Combining germline, tissue and liquid biopsy analysis by comprehensive genomic profiling to improve the yield of actionable variants in a real-world cancer cohort.

BACKGROUND: Comprehensive next-generation sequencing is widely used for precision oncology and precision prevention approaches. We aimed to determine the yield of actionable gene variants, the capacity to uncover hereditary predisposition and liquid biopsy appropriateness instead of, or in addition to, tumor tissue analysis, in a real-world cohort of cancer patients, who may benefit the most from comprehensive genomic profiling. METHODS: Seventy-eight matched germline/tumor tissue/liquid biopsy DNA and RNA samples were profiled using the Hereditary Cancer Panel (germline) and the TruSight Oncology 500 panel (tumor tissue/cfDNA) from 23 patients consecutively enrolled at our center according to at least one of the following criteria: no available therapeutic options; long responding patients potentially fit for other therapies; rare tumor; suspected hereditary cancer; primary cancer with high metastatic potential; tumor of unknown primary origin. Variants were annotated for OncoKB and AMP/ASCO/CAP classification. RESULTS: The overall yield of actionable somatic and germline variants was 57% (13/23 patients), and 43.5%, excluding variants previously identified by somatic or germline routine testing. The accuracy of tumor/cfDNA germline-focused analysis was demonstrated by overlapping results of germline testing. Five germline variants in BRCA1, VHL, CHEK1, ATM genes would have been missed without extended genomic profiling. A previously undetected BRAF p.V600E mutation was emblematic of the clinical utility of this approach in a patient with a liver undifferentiated embryonal sarcoma responsive to BRAF/MEK inhibition. CONCLUSIONS: Our study confirms the clinical relevance of performing extended parallel tumor DNA and cfDNA testing to broaden therapeutic options, to longitudinally monitor cfDNA during patient treatment, and to uncover possible hereditary predisposition following tumor sequencing in patient care.

Chediak-Higashi syndrome: Lessons from a single-centre case series.

BACKGROUND: Chediak-Higashi syndrome (CHS) is a rare and potentially fatal autosomal recessive disease characterized by frequent bacterial infections, bleeding tendency, oculocutaneous albinism, photosensitivity and progressive neurologic dysfunction. Owing to the rarity of this condition, the objective of this study was to describe patients with CHS. METHODS: Retrospective evaluation of patients followed in a paediatric tertiary centre of Allergy and Immunology of São Paulo, Brazil, between 1986 and 2018 with a confirmed diagnosis of CHS. Data were obtained from medical records. Demographic aspects, family history, clinical findings, laboratory data, diagnosis, treatment and outcome were described. RESULTS: A total of 14 patients (five male) were included. Clinical manifestations were first recognized at a median age of two months (at birth-20 months). Median age at diagnosis was 1.7 years (0-5 years). All patients had recurrent infections. Albinism was present in 13 patients and silvery or light hair was present in 14. Seven patients developed hemophagocytic lymphohistiocytosis (HLH); the median age at the diagnosis of HLH was 5.7 years (2.6-6.7 years) and the median interval between the diagnosis of CHS and HLH was 3.3 years (0-5 years). Four of the most recently diagnosed patients underwent bone marrow transplantation (BMT). Nine patients are deceased, and one was lost to follow-up. The median age of death was 6.7 years (3.8-22 years). Five patients died of HLH, one of lymphoma, and three of infection. All the patients who had HLH before the year of 2000 died of HLH. The two most recently diagnosed patients with HLH were able to cure the HLH, although they died of other causes. Four patients are alive, three of them after successful BMT. CONCLUSION: Thirty years of follow up showed an improvement in the prognosis in patients with CHS. The better understanding of the underlying biological mechanisms of HLH allowed the standardization of management protocols, resulting in survival improvement. BMT is the only treatment that can change CHS prognosis, which emphasizes the need for early identification of the disease.

Baked milk tolerant patient: Is there any special feature?

BACKGROUND: Determining whether patients with cow's milk allergy (CMA) can tolerate foods produced with baked milk could provide a better quality of life, a better prognosis, and an option for desensitization. OBJECTIVES: The aim of this study was to identify which patients over four years of age with persistent CMA could tolerate baked milk, to compare the clinical and laboratory characteristics of reactive and non-reactive groups and to describe their clinical evolution. MATERIALS AND METHODS: A cross-sectional study was conducted (January/13 to November/14) that included all the patients followed at a food allergy center who met the inclusion criteria. The patients underwent an oral food challenge (OFC) with a muffin (2.8g of cow's milk protein). To exclude cow's milk (CM) tolerance, the patients were subsequently challenged with unheated CM. RESULTS: Thirty patients met all the inclusion criteria. Fourteen patients (46.7%) were considered non-reactive to baked milk and reactive to unheated CM. When the groups that were reactive and non-reactive to baked milk were compared, no statistically significant differences in clinical features were found. The prick test for α-lactalbumin (p=0.01) and casein (p=0.004) and the serum specific IgE for casein (p=0.05) presented statistical differences. After one year, none of the patients who were reactive to baked milk were ingesting CM, while 28% of the tolerant patients were consuming fresh CM (p=0.037). CONCLUSIONS: Baked milk can be tolerated by patients with CMA, especially those with lower levels of casein and α-lactalbumin. This option can improve quality of life and accelerate tolerance.

Establishing a cut-off for the serum levels of specific IgE to milk and its components for cow's milk allergy: results from a specific population.

BACKGROUND: Cow's milk allergy diagnosis many times requires double-blind placebo-controlled food challenge (DBPCFC), which presents high accuracy but involves risks, specifically in infants and anaphylactic patients. The identification of the cut-off values for specific IgE to milk or its components would contribute to cow's milk allergy (CMA) diagnosis. The aim of this study was to compare discriminating concentration of a cow's milk specific IgE and its fractions (α-lactoalbumin, ß-lactoglobulin, casein) in children for the CMA diagnosis. METHODS: this study included 123 patients (M:F=1.3:1) median age at diagnosis=1.91 years, (3.5m to 13.21y) with CMA diagnosis via DBPCFC (n=26), proven anaphylaxis due to cow's milk (n=46) or a suggestive clinical history associated with a positive skin prick test (n=51) and open oral food challenge. The control group included 61 patients (1 male:1.1 female) ages ranging from 0.66 to 16.7 years (median=6.83 years). Receiver operator characteristics (ROC) curves were constructed to determine the best cut-offs that guarantees high specificity (>95%) for cow's milk and its components. RESULTS: considering 98% specificity, cut-off points were: 3.06 kU/L for cow's milk, 2.06 kU/L for α-lactalbumin, 1.85 kU/L for ß-lactoglobulin and 1.47kU/L for casein. The best ROC curve (area under the curve=0.929) was obtained evaluating cow's milk. CONCLUSION: this study showed that the cut-off point detected for whole cow's milk revealed a better discriminatory capacity for CMA diagnosis without the necessity of the milk components testing.

Curative immune checkpoint inhibitors therapy in patients with mismatch repair-deficient locally advanced rectal cancer: a real-world observational study.

BACKGROUND: Sustained clinical complete remissions were reported in all of 23 mismatch repair deficient/microsatellite instable (dMMR/MSI) locally advanced rectal cancer (LARC) patients treated with dostarlimab alone in a recent phase II study. These results led to off-label use of dostarlimab or other immune checkpoint inhibitors (ICIs) in dMMR/MSI-LARC even before regulatory approval. The present study [STAR(t)-IT-REDUCE] describes the outcome of dMMR/MSI-LARC patients treated with ICI in Italy. MATERIALS AND METHODS: Investigator-initiated, observational, retrospective-cohort, multicentric study of ICI treatment in dMMR/MSI-LARC. Patients were eligible if treated with ≥1 ICI dose from July 2022 to December 2023 (date of approval of dostarlimab for this indication in Italy). RESULTS: Seventeen dMMR/MSI-LARC patients (13 of 17 treatment-naïve) were eligible. Fourteen patients completed 6 months of treatment, two discontinued after four doses and one after five doses because of immune-related pneumonia, social constraints, or non-oncological bowel obstruction, respectively. Overall, 16 of 17 assessable patients [94.1%; 95% confidence interval (CI) 69.24% to 99.69%, 'ITT analysis'] achieved complete clinical response (cCR). Ten of 11 treatment-naïve patients completing 6 months of treatment had cCR (90.9%; 95% CI 57.12% to 99.52%, 'per-protocol analysis'). One patient with near-CR underwent rectal surgery and minimal residual intramucosal tumor was found. With a median follow-up of 9.5 months, no local relapse occurred. One patient developed unconfirmed lung metastases. Two grade 3 and no grade 4 adverse events were reported. CONCLUSION: The present STAR(t)-IT-REDUCE study documents the immunoablative and curative activity of ICI monotherapy in dMMR/MSI-LARC. Toxicity and compliance issues inherent to real-world practice are limited and do not affect achievement of initial complete tumor response but may limit response duration.

Double-blind, placebo-controlled food challenges in Brazilian children: adaptation to clinical practice.

BACKGROUND: A double-blind, placebo-controlled food challenge (DBPCFC) is considered the gold standard for diagnosing food allergy, but because of methodological difficulties it is rarely conducted in clinical practice, especially in paediatric patients. The purpose of the study was to propose a DBPCFC protocol that is adapted to our conditions for the diagnosis of an IgE-mediated cow's milk allergy (CMA) in a Brazilian reference centre for paediatric allergies. METHODS: This study includes the experimental phase (choice of materials, adjustments made to protocols described in the literature) and the test execution phase. DBPCFCs were performed in 58 patients aged 1-15 years who were separated into two groups: Group 1 (n=39), sex 1.6 M:F, 5.3 years median age, suggestive history of IgE-mediated CMA; and Group 2 (n=19), sex 1.4 M:F, 8.3 years median age with symptoms not associated with milk ingestion and laboratory data not compatible with IgE-mediated CMA. RESULTS: The materials were standardised for testing: containers and disposable products, low-lactose cow's milk (CM) and vehicles, such as natural fruit juice, vegetable soup and soybean-based beverages. Each DBPCFC was performed in a single day with two blind, randomised phases with a 2-h interval between them. The milk doses were gradually increased and offered in regular intervals of 15-30 min. Following negative or inconclusive results, patients underwent an open oral challenge test with 200 mL of low-lactose CM. CONCLUSIONS: The proposed adaptation for the DBPCFC allowed to implement this important test for the diagnosis of IgE-mediated CMA in a reference centre for paediatric allergies. It was considered feasible and safe if performed in an appropriate setting with physician supervision.

Common variable immunodeficiency associated with hepatosplenic T-cell lymphoma mimicking juvenile systemic lupus erythematosus.

Common variable immunodeficiency (CVID) is a heterogeneous disorder with susceptibility to infections, autoimmune manifestations, and cancer. To our knowledge, CIVD with T-cell lymphoma mimicking juvenile systemic lupus erythematosus (JSLE) was not described in the literature, and one case was reported herein. An 8-year-old female was admitted in our Pediatric Immunology Unit with a clinical history of hypogammaglobulinemia, recurrent upper respiratory infections, and pneumonias. She had a marked decrease of three serum immunoglobulin isotypes, and the diagnosis of CVID was established. At the age of 17 years, she presented with oral ulceration, nonerosive arthritis, nephritis, serositis, cytopenia, positive antiphospholipid antibodies, and positive antinuclear antibody fulfilling the American College of Rheumatology (ACR) criteria for SLE. She was treated with intravenous methylprednisolone for three consecutive days, and intravenous immunoglobulin, and maintenance therapy of chloroquine, azathioprine and prednisone 40 mg/day. Two months later, she died of septic shock secondary to acute pneumonia. The necropsy showed hepatosplenic T-cell lymphoma with diffuse involvement of bone marrow, spleen, liver, and lungs. The lymphoma cells were positive for CD3 immunostaining and negative for CD20 and lysozyme. In conclusion, the association of CVID and hepatosplenic T-cell lymphoma may simulate JSLE diagnosis.

Molecular characterization of patients with X-linked Hyper-IgM syndrome: description of two novel CD40L mutations.

Type 1, X-linked Hyper-IgM syndrome (HIGM1) is caused by mutations in the gene encoding the CD154 protein, also known as CD40 ligand (CD40LG). CD40L is expressed in activated T cells and interacts with CD40 receptor expressed on B lymphocytes and dendritic cells. Affected patients present cellular and humoral immune defects, with infections by intracellular, opportunistic and extracellular pathogens. In the present study we investigated the molecular defects underlying disease in four patients with HIGM1. We identified four distinct CD40L mutations, two of them which have not been previously described. P1 harboured the novel p.G227X mutation which abolished CD40L expression. P2 had a previously described frame shift deletion in exon 2 (p.I53fsX65) which also prevented protein expression. P3 demonstrated the previously known p.V126D change in exon 4, affecting the TNF homology (TNFH) domain. Finally, P4 evidenced the novel p.F229L mutation also located in the TNFH domain. In silico analysis of F229L predicted the change to be pathological, affecting the many hydrophobic interactions of this residue. Precise molecular diagnosis in HIGM syndrome allows reliable detection of carriers, making genetic counselling and prenatal diagnosis possible.

Prevalence of symptoms of asthma, rhinitis, and atopic eczema in Brazilian adolescents related to exposure to gaseous air pollutants and socioeconomic status.

OBJECTIVES: To evaluate the relationship between exposure to gaseous air pollutants (ozone [O3], carbon monoxide [CO], nitrogen dioxide [NO2], and sulfur dioxide [SO2]) socioeconomic status and the prevalence of symptoms of asthma, rhinitis and atopic eczema in adolescents. SUBJECTS AND METHODS: A sample of 16 209 adolescents from São Paulo West (SPW), São Paulo South (SPS), Santo André (SA), Curitiba (CR), and Porto Alegre (PoA) were enrolled. Data on air pollutants and socioeconomic status were compared to prevalence of symptoms with the Spearman correlation coefficient. RESULTS: Socioeconomic status was quite similar in all cities. The levels of O3 in SPW, SPS, and SA, and of CO in SA were higher than the acceptable ones. In relation to O3 and CO exposures, adolescents from SPW and SA had a significant risk of current wheezing, whereas living in SPW was associated with a high risk of rhinoconjunctivitis, eczema, and flexural eczema and living in CR to rhinitis. Exposure to NO2 was associated with a high risk of current wheezing in SPW and SA, and of severe asthma in SPW and PoA. Exposure to SO2 was associated with a high risk of current wheezing in SPW and SA, severe asthma in SPW and PoA, and nighttime cough, eczema, flexural eczema and severe eczema in SPW. Living in SPW, CR, or PoA was associated with a high risk of rhinitis, rhinoconjunctivitis, and severe rhinitis. CONCLUSIONS: Although we did not detect a characteristic pattern for all symptoms evaluated or a specific air pollutant, our data suggest a relationship between higher exposure to photochemical pollutants and high prevalence or risk of symptoms of asthma, rhinitis, and atopic eczema.

Prevalence of asthma, rhinitis and eczema in 6 - 7 years old students from the western districts of São Paulo City, using the standardized questionnaire of the "International Study of Asthma and Allergies in Childhood" (ISAAC)-phase IIIB.

UNLABELLED: The aims of the present work were the evaluation of allergic disease prevalence among 6 and 7 year-old students from the western districts of São Paulo city and the comparison of these data with those obtained in the International Study of Asthma and Allergies in Childhood (ISAAC) phase I, performed in the central-southern districts of São Paulo, using the ISAAC standardized written questionnaire. METHODS: 5,040 questionnaires were distributed and 3,312 were returned. Proportional differences were estimated by Chi square or Fisher exact tests. Odds Ratio and 95% confidence intervals between genders and allergic diseases were calculated. Values of p<0.05 were considered statistically significant. RESULTS: The corrected prevalences found were: asthma 24.4%, medical diagnosis of asthma 5.7%, rhinitis 25.7%, rhinoconjunctivitis 11.3%, medical diagnosis of rhinitis 20.0%, atopic eczema 9.2%. Significant associations between asthma and rhinitis (OR=3.3), asthma and eczema (OR=2.2), and rhinitis and eczema (OR=2.8) occurred. The male gender was prevalent regarding asthma and rhinitis. Compared to data from ISAAC phase I, higher asthma prevalence and severity, and lower values for rhinitis and eczema were observed in this study. CONCLUSIONS: The present study evidenced high prevalences for asthma and rhinitis compared to the children's medical diagnosis. The male gender predominated in all positive responses regarding asthma and rhinitis. The most frequent associations observed were between asthma and rhinitis and asthma and eczema. In the western districts of São Paulo, a higher prevalence of asthma symptoms and severity and lower prevalences for rhinitis and eczema occurred compared to the central-southern districts of the city.

Thyroid nodules with indeterminate cytology: prospective comparison between 18F-FDG-PET/CT, multiparametric neck ultrasonography, 99mTc-MIBI scintigraphy and histology.

PURPOSE: To evaluate the role of (18)F-flurodeoxiglucose positron emission tomography/computed tomography ((18)F-FDG-PET/CT) in predicting malignancy of thyroid nodules with indeterminate cytology. PATIENTS AND METHODS: We analysed 87 patients who have been scheduled to undergo surgery for thyroid nodule with indeterminate cytology. All patients underwent (18)F-FDG-PET/CT, multiparametric neck ultrasonography (MPUS), and (99m)Tc-methoxyisobutylisonitrile scintigraphy ((99m)Tc-MIBI-scan). Histopathology was the standard of reference. We compared the sensitivity (SE), specificity (SP), accuracy (AC), positive (PPV) and negative predictive (NPV) values of (18)F-FDG-PET/CT with those of (99m)Tc-MIBI-scan and MPUS in detecting cancer. Univariate and multivariate analyses evaluated the association between each diagnostic tool and histopathology. RESULTS: On histopathology, 69 out of 87 nodules were found to be benign and 18 to be malignant. The SE, SP, AC, PPV and NPV of (18)F-FDG-PET/CT were 94, 58, 66, 37 and 98% respectively. The SE, AC and NPV of (18)F-FDG-PET/CT were significantly higher than those of MPUS and (99m)Tc-MIBI-scan. The association of both positive (18)F-FDG-PET/CT and MPUS (FDG+/MPUS+) showed significantly lower SE (61% vs 94%) and NPV (88% vs 98%) than (18)F-FDG-PET/CT alone, but significantly higher SP (77% vs 58%). On univariate analysis, (18)F-FDG-PET/CT and the combination of FDG+/MPUS+ and of FDG+/MIBI- were all significantly associated with histopathology. On multivariate analysis, only FDG+/MIBI- was significantly associated with histopathology. CONCLUSION: The AC of (18)F-FDG-PET /CT in detecting thyroid malignancy is higher than that of (99m)Tc-MIBI-scan and MPUS. A negative (18)F-FDG-PET/CT correctly predicts benign findings on histopathology. The association of FDG+/MPS+ is significantly more specific than (18)F-FDG-PET/CT alone in identifying differentiated thyroid cancer. A positive (18)F-FDG-PET/CT is significantly associated with malignancy when qualitative (99m)Tc-MIBI-scan is rated as negative.

Measurement of lipid hydroperoxides in plasma lipoproteins by a new highly-sensitive 'single photon counting' luminometer.

The lipid hydroperoxide content of isolated, native human plasma lipoproteins, was measured, by the luminol-based chemiluminescent reaction, using a highly sensitive single photon counting instrument. The reaction was specific for lipid hydroperoxides since the signal completely disappeared after treatment with the selenoperoxidase specific for lipidic substrates. In this analytical procedure the whole kinetic of photon emission induced by lipid hydroperoxides and hemin in the presence of luminol is integrated, taking advantage of the mono-exponential fitting of the decay of photon emission. The addition of a detergent to the reaction mixture improved the precision of the measurements apparently by preventing oxidative chain reactions affecting the shape of the decay of photon emission. The sensitivity of the instrument allowed measurements on samples containing just a few picomoles of hydroperoxides, small enough to minimize the effect of antioxidants and quenchers possibly present in the sample (as in the case of lipoproteins). Thus, by using an internal calibration with a phospholipid hydroperoxide, the evaluation of the lipid hydroperoxide content in whole, native lipoproteins was possible without previous extraction and chromatographic separation. Data obtained from plasma lipoproteins isolated by different procedures suggest that lipid hydroperoxide content increases during ultracentrifugation.

Determination of lipid hydroperoxides in native low-density lipoprotein by a chemiluminescent flow-injection assay.

Lipid hydroperoxides have been implicated in the pathogenesis of atherosclerosis. This work was therefore set up to obtain a fast and specific chemiluminescent assay for measuring hydroperoxides in native low-density lipoprotein (LDL). The apparatus was a complete HPLC system including two pumps, an autosampler, a computer and a chemiluminescent detector with a T-mixing coil in the place of the column. Samples were injected from the autosampler and mixed with luminescent reagent (3 microM luminol and 1 microM microperoxidase in 0.1 M carbonate buffer (pH 10)) in the T-piece. To generate a calibration curve, linoleic acid hydroperoxide was obtained by incubating soybean lipoxygenase with linoleic acid. The calculated conjugated diene concentration was in good agreement with the nominal linoleic acid hydroperoxide concentration. The chemiluminescence was linear with the amount of linoleic acid hydroperoxide injected and the detection limit was about 3 pmol linoleic acid hydroperoxide. The chemiluminescence induced by copper-oxidized LDL was linear with concentration; the detection limit, when compared with linoleic acid hydroperoxide, was similar. The reproducibility of the linoleic acid hydroperoxide and of oxidized LDL hydroperoxide was examined in single pools. The coefficient of variation on the triplicates of each pool was about 3%. The titre of the linoleic acid hydroperoxide and oxidized LDL peroxides was quite stable for at least 10 days when stored under argon at 4 degrees C in the presence of EDTA. The mean value of the LDL hydroperoxides in 16 control subjects was 145.20 +/- 98.81 pmol/mg LDL protein. In conclusion, the microperoxidase-luminol-dependent chemiluminescence flow-injection assay is a rapid, sensitive and selective method for measuring lipid hydroperoxides in native LDL.

Kinetic analysis of lipid-hydroperoxides in plasma.

We increased the precision of chemiluminescent procedure for measuring lipid hydroperoxides in plasma or lipoproteins by (i) escaping from extraction and chromatography of lipids, (ii) using detergent dispersed lipids, and (iii) calculating the results by fitting the photon emission rate with the integrated equation, which describes the model of the series of reactions. The use of kinetics instead of the crude integration of cps increases precision because at each measurement the correct reaction pathway is tested. This was relevant for the optimization of the analytical procedure, contributing to the elimination of possible side reactions. The relationship between lipid hydroperoxide content in the sample and cps is not linear; thus, the calculation of results through internal calibration is carried out using an exponential equation. This is in agreement with the reaction mechanism and raises the point of the linear calibration previously reported in other chemiluminescent procedures. Although sensitive and precise, this procedure suffers for being time consuming, requiring approximately 30 min per sample. Moreover, since no chromatography is used, information about the hydroperoxides in different lipid classes is missing. Obviously this will be solved when a validated procedure for quantitatively extracting lipid hydroperoxides is available.

Effect of plasma on the degradation of hydroperoxides of unesterified linoleic acid and copper-peroxidized LDL.

The determination of lipid hydroperoxides in plasma and lipoproteins recently reached a clinical relevance in disorders such as atherosclerosis, where oxidative reactions have been suggested to play a fundamental pathogenetic role. The peroxide content of lipoproteins is usually measured after ultracentrifugation and extraction. During this procedure, some peroxides might decompose causing a too low recovery. To screen this possibility, the disappearance, in the presence of human plasma, of hydroperoxides of linoleic acid and Cu-oxidized low density lipoprotein (LDL) have been investigated, using both a iodometric titration and an enzymatic assay. While only in the presence of GSH plasma decomposes linoleic acid hydroperoxides quite rapidly, peroxides in Cu-oxidized LDL were stable both in presence as well as in absence of GSH. This indicated that lipid hydroperoxides are stable in plasma and that peroxides of Cu-oxidized LDL are not substrate for the glutathione-dependent peroxidase activity in plasma. The relevant decrease of the iodometric titre of LDL peroxides observed in the presence of elevated amounts of plasma was shown to be artifactual, since some compounds extracted from plasma do react with iodine generated by peroxides. Whole plasma itself, indeed, has been shown to reduce back to I- appreciable amount of free iodine.

Antioxidants inhibit the expression of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 induced by oxidized LDL on human umbilical vein endothelial cells.

The oxidative modification of low density lipoprotein (LDL) and the endothelial expression of adhesion molecules are key events in the pathogenesis of atherosclerosis. In this study we evaluated the effect of oxidized LDL on the expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on human umbilical vein endothelial cells (HUVECs). The hypothesis that oxidized LDL functions as a prooxidant signal was also evaluated, by studying the effect of different radical-scavenging antioxidants on expression of adhesion molecules. LDL was oxidized by using Cu2+, HUVECs or phospholipase A2 (PLA2)/ soybean lipoxygenase (SLO), the degree of oxidation being measured as thiobarbituric acid-reactive substances (TBARS) and conjugated dienes (CD). Exposure of 200 micrograms/ml of native LDL to 1 microns Cu2+, HUVECs and to PLA2/ SLO resulted in four- to fivefold higher levels of TBARS and CD than in native LDL. Cu(2+)-(1 microM), HUVEC-, and PLA2/SLO-oxidized LDL caused a dose-dependent, significant increase of ICAM-1 and VCAM-1 (p < .01). The expression of E-selectin did not change. LDL oxidized with a 2.5 and 5 microM Cu2+ did not increase ICAM-1 and VCAM-1 significantly. Both the Cu(2+)- and HUVEC-oxidized LDL, subjected to dialysis and ultrafiltration, induced ICAM-1 and VCAM-1 expression. After incubation with the ultrafiltrate, the expression of ICAM-1 and VCAM-1 was not significantly different from that obtained with native LDL. LDL pretreated with different antioxidants (vitamin E and probucol) and subjected to oxidation by Cu2+ and HUVECs induced a significantly lower expression of ICAM-1 and VCAM-1 than nonloaded LDL (p < .01). The pretreatment of HUVECs with vitamin E and probucol significantly reduced the expression of VCAM-1 on HUVECs induced by oxidized LDL (p < .01); the effect on ICAM-1 was much less evident. In conclusion, oxidized LDL can induce the expression of different adhesion molecules on HUVECs; this induction can be prevented by pretreating either the LDL or the cells with radical-scavenging antioxidant.

Predisposition to LDL oxidation in patients with and without angiographically established coronary artery disease.

Oxidative modification of low density lipoprotein (LDL) may play an important role in the mechanism of atherosclerotic damage to blood vessels. In the present study the LDL isolated from the plasmas of 73 coronary artery disease (CAD) patients, 28 valvular heart disease (VHD) patients, 59 subjects affected by type IIa hyperlipoproteinemia and 71 controls was oxidatively modified by incubation with copper ions. In 15 CAD and 15 Type IIa patients and 15 controls the LDL chemical composition and polyunsaturated fatty acid (PUFA) content were also measured. Differences in the LDL susceptibilities to lipid peroxidation were studied by measuring the changes of fluorescence intensity. The lag phase in the CAD patients was found to be significantly lower than in the VHD and controls (P < 0.001). The lag phase in the type IIa patients was significantly higher than in the CAD patients (P < 0.01), and significantly lower than the VHD and controls (P < 0.01). The LDL isolated from the type IIa patients had an increase in the relative content of free and esterified cholesterol (P < 0.05), while the CAD patients had a decrease in the relative content of free cholesterol (P < 0.05), and an increase in the relative content of protein (P < 0.05). The lowest value of the LDL cholesterol to protein ratio and LDL size, was found in the CAD patients (P < 0.05). When expressed in micrograms/mg LDL cholesterol, the concentration of the LDL PUFAs was significantly higher in the CAD group than in the others (P < 0.05). The LDL alpha-tocopherol concentration was quite similar in the different groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative effects of different dihydropyridines on the expression of adhesion molecules induced by TNF-alpha on endothelial cells.

OBJECTIVE: Lacidipine has already been demonstrated to reduce the expression of some adhesion molecules induced by pro-oxidant signals on endothelial cells. In order to verify if this effect is a peculiarity of this molecule, or belongs to other dihydropyridinic compounds (DHPs), the activity of lacidipine was compared with that of lercanidipine, amlodipine, nimodipine and nifedipine. DESIGN AND METHODS: The compounds were incorporated in human umbilical vein endothelial cells (HUVECs) using native low-density lipoprotein as a carrier. The drug concentrations in HUVECs were measured by mass spectrometry. Human recombinant tumour necrosis factor-alpha was then incubated with HUVECs for 7 h at 37 degrees C for adhesion molecule expression. RESULTS: The cellular amount of lacidipine, lercanidipine and amlodipine was similar, while nimodipine and nifedipine were almost undetectable or undetectable, respectively. Lacidipine, at any concentration, determined a dose-dependent significant decrease of the expression of intercellular adhesion molecule-1 (ICAM-1) ICAM-1, vascular cell adhesion molecule-1 (VCAM-1) VCAM-1 and E-selectin (P < 0.01). Lercanidipine and amlodipine determined variable decreases of adhesion molecules at the intermediate and highest concentrations. Nimodipine and nifedipine determined no effect on ICAM-1, VCAM-1 and E-selectin. The lowest IC50, i.e. the concentration determining the 50% reduction of ICAM-1, VCAM-1 and E-selectin expression was obtained with lacidipine for all the adhesion molecules considered (P < 0.01). CONCLUSIONS: It is concluded that the effect of the DHPs used in this study on adhesion molecule expression is determined first by their lipophilicity and then by their intrinsic antioxidant activity.

Oxidized low-density lipoprotein increases the production of intracellular reactive oxygen species in endothelial cells: inhibitory effect of lacidipine.

OBJECTIVE: The mechanisms by which oxidized low-density lipoprotein (ox-LDL) induces the expression of adhesion molecules on endothelial cells (HUVECs) are still not clear. The signal transduction pathways for these binding molecules include the translocation of the transcription factor NF-kB and the intracellular reactive oxygen species (ROS) are said to play a key role in this process. Aim of this study was (1) to evaluate the effect of ox-LDL on intracellular production of ROS in culture of HUVECs; (2) to evaluate if the intracellular increase of ROS induced by ox-LDL is mediated by the binding to a specific endothelial receptor; (3) to ascertain if lacidipine can decrease ox-LDL-induced ROS production in HUVECs. METHODS: Five microM Cu2+ ox-LDL were incubated with HUVECs for 5 min. 2',7'-Dichlorofluorescein (DCF) as an expression of intracellular ROS production, was measured by flow cytometry. RESULTS: ox-LDL induced a significant dose-dependent increase in DCF production (P < 0.001) through the binding to a specific receptor. The preincubation of HUVECs with radical scavengers compounds and lacidipine significantly reduced (P < 0.001) the ox-LDL-induced DCF production. CONCLUSIONS: ox-LDL increased the intracellular formation of ROS through the ligation to a specific endothelial receptor. Preincubation of HUVECs with lacidipine, a calcium antagonist with antioxidant properties, significantly reduced the intracellular ROS formation induced by ox-LDL. We propose that the effect of lacidipine on adhesion molecule expression and on NF-kB activation can be explained by its effect on intracellular ROS formation.