral whole blood was collected in sodium heparin tubes and incubated with LPS (from E. coli; 1 µg/ml) or placebo for 6 h at 37 °C, 5% CO2. To profile monocyte subsets we performed flow cytometry analysis based on HLA-DR and CD14/CD16 staining. HLA-DR + subsets of cells did not differ between PiZZ and PiMM COPD, and healthy controls (n = 7), used as a reference. Monocyte profiling, which express the CD14 and CD16, but not the HLA-DR (HLA-DR-) showed that intermediate monocytes subset was lowest in PiZZ group, and almost totally disappeared from blood treated with LPS. The non-classical subset was almost absent in PiZZ patients independently of LPS treatment. Recent studies demonstrate that non-classical monocytes exhibit a unique ability to protect the vascular endothelium under both homeostatic and inflammatory conditions whereas intermediate monocytes are recruited at a later stage of inflammation, and are associated with secretion of cytokines/chemokines and wound healing. Evident alterations in blood monocyte subsets together with a partial reduction of AAT levels, an important anti-inflammatory protein, can be key factors for the early manifestation of emphysema in some PiZZ AATD carriers.

rCBF pathology in Alzheimer's disease is associated with slow processing speed.

Decreased information processing speed (mental slowing) is a known sequelae of many brain disorders, and can be assessed by continuous naming tasks. Functional imaging studies have shown that pause and articulation times in continuous speech are normally associated with different brain regions, but knowledge about such association in dementia is lacking. We therefore tested the hypothesis that perfusion deficits in Alzheimer's disease (AD) are not only associated with slower processing, but also with these speech measures. Using regional cerebral blood flow (rCBF) measurements during the performance of a continuous colour and form-naming task, we found that naming speed was substantially slower in AD patients than in controls. This slower naming was exclusively determined by an increase in mean pause time, and only to a limited extent by articulation time. The increased pause time was uniquely associated with temporo-parietal rCBF reductions of the patients, while articulation was not. By contrast, the rCBF of healthy elderly control subjects was consistently accompanied by substantially shorter articulation and pause times, although the naming measures were not statistically associated with rCBF. These findings suggest that pause time (in contrast to articulation time) may serve as a sensitive measure in the assessment of information processing speed deficits in dementia, by virtue of its close association with brain pathology.

An association between Type 2 diabetes and alpha-antitrypsin deficiency.

AIMS: Alpha(1)-Antitrypsin (AAT) is a serine protease inhibitor which recently has been shown to prevent Type 1 diabetes development, to prolong islet allograft survival and to inhibit pancreatic B-cell apoptosis in vivo. It has also been reported that Type 1 diabetic patients have significantly lower plasma concentrations of AAT, suggesting the potential role of AAT in the pathogenesis of Type 1 diabetes. We have investigated whether plasma AAT levels are altered in Type 2 diabetes. METHODS: The study included patients with Type 2 diabetes (n = 163) and non-diabetic control subjects matched for age, sex and smoking habits (n = 158) derived from the population-based Malmö Diet and Cancer study. Plasma samples were analysed for AAT concentration and phenotype and serum glucose, insulin, C-reactive protein and lipid levels were measured. Glycated haemoglobin was also measured. RESULTS: In the diabetic group, the women had higher mean plasma AAT levels than men (P < 0.05). The mean plasma AAT levels did not differ between diabetic and control subjects. However, the number of individuals with low AAT levels (< 1.0 mg/ml) was 50% higher in the diabetic group (P < 0.05) and the frequency of AAT deficiency genotypes was 50% higher (NS) in diabetic compared with control subjects. In the group of diabetic patients with AAT < 1 mg/ml, AAT directly correlated with systolic blood pressure (P = 0.048) and inversely correlated with waist-hip ratio (P = 0.031). CONCLUSIONS: Our results provide evidence that deficiency of AAT may be associated with an increased risk of developing Type 2 diabetes.

Long-term augmentation therapy with alpha-1 antitrypsin in an MZ-AAT severe persistent asthma.

A young Caucasian female with severe bronchial asthma and Alpha1-antitrypsin (AAT) deficiency, MZ phenotype, experienced a quick and severe limitation of her physical capacity, which negatively affected her psychological state and social life, though she was under a strong antiasthmatic treatment. Given her declining health status and the significant chronic corticoid administration-related side-effects (including high reduction of muscle mass and bone density), a clinical trial with commercial intravenous AAT was proposed by the patient's doctors, and accepted by the Spanish Ministry of Health, although it this therapy was not approved for MZ phenotypes yet. This new therapy quickly stopped lung function decline rate, dramatically reduced the number of hospital admissions of the patient, suppressed the oral administration of prednisone, reversed the corticosteroid-related health adverse effects, significantly improving her quality of life. Thus, although AAT replacement therapy is not approved nor indicated for the treatment of bronchial asthma in MZ patients, its favourable effects observed in this isolated case support the hypothesis that bronchial asthma could be due to pathogenic mechanisms related to a protease-antiprotease imbalance, what which could open new perspectives for future research on the field.

Angiopoietin-like protein 4 and cardiovascular function in COPD.

INTRODUCTION: The coexistence of chronic obstructive pulmonary disease (COPD) and cardiovascular disease (CVD) is frequent and might be inter-related through inflammation-related processes reflected by specific markers. Here, we studied angiopoietin-like protein 4 (ANGPTL4), an upcoming cardiovascular marker, in stable COPD, and its relationship to cardiovascular function with respect to well-known CVD risk factors. METHODS: In a prospective COPD cohort study, we investigated serum ANGPTL4 levels, vascular status (ankle-brachial index (ABI)) and cardiac function (N-terminal pro-B-type natriuretic peptide (NT-proBNP)) as well as airflow limitation, objectively measured physical activity, the metabolic syndrome, high-sensitive C reactive protein (hs-CRP) and other CVD risk factors at 2 time points. We initially studied 74 stable COPD patients and 18 controls. For internal validation, we additionally studied 160 COPD patients of a former visit. RESULTS: ANGPTL4 was significantly elevated in COPD patients compared with controls (p=0.026). After correction for traditional CVD risk factors, including hs-CRP, higher levels of ANGPTL4 were independently associated with lower ABI (p=0.023) and higher NT-proBNP (p<0.001). These findings were confirmed in the internal validation analysis, which included echocardiographic assessments. CONCLUSIONS: Serum ANGPTL4 is independently associated with cardiovascular function in COPD and might qualify as a biomarker reflecting a pathogenic link between COPD and CVD.

Conformational properties of serine proteinase inhibitors (serpins) confer multiple pathophysiological roles.

Serine proteinase inhibitors (Serpins) are irreversible suicide inhibitors of proteases that regulate diverse physiological processes such as coagulation, fibrinolysis, complement activation, angiogenesis, apoptosis, inflammation, neoplasia and viral pathogenesis. The molecular structure and physical properties of serpins permit these proteins to adopt a number of variant conformations under physiological conditions including the native inhibitory form and several inactive, non-inhibitory forms, such as complexes with protease or other ligands, cleaved, polymerised and oxidised. Alterations of a serpin which affect its structure and/or secretion and thus reduce its functional levels may result in pathology. Serpin dysfunction has been implicated in thrombosis, emphysema, liver cirrhosis, immune hypersensitivity and mental disorders. The loss of inhibitory activity of serpins necessarily results in an imbalance between proteases and their inhibitors, but it may also have other physiological effects through the generation of abnormal concentrations of modified, non-inhibitory forms of serpins. Although these forms of inhibitory serpins are detected in tissues and fluids recovered from inflammatory sites, the important questions of which conditions result in generation of different molecular forms of serpins, what biological function these forms have, and which of them are directly linked to pathologies and/or may be useful markers for characterisation of disease states, remain to be answered. Elucidation of the biological activities of non-inhibitory forms of serpins may provide useful insights into the pathogenesis of diseases and suggest new therapeutic strategies.

The interaction of hydrophobic bile acids with the alpha 1-proteinase inhibitor.

An in vitro complex formation between cholesterol and human alpha 1-proteinase inhibitor (alpha 1-antitrypsin, alpha 1-Pi) has been described. Hydrophobic bile acids were studied for a similar interaction using lithocholic acid (LC) as a prototype of a hydrophobic acid. At a molar ratio of 5:1, LC induced conformational changes of alpha 1-Pi reflected in an abnormal gel-electrophoretic appearance, loss of anodal immunoreactivity on crossed immunoelectrophoresis, exposition of new antigenic determinant(s) on immunodiffusion, and loss of antiproteinase activity. After 6 h incubation, LC and alpha 1-Pi form a complex of approximately 200 kDa molecular mass seen following gel-filtration. After prolonged (24 h) interaction a series of large alpha 1-Pi polymers were seen on SDS-PAGE under reducing conditions followed by Western blotting. Glycolitho-, sulfolitho-, deoxycholic and 3-beta-hydroxy-5-cholenoic acids induced similar but less pronounced changes of alpha 1-Pi, whereas transferrin remained unaffected. Hydrophilic acids lacked effect on alpha 1-Pi. The results are compatible with a specific, irreversible interaction of alpha 1-Pi with hydrophobic bile acids affecting its physical and proteinase inhibitory properties. The cholestatic potency of the hydrophobic acids studied and their ability to induce alpha 1-Pi polymerization may be important in cholestatic conditions.

In vitro complex formation between cholesterol and alpha 1-proteinase inhibitor.

The in vitro interaction between human alpha 1-proteinase inhibitor (alpha 1-PI) and cholesterol was studied with electrophoretic and gel chromatographic methods. The addition of cholesterol (from 1 to 20 mol/mol alpha 1-PI) at 37 degrees C resulted in retarded electrophoretic mobility of alpha 1-PI towards the anode, diminished immunoreactivity and antiproteinase activity. At a molar ratio of 2:1 (cholesterol/alpha 1-PI), antitryptic activity was reduced by 15% but antielastase activity by 50%. At this ratio the gel filtration alpha 1-PI peak appeared at 67 kDa, as compared to 52 kDa for native alpha 1-PI. No size difference was noted on SDS-PAGE. These results suggest the occurrence of noncovalent complex formation between cholesterol and alpha 1-PI in vitro.

Conformational changes of the alpha 1-proteinase inhibitor affecting its cholesterol binding ability.

The effect of conformational changes of the alpha 1-proteinase inhibitor (alpha 1PI) on alpha 1PI-cholesterol complex (1:2 mol/mol) formation in vitro was studied with electrophoretic and gel chromatographic methods. Native alpha 1PI was modified by adding free thiol agents such as glutathione, cysteine HCl, or DL-homocysteine, by heating, or by cleavage with pancreatic elastase or trypsin. Conformational changes of the alpha 1PI molecule induced by these procedures were all accompanied by a loss of its ability to bind cholesterol in vitro under standard experimental conditions. The data suggest alpha 1PI-cholesterol binding to be affected by both direct and indirect modifications of the alpha 1PI-reactive center, that is situated on a mobile peptide loop.

C-terminal fragment of alpha1-antitrypsin activates human monocytes to a pro-inflammatory state through interactions with the CD36 scavenger receptor and LDL receptor.

Monocyte scavenger receptor, CD36 has been implicated in the pathogenesis of atherosclerosis as a major oxidised LDL receptor mediating lipid accumulation and foam cell formation. Previously, we found that treatment of monocyte cultures with the carboxyl terminal fragment of alpha1-antitrypsin (C-36) increases lipid binding and uptake, induces LDL receptor mRNA and CD36 receptor protein expression, and also significantly increases production of pro-inflammatory molecules. To assess the role of the CD36 receptor in proatherogenic monocyte activation by the C-36 fragment, we tested whether specific anti-CD36 receptor antibodies would block the effects of C-36 on monocyte activation. We find that pre-incubation of cells with anti-LDL and anti-CD36 receptor antibodies (10 microg/ml) blocks binding of 125I-C-36 by about 50%. Similarly, cells pre-incubated with oxidised LDL or native LDL at concentrations from 2.5 to 10 microg/ml showed a loss of 125I-C-36 binding (up to 49 and 57%) and uptake (up to 47 and 59.8%), respectively. In parallel experiments, monocytes were first incubated for 1 or 6 h with anti-CD36 antibodies (10 microg/ml) prior to adding C-36 peptide. Anti-CD36 antibodies suppressed C-36-induced production of gelatinase B, monocyte chemoattractant protein-1, interleukin-6 and cellular oxygen consumption to control levels, whereas levels of TNFalpha were unaffected. In contrast, saturation of LDL receptors with excess of anti-LDL (20 microg/ml) significantly inhibited C-36 induced TNFalpha levels. Results indicate that the C-36 peptide binds to both LDL and CD36 scavenger receptors which involves selective upregulation of pro-inflammatory molecules and activation of the respiratory burst in human monocytes. This also supports important roles for CD36 and LDL receptors in atherogenesis and suggests that blockade of CD36 receptor can be protective in pro-inflammatory activation of human monocytes.

Atherogenic properties of human monocytes induced by the carboxyl terminal proteolytic fragment of alpha-1-antitrypsin.

Atherosclerotic plaques contain a significant number of macrophage foam cells and are associated with an inflammatory state. Inflammation induces the secretion from monocytes and other cells of cytokines, reactive oxygen species, proteinases and proteinase inhibitors among many other molecular species. AAT is prominent among the serine proteinase inhibitors and is an important regulator of leukocyte elastase and proteinase-3. It has been shown that the stable AAT-proteinase complex can upregulate AAT biosynthesis, and we have shown that the shorter, carboxyl terminal peptide (C-36) resulting from proteinase cleavage of AAT polymerizes, and in its fibrillar form alters cellular metabolism. To test for a possible link between the inflammation-generated C-36 peptide and cellular processes associated with atherogenesis, we have studied the effects of the fibrillar form of this peptide at varying concentrations on human monocytes in culture. We have found that fibrillar C-36 at concentrations of greater than or equal to 5 micromol/l in monocyte cultures for 24 h significantly increases LDL binding and uptake, upregulates LDL receptors, induces cytokine production and glutathione reductase activity, and upregulates AAT synthesis. The expression of CD36 protein, LDL Scavenger receptor, is also upregulated by fibrillar C-36 and native LDL in the presence of C-36-activated monocytes is more oxidized than with unactivated control monocytes. The majority of monocytes cultured for 24 h in the presence of C-36 fibrils were transformed morphologically into macrophages. These data establish a direct molecular link, mediated by C-36 peptide of AAT, between inflammation and the oxidation and accumulation of lipid in monocyte-derived macrophages. This may be important for an understanding of the events conducive to atherogenesis.

Fibrillar Alzheimer's amyloid peptide Abeta(1-42) stimulates low density lipoprotein binding and cell association, free radical production and cell cytotoxicity in PC12 cells.

Amyloid forming peptides are known to disturb vital cellular functions and induce cell death. However, the mechanisms by which fibrillogenic peptides induce cytotoxic effects in various cells has not been established. In this study the effects on low density lipoprotein binding and uptake of fibrils of the Alzheimer's amyloid beta-peptide (Abeta(1-42)), which is known to play a central role in the pathogenesis of Alzheimer's disease, were investigated in pheochromocytoma PC12 cells. Fibrillar Abeta(1-42) at micromol concentrations increased low-density lipoprotein (LDL) binding and cell association by 460% and 200% respectively, and LDL degradation by about 62%. Approximately 49% and 34% of Abeta fibril stimulated LDL cell association and degradation was inhibited by anti-LDL receptor antibodies. The soluble form of Abeta had no effect on any of these measures of LDL metabolism. The observed increased glutathione reductase activity, DNA fragmentation (TUNEL assay) and decreased DNA synthesis ([(3)H] thymidine incorporation assay) in cells treated with Abeta(1-42) fibrils alone or together with LDL relative to controls, suggests that the interaction of fibrils with LDL receptors might be one possible pathway which contributes to fibril cytotoxicity.

Pravastatin down-regulates inflammatory mediators in human monocytes in vitro.

There is experimental evidence that pravastatin, which is designed to inhibit the rate-limiting enzyme of cholesterol synthesis, can affect cell metabolism and proliferation. We therefore studied the effects of pravastatin on the generation of inflammatory mediators in non-stimulated and stimulated primary human monocytes in vitro. In our experimental model, pravastatin induced a dose-dependent inhibition of monocyte cholesterol synthesis (up to 67%), up-regulation of low density lipoprotein receptor mRNA (by about 35%) and reduction in intracellular cholesterol accumulation. In parallel, exposure of non-stimulated monocytes to various doses of pravastatin resulted in inhibition of monocyte chemoattractant protein-1 protein expression (up to 15-fold), reduction of tumour necrosis factor alpha (TNF-alpha) levels (up to 2.4-fold) and a total loss of metalloproteinase-9 activity in stimulated cells. Pravastatin at concentrations of 5, 100 and 500 microM caused an inhibition of TNF-alpha-induced cellular oxygen consumption from 2. 4- to 5.5-fold. These data extend the findings of potential anti-inflammatory actions of statins and also suggest the possibility for pravastatin use in a broader spectrum of inflammatory situations.