Healing response of apicomarginal defects to two guided tissue regeneration techniques in periradicular surgery: a double-blind, randomized-clinical trial.

AIM: To compare healing responses to periosteal sliding grafts and polyglactin 910 periodontal mesh used as guided tissue regeneration (GTR) materials/techniques when both periapical and periradicular bone loss are present. METHODOLOGY: Thirty patients with suppurative chronic apical periodontitis with apicomarginal communication were selected and allocated randomly into two groups according to the barrier technique to be used during periradicular surgery: periosteal graft group (n = 15) and bioabsorbable membrane group (n = 15). Clinical and radiological evaluations were completed prior to surgery, a week later and every 3 months after surgery up to 12 months to measure the periodontal pocket depth (PD), clinical attachment level (CAL), gingival margin position (GMP), size of periapical lesion, percentage reduction of the periapical rarefaction, and periapical healing. RESULTS: Both groups showed highly significant (P < 0.001) reductions in periodontal PD, CAL and size of periapical lesion at 12 months whilst GMP was unaltered. No significant difference between the experimental groups was evident for these parameters, or for the percentage reduction of size of the periapical lesion and clinical-radiographic healing. CONCLUSION: Guided tissue regeneration applied to apicomarginal defects using sliding periosteal grafts and use of bioabsorbable membranes led to similar enhancements of the clinical outcome of periradicular surgery in terms of periapical healing, gain of periodontal support, PD reduction and minimal recession of the gingival margin.

The immunomodulatory factors of arthropod saliva and the potential for these factors to serve as vaccine targets to prevent pathogen transmission.

In general, attempts to develop vaccines for pathogens transmitted by arthropods have met with little or no success. It has been widely observed that the saliva of arthropods that transmit disease enhances the infectivity of pathogens the arthropod transmits to the vertebrate host. Indeed, it has been observed that vaccinating against components of the saliva of arthropods or against antigens expressed in the gut of arthropods can protect the host from infection and decrease the viability of the arthropod. These results suggest that multi-subunit vaccines that target the pathogen itself as well as arthropod salivary gland components and arthropod gut antigens may be the most effective at controlling arthropod-borne pathogens as these vaccines would target several facets of the lifecycle of the pathogen. This review covers known immunomodulators in arthropod salivary glands, instances when arthropod saliva has been shown to enhance infection and a limited number of examples of antiarthropod vaccines, with emphasis on three arthropods: sandflies, mosquitoes and hard ticks.

Periosteal grafts as barriers in periradicular surgery: report of two cases.

AIM: To describe the usefulness of periosteal grafts as barriers for bone regeneration in periradicular surgery when advanced periodontal breakdown occurs. SUMMARY: The treatment of advanced periodontal breakdown as a result of an associated endodontic lesion constitutes a multifaceted challenge to the clinician. If the source of the irritation cannot be removed by orthograde endodontic treatment, nonsurgical and surgical endodontic/periodontal intervention may be required. Two cases with suppurative chronic apical periodontitis with apicomarginal communication are described. Clinical and radiological evaluations were completed immediately prior to surgery, a week later and every 2 months after surgery for 10 months. Both patients were treated using split-thickness flaps and lateral displacement of the periosteum prior to suturing, in order to close the communication between the oral and the periapical surroundings. A remission of the clinical signs and symptoms, and successful healing in the short-term were achieved in these cases. KEY LEARNING POINTS: Periapical and periodontal lesions are closely related through pathways of communication. Disruption of the cortical plate and the presence of dentoalveolar sinus tracts can have a deleterious effect on the regeneration process after periradicular surgery. The adoption of supplementary periodontal surgical techniques may help to solve some of the difficulties in the healing process in periradicular surgery. Periosteal grafts have been shown to have the potential to stimulate bone formation when used as a graft material.

Characterisation of the carbohydrate components of Taenia solium metacestode glycoprotein antigens.

Human neurocysticercosis is caused by Taenia solium metacestodes. It usually affects the central nervous system of humans and can be confused with other brain pathologies. The Lens culinaris-binding glycoproteins from this parasite have been shown to be ideal targets for the development of a highly specific immunoassay for the diagnosis of neurocysticercosis. In the present study we characterised the carbohydrates associated with five antigenic glycoproteins of T. solium metacestodes in the range of 12-28 kilodaltons. Lectin-affinities and enzymatic deglycosylations suggested that each of the five antigens contain various glycoforms of asparagine-linked carbohydrates of the hybrid, complex and probably high mannose type. These carbohydrates accounted for at least 30-66% of the apparent molecular mass of the glycoconjugates. In contrast, there was no evidence for the presence of O-linked carbohydrates. Lectin affinity patterns suggested that the sugars are short and truncated in their biosynthetic route, and that some contain terminal galactose moieties. Elucidating the precise structure of the carbohydrates and establishing their role in antigenicity will be essential to design strategies to produce them in large and reproducible amounts for the development of improved immunoassays.

An analysis of the humoral immune response of dogs following vaccination with irradiated infective larvae of Dirofilaria immitis.

In this study, dogs were immunized with irradiated L3 larvae of Dirofilaria immitis. Following challenge with non-irradiated L3, vaccinated dogs had an average of 71% fewer adult worms compared to non-vaccinated animals. A comparative analysis of eosinophil and antibody responses of these two groups of dogs is presented. Vaccinated dogs preferentially recognized several larval (14, 20, 30, 34, 39 kDa), adult worm (20 kDa) and microfilarial (36, 38, 71, 84 kDa) antigens. To characterize these antigens, the extent of glycosylation was assessed. The data suggest that an earlier response to these antigens may be important in the protection induced in dogs by administration of irradiated L3 of D. immitis.

Mapping of a B-cell epitope present in the neuraminidase of Trypanosoma cruzi.

We have previously shown that a polyclonal (rabbit anti-TCNA) and a mouse monoclonal antibody (TCN-2) against the neuraminidase of Trypanosoma cruzi (TCNA) inhibit enzyme activity, immunoprecipitate active enzyme, enhance in vitro infection, and identify a subpopulation of extracellular trypomastigotes. We now report on the identification of a synthetic peptide that contains the epitope recognized by these antibodies. The synthetic peptide (TR) is a dodecamer (D-S-S-A-H-G-T-P-S-T-P-A) deduced from the DNA sequence of the long tandem repeat (LTR) domain present in the TCNA carboxyterminus. By ELISA, rabbit anti-TCNA bound to TR coupled to ovalbumin, and the binding was inhibited by soluble TR but not by BR (Y-S-V-D-D-G-E-T-W-E), a peptide derived from the N-terminal domain of the enzyme. TCN-2 recognized TR, and this reaction as well as TCN-2 binding to endogenous TCNA could be inhibited by soluble TR but not by BR. These results indicate that the rabbit anti-TCNA and TCN-2 react with the LTR region of TCNA. Antibodies to TR reacted by immunoblot with the TCNA of the Silvio X-10/4, MV-13 and Y-H6 strains, identifying the same molecular polymorphism previously observed with the rabbit anti-TCNA and TCN-2. Furthermore, anti-TR antibodies immunoprecipitated active enzyme and immunofluorescence analysis revealed that anti-TR and TCN-2 antibodies detected equally well the differential expression of their epitopes in intra- and extracellular trypomastigotes. Moreover, expression of TR and TCN-2 epitopes on the different stages of T. cruzi paralleled the stage-specificity of TCNA activity. TCN-2 prevented desialylation by TCNA of intact cells but not of soluble glycoconjugates, indicating that TCN-2 epitope is probably not associated with the enzyme catalytic site, in agreement with the predicted sequence of the TCNA gene. Finally, analysis of the humoral response of a Chagasic patient to different areas of the TCNA molecule indicated that the antibody response is predominantly against TR suggesting that the tandem repeat is the immunodominant domain of TCNA.

On the interaction of Trypanosoma cruzi neuraminidase and human lipoproteins.

Binding and penetration of Trypanosoma cruzi to host cells is a process that preludes infection and is mediated by specific recognition molecules. Neuraminidase is one of the parasite molecules involved in infection and, in this review, we describe some of its biochemical characteristics, its interaction with human lipoproteins and its effect on infection of mammalian cells.

The Trypanosoma cruzi neuraminidase contains sequences similar to bacterial neuraminidases, YWTD repeats of the low density lipoprotein receptor, and type III modules of fibronectin.

Trypanosoma cruzi expresses a developmentally regulated neuraminidase (TCNA) implicated in parasite invasion of cells. We isolated full-length DNA clones encoding TCNA. Sequence analysis demonstrated an open reading frame coding for a polypeptide of 1,162 amino acids. In the N-terminus there is a cysteine-rich domain containing a stretch of 332 amino acids nearly 30% identical to the Clostridium perfringens neuraminidase, three repeat motifs highly conserved in bacterial and viral neuraminidases, and two segments with similarity to the YWTD repeats found in the low density lipoprotein (LDL) receptor and in other vertebrate and invertebrate proteins. This domain is connected by a structure characteristic of type III modules of fibronectin to a long terminal repeat (LTR) consisting of 44 full length copies of twelve amino acids rich (75%) in serine, threonine, and proline. LTR is unusual in that it contains at least 117 potential phosphorylation sites. At the extreme C-terminus is a hydrophobic segment of 35 amino acids, which could mediate anchorage of TCNA to membranes via a glycosylphosphatidylinositol linkage. This is the first time a protozoan protein has been found to contain a YWTD repeat and a fibronectin type III module. The domain structure of TCNA suggests that the enzyme may have functions additional to its catalytic activity such as in protein-protein interaction, which could play a role in T. cruzi binding to host cells.

Trypanosoma cruzi: localization of neuraminidase on the surface of trypomastigotes.

Immunoelectron microscopy (IEM) using TCN-2, a monoclonal antibody specific for Trypanosoma cruzi neuraminidase (NA), was performed to determine the precise localization of the parasite enzyme. In agreement with previous observations, TCN-2 reacted with tissue culture trypomastigotes, but not with epimastigotes, amastigotes or intracellular forms in intermediate stages of development. NA was localized on the surface of tissue culture trypomastigotes and in the Golgi apparatus suggesting that the enzyme is modified post-translationally. In agreement with this suggestion, digestion of NA with N-Glycanase, an enzyme that releases N-linked oligosaccharides, decreased the molecular weight of the polypeptides that make up NA.

Differential expression of Trypanosoma cruzi neuraminidase in intra- and extracellular trypomastigotes.

The developmentally regulated expression of Trypanosoma cruzi neuraminidase in culture cells was monitored by immunofluorescence with a monoclonal antibody (TCN-2) against the enzyme. The results showed that TCN-2 reacted with all intracellular trypomastigote forms (NA+) but not with amastigotes. Immunoprecipitation of radiolabeled neuraminidase confirmed TCN-2 reactivity with trypomastigotes and the specificity of the antibody binding. During exiting from the host cells, all trypomastigotes were still NA+. However, when free in the extracellular environment, the relative proportion of NA+ parasites declined from 100% to about 20%, thereby establishing a subpopulation of trypomastigotes which did not express enzyme (NA-). The expression of neuraminidase in all intracellular trypomastigotes and in only a subpopulation of the extracellular counterpart suggests that the enzyme may play a role in parasite exiting from infected cells.

Monoclonal antibodies against Trypanosoma cruzi neuraminidase reveal enzyme polymorphism, recognize a subset of trypomastigotes, and enhance infection in vitro.

The identification and characterization of two murine mAb (TCN-1 and TCN-2) that react with the neuraminidase of Trypanosoma cruzi is reported. The mAb were identified based on their ability to inhibit enzyme activity and recognize neuraminidase in crude enzyme preparations. TCN-1 and TCN-2 recognized Ag in tissue culture trypomastigotes but not in the amastigotes, epimastigotes, or metacyclic trypomastigotes using immunoblot assays and immunofluorescence. In addition, clones Y-H6, MV-13, and Silvio X-10/4 of T. cruzi revealed a unique banding pattern characteristic of each clone. In Silvio X-10/4, the mAb recognized four distinct bands ranging from 121,000 to 203,000 whereas in Y-H6 and MV-13 they identified bands ranging from 138,000 to 222,000. Characterization of neuraminidase by two-dimensional PAGE revealed the polypeptides that make up the enzyme to have isoelectrical points ranging from 6.55 to 7.30. Immunofluorescence and C-mediated lysis assays showed that the mAb reacted with a subset of trypomastigotes representing 28% of the total parasite population. Functional studies showed that the mAb enhanced infection of cultured cells by trypomastigotes. Our experiments confirm previous findings with polyclonal Ab and are in accordance with the hypothesis that neuraminidase modulates infection through a negative control mechanism.