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Conjugation of a therapeutic agent to a polymer for enhanced delivery into target cells followed by its intracellular triggered release has proved to be an effective drug delivery approach. This approach is applied to the delivery of the immune-stimulatory unmethylated cytosine-phosphate-guanine (CpG) oligonucleotide for an anti-tumour immune response after intratumoral administration. On average four CpG-1668 molecules were covalently linked to a 40-kDa amino-functionalised dextran polymer via either a non-reversible (CpG-dextran) or an intracellular redox-responsive disulfide linkage (CpG-SS-dextran). Dynamic light scattering analysis showed that both conjugates had a similar particle size and surface charge of 17 nm and -10 mV, respectively. Agarose gel electrophoresis analysis showed that CpG-SS-dextran was stable in the extracellular low glutathione (GSH) concentration range (i.e. 10-20 μM) and was cleaved at the higher intracellular GSH concentration (5 mM), while CpG-dextran was stable in both GSH concentrations. Uptake and activation assays on bone-marrow-derived dendritic cells showed no significant difference between free CpG, CpG-dextran and CpG-SS-dextran. In a mouse subcutaneous colorectal tumour model the CpG-SS-dextran showed a statistically significantly greater inhibition of tumour growth (p < 0.03) and prolonged survival (p < 0.001) compared to CpG-dextran or free CpG. These results demonstrate that the redox-triggered intracellular release of CpG from a dextran polymer carrier has promise for intratumoral therapeutic vaccination against cancer., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Greg Walker reports equipment, drugs, or supplies was provided by University of Otago. Greg Walker reports a relationship with University of Otago that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

We develop a machine learning framework that integrates high content/high throughput image analysis and artificial neural networks (ANNs) to model the separation between chemical compounds based on their estrogenic receptor activity. Natural and man-made chemicals have the potential to disrupt the endocrine system by interfering with hormone actions in people and wildlife. Although numerous studies have revealed new knowledge on the mechanism through which these compounds interfere with various hormone receptors, it is still a very challenging task to comprehensively evaluate the endocrine disrupting potential of all existing chemicals and their mixtures by pure in vitro or in vivo approaches. Machine learning offers a unique advantage in the rapid evaluation of chemical toxicity through learning the underlying patterns in the experimental biological activity data. Motivated by this, we train and test ANN classifiers for modeling the activity of estrogen receptor-α agonists and antagonists at the single-cell level by using high throughput/high content microscopy descriptors. Our framework preprocesses the experimental data by cleaning, scaling, and feature engineering where only the middle 50% of the values from each sample with detectable receptor-DNA binding is considered in the dataset. Principal component analysis is also used to minimize the effects of experimental noise in modeling where these projected features are used in classification model building. The results show that our ANN-based nonlinear data-driven framework classifies the benchmark agonist and antagonist chemicals with 98.41% accuracy.

The effect of dextran molecular weight on the in vitro physicochemical and immune properties of cytosine-phosphate-guanine (CpG) oligodeoxynucleotide-amino-dextran conjugates is investigated. CpG-1668 was conjugated at the 3'-end to amino-dextran of differing molecular weight (20, 40, 70 or 110-kDa) via a stable bis-aryl hydrazone linkage. Conjugate formation was confirmed by agarose gel electrophoresis and dynamic light scattering measured the size and surface charge of conjugates. Uptake and immune-stimulatory activity of CpG-dextran by antigen-presenting cells was evaluated by flow cytometry and confocal microscopy. Degradation by DNase I was monitored by loss of the fluorescent signal from labelled CpG and changes in size and zeta potential. Hydrazone bond formation (UV 354 nm) showed on average four CpG molecules conjugated per polymer. CpG-dextran prepared from 20 or 40-kDa dextran had a size of 17 nm while 70 or 110-kDa was 30 nm. CpG-dextran was preferentially taken up by dendritic cells, followed by macrophages and then B-cells. Only the 20-kDa dextran conjugate significantly enhanced uptake by bone-marrow derived dendritic cells (BMDCs) compared to free CpG. Confocal microscopy showed that CpG and CpG-dextran accumulates in the endo-lysosomal compartment of BMDCs at 24 h. All conjugates upregulated activation markers (CD40, CD80 or CD86) of BMDCs to a similar level as for free CpG. CpG-dextran 40-kDa produced highest levels of cytokines (TNF-α, IL-6, and IL-12p70) secreted by BMDCs. Enzymatic protection assays showed that the conjugate made from dextran 20-kDa provided no protection for CpG while the higher molecular weight conjugates reduced degradation by DNase I. The 40-kDa dextran conjugate produced the greatest in vitro immune activity, this was due to the conjugate being relatively small in size for cell uptake while sufficiently large enough to protect CpG from nuclease attack. These in vitro studies identify the need to consider the molecular weight of the carrier in bioconjugate design., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Compression-induced destabilization was investigated in various celecoxib amorphous solid dispersions containing hydroxypropyl methylcellulose (HPMC), poly(vinylpyrrolidone)/vinyl acetate copolymer (PVP/VA), or poly(vinylpyrrolidone) (PVP) at a concentration range of 1-10% w/w. Pharmaceutically relevant (125 MPa pressure with a minimal dwell time) and extreme (500 MPa pressure with a 60 s dwell time) compression conditions were applied to these systems, and the changes in their physical stability were monitored retrospectively (i.e., in the supercooled state) using dynamic differential scanning calorimetry (DSC) and low-frequency Raman (LFR) measurements over a broad temperature range (-90 to 200 and -150 to 140 °C, respectively). Both techniques revealed similar changes in the crystallization behavior between samples, where the application of a higher compression force of 500 MPa resulted in a more pronounced destabilization effect that was progressively mitigated with increasing polymer content. However, other aspects such as more favorable intermolecular interactions did not appear to have any effect on reducing this undesirable effect. Additionally, for the first time, LFR spectroscopy was used as a viable technique to determine the secondary or local glass-transition temperature, T g,β , a major indicator of the physical stability of neat amorphous pharmaceutical systems.

Breast cancer (BC) is the most frequently diagnosed cancer in women, with many patients experiencing recurrence following treatment. Antigens delivered on virus-like particles (VLPs) induce a targeted immune response and here we investigated whether the co-delivery of multiple antigens could induce a superior anti-cancer response for BC immunotherapy. VLPs were designed to recombinantly express murine survivin and conjugated with an aberrantly glycosylated mucin-1 (MUC1) peptide using an intracellular cleavable bis-arylhydrazone linker. Western blotting, electron microscopy and UV absorption confirmed survivin-VLP expression and MUC1 conjugation. To assess the therapeutic efficacy of VLPs, orthotopic BC tumours were established by injecting C57mg.MUC1 cells into the mammary fat pad of mice, which were then vaccinated with surv.VLP-SS-MUC1 or VLP controls. While wild-type mice vaccinated with surv.VLP-SS-MUC1 showed enhanced survival compared to VLPs delivering either antigen alone, MUC1 transgenic mice vaccinated with surv.VLP-SS-MUC1 showed no enhanced survival compared to controls. Hence, while co-delivery of two tumour antigens on VLPs can induce a superior anti-tumour immune response compared to the delivery of single antigens, additional strategies must be employed to break tolerance when targeted tumour antigens are expressed as endogenous self-proteins. Using VLPs for the delivery of multiple antigens represents a promising approach to improving BC immunotherapy, and has the potential to be an integral part of combination therapy in the future.

Biologics can be combined with liquid polymer materials and electrospun to produce a dry nanofibrous scaffold. Unlike spray-drying and freeze-drying, electrospinning minimizes the physiological stress on sensitive materials, and nanofiber mat properties such as hydrophobicity, solubility, and melting temperature can be tuned based on the polymer composition. In this study, we explored the dry formulation of a virus-like particle (VLP) vaccine by electrospinning VLP derived from rabbit hemorrhagic disease virus modified to carry the MHC-I gp100 tumor-associated antigen epitope. VLP were added to a polyvinylpyrrolidone (PVP) solution (15% w / v ) followed by electrospinning at 24 kV. Formation of a nanofibrous mat was confirmed by scanning electron microscopy, and the presence of VLP was confirmed by transmission electron microscopy and Western blot. VLP from the nanofibers induced T-cell activation and interferon- (IFN-) γ production in vitro. To confirm in vivo cytotoxicity, Pmel mice treated by injection with gp100 VLP from nanofibers induced a gp100 specific immune response, lysing approximately 65% of gp100-pulsed target cells, comparable to mice vaccinated with gp100 VLP in PBS. VLP from nanofibers also induced an antibody response. This work shows that electrospinning can be used to dry-formulate VLP, preserving both humoral and cell-mediated immunity.

Cytosine-phosphate-guanine (CpG) oligonucleotides are commonly-used vaccine adjuvants to promote the activation of antigen-presenting cells (APCs). To mount an effective immune response, CpG needs to be internalized and bind to its endosomal Toll-like receptor 9 (TLR-9) inside the APCs. Using flow cytometry and fluorescence microscopy, this article presents the cellular uptake data of the amino-dextran nanoparticle (aDNP) and aDNP loaded with CpG immobilized on its surface by either electrostatic adsorption or covalent conjugation. The uptake of fluorescently-labelled aDNPs by murine splenic dendritic cells and macrophages was determined by flow cytometry and uptake by murine bone-marrow-derived dendritic cells was evaluated by fluorescence microscopy. The data presented in this paper correlates with the in vitro immune-stimulatory activity observed for the two different CpG loading methods in the research article " Nanoparticle system based on amino-dextran as a drug delivery vehicle: immune-stimulatory CpG-oligonucleotide loading and delivery " (Nguyen et al ., 2020) [1]. The data provide experimental evidence for a better understanding how the nanoparticle surface loading method of CpG influences the uptake of these nanoparticles by antigen-presenting cells as a step guide in the design of more effective vaccine formulations., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (© 2021 The Authors. Published by Elsevier Inc.)

A series of C-2 derivatized 8-sulfonamidoquinolines were evaluated for their antibacterial activity against the common mastitis causative pathogens Streptococcus uberis, Staphylococcus aureus and Escherichia coli, both in the presence and absence of supplementary zinc (50 µM ZnSO 4 ). The vast majority of compounds tested were demonstrated to be significantly more active against S. uberis when in the presence of supplementary zinc (MICs as low as 0.125 µg/mL were observed in the presence of 50 µM ZnSO 4 ). Compounds 5, 34-36, 39, 58, 79, 82, 94 and 95 were shown to display the greatest antibacterial activity against S. aureus (MIC ≤ 8 µg/mL; both in the presence and absence of supplementary zinc), while compounds 56, 58 and 66 were demonstrated to also exhibit activity against E. coli (MIC ≤ 16 µg/mL; under all conditions). Compounds 56, 58 and 66 were subsequently confirmed to be bactericidal against all three mastitis pathogens studied, with MBCs (≥3log 10 CFU/mL reduction) of ≤ 32 µg/mL (in both the presence and absence of 50 µM ZnSO 4 ). To validate the sanitizing activity of compounds 56, 58 and 66, a quantitative suspension disinfection (sanitizer) test was performed. Sanitizing activity (>5log 10 CFU/mL reduction in 5 min) was observed against both S. uberis and E. coli at compound concentrations as low as 1 mg/mL (compounds 56, 58 and 66), and against S. aureus at 1 mg/mL (compound 58); thereby validating the potential of compounds 56, 58 and 66 to function as topical sanitizers designed explicitly for use in non-human applications., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The aim of this study is to prepare and characterize an amino-dextran nanoparticle (aDNP) platform and investigate two loading strategies for unmethylated cytosine-phosphate-guanine (CpG) oligonucleotide. aDNP was prepared by desolvation of amino-dextran followed by the chemical crosslinking of amino groups. Size, surface charge, and surface morphology of aDNP was determined by dynamic light scattering and transmission electron microscopy. CpG was either loaded onto aDNP by adsorption (CpG-adsorbed-aDNP) or conjugated to aDNP (CpG-conjugated-aDNP). In vitro cytokine production by bone marrow-derived dendritic cells (BMDCs) was measured by flow cytometry. aDNPs size and zeta potential could be controlled to produce uniform particles in the size range of 50 to 300 nm, surface charge of -16.5 to +14 mV, and were spherical in shape. Formulation control parameters investigated included the anti-solvent, water-to-anti-solvent ratio, level of amine functionality of dextran, and the molar ratio of glutaraldehyde to amine. aDNP could be lyophilized without additional cryoprotectant. Unloaded cationic aDNP (+13 mV) showed acceptable in vitro hemolysis. Unloaded and CpG-loaded aDNPs showed no cytotoxicity on BMDCs. CpG-loaded nanoparticles stimulated cytokine production by BMDCs, the level of cytokine production was higher for CpG-conjugated-aDNP compared to CpG-absorbed-aDNP. aDNP is a promising new drug delivery platform as its offers versatility in loading and tuning of particle properties.

A series of substituted sulfonamide bioisosteres of 8-hydroxyquinoline were evaluated for their antibacterial activity against the common mastitis causative pathogens Streptococcus uberis, Staphylococcus aureus and Escherichia coli, both in the presence and absence of supplementary zinc. Compounds 9a-e, 10a-c, 11a-e, 12 and 13 were demonstrated to have MICs of 0.0625 µg/mL against S. uberis in the presence of 50 µM ZnSO 4 . Against S. aureus compounds 9g (MIC 4 µg/mL) and 11d (MIC 8 µg/mL) showed the greatest activity, whereas all compounds were found to be inactive against E. coli (MIC > 256 µg/mL); again in the presence of 50 µM ZnSO 4 . All compounds were demonstrated to be significantly less active in the absence of supplementary zinc. Compound 9g was subsequently confirmed to be bactericidal, with an MBC (≥3log 10  cfu/mL reduction) of 0.125 µg/mL against S. uberis in the presence of 50 µM ZnSO 4 . To validate the sanitising activity of compound 9g in the presence of supplementary zinc, a quantitative suspension disinfection (sanitizer) test was performed. In this preliminary test, sanitizing activity (>5log 10 reduction of CFU/mL in 5 min) was observed against S. uberis for compound 9g at concentrations as low as 1 mg/mL, validating the potential of this compound to function as a topical sanitizer against the major environmental mastitis-causing microorganism S. uberis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This article contains the uptake data of two reducible antigen-adjuvant conjugates with different sensitivities to the extracellular and intracellular reductive environment. Using a linker with different redox sensitivity the adjuvant cytosine-phosphate-guanine (CpG) was conjugated to the fluorescently labeled model tumour antigen ovalbumin (OVA). The uptake of the conjugates by dendritic cells in a total splenocyte culture was determined using flow cytometry. The data presented in this paper supports the finding in the research article "Intracellular cleavable CpG oligodeoxynucleotide - antigen conjugate enhances anti-tumour immunity" (Kramer et al., 2016) [1].

Background: A key challenge in the manufacturing of polymeric colloids is producing nanoparticles with good batch-to-batch consistency., Objective: Develop a robust microfluidics method for the preparation of PEG-PLGA nanoparticles using dimethyl sulfoxide (DMSO) as the organic phase solvent for the encapsulation of DMSO soluble agents., Methods: Microfluidic process parameters, total flow rate (10 mL/min), flow rate ratio (1:1) of the aqueous phase and the organic polymer solution, and polymer concentration (5 mg/ml). Polyvinyl alcohol (PVA) or human serum albumin (HSA) was included in the aqueous phase. Dynamic light scattering and transmission electron microscopy were used to investigate the size and morphology of particles., Results: PLGA nanoparticles made using DMSO with the aqueous solvent containing PVA (2%) had an average size of 60 nm while PLGA-PEG nanoparticles made with and without PVA (2%) had an average size of 70 and 100 nm, respectively. PLGA-PEG nanoparticles generated with or without PVA had a high batch-to-batch coefficient of variation for the particle size of 20% while for PLGA nanoparticles with PVA it was 4%. HSA added to the aqueous phase reduced the size and the zeta potential of PEG-PLGA nanoparticles as well the batch-to-batch coefficient of variation for particle size to < 5%. Nanoparticles were stable in solution and after lyophilized in the presence of sucrose., Conclusion: Albumin was involved in the self-assembly of PEG-PLGA nanoparticles altering the physicochemical properties of nanoparticles. Adding protein to the aqueous phase in the microfluidic fabrication process may be a valuable tool for tuning the properties of nanoparticles and improving batch-to-batch consistency., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Introduction: Virus-like particle (VLP) vaccines face significant challenges in their translation from laboratory models, to routine clinical administration. While some VLP vaccines thrive and are readily adopted into the vaccination schedule, others are restrained by regulatory obstacles, proprietary limitations, or finding their niche amongst the crowded vaccine market. Often the necessity to supplant an existing vaccination regimen possesses an immediate obstacle for the development of a VLP vaccine, despite any preclinical advantages identified over the competition. Novelty, adaptability and formulation compatibility may prove invaluable in helping place VLP vaccines at the forefront of vaccination technology., Areas Covered: The purpose of this review is to outline the diversity of VLP vaccines, VLP-specific immune responses, and to explore how modern formulation and delivery techniques can enhance the clinical relevance and overall success of VLP vaccines., Expert Commentary: The role of formation science, with an emphasis on the diversity of immune responses induced by VLP, is underrepresented amongst clinical trials for VLP vaccines. Harnessing such diversity, particularly through the use of combinations of select excipients and adjuvants, will be paramount in the development of VLP vaccines.

Targeted delivery and retention of drug formulations in the olfactory mucosa, the target site for nose-to-brain drug absorption is a major challenge due to the geometrical complexity of the nose and nasal clearance. Recent modelling data indicates that 10μm-sized microparticles show maximum deposition in the olfactory mucosa. In the present study we tested the hypothesis that 10μm-sized mucoadhesive microparticles would preferentially deposit on, and increase retention of drug on, the olfactory mucosa in a novel 3D-printed human nasal-replica cast under simulated breathing. The naturally occurring mucoadhesive polymer, tamarind seed polysaccharide (TSP) was used to formulate the microparticles using a spray drying technique. Physicochemical properties of microparticles such as size, morphology and mucoadhesiveness was investigated using a combination of laser diffraction, electron microscopy and texture-analysis. Furthermore, FITC-dextrans (5-40kDa) were incorporated in TSP-microparticles as model drugs. Size-dependent permeability of the FITC-dextrans was observed ex vivo using porcine nasal mucosa. Using the human nasal-replica cast, greater deposition of 10μm TSP-microparticles in the olfactory region was observed compared to TSP-microparticles 2μm in size. Collectively, these findings support our hypothesis that 10μm-sized mucoadhesive microparticles can achieve selective deposition and retention of drug in the olfactory mucosa., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The purpose of this study was to evaluate electrospun drug loaded nanofibers as a new matrix for minitablets. Prednisone, a poorly water-soluble drug, was loaded into povidone (polyvinylpyrrolidone, PVP) nanofibers using the process of electrospinning. The drug-loaded nanofiber mat was compressed into minitablets with a 2mm diameter and a height of 2.63±0.04mm. SEM analysis of the minitablet identified a nano-web structure with a nanofiber diameter in the range of 400-500nm. The minitablets met the requirements of the US Pharmacopeia with respect to content uniformity and friability. DSC and XRPD analysis of the minitablet indicated that the drug-polymer mixture was a one-phase amorphous system. XRPD analysis of the drug loaded nanofiber mat after 10-months of storage at ambient temperature showed no evidence of recrystallization of the drug. Solubility and dissolution properties of the drug formulated into a nanofiber mat and minitablet were evaluated. These results show that electrospun nanofibers may provide a useful matrix for the further development of minitablets., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Conjugation of CpG to an antigen induces a stronger immune response compared to that of the mixture. This study compares the in vitro immunostimulatory activity of CpG conjugated via either its 5' or 3' end to the model antigen ovalbumin (OVA). CpG modified with an amine at either the 5' or 3' end was conjugated to OVA via a stable bis-aryl hydrazone bond. Similar levels of CpG conjugation to OVA were observed for both conjugates on the basis of the absorbance at 360 nm for the formation of the bis-aryl hydrazone bond, which determined 2.8 ± 0.3 CpGs linked per OVA. Both the 5' and 3' CpG-OVA conjugates had similar size-exclusion chromatography elution profiles. The immunostimulatory properties of the conjugates were determined by dendritic cells (DCs) and T-cells isolated from mice. The activation of DCs was determined by the upregulation of activation markers CD86 and CD40. T-cells were co-cultured with stimulated DCs, and the immunogenicity was determined by measuring T-cell proliferation and interferon γ production. Both the CpG 5'- and 3'-linked conjugates induced the same level ( p > 0.5) of DC activation markers, which were significantly higher than those of the untreated control. Similarly, T-cell assays showed no significant difference ( p > 0.5) between the 5' and 3' conjugates with respect to T-cell proliferation and interferon γ production. The 5' and 3' conjugates induced T-cell activation significantly higher than the mixture of CpG and OVA. This study showed that the end at which CpG is conjugated to an antigen has no influence on the generation of a T-cell-based immune response in vitro., Competing Interests: The authors declare no competing financial interest.

Conjugation of a vaccine adjuvant to an antigen enhances anti-tumor immune responses. Direct chemical conjugation, however, may limit their processing by the antigen-presenting cell for immune stimulation. To test this hypothesis, antigen-adjuvant conjugates were designed to be cleaved by an intracellular trigger to release antigen and adjuvant from each other. The different reductive environment inside and outside antigen-presenting cells was used as a trigger for targeted intracellular release. Two redox-responsive disulphide linkers were used to conjugate the model antigen ovalbumin to CpG. In vitro stability assays with the reductant glutathione showed that one conjugate (SS) was cleaved by glutathione concentrations of the extra- and intracellular compartments. A second conjugate (HYN-SS) was only cleaved at the higher intracellular glutathione concentration. In vitro cell culture studies showed that high T cell responses were generated by the HYN-SS and the stable conjugate HYN. The SS conjugate induced a lower T cell response similar to a mixture of CpG and ovalbumin. An in vivo therapeutic tumor trial demonstrated a superior survival rate of 9/10 for mice vaccinated with HYN-SS conjugate compared to HYN (6/10), SS (2/10), and the mixture (2/10). This intracellular cleavable conjugation strategy represents a promising approach to improve cancer immunotherapy of soluble vaccines., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

PEGylation which is reversed after the therapeutic agent reaches the target cell presents an attractive feature for drug, protein or nucleic acid delivery. Amine-reactive, endosomal pH cleavable polyethylene glycol aldehyde-carboxypyridylhydrazone, N-hydroxysuccinimide esters (PEG-HZN-NHS) were synthesized and applied for bioreversible surface shielding of DNA polyplexes. Monofunctional mPEG-HZN-NHS was synthesized by reacting succinimidyl hydraziniumnicotinate with mPEG-butyraldehyde (20 kDa). Bifunctional OPSS-PEG-HZN-NHS was synthesized analogously via a omega-2-pyridyldithio-PEG (10 kDa) propionaldehyde intermediate. Polyethylenimine (PEI) polyplexes were reacted with the pH-sensitive (mPEG-HZN-NHS) or the corresponding stable (mPEG-NHS) reagent. Both types of polyplexes remained shielded at pH 7.4 as demonstrated by particle size and zeta potential measurements after 4h of incubation at 37 degrees C. Polyplex deshielding at endosomal pH 5 was observed only with the mPEG-HZN-NHS shielded particles. This was confirmed by fluorescence correlation spectroscopy using the analogous Alexa-488 fluorescently labeled bifunctional PEGylation reagents. Luciferase gene transfections with epidermal growth factor (EGF) containing polyplexes using EGF-receptor overexpressing hepatoma HUH7 cells showed an up to 16-fold enhancement in gene expression with the reversibly shielded polyplexes as compared to stably shielded polyplexes. Consistently, the reversibly shielded polyplexes mediated also an enhanced tumor specific in vivo transgene expression after intravenous administration in a subcutaneous HUH7 tumor model in SCID mice.

In this work, we design and investigate the complex formation of highly uniform monomolecular siRNA complexes utilizing block copolymers consisting of a cationic peptide moiety covalently bound to a poly(ethylene glycol) (PEG) moiety. The aim of the study was to design a shielded siRNA construct containing a single siRNA molecule to achieve a sterically stabilized complex with enhanced diffusive properties in macromolecular networks. Using a 14 lysine-PEG (K14-PEG) linear diblock copolymer, formation of monomolecular siRNA complexes with a stoichiometric 1:3 grafting density of siRNA to PEG is realized. Alternatively, similar PEGylated monomolecular siRNA particles are achieved through complexation with a graft copolymer consisting of six cationic peptide side chains bound to a PEG backbone. The hydrodynamic radii of the resulting complexes as measured by fluorescence correlation spectroscopy (FCS) were found to be in good agreement with theoretical predictions using polymer brush scaling theory of a PEG decorated rodlike molecule. It is furthermore demonstrated that the PEG coating of the siRNA-PEG complexes can be rendered biodegradable through the use of a pH-sensitive hydrazone or a reducible disulfide bond linker between the K14 and the PEG blocks. To model transport under in vivo conditions, diffusion of these PEGylated siRNA complexes is studied in various charged and uncharged matrix materials. In PEG solutions, the diffusion coefficient of the siRNA complex is observed to decrease with increasing polymer concentration, in agreement with theory of probe diffusion in semidilute solutions. In charged networks, the behavior is considerably more complex. FCS measurements in fibrin gels indicate complete dissociation of the diblock copolymer from the complex, while transport in collagen solutions results in particle aggregation.

p-Piperazinobenzaldehyde methoxy poly(ethylene glycol) (mPEG, 5 kDa) acetal was synthesized by the Buchwald-Hartwig coupling reaction from piperazine and p-bromobenzaldehyde mPEG acetal. Introduction of a maleimide moiety yielded a novel acetal-based PEGylation reagent (PEG-acetal-MAL) for pH-sensitive conjugation of PEG to thiol-functionalized biomolecules. For reversible shielding of polyplexes, PEG-acetal-MAL was conjugated to polyethylenimine (PEI). At 37 degrees C, the PEG-acetal-PEI conjugate had a half-life of 3 min at endosomal pH 5.5 and 2 h at physiological pH 7.4, respectively. PEI polyplexes containing PEG-acetal-PEI had a zeta potential of +3 mV and were stable to salt-induced aggregation for 2 h at pH 7.4. In contrast, at endosomal pH, the particles were deshielded and aggregated within 0.5 h. Epidermal growth factor or transferrin receptor-targeted polyplexes shielded with the pH-sensitive PEG-acetal mediated enhanced luciferase gene expression in receptor-expressing target cells (Renca-EGFR or K562) as compared to stably shielded control polyplexes. Thus, the novel PEG-acetal-MAL reagent may present a versatile tool for drug and gene delivery formulations when pH-sensitive PEGylation is preferred.

Fluorescence correlation spectroscopy (FCS) and gel electrophoresis measurements are performed to investigate both the number and size of complexes of linear double-stranded DNA (dsDNA) fragments with 1:1 diblock copolymers consisting of a cationic moiety, branched polyethyleneimine (bPEI) of 2, 10, or 25 kDa, covalently bound to a neutral shielding moiety, poly(ethylene glycol) (PEG; 20 kDa). By systematically decreasing the bPEI length, the PEG grafting density along the DNA chain can be directly controlled. For 25 and 10 kDa bPEI-PEG copolymers, severe aggregation is observed despite the presence of the shielding PEG. Upon decreasing the bPEI length to 2 kDa, controlled self-assembly of monomolecular DNA nanoparticles is observed. The resulting complexes are in quantitative agreement with a theoretical model based on a single DNA encased in a dense PEG polymer brush layer. The resulting PEGylated complexes show high stability against both salt and protein and hence are of potential use for in vivo gene delivery studies.

Background: Polycation (PC, polyplex), cationic lipid (CL, lipoplex), and a combination of PC/CL (lipopolyplex) formulations were investigated for gene transfer to slow-proliferating human colon carcinoma cell lines (COGA)., Methods: The luciferase reporter gene was complexed with either PC, CL, or PC/CL. PCs included linear (PEI22lin, 22 kDa) and branched polyethylenimine (PEI2k, 2 kDa; PEI25br, 25 kDa) and poly-L-lysine (PLL18 with 18 lysine monomers). CLs included DOCSPER, DOSPER and DOTAP. Lipopolyplexes were formed by either sequentially first mixing DNA with PC or CL, followed by addition of CL or PC, respectively, or simultaneously with both PC and CL. Particle size and zeta-potential were determined and gene transfer and cytotoxicity were quantified on COGA-3, -5, -12, HeLa and Sw480 cells., Results: The highest gene transfer was achieved when DNA was first complexed with PC followed by CL. At low ionic strength, particles were small (50-130 nm) with a zeta-potential of +20-40 mV. At physiological ionic strength, only lipoplexes of DOCSPER or DOSPER and their respective lipopolyplexes with PEI25br were stable to aggregation (140-220 nm). Lipopolyplexes of PEI25br were between 5- to 400-fold more efficient compared to the corresponding lipoplexes or polyplexes in all cases. Chloroquine did not significantly affect lipopolyplex-mediated gene transfer., Conclusions: Lipopolyplex formulations of PEI25br in combination with multivalent CLs (DOCSPER, DOSPER) are promising tools for in vitro and potentially also in vivo gene transfer to colorectal cancer cells., (Copyright 2005 John Wiley & Sons, Ltd.)

Nonviral vectors should undergo "virus-like" changes compatible with the steps of gene delivery. Poly(ethylene) glycol (PEG) shielding of DNA/polycation polyplexes protects from nonspecific interactions with the extracellular environment. pH-triggered removal of the shield within the endosome may be advantageous. Polycation and PEG were linked via acylhydrazides or pyridylhydrazines. The pyridylhydrazone prepared from polylysine and propionaldehyde-PEG showed the greatest acid-dependent hydrolysis; at pH 5, 37 degrees C for 10 min, 90% hydrolyzed, while at pH 7.4 the half-life was 1.5 h. Particle size and zeta potential measurements of the polyplexes showed complete deshielding within 1 h at pH 5, while at pH 7.4 the shield remained at 4 h, 37 degrees C. For gene transfection a targeting conjugate was also included in the polyplex, transferrin as ligand for K562 and Neuro2A cells and epidermal growth factor for HUH-7 and Renca-EGFR cells. Marker gene expression showed that the reversibly shielded polyplexes exhibited up to 2 log orders of magnitude higher gene expression in vitro and 1 log magnitude higher gene expression in an in vivo mouse model, compared to the stably shielded control polyplexes. Engineering of polyplexes with more dynamic domains is an encouraging new direction in nonviral vector design.

Objective: The aim of this study was to investigate the effect of various C-type natriuretic peptide (CNP) sequences (genomic DNA [CNPDNA], cDNA derived from mRNA [CNPcDNA], and sequence coding for 22 amino acids of the mature CNP [CNP22aa]) on the growth of primary porcine vascular cells., Methods and Results: Gene transfer was performed with cationic lipid DOCSPER or linear polyethylenimine. All 3 CNP sequences led to significant inhibition of smooth muscle cell (SMC) proliferation. In contrast, significant stimulation of cell growth was observed in endothelial cells (ECs) using CNPDNA or CNPcDNA but not CNP22aa. In a porcine restenosis model, a significant reduction in neointima hyperplasia was found 3 months after application of the CNPcDNA vector compared with the control transfection., Conclusions: The results demonstrate that the first intron in the CNP sequence does not contain any additional enhancer-binding sites. However, the signal sequence is indispensable for secretion of CNP and its appropriate physiological function. Furthermore, the results show for the first time the therapeutic effect of CNP using liposome-mediated gene transfer and local adventitial delivery. Advantages of the CNP gene are its dual effects with inhibition of SMC proliferation and simultaneous promotion of EC growth. Functional analysis of various C-type natriuretic peptide (CNP) sequences on growth of vascular cells. For the first time, dual therapeutic effects of CNP with inhibition of smooth muscle cell proliferation and stimulation of re-endothelialization were demonstrated in a pig restenosis model using liposome-mediated gene transfer and local adventitial delivery.

Unique properties of tumors, such as abnormalities in the cell cycle and apoptosis, migration and metastasis, neoangiogenesis or unique antigen profiles are targets for therapeutic anti-cancer strategies. Beyond the selection of such strategies, additional specificity for the targeted tumor tissue can be accomplished in cancer gene therapy in several ways. Upon systemic administration, appropriately packaged therapeutic nucleic acid may be preferentially transported into the tumor tissue (targeted delivery); formulation can mediate the intracellular uptake of the nucleic acid into the nucleus of target cells only (transductional targeting); and/or the use of specific promotor/enhancer elements can restrict transcription of therapeutic genes to the target cells only (transcriptional targeting). Options for physical and biological targeting of nucleic acid formulations into tumors and therapeutic approaches are reviewed.

Disulphide bonds between thiolated polymers (thiomers) and cysteine-rich subdomains of mucus glycoproteins are supposed to be responsible for the enhanced mucoadhesive properties of thiomers. This study set out to provide evidence for these covalent interactions using poly(acrylic acid)-cysteine conjugates of 2 and 450 kDa (PAA2-Cys, PAA450-Cys) displaying 402.5-776.0 micromol thiol groups per gram polymer. The effect of the disulphide bond breaker cysteine on thiomer-mucin disulphide bonds was monitored by (1) mucoadhesion studies and (2) rheological studies. Furthermore, (3) diffusion studies and (4) gel filtration studies were performed with thiomer-mucus mixtures. The addition of cysteine significantly (P<0.01) reduced the adhesion of thiomer tablets to porcine mucosa and G'/G" values of thiomer-mucin mixtures, whereas unthiolated controls were not influenced. These results indicate the cleavage of disulphide bonds between thiomer and mucus glycoproteins. Diffusion studies demonstrated that a 12.8-fold higher concentration of the thiomer (PAA2-Cys) remains in the mucin gel than the corresponding unmodified polymer. Gel filtration studies showed that PAA2-Cys was able to form disulphide bonds with mucin glycoproteins resulting in an altered elution profile of the mucin/PAA2-Cys mixture in comparison to mucin alone or mucin/PAA2 mixture. According to these results, the study provides evidence for the formation of covalent bonds between thiomer and mucus glycoproteins.

Tumor-targeting DNA complexes which can readily be generated by the mixing of stable components and freeze-thawed would be very advantageous for their subsequent application as medical products. Complexes were generated by the mixing of plasmid DNA, linear polyethylenimine (PEI22, 22 kDa) as the main DNA condensing agent, PEG-PEI (poly(ethylene glycol)-conjugated PEI) for surface shielding, and Tf-PEG-PEI (transferrin-PEG-PEI) to provide a ligand for receptor-mediated cell uptake. Within the shielding conjugates, PEG chains of varying size (5, 20, or 40 kDa) were conjugated with either linear PEI22 (22 kDa) or branched PEI25 (25 kDa). The three polymer components were mixed together at various ratios with DNA; particle size, surface charge, in vitro transfection activity, and systemic gene delivery to tumors was investigated. In general, increasing the proportion of shielding conjugate in the complex reduced surface charge, particle size, and in vitro transfection efficiency in transferrin receptor-rich K562 cells. The particle size or surface charge of the complexes containing the PEG-PEI conjugate did not significantly change after freeze-thawing, while complexes without the shielding conjugate aggregated. Complexes containing PEG-PEI conjugate efficiently transfected K562 cells after freeze-thawing. Furthermore the systemic application of freeze-thawed complexes exhibited in vivo tumor targeted expression. For complexes containing the luciferase reporter gene the highest expression was found in tumor tissue of mice. An optimum formulation for in vivo application, PEI22/Tf-PEG-PEI/PEI22-PEG5, containing plasmid DNA encoding for the tumor necrosis factor (TNF-alpha), inhibited tumor growth in three different murine tumor models. These new DNA complexes offer simplicity and convenience, with tumor targeting activity in vivo after freeze-thawing.

Oral delivery of peptide and protein drugs has potential advantages for the aquaculture industry. The bioavailability of proteins and peptides from the intestinal tract is very low. This can be attributed in part to the proteolytic activities of the intestine. Bovine serum albumin (BSA), human (hLHRH) and salmon (sLHRH) luteinizing-hormone releasing hormones were used to evaluate the proteolytic activity of anterior, middle and posterior sections of the Quinnat salmon (Oncorhynchus tshawytscha) intestinal tract. The lumenal proteolytic activities of the posterior intestinal section towards BSA were approximately half that of the anterior and middle sections. The half-lives of the LHRH analogues in the posterior were twofold longer than for the anterior and middle sections. Proteolytic activity of the posterior mucosal homogenates towards BSA was fourfold higher than the middle mucosal homogenates. LHRH analogues were hydrolysed by the posterior mucosal homogenate, whereas in the middle mucosal homogenate they were stable. Soybean trypsin inhibitor was shown to be the most effective inhibitor of lumenal proteolytic activity towards LHRH analogues. Sodium deoxycholate, EDTA and bestatin significantly inhibited the posterior mucosal hydrolytic activity towards the LHRH analogues. The posterior intestine of salmon is the most favourable site for the delivery of BSA and LHRH analogues with respect to the lumen, however the higher proteolytic activity of the posterior mucosa has to be overcome.

A rapid and simple capillary electrophoresis (CE) assay for measuring the stability of luteinizing hormone-releasing hormone (LHRH) analogues in the presence of intestinal enzymes has been developed and validated. Buffer pH and sample stacking were important factors in controlling resolution and reproducibility. The CE assay for human (h) and salmon LHRH analogues between 0.05 and 0.25 mM was linear for peak height versus concentration (r2>0.99). Analysis of hLHRH at 0.1 mM had an intra-day relative standard deviation of 1.25% and an inter-day relative standard deviation of 5.0%. The method was applied to the stability of LHRH analogues in salmon intestinal digests.

Peptide drugs in buccal bioadhesive delivery systems are exposed to the surface of the buccal mucosa at high concentrations over long periods of time. The peptidase activity on the surface of the buccal mucosa has not been evaluated as a barrier to peptide buccal delivery. The in vitro stability of various synthetic substrates on the surface of intact porcine buccal mucosa was determined. No carboxypeptidase or dipeptidyl peptidase IV activity was detected on the buccal mucosa, while aminopeptidase N activity was detected using Leu-p-nitroanilide. No endopeptidase activity was observed towards the peptide substrates. Insulin and insulin B-chain were intact at the 2 h time point at 37 degrees C, while the percent of parent Leu-enkephalin remaining was 18+/-9 (mean+/-S.D., n=9). In the presence of aminopeptidase inhibitors, amastatin, sodium deoxycholate and EDTA, the degradation of Leu-enkephalin was dramatically reduced. This work suggests that the buccal route maybe advantageous for the delivery of peptides that are susceptible to such activities. The inclusion of aminopeptidase inhibitors in buccal bioadhesive delivery systems could improve buccal bioavailability of Leu-enkephalin. We suggest that compared with the existing in vitro metabolism methods, the analysis of peptide or protein metabolism on intact buccal mucosa could better predict the degradation of the drug as it crosses the tissue.

The purpose of this study was to evaluate the potential of polycarbophil-cysteine conjugates (PCP-Cys) as an oral excipient to protect leucine enkephalin (leu-enkp) from enzymatic degradation by the intestinal mucosa. Cysteine was covalently linked to polycarbophil by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC). Inhibitory activity was tested towards isolated aminopeptidase N and excised intact pig intestinal mucosa, with native mucus. Aminopeptidase N activity was assayed spectrophotometrically using L-leucine p-nitroanilide (leu-pNA) as a synthetic substrate and against the model peptide drug leu-enkp, by high-performance liquid chromatography (HPLC). Free cysteine at 6.3 and 63 microM (pH 6) significantly (p < 0.05) inhibited aminopeptidase N activity, and PCP-Cys (0.25% w/v, pH 6) had a significantly (p < 0.05) greater inhibitory effect than PCP on the aminopeptidase N activity towards both substrates. PCP-Cys completely protected leu-enkp against aminopeptidase N activity over a 2-h incubation period, whereas 83 +/- 4 and 60 +/- 7% remained stable in the presence of PCP and buffer only, respectively. Leu-enkp in the absence and presence of PCP (0.25% w/v) at pH 6 was completely digested by the intact intestinal mucosa at the 60- and 90-min incubation time points, respectively, whereas in the presence of PCP-Cys (0.25% w/v, pH 6) 11 +/- 3.5% of leu-enkp remained at the 120-min time point. Thiolation of PCP increased the stability of leu-enkp against the enzymatic degradation by aminopeptidase N and the intact intestinal mucosa, identifying a promising new excipient for peroral delivery of peptides., (Copyright 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association)

LHRH and its analogues have low oral bioavailability; this is in part due to their degradation by peptidases present in the intestinal lumen. To determine the appropriate inhibitors to co-administer with LHRH oral formulations, the peptidases involved in their digestion have to be identified. Human (hLHRH) and salmon (sLHRH) LHRH analogues contain a number of potential cleavage sites for the lumenal pancreatic secreted serine endopeptidases: chymotrypsin, trypsin and elastase. The rate of LHRH degradation by equimolar concentrations of chymotrypsin, trypsin and elastase were examined separately in vitro, at pH 8.0, 15 degrees C. At a molar ratio of 1:1000 (enzyme:LHRH), both LHRH analogues were rapidly hydrolysed by alpha-chymotrypsin with half-lives of 2.5+/-0.3 and 2.7+/-0.4 min (mean+/-S.D., n=3), respectively, whereas in the presence of elastase both LHRH analogues were slowly hydrolysed with half-lives of 90+/-15 and 114+/-21 min (mean+/-S.D., n=3), respectively. Trypsin had no activity towards either LHRH analogues after 2 h incubation. The degradation of the LHRH analogues by elastase is likely to be a property of the chymotrypsin impurity. It is concluded that protection of the LHRH analogues from alpha-chymotrypsin is a requirement for the development of oral absorbable product.

Purpose: To determine the mechanism by which Carbomer inhibits the enzymatic activity of trypsin in hydrolysis of N-alpha-benzoyl-L-arginine ethyl ester (BAEE) and luteinizing hormone-releasing hormone (LHRH)., Methods: Inhibition of enzymatic activity was studied by measuring the formation of metabolites from LHRH and BAEE. Binding of trypsin and substrates to 0.35% (w/v) Carbomer at pH 7.0 was studied by centrifugal filtration. Gel filtration and reverse phase HPLC was used to determine the stability of trypsin., Results: Carbomer reduced the rate of hydrolysis of BAEE and LHRH by trypsin to 34% and 28% of the control activity, respectively. The rate of metabolite formation for both substrates followed pseudo-zero order kinetics in the presence and absence of carbomer. Binding studies showed that 68% of the trypsin protein and 10% of BAEE was bound to carbomer, but no LHRH was bound. No low molecular weight autolysis products of trypsin could be identified by gel filtration. Reverse phase HPLC analysis of the unbound carbomer-treated-trypsin suggests a number of conformational forms of trypsin. The equilibrium binding capacity was 30 microg of trypsin to 1000 microg of carbomer., Conclusions: Decreased hydrolysis of LHRH and BAEE by trypsin in the presence of carbomer is due to enzyme-polymer interaction.

The structures of uniformly 13C-labeled beta-glucan octa- and undeca-oligosaccharides enzymatically prepared by the yeast cell wall glucanosyl transferase of Candida albicans were characterized by using a combination of HCCH-COSY, HCCH-TOCSY, and HMBC experiments. The oligosaccharide structures indicate that the cell wall glucanosyl transferase cleaves two glucosyl units from the reducing end of the initial linear beta(1-->3) penta-oligosaccharide and subsequently transfers the remainder to another oligosaccharide at the nonreducing end via a beta(1-->6) linkage. These results indicate that the combined action of cell wall glucanase and glucanosyl transferase activities could not only introduce intrachain beta(1-->6) linkages within a single glucan strand, but also result in cross-linking of two initially separate glucan strands with concurrent introduction of intrachain beta(1-->6) linkages. Since isolated fungal membranes only synthesize linear beta(1-->3) glucan strands, wall-associated enzymes probably participate in the assembly of the final wall glucan structure during cell growth and division.

In eighteen subjects assigned for immediate complete upper and lower dentures, roentgen cephalometric recordings were made before extraction of the residual anterior dentition and 3 weeks, 3 months, 6 months and 1 year after denture insertion. The cephalometric analysis was based on electronic measurements of linear and angular morphological variables and computer head plots generated from 177 reference points (Walker, 1967), derived for each subject for each of the five observation stages. The reduction of the alveolar ridges was most rapid during the first 3 months of denture wear and particularly during the post-extraction period of 3 weeks. The reduction in anterior height of the lower ridge was on average twice as great as that of the upper ridge. The ridge resorption and the accompanying settling of the dentures on the basal seats, measured from lead shots inserted in the dentures, brought about an upward rotation of the mandible with a resulting decrease in occlusal vertical dimension and reduction in overjet of the dentures. In accordance with the amount of ridge reduction, these changes showed great individual variation.

A cephalometric study of the relation of the malar eminence to the A point has been made. It is proposed that the orbital-NA distance is of clinical importance in patients with SNA of less than 79 degrees in whom a Le Fort I osteotomy procedure is contemplated. The measurement of the angle SNO is a significant guide to the malar-maxillary relationship and will assist in complete cephalometric evaluation of these patients.

Three principal components, explaining 83 percent of the common variation for 999 males and females between 6 and 22 years of age, describe ontogenetic patterns of relationship for seven facial dimensions, including sella-nasion, sella-basion, nasion-prosthion, infradentale-menton, articulare-gnathion, gonion-gnathion, and articulare-gonion. Accounting for 65 percent of the variation, a general component associated with both size and shape defines size-required changes in proprotion during growth. Independent patterns of regional variation associated with alveolar remodeling (second component) and condylar growth (third component) describe specific sources of facial modification. Mean multivariate component scores reveal that sexual dimorphism, which progressively favors males over females with age, results from accumulating differences in size and related proportional changes in shape. The timing of the condylar growth spurt, as evident from variation in ramus height, produces secondary dimorphism which diminishes following the adolescent phase in males. Significant age effects are indicated for alveolar remodeling and mandibular growth of the condyle.

To establish the race-specific norms and investigate the underlying etiological basis of racial differences in malocclusion, cephalometric measurements of Steiner were analyzed for five major racial groups in Hawaii: Caucasians, Japanese, Chinese, Filipinos, and Hawaiians. Subjects consisted of 210 healthy and orthodontically normal individuals of both sexes aged 15 years or older. Analysis of age- and sex-adjusted cephalometric values showed that bone-to-bone relationships are largely comparable among races with an exception of GoGn/SN. Significant racial differences are incisal inclinations in relation to the maxilla, the mandible, or the opposite incisors, with the least inclination for Caucasians and the greatest for Chinese. The result is general tendency of bimaxillary protrusion of non-Caucasians. No significant differences were detected in arch lengths of both jaws among these groups. It is suggested that the observed higher prevalence of mesioclusion in non-Caucasians--especially Orientals--is due to an imbalance of tooth dimension to the alveolar bone.

Dentist and patient preferences are often used to select replacement teeth in prosthodontics. Face shape compared with inverted tooth form classifications based on Leon William's work are currently used. Shapes of teeth and faces have been referred to as square, ovoid or tapered, or some combination of these. Six patients, three male and three female, were selected as being classically square, tapered or ovoid in facial form. Three sets of dentures had been made for each patient with tapering, ovoid and square denture teeth. Using a standardized photographic technique, full face views with profiles and close-ups of the teeth were taken. Then from standardized enlarged tracings, key anatomic and derived points were marked, digitized and computer analysed. The face shapes and inverted tooth forms were digitized in the same manner. A comparison of tooth moulds versus the actual denture teeth shows a highly significant difference (P less than 0.001) between set and unset denture teeth. There is also a significant difference (P less than 0.001) between facial form and denture teeth using temporal zygomatic and gonial widths for faces, compared with incisal, contact, and cervical widths for the teeth.

An unidentified female mummy found in a cache of great kings and queens in 1898 in the Valley of the Kings was examined from the viewpoint of Egyptology, x-ray cephalometry, biostatistics, and biochemistry. The result was the identification of Queen Tiye, of the Eighteenth Dynasty, wife of Amenhotep III and mother of Akhenaton.

In a group of 18 partially edentulous patients provided with immediate complete dentures, changes in hyoid bone position and craniocervical posture were examined on cephalometric radiographs made during 1 year of denture use. The findings indicated that the changes in hyoid bone position largely followed the pattern of forward-upward rotation of the mandible due to ridge resorption. During this course the hyoid position in relation to the cervical spine showed a mean increase. The hyocervical changes, however, showed less variability than the hyomaxillary and hyomandibular changes. The posture of the head and cervical column showed no definite mean changes during the 1-year period. On the other hand, analysis of individual changes revealed that a pronounced decrease in mandibular inclination due to ridge resorption was associated with retroclination of the cervical column and decreased craniocervical angulation. These postural changes may be regarded as adaptive changes to a marked initial change in mandibular position.

The large number of persons with Down's syndrome among the group of physically and mentally retarded demands that continuing study be directed toward its causes and effects. Much conflicting data are reported in the literature concerning the facial and cranial effects of this syndrome. The cephalic proportions, in profile, of a group of male Caucasian trisomic Mongoloids were compared with the proportions of a control group of male Caucasians of similar age range. Using the areas of the midface, mandible, and endocranium obtained from cephlograms of the two groups, the following ratios were studied: (1) midfacial area/endocranial area (2) mandibular area/endocranial area, and (3) midfacial area/mandibular area. A significant degree of deficiency in midfacial area, mandibular area, and endocranial area was found in the Mongoloid group. In studying the facial proportions, we found that the Down's syndrome group's ratios were significantly smaller in all three areas. The magnitude of the deficiency in the Mongoloid midface, both in gross area and in relation to endocranial area, remained nearly constant with age. The ratio of midfacial area to mandibular area in Mongoloids was much more comparable to that of the normal group than the other two ratios studied. In both groups the ratio of the midfacial area to the mandibular area became smaller with age. The decrease was more rapid in the Down's syndrome group. The findings of this study imply that all areas of the face and skull are deficient in persons with Down's syndrome. The data point to the possibility that the characteristics of this syndrome and the deficiencies described are polygenic in origin. Also, contrary to the majority of reports in the literature, the mandible and the midface grow in approximately the same proportion in both study and control groups