Preliminary report of two-stage secondary alveolar bone grafting for patients with bilateral cleft lip and palate.

OBJECTIVE: To overcome the difficulties of one-stage secondary alveolar bone grafting for patients with bilateral cleft lip and palate (BCLP) who have a broad alveolar cleft, the value of two-stage alveolar bone grafting was examined. PATIENTS: Three patients (2 girls and 1 boy) with BCLP were treated by two-stage alveolar bone grafting. The procedure consisted of a first-stage surgery (mean age 8 year 6 months +/- 7.8 months), which consisted of alveolar bone grafting for one side of the BCLP, and second-stage surgery for the contralateral side several months later. For the postoperative assessment, radiographs of the operated site were examined. RESULTS: The average amount of implanted bone per operation and per patient was 6.8 +/- 1.1 g and 13.7 +/- 1.0 g, respectively. Postoperative clinical and radiographic examinations revealed that an appreciable alveolar bone ridge had formed, and there were no major complications. CONCLUSIONS: Two-stage alveolar bone grafting, which makes it possible to reduce the amount of implanted bone, could be an optional surgical procedure for patients with BCLP and a broad alveolar cleft.

Incidence of cleft lip or palate in 303738 Japanese babies born between 1994 and 1995.

To investigate the incidence of cleft lip or palate or both (CLP) in Japan, 303738 babies born in 1532 institutions between 1994 and 1995 were examined and 437 (0.14%) were found to have abnormalities. Of these babies, 32.1% had cleft lip, 43.3% had cleft lip and palate, and 24.8% had cleft palate (Table 2). These results show that the incidence of cleft lip and palate has declined compared with the period from 1981 to 1982.

Treatment of hypoglossia-hypodactyly syndrome without extremeity anomalies.

Three cases of hypoglossia-hypodactyly syndrome without limb deformities are reported. All exhibited different degrees of tongue hypoplasia, micrognathia, retrognathia with a very narrow space between the left and right halves of the mandible, constricted isthmus, and only one lower incisor. Bone lengthening for the midline mandibular hypoplasia and orthodontic treatment were performed in the three cases with satisfactory results.

Dental rehabilitation using endosseous implants and orthognathic surgery in patients with cleft lip and palate: report of two cases.

We describe the use of endosseous implants in the autogenous particulate cancellous bone and marrow grafted alveoli after orthognathic surgery for dental rehabilitation of patients with cleft lip and palate. This procedure has been applied to two patients and produced good results functionally and aesthetically. The results are encouraging and indicate that implant placement after orthognathic surgery is useful for patients both with congenital missing teeth and retrognathic maxillae.

Induction of apoptosis in mouse brain capillary endothelial cells by cyclosporin A and tacrolimus.

Although cyclosporin A and tacrolimus are used clinically as potent immunosuppressants, there have been reports of neurotoxicity and encephalopathy. A possible mechanism is that these drugs damage the blood-brain barrier (BBB), inducing dysfunction and increased permeability, and are then able to enter the brain. We studied the cytotoxicity of cyclosporin A and tacrolimus, focused on apoptosis induction, using an immortalized cell line established from BALB/c mouse cerebral microvessel endothelial cells (MBEC4). We found that these two drugs induced cell shrinkage, chromatin condensation and DNA fragmentation, which are characteristics of apoptosis. Our data suggest that the induction of apoptosis on the brain capillary endothelial cells may be at least partly involved in the occurrence of immunosuppressant-induced encephalopathy.

Development of a DeltaglnA balanced lethal plasmid system for expression of heterologous antigens by attenuated vaccine vector strains of Vibrio cholerae.

We have previously shown that more prominent immune responses are induced to antigens expressed from multicopy plasmids in live attenuated vaccine vector strains of Vibrio cholerae than to antigens expressed from single-copy genes on the V. cholerae chromosome. Here, we report the construction of a DeltaglnA derivative of V. cholerae vaccine strain Peru2. This mutant strain, Peru2DeltaglnA, is unable to grow on medium that does not contain glutamine; this growth deficiency is complemented by pKEK71-NotI, a plasmid containing a complete copy of the Salmonella typhimurium glnA gene, or by pTIC5, a derivative of pKEK71-NotI containing a 1. 8-kbp fragment that directs expression of CtxB with a 12-amino-acid epitope of the serine-rich Entamoeba histolytica protein fused to the amino terminus. Strain Peru2DeltaglnA(pTIC5) produced 10-fold more SREHP-12-CtxB in supernatants than did ETR3, a Peru2-derivative strain containing the same fragment inserted on the chromosome. To assess immune responses to antigens expressed by this balanced lethal system in vivo, we inoculated germfree mice on days 0, 14, 28, and 42 with Peru2DeltaglnA, Peru2DeltaglnA(pKEK71-NotI), Peru2(pTIC5), Peru2DeltaglnA(pTIC5), or ETR3. All V. cholerae strains were recoverable from stool for 8 to 12 days after primary inoculation, including Peru2DeltaglnA; strains containing plasmids continued to harbor pKEK71-NotI or pTIC5 for 8 to 10 days after primary inoculation. Animals were sacrificed on day 56, and serum, stool and biliary samples were analyzed for immune responses. Vibriocidal antibody responses, reflective of in vivo colonization, were equivalent in all groups of animals. However, specific anti-CtxB immune responses in serum (P

Effect of cyclosporin A or tacrolimus on the function of blood-brain barrier cells.

Recently, it has been reported that continuous treatment with cyclosporin A or tacrolimus induces encephalopathy in transplant patients. The mechanism of immunosuppressant-induced encephalopathy is unclear. We investigated the cytotoxicity to brain capillary endothelial cells and the effect of these two drugs on P-glycoprotein function using mouse brain capillary endothelial (MBEC4) cells. The transcellular transport of [3H]sucrose was significantly increased and the cellular viability, based on 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and trypan blue exclusion test, was decreased by cyclosporin A (approximately 50% at 5 microM; P<0.005), while tacrolimus showed a much smaller effect. These findings indicate that the toxicity of cyclosporin A was greater than that of tacrolimus. The uptake of [3H]vincristine, a substrate of P-glycoprotein, was increased by these two drugs. The expression of P-glycoprotein in MBEC4 cells was reduced, but there was no effect on mdr1b mRNA levels. The decrease in the expression of P-glycoprotein may be due to the inhibition of the turnover of P-glycoprotein, which involves translation. In conclusion, the direct cytotoxic effect on the brain capillary endothelial cells and the inhibition of P-glycoprotein may be partly involved in the occurrence of immunosuppressant-induced encephalopathy.

Placement of endosseous implants into bone-grafted alveolar clefts: assessment of bone bridge after autogenous particulate cancellous bone and marrow graft.

The objective of this study was to evaluate the bone quantity of alveoli grafted with autogenous particulate cancellous bone and marrow for implant placement in patients with alveolar clefts. Bone height, bone width, and interdental alveolar crest level were evaluated using computed tomography and periapical radiographs. The grafted alveoli underwent resorption 3-dimensionally, and the interdental alveolar crest level also decreased. The latter seemed to be the critical factor for implant surgery, as almost half of the grafted alveoli required another bone graft within 24 months after the original bone graft to increase the interdental alveolar crest level for endosseous implant placement. These data suggest that alveoli grafted with particulate cancellous bone and marrow are suitable for implant placement, but that the loss of width and height of the bone bridge must also be considered.

Placement of endosteal implants combined with chin bone onlay graft for dental reconstruction in patients with grafted alveolar clefts.

Endosteal implants were inserted into grafted alveoli after particulate cancellous bone and marrow grafting in seven patients with cleft lip or palate in conjunction with simultaneous chin bone onlay grafting. In these patients, the alveolar bone height of the bony bridge was insufficient when evaluated by both computed tomographic and periapical radiographic images. The age at first implant surgery ranged from 14 to 28 years. Although four of the seven patients had an uneventful course, three had wound dehiscence, and in all but one of them the exposed chin bone underwent partial or total necrosis. Ultimately all seven implants integrated into the bone, and the alveolar bone height was increased in all but one patient. The results indicate that chin bone onlay grafting with simultaneous implant insertion is useful in patients with cleft lip or palate with insufficient alveolar bone height.

Surgical repositioning of the premaxilla in combination with two-stage alveolar bone grafting in bilateral cleft lip and palate.

OBJECTIVE: This paper introduces a surgical technique for premaxillary repositioning in combination with two-stage alveolar bone grafting for the correction of the premaxillary deformity of patients with bilateral cleft lip and palate (BCLP). The paper also reports on two patients with BCLP who underwent this surgical management. SURGICAL PROCEDURE: The operation is usually performed when the patient is 8 to 14 years of age. In the first stage of surgery, the side more accessible to the septo-premaxillary junction is selected, and an osteotomy of the premaxilla and unilateral alveolar bone grafting are performed. Approximately 4 to 12 months after the first stage of surgery, contralateral alveolar bone grafting is carried out. CONCLUSION: We have found that this surgical procedure is highly effective, because it ensures the blood supply to the premaxilla and minimizes the potential for surgical failure. Moreover, it affords wide exposure of the premaxillary bone surface, facilitating sufficient boney bridging and allowing for orthodontic tooth movement.

Use of endosseous implants for dental reconstruction of patients with grafted alveolar clefts.

PURPOSE: The purpose of this study was to investigate the clinical application of endosseous implants placed into grafted alveolar clefts and to evaluate the short-term outcome. PATIENTS AND METHODS: Nineteen patients (6 males and 13 females; mean age, 17.9 years; range, 9.7 to 33.6 years at first implant surgery), including 11 with unilateral cleft lip and palate, and eight with unilateral cleft lip and alveolus, were studied. All patients except for one who underwent periosteoplasty received grafts of autogenous particulate cancellous bone and marrow (PCBM) obtained from the llium. After bone bridge formation, orthodontic treatment and preparation for implant placement were performed. RESULTS: A total of 21 implants were placed in the bone-grafted alveoli of the 19 patients. The most frequently used length was 15 mm. In five patients with insufficient alveolar bone height, a chin bone onlay graft was combined with simultaneous implant insertion. The follow-up period ranged from 1 year to almost 3 years after implant placement, and the clinical outcome was excellent in all except one patient. In this short-term study, the overall survival rate was 90.5%. CONCLUSION: The grafted alveoli were well suited to the placement of endosseous implants, and this treatment was shown to be a viable option for the dental reconstruction of alveolar clefts. However, the interdental alveolar bone height was insufficient for implant installation in a few patients. Further longitudinal studies are required to determine the optimal timing between secondary bone grafting and implant placement.

Use of an osseointegrated implant for dental rehabilitation after cleft repair by periosteoplasty: a case report.

OBJECTIVE: We report here on a patient with unilateral cleft lip and alveolus who underwent dental rehabilitation of cleft alveolus using an osseointegrated implant after cleft repair by periosteoplasty. The patient, whose lateral incisor was congenitally missing, had periosteoplasty, followed by excellent bone formation at the cleft alveolus within 2 years. After the completion of orthodontic alignment of the maxillary dental arch, a Brånemark single-tooth implant was placed into the bone-formed alveolus. CONCLUSION: This treatment procedure offers an option of dental rehabilitation for the alveolar clefts of patients with cleft lip and/or palate.

Immunological and functional comparison between Clostridium perfringens iota toxin, C. spiroforme toxin, and anthrax toxins.

Clostridium perfringens iota and C. spiroforme toxins consist of two separate proteins. One is the binding component and the other the enzymatic component. The two toxins secreted by Bacillus anthracis are composed of binary combinations of three proteins: protective antigen, lethal factor, and edema factor. As shown by Western blotting and ELISA, the binding component of anthrax toxin shares common epitopes with that of iota toxin and C. spiroforme toxin which are closely related immunologically. However, no functional complementation was observed between iota toxin and anthrax toxin components. The binding components can form toxins active on macrophages only in combination with their respective enzymatic components. Agents which prevent acidification of endosomes do not have the same effects on anthrax toxin activity as they do on iota and C. spiroforme toxins. Therefore, the mechanisms of entry into the cells are presumably different. Since the binding components of anthrax toxins and iota toxin share a conserved putative translocation domain, these binding components could have a common mode of insertion into the cell membranes.

Zinc content of the Bacillus anthracis lethal factor.

We present evidence that the anthrax toxin lethal factor binds multiple zinc atoms. Results from atomic adsorption spectroscopy indicate that lethal factor contains approximately three zinc atoms per toxin molecule. Lethal factor treated with EDTA and o-phenanthroline contained a similar number of zinc atoms, indicating that all three zinc atoms are tightly bound to the protein. Lethal factor contains the highly conserved zinc-binding consensus sequence, HExxH, that is present in all known zinc metalloproteases. In addition, lethal factor contains an inverted form of this motif, HxxDH, which may also be involved in zinc binding. Using a large array of protease model substrates, however, we were unable to detect an endogenous protease activity for lethal factor.

The effects of pH on the interaction of anthrax toxin lethal and edema factors with phospholipid vesicles.

Bacillus anthracis secretes three distinct proteins which interact in binary combinations to produce two toxins. The two effector moieties, edema factor (EF) and lethal factor (LF), interact competitively with the cell receptor-binding moiety, protective antigen (PA), to produce biologically distinct effects. The passage of the toxins through an acidified endosomal compartment is an essential step in the intoxication process, and it has been shown that low pH triggers the insertion of the activated form of PA, PA63, into model lipid bilayers. In this study, we have examined the effects of pH on the interaction of LF and EF with a model membrane system. Protein labeling by radioactive phospholipid probes indicated that both LF and EF are able to insert into asolectin lipid bilayers in a pH-dependent manner. For LF, the extent of insertion into the bilayer was accompanied in parallel by the release of calcein from preloaded LUV (large unilamellar vesicles). The transition pH for protein insertion, however, was somewhat higher than that for membrane destabilization. The extent of protein radiolabeling and the release of calcein from LUV incubated with EF was similar to that seen with LF; however, the pH dependency was significantly less. Low pH-induced membrane insertion by both proteins was accompanied by only a minimal change in conformation. These results suggest that LF and EF may be actively involved in the process of toxin translocation.

DNA fragmentation and cytolysis in U937 cells treated with diphtheria toxin or other inhibitors of protein synthesis.

We treated the human monoblastoid cell line, U937, with various cytotoxic proteins or drugs that specifically inhibit protein synthesis and monitored the cells for degradation of chromosomal DNA and other changes. In cells treated with diphtheria toxin (DT), Pseudomonas aeruginosa exotoxin A, ricin toxin, and abrin toxin the chromosomal DNA was degraded into oligonucleosome-sized fragments, the chromatin became condensed, and the cell nuclei fragmented. All of these changes are characteristic of cells undergoing apoptosis, or programmed cell death. Various drugs, including puromycin, cycloheximide, emetine, and anisomycin produced similar changes. An enzymically attenuated mutant of DT, DT-E148S, produced effects identical to those produced by the native toxin, except that a higher concentration of toxin was required, corresponding to the reduction in ADP-ribosylation activity. In all cases, DNA degradation and other changes were observed only after the rate of protein synthesis was reduced to low levels, approximately 10% or less of normal levels. These results imply that inhibition of protein synthesis in U937 cells induces apoptosis, regardless of the mechanism of action of the inhibitor. Differences in the kinetics of induction of apoptosis by the various inhibitors may reflect secondary effects on other aspects of cellular physiology.

Facilitation of complement-dependent killing of the Lyme disease spirochete, Borrelia burgdorferi, by specific immunoglobulin G Fab antibody fragments.

In the absence of specific antibody, Borrelia burgdorferi is resistant to the bactericidal action of complement, despite the capacity of the spirochete to activate complement. Complement-mediated killing of B. burgdorferi requires the presence of antiborrelial immunoglobulin G (IgG). The effect of bactericidal IgG takes place after formation of the C5 convertase. Therefore, we examined the ability of Fab fragments from bactericidal IgG to mediate killing of B. burgdorferi by complement. The complement-activating domain of IgG, the Fc fragment, was not required for killing of borreliae, as monovalent Fab fragments prepared from immune IgG were also able to mediate killing. However, the killing efficiency of the Fab fragments was less than that of intact IgG, suggesting that the bactericidal activity of IgG is enhanced by divalency. IgG Fab-mediated killing occurred without increased complement activation or C3 fluid-phase consumption. Cell killing proceeded via the classical complement pathway, as no killing of Fab fragment-sensitized cells was observed in human serum deficient in C2. These results demonstrate directly that the bactericidal effect of anti-B. burgdorferi IgG is independent of the complement-activating properties of the antibody.

Biochemical and physiological changes induced by anthrax lethal toxin in J774 macrophage-like cells.

Experiments were performed to probe the mechanism by which Bacillus anthracis Lethal Toxin (LeTx) causes lysis of J774 macrophage-like cells. After incubation of cells with saturating concentrations of the toxin, two categories of effects were found, which were distinguishable on the basis of chronology, Ca(2+)-dependence, and sensitivity to osmolarity. The earliest events (category I), beginning 45 min postchallenge, were an increase in permeability to 22Na and 86Rb and a rapid conversion of ATP to ADP and AMP. Later events (category II) included alterations in membrane permeability to 45Ca, 51Cr, 36Cl, 35SO4, 3H-amino acids, and 3H-uridine, beginning at 60 min; inhibition of macromolecular synthesis, leakage of cellular lactate dehydrogenase and onset of gross morphological changes, at approximately 75 min; and cell lysis, beginning at 90 min. Category II events exhibited an absolute requirement for extracellular Ca2+ and were blocked by addition of 0.3 M sucrose to the medium, whereas category I events were attenuated, but not blocked, by either of these conditions. On the other hand, both ATP depletion and the category II events were blocked in osmotically stabilized medium that was also isoionic for Na+ and K+. This suggests that permeabilization of the plasma membrane to monovalent cations and water may be the earliest of the physiological changes described here. The resulting influx of Na+ and efflux of K+ would be expected to cause depletion of ATP, via increased activity of the Na+/K+ pump. Subsequently the influx of Ca2+, induced by depletion of ATP, imbalances in monovalent cautions, and/or more dramatic changes in permeability due to influx of water, would be expected to trigger widespread changes leading ultimately to cytolysis.

Complement-mediated killing of the Lyme disease spirochete Borrelia burgdorferi. Role of antibody in formation of an effective membrane attack complex.

Lyme disease is a multisystemic illness caused by the spirochete Borrelia burgdorferi. In the absence of specific antibody, the spirochete is resistant to the bactericidal activity of C, despite the capacity of B. burgdorferi to activate both C pathways. We examined the mechanism of serum resistance by measuring the deposition of C3 and terminal C components on B. burgdorferi in the presence and absence of immune IgG. In normal human serum antibody-sensitized borreliae bound similar amounts of C3, and similar or increased amounts of C8 and C9, in comparison to unsensitized bacteria. However, at comparable levels of C3, C8, or C9 uptake, only sensitized bacteria were killed. The requirement of antibody for killing could not be explained by differences in the rate of C deposition or by differences in the C9 to C8 ratio in the membrane attack complex (MAC). We found that bacteria incubated in C5-depleted human serum, but not in C6-depleted serum, were killed when this treatment was followed by antibody and the missing C components. Bacteria were also killed by reactive lysis (C5b-9) provided that antibody was present. Therefore, the effect of bactericidal IgG occurred at the stage of C5b binding to the bacterial surface. Elution studies of bound C9 indicated that the MAC was stably bound to the outer membrane of B. burgdorferi, whether or not the bacteria were treated with antibody. However, treatment with 0.1% trypsin released 48% of 125I-C9 from the surface of unsensitized borreliae and 24% from IgG-sensitized cells, demonstrating that the presence of the antibody changed the accessibility to trypsin of C9 in the MAC. These results indicate that the effect of antibody in the killing process is not to enhance the rate or extent of initial or terminal component binding, but rather to alter the bacterial outer membrane to allow effective MAC formation.