Evaluating very high energy electron RBE from nanodosimetric pBR322 plasmid DNA damage.

This paper presents the first plasmid DNA irradiations carried out with Very High Energy Electrons (VHEE) over 100-200 MeV at the CLEAR user facility at CERN to determine the Relative Biological Effectiveness (RBE) of VHEE. DNA damage yields were measured in dry and aqueous environments to determine that ~ 99% of total DNA breaks were caused by indirect effects, consistent with other published measurements for protons and photons. Double-Strand Break (DSB) yield was used as the biological endpoint for RBE calculation, with values found to be consistent with established radiotherapy modalities. Similarities in physical damage between VHEE and conventional modalities gives confidence that biological effects of VHEE will also be similar-key for clinical implementation. Damage yields were used as a baseline for track structure simulations of VHEE plasmid irradiation using GEANT4-DNA. Current models for DSB yield have shown reasonable agreement with experimental values. The growing interest in FLASH radiotherapy motivated a study into DSB yield variation with dose rate following VHEE irradiation. No significant variations were observed between conventional and FLASH dose rate irradiations, indicating that no FLASH effect is seen under these conditions.

Poly(trimethylene carbonate-co-L-lactide) electrospun scaffolds for use as vascular grafts.

Currently, there is great clinical need for suitable synthetic grafts that can be used in vascular diseases. Synthetic grafts have been successfully used in medium and large arteries, however, their use in small diameter vessels is limited and presents a high failure rate. In this context, the aim of this study was to develop tissue engineering scaffolds, using poly(trimethylene carbonate-co-L-lactide) (PTMCLLA), for application as small diameter vascular grafts. For this, copolymers with varying trimethylene carbonate/lactide ratios - 20/80, 30/70, and 40/60 - were submitted to electrospinning and the resulting scaffolds were evaluated in terms of their physicochemical and biological properties. The scaffolds produced with PTMCLLA 20/80, 30/70, and 40/60 showed smooth fibers with an average diameter of 771±273, 606±242, and 697±232 nm, respectively. When the degradation ratio was evaluated, the three scaffold groups had a similar molecular weight (Mw) on the final day of analysis. PTMCLLA 30/70 and 40/60 scaffolds exhibited greater flexibility than the PTMCLLA 20/80. However, the PTMCLLA 40/60 scaffolds showed a large wrinkling and their biological properties were not evaluated. The PTMCLLA 30/70 scaffolds supported the adhesion and growth of mesenchymal stem cells (MSCs), endothelial progenitor cells, and smooth muscle cells (SMCs). In addition, they provided a spreading of MSCs and SMCs. Given the results, the electrospun scaffolds produced with PTMCLLA 30/70 copolymer can be considered promising candidates for future applications in vascular tissue engineering.

Poly(trimethylene carbonate-co-L-lactide) electrospun scaffolds for use as vascular grafts

Currently, there is great clinical need for suitable synthetic grafts that can be used in vascular diseases. Synthetic grafts have been successfully used in medium and large arteries, however, their use in small diameter vessels is limited and presents a high failure rate. In this context, the aim of this study was to develop tissue engineering scaffolds, using poly(trimethylene carbonate-co-L-lactide) (PTMCLLA), for application as small diameter vascular grafts. For this, copolymers with varying trimethylene carbonate/lactide ratios - 20/80, 30/70, and 40/60 - were submitted to electrospinning and the resulting scaffolds were evaluated in terms of their physicochemical and biological properties. The scaffolds produced with PTMCLLA 20/80, 30/70, and 40/60 showed smooth fibers with an average diameter of 771±273, 606±242, and 697±232 nm, respectively. When the degradation ratio was evaluated, the three scaffold groups had a similar molecular weight (Mw) on the final day of analysis. PTMCLLA 30/70 and 40/60 scaffolds exhibited greater flexibility than the PTMCLLA 20/80. However, the PTMCLLA 40/60 scaffolds showed a large wrinkling and their biological properties were not evaluated. The PTMCLLA 30/70 scaffolds supported the adhesion and growth of mesenchymal stem cells (MSCs), endothelial progenitor cells, and smooth muscle cells (SMCs). In addition, they provided a spreading of MSCs and SMCs. Given the results, the electrospun scaffolds produced with PTMCLLA 30/70 copolymer can be considered promising candidates for future applications in vascular tissue engineering.

Electrospun scaffolds functionalized with heparin and vascular endothelial growth factor increase the proliferation of endothelial progenitor cells.

In severe cases of peripheral arterial disease, tissue loss can occur and the use of vascular grafts can be necessary. However, currently, there are no suitable substitutes for application in small diameter vessels. The aim of this work has been to produce scaffolds with adequate properties for application as vascular substitutes. Polycaprolactone scaffolds were produced by the electrospinning technique. The surface of the scaffolds was functionalized with heparin and vascular endothelial growth factor (VEGF) and their physical-chemical properties were characterized. Human endothelial progenitor cells (EPCs) or mesenchymal stem cells (MSCs) were seeded onto the surface of the scaffolds in order to create an endothelial layer. The electrospun scaffolds exhibited mechanical properties compatible with the native arteries. The presence of heparin prevented blood coagulation on the scaffold surface. The presence of heparin and VEGF favored the adaptation of MSCs and EPCs on the scaffolds in relation to the non functionalized scaffolds. In addition, the EPCs cultivated on the scaffolds maintained the expression of CD31, CD34 and VE-cadherin genes. The results obtained in the present study suggest that electrospun scaffolds functionalized with heparin and VEGF can be applied in vascular tissue engineering. These scaffolds exhibited antithrombogenic properties and favored the development of cells on their surface. The association of heparin and VEGF with electrospun scaffolds increased EPC proliferation, favoring the formation of the endothelial layer and the regeneration of damaged vessels.

Early induction of labor with PGE2-intravaginal gel in premature rupture of membranes.

Results of induction of labor with PGE2-intravaginal gel in PROM, were evaluated considering the best management.

Benefits of antibiotic prophylaxis in laparoscopic gynaecological surgery.

The prevention of infectious complications by antibiotic prophylaxis has made traditional or laparoscopic surgery much safer but at the same time has contributed to an uncontrolled and often irrational use of every kind of antibiotic. In this study we wanted to show that often mini-invasive surgery like laparoscopy can be practised without the use of antibiotics. Thus, postoperatively several patients undergoing laparoscopic, diagnostic and operative interventions were followed-up. The results showed that subjects without antibiotic therapy did not have any symptomatology that could be ascribed to bacterial infections. In conclusion this study has demonstrated that laparoscopic surgery, especially without any complications, should follow the elementary rules of surgical techniques and surgical asepsis and that antibiotic prophylaxis is not always necessary.

Diagnostic potential of Mab-based ELISAs for antibodies to non-structural proteins of foot-and-mouth disease virus to differentiate infection from vaccination.

This paper summarises the development of monoclonal antibody (Mab)-based immunoassays measuring antibodies to non-structural proteins of FMDV to differentiate infection from vaccination. Of the three non-structural proteins 2C, 3C and 3ABC evaluated in this study, the polypeptide 3ABC was the most immunogenic. Three ELISAs for the detection of antibodies to 3ABC were developed. Two assays rely on the competition of test sera against either a anti-3A Mab or against antisera to 3ABC raised in rabbits and guinea-pigs. The third, 3ABC Mat-ELISA, based on the direct binding of antibodies to the 3ABC trapped by a specific Mab, provided the best combination of specificity and sensitivity. The 3ABC Mat-ELISA was extensively validated for cattle, either in experimental and in field conditions, showing specificity of 99% in vaccinated and in naive cattle and the capacity to detect silent infections in FMD-vaccinated populations. The test showed similar specificity and sensitivity in experimentally vaccinated and infected sheep.

Gamma nailing of pertrochanteric and subtrochanteric fractures: clinical results of a series of 63 consecutive cases.

OBJECTIVE: To analyze the use of the gamma nail in the treatment of pertrochanteric fractures. DESIGN: Prospective. SETTING: University. PATIENTS: Sixty-three fractures in sixty-three patients treated with gamma nails. RESULTS: Forty-five of the sixty-three patients (71 percent) were followed until the end of treatment, for an average follow-up period of 7.2 months. Of the remaining eighteen, eleven died and seven were lost to follow-up. Reduction was classified as good in thirty-eight cases, acceptable in nineteen, and unsatisfactory in six. CONCLUSION: The findings from this series indicate that, compared with other methods, the gamma nail enables the surgeon to treat more types of hip fractures with a less invasive technique and achieve equal or better results.

Development of two novel monoclonal antibody-based ELISAs for the detection of antibodies and the identification of swine isotypes against swine vesicular disease virus.

Two novel formats of ELISA for the detection of antibodies against swine vesicular disease (SVD) virus were developed. One of the tests described is a monoclonal antibody-based competitive ELISA (MAC-ELISA). In this test, specific antibodies in serum are detected due to their ability to compete with a neutralizing monoclonal antibody (MAb). The second is an indirect trapping ELISA which employs isotype-specific MAbs to detect swine IgG or IgM specific for SVD virus. The diagnostic sensitivity and specificity of the MAC-ELISA was studied on 5671 field sera of known origin, enabling the cut-off level to be defined. Using the MAC-ELISA, 100% of sera from infected pigs were found positive, whereas only 0.45% of negative sera gave a false-positive result. A positive correlation between MAC-ELISA and virus neutralizing titres was recorded for pig sera collected sequentially after experimental infections. The results from the isotype-specific ELISA revealed the dynamics of the antibody response to SVD virus in pigs. The first antibodies were detectable as early as 3 days after experimental infection. Up to the 10th day, demonstrable antibodies were exclusively of the IgM class. IgG developed later, between 11 and 14 days postinfection and remained at a plateaux level throughout the whole investigation period. The two tests satisfy different diagnostic requirements: the MAC-ELISA is useful as a screening test, the isotype-specific ELISA has potential application for the determination of stage of infection. Both tests benefit from the use of MAbs in terms of specificity and standardization and have advantages over the virus neutralization test.

[Treatment by external fixator of open Gustilo stage III A and III B leg fractures]. / Traitment par fixateur externe des fractures ouvertes des jambes stade III A et III B selon Gustilo.

From June 1992 to July 1993, we treated 10 consecutive open tibial fractures (6 III A and 4 III B according to the classification of Gustilo). All fractures were treated by external fixation (9 Hoffmann external fixators and 1 Orthofix). Only one patients needed a primary facsiocutaneous flap; the remaining patients were treated by skin grafting (7 cases), secondary wound (1 cases) and granulation (1 case). In 8 cases we realised a decortication and autologous bone grafting. Consolidation time was inferior or equal to 6 months in 5 patients, equal to 8 months in one and equal to 11 months in one. One patient was lost to follow-up and one ist still in treatment. In 2 patients we changed the stabilisation system and we used the Ilizarov technique, once for pseudoarthosis at 7.5 months from injury and once for bone-transfer at 3.5 months. We do not deplore any other case of pseudoarthrosis nor any case of osteitis. None of our patients needed amputation. Early and adequate soft tissue treatment is essential and decoration and bone grafting, which we used 8 times at mean of 12 weeks from injury, should be done earlier.

The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication.

The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.

Improvements in the diagnosis of contagious bovine pleuropneumonia through the use of monoclonal antibodies.

Seventeen monoclonal antibodies (MAbs) raised against Mycoplasma mycoides ssp. mycoides small colony type (Mmm SC), the causative agent of contagious bovine pleuropneumonia (CBPP), were partially characterised. Six MAbs recognising a main protein of 70 kDa and showing reciprocal competition were found to be specific for Mmm SC, while the other MAbs showed different patterns of reactivity with Mycoplasma spp. within the mycoides "cluster". Sandwich enzyme-linked immunosorbent assay (ELISA) performed with different combinations of MAbs enabled the detection of Mycoplasma of the mycoides cluster in pathology samples and the specific identification of Mmm SC from the initial culture passages, overcoming the necessity for difficult and time-consuming biochemical and growth inhibition assays. A competitive ELISA based on Mmm SC-specific MAbs was able to measure anti-CBPP antibodies in cattle sera, and detected only the set of antibodies directed at Mmm SC-specific epitopes, thus avoiding the false-positive reactions occasionally observed with the complement fixation test.

Binding characteristics of monoclonal antibodies raised against bovine growth hormone.

Monoclonal antibodies have been raised against pituitary bovine growth hormone using the hybridoma procedure. The binding characteristics of the seven selected monoclonal antibodies toward the antigen molecule in its native, chemically or enzymatically treated form have been studied. The reactivities of the monoclonal antibodies with growth hormones from other species and bovine prolactin have also been investigated. The epitopes recognized by four of the produced monoclonal antibodies are conformational, whereas two other monoclonal antibodies bind to sequential determinants. Three antibodies define immunological sites located between residues 6-124 of the bovine growth hormone molecule, and one of this antibody shows higher affinity to human than bovine growth hormone. The immunoreactivity of one monoclonal antibody is enhanced by the previous binding of the antigen to polyclonal antibodies, probably because of a localized conformational change of the bovine growth hormone molecule. This antibody also shows cross-reactivity with all the homologous hormones tested, indicating to recognize a highly conserved antigenic determinant.

Immunochemical characterization of human liver and heart ferritins with monoclonal antibodies.

A library of 27 murine monoclonal antibodies was obtained by using human liver and heart ferritins as immunogens. The specificity of the antibodies for the two ferritins and their subunits was studied with five different methods. The antibodies elicited by the liver ferritin bound preferentially the immunogen and were specific for the L subunit. Some antibodies elicited by the heart ferritin had characteristics similar to the anti-liver antibodies, other ones bound preferentially the heart over the liver ferritin and were specific for the H subunit. Only two antibodies were able to bind both ferritins and subunits. Some anti-H and anti-L chain antibodies were used to develop and compare four types of immunoassay to quantitate isoferritins. The results indicate that heart ferritin is immunologically more heterogeneous than liver, the H and L subunits having large immunological differences with few, if any, identical epitopes; and that that the architecture of the immunoassays have a strong influence on the crossreactivity of the antibodies with the two isoferritins, probably because H and L chains are not arranged randomly in the assembled protein.

A new bacterial dehydrogenase oxidizing the lignin model compound guaiacylglycerol beta-O-4-guaiacyl ether.

A lignin model compound, named in short guaiagylglycerol beta-guaiacyl ether (GGE), contains the beta-0-4 ether linkage that is common in the chemical structure of lignin. A Pseudomonas sp. (GU5) had been isolated as an organism able to grow with GGE as the sole source of carbon and energy. When grown on vanillate, the bacteria contained a NAD+ -dependent dehydrogenase converting GGE to a 355 nm absorbing product. The enzyme, named GGE-dehydrogenase, was purified about 160-fold using gel permeation, ion exchange on DEAE-Sephadex, and dye-ligand affinity chromatography. The new protein was about 52 kDa in apparent size with but one polypeptide chain after denaturation and reduction. According to several criteria, the product of GGE oxidation (Km = 12 microM) was identified as the corresponding conjugated ketone at the alpha-carbon of the C3 side-chain. The secondary alcohol function in GGE was apparently the sole target of the enzyme action. However the conversion of GGE into ketone catalyzed by the enzyme was only partial, and did not exceed 50%, probably because only one of the alpha-enantiomers was susceptible to enzyme attack. In contrast the ketone, either made by organic synthesis or by enzymic oxidation of GGE, could be totally reduced back to GGE (Km = 13 microM at pH 8.4, 8 microM at neutral pH), with NADH as the reductant, as confirmed by UV absorption and NMR spectra. Other model compounds with no primary alcoholic function, ether linkage or phenolic group were also substrates for the enzyme, confirming the specificity of GGE-dehydrogenase for the alpha-carbon position. Conjugation of the alpha-ketone with an adjacent phenolic nucleus interfered strongly with equilibrium constants and redox potentials of the system according to pH, and the enzyme displayed widely different optima with pH over 9 when oxidizing GGE, below 7 when reducing the ketone. Equilibrium studies showed that the ketone/GGE potential was -0.37 volt at pH 8.7, -0.23 volt at pH 7 (30 degrees C). The significance of this new dehydrogenase and its properties are discussed, especially in the general concern of lignin biodegradation.