Application of forensic luminol for blood detection in endodontic files / Aplicação do luminol forense para detecção de sangue em limas endodônticas

Introduction: Blood is a biological material with high potential of infectious transmission in dental environments, including herpes simplex, hepatitis and AIDS. Aim: To investigate the efficacy of luminol in detecting blood in endodontic files before and after the sterilization process. Material and method: Luminol was used to investigate the presence or absence of traces of blood tissue in 50 endodontic files, visible to naked eye or not, after performing endodontic treatment and after the cleaning/sterilization process. The results obtained were tabulated and statistically analyzed by using the Friedman's test at a significance level of 5% (p<0.05). Result: By naked eye, it was found that 31/50 files showed no trace of blood, 8/50 showed a slight presence of blood and 11/50 showed a considerable presence of blood after endodontic treatment. After the use of luminol, however, 16/50 endodontic files showed no trace of blood, 19/50 showed a slight presence of blood and 15/50 showed a considerable presence of blood. After the cleaning and sterilization process, no blood was detected in the files. Conclusion: It was concluded that the luminol solution is effective in detecting blood tissue in endodontic files as well as in validating the cleaning/sterilization process.

Introdução: Sangue é um material biológico com alto potencial de transmissão de infecção em ambientes odontológicos, incluindo herpes simples, hepatites e AIDS. Objetivo: Investigar a eficácia do luminol em detector sangue em limas endodônticas antes e após o processo de esterilização. Material e método: Luminol foi utilizado para investigar a presença ou ausência de vestígios tecido sanguíneo em 50 limas endodônticas, visíveis ou não à olho nu, após a realização do tratamento endodôntico e após o processo de limpeza/esterilização. Os resultados obtidos foram tabulados e analisados estatisticamente utilizando o teste de Friedman com nível de significância de 5% (p<0,05). Resultado: A olho nú, foi observado que 31/50 limas não apresentaram vestígios de sangue, 8/50 apresentaram uma leve presença de sangue e 11/50 apresentaram uma presença considerável de sangue após o tratamento endodôntico. Após a utilização do luminol, entretanto, 16/50 limas endodônticas não apresentaram vestígios de sangue, 19/50 apresentaram uma leve presença de sangue e 15/50 apresentaram uma presença considerável de sangue. Após o processo de limpeza e esterilização não foi detectado sangue nas limas endodônticas. Conclusão: A solução de luminol é efetiva na detecção de tecido sanguíneo em limas endodônticas, validando o processo de limpeza/esterilização.

WT-1 mRNA expression is modulated by nitric oxide availability and Hsp70 interaction after neonatal unilateral ureteral obstruction

Wilms tumor gene 1 (wt-1), a key regulator of mesenchymal-epithelial transformation, is downregulated during congenital obstructive nephropathy, leading to apoptosis. There is a functional interaction between WT-1 and inducible nitric oxide synthase (iNOS). In this regard, we reported that after neonatal unilateral ureteral obstruction, rosuvastatin prevents apoptosis through an increase in nitric oxide bioavailability, which in turn is linked to higher Hsp70 expression. Hence, the goal of this study was to determine whether a nitric oxide/Hsp70 interaction is involved in changes in WT-1 mRNA expression after ureteral obstruction. Neonatal rats submitted to experimental ureteral obstruction were treated with either vehicle or rosuvastatin for 14 days. Decreased nitric oxide and iNOS/Hsp70 expression associated wit h WT-1 low expression was shown in obstructed kidneys. Apoptosis was induced and it was associated with an increased Bax/BcL2 ratio. Conversely, iNOS/Hsp70 upregulation and an increased WT-1 mRNA expression, without an apoptotic response, were observed in the cortex of obstructed kidneys of rosuvastatin-treated rats. Nitric oxide also modulated Hsp70 and WT-1 mRNA expression in MDCK cells. Finally, in vivo experiments with nitric oxide modulators support our hypothesis that WT-1 mRNA expression is associated with nitric oxide level. Results suggest that rosuvastatin may modulate WT-1 mRNA expression through renal nitric oxide bioavailability, preventing neonatal obstruction-induced apoptosis associated with Hsp70 interaction.

Increased oxygen free radical production from isolated human pmnls and whole blood by luminol-enhanced chemiluminescence in autistic children

To investigate the estimated capacity of polymorphonuclear leucocytes [PMNLs] and whole blood to produce reactive oxygen species [ROS] in children with autism and mental retardation, and compare it with normal children. Cohort study conducted between January and April 2007. Chemiluminescence laboratory [CL], Department of Physiology, Faculty of Medicine, King Khaled University Hospital, Riyadh, Saudi Arabia. Forty autistic and eight mentally retarded children. Oxygen free radical production [O[2]-, H[2]O[2], OH-] was detected by luminol-enhanced chemiluminescence, from isolated PMNLs and whole blood, stimulated by phorbol myristate acetate [PMA] and opsinized zymazan [OPZ]. Oxygen free radical production from whole blood and PMNLs. Forty autistic [35 male and fivefemale],andeightmentally retarded children [study group] were compared with forty six normal Saudi children [control group]. The mean age was 7.4 +/- 0.5 years. The CL peak response of whole blood and PMNLs stimulated with PMA and OPZ, in autistic children was significantlyhigher [p < 0.05]compared to control children. However, the CL peak response in children with mental retardation did not show any significantdifferenceswhencomparedtothecontrolgroup. There is an increase in oxygen free radicals production from whole blood and from PMNLs in autistic children. Therefore, an increase the antioxidant consumption in autistic children is strongly recommended

Potencial antioxidativo da lágrima de adultos jovens / Tear antioxidant potential in young adults

OBJETIVO: Estudar os antioxidantes da lágrima humana, estimulada pelo corte de cebola e a possível influência dos hábitos de vida sobre estas medidas. MÉTODOS: A amostra consistiu de dez adultos jovens, que responderam questionário sobre o hábito de fumar, de ingerir bebidas alcoólicas, frutas, vegetais e cereais, de usar vitaminas e/ou drogas e de praticar exercícios. O potencial reativo antioxidante total (TRAP) foi analisado por meio da quimioluminescência do luminol, a superóxido dismutase (SOD) foi medida pela inibição do piragolol e a absorbância de H2O2 a 240 nm foi utilizada para identificar a catalase. RESULTADOS: A média ± DP dos valores de potencial reativo antioxidante total foi 33,8±11,5 µM e de superóxido dismutase foi 10,8±1,9 U/mL. Não foi identificada atividade da catalase. Detectou-se associação entre a prática regular de exercícios e aumento nos valores de potencial reativo antioxidante total (p=0,021), com diferença de 18,6 µM entre os indivíduos que se exercitavam pelo menos uma vez por semana e os sedentários. Sexo masculino e aumento na concentração de potencial reativo antioxidante total também se associaram estatisticamente (p=0,013), com diferença de 16,3 µM entre os sexos. Houve associação entre hábito tabágico e aumento na superóxido dismutase (p=0,041), com diferença de 3,3 U/mL entre fumantes de mais de cinco cigarros/dia e não fumantes. O uso de vitamina C também demonstrou associação com a superóxido dismutase (p=0,018); a diferença para os que tomavam vitamina C foi de 3,3 U/mL a mais. CONCLUSÃO: Os antioxidantes do lacrimejamento reflexo foram mensuráveis em adultos jovens, e diferentes variáveis parecem ter influenciado nos resultados.

PURPOSE: To study the antioxidant status of human tears, stimulated by onion fumes and the possible influence of the life habits thereon were measured. METHODS: Subjects were ten healthy young adults, who answered a questionnaire about smoking, alcohol ingestion, fruit, vegetable, cereal, and vitamin intake and/or intake of other drugs, and physical exercise habits. Chemoluminescensce of luminol was used to analyze the total reactive antioxidant potential (TRAP), inhibition of piragollol was used to measure superoxide dismutase (SOD) and absorbance of H2O2 at 240 nm was used to identify catalase. RESULTS: Mean ± SD value for total reactive antioxidant potential was 33.8±11.5 µM and for superoxide dismutase 10.8±1.9 U/mL. Catalase was not identified. Regular exercise was associated with increased total reactive antioxidant potential values (p=0.021), with a difference of 18.6 µM between individuals who exercise at least once a week and sedentary individuals. Male gender and total reactive antioxidant potential values were statistically associated (p=0.013), with a difference of 16.3 µM between genders. There was an association between smoking and increased superoxide dismutase values (p=0.041), with a difference of 3.3 U/mL between smokers of more than five cigarettes/day and non-smokers. Also, vitamin C intake and superoxide dismutase values were associated (p=0.018); the difference for vitamin C takers was 3.3 U/mL. CONCLUSION: Reflex tearing antioxidants were measurable in healthy young adults, and different variables apparently influenced their values.

Monitoring sub-nanogram amount of acetylspiramycin in human urine using flow injection analysis with chemiluminescence detection / 药学学报

<p><b>AIM</b>To establish a new and simple flow injection method for the rapid determination of acetylspiramycin (ASPM).</p><p><b>METHODS</b>ASPM was determined by chemiluminescence (CL) method combined with flow injection (FI) technology, which was based on the inhibitive effect of ASPM on the chemiluminescence reaction of the luminol-K3Fe (CN)6 system.</p><p><b>RESULTS</b>The decrease of chemiluminescence intensity was proportional to the logarithm of ASPM concentration (0.1-100) microgram.L-1, the detection limit was 40 ng.L-1 (3 sigma). The whole process, including sampling and washing, could be completed in 0.5 min with a RSD less than 3.0% (n = 5).</p><p><b>CONCLUSION</b>The FI-CL method is of both high sensitivity and good selectivity giving a throughput of 120 h-1. The proposed method was applied successfully to the determination of ASPM in pharmaceutical preparations and human urine without any pre-treatment. It was found that the ASPM concentration reached its maximum after being orally administrated for two hours.</p>

Three model systems measure oxidation/nitration damage caused by peroxynitrite.

Diseases activate the innate immune response which causes ancillary damage to the human body. Peroxynitrite (OONO-) or its carbon dioxide derivatives cause oxidation/nitration and hence mutation to various body polymers e.g. DNA, RNA, protein, lipids and sugars. The control of the ancillary damage can come from antioxidants which inhibit control the amount of peroxynitrite available for damage. In this paper we have developed three different levels of antioxidant screening: (i) Peroxynitrite or SIN-1 reaction with luminol to produce light, and the inhibition of light by substances therefore represents antioxidation. (ii) Nicking of plasmid DNA occurs via oxidants: and is prevented by antioxidants. (iii) Detection of plasmid luciferase activity post-oxidation and infection indicates either prevention or repair of damage: via antioxidants. We found green tea and a number of its polyphenolic constituents effective only at the first level of antioxidation, while extracts of various fruit help at all levels antioxidation. In the final analysis, a combination of green tea extracts and fruits is suggested to produce more complete antioxidant protection.

Chemiluminescence flow-through sensor for the determination of vitamin B2 using controlled-reagent-release technology / 药学学报

<p><b>AIM</b>To establish a simple and novel method for the determination of vitamin B2 rapidly in pharmaceutical preparations.</p><p><b>METHODS</b>Vitamin B2 was determined by a chemiluminescence (CL) sensor combined with flow-injection (FI) technology. The analytical reagents involved in the CL reaction, luminol and hexacyanoferrate (III), were both immobilized on an anion-exchange resin column in FI system. The CL signal produced by the reaction between luminol and hexacyanoferrate (III), which were eluted from the column through sodium phosphate injection, decreased in the presence of vitamin B2.</p><p><b>RESULTS</b>The decreased CL intensity was linearly correlated with the vitamin B2 concentration in the range of 0.01-1.0 microgram.mL-1, the detection limit was 4.0 ng.mL-1 vitamin B2 (3 sigma). At a flow rate of 2.0 mL.min-1, the procedure including sampling and washing could be performed in 2 min with a relative standard deviation of less than 3.0%.</p><p><b>CONCLUSION</b>The flow sensor exhibited both good sensitivity and stability. It could be reused more than 450 times and has been applied successfully to the analysis of vitamin B2 in pharmaceutical preparations.</p>

In vitro effect of different non-steroidal anti-inflammatory drungs on human polymorphonuclear leukocyte activity measured by luminol-dependent chemiluminescence of the whole blood

To define the well-known variability in the effects of non-steroidal anti-inflammatory drugs and to search for predictors of such variability using an in vitro model. Polymorphonuclear leukocyte activity was measured by luminol-dependent chemiluminescence of the whole blood using barium sulphate as a stimulator. Blood was taken from 40 apparently healthy volunteers [22 males and 18 females; their age ranged from 20-50 years]. Drugs [indomethacin 10ug/ml, aspirin 300ug/ml, ibuprofen 25ug/ml or diclofenac 8ug/ml] were added into the blood of each individual in vitro. The chemiluminescence was measured in a photon counting system. There was a marked inter and intra individual variation in the chemiluminescence response to the 4 non-steroidal anti-inflammatory drugs, added in vitro. The variation exhibited a continuous pattern. No statistically significant correlation was found between the in vitro effect of one non-steroidal anti-inflammatory drug and the other 3 drugs, nor between the effect of each drug and factors like age, sex, weight, height, packed cell volume, hemoglobin percentage and white blood cell count. Subjects with hemoglobin-AS type [number = 9] responded mainly by enhancement to indomethacin and diclofenac. When the number of subjects rather than the average net effect was compared according to blood groups, those with blood group A showed chemiluminescence responses towards enhancement with indomethacin and diclofenac and blood group O with aspirin. A consistent pattern of enhancement and inhibition was evident; enhancements and inhibitions by any 2 drugs involve a seemingly constant proportion of subjects. Luminol-dependent chemiluminescence responses of polymorphonuclear leukocyte activity could be a good in vitro model to study the variability in response to non-steroidal anti-inflammatory drugs. Characteristics of each individual are not able to predict the pattern of variability. Abnormal hemoglobin and the type of blood group seem to be an interesting area for research

La alfa hemolisina de Escherichia coli potencia el metabolismo oxidativo de neurotrófilos humanos en respuesta al péptido quiomiotáctico FMLP: comparación con el ionóforo de calcio A23187 / The alpha hemolysin of E. coli and oxidative metabolism of human neutrophils in response to the chemotactic peptide FMLP: comparation with the ionophores of calcium A23187

El aumento en el calcio citoplásmatico inducido por el ionóforo de calcio ionomicina en leucocitos polimorfonucleares (PMN) incrementa la producción de superóxido en respuesta a estimulación secundaria con el péptido quimiotáctico FMLP (Helman Finkel, T. et al J. Biol Chem 1987; 262:12589-12596). En esta investigación se estudió el efecto del tratamiento de PMN con alfa hemolisina (AH) o el ionóforo de calcio. A23187 (que incrementan el calcio intracelular) en el metabolismo oxidativo de PMN (estimado por medio quimioluminiscencia) en respuesta a la estimulación secundaria con FMLP. Tanto el A23187 como la AH potenciaron la respuesta quimioluminiscente (amplificada con luminol) a estimulación posterior con FMLP indicando sobreestimulación del metabolismo oxidativo en PMN. Experientos adicionales, con lucigenina como amplificador de luminiscencia, mostraron que el ionóforo de calcio A23187 potenció la liberación de superóxido, de manera similar a la reportada para la ionomicina, pero que la AH solo causó liberación ínfima de superóxido al espacio extracelular. Los resultados se comentan en referencia a procesos infecciosos causados por cepas hemolíticas de Escherichia coli. (Rev Cost Cienc Med 2000; 21(3-4): 141-59). Palabras clave: leucocito polimorfonuclear, quimioluminiscencia, luminol, lucigenina, A23187, alfa hemolisina, FMLP.

Caracterización de la respuesta quimioluminiscente de neutrófilos humanos a la hemolisia alfa de Eschericia coli / Caracterization os the chemiluminescence response of human neutrophils to hemolysins-alpha of escherichia coli

Leucocitos polimorfonucleares humanos (PMN) expuestos a la alfa hemolisina (AH) de Escherichia coli manifestaron quimioluminiscencia (QL), amplificada mediante el uso de luminol. Los parámetros cinéticos de la QL mostraron semejanza a los de la respuesta quimioluminiscente de PMN al ionófor de calcio A23187. Dado que las respuestas de PMN y AH y al A23187 se inhiben de manera similar cuando se preincuban con A63612, un derivado del ácido hidroxámico e inhibidor de la lipooxigenasa, es probable que ambas compartan un mecanismo común, el cual es activación de la síntesis de leucotrienos, debida a la entrada de calcio a la célula causada por la hemolisina y el ionófor. La QL en respuesta a AH es inhibida además por el DMSO, el cual reacciona con activación del metabolismo del araquidonato. Esto en evidencia adicional de la relación entre la QLL causada por AH y A23187, y el metabolismo del araquidonato (Rev Cost Cienc Med 2000; 21(1-2): 31-42). Palabras clave: Leucocito polimorfonuclear, Quimioluminiscencia, Alfa hemolisina

A comparison of methods employed to evaluate antioxidant capabilities

Three different methodologies frequently employed to evaluate the indexes that report the antioxidant capabilities of pure compounds and/or complex mixtures of antioxidants are applied to a series of mono- and polyphenols, as well as to two wine (red and white) samples. These methodologies are based on the bleaching of a stable radical, the effect of the additive upon luminol chemiluminescence induced by peroxyl radicals, and the effect of the additive upon the bleaching of the fluorescence from a dye molecule. Widely different responses are obtained from the different methodologies. These differences are interpreted in terms of the different factors (stoichiometric factors and/or reactivities) that determines the indexes evaluated by these different methodologies.

Inhibition of luminol-dependent chemiluminescence of human granulocytes by low doses of inorganic mercury

HgCl2, added in vitro to human granulocytes in whole blood, caused a marked inhibitory effect on the luminol-dependent chemiluminescence induced by BaS04 crystals in suspension of these cells. The effect was both dose- and time-dependent when BaS04 was used to stimulate the oxidative burst in granulocytes. Incubation with the highest concentration of HgCl2 used [10 mmol/L], however, did not cause disruption of the membranes of granulocytes. The effect of HgCl2 on the granulocytes was irreversible following washing of the HgCl2-treated cells with phosphate buffered saline. HgCl2 did not affect chemiluminescence produced when luminol was excited by oxidative hydrogen peroxide in a cell-free medium. These results suggest that some of the toxicity of HgCl2 may be greater than mediated by an action on the phagocytic immune system

Phenotypic and Functional Differentiation of Promyelocytic Cell Line HL-60 by N-N-dimethylformamide

PURPOSE: HL-60 is a promyelocytic cell line. Fc receptors and complement receptor 3 (CR3) play important role in the protective response of granulocytes and monocytes against microbial infection. We quantified the expression of Fc I, Fc II, Fc III, and CD11b/CD18 during differentiation using HL-60 cells by N-N-dimethylformamide (DMF). Functional studies, such as phagocytic activity, respiratory burst and ADCC, were also performed. METHODS: HL-60 cells were induced to differentiate by adding 0.8% DMF. On the 4th and 7th day after stimulation as well as before stimulation, the cells were analyzed for phenotypic and functional differentiaton. Phenotypic analysis was performed by flow cytometry after staining the cells with PE-conjugated anti-human CD64, CD32, CD16, CD11b, CD18, and isotype controls. And the measured fluorescent intensity was transformed into Molecules of Equivalent Soluble Fluorochromes (MESF). Phagocytic activity was also measured by flow cytometry after incubation with fluorochrome-conjugated beads. Respiratory burst was measured by chemiluminescence assay of cells incubated with luminol after stimulation with PMA. ADCC was measured by hemoglobin release assay. RESULTS: The expression of CD11b, CD18 and CD64 on HL-60 cells markedly increased on the 4th day and slightly decreased on the 7th day. Expression of CD32 was already induced before differentiation induction and slightly increased by DMF. CD16 was not expressed during differentiation. In phagocytic assay, the phagocytic cell fraction increased by stimulation on 4th and 7th day. Chemiluminescence showed the DMF increased the respiratory burst of HL-60 cells on the 4th and 7th day. In ADCC, DMF increased the target cell lysis continuously. CONCLUSION: HL-60 cells which were differentiated with DMF for are good models for studying opsonophagocytic assay of immunized sera.

comparative study of hemoglobin estimated by the traditional [WHO] technique and chemiluminescence method [research note]

The addition of hemoglobin [Hb] caused an inhibitory effect on the luminol-dependent chemiluminesence [CL] induced by the excitation of luminol by the oxidative metabolic, hydrogen peroxide, In a cell free medium. The CL was detected with an ultra-high sensitive photon counting system designed and built in the department of physiology. The Inhibitory effect produced by various Hb levels was dose dependent, reproducible and linear with an r=0.997. Hb concentration curves constructed by CL and standard Cyanmethaemoglobin [HiCN] methods were parallel. A comparison between the inhibited CL [area under the curves], and the optical density [HiCN method] produced by same Hb levels was linear with an r=0.990. There was no significant difference [0.1 > P < 0.5] between Rb level measured by CL and HICN method in healthy adults samples. A point of importance, turbidity due to high leukocytes count [250 x 109 CIL] has no significant effect on Hb levels measured by CL and the modified HICN methods. These results suggest that, CL method may provide an additional reliable method for Hb estimation

Elevated polymorphonuclear phagocytic function in thalassemia patients by chemiluminescence.

Twenty five patients with beta thalassemia major, with no evidence of infection were evaluated for their polymorphonuclear cell (PMN) metabolic function and serum opsonic activity by chemiluminescence assay. These were divided into Group I of normal adults (n = 21), Group II thalassemia major < 5 years (n = 9) and Group III thalassemia major > 5 years (n = 16). The ability of the chemiluminescence assay (CL) to reflect opsonic and phagocytic dysfunction suggested its potential application in the evaluation of phagocytic function. The peak count of Group I was (1.07 +/- 0.24 x 10(-5)), Group II (1.60 +/- 0.83 x 10(-5)) and Group III was (2.71 +/- 0.98 x 10(-5)) respectively in the presence of autologous sera. The peak count compared between Group I and III was found to be statistically significant (p < 0.05). The peak count of Group I and II when compared showed a trend in the increase activity not statistically significant. The polymorph function of all the groups were compared with autologous serum as well as normal serum. There was no increase in polymorph function of Group III in the presence of thalassemia serum, nor any decrease in the polymorph function of thalassemia patients of Group II and III. This concluded that polymorphs of thalassemia patients are active in the presence of autologous as well as normal serum. The increased activity of thalassemia polymorphs may be due to antigenic stimulation which may be due to multiple transfusion and not due to circulating iron load.

Enhanced eosinophil luminol-dependent chemiluminescence and complement receptor expression by platelet-activating factor and interleukin-5.

The cytokine interleukin-5 (IL-5) and the lipid mediator platelet-activating factor (PAF) have both been shown to be involved in eosinophil differentiation and activation. We have measured and compared the effect of PAF and IL-5 on human eosinophils in terms of their luminol-dependent chemiluminescence (CL) response and their expression of complement receptors, CR1 and CR3. Both IL-5 and PAF enhanced the eosinophil CL response. The optimal concentrations were 40 U/ml for IL-5, and 10(-6) M for PAF. The priming effect of IL-5 was slow and reached a maximal response after 90 minutes incubation. In contrast, the effect of PAF peaked early and declined during incubation. In the complement receptor study, only PAF was able to enhance CR3 expression (p < 0.05) while the effect of IL-5 on eosinophil complement receptor expression was negligible. These results provide evidence that both inflammatory mediator (PAF) and cytokine (IL-5) can activate eosinophils but the effects of IL-5 and PAF on eosinophil CL response appear to be distinct. The activation of eosinophils by PAF and IL-5 may occur through different mechanisms.

Galactosamine [GaIN] inhibits luminol-dependent chemiluminescence of human polymorphonuclear leukocytes

The aim of the present study was to examine the effect of D- galactosamine HCl [GaIN] on the oxidative respiratory burst of isolated human polymorphonuclear leukocyte [PMNs]. GaIN added in vitro to PMNs caused a marked inhibitory effect on the luminol- dependent chemiluminescene [CL] induced by phorbol myristate acetate [PMA] on PMNs. The inhibitory effect produced by GaIN was both dose and time dependent when PMA was used to stimulate the oxidative burst of PMNs. The effect of GaIN on the isolated PMNs was partially irreversible, following washing of the GaIN-treated PMNs with phosphate buffered saline [PBS]. PMNs viability was not significantly altered by incubation with GaIN. Addition of lipopolysaccharide [endotoxin] to isolate PMNs in the presence or absence of GaIN did not alter the oxidative burst of PMNs. Addition of oxygen-free radical scavengers enhanced the inhibitory effect induced by GaIN on PMNs CL. In a cell free medium, GaIN has no inhibitory effect on CL induced by luminol, H2O2 and horse radish peroxidase. As a conclusion, results suggested that in vitro, GaIN has a remarkable inhibitory effect on the release of oxygen products from stimulated PMNs