Salmonella spp. contamination in commercial layer hen farms using different types of samples and detection methods.

The performance of detection methods (culture methods and polymerase chain reaction assay) and plating media used in the same type of samples were determined as well as the specificity of PCR primers to detected Salmonella spp. contamination in layer hen farms. Also, the association of farm characteristics with Salmonella presence was evaluated. Environmental samples (feces, feed, drinking water, air, boot-swabs) and eggs were taken from 40 layer hen houses. Salmonella spp. was most detected in boot-swabs taken around the houses (30% and 35% by isolation and PCR, respectively) follow by fecal samples (15.2% and 13.6% by isolation and PCR, respectively). Eggs, drinking water, and air samples were negative for Salmonella detection. Salmonella Schwarzengrund and S. Enteritidis were the most isolated serotypes. For plating media, relative specificity was 1, and the relative sensitivity was greater for EF-18 agar than XLDT agar in feed and fecal samples. However, relative sensitivity was greater in XLDT agar than EF-18 agar for boot-swab samples. Agreement was between fair to good depending on the sample, and it was good between isolation and PCR (feces and boot-swabs), without agreement for feed samples. Salmonella spp. PCR was positive for all strains, while S. Typhimurium PCR was negative. Salmonella Enteritidis PCR used was not specific. Based in the multiple logistic regression analyses, categorization by counties was significant for Salmonella spp. presence (P-value = 0.010). This study shows the importance of considering different types of samples, plating media and detection methods during a Salmonella spp. monitoring study. In addition, it is important to incorporate the sampling of floors around the layer hen houses to learn if biosecurity measures should be strengthened to minimize the entry and spread of Salmonella in the houses. Also, the performance of some PCR methods and S. Enteritidis PCR should be improved, and biosecurity measures in hen farms must be reinforced in the region of more concentrated layer hen houses to reduce the probability of Salmonella spp. presence.

A comparative study of culture methods and PCR assay for Salmonella detection in poultry drinking water.

The present work compared 2 culture methods and PCR assays for motile and nonmotile Salmonella detection using artificially contaminated poultry drinking water. The specificity was 1 for all methods studied. The accuracy and sensitivity were 1 for all motile strains, whereas these parameters were between 0 and 0.7 for nonmotile Salmonella strains. The positive predictive value and negative predictive value were 1 for all motile Salmonella strains in the 3 methods used. Nonmotile Salmonella strains showed a positive predictive value of 1 in the PCR method. However, the positive predictive value was indeterminate in the tetrathionate (TT) methods for both strains tested and in the modified semisolid Rappaport-Vassiliadis (MSRV) method for Salmonella Pullorum. On the other hand, the negative predictive value was between 0.20 and 0.43 for the 3 methods. The detection level of motile strains was 4 to 7 cfu/25 mL for all methods. Nonmotile Salmonella strains could not be detected in the TT method, whereas only Salmonella Gallinarum could be recovered from 1.1 × 10(1) cfu/25 mL in the MSRV method. In relation to the molecular methods, PCR could detect these strains from 1.1 × 10(4) cfu/25 mL. Extending incubation time of the enrichment medium to 6 d in the TT method did not improve the isolation rates. In general, all selective plating media did not show any statistical differences in the parameters of performance studied. The kappa coefficient showed that there was an excellent agreement between the 3 methods for motile strains. For nonmotile strains, the agreement was poor between the MSRV and the PCR; there was no agreement when the TT method was compared with the MSRV and the PCR methods. The difference in detection levels obtained with the methods used for motile and nonmotile Salmonella strains and the difficulty in detecting these last strains represents a potential problem when a poultry water sample is considered negative for the presence of Salmonella.

Exposición a fármacos citotóxicos en el personal sanitario / Exposure to cytotoxic drugs among health care professionals

Objetivo: Cuantificar los niveles de exposición del personal sanitario a fármacos citotóxicos con el fin de establecer el nivel umbral de exposición e implantar medidas para incrementar la protección y seguridad. Material y método La cuantificación de la contaminación de 5-fluorouracilo, gemcitabina y ciclofosfamida se llevó a cabo en las superficies de las siguientes áreas: cabina de seguridad biológica clase II tipo B3 (S1), mesa de preparación de tratamientos en antecámara (S2) y mesa de la sala de administración en hospital de día (S3). Se tomaron muestras de las superficies con un paño absorbente a tiempo t0, previo inicio de la sesión de trabajo, y t1, tras 3 h de trabajo mediante arrastre. En cada superficie se calculó el valor de la masa mediana respecto al valor basal y los percentiles 90, 75, 50 y 25 para cada citotóxico en μg/m2.Se comprobó la normalidad de la distribución con la prueba Shapiro-Wilk. El análisis estadístico incluyó las pruebas U de Mann-Whitney, Kruskal-Wallis y Wilcoxon. Se fijó el nivel de significación estadística para valores de p < 0,05.ResultadosSe recogieron un total de 90 muestras en total, 30 muestras por cada superficie de estudio. La masa media registrada de cualquier compuesto citotóxico fue superior en S1 y t1, con un valor de p = 0,017 y p = 0,004, respectivamente. Para cada fármaco citotóxico se fijó como valor objetivo el percentil 25 donde se obtuvieron valores de contaminación indetectables. Conclusiones La introducción de un programa de monitorización continua de superficies de diversos compuestos citotóxicos es esencial para fijar unos niveles aceptables de contaminación residual y reducir la exposición ocupacional (AU)

Objective: To quantify levels of exposure to cytotoxic drugs among health professionals in order to establish an exposure threshold and implement measures to increase protection and safety Material and method: Contamination with 5-fluorouracil, gemcitabine and cyclophosphamide was measured on work surfaces in the following areas: a class II type B3 biological safety cabinet(S1), a treatment prep table in an antechamber (S2) and a desk from the administrative room in the Outpatient Unit (S3). We took samples from the work surfaces by wiping them with an absorbent cloth at time t0, prior to the work session, and at t1 after three hours of work. For each surface, we calculated the median mass value with respect to the baseline value and the90th, 75th, 50th and 25th percentiles for each cytotoxin in g/m2.Distribution normality was assessed using the Shapiro-Wilk test. Statistical analysis included the Kruskal-Wallis and Mann-Whitney-Wilcoxon tests. Statistical significance was established for values of P<.05.Results: We gathered a total of 90 samples, 30 from each of the studied work surfaces. The mean recorded mass of any of the cytotoxic compounds was higher for S1 and t1, with values of P=.017 and P=.004 respectively. The target value for each cytotoxic drug was established at the 25th percentile, where undetectable contamination values were obtained. Conclusions: Introducing a continuous programme to monitor work surfaces for an array of cytotoxic compounds is fundamental in order to establish acceptable levels of residual contamination and reduce exposure in the workplace (AU)

A comparative study of culture methods and polymerase chain reaction for Salmonella detection in egg content.

The present work compared 2 culture methods and a PCR assay applied with 2 enrichment methods for the detection of motile and nonmotile Salmonella strains using artificially contaminated egg content. The specificity (Sp) was 1 in all methods. The sensitivity (Se), accuracy (Ac), positive predictive value (PPV), and negative predictive value (NPV) were 1 in both culture methods for motile and nonmotile strains. In reference to the PCR methods, Se and PPV were between 0 and 1, whereas Ac and NPV were between 0.14 and 1. The detection level of motile and nonmotile strains was 5 to 54 cfu per 25 mL for both culture methods, but some strains could not be detected by the PCR methods. Extending incubation time of the enrichment medium to 5 d in the tetrathionate broth (TT), and Muller-Kauffmann tetrathionate-novobiocin broth (MKTTn) methods did not improve the isolation rates. All selective plating media did not show any statistical differences in the parameters of performance studied. Kappa coefficients showed that there was an excellent agreement between the bacteriological methods for all Salmonella strains. The agreement was very good and good between the PCR methods, for motile and nonmotile strains, respectively. However, there was a poor agreement when the PCR and bacteriological methods were compared for motile and nonmotile Salmonella strains. The TT and MKTTn methods are similar in terms of Ac, Se, Sp, PPV, and NPV for different Salmonella strains in egg content. The use of the PCR method cannot improve the same parameters, described before, in this matrix. So, further studies are needed to improve the performance parameters and limit of detection in egg content for the PCR methods, so that test can be used in poultry and food industry.

[Exposure to cytotoxic drugs among health care professionals]. / Exposición a fármacos citotóxicos en el personal sanitario.

OBJECTIVE: To quantify levels of exposure to cytotoxic drugs among health professionals in order to establish an exposure threshold and implement measures to increase protection and safety. MATERIAL AND METHOD: Contamination with 5-fluorouracil, gemcitabine and cyclophosphamide was measured on work surfaces in the following areas: a class II type B3 biological safety cabinet (S(1)), a treatment prep table in an antechamber (S(2)) and a desk from the administrative room in the Outpatient Unit (S(3)). We took samples from the work surfaces by wiping them with an absorbent cloth at time t(0), prior to the work session, and at t(1) after three hours of work. For each surface, we calculated the median mass value with respect to the baseline value and the 90th, 75th, 50th and 25th percentiles for each cytotoxin in µg/m(2). Distribution normality was assessed using the Shapiro-Wilk test. Statistical analysis included the Kruskal-Wallis and Mann-Whitney-Wilcoxon tests. Statistical significance was established for values of P<.05. RESULTS: We gathered a total of 90 samples, 30 from each of the studied work surfaces. The mean recorded mass of any of the cytotoxic compounds was higher for S(1) and t(1), with values of P=.017 and P=.004 respectively. The target value for each cytotoxic drug was established at the 25th percentile, where undetectable contamination values were obtained. CONCLUSIONS: Introducing a continuous programme to monitor work surfaces for an array of cytotoxic compounds is fundamental in order to establish acceptable levels of residual contamination and reduce exposure in the workplace.

Comparison of 2 culture methods and PCR assays for Salmonella detection in poultry feces.

The present work compared 2 culture methods and the combinations of pre-enrichment and enrichment culture methods with PCR assays [buffered peptone water-PCR and tetrathionate-PCR or modified semisolid Rappaport-Vassiliadis (MSRV)-PCR] for motile and nonmotile Salmonella strain detection using artificially contaminated poultry feces. The specificity and positive predictive values were equal to one in both culture methods. Specificity and positive predictive values, accuracy, sensitivity, and negative predictive values were higher for motile than nonmotile Salmonella strains in culture methods. Only Salmonella enterica serovar Gallinarum was detected by the MSRV method with low accuracy, sensitivity, and negative predictive value. The detection level of motile strains was 2 ×10(0) to 22 × 10(2) cfu per 25 g for these methods, whereas it was 6.9 × 10(2) cfu per 25 g in culture methods for Salmonella Gallinarum. Extending the incubation time of the enrichment medium to 6 d in the TT method did not improve the isolation rates. In general, all selective plating media did not show any statistical differences in the parameters of performance studied. On the other hand, accuracy and sensitivity values were higher in MSRV-PCR and tetrathionate-PCR methods than in the buffered peptone water-PCR method. Specificity and positive predictive values were equal to one in most of the cases. In terms of detection limits, motile Salmonella strains were recovered from 5 × 10(0) cfu per 25 g in MSRV-PCR and tetrathionate-PCR methods, whereas the detection limit was better for nonmotile Salmonella in MSRV-PCR methods than in the tetrathionate-PCR method. Kappa coefficients showed that there was a very good agreement between tetrathionate and MSRV methods for motile Salmonella strains, whereas these methods did not show any concordance for nonmotile Salmonella strains. When buffered peptone water-PCR was compared with both tetrathionate-PCR and MSRV-PCR, agreement was poor for motile Salmonella strains and slight to fair for nonmotile Salmonella strains. The difference in isolation rate obtained with the methods used for motile and nonmotile Salmonella strains must be taken into account when a poultry fecal sample is considered negative for the presence of Salmonella.

A comparative study of culture methods and polymerase chain reaction assay for Salmonella detection in poultry feed.

The present work compared 2 culture methods and PCR assay for the detection of motile and non-motile Salmonella strains using artificially contaminated poultry feed. The specificity was 1 in all methods. The accuracy and sensitivity were between 0.5 and 1 for motile Salmonella strains, whereas these parameters were between 0 and 0.6 for non-motile Salmonella strains. The positive predictive value was 1 for tetrathionate (TT), PCR, and modified semisolid Rappaport-Vassiliadis (MSRV) methods in most of the strains studied. The negative predictive value of each method was very low for non-motile Salmonella strains. The detection level of motile strains was 8 to 20 cfu/25 g for all methods, whereas it was ≥10(4) cfu/25 g in culture methods for non-motile Salmonella strains. In general, the PCR method detected lower non-motile Salmonella contamination levels in feed than did culture methods. Extending incubation time of the enrichment medium to 6 d in the TT method did not improve the isolation rates. All selective plating media did not show any statistical differences in the parameters of performance studied. Kappa coefficients showed that there was good agreement between TT and MSRV methods, and MSRV and PCR methods for motile Salmonella strains in poultry feed samples. The agreement was fair between TT and PCR methods for these strains. For non-motile Salmonella strains, there was poor (TT and MSRV methods), slight (PCR and TT methods), and fair (MSRV and PCR methods) agreement. The TT, MSRV, and PCR methods are similar in terms of accuracy, sensitivity, specificity, positive predictive value, and negative predictive value for different motile Salmonella strains in poultry feed. For non-motile Salmonella strains, the use of the PCR method improves the same parameters, described before, in this matrix. The difference in detection levels obtained with the methods used for motile and nonmotile Salmonella strains and the difficulty to detect these last strains represent a potential problem, when a poultry feed sample is considered negative for the presence of Salmonella.

[Cutaneous toxicity associated with cetuximab treatment in metastatic colorectal cancer]. / Toxicidad cutánea asociada a cetuximab en cáncer colorrectal metastásico.

OBJECTIVE: To evaluate the impact and type of side-effects in patients treated with cetuximab and provide a description of the general measures and treatment. METHODS: Retrospective safety study. We included all patients that received cetuximab from January to December 2009. All information was obtained from the Pharmacy and Oncology Department's Access databases and reviewed the patient's medical history. All data was registered in an Excel workbook. Skin toxicity was graded by the current National Cancer Institute-Common Toxicity Criteria (NCI-CTC). RESULTS: During the study period 43 patients received treatment with cetuximab. Acneiform eruption was present in 30 of the cases (69.8%): 14 patients with grade 1 (48.3%), 13 with grade 2 (44.8%) and 3 with grade 3 (10.3%). These adverse effects appeared in a median of seven (4-28) days. In a median of 40 (20-56) days, ten patients (23.3%) presented xerosis, and three (7%) suffered painful fissures in hands and feet after a median of 28 (21-35) days. Paronychia was present in two patients after a median of 42 (35-49) days. Finally, an alteration in hair growth was observed in two patients with overgrowth of facial hair and one patient with overgrowth of the eyelashes. Five patients presented important conjunctivitis. Three infusion reactions occurred. A grade-based treatment algorithm was used for all patients that presented cutaneous toxicity. CONCLUSIONS: A considerable number of patients treated with cetuximab develop dermatological side-effects which left untreated could represent a threat to the efficacy of the therapy. Therefore effective management is mandatory, patient education and immediate treatment based on a grade-based algorithm to alleviate symptoms is necessary, so that patient compliance is guaranteed.

Aceptación de recomendaciones en el paciente con oportunidades de mejora farmacoterapéutica / Doctors’ acceptance of recommendations for patients with the opportunity for pharmacotherapy improvement

Objetivo: Identificar y cuantificar la influencia de diferentes variables en la implantación de medidas de optimización farmacoterapéutica en pacientes ingresados. Método Estudio descriptivo, transversal. Período: 2000–2007. Ámbito: hospital general universitario público (25.000 pacientes ingresados/año).El Programa de Mejora de la Calidad de la Farmacoterapia y la Seguridad del Paciente implantado da cobertura al 30% de los pacientes. A partir de los registros del aplicativo Atefarm® Farmis, se analizaron las recomendaciones farmacoterapéuticas (RF) realizadas por los farmacéuticos al médico. Las variables seleccionadas fueron las siguientes: riesgo del medicamento (problema relacionado con el medicamento [PRM]) (0, bajo; 1, alto), categoría del PRM, (0, indicación; 1, efectividad; 2, seguridad), gravedad potencial (escala 1–5), impacto de la RF (0, efectividad; 1, seguridad; 2, eficiencia) e implantación de la RF (sí/no).Se calculó la frecuencia (%) y el intervalo de confianza del 95% (IC95%) de las variables categóricas y se realizó un análisis de regresión logística multivariante para identificar el grado de influencia de las variables en la implantación de las RF. Resultados Se identificaron 7.920 PRM en 4.680 pacientes. En el 85% (6.762) de los PRM se realizó una RF, que se implantó en el 83% (IC95%: 74,2–89,8). La gravedad potencial del PRM superior o igual a 2 (OR: 1,57; IC95%: 1,27–1,94) y la categoría del PRM de efectividad y seguridad (OR: 1,19; IC95%: 1,02–1,39) se manifestaron como determinantes de la implantación de la RF en el paciente. Conclusiones La probabilidad de implantación de RF en el paciente está relacionada con la gravedad potencial y la categoría del PRM identificado. Así, las recomendaciones orientadas a mejorar la efectividad de la farmacoterapia o la seguridad del paciente, y con consecuencias clínicas potenciales presentan mayor éxito en su aplicación al paciente(AU)

Objective: To identify and quantify the influence of different variables on the implementation of pharmacotherapy optimisation measures in hospitalised patients. Method: Descriptive transversal study. Period: 2000—2007. Environment: public university general hospital (25,000 patients admitted/year).The Programme implemented to improve pharmacotherapy quality and patient safety covers30% of all patients. Using records from the Atefarm®Farmis application, we analysed pharmacotherapy recommendations (PRs) made by pharmacists to doctors. The selected variables were the following: Risk of the medication for ADE (1-high, 0-low), ADE category, (0-indication, 1-effectiveness, 2-safety), potential severity (scale of 1 to 5), impact of the PR (0-effectiveness,1-safety, 2-efficiency) and implementation of the PR (yes/no).We calculated the frequency (%) and 95% CI for the categorical variables and performeda multivariate logistical regression analysis to identify the variables’ degree of influence onimplementing the PRs. Results: We identified 7920 ADEs in 4680 patients. A PR was issued in 85% of the cases (6762),and it was implemented in 83% (95% CI 74.2—89.8). Potential severity of the ADE ≥2 (OR 1.57;95% CI 1.27—1.94), and ADE category for effectiveness and safety (OR 1.19; 95% CI 1.02—1.39)were shown to be determining factors for implementing the PR for the patient. Conclusions: The probability that a PR will be implemented for a patient is related to the potential severity and the category of the identified ADE. Therefore, recommendations intended to improve effectiveness of pharmacotherapy or patient safety, and those with potential clinical consequences have a greater chance of being applied to a patient (AU)

Doctors' acceptance of recommendations for patients with the opportunity for pharmacotherapy improvement.

OBJECTIVE: To identify and quantify the influence of different variables on the implementation of pharmacotherapy optimisation measures in hospitalised patients. METHOD: Descriptive transversal study. PERIOD: 2000-2007. ENVIRONMENT: public university general hospital (25,000 patients admitted/year). The Programme implemented to improve pharmacotherapy quality and patient safety covers 30% of all patients. Using records from the Atefarm(®) Farmis application, we analysed pharmacotherapy recommendations (PRs) made by pharmacists to doctors. The selected variables were the following: Risk of the medication for ADE (1-high, 0-low), ADE category, (0-indication, 1-effectiveness, 2-safety), potential severity (scale of 1 to 5), impact of the PR (0-effectiveness, 1-safety, 2-efficiency) and implementation of the PR (yes/no). We calculated the frequency (%) and 95% CI for the categorical variables and performed a multivariate logistical regression analysis to identify the variables' degree of influence on implementing the PRs. RESULTS: We identified 7,920 ADEs in 4,680 patients. A PR was issued in 85% of the cases (6,762), and it was implemented in 83% (95% CI 74.2-89.8). Potential severity of the ADE ≥2 (OR 1.57; 95% CI 1.27-1.94), and ADE category for effectiveness and safety (OR 1.19; 95% CI 1.02-1.39) were shown to be determining factors for implementing the PR for the patient. CONCLUSIONS: The probability that a PR will be implemented for a patient is related to the potential severity and the category of the identified ADE. Therefore, recommendations intended to improve effectiveness of pharmacotherapy or patient safety, and those with potential clinical consequences have a greater chance of being applied to a patient.

Genetic polymorphisms and individualized tacrolimus dosing.

BACKGROUND: Genetic polymorphisms of metabolism enzymes or intestinal drug transporters may affect pharmacokinetic responses to immunosuppressive drugs in renal transplant recipients. We sought to identify the frequency of genetic polymorphisms and their importance for individualization of tacrolimus doses. PATIENTS AND METHODS: We performed an observational study in 35 renal transplant recipients treated with tacrolimus, mycophenolate mofetil, and corticosteroids. Tacrolimus concentrations were determined by immunoanalysis (IMx method; Abbott Diagnostics, Abbott Park, Ill), on 11 blood samples per patient during the first 6 weeks after renal transplantation. For each patient, we calculated the mean value and its standard error (SEM) of the concentration/dose ratio (ng/mL/mg) of tacrolimus. The pharmacogenetic analysis included single nucleotide polymorphisms (SNPs) in the CYP3A5 (CYP3A5*3 (A6986G), CYP3A5*6 (G14690A), MDR1 (C3435T and G2677T/A) and PXR (C-25385T) genes. RESULTS: Of the patients, 62.8% (n=22) were men and the overall mean age was 55 years (95% confidence interval, 48.7-62.7). The SNP distribution was: CYP3A5*3: G/G=82.9%, A/G=17.1%; CYP3A5*6: G/G=88.6%, G/A=11.4%; MDR1 C3435T: C/C=25.7%, C/T=62.9%, T/T=11.4%; for MDR1 G2677T/A: G/G=22.9%, G/T=65.7%, T/T=11.4% and for PXR: C/T=85.7%, T/T=14.3%. Tacrolimus concentration/dose ratios in heterozygote patients for CYP3A5*3 genotypes was >120% lower than for the homozygote CYP3A5*3 genotype (0.65±0.04 vs 1.45±0.05; P<.0001). Wild-type MDR1 (3435 C/C) genotype patients showed up to 40% lower concentration/dose ratios compared with heterozygote and homozygote genotypes (C/C; 1±0.07 vs C/T; 1.4±0.06 vs T/T; 1.37±0.09; P<.0001). CONCLUSION: Intestinal absorption and metabolism of tacrolimus was significantly affected by the SNPs in the CYP3A5 and MDR1 genes, which may offer a useful tool to optimize tacrolimus dosing after renal transplantation.

Predicting risk of acute rejection in patients with kidney transplants.

OBJECTIVE: Create a model to predict the risk of acute rejection of kidney transplant considering variables related to the immunosuppressant agent used, the receiver, the donor and the transplanted organ. METHODS: Cohort study in a population of 68 patients with kidney transplants being treated with tacrolimus triple therapy. Predicting the risk of acute rejection was carried out with a logistic regression analysis using age, sex, re-transplant status, number of HLA incompatibilities, cold ischaemia time, acute tubular necrosis, induction with basiliximab or thymoglobulin and treatment type as explanatory variables. The contribution of variables associated with determining the blood concentration of tacrolimus was also evaluated; these variables include the average blood concentration, the number of values below and included in the pre-defined therapeutic interval, and the time during which those values remained within that interval. RESULTS: The logistic regression analysis indicates that the risk of acute rejection depends on the acute tubular necrosis (OR: 3; CI 95%, 0.7 to 13.2) and on the time that the blood concentrations of tacrolimus remains within the therapeutic interval (OR: 0.8; CI 95%, 0.7 to 0.9). The final model presents an optimal discrimination power (AUCROC: 77%; CI 95%, 62% to 92%). For the selected cut-off point (probability greater than or equal to 0.24) the model shows a sensitivity of 83% (CI 95%, 74 to 90%) and a specificity of 71% (CI 95%, 61 to 80%). CONCLUSIONS: In patients with kidney transplants, the presence of acute tubular necrosis, together with the time the blood concentration of tacrolimus remained within the predetermined therapeutic interval, permitted the identification of patients with a higher probability of having an acute rejection episode during the first two weeks following the transplant.

Predicción de riesgo de rechazo agudo en pacientes con trasplante renal / Predicting risk of acute rejection in patients with kidney transplants

Objetivo: Construir un modelo para predecir el riesgo de rechazo agudo al trasplante renal considerando variables relacionadas con el tratamiento inmunosupresor instaurado, el receptor, el donante y el órgano trasplantado. Método: Estudio de cohortes en una población de 68 pacientes con trasplante renal en tratamiento con tacrolimus en triple terapia. La predicción del riesgo de rechazo agudo se realizó mediante un análisis de regresión logística utilizando como variables explicativas la edad, sexo, presencia de retrasplante, número de incompatibilidades HLA, tiempo de isquemia fría, necrosis tubular aguda, inducción con basiliximab o timoglobulina y tipo de tratamiento. También se evaluó la contribución de variables asociadas a la determinación de la concentración sanguínea de tacrolimus, entre ellas la media de la concentración sanguínea, el número de valores por debajo e incluidos en el intervalo terapéutico predefinido, y el tiempo que dichos valores permanecían en las condiciones referidas. Resultados: El análisis de regresión logística indica que el riesgo de rechazo agudo depende de la necrosis tubular aguda (odds ratio [OR] = 3; intervalo de confianza [IC] del 95 %, 0,7 a 13,2) y del tiempo que las concentraciones sanguíneas de tacrolimus permanecen dentro del intervalo terapéutico (OR = (..) (AU)

Objective: Create a model to predict the risk of acute rejection of kidney transplant considering variables related to the immunosuppressant agent used, the receiver, the donor and the transplanted organ. Methods: Cohort study in a population of 68 patients with kidney transplants being treated with tacrolimus triple therapy. Predicting the risk of acute rejection was carried out with a logistic regression analysis using age, sex, re-transplant status, number of HLA incompatibilities, cold ischaemia time, acute tubular necrosis, induction with basiliximab or thymoglobulin and treatment type as explanatory variables. The contribution of variables associated with determining the blood concentration of tacrolimus was also evaluated; these variables include the average blood concentration, the number of values below and included in the pre-defined therapeutic interval, and the time during which those values remained within that interval. Results: The logistic regression analysis indicates that the risk of acute rejection depends on the acute tubular necrosis (OR: 3; CI 95 %, 0.7 to 13.2) and on the time that the blood concentrations of tacrolimus remains within the therapeutic interval (OR: 0.8; CI 95 %, 0.7 to 0.9).The final model presents an optimal discrimination power (AUCROC:77 %; CI 95 %, 62 % to 92 %). For the selected cut-off point (probability greater than or equal to 0.24) the model shows a sensitivity of 83 % (CI 95 %, 74 to 90 %) and a specificity of 71 % (CI 95 %, 61 to 80 %).Conclusions: In patients with kidney transplants, the presence of acute tubular necrosis, together with the time the blood concentration of tacrolimus remained within the predetermined therapeutic interval, permitted the identification of patients with a higher probability of having an acute rejection episode during the first two weeks following the transplant (AU)

Long-term NSAID treatment directly decreases COX-2 and mPGES-1 production in the articular cartilage of patients with osteoarthritis.

OBJECTIVE: To simultaneously study the effect of a selective cyclooxygenase-2 (COX-2) inhibitor and that of a classic non-steroidal anti-inflammatory drug (NSAID) on the expression of pro-inflammatory genes in the cartilage of patients with severe knee osteoarthritis (OA) and in cultured human OA chondrocytes. METHODS: A 3-month clinical trial was carried out on 30 patients with severe knee OA scheduled for knee replacement surgery. Patients were randomized into two groups: patients treated with celecoxib (CBX) and patients treated with aceclofenac (ACF). OA patients who did not want to be treated served as the control group. After surgery, cartilage was processed for molecular biology studies. We also employed cultured chondrocytes from different OA patients to examine NSAID effects on pro-inflammatory gene expression in cells stimulated with interleukin (IL)-1beta. RESULTS: Both CBX and ACF inhibited COX-2, microsomal prostaglandin E synthase-1 (mPGES-1) and inducible nitric oxide synthase (iNOS) synthesis in the articular cartilage of OA patients. In cultured chondrocytes, both NSAID decreased COX-2 and mPGES-1 synthesis and prostaglandin E2 (PGE2) release induced by IL-1beta, while no effect was observed on nitric oxide or iNOS synthesis. In OA patients, only CBX decreased tumor necrosis factor alpha and IL-1beta expression in the cartilage, while both NSAID diminished IL-1beta induced cytokine synthesis in cultured OA chondrocytes. CONCLUSIONS: Both NSAID diminished PGE2 release and induced a decrease in COX-2 and mPGES-1 synthesis in the cartilage from OA patients and in OA chondrocytes. These data suggest that prolonged therapy with PGE2 blocking agents decreases PGE2 production not only by direct inhibition of COX-2 activity, but also by down-regulating COX-2 and mPGES-1 synthesis in the cartilage. However, CBX and ACF seem to have a different anti-inflammatory profile in controlling pro-inflammatory gene expression in the cartilage.

Diacerein has a weak effect on the catabolic pathway of human osteoarthritis synovial fibroblast--comparison to its effects on osteoarthritic chondrocytes.

OBJECTIVES: Synoviocytes play a crucial role in the inflammatory response leading to structural damage in OA. Our aim was to assess the effects of diacerein and NSAIDs on cellular responses of synoviocytes associated with inflammation and structural integrity of cartilage in OA. METHODS: The effects of diacerein, celecoxib, diclofenac, meloxicam and indomethacin on prostaglandin (PG) E2 production, cyclo-oxygenase-2 (COX-2) protein expression, nitrite levels, presence of MMP-1 and -13, and activation of nuclear factor-kappaB (NF-kappaB) were studied on stimulated OA synoviocytes and chondrocytes. RESULTS: Diacerein and NSAIDs inhibited IL-1beta-stimulated NF-kappaB activation in synoviocytes and chondrocytes except indomethacin in synoviocytes. Diacerein further increased COX-2 protein expression and PGE2 synthesis in synoviocytes stimulated with IL-1beta, while no effect was observed on stimulated chondrocytes. NSAIDs diminished until almost basal levels PGE2 release in both cells and, surprisingly, these drugs also diminished COX-2 protein expression both in synoviocytes and chondrocytes. With regard to structural mediators, diacerein decreased MMP-13 levels in synoviocytes but did not modify MMP-1 presence. NSAIDs induced a significant increase in MMP-1 levels in both cell types and in MMP-13 levels in chondrocytes. CONCLUSIONS: Diacerein does not seem to reduce but rather increase inflammatory mediators in synoviocytes, while it does not overall affect chondrocyte inflammatory profile.

Tomato genotype and Azospirillum inoculation modulate the changes in bacterial communities associated with roots and leaves.

AIMS: To evaluate the effect of plant variety and Azospirillum brasilense inoculation on the microbial communities colonizing roots and leaves of tomato (Lycopersicon esculentum Mill.) plants. METHODS AND RESULTS: Seeds of cherry and fresh-market tomato were inoculated with A. brasilense BNM65. Sixty days after planting, plants were harvested and the microbial communities of the rhizoplane and phyllosphere were analysed by community-level physiological profiles (CLPP) using BIOLOG EcoPlates and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Differences on the rhizoplane and phyllosphere bacterial communities between the two tomato types were detected by principal component analysis of the CLPP; DGGE fingerprints also showed differences at the phyllosphere level. Fresh-market tomato had a more complex phyllosphere bacterial community than cherry tomato, as determined by DGGE profiles. Physiological and genetic changes on phyllosphere and rhizoplane bacterial communities by Azospirillum seed inoculation were evident only on cherry tomato. CONCLUSIONS: Tomato genotype affects the response of native bacterial communities associated with the roots and leaves to A. brasilense seed inoculation. SIGNIFICANCE AND IMPACT OF THE STUDY: The successful implementation of Azospirillum inoculation requires not only the consideration of the interactions between A. brasilense strains and plant genotypes, but also the plant-associated microflora.

Expression of the peptide C4b-binding protein beta in the arthritic joint.

BACKGROUND: C4b-binding protein (C4BP) is a plasma oligomeric glycoprotein that participates in the regulation of complement and haemostasis. Complement-regulatory activity depends on the C4BPalpha-polypeptide, whereas the C4BPbeta-polypeptide inactivates protein S, interfering with the anti-coagulatory protein C-dependent pathway. OBJECTIVE: To investigate the expression of C4BPbeta in the rheumatoid joint. METHODS: Expression of C4BP was studied in synovial explants from patients with rheumatoid arthritis, osteoarthritis and healthy controls, using immunohistochemistry and in situ hybridisation. C4BP isoforms and free C4BPbeta were studied in synovial effusions from patients with rheumatoid arthritis, osteoarthritis and microcrystalline arthritis (MCA) by immunoblotting; total and free protein S levels were studied by enzyme immunoassay. RESULTS: C4BPbeta was overexpressed in the synovial membranes of patients with rheumatoid arthritis, in close association with the severity of synovitis and the extension of interstitial fibrin deposits. As many as 85% fluids from patients with rheumatoid arthritis contained free C4BPbeta, whereas this unusual polypeptide was present in 50% fluids from patients with MCA and 40% fluids from patients with osteoarthritis. Free protein S at the effusions was pathologically reduced in patients with rheumatoid arthritis and MCA, and remained normal in patients with osteoarthritis. CONCLUSION: C4BPbeta is expressed by the inflamed synovial tissue, where it can participate in processes of tissue remodelling associated with invasive growth.

Long term NSAID treatment inhibits COX-2 synthesis in the knee synovial membrane of patients with osteoarthritis: differential proinflammatory cytokine profile between celecoxib and aceclofenac.

OBJECTIVE: To compare the effect of celecoxib with that of a classic non-steroidal anti-inflammatory drug (NSAID) on synovial inflammation and on the synovial expression of proinflammatory genes in patients with knee osteoarthritis (OA). METHODS: 30 patients with severe knee OA scheduled for total knee replacement surgery were included in a 3 month clinical trial. They were randomised to two groups: patients treated with celecoxib (CBX) (200 mg/24 h) and patients treated with aceclofenac (ACF) (100 mg/12 h). Those patients with OA who did not want to be treated with NSAIDs served as a control group. During knee surgery, synovial fluid (SF) and synovial membrane (SM) were collected. A SM specimen was fixed and embedded in paraffin and another part was frozen for molecular biology studies. RESULTS: At the end of study both CBX and ACF treated patients showed a significant improvement in pain and knee function compared with controls. Both drugs significantly reduced prostaglandin E(2) (PGE(2)) SF concentration and down regulated COX-2 mRNA and protein expression at the SM. However, synovial macrophage infiltration (CD68 antigen staining) and expression of proinflammatory mediators, such as interleukin 1beta and tumour necrosis factor alpha, were decreased only by CBX treatment. CONCLUSION: Both drugs improved joint pain and function, inhibited SF PGE(2) concentration, and induced a decrease in synovial COX-2 expression and synthesis not related to the tissue inflammatory status. These data suggest that PGE(2) blocking agents may decrease PGE(2) production not only by direct COX-2 inhibition but also by down regulating COX-2 expression and synthesis. However, CBX and ACF appear to have different anti-inflammatory profiles in controlling OA synovial macrophage infiltration and proinflammatory expression.

EP2/EP4 signalling inhibits monocyte chemoattractant protein-1 production induced by interleukin 1beta in synovial fibroblasts.

BACKGROUND: Besides its proinflammatory properties, prostaglandin E(2) (PGE(2)) acts as a regulator of the expression of inducible genes. Inhibition of PGE(2) synthesis might thus result in a paradoxical deleterious effect on inflammation. OBJECTIVE: To examine the effect of PGE(2) on monocyte chemoattractant protein-1 (MCP-1) expression in cultured synovial fibroblasts (SF) stimulated with interleukin (IL)1beta. METHODS: MCP-1 expression was assessed in SF stimulated with IL1beta in the presence of PGE(2) or different NSAIDs by RT-PCR or northern blot and immunocytochemistry. Expression of cyclo-oxygenase (COX) isoforms was studied by western blot techniques. The role of PGE(2) receptors (EP) in PGE(2) action was assessed employing EP receptor subtype-specific agonists. RESULTS: PGE(2) significantly inhibited IL1beta induced MCP-1 expression in SF in a dose dependent manner. IL1beta increased COX-2 and did not alter COX-1 synthesis in SF. 11-Deoxy-PGE(1), an EP(2)/EP(4) agonist, reproduced PGE(2) action on MCP-1 expression. Butaprost, a selective EP(2) agonist, was less potent than PGE(2). Sulprostone, an EP(1)/EP(3) agonist, had no effect on IL1beta induced MCP-1 expression. Inhibition of endogenous PGE(2) synthesis by NSAIDs further enhanced MCP-1 mRNA expression in IL1beta stimulated SF, an effect prevented by addition of exogenous PGE(2). CONCLUSION: Activation of EP(2)/EP(4) receptors down regulates the expression of MCP-1 in IL1beta stimulated SF, while PGE(2) pharmacological inhibition cuts off this signalling pathway and results in a superinduction of MCP-1 expression. The data suggest that NSAIDs may intercept a natural regulatory circuit controlling the magnitude of inflammation, which questions their continuous administration in inflammatory joint diseases.

A fibrin based model for rheumatoid synovitis.

Intracavitary fibrin clots may initiate pannus formation and the immunopathology of RA. Two critical steps, probably host dependent, may determine the development of RA: an altered regulation of extravascular haemostasis or an aberrant reactivity of synovial fibroblasts to the adhered fibrin clots. Current treatments for RA target events downstream of fibrin deposition, perhaps agents acting at an earlier stage should be tried.