Decreased platelet responsiveness to clopidogrel correlates with CYP2C19 and PON1 polymorphisms in atherosclerotic patients.

Clopidogrel and aspirin are the most commonly used medications worldwide for dual antiplatelet therapy after percutaneous coronary intervention. However, clopidogrel hyporesponsiveness related to gene polymorphisms is a concern. Populations with higher degrees of genetic admixture may have increased prevalence of clopidogrel hyporesponsiveness. To assess this, we genotyped CYP2C19, ABCB1, and PON1 in 187 patients who underwent percutaneous coronary intervention. Race was self-defined by patients. We also performed light transmission aggregometry with adenosine diphosphate (ADP) and arachidonic acid during dual antiplatelet therapy. We found a significant difference for presence of the CYP2C19*2 polymorphism between white and non-white patients. Although 7% of patients had platelet resistance to clopidogrel, this did not correlate with any of the tested genetic polymorphisms. We did not find platelet resistance to aspirin in this cohort. Multivariate analysis showed that patients with PON1 and CYP2C19 polymorphisms had higher light transmission after ADP aggregometry than patients with native alleles. There was no preponderance of any race in patients with higher light transmission aggregometry. In brief, PON1 and CYP2C19 polymorphisms were associated with lower clopidogrel responsiveness in this sample. Despite differences in CYP2C19 polymorphisms across white and non-white patients, genetic admixture by itself was not able to identify clopidogrel hyporesponsiveness.

Blastocele fluid from in vitro- and in vivo-produced equine embryos contains nuclear DNA.

Normal mammalian early embryonic development involves apoptosis of blastomeres as a remodeling process during differentiation, starting at the blastocyst stage. Genomic DNA has been recently detected in the blastocele fluid of human embryos and has been amplified by real-time polymerase chain reaction (PCR) to diagnose the sex of in vitro-produced human embryos. This new approach varies from conventional preimplantation genetic diagnosis in that no cells are extracted from the embryo and only the blastocele fluid is aspirated and used as a DNA sample for diagnosis. In the present work, we investigated whether the blastocele fluid of equine preimplantation embryos contains nuclear DNA and whether this DNA could be used to diagnose the sex of the embryos by conventional PCR, using specific primers that target the TSPY and AMEL equine genes. The sex of 11 of 13 in vivo-produced embryos and of four of five in vitro-produced embryos was successfully diagnosed. The PCR amplification product was analyzed using genetic sequencing reporting that the DNA present in blastocele fluid was genomic. Additionally, after polyacrylamide gel electrophoresis and silver staining, the blastocele fluid from three different embryos produced a ladder pattern characteristic of DNA fragmented during apoptosis. Therefore, the results presented in this work report that blastocele fluid from in vivo- and in vitro-produced equine embryos contains nuclear DNA which is probably originated by apoptosis of embryonic cells, and this DNA could be used to diagnose the sex of preimlpantation embryos by conventional PCR.

Correlation of the in vitro antifungal drug susceptibility with the in vivo activity of fluconazole in a murine model of cerebral infection caused by Cryptococcus gattii.

Forty Cryptococcus gattii strains were submitted to antifungal susceptibility testing with fluconazole, itraconazole, amphotericin B and terbinafine. The minimum inhibitory concentration (MIC) ranges were 0.5-64.0 for fluconazole, <0.015-0.25 for itraconazole, 0.015-0.5 for amphotericin B and 0.062-2.0 for terbinafine. A bioassay for the quantitation of fluconazole in murine brain tissue was developed. Swiss mice received daily injections of the antifungal, and their brains were withdrawn at different times over the 14-day study period. The drug concentrations varied from 12.98 to 44.60 µg/mL. This assay was used to evaluate the therapy with fluconazole in a model of infection caused by C. gattii. Swiss mice were infected intracranially and treated with fluconazole for 7, 10 or 14 days. The treatment reduced the fungal burden, but an increase in fungal growth was observed on day 14. The MIC for fluconazole against sequential isolates was 16 µg/mL, except for the isolates obtained from animals treated for 14 days (MIC = 64 µg/mL). The quantitation of cytokines revealed a predominance of IFN-γ and IL-12 in the non-treated group and elevation of IL-4 and IL-10 in the treated group. Our data revealed the possibility of acquired resistance during the antifungal drug therapy.

Glycoconjugates and polysaccharides of fungal cell wall and activation of immune system / Glicoconjugados e polissacarídios de parede celular de fungos e ativação do sistema imune

The fate of organochlorine 14C-dicofol in activated sludge process was investigated. Results showed that the major part of radioactivity remained adsorbed on biological sludge. Consequently, its final disposal deserves special attention. The small amounts of dicofol, biotransformed or not, which remained in the treated effluent could contaminate receiving bodies.

Glicoproteínas, glicoesfingolipídios e polissacarídios, expostos nas camadas mais externas da parede celular dos fungos, estão envolvidos em diferentes tipos de interações com o ambiente extracelular. Essas moléculas são componentes essenciais desses organismos, contribuindo para a estrutura, integridade, crescimento celular, diferenciação e sinalização. Alguns são compostos imunologicamente ativos com potencial para regular a patogênese e a resposta imune do hospedeiro, Algumas dessas estruturas podem ser especificamente reconhecidas por anticorpos presentes no soro de pacientes, sugerindo uma possível utilização como ferramenta no diagnóstico das infecções fúngicas.

The opportunistic fungal pathogen Scedosporium prolificans: carbohydrate epitopes of its glycoproteins.

Isolated from the mycelium of Scedosporium prolificans were complex glycoproteins (RMP-Sp), with three structurally related components (HPSEC). RMP-Sp contained 35% protein and 62% carbohydrate with Rha, Ara, Man, Gal, Glc, and GlcNH(2) in a 18:1:24:8:6:5 molar ratio. Methylation analysis showed mainly nonreducing end- of Galp (13%), nonreducing end- (9%), 2-O- (13%), and 3-O-subst. Rhap (7%), nonreducing end- (11%), 2-O- (10%), 3-O- (14%), and 2,6-di-O-subst. Manp units (13%). Mild reductive beta-elimination of RMP-Sp gave alpha-l-Rhap-(1-->2)-alpha-l-Rhap-(1-->3)-alpha-l-Rhap-(1-->3)-alpha-d-Manp-(1-->2)-d-Man-ol, with Man-ol substituted at O-6 with beta-d-Galp units, a related pentasaccharide lacking beta-d-Galp units, and beta-d-Galp-(1-->6)-[alpha-d-Manp-(1-->2)]-d-Man-ol in a 16:3:1w/w ratio. Traces of Man-ol and Rha-ol were detected. ESI-MS showed HexHex-ol and Hex(3-6)Hex-ol components. Three rhamnosyl units were peeled off successively from the penta- and hexasaccharide by ESI-MS-MS. The carbohydrate epitopes of RMP-Sp differ from those of the glycoprotein of Pseudallescheria boydii, a related opportunistic pathogen.

Glycoconjugates and polysaccharides of fungal cell wall and activation of immune system.

Glycoproteins, glycosphingolipids and polysaccharides exposed at the most external layers of the wall are involved in several types of interactions of fungal cells with the exocellular environment. These molecules are fundamental building blocks of organisms, contributing to the structure, integrity, cell growth, differentiation and signaling. Several of them are immunologically active compounds with potential as regulators of pathogenesis and the immune response of the host. Some of these structures can be specifically recognized by antibodies from patients' sera, suggesting that they can be also useful in the diagnosis of fungal infections.

Postoperative deep wound infections in adults after spinal fusion: management with vacuum-assisted wound closure.

OBJECTIVE: Vacuum-assisted wound closure (VAC) exposes the wound bed to negative pressure, resulting in removal of edema fluid, improvement of blood supply, and stimulation of cellular proliferation of reparative granulation tissue. It has been used to treat open wounds in the extremities, open sternal wounds, pressure ulcers, and abdominal wall wounds. This study retrospectively reviewed instrumented spine fusions complicated by surgical wound infection and managed by a protocol including the use of VAC in order to evaluate the efficacy of applying vacuum therapy on patients with deep spine infections and exposed instrumentation. METHODS: Twenty consecutive patients with deep wound infections after undergoing spinal fusion procedures were studied. There were 12 men and 8 women with an average age of 55 years (31-81 years). Eight patients had undergone concomitant anterior and posterior arthrodesis, nine patients had a posterior spinal fusion, and three patients had a transforaminal lumbar interbody fusion. Seven patients had a decompression with exposed dura. Sixteen patients presented with a draining wound within the first 6 weeks postoperatively (average 24 days). There were four patients who presented with back pain and temperature after 1 year postoperatively (average 3 years). All patients were taken to the operating room for irrigation and debridement followed by placement of the VAC with subsequent delayed closure of the wound. RESULTS: There was an average of 1.8 (1-8) irrigation and debridement procedures prior to placement of the VAC. Once the VAC was initiated, there was an average of 2.2 (2-3) procedures until and including closure of the wound. The wound was closed an average of 7 days (5-14 days) after the placement of the initial VAC in the wound. All patients tolerated the VAC without adverse effects. All patients were kept on a 6-week course of intravenous antibiotic therapy. The average follow-up was 10 months (6-24 months). There were no cases of uncontrolled sepsis once the VAC was initiated. All patients achieved a clean closed wound without removal of instrumentation at a minimum follow-up of 6 months. CONCLUSION: VAC therapy is an effective adjunct in closing complex deep spinal wounds with exposed instrumentation.

Supernumerary ring chromosome 20 in a mother and her child.

The authors report a familial case of mosaicism for an extra ring 20, identified by fluorescence in situ hybridization (FISH), in a mother and her child. In spite of the fact that both patients had clinical abnormalities, the more severe phenotype present in the child was probably due to the higher percentage of abnormal cells. To our knowledge, this is the first report of a familial extra ring 20 mosaicism.

Pre-transplant prognostic factors for patients with high-risk leukemia undergoing an unrelated cord blood transplantation.

From July 1995 to December 2001, 42 patients with leukemia aged 1-42 years underwent cord blood transplant (CBT) from unrelated, < or = 2 antigen HLA mismatched donors. In all, 26 patients were in < or = 2nd complete remission and 16 in more advanced phase. Conditioning regimens, graft-versus-host disease (GVHD) prophylaxis and supportive policy were uniform for all patients. The cumulative incidence of engraftment was 90% (95% CI: 0.78-0.91). The cumulative incidence of III-IV grade acute- and chronic-GVHD was 9% (95% CI: 0.04-0.24) and 35% (95% CI: 0.21-0.60), respectively. The 4-year cumulative incidence of transplant-related mortality (TRM) and relapse was 28% (95% CI: 0.17-0.47) and 25% (95% CI: 0.14-0.45), respectively. The 4-year overall survival (OS), leukemia-free survival (LFS) and event-free survival (EFS) were 45% (95% CI: 0.27-0.63), 47% (95% CI: 0.30-0.64) and 46% (95% CI: 0.30-0.62), respectively. In multivariate analysis, the most important factor affecting outcomes was the CFU-GM dose, associated with CMV serology (P=0.003 and 0.04, respectively) in influencing OS and with patient sex (P=0.008 and 0.03, respectively) in influencing LFS. Finally, CFU-GM dose was the only factor that affected EFS significantly (P=0.02). In conclusion, the infused cell dose expressed as in vitro progenitor cell growth is highly predictive of outcomes after an unrelated CBT and should be considered the main parameter in selecting cord blood units for transplant.

A molecular phylogeographic study based on DNA sequences from individual metacercariae of Paragonimus mexicanus from Guatemala and Ecuador.

A molecular phylogeographic study of Paragonimus mexicanus collected from Guatemala and Ecuador was performed. Genomic DNA was extracted from individual metacercariae, and two gene regions (partial mitochondrial cytochrome c oxidase subunit 1 (CO1) and the second internal transcribed spacer of the nuclear ribosomal gene repeat (ITS2)) were amplified by the polymerase chain reaction (PCR). Sequences segregated in a phylogenetic tree according to their geographic origins. ITS2 sequences from Ecuador and Guatemala differed at only one site. Pairwise distances among CO1 sequences within a country were always lower than between countries. Nevertheless, genetic distances between countries were less than between geographical forms of P. westermani that have been suggested to be distinct species. This result suggests that populations from Guatemala and Ecuador are genetically differentiated perhaps at the level of subspecies.

Ergonomics, gerontechnology, and design for the home-environment.

An ergonomic approach could improve the quality of life and activities in daily living. Gerontechnology reduces the effects of age-related impairments with technological devices and particular design for the home-environment. Physiological decline with increasing age renders the daily activities at home more difficult. This paper highlights some "common sense" and specific design suggestions in the entrance and kitchen, aimed to increase the self-sufficiency of elderly people. We suggest that gerontechnology may have a particular role in the improvement of comfort and safety for aged people.

Postoperative deep wound infection in adults after posterior lumbosacral spine fusion with instrumentation: incidence and management.

The authors reviewed 817 instrumented lumbosacral fusions in adults and found an incidence of 3.2% deep wound infections. The primary focus of this study was the management of these infections, with particular attention to whether the implants needed to be removed. A consulting infectious disease specialist indicated that an acute infection of a low back fusion wound could not be healed without removal of the metallic implants. This opinion was in contrast to the authors' daily experience and prompted this study. The authors identified and reviewed 817 cases of instrumented posterior lumbosacral arthrodeses in adults. A detailed analysis of any case with a deep wound infection was performed and yielded and infection rate of 3.2% (26 patients). Of these, 24 achieved a clean, closed wound without removal of instrumentation through a protocol of aggressive debridement and secondary closure. Instrumentation removal is not necessary to obtain a clean, closed wound using an aggressive approach with early diagnosis, vigorous debridement in the operative room under general anesthesia, delayed primary or secondary closure, and appropriate antibiotic coverage.

Complications associated with pedicle screws.

BACKGROUND: The safety and the effectiveness of pedicle-screw instrumentation in the spine have been questioned despite its use worldwide to enhance stabilization of the spine. This review was performed to answer questions about the technique of insertion and the nature and etiology of complications directly attributable to the screws. METHODS: We performed a retrospective review of all of the pedicle-screw procedures that were done by us from January 1, 1984, to December 31, 1993. We inserted 4790 screws during 915 operative procedures on 875 patients; 668 (76.3 percent) of the patients had a lumbosacral arthrodesis. The mean duration of follow-up was three years (range, two to five years). The accuracy of screw placement was assessed on intraoperative, immediate postoperative, and follow-up radiographs with use of a technique that was developed by one of us (F. D.); this technique has yet to be validated to determine the prevalence of various types of error. RESULTS: Of the 4790 screws, 4548 (94.9 percent) had been inserted within the pedicle and the vertebral body. One hundred and thirty-four (2.8 percent) of the screws had perforated the anterior cortex, and this was the most common type of perforation. One hundred and fifteen (2.4 percent) of the screws were associated with complications that could be ascribed to the use of pedicle screws. The most common problem was late-onset discomfort or pain related to a pseudarthrosis or perhaps to the screws; this problem was associated with 1102 (23.0 percent) of the screws, used in 222 (24.3 percent) of the procedures. The symptoms necessitated removal of the instrumentation with or without repair of the pseudarthrosis. A pseudarthrosis was found during forty-six (20.7 percent) of the 222 procedures. Irritation of a nerve root occurred after nine procedures (1.0 percent) and was caused by eleven screws (0.2 percent); it was more commonly caused by medially placed screws. Three patients had residual neurological weakness despite removal of the screws. Twenty-five screws (0.5 percent), used in twenty procedures (2.2 percent), broke. The screws that broke were of an early design. A pseudarthrosis was found in thirteen of twenty patients who had broken screws. Sixteen of the twenty patients had an exploration; three of them were found to have a solid fusion, and thirteen were found to have a pseudarthrosis. The remaining four patients had evidence of a solid fusion on radiographs and had no pain. CONCLUSIONS: There are few problems associated with the insertion of screws, provided that the surgeon is experienced and adheres to the principles and details of the operative technique. Our review revealed a low rate of postoperative complications related to pedicle screws. The problem of late-onset pain may be related to the implants or to the stiffness of the construct; however, it is difficult to accurately identify its exact etiology.

Follicle cell proteasome activity and acid extract from the egg vitelline coat prompt the onset of self-sterility in Ciona intestinalis oocytes.

In the hermaphrodite ascidian Ciona intestinalis, the egg vitelline coat (VC) controls gamete self-nonself discrimination. Oocytes, after germinal vesicle breakdown, can be fertilized by both self and nonself sperm. However, a barrier to fertilization by self sperm progressively develops in the VC in the 3 hours after germinal vesicle breakdown. During this period, follicle cells attached to the outer surface of the VC release self-sterility factors that bind to the VC. Within the follicle cells, these factors (possibly peptides) are thought to be shuttled to the cell membrane by an hsp70 homolog (Cihsp70). In fact, antibodies to hsp70 block the development of self-sterility. Proteasomes are central to the production of antigen peptides. Specific inhibition of proteasome activity with clasto-lactacystin beta-lactone (CLbetaL) prevented the onset of self-sterility, but had no effect once this process had started. CLbetaL did not block fertilization by nonself sperm. The self-sterility factors were removed from mature oocytes by exposure to acidified media, and their biological activity was transferred to immature oocytes treated with CLbetaL. The obvious high multiplicity of self-nonself recognition alleles involved in fertilization, and the involvement of an hsp70 and a proteasome in processing self-sterility factors, suggests that this system may be evolutionarily related to the vertebrate immune system.

Plasma membrane association and preliminary characterization of Drosophila sperm surface glycosidases.

Previous studies have identified beta-N-acetylglucosaminidase (GlcNAc'ase) and (alpha-mannosidase activities on the Drosophila melanogaster sperm surface which may have a role in fertilization. The aim of this study was to investigate their linkage to the sperm plasma membrane. We verified that glycosidases are not peripherally adsorbed to the cell surface by evaluating their resistance to release by KI, by buffered salt solutions of high ionic strength or alkaline buffers. Glycosidases were released from the sperm surface by detergents and, only to a minor extent, by mild proteolysis. Differential detergent solubilization pointed out that Triton X-114 was the most effective releasing agent for GlcNAc'ase and CHAPS for mannosidase. No activity was released from the membrane by a phosphatidylinositol-specific phospholipase C (PI-PLC). The released forms were quite hydrophilic in phase separation experiments with Triton X-114. This finding indicates the presence of a hydrophobic domain limited to a single transmembrane helix or/and the presence of an extensive glycosylation. The use of a Con-A binding assay demonstrated that both the enzymes are glycosylated. The molecular weight of the released glycosidases estimated by gel filtration was 158 kDa for GlcNAc'ase and 317 kDa for mannosidase. These results suggest that Drosophila melanogaster GlcNAc'ase and mannosidase are mannosylated integral membrane proteins that would function as exoenzymes with their active sites accessible in the extracellular space.

Identification and developmental expression of Ci-isl, a homologue of vertebrate islet genes, in the ascidian Ciona intestinalis.

Here we describe the cloning and expression pattern of Ci-isl, a homologue of vertebrate genes, in the ascidian. Early in development, Ci-isl expression occurs in the primordia of palps and brain vesicle, then in the tailbud embryo it is transiently extended to the notochord cells. At larva stage, the expression is down-regulated in the notochord, and it persists predominantly in the compartments of the nervous system. These observations indicate that also in invertebrates, islet genes show an expression pattern during differentiation of the nervous system.