Interphase effects in dental nanocomposites investigated by small-angle neutron scattering

Small-angle and ultrasmall-angle neutron scattering (SANS and USANS) were used to characterize silica nanoparticle dispersion morphologies and the interphase in thermoset dimethacrylate polymer nanocomposites. Silica nanoparticle fillers were silanized with varying mass ratios of 3-methacryloxypropyltrimethoxysilane (MPTMS), a silane that interacts with the matrix through covalent and H-bonding, and n-octyltrimethoxysilane (OTMS), a silane that interacts through weak dispersion forces. Interphases with high OTMS mass fractions were found to be fractally rough with fractal dimensions, D(s), between 2.19 and 2.49. This roughness was associated with poor interfacial adhesion and inferior mechanical properties. Mean interparticle distances calculated for composites containing 10 mass % and 25 mass % silica suggest that the nanoparticles treated with more MPTMS than OTMS may be better dispersed than OTMS-rich nanoparticles. The results indicate that the covalent bonding and H-bonding of MPTMS-rich nanoparticles with the matrix are necessary for preparing well-dispersed nanocomposites. In addition, interphases containing equal masses of MPTMS and OTMS may yield composites with overall optimal properties. Finally, the combined SANS/USANS data could distinguish the differences, as a function of silane chemistry, in the nanoparticle/silane and silane/matrix interfaces that affect the overall mechanical properties of the composites.     

Material model measurements and predictions for a random pore poly(epsilon-caprolactone) scaffold

We investigated material models for a polymeric scaffold used for bone. The material was made by co-extruding poly(epsilon-caprolactone) (PCL), a biodegradable polyester, and poly(ethylene oxide) (PEO). The water soluble PEO was removed resulting in a porous scaffold. The stress-strain curve in compression was fit with a phenomenological model in hyperbolic form. This material model will be useful for designers for quasi-static analysis as it provides a simple form that can easily be used in finite element models. The ASTM D-1621 standard recommends using a secant modulus based on 10% strain. The resulting modulus has a smaller scatter in its value compared with the coefficients of the hyperbolic model, and it is therefore easier to compare differences in material processing and ensure quality of the scaffold. A prediction of the small-strain elastic modulus was constructed from images of the microstructure. Each pixel of the micrographs was represented with a brick finite element and assigned the Young's modulus of bulk PCL or a value of 0 for a pore. A compressive strain was imposed on the model and the resulting stresses were calculated. The elastic constants of the scaffold were then computed with Hooke's law for a linear-elastic isotropic material. The model was able to predict the small-strain elastic modulus measured in the experiments to within one standard deviation. Thus, by knowing the microstructure of the scaffold, its bulk properties can be predicted from the material properties of the constituents.     

Expression of phosphorylcholine-specific B cells during murine development

The TEPC 15 (T15) clonotype, a putatively germline antibody specificity, does not appear in the neonatal B-cell repertoire until approximately 1 wk of age. This report extends this observation by the demonstration that (a) the T15 clonotype follows similar kinetics of appearance in germfree as well as conventionally-reared mice; (b) maternal influences and genetic background play a minor role in the development of the T15 clonotype since CBFI neonates raised by C57BL/6 or BALB/c mothers acquire the T15 clonotype at the same time in ontogeny as BALB/c neonates; (c) the lack of phosphorylcholine (PC)-specific B cells shortly after birth is reflected in a dearth of PC-binding cells in the neonate as well; and (d) no PC-specifc B cells are found in 19-day fetal liver or in bone marrow until 7 days of life, coincident with their appearance in the spleen. These findings, along with a previous report that PC-specific splenic B cells are tolerizable as late as day 10 after birth, confirm the invariant, late occurrence of the T15 clonotype and support a highly- ordered, rigorously predetermined mechanism for the acquisition of the B- cell repertoire. The results are discussed in light of other studies on the ontogeny of B-cell specificity, and in terms of the implications on the mechanism by which antibody diversity is generated.     

Controlled tetra-Fc sialylation of IVIg results in a drug candidate with consistent enhanced anti-inflammatory activity

Despite the beneficial therapeutic effects of intravenous immunoglobulin (IVIg) in inflammatory diseases, consistent therapeutic efficacy and potency remain major limitations for patients and physicians using IVIg. These limitations have stimulated a desire to generate therapeutic alternatives that could leverage the broad mechanisms of action of IVIg while improving therapeutic consistency and potency. The identification of the important anti-inflammatory role of fragment crystallizable domain (Fc) sialylation has presented an opportunity to develop more potent Ig therapies. However, translating this concept to potent anti-inflammatory therapeutics has been hampered by the difficulty of generating suitable sialylated products for clinical use. Therefore, we set out to develop the first, to our knowledge, robust and scalable process for generating a well-qualified sialylated IVIg drug candidate with maximum Fc sialylation devoid of unwanted alterations to the IVIg mixture. Here, we describe a controlled enzymatic, scalable process to produce a tetra-Fc–sialylated (s4-IVIg) IVIg drug candidate and its qualification across a wide panel of analytic assays, including physicochemical, pharmacokinetic, biodistribution, and in vivo animal models of inflammation. Our in vivo characterization of this drug candidate revealed consistent, enhanced anti-inflammatory activity up to 10-fold higher than IVIg across different animal models. To our knowledge, this candidate represents the first s4-IVIg suitable for clinical use; it is also a valuable therapeutic alternative with more consistent and potent anti-inflammatory activity.Intravenous immunoglobulin (IVIg) is a therapeutic blood product prepared from the pooled plasma of 3,000–60,000 healthy donors per batch (1–3). It is a complex heterogeneous mixture of IgG subclasses and low amounts of IgA, IgM, and other plasma proteins (4). IVIg contains a wide array of antibodies expected to be present in human serum, and the large number of donors ensures diversity in the Ig repertoire that far exceeds that of an individual donor (5).IVIg has been used for more than 30 years for the treatment of a variety of acute and chronic autoimmune and systemic inflammatory diseases (2–5). Although efficacious in these diseases, the precise mechanism of action of IVIg is not well-understood. Various studies have documented a series of nonmutually exclusive mechanisms modulating components of the innate and adaptive immune system (4, 6). For example, IVIg has been shown to mediate anti-inflammatory responses through its action on dendritic cells, natural killer cells, regulatory T cells, B cells, and the monocyte/macrophage system, and through its suppression or neutralization of soluble factors, such as inflammatory cytokines, chemokines, and pathogenic autoantibodies (5–7).Although beneficial in numerous indications, IVIg preparations have distinct limitations, such as variable efficacy, clinical risks, high costs, and finite supply (2, 3, 8). Different IVIg preparations are frequently treated as interchangeable products clinically, but it is well-known that significant differences in product preparations exist that may impact tolerability and activity in selected clinical applications (9). At the current maximal dosing regimens, only partial and unsustained responses are obtained in many instances (2, 4). In addition, the long infusion times (4–6 h) associated with the high volume of IVIg treatment consume significant resources at infusion centers (8) and negatively affect patient-reported outcomes, such as convenience and quality of life (10). Developing therapeutic alternatives that could leverage the broad biological activities of IVIg and simultaneously, provide more consistent and potent anti-inflammatory activity with minimal inconvenience would be highly valuable to clinicians and patients.The IgGs in IVIg are composed of a fragment antigen-binding domain (Fab fragment) that facilitates the selective interaction with specific antigens and a fragment crystallizable domain (Fc fragment) that interacts with cellular receptors (Fc receptors) known to play critical functions in modulating the activation state of immune cells (11). The IgG Fc fragment contains a conserved N-linked glycan at position N297. The core of the N297 N-linked glycan is composed of two GlcNAc and three mannose residues. Typically, this core can be further extended with fucose, galactose, sialic acid, and bisecting GlcNAc monosaccharides through selective enzymatic glycosylation reactions (12, 13). Alterations to the N297 glycan composition have been shown to have a significant impact in modulating the interaction between the IgG Fc fragment and the Fc receptors. For example, removal of fucose from the IgG N-glycan core has been shown to increase its affinity for Fc gamma receptor IIIa (FcγRIIIa), leading to enhanced antibody-dependent cellular cytotoxicity (14). Sialylation, the addition of terminal sialic acid to N297 glycan, has also been shown to decrease the affinity for type I Fc receptors and increase the affinity for type II Fc receptors (11).In the past three decades, a wealth of reports has documented alterations in antibody glycosylation associated with different diseases in humans. Among these reports, changes in antibody sialylation have been associated with the evolution of autoimmune and inflammatory diseases. For example, rheumatoid arthritis and juvenile idiopathic arthritis are associated with decreased levels of IgG sialylation (15, 16). Additional studies have shown that this translates particularly to the pathogenic antibodies in inflamed joints of arthritis patients (17). It has also been shown that IgG sialylation increases during pregnancy and that this increase may be associated with the remission of rheumatoid arthritis during pregnancy (18, 19). In addition, pathogenic proteinase 3 autoantibodies are less sialylated in patients with active Wegener’s vasculitis (granulomatosis with polyangiitis) (20).Alterations in endogenous IgG sialylation have been associated with treatment response in inflammatory/autoimmune diseases. For example, in patients with Kawasaki disease treated with IVIg, increased levels of endogenous human IgG sialylation decreased the likelihood of IVIg treatment resistance (defined as persistent or recrudescent fever at least 36 h after the completion of IVIg infusion) (21). Similarly, in patients with Guillain–Barré syndrome, those with more severe forms of the disorder showed a lower level of IgG sialylation, despite IVIg treatment (22).Translation of these natural observations in humans to therapeutic options was first realized in 2006, when it was proposed that high doses (>1 g/kg) of unfractionated IVIg were required to elicit sufficient anti-inflammatory activity because of a limited concentration of sialylated IgG in the total IVIg preparation. This theory was substantiated in a mouse model of arthritis that showed similar levels of anti-inflammatory activity when using 1 g/kg IVIg and 0.1 g/kg sialic acid-enriched IVIg, therefore indicating a 10-fold enhancement with the addition of terminal sialic acids (23, 24). Additional results supporting this theory were subsequently reported in other independent studies and other animal models (25, 26). It was further shown that the anti-inflammatory activity of sialylation can be recapitulated using a sialylated Fc fragment derived from IVIg or an IgG1 recombinant antibody (after in vitro sialylation of the Fc) at a 30-fold lower dose than IVIg (24, 27). These results opened the possibility of developing sialylated IVIg and other sialylated antibodies with enhanced anti-inflammatory properties.In recent years, different reports have debated the anti-inflammatory benefits of sialylation. For example, studies using desialylated IVIg in immune thrombocytopenic purpura (ITP) models (28) and Sambucus nigra agglutinin (SNA) -enriched IVIg in arthritis models (29) have shown that sialylation does not enhance IVIg activity or that it may even be dispensable for its therapeutic effects. On the contrary, more comprehensive studies performed by several independent laboratories testing desialylated or hypersialylated IVIg across different animal models under preventive and therapeutic treatment modalities have shown that sialylation is critical for the anti-inflammatory activity of IVIg (24–27, 30). Furthermore, T cell-independent vaccinations resulted in the generation of hypersialylated immunomodulatory antibodies that were able to modulate other immune responses, establishing a broad relevance of these sialylated IgG glycoforms as modulators of immune responses (31). Notably, none of the previous studies used precisely the same protocols for enriching or depleting sialic acid-containing IgG glycoforms, which may partially explain the discrepancies of these studies. Thus, the major aim of this study was to use rigorous, controlled processes and quality controls to investigate the potential of hypersialylated IVIg as a drug candidate with enhanced therapeutic activity.We describe a robust, controlled sialylation process to generate tetra-Fc–sialylated IVIg and show that this process yields a product with consistent enhanced anti-inflammatory activity. Specifically, we first observed that the sialylated IVIg was at least 10 times more potent than the parent IVIg product in a model of collagen antibody-induced arthritis (CAIA) using a prophylactic dose. We further confirmed this enhanced anti-inflammatory activity with therapeutic dosing in models of K/BxN serum-induced arthritis and ITP, and a prophylactic model of skin-blistering disease. Importantly, we noted that, without a tightly controlled process of sialylation, unwanted side products can accumulate that may be responsible for the inconsistent activity observed in previous studies. Therefore, the process that we describe combined with sensitive controls to generate a tetra-Fc–sialylated IVIg (s4-IVIg) devoid of undesired modifications has been critical to obtain the consistent enhanced anti-inflammatory activity and serves as the first example, to our knowledge, of a therapeutic candidate for product development.     

A new method to combine contrast-enhanced magnetic resonance imaging during live ultrasound of the breast using volume navigation technique: a study for evaluating feasibility, accuracy and reproducibility in healthy volunteers

Objectives

To evaluate feasibility, accuracy and reproducibility of combined US-MR of the breast using volume navigation technique.

Subjects and methods

Five healthy females underwent bilateral contrast-enhanced MR (CE-MR) of the breast in supine position, after positioning three couples of markers on the breast. After CE-MR data uploading in the ultrasound (US) database, manual co-registration was obtained during live US of the breast by means of an electromagnetic transmitter positioned near the subject under examination and two electromagnetic sensors were mounted on the transducer bracket. Transmitter and sensors were connected to a position-sensing unit embedded in the US equipment allowing to track probe position and orientation within the electromagnetic field. Live US image were co-registered to the previously loaded breast CE-MR volume by coupling markers. For each subject, two independent radiologists recorded the examination time and verified twice image alignment using five fixed checkpoints. Pair t Student test and Wilcoxon test were used for statistical analysis.

Results

In all subjects US and CE-MR images were successfully combined. The examination time was 10 ± 2 vs. 9 ± 4 min, respectively (p = 0.642; NS). A total of one hundred measurements of images misalignment were performed: the measurements recorded between the two operators were 0.42 ± 0.32 cm and 0.58 ± 0.41 cm (p = 0.161; NS), and 0.50 ± 0.32 cm and 0.56 ± 0.52 cm (p = 0.928; NS), respectively.

Discussion

In our preliminary experience, volume navigation technique appears to be a accurate and reproducible method to combine CE-MR image during unilateral US of the breast.     

Tumor necrosis factor alpha-induced endothelial tissue factor is located on the cell surface rather than in the subendothelial matrix

Because there is no consensus regarding the precise distribution of induced endothelial tissue factor (TF), we studied TF activity in and on tumor necrosis factor alpha-stimulated cultured human umbilical vein endothelial cells (ECs) and their underlying matrix. TF was mainly expressed on the cell surface. Only small traces were found on the apical surface suggesting that TF is predominantly located on the basolateral side of the cell membrane. The presence of TF on the cell surface was confirmed by flow cytometry. Subendothelial TF activity appeared to be dependent upon the procedure used to remove the stimulated EC monolayer. Whereas ammonium hydroxide or hypotonic lysis resulted in relatively high levels of matrix-associated TF, virtually no TF was found on the matrix after mild enzymatic detachment of stimulated ECs. Cell removal with EDTA resulted in intermediate levels of matrix-associated TF. Neither the enzymatic treatment nor EDTA degraded or removed this TF activity. Similar patterns were observed for matrix-associated TF antigen and EC surface markers. Electron microscopic analysis showed cell fragments on the matrix after monolayer lysis. The findings strongly suggest that induced endothelial TF associated with the subendothelial matrix actually represents TF on EC remnants.     

Early results of a prospective study on the pyrolytic carbon (pyrocarbon) Amandys? for osteoarthritis of the wrist

INTRODUCTION

The preliminary results of a pyrocarbon interpositional radiocarpal implant in a small cohort of patients were reviewed. As it is currently only a limited release product, we describe to potential users early complications and negative outcomes.

METHODS

Patients were assessed using pain levels, ranges of motion, grip strength, type of and time to return to work as well as pre-operative and post-operative DASH (Disabilities of the Arm, Shoulder and Hand) scores. Radiographs were taken and patient satisfaction was recorded.

RESULTS

All six patients were contacted. One was not satisfied. Three had reduced motion. None experienced squeaking. There were no immediate or late post-operative complications. There was one early volar displacement of an implant.

CONCLUSIONS

Although our early results are somewhat encouraging, further and longer studies are warranted before supporting the use of this particular pyrocarbon implant as a primary procedure.