Evaluación in Vitro de la Biocompatibilidad de Cuatro Cementos Selladores con Osteoblastos Humanos / In vitro Evaluation of Four Sealer Cements Biocompatibility with Human Osteoblasts

RESUMEN: El correcto sellado apical es un paso importante durante el tratamiento de conductos, para esto, se utilizan puntas de gutapercha y cemento sellador, de este último existen diversas formulaciones químicas en el mercado, por lo cual es importante tomar en cuenta los efectos que estas pueden tener en el proceso de cicatrización periapical. El propósito de este estudio fue evaluar la biocompatibilidad de cuatro cementos selladores con diferente composición química con osteoblastos humanos. Se prepararon extractos de cementos selladores a con dos concentraciones (10 mg/mL y 40 mg/mL) y dos tiempos de exposición (10 min y 8 h), estos fueron colocados en contacto con osteoblastos humanos para evaluar la proliferación y citotoxicidad a 24, 72 y 96 h con sus respectivos controles y blancos. Se realizó un análisis estadístico con ANOVA de un factor y la prueba de comparaciones múltiple de Bonferroni. Los resultados obtenidos, tanto en el ensayo de citotoxicidad como en el de proliferación, indicaron que el cemento a base de resina no es biocompatible con osteoblastos. El cemento a base de poli-dimetilxilosano fue el único que no mostró citotoxicidad a ningún de tiempo de exposición y concentración examinadas en este estudio.

ABSTRACT: Correct apical sealing is an important step during root canal treatment, hence, gutta-percha points and sealant are used. There are several chemical compositions on the market, so it is important to evaluate the effects of these in the periapical healing process. The aim of this study was to evaluate the biocompatibility of four sealer cements with different chemical composition placed in contact with human osteoblast. Different extracts were prepared at two concentrations (10 mg/mL and 40 mg/mL) and two exposure times (10 min and 8 h) these were placed in contact with human osteoblast to evaluate cytotoxicity and proliferation at 24, 48 and 72 h with their respective controls and blanks. A statistical analysis was performed with ANOVA of one factor and Bonferroni post hoc. Results obtained in cytotoxicity and proliferation assays, indicated that the resinbased cement is not biocompatible with osteoblast. The poly-dimethylxilosanbased cement was the only that did not show cytotoxicity at any time of exposure and concentration examined in this study.

Glucose-6-phosphate dehydrogenase deficiency in northern Mexico and description of a novel mutation.

Glucose-6-phosphate dehydrogenase deficiency (G6PD) is the most common enzyme pathology in humans; it is X-linked inherited and causes neonatal hyperbilirubinaemia, chronic nonspherocytic haemolytic anaemia and drug-induced acute haemolytic anaemia. G6PD deficiency has scarcely been studied in the northern region of Mexico, which is important because of the genetic heterogeneity described in Mexican population. Therefore, samples from the northern Mexico were biochemically screened for G6PD deficiency, and PCR-RFLPs, and DNA sequencing used to identify mutations in positive samples. The frequency of G6PD deficiency in the population was 0.95% (n = 1993); the mutations in 86% of these samples were G6PD A(-202A/376G), G6PDA(-376G/968C) and G6PD Santamaria(376G/542T). Contrary to previous reports, we demonstrated that G6PD deficiency distribution is relatively homogenous throughout the country (P = 0.48336), and the unique exception with high frequency of G6PD deficiency does not involve a coastal population (Chihuahua: 2.4%). Analysis of eight polymorphic sites showed only 10 haplotypes. In one individual we identified a new G6PD mutation named Mexico DF(193A>G) (rs199474830), which probably results in a damaging functional effect, according to PolyPhen analysis. Proteomic impact of the mutation is also described.

[« Double flag ¼ flap nipple reconstruction. Clinical evaluation on 70 cases]. / Reconstruction mamelonnaire par lambeau en « double drapeau ¼. Évaluation clinique à propos de 70 cas.

BACKGROUND: Since 2004, we have chosen a nipple reconstruction with a "double flag" local flap. We have retrospectively assessed its results in the long term and identified the factors likely to influence them. METHODS: Seventy reconstructions have been analyzed through a subjective study and objective one through measures of size and the projection of the nipple. RESULTS: They were examined in an average of 15months. The result was found satisfying for 74% of the patients and 66% of the clinician. The diameter of new nipple was 12.6mm (less 1mm than controlateral). The average height was 4.9mm and an average projection ratio was 0.8 (between reconstructed and controlateral). The limit of satisfaction of the patients and of the surgeon corresponded to a 0.7 projection ratio. We observed 4% of complete necroses. CONCLUSIONS: These results, compared to those of the literature, are very encouraging. The tegument quality of the reconstructed breast has a great influence on that of the nipple.

Parasitaemia levels in Plasmodium chabaudi infected-mice modify IFN-gamma and IL-10 expression after a homologous or heterologous challenge.

CB6F1 mice infected with the nonlethal Plasmodium chabaudi chabaudi AS suffer parasitaemia levels up to 40% (full parasitaemia, FP) and develop both homologous and heterologous (against the lethal Plasmodium yoelii 17XL) protective immunity. However, if mice are treated with anti-malarial drug when parasitaemia is below 10% (low parasitaemia, LP), they only develop homologous immunity. For the better understanding of this interesting dissociation related to the degree of parasitaemia, in this work, we studied the genetic expression of some cytokines. We found that during primary parasitaemia both FP and LP mice showed at first a TNF-alpha, IL-2 and IFN-gamma response which is followed by an IL-4 and IL-10 response. When FP and LP mice were challenged with either the homologous (FP + AS and LP + AS mice) or the heterologous parasite (FP + 17XL and LP + 17XL mice), we observed that LP + 17XL mice, which failed to develop heterologous immunity and succumbed to the challenge, showed a stronger IFN-gamma and a weaker IL-10 expression than FP + 17XL mice, which developed heterologous immunity and survived the challenge. The importance and the possible implications of these findings are discussed.

The effect of an anti-inflammatory pentapeptide produced by Entamoeba histolytica on gene expression in the U-937 monocytic cell line.

OBJECTIVE: Evaluate the Monocyte Locomotion Inhibitory Factor (MLIF) effect upon the expression of genes encoding human cytokines, receptors and related factors in the human cell line U-937. MLIF (Met-Gln-Cys-Asn-Ser) is an anti-inflammatory pentapeptide produced by Entamoeba histolytica that inhibits many human monocyte functions. MATERIAL AND METHODS: U-937 cell line cultured (24 hrs/RPMI). RNA extracted by Trizol method. 385 genes were analyzed on microarray membranes, complement by real-time RT-PCR and protein expression of some affected genes. RESULTS: MLIF had a preferentially inhibitory effect on gene expression; four genes were over-expressed and 13 underexpressed in MILF vs. simple medium - constitutive expression. Three genes are over-expressed and 19 under-expressed in MLIF/PMA vs. PMA - induced expression. CONCLUSIONS: Many modified genes are products regulated by the Nuclear Factor-kappaB and Mitogen Activated Protein Kinase pathways, suggesting MLIF involvement with these two major pathways for the modulation of the inflammation and immune responses.

[Early detection of leptomeningeal metastasis in patients with metastatic breast carcinoma: validation of CA 15-3 measurement in cerebrospinal fluid]. / Détection précoce des métastases leptoméningées chez les patientes atteintes de cancer du sein métastatique: validation du dosage du CA 15-3 dans le liquide céphalorachidien.

Fifteen per cent of metastatic breast cancer will develop symptomatic leptomeningeal metastases. The introduction of trastuzumab (Herceptin) therapy has improved the response rates of survival of patients with metastatic breast cancer overexpressing HER2. Although previous studies are retrospective and of limited number, involving small study groups and different types of patient management, several authors have reported a 30% incidence of leptomeningeal metastases in patients with metastatic breast cancer overexpressing HER2 who were treated with trastuzumab, while 70 to 80% of cases of the disease were controlled systemically. In order to improve control of the disease at the level of the central nervous system (CNS), routine detection of leptomeningeal metastases in high-risk patients could be offered. CA 15-3 in cerebrospinal fluid (CSF) detection might be useful in helping to diagnose CNS metastases, particularly where cytology results are negative--which applies to 30% of cases--because tumor markers are more sensitive in detecting the tumor process. Our study validate CA 15-3 measurement in CSF and reference values were given.

[Retrospective analysis of 108 ductal carcinomas in situ of the breast treated by radiosurgery association]. / Analyse rétrospective de 108 carcinomes canalaires in situ mammaires traités par association radiochirurgicale.

PURPOSE: To evaluate survival and prognostic factors of 108 patients with clinically or mammographically detected ductal carcinoma in situ (DCIS), treated from 1980 to 1996 by complete local excision followed by external irradiation. PATIENTS AND METHODS: The median age was 51 (range 37-80). All the patients underwent surgery consisting of a wide resection of the mammary gland harbouring the tumour. The surgical specimens were sent to the pathologists to get information on histology and margin clearance; all the slides were reviewed by one of us to assess the tumoral diameter. External beam therapy was delivered within 8 weeks after surgery. The prescribed irradiation dose was 50 Gy in 25 fractions to be given in 5 weeks. The median duration of follow-up was 93 months (range 40-173). RESULTS: There were nine patients with local recurrence (8.3%); three patients had local recurrence of DCIS and six patients developed invasive breast cancer. The treatment of local recurrence consisted of mastectomy with or without axillary dissection (eight cases) and quadrantectomy (one case). The 5-year and 10-year ipsilateral recurrence-free rate was respectively 92 and 89%. The 10-year cause specific survival was 100%. In univariate analysis, size>or=10 mm, age<45 years old and margin status were significant P=0,02, P=0,03, P=0,005; margin status was significant in multivariate analysis (P<0,02). CONCLUSION: These results are in keeping with those of the literature. They could be improved by the mass screening campaign, which is going on since January 1990 among women aged 50-74 years.

Differential pattern of cytokine expression by macrophages infected in vitro with different Mycobacterium tuberculosis genotypes.

It has been shown recently that different genotypes of Mycobacterium tuberculosis induce distinct immune responses in the host, as reflected by variations in cytokine and iNOS expression. Because these molecules are probably regulated by multiple factors in vivo this complex phenomenon was partially analysed by assessing cytokine and iNOS expression by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) in an in vitro model of bone marrow-derived macrophages infected with three different M. tuberculosis genotypes: Canetti, H37 Rv and Beijing. Although the three genotypes induced production of iNOS and the different cytokines tested at 24 h post-infection, macrophages infected with the Beijing isolate expressed the highest levels of mRNA for iNOS, interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, IL-12 cytokines and lower levels of IL-10 compared with cells infected with other genotypes. This expression pattern has been associated with infection control, but during infection in vivo with the Beijing genotype it is lost upon progression to chronic phase. The failure to control infection is likely to be influenced by cytokines produced by other cell types and bacterial molecules expressed during the course of disease. Results presented in this work show that each genotype has the ability to induce different levels of cytokine expression that could be related to its pathogenesis during infection.

Quantification of cytokine gene expression using an economical real-time polymerase chain reaction method based on SYBR Green I.

Assessment of cytokine expression has become crucial to understand host responses to infections as well as autoimmunity. Several approaches including Northern blot, RNase protection assay and enzyme-linked immunosorbent assay have been used for this purpose, but they are time consuming, labour intense, and relatively large quantity of the samples is usually required. Recently, a technique termed real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to determine genetic expression with great sensitivity and specificity; however, specialized instrumentation and costly reagents are usually needed. We aimed at using low-cost reagents for real-time PCR. This was achieved by adapting a conventional RT-PCR protocol to the quantitative real-time format, by the addition of the SYBR Green I reagent. We validated the approach by assessing the cytokine gene expression of murine splenocytes upon stimulation with phorbol 12-myristate 12-acetate (PMA)-ionomycin. The results using this technique were compared with those obtained with the well-established gene array method. We conclude that the use of the SYBR Green I reagent during real-time RT-PCR provides a highly specific and sensitive method to quantify cytokine expression with accuracy and no post-PCR manipulation.

Tubulin detyrosination is a frequent occurrence in breast cancers of poor prognosis.

Tubulin, the dimeric subunit of microtubules, is a major cell protein that is centrally involved in cell division. Tubulin is subject to specific enzymatic posttranslational modifications including cyclic tyrosine removal and addition at the COOH terminus of the alpha-subunit. Tubulin is normally extensively tyrosinated in cycling cells. However, we have previously shown that detyrosinated tubulin accumulates in cancer cells during tumor progression in nude mice. Tubulin detyrosination, resulting from suppression of tubulin tyrosine ligase and the resulting unbalanced activity of tubulin-carboxypeptidase, apparently represents a strong selective advantage for cancer cells. We have now analyzed the occurrence and significance of tubulin detyrosination in human breast tumors. We studied a total of 134 breast cancer tumors from patients with or without known complications over a follow-up period of 31 +/- 10 months. The mean age of the patients at the time of diagnosis was 57 years. For each patient, detailed data concerning the histology and extension of the tumor were available. Tumor cells containing detyrosinated tubulin were visualized by immunohistochemical staining of paraffin-embedded tissue sections. Cancer cells with detyrosinated tubulin were observed in 53% of the tumors and were predominant in 19.4% of the tumors. Tubulin detyrosination correlated to a high degree of significance (P < 0.001) with a high Scarf-Bloom-Richardson (SBR) grade, a known marker of tumor aggressiveness. Among SBR grade 1 tumors, 3.8% were strongly positive for tubulin detyrosination compared with 65.4% of the SBR grade 3 tumors. The SBR component showing the strongest correlation with tubulin detyrosination was the mitotic score. In the entire patient population, neither the SBR grade nor the detyrosination index had significant prognostic value (P = 0.11, P = 0.27, respectively), whereas a combined index was significantly correlated with the clinical outcome (P = 0.02). A preliminary subgroup analysis indicated that tubulin detyrosination may define high- and low- risk groups in breast cancer tumors with an SBR grade of 2. Our study shows that tubulin detyrosination is a frequent occurrence in breast cancer, easy to detect, and linked to tumor aggressiveness.

[Evaluation of the diagnostic usefulness of CA125 immunoscintigraphy for ovarian carcinoma follow-up after treatment: contribution of this technique in Grenoble University Medical Center]. / Evaluation de l'utilité diagnostique de l'immunoscintigraphie au CA.125 dans le suivi des carcinomes du revêtement épithélial de l'ovaire après traitement: apport décisionnel de la technique au CHU de Grenoble.

Immunoscintigraphy using indium-111-labeled OC125 monoclonal antibody F(ab')2 fragments is a technic complementary of morphological imaging (i.e. ultrasonography and computed tomography). It allows early detection of recurrences of ovarian carcinomas. We performed immunoscintigraphy 30 times in 26 patients who previously underwent radical treatment for ovarian carcinoma, and were suspected to have a recurrence. Our purposes were appreciation of diagnostic accuracy of the method, and above all its impact on clinical decisions and evolution of the patients. There were, after reevaluation of the results, 18 true positives, 7 true negatives, 3 false negatives and 2 false positive cases (sensitivity 85.7%, specificity 77.8%). Bayesian analysis showed positive and negative predictive values of 86% and 87% when probability of recurrence a priori was 50%, and 80% and 58% when probability of recurrence a priori was 70%. The result of immunoscintigraphy contributed to clinical decisions in 24 cases out of 30, and led to a correct decision for the patient in 21 cases. Conversely, for the 6 cases in which the result has not been considered, to take this result into account would have been beneficial in 4 cases, but harmful in 2. Finally, survival tended to be longer when immunoscintigraphy was negative, which could be associated with a better prognosis. We conclude that OC125-immunoscintigraphy may be useful for ovarian carcinoma follow-up and may contribute to a better therapeutic strategy.

Prognostic value of quantitative cytometry in a series of 415 T1T2/N0N1/M0 breast cancer patients--preliminary results.

Identifying prognostic markers in local regional breast carcinomas remains an important challenge today. DNA content obtained by flow cytometry, has been found to be of prognostic value; results with other methods remain less clear. This report describes DNA image cytometry patterns which are assessed with respect to disease-free survival. From June 1982 to December 1992, 415 patients under 75 years of age, without any previous or synchronous carcinoma, suffering from an invasive breast cancer classified as T1 (52.8%), T2 (47.2%), N0 (65.1%) N1 (34.9%), MO according to clinical TNM staging, were enrolled in this study. The median age was 53 (28-75) and 58.8% of the patients were premenopausal; 85.3% underwent a breast conservative procedure and 14.7% a modified radical mastectomy followed by postoperative irradiation. Histological axillary lymph node status, Scarff-Bloom grade and/or cytological grade and, oestrogen receptor content were used in decision-making for adjuvant treatment: hormonotherapy (48%) or chemotherapy (18.8%). Imprints were taken from the macroscopically visible lesion at the time of surgery, and a Feulgen staining was carried out on air dried smears to be analysed using the Samba 200 cell image processor (Alcatel TITN, France). Five parameters were systematically assessed: proliferation index; DNA histogram, integrated optical density, DNA malignancy grade, ploidy balance. With a median follow-up of 36 months (0-105), proliferation index (P = 0.0008), DNA histogram (P = 0.0017), integrated optical density (IOD) (P = 0.018) and DNA malignancy grade (P = 0.017) had a significant prognostic value on disease-free survival estimated by the Kaplan-Meier method. When these parameters were included in a Cox proportional regression hazards model, PR (P = 0.01), Scarff-Bloom histological grading (P = 0.02), axillary clearance (P = 0.04) were significant; however, in the same model, taking into account the axillary lymph node histological status, IOD was significant for pN- patients (P = 0.03), and proliferation index (P = 0.03) was significant for pN+. Such results need to be updated with a longer median follow-up, but they suggest that the mean DNA content, as measured by the integrated optical density (IOD), should be considered when deciding on medical adjuvant treatment with respect to patients with a negative axillary clearance.

[Prognostic value of quantitative DNA analysis, expressed in integrated optical density, in a series of 415 T1-T2, N0N1, M0 UICC breast cancers]. / Valeur pronostique du contenu en ADN, exprimé dans la densité optique intégrée, dans une série de 415 cancers du sein T1-T2, N0N1, M0 UICC.

From June 1982 to December 1992, 415 patients less than 75 years of age, without any previous or synchronous carcinoma, suffering from an invasive breast cancer classified as T1 (52.8%), T2 (47.2%), NO (65.1%) N1(34.9%), MO according to clinical TNM staging, were enrolled in this study. The median age was 53 (28-75), and 58.8% of the patients were menopaused; 85.3% underwent a breast conservative procedure and 14.7% a modified radical mastectomy followed by postoperative irradiation. Histological axillary lymph node status, Scarff-Bloom grade and/or cytological grade, estradiol receptor content, were used to set up medical adjuvant treatment: hormonotherapy (52%) or chemotherapy (18.8%). Imprints were taken from the macroscopically visible lesion at the time of surgery, and a Feulgen staining was done on air dried smears to be analyzed using the Samba 200 cell image processor (Alcatel TITN, France). Five parameters were systematically assessed: proliferation index, DNA histogram, integrated optical density, DNA malignancy grade, and policy balance. With a median follow-up of 36 months (0-105), proliferation index (P = 0.0008), DNA histogram (P = 0.0017), integrated optical density (P = 0.018), DNA malignancy grade (P = 0.017) have a significant prognostic value on disease free survival estimated by the Kaplan-Meir method. When these parameters were included in a Cox proportional regression hazards model, Scarff-Bloom histological grading (P = 0.002), positives nodes (P = 0.02), optical integrated density (P = 0.045) were significant. Such results need to be updated with a longer follow-up, but they suggest that the mean DNA content, as measured by the integrated optical density (IOD), has to be considered when deciding on medical adjuvant treatment with respect to patients with a negative axillary clearance.

[Importance of the systematic cytologic examination of breast secretions: 60 cancers diagnosed in 2120 discharges examined]. / Intérêt de l'examen cytologique systématique des sécrétions mammaires: 60 cancers diagnostiqués sur 2 120 écoulements examinés.

The systematic collection of breast secretions by manual expression of the breast and nipple in 2 120 patients examined for breast diseases has led to the following results: 529 (25%) disclose epithelial papillary hyperplasia, 136 of which underwent surgical control. Epithelial hyperplasia without cellular atypias was found in 9 fibroadenomas, 42 fibrocystic diseases, 29 papilloma/papillomatosis and 21 cancers. Atypical or malignant hyperplasia was observed in 42 cases. Out of 60 cancers, thus investigated, 31 (51%) had malignant cells in such induced nipple discharge, 8 (13%) atypical cells and 21 (35%) concomitant papillary hyperplasia. Systematic cytological examination of breast secretion allows to control intraductal epithelial hyperplasia and to eventually disclose malignant tumor.