Three-Dimensional Cephalometric Analysis: The Changes in Condylar Position Pre- and Post-Orthognathic Surgery With Skeletal Class III Malocclusion.

ABSTRACT: The study includes 21 adult patients with skeletal class III malocclusion who underwent orthognathic surgery and had computed tomography images records presurgery (T0) up to 6 months after the surgery (T1). The computed tomography images were analyzed three-dimensionally using the Proplan CMF 3.0 software. Different skeletal and dental parameters were used in analyzing the cephalometric analysis of the patients. The change in the condylar axis angle was evaluated on 3 planes: axial, coronal, and sagittal. The anteroposterior position of the condyle in relation to the glenoid fossa was evaluated in the sagittal plane. ∠SNB, ∠ANB, ∠Left Y-axis, ∠Right Y-axis were statistically significant (P < 0.01). Significant differences on the condylar axis angle were found between the groups on the sagittal plane (P < 0.05) whereas no significant differences were noted on the axial and the coronal plane. In the anteroposterior condylar position related to the glenoid fossa, the condyle exhibited different displacement on different condyles. The right condyle exhibited more of the posterior displacement whereas the left condyle exhibited more of anterior displacement of the condyle in relation to the glenoid fossa. Numerous studies have done regarding the changes after postsurgery using the two-dimensional cephalometric analysis. Using the 3D techniques helps us to identify the cephalometric point more accurately which thus enhances the accuracy in the cephalometric analysis. However, care should be taken not to change the axis of rotation of the condyle to prevent from the treatment relapse and to avoid temporo-mandibular disorders.

Novel Sperm and Gonadotropin-releasing Hormone-based Recombinant Fusion Protein: Achievement of 100% Contraceptive Efficacy by Co-immunization of Male and Female Mice.

Improvements in long-term female contraception can be achieved by vaccinating with sperm-derived proteins. Here, recombinant proteins comprising either (i) N- (amino acid residues 1-80) or C- (amino acid residues 76-126) terminal fragments of mouse sperm protein 17 (Sp17) fused to the promiscuous T non-B cell epitope of tetanus toxoid (TT), amino acid residues 830-844 followed by di-lysine linker (KK) (TT-KK-Sp17N or TT-KK-Sp17C , respectively) or (ii) mouse equatorin (amino acid residues 21-185) fused to the T non-B cell epitope of bovine RNase (amino acid residues 94-104) were expressed in Escherichia coli. Immunization of female FVB/J mice, using alum as an adjuvant, led to the generation of high antibody titers against the above proteins. Antibodies against both N- and C-terminal fragments of Sp17 reacted with the entire capacitated mouse spermatozoa, whereas those against equatorin reacted exclusively with the equatorial region. Despite the reactivity of all immune sera, only sera from mice immunized with TT-KK-Sp17N and TT-KK-Sp17C significantly reduced mouse in vitro fertilization. Mating studies of the immunized females with un-immunized male mice revealed the highest infertility in the TT-KK-Sp17C -immunized group. In an attempt to further boost the immune response, the C-terminal fragment of Sp17 was expressed as fusion protein with a tandem repeat of gonadotropin-releasing hormone (GnRH) (Sp17C -GnRH2 ). Immunization of both male and female mice with Sp17C -GnRH2 led to higher contraceptive efficacy compared to mice immunized with TT-KK-Sp17C . Interestingly, mating studies wherein partners were both immunized with Sp17C -GnRH2 showed a complete failure of female mice to conceive. Thus, immunization of both males and females with Sp17C -GnRH2 has the potential to increase contraceptive efficacy. Mol. Reprod. Dev. 83: 1048-1059, 2016. © 2016 Wiley Periodicals, Inc.

Contraceptive efficacy of recombinant fusion protein comprising zona pellucida glycoprotein-3 fragment and gonadotropin releasing hormone.

Contraceptive vaccines have been used for the management of wildlife population. In the present study, we have examined the contraceptive potential of Escherichia coli-expressed recombinant fusion protein comprising of 'promiscuous' T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker (KK), dog ZP3 fragment (aa residues 307-346), triglycine spacer (GGG), T cell epitope of bovine RNase (bRNase; aa residues 94-104), GnRH, T cell epitope of circumsporozoite protein of Plasmodium falciparum (CSP; aa residues 362-383), and GnRH. SDS-PAGE analysis of the purified refolded protein revealed a dominant ∼12 kDa band, which in Western blot reacted with mouse polyclonal antibodies against dog ZP3 fragment and mouse monoclonal antibodies against GnRH. Immunization of female FvB/J mice following two booster schedule with the above recombinant protein supplemented with alum led to high antibody titres against the immunogen as well as ZP3 and GnRH as determined by ELISA. The immune sera reacted with zona pellucida of mouse oocyte and also inhibited in-vitro fertilization. The qRT-PCR studies showed decrease in the ovarian GnRH receptor in mice immunized with the recombinant fusion protein. Mating studies revealed high contraceptive efficacy of the recombinant protein as in two independent experiments, 90% of the immunized female mice failed to conceive. Following one booster immunization schedule, 50% of the immunized female mice failed to conceive. However, in adjuvanted controls, all the female mice became pregnant. To conclude, the recombinant protein described herein has a good potential to be developed as candidate contraceptive vaccine.

Canine zona pellucida glycoprotein-3: up-scaled production, immunization strategy and its outcome on fertility.

Zona pellucida (ZP) glycoproteins based contraceptive vaccines have been proposed for the management of wildlife population. In the present study, a fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by a dilysine linker and an ectodomain of dog ZP3 (ZP3; aa residues 23-348) without any affinity tag (TT-KK-ZP3) has been expressed in Escherichia coli. The recombinant protein was successfully produced in fed-batch fermentor and purified. The average yield of purified refolded protein was 12.20 ± 0.61 mg/2g wet cell pellet. Female FvB/J mice immunized with the varying doses of recombinant TT-KK-ZP3 supplemented with alum/PetGel A as adjuvants following a three injection schedule, showed dose dependent increase in serum IgG titer. Antibodies against TT-KK-ZP3 recognized native mouse/dog ZP and significantly inhibited mouse in-vitro fertilization (p=0.012). Immunized mice showed significant reduction in fertility (p<0.05). Higher antibody titers were associated with a decrease in the number of pups born to the immunized female mice. To reduce the number of injections, two injection schedule using various dose combinations of TT-KK-ZP3 supplemented with alum revealed lower immunogenicity and contraceptive efficacy as compared to the three injection schedule. To overcome this, CpG motif was included in addition to alum and both intraperitoneal and intranasal route of immunization following the two injection schedule was investigated. Inclusion of CpG significantly enhanced the antibody titer and improved contraceptive efficacy. In the mice immunized following intraperitoneal route, serum/vaginal IgG and in the mice immunized through intranasal route, vaginal IgA seemed to be important for curtailment in fertility. To conclude, the recombinant protein described herein may be a good candidate for developing contraceptive vaccine for the wildlife population management, in particular street dogs.

Milestones in contraceptive vaccines development and hurdles in their application.

Contraceptive vaccines have been proposed for controlling the growing human population and wildlife population management. Multiple targets such as gonadotropin releasing hormone (GnRH), luteinizing hormone, follicle stimulating hormone, gonadotropin receptors, sperm-specific proteins and zona pellucida glycoproteins have been exploited to develop contraceptive vaccine and their efficacy investigated and shown in various experimental animal models. Vaccines based on GnRH have found application in immuno-castration of male pigs for prevention of boar-taint. Vaccines based on zona pellucida glycoproteins have shown promising results for population management of wild horses and white-tailed deer. Phase II clinical trials in women with ß-human chorionic gonadotropin (ß-hCG)-based contraceptive vaccine established proof of principle that these can be developed for human application. Block in fertility by ß-hCG contraceptive vaccine was reversible. Further research inputs are required to establish the safety of contraceptive vaccines, improve their immunogenicity and to develop novel vaccine delivery platforms for providing long lasting immunity.

Evaluation of recombinant fusion protein comprising dog zona pellucida glycoprotein-3 and Izumo and individual fragments as immunogens for contraception.

Vaccines based on gamete specific proteins have been proposed for fertility inhibition. In the present study, immunogenicity and contraceptive potential of E. coli-expressed recombinant fusion protein TT-KK-ZP3-GGG-Iz, comprising promiscuous T cell epitope of tetanus toxoid (TT) followed by dilysin linker (KK), a fragment of dog zona pellucida glycoprotein-3 (ZP3), triglycine spacer (GGG) and a fragment of dog Izumo (Iz) without any affinity tag has been evaluated in female FvB/J mice. In addition, recombinant TT-KK-ZP3 and Izumo linked to the promiscuous T cell epitope of bovine RNase (bRNase-KK-Iz) and their physical mixture were also used. SDS-PAGE and immunoblot studies revealed ∼32kDa band corresponding to TT-KK-ZP3-GGG-Iz, ∼22kDa band of TT-KK-ZP3 and ∼11kDa band of bRNase-KK-Iz. Groups of mice immunized with the above recombinant proteins led to the generation of high antibody titres against the respective proteins. Immunization with recombinant TT-KK-ZP3-GGG-Iz generated higher antibody titre as compared to mice immunized with physical mixture of TT-KK-ZP3 and bRNase-KK-Iz. Antibodies against TT-KK-ZP3-GGG-Iz and TT-KK-ZP3 recognized mouse and dog ZP and those against TT-KK-ZP3-GGG-Iz and bRNase-KK-Iz recognized mouse and dog acrosome-reacted sperm in an indirect immunofluorescence assay. Immune sera from groups of mice immunized with the above recombinant proteins led to a significant reduction in mouse in vitro fertilization. Mating studies revealed significant reduction in fertility as compared to adjuvant control group. Highest infertility was observed in group of mice immunized with TT-KK-ZP3 followed by TT-KK-ZP3-GGG-Iz. Infertility was associated with the antibody titres against ZP3, whereas no association in the inhibition of fertility and antibody titres against Izumo was observed. In conclusion, these studies revealed the contraceptive potential of ZP3, which could not be further enhanced by the inclusion of Izumo.

Production of tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid and dog zona pellucida glycoprotein-3 for contraceptive vaccine development.

Affinity tags can interfere in various physicochemical properties and immunogenicity of the recombinant proteins. In the present study, tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker and dog zona pellucida glycoprotein-3 (ZP3; aa residues 23-348) (TT-KK-ZP3) was expressed in Escherichia coli. The recombinant protein, expressed as inclusion bodies (IBs), was purified by isolation of IBs, processed to remove host cell proteins, followed by solubilization and refolding. A specific 39 kDa protein including ZP3 was identified by SDS-PAGE. CD spectra showed the presence of α-helices and ß-sheets, and fluorescent spectroscopy revealed emission maxima of 265 A.U. at 339 nm for refolded protein and showed red shift in the presence of 6 M guanidine hydrochloride. Immunization of inbred FvB/J female mice with purified recombinant TT-KK-ZP3 (25 µg/animal) led to generation of high antibody titers against the recombinant protein. The antibodies reacted specifically with ZP matrix surrounding mouse oocytes. Immunized mice showed significant reduction in fertility as compared to the control group. The studies described herein provide a simple method to produce and purify tag-free recombinant protein for the development of a contraceptive vaccine.

Mammalian zona pellucida glycoproteins: structure and function during fertilization.

Zona pellucida (ZP) is a glycoproteinaceous translucent matrix that surrounds the mammalian oocyte and plays a critical role in the accomplishment of fertilization. In humans, it is composed of 4 glycoproteins designated as ZP1, ZP2, ZP3 and ZP4, whereas mouse ZP is composed of ZP1, ZP2 and ZP3 (Zp4 being a pseudogene). In addition to a variable sequence identity of a given zona protein among various species, human ZP1 and ZP4 are paralogs and mature polypeptide chains share an identity of 47%. Employing either affinity purified native or recombinant human zona proteins, it has been demonstrated that ZP1, ZP3 and ZP4 bind to the capacitated human spermatozoa and induce an acrosome reaction, whereas in mice, ZP3 acts as the putative primary sperm receptor. Human ZP2 only binds to acrosome-reacted spermatozoa and thus may be acting as a secondary sperm receptor. In contrast to O-linked glycans of ZP3 in mice, N-linked glycans of human ZP3 and ZP4 are more relevant for induction of the acrosome reaction. Recent studies suggest that Sialyl-Lewis(x) sequence present on both N- and O-glycans of human ZP play an important role in human sperm-egg binding. There are subtle differences in the downstream signaling events associated with ZP3 versus ZP1/ZP4-mediated induction of the acrosome reaction. For example, ZP3 but not ZP1/ZP4-mediated induction of the acrosome reaction is dependent on the activation of the Gi protein-coupled receptor. Thus, various studies suggest that, in contrast to mice, in humans more than one zona protein binds to spermatozoa and induces an acrosome reaction.

Contraceptive vaccines based on the zona pellucida glycoproteins for dogs and other wildlife population management.

Zona pellucida (ZP) glycoproteins, by virtue of their critical role in fertilization, have been proposed as candidate antigens for the development of contraceptive vaccines. In this review, the potential of a ZP-based contraceptive vaccine for the management of wildlife population, with special reference to street dogs, is discussed. Immunization of various animal species, including female dogs, with native porcine ZP led to inhibition of fertility, which was associated with the ovarian dysfunction. Immunization of female dogs with Escherichia coli-expressed recombinant dog ZP glycoprotein-3 (ZP3) either coupled to diphtheria toxoid or expressed as fusion protein with 'promiscuous' T non-B-cell epitope of tetanus toxoid also led to inhibition of fertility. To improve the contraceptive efficacy of ZP-based contraceptive vaccine, various groups are working on improving the immunogen, use of DNA vaccine as prime-boost strategy, and delivering the zona proteins/peptides presented on either virus-like particles or entrapped in microsphere. Host-specific live vectors such as ectromelia virus and cytomegalovirus have also been used to deliver mouse ZP3 in mice. Various studies show the enormous potential of the ZP-based vaccine for the management of wildlife population, where permanent sterilization may be desirable.