Radiological assessment of mandibular invasion in squamous cell carcinoma of the oral cavity and oropharynx.

BACKGROUND: Preoperative assessment of mandibular bone invasion in squamous cell carcinoma of the oral cavity and oropharynx is crucial for optimizing bone resection. The principal aim of this study was to evaluate the diagnostic value of CT and MR imaging for the diagnosis of mandibular bone invasion compared to the histological reference. In addition, we assessed the survival impact of bone invasion. PATIENTS AND METHODS: A single-center retrospective study included all consecutive patients treated by mandibular bone interruption for squamous cell carcinoma of the oral cavity and/or oropharynx. RESULTS: Sixty-eight patients were included. Prevalence of bone invasion on histology was 43%. Sensitivity, specificity and positive and negative predictive value were respectively 70%, 71%, 66% and 76% for CT compared with histologic analysis, 83%, 50%, 59% and 78% for MRI, and 83%, 62% 62%, 83% for associated CT and MRI. The two tests showed good agreement, with kappa index 0.69 (95% CI, 0.49-0.89) (P<0.0001). There was no difference in overall survival (log-rank>0.70) between the groups with and without bone invasion. CONCLUSION: CT and MRI are complementary for preoperative assessment of mandibular bone invasion, be it cortical and/or medullary, and in some cases may allow mandibular bone-sparing.

Genetic and non-genetic parameters related to embryo production in superovulated Large White (LW) gilts.

The aim of this study was to identify genetic and non genetic factors which might affect results of embryo production of Large White (LW) cyclic gilts from data collected in one herd during 6 years. Donors (n=1060) were synchronized with a progestogen treatment and luteolysis was induced 13-15 days later by 2 injections of cloprostenol. To stimulate follicular development 800IU eCG was then injected 24h later, followed by 500IU hCG 48h later. Donors were inseminated twice; depending on the onset of oestrus, the interval between hCG treatment and first insemination (hCGAI1) was either 24 or 41 h. Embryos were collected at 5-6 days after the 1st AI by flushing uterine horns. Traits of interest were the number of corpora lutea (CL), the number of flushed embryos (FE), the number of transferable embryos (TE) and the number of unfertilized embryos (UE). The average number of TE was 18.8 ± 9.0. The main sources of variation for CL, FE and TE were the season (P≤0.002) and hCGAI1 (P≤0.001) effects. For the interval of 24h of hCGIA1 the number of TE was increased by 4 compared with the TE obtained for the 41 h interval of hCGIA1. Maternal and paternal genetic effects were estimated using restricted maximum likelihood methodology applied to the univariate animal model, whereas genetic covariance components were estimated in bivariate models. Estimates of maternal heritability were 0.45 for CL, 0.32 for FE, 0.29 for TE and 0.05 for UE whereas for the paternal effect, heritabilities were very low (<0.06). Genetic correlation between CL, FE and TE variables were very high (>0.89) for the maternal effect. A breeding scheme based on CL selection in response to superovulation could thus improve the number of transferable embryos.

Development of OPS vitrified pig blastocysts: effects of size of the collected blastocysts, cryoprotectant concentration used for vitrification and number of blastocysts transferred.

Unhatched blastocysts from Large White hyperprolific gilts (n=103) were identified, measured and vitrified using the Open Pulled Straw (OPS) technique to evaluate the effects of the collected blastocyst size and cryoprotectant concentrations used for vitrification, and the number of embryos transferred per recipient. Vitrified/warmed blastocyst viability was estimated in vitro, as the percentage of embryos developing after 72h, and in vivo, on pregnancy Day 30. In the in vitro study, we compared the use of three cryoprotectant concentrations (16.5, 18, or 20% DMSO+16.5, 18, or 20% EG+0.4M sucrose). Survival rates differed significantly between the control (98.3%) and the three cryoprotectant concentrations (67, 62.3, and 57%, respectively). Blastocyst size at vitrification determined the further in vitro development of embryos (26% survival for blastocysts 126-144microm versus 100% for blastocysts >199microm). For the in vivo study, blastocysts were vitrified using cryoprotectant concentrations of 16.5 or 18% DMSO+EG and transferred surgically in groups of 20 or 30 per recipient (n=40). Recipients were slaughtered on pregnancy D30. No significant differences were detected in gestation rates (50-70%) and embryo survival rates (14.7-25%), although survival was higher (P=0.0003) when 20 blastocysts were transferred compared to 30 (24.7% versus 15.5%). Our findings indicate that best results, in terms of subsequent in vivo embryo survival, were achieved after transferring 20 embryos at the blastocyst or expanded blastocyst stage, previously vitrified using cryoprotectant concentrations of 16.5 or 18%.

The effectiveness of the stereomicroscopic evaluation of embryo quality in vitrified-warmed porcine blastocysts: an ultrastructural and cell death study.

The objective of this study was to analyze the validity of the stereomicroscopic evaluation of vitrified-warmed (V-W) porcine blastocysts. Unhatched blastocysts were obtained from Large-white gilts (n=10). Blastocysts (n=156) were vitrified using the Open Pulled Straw technology. After warming, V-W blastocysts were cultured for 24h (V24). Then, their developmental progression was morphologically assessed by stereomicroscopy and classified as: V24 viable re-expanded blastocysts; V24 viable hatched blastocysts or V24 degenerated. Blastocysts which re-expanded or hatched after warming were considered viable. Some fresh blastocysts were not vitrified and were evaluated after 24h in culture (F24). By stereomicroscopic analysis all the fresh blastocysts were considered viable. Some F24, V24 re-expanded viable, V24 hatched viable and V24 degenerated blastocysts were processed for transmission electron microscopy (n=13, 19, 9 and 9, respectively) or assessed by TUNEL for cell-death evaluation (n=16, 21, 11 and 21, respectively). All V24 hatched blastocysts showed similar ultrastructure to fresh blastocysts. However, some V24 re-expanded blastocysts considered viable (6/19) revealed ultrastructural alterations. Degenerated V24 blastocysts showed ultrastructural disintegration. Hatched V24 blastocysts did not differ (p>0.05) from F24 hatched blastocysts with regard to the ratio of dead cells (2.8+/-0.5% versus 1.9+/-0.3%, respectively). However, V24 expanded blastocysts had higher (p<0.01) cell death levels (4.3+/-3.4%) than those observed in the F24 expanded blastocysts (1.1+/-0.3%). The degenerated blastocysts showed the highest cell-death index (19.4+/-6.3%). In summary, V-W blastocyst hatching during in vitro culture appears to coincide with good ultrastructure and low cell-death index, suggesting that the hatching rate assessed by stereomicroscopy is more appropriate than embryo re-expansion for an evaluation of V-W blastocyst quality.

[Cryopreservation and transfer of pig in vivo embryos: State of the art]. / Cryopréservation et transfert des embryons porcins produits in vivo: état des lieux.

The present review article describes the development of porcine cryopreservation and transfer of embryos and the sanitary regulations related to these technologies. Rapid progress has been made during the last decade in cryopreservation by using vitrification which involves a very rapid cooling rate. Currently, this technology is suitable for morulae and blastocysts and to date, more than 300 piglets are born after surgical transfers of vitrified embryos. Moreover, farrowings are obtained after non-surgical transfers of fresh or vitrified embryos. However, further improvements are required to permit the dissemination of these technologies in the near future. According to the recommendations of the International Embryo Transfer Society (IETS) regarding international transportation, embryos must be washed and cryopreserved with an intact zona pellucida. Transmission by embryo transfer of virus or bacteria to swine has been studied and rather converging results are now published. The risk of disease transmission by the embryo transfer is reduced. The major advantage of these technologies is the possibility to transport and store genetic material whilst reducing the risks of pathogen transmission.

Piglets born after non-surgical deep intrauterine transfer of vitrified blastocysts in gilts.

The aims of this study were: (1) to evaluate the effect of the number of previous estrus of recipient gilts on effectiveness of intrauterine insertion of a flexible catheter designed for non-surgical deep intrauterine catheterization during diestrus in pigs; and (2) to determine the farrowing rate and the litter size after non-surgical deep intrauterine embryo transfer (ET) of porcine blastocysts vitrified by the open pulled straw (OPS) method. In experiment 1, 27 large white hyperprolific gilts (LWh) with 2-6 previous estrus were used. Intrauterine insertions of the flexible catheter were carried out at day 5.5-6 of the estrous cycle (D0=onset of estrus). During insertions, no or only moderate reactions were observed in 88.9% of gilts and was not related (P >0.05) to the number of estrus prior to the insertion periods. The number of the estrus had a significant effect (P <0.05) on the difficulties found during the procedure. In the 100% of gilts with two estrus (N=6) it was not possible to insert the flexible catheter through the cervix. In gilts with three or more estrus, it was possible to pass the cervix and to progress along a uterine horn in 80.9% of the cases. In 86.7% of the gilts, the tip of the flexible catheter achieved the second or third quarter of the uterine horn. In experiment 2, following non-surgical deep intrauterine transfer of 20 vitrified/warmed blastocysts, 9 Meishan recipients (42.9%) farrowed an average of 5.4 +/- 0.8 piglets (range 3-9) of which 0.6 +/- 0.3 piglets (range 0-2) were born dead. In conclusion, this study shows that it is possible to obtain birth of piglets following non-surgical deep intrauterine embryo transfer (ET) of vitrified/warmed blastocysts. Non-surgical deep intrauterine ET and OPS vitrification methods are promising procedures to be used together for the introduction of new genetic material in a farm.

Birth of piglets after OPS vitrification and transfer of compacted morula stage embryos with intact zona pellucida.

In swine, five to six days post-insemination, morulae and blastocysts are collected together after uterine flushing. The purpose of this study was to vitrify zona pellucida-intact morulae with Open Pulled Straw (OPS) technology and obtain piglets after transfer. Morulae (200) were vitrified after a two-step equilibration in ethylene glycol, dimethyl sulfoxide and sucrose in Hepes-buffered TCM199 + 20% NBCS medium (TCM). 2-6 morulae were loaded into OPS and plunged into liquid nitrogen. At embryo warming, a three-step dilution with decreasing concentrations of sucrose was applied. In each of 10 recipients, 20 morulae were transferred surgically. Day 25, gestation rate and the farrowing rate were 80% and 70%, respectively. The pregnant recipients farrowed from 1 to 8 piglets and the survival of total transferred embryos was 13%. Although survival rates are still compromised, OPS technology is therefore appropriate to cryopreserve porcine morulae with intact zona pellucida.

Piglets born after vitrification of embryos using the open pulled straw method.

Morulae and unhatched blastocysts from Large White hyperprolific (LWh) and Meishan (MS) gilts were selected to test an ultrarapid open pulled straw (OPS) vitrification method with two media. The viability of vitrified/warmed embryos was estimated by the percentage of embryos that developed to the hatched blastocyst stage in vitro or by birth after transfer. In Experiment 1, two cryoprotectant dilution media were compared for cryopreservation of MS and LWh blastocysts: TCM was a standard Hepes-buffered TCM199 + 20% NBCS medium and PBS was a PBS + 20% NBCS medium. After a two-step equilibration in ethylene glycol, dimethyl sulfoxide, and sucrose, 2-5 blastocysts were loaded into OPS and plunged into liquid nitrogen. Embryos were warmed; a four-step dilution with decreasing concentrations of sucrose was applied. In PBS, LWh blastocysts (27%) had a lower viability in vitro than MS blastocysts (67%; P = 0.001). In TCM, no significant difference was observed between genotypes (41% for LWh and 43% for MS blastocysts) and both viability rates were lower than that of the control groups. In Experiment 2, morula-stage LWh and MS embryos were vitrified and warmed using PBS. The viability rate was low and did not differ between LWh (11%) and MS (14%). In Experiment 3, 200 MS and 200 LWh blastocysts were vitrified/warmed as described in Experiment 1 (PBS). In each of 20 MS recipients, 20 embryos were transferred. The farrowing rate was 55% and recipients farrowed four and five piglets (median) for MS and LWh blastocysts, respectively. The OPS method is therefore appropriate for cryopreservation of unhatched porcine blastocysts.

Effects of pressure ramp slope values on the work of breathing during pressure support ventilation in restrictive patients.

OBJECTIVE: To investigate, in restrictive patients, the influence of pressure ramp slope values on the efficacy of pressure support ventilation. DESIGN: Prospective study. SETTING: A university hospital medical intensive care unit. PATIENTS: Twelve intubated restrictive patients. INTERVENTIONS: Patients were randomly assigned to four sequences in which the values of the slope of the pressure ramp increase were modulated so that the plateau pressure was reached within a predetermined time: 0.1, 0.50, 1, or 1.50 secs. The more rapidly the pressure plateau was achieved, the higher was the initial flow rate. For convenience, these four different ventilatory settings were termed T 0.1, T 0.5, T 1, and T 1.5. MEASUREMENTS AND MAIN RESULTS: We measured the following parameters 10 mins after application of each pressure ramp slope: inspiratory work of breathing, breathing pattern, and intrinsic PEEP (PEEPi). Work of breathing was evaluated using Campbell's diagram, and expressed as a percentage of the values observed under spontaneous ventilation. A marked interindividual variation of the values for work of breathing was observed under spontaneous ventilation; the mean value for work of breathing was 1.97 +/- 0.82 joule/L, with a range of 1.22 to 4.10 joule/L. Comparison between the means for each sequence and each variable measured was performed by two-way analysis of variance with internal comparisons between sequences by Duncan's test. Between the first (T 0.1) and the last (T 1.5) sequence, the reduction of values of the pressure ramp slope induced a progressive increase in the values for work of breathing, regardless of the mode of expression (in joule, joule/L, or joule/min). The values for work of breathing (joule/ L), expressed as a percentage of the values observed under spontaneous ventilation, increased from 44.2 +/- 14.4% to 78.3 +/- 17.8% (p < .001). In contrast, the reduction of the pressure ramp slope values and initial flow rate did not induce any significant change in tidal volume, respiratory frequency, and PEEPi. CONCLUSION: Among the four tested slope values, the steepest was that which induced the lowest possible work of breathing in restrictive patients ventilated by pressure support ventilation. In this type of patient, we therefore suggest that the programmed pressure value should be reached by using a steep pressure ramp slope.

Variability in the resistance of four chicken lines to experimental intravenous infection with Salmonella enteritidis phage type 4.

The capacity of four chicken lines (Y11, L2, B13, PA12) to control Salmonella enteritidis (SE) phage type 4 (PT4) systemic colonization was investigated. Thirteen-week-old chickens were intravenously inoculated with 10(6) SE colony-forming units, and the levels of SE colonization were determined at various time intervals after inoculation in liver, spleen, genital organs, and ceca. The course of SE infection showed a rapid contamination of liver, spleen, and genital organs, whereas the ceca were infected later. A significant (P < 0.001) effect of the chicken line on levels of SE was detected on day 3 postinoculation (PI) in liver and ceca, on day 10 PI in ceca, and on day 15 PI in spleen. Because an early control of systemic Salmonella infection by the Ity/Nramp1 gene has been demonstrated in mice, we aimed to study the early resistance of chickens to SE. As a consequence, we then focused our study on the between- and within-line variabilities of SE levels on day 3 PI. According to the SE levels in liver on day 3 PI, the chicken lines could be classified as susceptible (Y11 and L2) or resistant (PA12 and B13). This early variability was explored in resistant B13 and susceptible L2 lines. Differences between these two lines were confirmed in liver but not in ceca. A large within-line variability was observed in all organs of these two lines. The genetic origin of this variability will have to be determined as a prerequisite to an eventual selection.

Estimated heritability of the resistance to cecal carrier state of Salmonella enteritidis in chickens.

Previously, we have shown differences in susceptibility to the cecal carrier state in chicks orally infected with Salmonella enteritidis (SE) at 1 wk of age for four outbred lines: L2, B13, PA12, and Y11. The egg-type line L2 was one of the most susceptible lines and presented a large variability in cecal SE colonization. The heritability (h2) of the resistance to SE colonization in ceca was estimated in L2 chickens to determine whether genetic factors might be involved in its control. In three independent trials, a total of 819 L2 chicks produced from 88 sires and 232 dams were challenged orally with SE at 1 wk of age. Each week after inoculation, the frequency of cecal colonization was estimated. When this value had fallen to 50%, all the remaining animals were killed. The extent of cecal colonization by SE was estimated directly by counting the viable organisms in organs and determining the numbers of positive ceca. Enrichment culture was used in Trials 2 and 3. The effects of trial, of room within trial, and of cage within room on the frequency of SE contaminated ceca were often significant. No significant effect of sex was observed. Estimation of h2 using the frequency of SE positive ceca was low, 0.06 +/- 0.07, when results of direct culture were considered. In contrast, when considering the frequency obtained after enrichment, the h2 was estimated at 0.20 +/- 0.12. This result suggests a genetic basis for the expression of the resistance to colonization. An experiment of selection for resistance to SE carrier state in the chicken ceca should definitively confirm the genetic origin of the resistance.

Differences in frequency, level, and duration of cecal carriage between four outbred chicken lines infected orally with Salmonella enteritidis.

Four chicken lines, L2, B13, PA12 (egg-type), and Y11 (meat-type), were tested for experimental carrier state of Salmonella enteritidis (SE) in two identical trials. After oral inoculation of SE at 1 wk of age with 5 x 10(4) SE colony-forming units (CFU), 10 chickens per line were necropsied weekly for 6 wk and then every 8 or 15 days until the 12th week postinoculation (PI). Liver, spleen, ovary, and ceca were examined for level of SE colonization. Numbers of positive livers and spleens and levels of the challenge strain in these organs differed little between the four chicken lines. Only three positive ovaries were detected. According to the chicken line, ceca exhibited generally significant (P < 0.05) differences in the number of positive organs during weeks 5-11 PI, in the SE CFU levels (P < 0.05) in the first 5 wk PI and during weeks 8 and 10 PI, and in the duration of colonization. L2 and B13 chickens generally carried SE in their ceca at higher levels, in more animals, and for a longer time than PA12 and Y11 chickens. Y11 chickens were the most resistant to SE cecal colonization.

Effects of oxygen, CO2/pH and medium on the in vitro development of individually cultured porcine one- and two-cell embryos.

The gas atmosphere and medium composition are critical factors in the in vitro development of one- and two-cell embryos of several species. The present study evaluated the effect of different O2/CO2 concentrations (2/5, 2/10, 5/2.5, 5/5, 5/10, 10/10 and 21/5) on pig one- and two-cell embryo development. The embryos were individually cultured, for 6 days at 39 degrees C in a medium rich in bicarbonate and glutamine and containing pyruvate and lactate but lacking glucose. When the CO2 levels increased from 2.5% to 10%, the pH of the medium decreased from 8.2 to 7.5 and the development of the embryos was affected, but this depended mainly on the O2 levels. Pig embryo development was inhibited by 2 and 21% O2 levels. The optimum level for pig embryo development was 5% O2 and 5% CO2, whatever the criteria used to evaluate embryo development. At these optimal levels, the mean number of cells per embryo was 26 +/- 1.7 (ls mean +/- SE), and 50% of the one- and two-cell embryos developed to blastocysts. The substitution of 0.5% bovine serum albumin (BSA) in the medium by 0.3% polyvinyl-pyrrolidone (PVP) significantly decreased the one- and two-cell embryo development. When the calcium and chloride contents of the medium with PVP were reduced, however, the embryo development was similar to that observed in the medium containing BSA. Pig embryo development in vitro was found to be optimal under an atmosphere of 5% O2 and 5% CO2 and PVP could replace BSA as the high molecular weight supplement.

Performance of European laboratories testing serum samples for Toxoplasma gondii.

One hundred twenty-nine European laboratories participated in a collaborative, multicentre study designed to evaluate the overall reliability of different serological techniques for diagnosis of Toxoplasma gondii infection. Five freeze-dried reference sera were distributed to each laboratory, each of which analysed the sera with its routine methods. The enzyme-linked immunosorbent assay was the technique used most frequently, followed by the immunofluorescent antibody technique. Only nine laboratories performed the Sabin-Feldman dye test. In general, there was good concordance between qualitative results, but for sera with low concentrations of Toxoplasma gondii-specific IgG antibodies, some false-negative results were found. For specific IgM and IgA antibodies, the immunosorbent agglutination assay proved the most sensitive. The present study demonstrates the need for regular assessment of laboratory serodiagnosis of Toxoplasma gondii infection.

[Multicenter study of toxoplasma serology using various commercial ELISA reagents. Work Group Toxoplasmosis of the National Agency for Quality Control in Parasitology]. / Etude multicentrique de la sérologie toxoplasmique par différents réactifs ELISA commercialisés. Groupe de travail Toxoplasmose du Contrôle National de Qualité en Parasitologie.

A collaborative study conducted by the French National Agency for Quality Control in Parasitology (CNQP) and various manufacturers of ELISA kits, represented by the Association of Laboratory Reagent Manufacturers (SFRL) compared the toxoplasmosis IgG antibody titres obtained with different ELISA-IgG kits and determined the relationships between the titres obtained by these techniques and the titre defined in international units (IU). Fifty-one serum samples with toxoplasmosis antibody titres ranging from 0 to 900 IU were tested in two successive studies with 16 ELISA-IgG kits. For the negative sera, false-positive reactions were observed with one kit. For the positive sera, the titres observed in ELISA were generally higher than those expressed in IU. Above 250 IU, the very wide variability of the titres found with the different ELISA kits renders any comparative analysis impossible. For titres below 250 IU, the results are sufficiently homogeneous to permit the use of regression analysis to study how the results for each ELISA kit compare with the mean results for the other kits. The slope of the line of regression shows a tendency to over-titration or under-titration compared with the results of the other manufacturers; the ordinate at the origin reflects the positivity threshold of the reaction and can be used to assess the risk of a lack of sensitivity (high threshold) or of specificity (threshold too low). On the whole, the trends revealed for a given manufacturer are constant from one study to the other. Within this range of titres, regression analysis also reveals the general tendency of ELISA kits to overestimate the titres by comparison with immunofluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological and functional features of ovine follicles in perifusion with pulsatile hormone delivery.

Large follicles were obtained from sheep ovaries during the follicular phase, dissected and incubated for 24 h in a perifusion system. Continuous flow of B2 medium gassed with O2 and CO2 and supplemented with FSH/LH pulses every other hour enabled us to measure the steroid secretion rates of each follicle. At the end of the perifusion, the follicles were processed for histological examination. It was demonstrated that 70% of the follicles were healthy after 24 h of perifusion. This was associated with a high secretion rate of oestradiol compared to atretic follicles. In contrast testosterone and progesterone secretion rates were similar in healthy and atretic follicles. In both healthy and atretic follicles, repeated gonadotrophin pulses produced increases in steroid production. Such a perifusion system might be a valuable tool to study between and within-follicle interactions to get new insights in paracrine and autocrine regulations in the ovary.

Effects on mitochondrial metabolism of CCA, one inducer of neuroblastoma differentiation.

CCA, a potent neuroblastoma differentiation inducer, was shown by oxygraphic measurements to reduce significantly the O2 consumption of whole neuroblastoma cells as of mitochondria purified from neuroblastoma or mouse cortex. The effect of CCA on the respiration was compared to those of oligomycin. Our results suggest that the molecular target of CCA is the matrix F1 catalytic component of the F0F1 mitochondrial ATPase.