Leukotriene B(4)-loaded microspheres: a new therapeutic strategy to modulate cell activation

Background: Leukotriene B(4) (LTB(4)) is a potent inflammatory mediator that also stimulates the immune response. In addition, it promotes polymorphonuclear leukocyte phagocytosis, chemotaxis, chemokinesis and modulates cytokines release. Regarding chemical instability of the leukotriene molecule, in the present study we assessed the immunomodulatory activities conferred by LTB(4) released from microspheres (MS). A previous oil-in-water emulsion solvent extraction-evaporation method was chosen to prepare LTB(4)-loaded MS. Results: In the mice cremasteric microcirculation, intraescrotal injection of 0.1 ml of LTB(4)-loaded MS provoked significant increases in leukocyte rolling flux, adhesion and emigration besides significant decreases in the leukocyte rolling velocity. LTB(4)-loaded MS also increase peroxisome proliferator-activated receptor-alpha (PPAR alpha) expression by murine peritoneal macrophages and stimulate them to generate nitrite levels. Monocyte chemoattractant protein-I (MCP-I) and nitric oxide (NO) productions were also increased when human umbilical vein and artery endothelial cells (HUVECs and HUAECs, respectively) were stimulated with LTB(4)-loaded MS. Conclusion: LTB(4)-loaded MS preserve the biological activity of the encapsulated mediator indicating their use as a new strategy to modulate cell activation, especially in the innate immune response.

Manipulating avocado fruit ripening with 1-methylcyclopropene

Previous investigations with 1-methylcyclopropene (1-MCP) on avocado (Persea americana Mill.) fruit have focussed mainly on improving storage life by reducing the severity of disorders causing discolouration of the flesh. Development of 1-MCP and ethylene treatments, which also help control the time to reach the eating ripe stage, may confer additional practical benefits. In this context, the current study investigated the potential of 1-MCP to accurately manipulate ripening of non-stored 'Hass' avocado fruit by treatment before or after ethylene and at different times during ripening. To investigate this, 500 nL L-1 1-MCP was applied within 1 day after harvest, followed by ethylene 0-14 days after 1-MCP. In addition, fruit were treated with ethylene, then 1-MCP 0-8 days after ethylene. Treatment of fruit with 500 nL L-1 1-MCP for 18 h at 20 degreesC provided the maximum effect by increasing the days from harvest to ripe (DTR) from 8 (with no 1-MCP) to 20. Fruit treated with 500 nL L-1 1-MCP for 18 h at 20 degreesC remained insensitive to 100 muL L-1 ethylene applied between 0 and 14 days after 1-MCP for 24 h at 20 degreesC. Ripening of fruit exposed to 100 muL L-1 ethylene for 24 h at 20 degreesC could be delayed by up to 3.3 days by applying 500 nL L-1 1-MCP for 18 h at 20 degreesC up to 2 days after ethylene treatment. However, once the fruit started to soften (sprung) there was little effect of 1-MCP on DTR, compared with no 1-MCP. 1-MCP treatment was associated with increased severity of body rots (caused mainly by Colletotrichum spp.) and stem-end rots (caused mainly by Dothiorella spp.), which was likely due to the increased DTR in these treatments. Significant differences in disease severity were found between orchards (replications), with replicates with low disease severity being less affected by 1-MCP treatment. These results indicate that 1-MCP can delay ripening, but careful sourcing of fruit is required to reduce the risk of diseases in ripe fruit. There is some capacity to delay ripening using 1-MCP after ethylene. There is little potential to control ripening using ethylene after treatment with 500 nL L-1 1-1-MCP, but lower concentrations may be more effective. (C) 2004 Elsevier B.V. All rights reserved.

Association of the MCP-1-2518 A/G Polymorphism and No Association of Its Receptor CCR2-64 V/I Polymorphism with Lupus Nephritis

Objective. To evaluate whether the A/G polymorphism at position 2518 in the regulatory region of the monocyte chemoattractant protein-1 (MCP-1) or the V/I polymorphism at position 64 of the receptor. CCR2, are associated with lupus nephritis (LN) or any clinical characteristics of the disease or with renal survival in a patient population. Methods. We selected 197 patients with lupus nephritis and 220 matched healthy controls for study. MCP-1 and CCR2 genotyping was performed by polymerase chain reaction. Clinical and laboratory data were compiled from patients` charts over followup that ranged from 6 months to 10 years. Results. The GIG genotype of MCP-1 was more common in LN patients (p = 0.019), while the A allele was associated with healthy controls (p = 0.007) as was the V allele of CCR2 (p = 0.046) compared to LN patients. Clinical index measures [SLE Disease Activity Index (SLEDAI)], immunological markers, renal histology, renal function at enrollment, and renal survival were not influenced by these polymorphisms. A less aggressive renal disease, measured by renal SLEDAI index, was associated with the V allele of the CCR2 gene polymorphism. Conclusion. These findings support that MCP-1 2518 GIG is associated with LN but there was no association of this genotype with renal function or renal survival. When studying CCR2 64 V/I polymorphism we showed a positive association of the V allele with healthy controls but no association of the genotype with LN patients. (First Release March 152010; J Rheumatol 2010;37:776-82; doi:10.3899/jrheum.090681)

Genetic polymorphisms of RANTES, IL1-A, MCP-1 and TNF-A genes in patients with prostate cancer

BACKGROUND Inflammation has been implicated as an etiological factor in several human cancers, including prostate cancer. Allelic variants of the genes involved in inflammatory pathways are logical candidates as genetic determinants of prostate cancer risk. The purpose of this study was to investigate whether single nucleotide polymorphisms of genes that lead to increased levels of pro-inflammatory cytokines and chemokines are associated with an increased prostate cancer risk. METHODS A case-control study design was used to test the association between prostate cancer risk and the polymorphisms TNF-A-308 A/G (rs 1800629), RANTES-403 G/A (rs 2107538), IL1-A-889 C/T (rs 1800587) and MCP-1 2518 G/A (rs 1024611) in 296 patients diagnosed with prostate cancer and in 311 healthy controls from the same area. RESULTS Diagnosis of prostate cancer was significantly associated with TNF-A GA + AA genotype (OR, 1.61; 95% CI, 1.09-2.64) and RANTES GA + AA genotype (OR, 1.44; 95% CI, 1.09-2.38). A alleles in TNF-A and RANTES influenced prostate cancer susceptibility and acted independently of each other in these subjects. No epistatic effect was found for the combination of different polymorphisms studied. Finally, no overall association was found between prostate cancer risk and IL1-A or MCP-1 polymorphisms. CONCLUSION Our results and previously published findings on genes associated with innate immunity support the hypothesis that polymorphisms in proinflammatory genes may be important in prostate cancer development.

1- metilciclopropeno (1-mcp) e atmosfera modificada em manga ‘Tommy Atkins’ e quantificação de antioxidantes em frutas e hortaliças

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Induktion und Signaltransduktion bei der Expression des Chemokins MCP-1 in Monozyten

Das Chemokin 'Monocyte Chemoattractant Protein-1' (MCP-1) spielt bei inflammatorischen Erkrankungen eine wesentliche Rolle. Verschiedene Zelltypen produzieren MCP-1. Es interessierte, welche Stimuli in Monozyten MCP-1 induzieren können und welche Signaltransduktionskaskaden daran beteiligt sind. Darüber hinaus sollte die Rolle einzelner Transkriptionsfaktoren und Promotorregionen des MCP-1-Gens untersucht werden.Komponenten Gram-positiver und -negativer Bakterien, Phorbolester und Substanzen, die die intrazelluläre Calciumkonzentration erhöhen, induzierten die MCP-1-Expression in einer humanen myelomonozytären Zellinie (THP-1) und in frisch isolierten Monozyten. Die mit Lipopolysaccharid (LPS)-induzierte MCP-1-Expression war stark von der MAPK/ERK-Kinase (MEK)-1/-2 und von I-kappaB Kinasen beziehungsweise NF-kappaB abhängig, dagegen scheinen Calcineurin, Calmodulinkinasen und die 'Mitogen-Activated Protein Kinase' p38 keine entscheidende Rolle zu spielen. Die Thapsigargin (TG)-induzierte MCP-1-Bildung durch Erhöhung der intrazellulären Calciumkonzentration war zusätzlich von Calcineurin und Calmodulinkinasen abhängig. Als nukleäre Transkriptionsfaktoren wurden bei der LPS-Stimulation NF-kappaB sowie AP-1 und zusätzlich NF-ATc3 bei Stimulation durch TG nachgewiesen. Die Untersuchung des MCP-1-Promotors konnte eine Bindung von NF-kappaB- und AP-1-Mitglieder an eine bislang nicht untersuchte distale Region und eine AP-1-Bindung an eine proximale Region nachweisen. Die Ergebnisse lassen den Schluß zu, daß die Aktivierung der MCP-1-Expression durch verschiedene Stimuli unter Beteiligung teilweise unterschiedlicher Signaltransduktionswege abläuft und sowohl eine proximale als auch eine distale Promotorregion des MCP-1-Gens daran beteiligt ist.

Caratterizzazione di un rivelatore MCP-PMT per misure di risoluzione temporale e spaziale.

Questa tesi descrive una prima serie di misure effettuate per caratterizzare le prestazioni di un rivelatore di fotoni MCP-PMT a multi anodo, con particolare enfasi sulla risoluzione temporale e spaziale. I risultati sono stati confrontati con quelli relativi a tre ulteriori rivelatori (un MCP-PMT a singolo anodo e due fotomoltiplicatori a silicio). Le misure sono state effettuate presso i Laboratori dell’Istituto Nazionale di Fisica Nucleare (INFN - Sezione di Bologna) per conto del Dipartimento di Fisica dell’Università di Bologna. La rivelazione della luce ha sempre occupato un posto fondamentale nella Fisica Nucleare e Subnucleare. Il principio base della rivelazione consiste nel trasformare la luce incidente in un segnale elettrico misurabile e i primi strumenti storicamente utilizzati furono i fotomoltiplicatori. Successivamente furono introdotti dei nuovi rivelatori di fotoni chiamati Micro Channel Plates, composti da un array di canali di dimensioni microscopiche, ciascuno in grado di moltiplicare gli elettroni prodotti per effetto fotoelettrico dai fotoni incidenti. Questo nuovo modello presenta ottime prestazioni in particolare per quanto riguarda la risoluzione temporale, e questa insieme ad altre caratteristiche (come la segmentazione degli anodi, che permette una risoluzione spaziale migliore), ha spinto a studiarne il funzionamento.

Efeito do 1-MCP no perfil de transcritos diferencias de genes da parede celular, e fotossíntese em maçã cv. Gala armazenada em atmosfera controlada.

Maçãs cv. Gala são conhecidas por apresentarem bom potencial de armazenamento em atmosfera controlada (AC) associada a refrigeração. Condições de 1% de O2; 2% de CO2; 0°C ± 0,5; 90% UR ± 5% são relatadas por preservarem a firmeza de polpa por até nove meses. Entretanto, logo após a saída da AC, uma perda da firmeza é observada, resultando em perdas na qualidade dos frutos durante o transporte e comercialização. Desse modo, o 1-metilciclopropeno (1-MCP) é utilizado com sucesso em conjunto com a AC, por ser capaz de manter a firmeza de polpa de maçãs mesmo após a saída do armazenamento. O objetivo do trabalho foi elucidar mecanismos pelo qual o 1-MCP preserva a firmeza de polpa da maçã armazenada em AC.

Efeito do 1-MCP no desenvolvimento de podridões em maçã.

O objetivo deste trabalho foi avaliar o efeito do 1-MCP em maçãs MaxiGala expostas a diferentes atmosferas (ATM), no progresso da doença ocasionada por Botrytis cinerea e Cryptosporiopsis brasiliensis.

An Isoflavone From Dipteryx Alata Vogel Is Active Against The In Vitro Neuromuscular Paralysis Of Bothrops Jararacussu Snake Venom And Bothropstoxin I, And Prevents Venom-induced Myonecrosis.

Snakebite is a neglected disease and serious health problem in Brazil, with most bites being caused by snakes of the genus Bothrops. Although serum therapy is the primary treatment for systemic envenomation, it is generally ineffective in neutralizing the local effects of these venoms. In this work, we examined the ability of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM), an isoflavone from Dipteryx alata, to neutralize the neurotoxicity (in mouse phrenic nerve-diaphragm preparations) and myotoxicity (assessed by light microscopy) of Bothrops jararacussu snake venom in vitro. The toxicity of TM was assessed using the Salmonella microsome assay (Ames test). Incubation with TM alone (200 μg/mL) did not alter the muscle twitch tension whereas incubation with venom (40 μg/mL) caused irreversible paralysis. Preincubation of TM (200 μg/mL) with venom attenuated the venom-induced neuromuscular blockade by 84% ± 5% (mean ± SEM; n = 4). The neuromuscular blockade caused by bothropstoxin-I (BthTX-I), the major myotoxic PLA2 of this venom, was also attenuated by TM. Histological analysis of diaphragm muscle incubated with TM showed that most fibers were preserved (only 9.2% ± 1.7% were damaged; n = 4) compared to venom alone (50.3% ± 5.4% of fibers damaged; n = 3), and preincubation of TM with venom significantly attenuated the venom-induced damage (only 17% ± 3.4% of fibers damaged; n = 3; p < 0.05 compared to venom alone). TM showed no mutagenicity in the Ames test using Salmonella strains TA98 and TA97a with (+S9) and without (-S9) metabolic activation. These findings indicate that TM is a potentially useful compound for antagonizing the neuromuscular effects (neurotoxicity and myotoxicity) of B. jararacussu venom.

Quantitative Proteomic Analysis Reveals Metabolic Alterations, Calcium Dysregulation, And Increased Expression Of Extracellular Matrix Proteins In Laminin α2 Chain-deficient Muscle.

Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain-deficient dy(3K)/dy(3K) mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain-deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978).

Nebivolol Reduces Central Blood Pressure In Stage I Hypertensive Patients: Experimental Single Cohort Study.

Assessment of central blood pressure (BP) has grown substantially over recent years because evidence has shown that central BP is more relevant to cardiovascular outcomes than peripheral BP. Thus, different classes of antihypertensive drugs have different effects on central BP despite similar reductions in brachial BP. The aim of this study was to investigate the effect of nebivolol, a β-blocker with vasodilator properties, on the biochemical and hemodynamic parameters of hypertensive patients. Experimental single cohort study conducted in the outpatient clinic of a university hospital. Twenty-six patients were recruited. All of them underwent biochemical and hemodynamic evaluation (BP, heart rate (HR), central BP and augmentation index) before and after 3 months of using nebivolol. 88.5% of the patients were male; their mean age was 49.7 ± 9.3 years and most of them were overweight (29.6 ± 3.1 kg/m2) with large abdominal waist (102.1 ± 7.2 cm). There were significant decreases in peripheral systolic BP (P = 0.0020), diastolic BP (P = 0.0049), HR (P < 0.0001) and central BP (129.9 ± 12.3 versus 122.3 ± 10.3 mmHg; P = 0.0083) after treatment, in comparison with the baseline values. There was no statistical difference in the augmentation index or in the biochemical parameters, from before to after the treatment. Nebivolol use seems to be associated with significant reduction of central BP in stage I hypertensive patients, in addition to reductions in brachial systolic and diastolic BP.

Predictive Value Of Phase I Trials For Safety In Later Trials And Final Approved Dose: Analysis Of 61 Approved Cancer Drugs.

Phase I trials use a small number of patients to define a maximum tolerated dose (MTD) and the safety of new agents. We compared data from phase I and registration trials to determine whether early trials predicted later safety and final dose. We searched the U.S. Food and Drug Administration (FDA) website for drugs approved in nonpediatric cancers (January 1990-October 2012). The recommended phase II dose (R2PD) and toxicities from phase I were compared with doses and safety in later trials. In 62 of 85 (73%) matched trials, the dose from the later trial was within 20% of the RP2D. In a multivariable analysis, phase I trials of targeted agents were less predictive of the final approved dose (OR, 0.2 for adopting ± 20% of the RP2D for targeted vs. other classes; P = 0.025). Of the 530 clinically relevant toxicities in later trials, 70% (n = 374) were described in phase I. A significant relationship (P = 0.0032) between increasing the number of patients in phase I (up to 60) and the ability to describe future clinically relevant toxicities was observed. Among 28,505 patients in later trials, the death rate that was related to drug was 1.41%. In conclusion, dosing based on phase I trials was associated with a low toxicity-related death rate in later trials. The ability to predict relevant toxicities correlates with the number of patients on the initial phase I trial. The final dose approved was within 20% of the RP2D in 73% of assessed trials.

Pyrimidine-5'-nucleotidase Campinas, A New Mutation (p.r56g) In The Nt5c3 Gene Associated With Pyrimidine-5'-nucleotidase Type I Deficiency And Influence Of Gilbert's Syndrome On Clinical Expression.

Pyrimidine-5'-nucleotidase type I (P5'NI) deficiency is an autosomal recessive condition that causes nonspherocytic hemolytic anemia, characterized by marked basophilic stippling and pyrimidine nucleotide accumulation in erythrocytes. We herein present two African descendant patients, father and daughter, with P5'N deficiency, both born from first cousins. Investigation of the promoter polymorphism of the uridine diphospho glucuronosyl transferase 1A (UGT1A) gene revealed that the father was homozygous for the allele (TA7) and the daughter heterozygous (TA6/TA7). P5'NI gene (NT5C3) gene sequencing revealed a further change in homozygosity at amino acid position 56 (p.R56G), located in a highly conserved region. Both patients developed gallstones; however the father, who had undergone surgery for the removal of stones, had extremely severe intrahepatic cholestasis and, liver biopsy revealed fibrosis and siderosis grade III, leading us to believe that the homozygosity of the UGT1A polymorphism was responsible for the more severe clinical features in the father. Moreover, our results show how the clinical expression of hemolytic anemia is influenced by epistatic factors and we describe a new mutation in the P5'N gene associated with enzyme deficiency, iron overload, and severe gallstone formation. To our knowledge, this is the first description of P5'N deficiency in South Americans.

Effects Of Partial Inhibition Of Respiratory Complex I On H2o 2 Production By Isolated Brain Mitochondria In Different Respiratory States.

The aim of this work was to characterize the effects of partial inhibition of respiratory complex I by rotenone on H2O2 production by isolated rat brain mitochondria in different respiratory states. Flow cytometric analysis of membrane potential in isolated mitochondria indicated that rotenone leads to uniform respiratory inhibition when added to a suspension of mitochondria. When mitochondria were incubated in the presence of a low concentration of rotenone (10 nm) and NADH-linked substrates, oxygen consumption was reduced from 45.9 ± 1.0 to 26.4 ± 2.6 nmol O2 mg(-1) min(-1) and from 7.8 ± 0.3 to 6.3 ± 0.3 nmol O2 mg(-1) min(-1) in respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration), respectively. Under these conditions, mitochondrial H2O2 production was stimulated from 12.2 ± 1.1 to 21.0 ± 1.2 pmol H2O2 mg(-1) min(-1) and 56.5 ± 4.7 to 95.0 ± 11.1 pmol H2O2 mg(-1) min(-1) in respiratory states 3 and 4, respectively. Similar results were observed when comparing mitochondrial preparations enriched with synaptic or nonsynaptic mitochondria or when 1-methyl-4-phenylpyridinium ion (MPP(+)) was used as a respiratory complex I inhibitor. Rotenone-stimulated H2O2 production in respiratory states 3 and 4 was associated with a high reduction state of endogenous nicotinamide nucleotides. In succinate-supported mitochondrial respiration, where most of the mitochondrial H2O2 production relies on electron backflow from complex II to complex I, low rotenone concentrations inhibited H2O2 production. Rotenone had no effect on mitochondrial elimination of micromolar concentrations of H2O2. The present results support the conclusion that partial complex I inhibition may result in mitochondrial energy crisis and oxidative stress, the former being predominant under oxidative phosphorylation and the latter under resting respiration conditions.