Variations in caries diagnoses and treatment recommendations and their impacts on the costs of oral health care.

OBJECTIVE: To evaluate the reproducibility of caries detection and treatment planning among public health dentists and estimate the possible impact of their decisions on financial costs. RESEARCH DESIGN AND SETTINGS: Thirty nine dentists working in the public health service of Piracicaba, São Paulo, Brazil made a combined visual-radiographic caries examination of 40 occlusal surfaces of extracted permanent teeth mounted on two dental mannequins and proposed treatment plans for each tooth. Histological validation then evaluated the diagnoses validity and the suitability of the treatment plans. OUTCOME MEASURES: Inter-examiner agreement was calculated by Cohen's Kappa statistics. The sensitivity and specificity of caries detection and treatment decision were calculated. The costs of dental treatment plans for public health system were calculated from a Brazilian public health service fee scale. RESULTS: Inter-examiner agreement for caries detection was moderate (kappa = 0.42) while for treatment decisions it was fair (kappa = 0.29). The sensitivity and specificity were 0.69 and 0.65 for caries detection and 0.56 and 0.65 for treatment decision respectively. Dentists overestimated the presence and depth of carious lesions and there was a tendency to treat enamel lesions using invasive therapeutic procedures. Mean treatment cost across the two cases was 32US$ (range 9-65) while the histologically validated cost was 23US$. CONCLUSION: The variability in caries detection and treatment decision negatively affected the cost of the dental treatment.

Expression of heterologous promoters in Lutzomyia longipalpis and Phlebotomus papatasi (Diptera: Psychodidae) cell lines.

To establish a transient expression system for genes introduced into sand fly cell lines, we tested the expression of the luciferase reporter gene under control of different promoters. Towards this end, we lipofected cell lines obtained from New and Old World sand flies, LL-5 from Lutzomyia longipalpis Lutz & Neiva and PP-9 from Phlebotomus papatasi Scopoli, respectively. The relative levels of luciferase expression were studied under control of Drosophila melanogaster Meigen heat shock protein 70 (hsp70), human cytomegalovirus, simian virus 40 or Junonia coenia (Hübner) densovirus (P9) promoters. The Drosophila heat shock protein 70 promoter, originating from insect genes, functioned as a strong promoter in both cell lines. Promoters from the different virus genes also were capable of driving transgene expression in both cell lines.

Promoters from Drosophila heat shock protein and cytomegalovirus drive transient expression of luciferase introduced by particle bombardment into embryos of the oyster Crassostrea gigas.

Using high velocity particle bombardment, we transferred a reporter gene into early stages of the oyster Crassostrea gigas and showed the expression of the introduced genes in these embryos at later stages of development. We tested two promoters: (1) the heat shock protein 70 promoter of Drosophila; (2) the cytomegalovirus early promoter, both linked to the luciferase reporter gene. The hsp 70-luc (pDrluc) construct allowed an expression level up to 55-fold higher than the control in a heat inducible fashion. The CMV-luc (pCMVL) construct constitutively gave a 4-fold higher expression than the control. This confirms the suitability of particle bombardment for transfecting genes into eggs, zygotes and trochopores of bivalves and demonstrates the functionality of two heterologous expression vectors in C. gigas.

Microinjection of bivalve eggs: application in genetics.

New strategies for embryonic manipulation have been developed in recent years through plant and animal research. However, research on marine invertebrate embryos has suffered from a lack of basic tools, such as microinjection. Here we present a technique developed for microinjecting eggs and embryos of the oyster Crassostrea gigas and the mussel Mytilus edulis. In experimental trials, approximately 40% of microinjected embryos survived. This technique was used to microinject beta-galactosidase, for which specific detection techniques were developed. A reporter construct (CMV-beta) based on a promoter of cytomegalovirus linked to the beta-galactosidase-encoding gene was then microinjected, and the expression level of this construct was monitored. The suitability of this technique is discussed in terms of its application to the manipulation of bivalve mollusks in pathology and genetics.

[Endovascular treatment of abdominal aorta aneurysms using the Stentor device. Preliminary experience]. / Trattamento endovascolare degli aneurismi dell'aorta addominale con il dispositivo Stentor. Esperienza preliminare.

PURPOSE: The aim of this report is to describe our experience with the Stentor device for endovascular treatment of the abdominal aortic infrarenal aneurysms also extending to the bifurcation and the common iliac arteries. Stentor is a thermal memory (Nitinol) self-expanding graft, covered by an external 0.1 mm Dacron material. METHODS: Between December 1994 and July 1995 endoluminal repair of infrarenal aneurysmal disease was undertaken in 6 patients at high surgical risk. The lesions include 2 infrarenal abdominal aorto-aortic aneurysms, 2 infrarenal abdominal aortic aneurysms extended to the common iliac arteries and 2 false aortic aneurysms in patients with previous aorto-bifemoral graft. Straight grafts were implanted in 4 patients and bifurcated in 2. Repair was done in the operating room using general anesthesia. The endograft was placed through remote arteriotomies and advanced under fluoroscopic guidance to his predetermined site. Three-dimensionally reconstructed spiral CT scan and arteriography were performed before the procedure for a preoperative accurate measurement for endograft preprocedural adaptation in length and diameter. RESULTS: All endografts were successfully deployed. Intraoperative arteriography at the end of the procedure revealed a distal "leak" into an aneurysmal common iliac artery, due to diameter mismatch, in a bifurcated device. There was no instance of embolism or graft migration. No patient required conversion to an open operation. There were no instances of embolism or graft migration. No patient required conversion to an open operation. There were no coagulative disorders. Minor complications were: groin haematoma (1), fever (1), intestinal paralysis (1), pelvic pain (1). Follow-up with spiral CT-scan and echo color-Doppler confirmed normal blood flow through the graft in 5 patients and persistence of distal leak in 1 patient. CONCLUSIONS: These preliminary results demonstrate the accuracy of implantation and device's adaptability to the particular anatomy of the aneurysmal aorta and iliac arteries. Proximal fixation to the aortic wall, secure seal at the proximal and distal fixation point present the critical aspects of this new surgical technique. More detailed preoperative measurements of aneurysmal disease are required rather than for traditional surgery. Presently we prefer to treat the no operable patients with this endovascular technique in relation with shortness of the follow-up.

Future of biotechnology-based control of disease in marine invertebrates.

Infectious disease is the single most devastating problem in mollusc and shrimp aquaculture. Pathogens causing the greatest problems have been identified as viruses, prokaryotes, and protozoans. Two approaches employing methods of biotechnology have been proposed to prevent, manage, and control mollusc and shrimp diseases. The first is development of a diagnostic scheme for detection and identification of pathogens, using molecular probes. This offers the opportunity for prophylactic measures to be taken. Molecular probes have been prepared for the major pathogens of molluscs, but in the case of shrimp pathogens, only a few are available. Monoclonal antibodies have also been prepared and are used in immunodiagnosis, e.g., immunofluorescence detection. Such diagnostic tools are relatively new to aquaculture, but have enormous potential. A second approach to the control of disease in marine invertebrates, notably shrimp, involves use of genetically transformed strains resistant to specific pathogens. Pathogen-resistant transgenic animals have been developed, but such research has only just begun for molluscs and shrimp. Transfection methods applied to mollusc and shrimp embryos have been successful, with preliminary data showing efficiency of heterologous promoters in controlling expression of reporter genes. Other transformation systems also show promise, including transposable elements and densoviruses.

Characterisation of shrimp haemocytes and plasma components by monoclonal antibodies.

Various haemolymph components of the shrimp Penaeus japonicus were identified and characterised by monoclonal antibodies. Three groups of monoclonal antibodies were raised. Their reactivity to haemocyte types and/or secreted molecules was determined by immunofluorescence and the molecular masses of the antigens were analysed by western-blotting. A 170 kDa protein, in reducing conditions, was recognized by four panhaemocytic monoclonal antibodies from group 1. This protein was present both in the plasma and in the haemocytes from which it appears to be secreted. The shrimp haemocytes were separated by isopycnic centrifugation on a Percoll gradient and the different subpopulations were antigenically analysed using the two monoclonal antibodies, 40E2-2A and 40E10-2B, from group 2. The granular cells were labelled by 40E2-2A which was specific for a protein of 142 kDa also present in plasma. By comparison, the 40E10-2B monoclonal antibody was assumed to be the marker for small hyaline and semigranular cells since the granular ones were not labelled. Moreover, the antigen immunoprecipitated by this monoclonal antibody was shown to have different molecular masses of 250, 150, 66 and 27 kDa under nonreducing conditions. It appeared to be secreted by the haemocytes. Some plasma proteins were recognized by the third group of monoclonal antibodies. The antibodies, designated 41D11-3A, 42C11-3B and 42E8-3C, all immunoprecipitated a protein with an apparent molecular mass of 180 kDa under reduced conditions. The 44E6-3D antibody was specific for a 75 kDa protein under reduced conditions and was shown to be immunoreactive against P. japonicus haemocyanin extract.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro activity of the antimicrobial peptide magainin 1 against Bonamia ostreae, the intrahemocytic parasite of the flat oyster Ostrea edulis.

Magainins are peptide antibiotics with broad antibacterial and antiparasitic activities, originally extracted from the skin of Xenopus laevis. We investigated the effects of magainin 1 against Bonamia ostreae, the intrahemocytic parasite of the flat oyster Ostrea edulis. Viability of purified protozoa was assessed microscopically by the uptake of the vital dyes acridine orange and ethidium bromide. Following exposure to magainin 1, Bonamia viability was reduced in a dose-dependent manner. Within the peptide concentration of 500 micrograms/ml, the parasite viability was reduced by 94%. Electron microscopy showed membrane damage and release of cytoplasmic organelles in the injured Bonamia. The study of magainin 1 activity against Ostrea edulis hemocytes did not show any morphological change in the host cells, and the peptide did not impair the capabilities of hemocytes to produce chemiluminescence when stimulated to phagocytize zymosan particles. The possibility to genetically transform molluscs to generate disease-resistant organisms is currently under investigation. Antimicrobial peptides such as magainins may provide effective gene sequences to be manipulated.

Identification of insulin-like peptides in cerebral ganglia neurosecretory cells of the mussel Mytilus edulis.

The immunostaining patterns of cerebral ganglia sections from the mussel Mytilus edulis with monoclonal antibodies raised against cerebral ganglia (CG) extracts were compared to those obtained with various polyclonal anti-insulin-like antibodies. One of the monoclonal antibodies (MAB 46) revealed clusters of positive cells in localization comparable to those revealed by the polyclonal antibodies. The nature of the antigen recognized by MAB 46 and the polyclonal antibodies was compared by gel filtration-HPLC of a cerebral ganglia extract. Similar peaks were revealed by the monoclonal and polyclonal antibodies. MAB 46 significantly inhibited the cerebral ganglia induced stimulation of amino-acid incorporation by mantle edge cell suspensions, suggesting that the antigen recognized by MAB 46 is involved in the control of growth.

Use of monoclonal antibodies for identification of growth-controlling neuropeptides in the mussel Mytilus edulis (Mollusca:Bivalvia).

Monoclonal antibodies were developed against cerebral ganglia (CG) of the mussel Mytilus edulis by the immunization of mice with unpurified homogenates of these organs. The screening protocol of hybridoma was based upon immunohistological observations of cytocentrifugated ganglia cells. A panel of 29 monoclonal antibodies (MABs) specific of CG epitopes was harvested and subsequently used for the immunocytochemical study of CG cells. Several subpopulations of ganglia cells were specifically revealed by MABs. Identification of epitopes involved in growth control was approached via the application of a bioassay allowing the assessment of protein synthesis stimulation. MAB 42 and 46 affected amino acid incorporation induced by CG extract. These results lead to the conclusion that the epitopes recognised by these antibodies are involved in growth control. An immunoenzymatic assay was performed with CG extracts for quantitative analyses of epitopes.

Biolistic techniques for transfection of mosquito embryos (Anopheles gambiae).

To compensate for the extremely low rates of transformation by DNA microinjection into mosquito embryos of Anopheles gambiae, biolistic techniques were evaluated for introduction of DNA into large numbers of mosquito embryos. Biolistic experiments were first performed with a commercially available instrument intended for this purpose, according to the recommended procedure. The amount of DNA delivered was measured by the expression of luciferase under the control of the Drosophila heat shock protein (hsp) 70 promoter. Despite attempts to optimize biolistic parameters, the level of luciferase activity was low and highly variable. Two other methods of biolistic delivery of DNA-coated particles in aqueous suspension were then evaluated. One method used the gas explosion of the commercially available instrument (mentioned above) to drive an aqueous suspension of DNA-coated particles at high pressure. This method reproducibly increased the level of expression about 100-fold without greatly reducing embryo viability. Another method, which was recently described for plant transfection, uses lower pressure to deliver the aqueous suspension of DNA-coated particles. The level of expression of luciferase and the survival of embryos were equivalent to that obtained with the instrument modified for aqueous delivery of particles. Thus, both aqueous methods offer the advantages of reproducibly delivering more DNA to the embryos. Moreover, these methods could be suitable for delivering DNA mixed with proteins, such as restriction endonucleases and integrases, that may be destroyed by ethanol precipitation used in the standard PDS-1000/He method.

Phagocytosis associated chemiluminescence of hemocytes in Mytilus edulis (Bivalvia).

The respiratory burst associated with phagocytosis by Mytilus edulis hemocytes was investigated by measurement of luminol-dependent chemiluminescence (LDCL). After experimental parameters (number of cells, quantity of stimulus) were determined, the biochemical mechanisms involved in the chemiluminescent process were investigated using inhibitors of oxygen radicals and enzymes. In particular, catechol-like phenols suggested the involvement of NADPH-oxidase and peroxidase in oxidative metabolism of mussel hemocytes. The variability of LDCL response observed among individuals and separated hemocyte subpopulations strongly suggests a variable immunocapacity depending on hemogram composition. Using a specific monoclonal antibody to discriminate different hemocyte types, the eosinophilic granulocytes appeared to exhibit the highest LDCL activity.

Restriction maps and sequence homologies of two densovirus genomes.

The genomes of Junonia coenia densonucleosis virus (JcDNV) and Galleria mellonella densonucleosis virus (GmDNV) were analysed by restriction endonuclease analysis and Southern blot hybridization. A total of 37 and 33 restriction sites were mapped on JcDNV and GmDNV DNA, respectively. BglI, HaeII and BstEII were site-specific for JcDNV DNA, and BglII and ClaI for GmDNV DNA. The two genomes had nearly identical maps for several restriction endonucleases and Southern blot hybridization using a total genomic JcDNV probe indicated extensive DNA sequence homologies spanning the entire length of the two genomes. Symmetrical cleavage sites, mapping at the extremities of both genomes, confirmed the presence of inverted terminal repeats of at least 420 to 440 bases in length.

Evidence of neutralizing activity against T3 coliphage in oyster Crassostrea gigas hemolymph.

To investigate defense reactions of bivalve molluscs against viruses, experimental in vitro assays have been developed using T3 coliphage as a test virus. A native neutralizing factor in oyster Crassostrea gigas serum showed high individual variability and was enhanced significantly by repeated sampling of hemolymph from the same oysters. The responsible factor is apparently thermolabile and sensitive to EDTA treatment. Because of an inhibitory effect by the enzymatic inhibitor, phenylmethylsulphonyl fluoride (PMSF), the T3-neutralizing factor may be related to serine protease.

[The schizogony of Polychromophilus (Haemoproteidae), cosmopolitan parasite of Microchiroptera]. / La schizogonie de Polychromophilus (Haemoproteidae) parasite cosmopolite de Microchiroptères.

The schizogony of Polychromophilus was partially known by the findings of Schingarew, Mer and Goldblum who demonstrated microschizonts in the reticulo-endothelial tissue. The present work completes these data by showing the presence of macroschizonts in the lungs of both African and European species. These schizonts are characterized by their large size, the hypertrophy of the host cell and its nucleus and the evolution of the parasite apparently slow when young, becoming rapid later.

[Description of Hepatocystis bainae n.sp., parasite of Hipposideros galeritus (Cantor), Microchiroptera, in Malaysia (author's transl)]. / Description d'Hepatocystis bainae n. sp. (Haemoproteidae) parasite d'Hipposideros galeritus Microchiroptère de Malaisie.

Hepatocystis bainae n. sp., parasite on the Microchiropteran bat, Hipposideros galeritus is described and differentiated from Hepatocystis rodhaini; it is characterized by the type of the microgametocytes ("diffuse"), the small size of the hepatic schizonts and the repartition of the colloide.