Thiol/disulfide exchange occurs in rotavirus structural proteins during contact with intestinal villus cell surface.

Protein disulfide isomerase (PDI) is an enzyme that catalyzes disulfide bond reduction or formation and rearrangements of disulfide bridges, and also functions as a chaperone. During entry of some of the viruses PDI participates in thiol-disulfide exchange. Previous reports show that rotavirus entry is interfered by impermeant thiol/disulfide exchange inhibitors and antibodies against PDI. Our objective was to assess the interaction between PDI and triple-layered particles (TLPs) from rotavirus strains ECwt and RRV and from a human rotavirus isolate (HI) during the early steps of virus entry in a system of isolated small intestinal villi. Purified soluble PDI was incubated with either isolated intestinal villi or cell membrane-enriched fractions in the presence or absence of thiol/disulfide inhibitors such as bacitracin, DTNB or N- ethylmaleimide followed by the assessment of the PDI interactions with TLPs and rotavirus structural proteins in terms of their redox state changes. Soluble and membrane-bound PDI was found to interact with TLPs from all the rotaviruses assayed and also with the isolated structural proteins represented by the recombinant rVP5* (a tryptic cleavage product of VP4), rVP6 and the native VP7. PDI interaction with TLPs and rotavirus structural proteins was decreased by the presence of thiol/disulfide exchange inhibitors. Interactions of cell membrane-enriched fractions with TLPs produced rearrangements in the disulfide bridges of rotavirus structural proteins. We conclude that PDI interacts with rotavirus virions through redox reactions that could facilitate the rotavirus entry into the host cell. Keywords: cell surface PDI; thiol-disulfide exchange; rotavirus TLPs; virus entry; bacitracin; DTNB.

Rotavirus receptor proteins Hsc70 and integrin αvß3 are located in the lipid microdomains of animal intestinal cells.

Several cell surface molecules such as integrins, sialic acid and Hsc70 have been reported to participate in the process of adsorption and penetration of rotaviruses into cells. Some of them have been found in susceptible cell lines but not in epithelial cells of natural hosts so that their real role in the infection process is unclear. In this study, we attempted to confirm the presence of Hsc70 and integrin αvß3 in the cytoplasmic membrane of isolated intestinal epithelial cells of pig, mouse and cow. Using immunocytochemistry, immunofluorescence, co-immunoprecipitation, ELISA, Western blot analysis and flow cytometry we established that in these cells, (i) Hsc70 and integrin αvß3 formed a complex in lipid raft microdomains of the cytoplasmatic membrane and (ii) Hsc70 levels increased after rotavirus infection. These results indicate that these molecules act as receptors of rotaviruses in susceptible cells.

Identification of two immunoreactive peptides useful for the detection of porcine astrovirus.

Porcine astroviruses (PAstVs) are small RNA viruses associated with gastroenteritis. The capsid polyprotein of PAstVs, human (HAstVs) and feline (FAstVs) AstVs has a high similarity at the N-terminus before residue 415. Previous results showed the cross-detection of PAstVs and HAstVs from diarrheal samples using a commercial ELISA test that uses a monoclonal antibody to capture HAstVs, suggesting the existence of common immunoreactive epitopes between these two virus types. In this study, we seeked immunoreactive peptides located in the PAstV capsid that may be potentially used for their specific detection. The variability and hydrophobicity of a short fragment of 132 amino acids were analyzed using several capsid sequences of PAstVs and HAstVs. Peptides TATL, SLNP and IDIV were selected, synthesized and inoculated into rabbits. Pre- and hyperimmune sera were collected and their reactivity was examined by immunoassay and immunofluorescence against two wild-type strains of PAstV adapted to grow in cell culture, observing reactivity in two of the sera. Finally, the possible cross-reactivity of the sera against HAstVs was partially ruled out using HAstV8. Our data suggest that TATLGTIGSNSSGKTELEAC and IDIVVGKAATFNLKASDLSGP peptides represent immunoreactive regions useful for the specific detection of PAstVs.

Amino acid domains 280-297 of VP6 and 531-554 of VP4 are implicated in heat shock cognate protein hsc70-mediated rotavirus infection.

The rotavirus infection mechanism seems to be a multi-step process which is still not fully understood. The heat shock cognate protein hsc70 has been proposed as being a co-receptor molecule for rotavirus entry into susceptible cells. In this work, an attempt was made to determine the existence of possible domains for VP4 and VP6 binding to hsc70. We selected amino acid sequences 531-554 from VP4 and 280-297 from VP6 on the basis of already recognized sequences for binding to hsc70. This study determined that DLPs and synthetic peptides from VP6 (aa 280-297) and VP4 (aa 531-554), individually or in combination, inhibited rotavirus RRV, YM and WA entry into MA104 and Caco-2 cells in an additive and dose-dependent manner. Hyperimmune sera against these synthetic peptides blocked infection by infectious TLPs. Capture ELISA results showed that DLPs interact with hsc70, probably through VP6 as the specific interaction between hcs70 and DLPs was disrupted by a VP6 peptide. These results suggest that VP6 takes part during rotavirus cell entry by binding to hsc70. This, as well as previous work, provides insight concerning the function of hsc70 within a multi-step model of rotavirus entry.

Early events of rotavirus infection: the search for the receptor(s).

The entry of rotaviruses into epithelial cells seems to be a multistep process. Infection competition experiments have suggested that at least three different interactions between the virus and cell surface molecules take place during the early events of infection, and glycolipids as well as glycoproteins have been suggested to be primary attachment receptors for rotaviruses. The infectivity of some rotavirus strains depends on the presence of sialic acid on the cell surface, however, it has been shown that this interaction is not essential, and it has been suggested that there exists a neuraminidase-resistant cell surface molecule with which most rotaviruses interact. The comparative characterization of the sialic acid-dependent rotavirus strain RRV (G3P5[3]), its neuraminidase-resistant variant nar3, and the human rotavirus strain Wa (G1P1A[8]) has allowed us to show that alpha 2 beta 1 integrin is used by nar3 as its primary cell attachment site, and by RRV in a second interaction, subsequent to its initial contact with a sialic acid-containing cell receptor. We have also shown that integrin alpha V beta 3 is used by all three rotavirus strains as a co-receptor, subsequent to their initial attachment to the cell. We propose that the functional rotavirus receptor is a complex of several cell molecules most likely immersed in glycosphingolipid-enriched plasma membrane microdomains.

Positional changes and stability of bone segments during simultaneous bilateral mandibular lengthening and widening by distraction.

The purpose of this study was to analyse the skeletal changes and stability of the distracted segments during and after simultaneous widening and bilateral lengthening of the mandible in baboons with a miniaturized intraoral bone-borne osteodistractor. Distraction appliances were activated 5 days after vertical posterior body and midsymphyseal osteotomies at a rate of 0.9 mm/day for 10 days. The appliances were then stabilized for a period of 8 weeks, after which the animals were killed. The distraction gaps and gingival tissues were studied clinically and on standardized radiographs. The proportional movement of the distracted segments that we found supports the clinical use of the miniaturized intraoral bone-borne distraction appliance to widen and lengthen the mandible selectively. It also supports the concept of positioning the mandibular osteodistractors parallel to the common vector of distraction, which should parallel the corrected maxillary occlusal plane.

Integrin alpha2beta1 mediates the cell attachment of the rotavirus neuraminidase-resistant variant nar3.

It was previously reported that integrins alpha2beta1, alpha4beta1, and alphaXbeta2 are involved in rotavirus cell infection. In this work we studied the role of integrin subunits alpha2, alpha4, and beta2 on the attachment of rotaviruses RRV and nar3 to MA104 cells. Integrin alpha2beta1 was found to serve as the binding receptor for the neuraminidase-resistant virus nar3, whereas the neuraminidase-sensitive strain RRV interacted with this integrin at a postattachment step. It was shown that nar3 binds alpha2beta1 through the DGE integrin-recognition motif located in the virus surface protein VP5. Integrin subunits alpha4 and beta2 do not seem to be involved in the initial cell binding of either virus.

Integrin alpha(v)beta(3) mediates rotavirus cell entry.

Rotavirus strains differ in their need for sialic acid (SA) for initial binding to the cell surface; however, the existence of a postattachment cell receptor, common to most, if not all, rotavirus strains, has been proposed. In the present study, antibodies to the alpha(v) and beta(3) integrin subunits, and the alpha(v)beta(3) ligand, vitronectin, efficiently blocked the infectivity of the SA-dependent rhesus rotavirus RRV, its SA-independent variant nar3, and the neuraminidase-resistant human rotavirus strain Wa. Vitronectin and anti-beta(3) antibodies, however, did not block the binding of virus to cells, indicating that rotaviruses interact with alpha(v)beta(3) at a postbinding step, probably penetration. This interaction was shown to be independent of the tripeptide motif arginine-glycine-aspartic acid present in the natural ligands of this integrin. Transfection of CHO cells with alpha(v)beta(3) genes significantly increased their permissiveness to all three rotavirus strains, and the increment of virus infectivity was reverted by incubation of these cells either with antibodies to beta(3) or with vitronectin. These findings implicate alpha(v)beta(3) integrin as a cellular receptor common to neuraminidase-sensitive and neuraminidase-resistant rotaviruses, and support the hypothesis that this integrin could determine, at least in part, the cellular susceptibility to rotaviruses.

Long-term skeletal and dental effects of mandibular symphyseal distraction osteogenesis.

The purpose of this study was to evaluate the effects of mandibular symphyseal distraction osteogenesis using a tooth-borne expansion device. The sample included 20 Hispanic nonsyndromic patients (11 males and 9 females) between 13.5 years and 37.3 years of age. Predistraction (1.5 months before surgery), postdistraction (1 month after surgery), and long-term follow-up (1.3 year after surgery) records included posteroanterior, lateral, and panoramic radiographs and models. Postdistraction radiographic evaluation showed that symphyseal distraction osteogenesis produced insignificant increases in the bicondylar, bigonion, and biantegonion widths; intermolar and, especially, intercanine widths increased significantly and a distraction gap was observed in the symphyseal region. Follow-up model analysis showed the largest width increases between the first molars and second premolars and the smallest width increases between canines and first premolars. The difference between the postdistraction and long-term follow-up width changes was explained by the postdistraction orthodontic effect, which modified the shape of the dental arch. A disproportionate pattern of distraction, characterized by significantly greater dental than skeletal widening, was observed in the second molar and antegonion region. Distraction osteogenesis without presurgical orthodontic treatment produced significant proclination of the mandibular incisors; no proclination was observed in cases with predistraction orthodontic treatment. Dental crowding was resolved by the movement of teeth into the distraction regenerate and concomitant orthodontic treatment. Follow-up radiographs showed transverse skeletal stability of the distraction procedure. We conclude that mandibular symphyseal distraction osteogenesis increased mandibular arch width and partially corrected dental crowding, with a potential for disproportionate distraction patterns and proclination of the mandibular incisors.

Biochemical characterization of rotavirus receptors in MA104 cells.

We have tested the effect of metabolic inhibitors, membrane cholesterol depletion, and detergent extraction of cell surface molecules on the susceptibility of MA104 cells to infection by rotaviruses. Treatment of cells with tunicamycin, an inhibitor of protein N glycosylation, blocked the infectivity of the SA-dependent rotavirus RRV and its SA-independent variant nar3 by about 50%, while the inhibition of O glycosylation had no effect. The inhibitor of glycolipid biosynthesis d, l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) blocked the infectivity of RRV, nar3, and the human rotavirus strain Wa by about 70%. Sequestration of cholesterol from the cell membrane with beta-cyclodextrin reduced the infectivity of the three viruses by more than 90%. The involvement of N-glycoproteins, glycolipids, and cholesterol in rotavirus infection suggests that the virus receptor(s) might be forming part of lipid microdomains in the cell membrane. MA104 cells incubated with the nonionic detergent octyl-beta-glucoside (OG) showed a ca. 60% reduction in their ability to bind rotaviruses, the same degree to which they became refractory to infection, suggesting that OG extracts the potential virus receptor(s) from the cell surface. Accordingly, when preincubated with the viruses, the OG extract inhibited the virus infectivity by more than 95%. This inhibition was abolished when the extract was treated with either proteases or heat but not when it was treated with neuraminidase, indicating the protein nature of the inhibitor. Two protein fractions of around 57 and 75 kDa were isolated from the extract, and these fractions were shown to have rotavirus-blocking activity. Also, antibodies to these fractions efficiently inhibited the infectivity of the viruses in untreated as well as in neuraminidase-treated cells. Five individual protein bands of 30, 45, 57, 75, and 110 kDa, which exhibited virus-blocking activity, were finally isolated from the OG extract. These proteins are good candidates to function as rotavirus receptors.

Intraoral mandibular distraction osteogenesis.

During recent years, distraction osteogenesis has gained in popularity for the treatment of various bone deficiencies either in the vertical, transverse, or anteroposterior dimension. Distraction osteogenesis has been shown to be an effective technique for mandibular widening and lengthening where traditional orthognathic surgery has important limitations. The intraoral approach to these procedures prevents damage to the inferior alveolar nerve and the developing dental follicles, and eliminates hypertrophic facial scars. Intraoral distraction osteogenesis also avoids donor-site morbidity, and minimizes the need for blood transfusion or prolonged fixation. This intraoral application provides for enhanced patient acceptance and reduces the potentially negative psychosocial effects of wearing an extraoral distraction appliance.

Intraoral widening and lengthening of the mandible in baboons by distraction osteogenesis.

PURPOSE: The purpose of this study was to analyze the skeletal and dental positional changes and histomorphology of the distraction regenerates and mucogingival periosteal tissues that occurred after simultaneous widening and bilateral lengthening of the mandible in baboons by a miniaturized intraoral bone-borne distraction appliance. MATERIALS AND METHODS: Distraction appliances were activated 5 days after vertical ramus and symphyseal osteotomies at a rate of 0.9 mm/d for 10 days. The appliances were then stabilized for 8 weeks, after which the animals were killed. The distraction gaps and gingival tissues were analyzed clinically, histologically, and by standardized radiographic studies. RESULTS: Positional changes of the canines and incisor apices were proportional to the skeletal movements. Tipping of both incisors toward the center of the distraction gap was observed. Proportionate movement of the superior and inferior portion of the distracted segments was noted. Newly formed longitudinal trabecular columns parallel to the vector of distraction originated from the intact margins of alveolar bone contiguous with the adjacent teeth. Active histogenesis occurred in the stretched mucogingival periosteal tissues located in the distraction gaps. CONCLUSIONS: The results of this investigation support the clinical use of the miniaturized intraoral bone-borne distraction appliance to selectively widen and lengthen the mandible. The orientation of the mandibular distractors must be parallel to the common vector of distraction, which should be parallel to the maxillary occlusal plane. The formation of a bone regenerate in the alveolar region depends on the presence of an adequate bone interface on either side of the distraction gap.

Surgical implant repositioning: a clinical report.

Esthetically compromised or nonrestorable implants present major clinical problems. Of 3,850 implants placed, 10 osseointegrated implants in 6 patients were surgically repositioned using maxillary or mandibular osteotomies and rigidly fixated, under intravenous sedation. The segments were predictably changed in a vertical, anteroposterior, transverse, or axial inclination manner. Excellent healing of bone and soft tissue was observed. This simple, reliable technique allowed these 10 implants to be esthetically and functionally restored with permanent prostheses.

Mandibular widening by intraoral distraction osteogenesis.

Transverse mandibular deficiency with crowding of the mandibular anterior teeth is frequently present in patients with Class I and II malocclusions. The hallmarks of treatment by compensating orthodontics, functional appliances or orthopaedic devices are instability, compromised periodontium and compromised facial aesthetics. A new surgical technique has been developed to widen the mandible. The method is based upon gradual osteodistraction following vertical interdental symphyseal osteotomy. Ten patients with transverse mandibular deficiency and significant dental crowding were treated by symphyseal distraction and subsequent non-extraction decompensating orthodontic treatment. Either an intraoral tooth-borne Hyrax appliance or a new custom-made bone-borne osteodistractor was used to gradually widen the mandible. The surgical procedures were accomplished under local anaesthesia and intravenous sedation in an ambulatory surgical setting using an individualized distraction protocol. The appliances were activated 7 days after symphyseal osteotomies, once each day at a rate of 1 mm per day and stabilized for 30-40 days after distraction. After the segments were distracted, non-extraction orthodontic alignment of the mandibular anterior teeth was accomplished. The symphyseal distraction gaps were bridged by new bony regenerate. Distraction osteogenesis provided an efficient surgical alternative to orthognathic surgery for widening the mandible and treatment of transverse mandibular deficiency without extraction of teeth.