MP2 Study of Physisorption of Molecular Hydrogen onto Defective Nanotubes: Cooperative Effect in Stone-Wales Defects.

We use large-scale MP2 calculations to investigate the physisorption of molecular hydrogen on (9,0) defective carbon nanotubes (CNTs) of C72H18. These large (supra)molecular systems are typically studied using conventional DFT methods, which do not describe well the van der Waals interactions responsible for this process. Here we use CCSD(T)-calibrated MP2 calculations to estimate binding energies by considering four defective structures (hydrogenated divacancy, octagon-pentagon, and two Stone-Wales defects). The largest physisorption energies for the nondefective CNT are for configurations in which H2 points toward the center of one ring. The computed interaction energies for defect-free CNT are in the range 5.7 to 5.9 kJ/mol, in good agreement with the experimental value of 5.98 kJ/mol. The defects introduced in the (9,0)-CNT increase the surface area of the nanotube, such that the largest surface in found in the 55-77 Stone-Wales defective CNT that furthermore is the most aromatic. Only that defect enlarges the physisorption binding energy, which can become >25% larger. Moreover, a cooperative effect in the adsorption of H2 not appearing in the regular structure is found.

Eficacia de un dentífrico con revelador en la eliminación de placa bacteriana denta / Efficacy of a dentifrice containing a developer in the removal of dental plaque

Dos de las enfermedades más prevalentes en el mundo y en nuestro país son la caries dental y la enfermedad periodontal. Pueden darse en cualquier persona y son la causa más importante de pérdida de los dientes, afectando la salud general y la calidad de vida de los individuos. Son producidas por microorganismos que forman la placa bacteriana. Para eliminarla es necesario realizar una correcta higiene oral. Como la placa es invisible inicialmente, para visualizarla se utilizan colorantes (reveladores de placa). Con el objeto de comprobar la eficacia de un dentífrico que ya tiene incorporada una sustancia natural obtenida de la Bixa Orellana como revelador de placa, se planteó un ensayo clínico aleatorizado, en 139 niños de 5 a 12 años de edad, pacientes de la Facultad de Odontología de la UNA y de clínicas privadas de Asunción en el año 2008, los cuales fueron divididos en dos grupos cada uno de los cuales utilizó, previa tinción de placa y enseñanza de cepillado, un tipo de pasta dental (con o sin revelador) durante una semana al cabo de la cual se midió el índice de placa. Se invirtió la intervención en la segunda semana y se volvió a medir el índice de placa, para ver persistencia de efectos. Se comprobó la reducción significativa de la placa bacteriana, llegando a un promedio de 6 a 14% del índice de O´Leary (compatible con salud), lográndose una reducción de 56 a 70% en los grupos que utilizaron la pasta con el colorante

Clinical study on Alternaria spores sensitization.

BACKGROUND: sensitization to fungi spores is often associated with sensitization to other allergens and so it is difficult to estabilish the exact cause of illness. OBJECTIVE: the aim of the study was to evaluate a group of patients monosensitized to Alternaria and to establish the prevalence, periodicity and clinical profile of this kind of sensitization. METHODS: 37 subjects who were monosensitized to Alternaria (prick-test positive), according to a previous epidemiological study were evaluated. Clinical symptoms and the period of their occurrence were taken into consideration as well as immunological parameters (RAST). RESULTS: 20 patients (over 50%) included in the study proved to be affected by asthma associated with other allergic symptoms and 22 patients (60%) presented perennial symptoms. The RAST carried out on 34 monosensitized subjects proved positive in 11 and negative in 23. CONCLUSIONS: Alternaria sensitization is characterized by a perennial periodicity with severe respiratory symptoms (asthma) which occur primarily in children. Prick test is preferable to and more reliable than RAST as a diagnostic test.

[Treatment of pelvic endometriosis with goserelin acetate or nafarelin acetate. Comparative study]. / Tratamiento de la endometriosis pélvica con acetato de goserelina o acetato de nafarelina. Estudio comparativo.

The medical treatment of the pelvic endometriosis includes the hormonal therapy that attacks directly to the endometriosis lesions or indirectly it inhibits the endometrial proliferation and it induces atrophy through the estrogenic deprivation, or to inclination of a combination of these effects. The objective of the present study was to evaluate the effects of two gonadotrophin release analogs in the treatment of the endometriosis. 15 patients were studied in who the diagnosis settled down of endometriosis by means of laparoscope; these were formed two groups in aleatory form and there were administered to each one them nafarelin acetate in dose of 200 or 400 micrograms each 12 hours or goserelin acetate in dose of 3.6 mg every 21 days; the serum levels of the stimulating follicle, luteinizing, estradiol and prolactin hormones were determined at intervals of four weeks and the secondary effects of these drugs were reported. There were carried out a second look laparoscope in 10 patients. The statistical analysis was carried out by means of the test of hypothesis of Wilcoxon and X2. Inside those results were found that as much the nafarelin as the Goserelin are able to diminish the gonadotropins and estradiol serum levels. Both analogs are effective for the control of the endometriotic lesions. The secondary symptoms are smaller and better supported by the patients using nafarelin. In conclusion as much the nafarelin as the goserelin are appropriate medications for the treatment of the pelvic endometriosis and the election between one or another one can settle down for the comfort of application, secondary effects and cost.

Individual V(H) promoters vary in strength, but the frequency of rearrangement of those V(H) genes does not correlate with promoter strength nor enhancer-independence.

The process of V(D)J recombination is highly regulated. Germline transcription of unrearranged gene segments precedes V(D)J rearrangement, and the correlation between germline transcription and accessibility for recombination is strong; thus it has been suggested that germline transcription may be required for rearrangement. If germline transcription is essential for rearrangement, then the level of transcription of individual gene segments might affect the relative frequency of recombination of those genes. Also, since the intronic enhancer, E(mu), is very distant from V(H) genes before they rearrange, then any promoters which were enhancer dependent might have a transcriptional advantage. Here we study in luciferase vectors the promoters of three functional genes of the V(H)S107 family, and compare them to that of the most frequently rearranging gene in the mouse I(g)H locus, V(H)81X, and to a V(H)J558 gene. Within the V(H)S107 family, the three V(H) genes rearrange with very different relative frequencies, with V1 rearranging the most, and V13 seldom rearranging. We show that only the strong V(H)J558 promoter has significant luciferase reporter gene activity in the absence of E(mu). V1 has only 20% as much activity as J558 in the absence of E(mu), and the other promoters have less than 8% of the activity of J558. Notably, the 81X promoter has essentially no enhancer-independent activity. In the presence of E(mu), V1 has equivalent activity to J558, while the other promoters show much less activity. Again, 81X is the weakest promoter of all, despite being the most frequently rearranging gene. Finally, we show that the steady state level of V(H)S107 and V(H)7183 germline transcripts in vivo is very low. Thus, these data show little correlation between the strength or enhancer-independence of these V(H) promoters and the relative frequency of recombination of the corresponding V(H) genes. In addition, the data show that individual V(H) promoters have different strengths even in the presence of E(mu), demonstrating that even promoters within a single V(H) family can be quite heterogeneous.

Clinical study on Alternaria spores sensitization

Background: sensitization to fungi spores is often associated with sensitization to other allergens and so it is difficult to estabilish the exact cause of illness. Objective: the aim of the study was to evaluate a group of patients monosensitized to Alternaria and to establish the prevalence, periodicity and clinical profile of this kind of sensitization. Methods: 37 subjects who were monosensitized to Alternaria (prick-test positive), according to a previous epidemiological study were evaluated. Clinical symptoms and the period of their occurrence were taken into consideration as well as immunological parameters (RAST). Results: 20 patients (over 50%) included in the study proved to be affected by asthma associated with other allergic symptoms and 22 patients (60%) presented perennial symptoms. The RAST carried out on 34 monosensitized subjects proved positive in 11 and negative in 23. Conclusions: Alternaria sensitization is characterized by a perennial periodicity with severe respiratory symptoms (asthma) which occur primarily in children. Prick test is preferable to and more reliable than RAST as a diagnostic test (AU)

Introducción: la sensibilización a esporas de hongos se asocia frecuentemente con la sensibilización a otros alergenos, con lo que es difícil establecer la causa exacta de la enfermedad. Objetivo: la finalidad del estudio fue evaluar a grupos de pacientes monosensibilizados a Alternaria y establecer la prevalencia, periodicidad y perfil clínico de esta clase de sensibilización. Métodos: se estudiaron 37 pacientes monosensibilizados a Alternaria (prick test positivo) seleccionados en un estudio epidemiológico previo. También se consideraron los síntomas clínicos y su frecuencia, así como algunos parámetros inmunológicos (RAST). Resultado: 20 pacientes (por encima del 50 por ciento) padecían asma asociada con otros síntomas alérgicos y 22 pacientes (60 por ciento) presentaban síntomas perennes. El RAST llevado a cabo en 34 pacientes monosensibilizados, fue positivo en 11 y negativo en 23. Conclusiones: la sensibilización a Alternaria se caracterizó por ser perenne, cursar con síntomas respiratorios graves (asma) y tener lugar principalmente en niños. Es preferible y más fiable como test diagnóstico el test cutáneo que el RAST. (AU)

Decreased frequency of rearrangement due to the synergistic effect of nucleotide changes in the heptamer and nonamer of the recombination signal sequence of the V kappa gene A2b, which is associated with increased susceptibility of Navajos to Haemophilus influenzae type b disease.

Navajos and genetically related populations have a 10-fold increased incidence of Haemophilus influenzae type b (Hib) disease compared with control populations. The Vkappa gene A2 is used to encode the majority of anti-Hib Abs, and these are the highest affinity anti-Hib Abs. Navajos carry a different allele of the A2 gene segment (A2b) that is defective in its ability to undergo V-J recombination. The A2b allele has only three nucleotide changes from the commonly occurring A2a allele, two of which could potentially affect its ability to recombine. In this study we used two independent in vitro assays to test whether the nucleotide change found in the A2b promoter and/or in the A2b recombination signal sequence (RSS) might be responsible for the decrease in recombination frequency observed in vivo. Using a luciferase reporter gene assay, we found no significant difference between A2a and A2b promoter activities. However, the competition recombination substrate assay showed a 4.5-fold reduction in the relative frequency of recombination of the A2b RSS compared with A2a. We show that this decreased frequency is due to a synergistic effect of the unique nucleotide change present in the heptamer of the A2b RSS and the shared nucleotide change present in the nonamer of both A2b and A2a. This in vitro relative frequency of rearrangement is not significantly different from that observed in vivo; therefore, the A2b RSS is probably the factor associated with the increased susceptibility to Hib disease among individuals carrying the A2b allele.

Sequence of the spacer in the recombination signal sequence affects V(D)J rearrangement frequency and correlates with nonrandom Vkappa usage in vivo.

Functional variable (V), diversity (D), and joining (J) gene segments contribute unequally to the primary repertoire. One factor contributing to this nonrandom usage is the relative frequency with which the different gene segments rearrange. Variation from the consensus sequence in the heptamer and nonamer of the recombination signal sequence (RSS) is therefore considered a major factor affecting the relative representation of gene segments in the primary repertoire. In this study, we show that the sequence of the spacer is also a determinant factor contributing to the frequency of rearrangement. Moreover, the effect of the spacer on recombination rates of various human Vkappa gene segments in vitro correlates with their frequency of rearrangement in vivo in pre-B cells and with their representation in the peripheral repertoire.

Prevalence of sensitization to Alternaria in allergic patients in Italy.

BACKGROUND: The actual prevalence of sensitization to Alternaria is not known, partly due to the unreliability of diagnostic extracts. OBJECTIVE: To assess skin positivity to extracts of Alternaria in a wide population of Italian patients suffering from respiratory symptoms using a biologically standardized extract. METHODS: A total of 2942 patients were skin prick tested with Alternaria, and a panel of common inhalant allergens. Blood samples for specific IgE quantitation were taken both from patients positive and from patients negative (control group) to Alternaria extract. RESULTS: Three hundred six patients (10.4%, ranging from 1.8% in Turin to 29.3% in Cagliari) were positive to Alternaria; 37 were sensitized to only this mold, while the remaining 269 were sensitized to at least one other allergen. Of the Alternaria-positive patients, 79.7% suffered from rhinitis and 53.3% from asthma, either alone or associated with other symptoms. CONCLUSIONS: We suggest that, at least in Italy and in countries with similar climatic and environmental situations, standardized Alternaria extract should be included in the panel commonly used in investigating the allergen responsible in patients suffering from respiratory allergy.

Amikacin Bayesian forecasting in critically ill patients with sepsis and cirrhosis.

This study was designed to determine the population pharmacokinetic parameters of amikacin in two subpopulations of intensive care unit patients with sepsis and cirrhosis and sepsis without cirrhosis. The authors evaluated the usefulness of the obtained parameters to forecast the serum amikacin concentrations in a validation group of patients with sepsis and cirrhosis when used as a priori distribution in a Bayesian forecaster. The population parameters were estimated by a nonparametric expectation maximization algorithm (NPEM), and the accuracy of the predictions were evaluated through a prediction error analysis. Significant differences (p < 0.05) were found in Vd (0.668 versus 0.470 l/kg) and K (0.0701 versus 0.161 h-1) between subpopulations of patients with and without cirrhosis. The model derived for patients with cirrhosis used as a priori distribution, with and without feedback, was superior to the model derived for patients with sepsis in forecasting amikacin serum concentrations. The results show the relevance of using the specific model for the subgroup with cirrhosis as a priori distribution in a Bayesian forecaster to obtain a nonbiased prediction with an acceptable precision.

Relationship between hemodynamic and vital support measures and pharmacokinetic variability of amikacin in critically ill patients with sepsis.

OBJECTIVE: To examine the relationship between aminoglycoside disposition kinetics and hemodynamic response to sepsis, as well as vital support therapy, in critically ill patients with sepsis. DESIGN: Cross-sectional study of critically ill patients with sepsis undergoing physiologic and aminoglycoside pharmacokinetic monitoring. SETTING: Ten-bed general intensive care unit in a tertiary care center. PATIENTS: Thirty consecutive critically ill patients who had Gram-negative sepsis treated with amikacin and who were undergoing hemodynamic monitoring. INTERVENTIONS: Clinical, hemodynamic, oxygenation, and amikacin pharmacokinetic data were obtained simultaneously in each patient during aminoglycoside therapy. MEASUREMENTS AND MAIN RESULTS: Aminoglycoside pharmacokinetic values were estimated from serum amikacin concentration-time data using a nonlinear least squares regression computer program, assuming a one-compartment infusion pharmacokinetic model. Individual pharmacokinetic values were subjected to statistical analysis to explain their variability. Selection of the subset of variables to be used in the final model was performed by combining principal component analysis and multiple stepwise linear regression. The mean prediction error and the root mean square error, as expressions of bias and precision, were estimated. Mean volume of distribution was 0.47 L/kg, with a coefficient of variation of 35%. Mean serum amikacin clearance was 60.2 mL/min, with a coefficient of variation of 34%. Seventy-six percent of the variability in volume of distribution was explained by three covariates: body weight (p < .0001); oxygen extraction (p < .001); and serum albumin (p < .001). For serum amikacin clearance, 70% of the variability was explained by three covariates: creatinine clearance (p < .001); positive end-expiratory pressure (p < .01); and use of catecholamines as vital support therapy (p < .05). CONCLUSIONS: Factors related to hemodynamic response and vital support measures have a significant influence on the disposition kinetics of amikacin in severely ill patients with sepsis. Consideration of hemodynamic response and vital support measures, in addition to other previously described covariates, can be of great value in the design of initial dosing regimens.

[Sensitization and allergic pathology in a group of workers employed in insect breeding for biological pest control]. / Sensibilizzazioni e patologia allergica in un gruppo di lavoratori addetti all'allevamento di insetti utili per la lotta biologica.

On the basis of the results of a previous study and immuno-allergological tests, a group of workers employed in breeding insects for biological pest control were studied to ascertain whether any new sensitizations to arthropods recently included in the work cycle occurred and whether any new allergic diseases were observed. The results confirmed a high sensitization to Ephestia kuehniella and to Orius, the latter probably due to contamination; sensitization to Tetranicus urticae and to Planococcus citri were observed, although to a lesser degree. It is concluded that the insects used in biological pest control are highly allergogenic and that the protective devices used up to the present were ineffective; the authors therefore recommend the use of closed or semi-closed cycles along with local ventilation equipment.

Human cord blood kappa repertoire.

To determine the Vkappa gene utilization in cord blood, we made libraries of Igkappa sequences from two cord blood cDNA samples. The rearranged sequences were amplified using random amplification of cDNA ends PCR, ensuring unbiased amplification of all Vkappa genes. Although the human kappa locus contains approximately 38 potentially functional V genes, we observed that approximately 75% of the 146 sequences from our two samples used only nine Vkappa genes. Using leader-specific primers, we also amplified VkappaI and VkappaIII rearrangements from genomic DNA from one of these individuals. Nonproductive rearrangements give an approximation of the relative frequency of gene rearrangement. Some of the genes that were overused in the cDNA libraries were also observed to rearrange frequently, but others did not show high rearrangement frequencies, suggesting that cellular selection caused their increase in the periphery. Surprisingly, we observed a high frequency of rearrangements using L9, which has been reported to be a defective Vkappa gene. Sequence analysis of the unrearranged gene revealed two new functional alleles of this gene. We observed that N nucleotides were present in 29% of the productive sequences from cord blood DNA and RNA. To determine the actual rate of N region addition, we analyzed V-J junctions of rearrangements of two nonfunctional V genes. Forty-six percent of those cord blood sequences contained N regions. In comparison, 57% of junctions of the rearranged nonfunctional gene from adult PBMC contained N regions. Finally, we observed that CDR3 length heterogeneity was more pronounced for VkappaIII genes than for all of the other Vkappa families.

A defective Vkappa A2 allele in Navajos which may play a role in increased susceptibility to haemophilus influenzae type b disease.

The antibody response to H. influenzae type b (Hib) is pauciclonal, and is dominated by antibodies using the VkappaA2 gene. Navajos have a 5-10-fold increased incidence of Hib disease compared with control populations. We hypothesized that a polymorphism in one of the genes in this oligoclonal response may lead to increased disease susceptibility. Since the predominant A2+ anti-Hib antibodies have high avidity for Hib and can be unmutated, the A2 Vkappa gene was analyzed. Over half of the Navajos studied, but only one control individual, had a new allele of A2, termed A2b, with three changes from the published A2 germline sequence. One of the changes was in the recombination signal sequence, suggesting that the A2b allele might not undergo V-J rearrangement very frequently. This possibility was confirmed by analyzing the relative frequency of non-productive A2 rearrangements in A2a/b heterozygous Navajos. Many fewer A2b rearrangements were observed, showing that the A2b allele is defective in its ability to undergo rearrangement. The prevalence of this allele in Navajos may play a role in their increased susceptibility to invasive Hib disease. If so, it would underscore the importance of the germline Ig repertoire for protective antibody responses to pathogenic bacteria in unimmunized children.

A new risk of occupational disease: allergic asthma and rhinoconjunctivitis in persons working with beneficial arthropods.

Allergic asthma and rhinoconjunctivitis have been reported in workers who are constantly in close contact with arthropods during the course of breeding and research. We investigated a group of ten people working with beneficial arthropods in a "bio-factory." The following tests were carried out: skin prick test with allergens present in the workplace, baseline pulmonary function test, aspecific bronchial provocation test with ultrasonically nebulized distilled water, and pulmonary function test after workplace exposure in two subjects. The results demonstrated a good correlation between skin prick test, previous RAST positive reactions, clinical symptoms, and exposure. Pulmonary function test after workplace exposure proved positive in one asthmatic subject and in one subject with rhinoconjunctivitis. It is concluded that the evidence supports the occupational nature of these allergic diseases and that there is a need for careful surveillance and environmental monitoring within the workplace.

Escherichia coli adherence to HEp-2 cells with prefixed cells.

We describe a new method which uses cold absolute methanol-prefixed cells for adherence of enteropathogenic Escherichia coli to HEp-2 cells. We found that a method using bacteria grown in Penassay broth to 10(6) to 10(7) CFU/ml and incubated with prefixed cells for 3 h at 37 degrees C, showed 100% sensitivity and specificity against a method using live cells.

A new risk of occupational disease: allergic asthma and rhinoconjunctivitis in persons working with beneficial arthropods. Preliminary data.

For some years, biological pest control has been re-evaluated as an alternative to the indiscriminate use of pesticides. The number of "bio-factories" in Europe has been constantly increasing and today more than 100 technicians work in mass rearing of arthropods. Seven cases of asthma and allergic rhinitis are reported in 13 workers engaged in the production of beneficial arthropods. The subjects suffered from attacks of asthma and rhinitis when they came into contact with eggs and scales or waste of Chrysoperla carnea, Leptinotarsa decemlineata, Ostrinia nubilalis and Ephestia kuehniella. These attacks occurred after an average exposure period of 18 months. A RAST test, prepared by Lofarma, was carried out on subjects' serum against the above-mentioned allergens and was positive in all symptomatic subjects (7 of 13) and negative in the others. This demonstrates IgE direct sensitization mediated by complete antigens.

Relationship between oxygen consumption and oxygen delivery during anesthesia in high-risk surgical patients.

OBJECTIVE: To identify critical oxygen delivery (DO2) and oxygen extraction ratio in high-risk surgical patients studied preoperatively and intraoperatively. DESIGN: Prospective study. Consecutive series of intraoperatively monitored patients. SETTING: The surgical ICU in a tertiary care center. PATIENTS: High-risk surgical patients undergoing major noncardiac surgery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Twenty high-risk patients were studied during the preoperative and intraoperative periods. All patients were monitored with triple-lumen thermodilution catheters. Hemodynamic profiles consisted of determinations of intravascular pressures, cardiac output, and arterial and venous pulmonary gases. Oxygen transport variables were calculated by standard formulas. Multiple determinations were carried out during the perioperative period in each patient. The critical levels of DO2, determined by a polynomial regression method, were 375 and 390 mL/min/m2 in the preoperative and intraoperative periods, respectively. Oxygen extraction reached at the critical level of DO2 was significantly (p < .01) lower during the intraoperative period (31 +/- 4.5% vs. 18 +/- 2.3%). Critical mixed venous oxygen tension was significantly (p < .01) higher in the intraoperative period (36 +/- 5 vs. 46 +/- 4 torr [4.8 +/- 0.7 vs. 6.1 +/- 0.5 kPa]). CONCLUSIONS: Our data show that the intraoperative period might be associated with a reduction in tissue ability to extract oxygen. If this reduction in oxygen extraction is proportionately higher than the reduction in metabolic oxygen demand produced by anesthetic agents and hypothermia, then the critical value for DO2 may be similar to, or higher than, that value in the preoperative period. Thus, the intraoperative period represents, for this patient population, a high-risk condition for the development of a tissue oxygenation debt in the presence of a limitation in DO2. Cautious dosing of inhaled agents, maintenance of normothermia, and early optimization of the oxygen delivery/oxygen consumption relationship seem to constitute reasonable measures in the intraoperative handling of these patients in order to reduce perioperative morbidity and mortality.

Local nasal immunotherapy in allergic rhinitis to Parietaria. A double-blind controlled study.

Preseasonal local nasal immunotherapy (LNIT) by means of an extract in macronized powder form has been studied in allergic rhinitis to parietaria. Twenty-four Parietaria-sensitive patients have been studied for 18 weeks in a double-blind controlled trial. Subjects were selected on the basis of a positive skin test, RAST and intranasal challenge to Parietaria antigen. Three eight-patient groups were randomly planned: the first group was given native Parietaria product, the second modified Parietaria product, and the third placebo. During the pollen season no difference was observed in mean weekly symptom score between the three groups, while the mean weekly medication score was significantly lower in the treated groups than the control group. Only the treated groups showed a significant increase in specific nasal threshold to Parietaria after treatment. Adverse reactions to LNIT, limited to the upper respiratory tract, occurred rarely and did not interfere with the dose schedule. This study indicates that LNIT in powder form may be a suitable alternative to the traditional subcutaneous immunotherapy in terms of clinical efficacy and safety.

Magnesium pidolate in the treatment of seasonal allergic rhinitis. Preliminary data.

This paper describes how the authors, after examining the calcium modulating action of the Mg ion and its antihistamine-like action, tested a salt containing Mg (Mg pidolate, MAG2) in the clinical treatment of seasonal allergic rhinitis. A total of 38 hayfever subjects were studied. They were given 1.5 g Mg salt three times a day for one month (oral MAG2 vials); placebo was used as a control in a randomized double-blind study. The efficacy of the product was assessed using the following criteria: intensity of rhinorrhoea, daily consumption of Kleenex tissues, number of sneezes in a day, intensity of lacrimation. The results were analysed using Student's t test and demonstrated the clinical efficacy of the product in the control of seasonal allergic rhinitis symptoms (P less than 0.001).