Maxillofacial reconstruction with prefabricated osseous free flaps: a 3-year experience with 24 patients

Between January of 1998 and May of 2002, 25 prefabricated osseous free flaps (23 fibula and two iliac crest flaps) were transferred in 24 patients to repair maxillary (six flaps) or mandibular (eight flaps) defects after tumor resection, severe maxillary (four flaps) or mandibular (one flap) atrophy (Cawood VI), maxillary (one flap) or mandibular (three flaps) defects after gunshot injury, and maxillary (two flaps) defects after traffic accidents. Prefabrication included insertion of dental implants, positioned with a drilling template in a preplanned position, and split-thickness grafting. Drilling template construction was based on the prosthetic planning. The template determined the position of the implants and the site and angulation of osteotomies, if necessary. The mean delay between prefabrication and flap transfer was 6 weeks (range, 4 to 8 weeks). While the flap was harvested, a bar construction with overdentures was mounted onto the implants. The overdentures were used as an occlusal key for exact three-dimensional positioning of the graft within the defect. The bar construction also helped to stabilize the horseshoe shape of the graft. The follow-up period ranged from 2 months to 4 years (mean, 21 months), during which time two total and three partial flap losses occurred. One total loss was due to thrombosis of the flap veins during the delay period, whereas the other total loss was caused by spasm of the peroneal artery. Two partial losses were due to oversegmentation of the flaps with necrosis of the distal fragment, whereas one partial loss was caused by disruption of the vessel from the distal part. Of the 90 implants that were inserted into the prefabricated flaps during the study period, 10 were lost in conjunction with flap failure; of the remaining 80 implants, four were lost during the observation period, for a success rate of 95 percent. Flap prefabrication based on prosthetic planning offers a powerful tool for various reconstructive problems in the maxillofacial area. Although it involves a two-stage procedure, the time for complete rehabilitation is shorter than with conventional procedures.     

Laparoscopical intrauterine insemination with different doses of fresh,conserved, and frozen semen for the production of ovine zygotes

The objective of the present study was to increase the efficiency in the production of ovine zygotes suitable for microinjection via laparoscopical intrauterine insemination. In the first part of the study, 71 ewes of three different breeds were inseminated with one of two different insemination doses (50 x 10(6) or 300 x 10(6) sperm per inseminate) and semen was either freshly diluted, liquid conserved, or frozen/thawed, or females were mated by a fertile ram (controls). In the second part, a total of 46 ewes was inseminated with 300 x 10(6) freshly diluted sperm to verify the findings from part 1 and to unravel effects of breed and age of donor ewe. The oviducts were flushed 24-26 h after insemination and the success of insemination was assessed by microscopical examination. Recovery rates were 78.0+/-26.4 and 72.1+/-24.6% in parts 1 and 2 of the study, respectively. Of these oocytes 62.3 and 62.8% (parts 1 and 2, respectively) were fertilized. In part 1, the highest proportion (64.7%) of pronuclear stages was observed in the group inseminated with 300 x 10(6) freshly diluted semen and was significantly higher compared to the groups inseminated with 50 x 10(6) freshly diluted semen (25.5%, P<0.001), 300 x 10(6) liquid conserved semen (49.0%, P<0.001), or 50 x 10(6) frozen/thawed semen (39.6%, P<0.05). In the control group, the proportion of pronuclear stages amounted to 60.2%. Irrespective of the type of sperm conservation, the overall fertilization rate (zygotes plus 2-cell stages) was higher (P<0.05) following insemination with 300 x 10(6) sperm (68.2%) compared to 50 x 10(6) sperm (56.8%). In part 2, the proportion of pronuclear stages reached 54.2% with an overall fertilization rate of 62.9%. These results were affected by breed and age of the donor as crossbred and younger (<3 years) animals yielded the highest proportion of pronuclear stages. The present study shows that freshly diluted semen at a dosage of 300 x 10(6) sperm yields the highest fertilization rates, the greatest proportion of pronuclear stages and the lowest proportion of mature unfertilized oocytes. Further increases in yields of pronuclear stages can possibly be achieved by selection of sheep from the best suited breed and younger than 3 years of age.     

Comparative study of different fluorescent dyes for the detection of proteins on membranes using the peroxyoxalate chemiluminescent reaction

We have previously shown that the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H(2)O(2) chemiluminescent reaction in acetone can be used for the detection of proteins labeled with the fluorescent reagent 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) on polyvinylidene difluoride (PVDF) membranes. To improve this method, in this work we have designed and constructed a cell that allows us to perform this chemiluminescent reaction on PVDF membranes with a homogeneous distribution of the reagents. Using this cell we have examined the analytical properties of several recently developed fluorescent protein dyes chemically different from MDPF. We have found that the metal chelate dye SYPRO Ruby can also be excited by the high-energy intermediate produced in the TCPO-H(2)O(2) reaction.     

Isolation,structural characterization,and antiviral activity of positional isomers of monopegylated interferon alpha-2a (PEGASYS)

Interferon alpha-2a plays an essential role in the treatment of chronic hepatitis C, but it is limited in its efficacy by the short in vivo half-life. To improve the half-life and efficacy, interferon alpha-2a is conjugated with a 40-kDa branched polyethylene glycol moiety (PEG-IFN, PEGASYS). From this preparation the positional PEG-IFN isomers were isolated and characterized by different analytical methods and antiviral assay. Two chromatographic steps were used to separate and purify nine isomers. The analytical methods IE-HPLC, RP-HPLC, SE-HPLC, SDS-PAGE, and MALDI-TOF MS indicated that each of these nine isomers is conjugated to the branched polyethylene glycol chain at a specific lysine. No isomer with a modification at the amino terminus was observed. All positional isomers induced viral protection of MDBK cells in the antiviral assay. When comparing the quantitative potency of the individual isomers with the whole mixture of PEG-IFN, significant differences in the specific activities were observed: PEG-Lys(31) and PEG-Lys(134) showed higher activities than the mixture, PEG-Lys(164) was equal to the mixture, whereas the activities of PEG-Lys(49), PEG-Lys(70), PEG-Lys(83), PEG-Lys(112), PEG-Lys(121), and PEG-Lys(131) were lower.     

Caffeine induces a second wave of apoptosis after low dose-rate gamma radiation of HL-60 cells

Most cell lines that lack functional p53 protein are arrested in the G₂ phase of the cell cycle due to DNA damage. It was previously found that the human promyelocyte leukemia cells HL-60 (TP53 negative) that had been exposed to ionizing radiation at doses up to 10 Gy were arrested in the G₂ phase for a period of 24 h. The radioresistance of HL-60 cells that were exposed to low dose-rate gamma irradiation of 3.9 mGy/min, which resulted in a pronounced accumulation of the cells in the G₂ phase during the exposure period, increased compared with the radioresistance of cells that were exposed to a high dose-rate gamma irradiation of 0.6 Gy/min. The D₀ value (i.e. the radiation dose leading to 37% cell survival) for low dose-rate radiation was 3.7 Gy and for high dose-rate radiation 2.2 Gy. In this study, prevention of G₂ phase arrest by caffeine (2 mM) and irradiation of cells with low dose-rate irradiation in all phases of the cell cycle proved to cause radiosensitization (D₀=2.2 Gy). The irradiation in the presence of caffeine resulted in a second wave of apoptosis on days 5–7post-irradiation. Caffeine-induced apoptosis occurring later than day 7 post-irradiation is postulated to be a result of unscheduled DNA replication and cell cycle progress.     

Streptococcus pneumoniae heat shock protein 70 does not induce human antibody responses during infection

Mouse monoclonal antibodies (mAbs) were developed against Streptococcus pneumoniae in search for potential common pneumococcal proteins as vaccine antigens. mAb 230,B-9 (IgG1) reacted by immunoblotting with a 70-kDa protein which was isolated by immunoaffinity chromatography and subsequent preparative electrophoresis. N-terminal amino acid sequencing showed homology to that of heat shock protein 70 (hsp70). The hsp70 epitope reactive with mAb 230,B-9 was found in all the pneumococci examined as well as in other streptococci and enterococci. The epitope was not expressed in several other examined Gram-positive or -negative bacteria. Pneumococcal hsp70 has by other investigators been proposed to be a vaccine candidate. Binding experiments using flow cytometry showed that the epitope was not surface-exposed on live exponential phase grown S. pneumoniae. Human patient sera did not react with affinity-purified pneumococcal hsp70. Therefore the pneumococcal hsp70 does not seem to be of special interest in a vaccine formulation. The human sera contained antibodies to high molecular proteins co-purified with hsp70. Some of these proteins could be the pneumococcal surface protein A.     

G Proteins Modulate D2 Receptor-Coupled K(ATP) Channels in Rat Dopaminergic Terminals

The presynaptic dopamine (DA) D2 receptor-mediated regulation of ATP-sensitive potassium (K⁺ _ATP) channels was examined in slices of the rat caudate-putamen. When slices were incubated with the specific D2 receptor antagonist (–)-sulpiride (SLP), a concentration-dependent increase of extracellular DA release was observed. SLP-induced enhancement was completely antagonized by coincubation with the K⁺ _ATP channel opener diazoxide (DIA). Treatment of slices with the D2 receptor agonist quinpirole (QUI) almost completely inhibited DA outflow induced by the K⁺ _ATP channel blocker butanedione-monoxime (BDM). Coincubation of SLP and guanosine triphosphate (GTP) or its non-hydrolizable analogue guanylyl-5-imidodiphosphate [Gpp(NH)p], significantly reduced the SLP-induced effect on DA levels. Furthermore, we observed that BDM-induced DA outflow was markedly inhibited by G protein activators suggesting an additional receptor-independent regulation of K⁺ _ATP channel gating. Our results suggest that PTX-sensitive G proteins are involved in the signal transduction between D2 receptors and K⁺ _ATP channels. Furthermore, K⁺ _ATP channels can be modulated in a receptor-independent mechanism by G protein activators.