Diagnostic and Practical Challenges in Applying National Comprehensive Cancer Network Guidelines for Suspected Pathogenic TP53 Mosaicism.

Benefits and limitations in using NCCN guidelines to distinguish TP53 CH from mosaic LFS.

In vivo migration of endogenous brain progenitor cells guided by an injectable peptide amphiphile biomaterial.

Biomaterials hold great promise in helping the adult brain regenerate and rebuild after trauma. Peptide amphiphiles (PAs) are highly versatile biomaterials, gelling and forming macromolecular structures when exposed to physiological levels of electrolytes. We are here reporting on the first ever in vivo use of self-assembling PA carrying a Tenascin-C signal (E2 Ten-C PA) for the redirection of endogenous neuroblasts in the rodent brain. The PA forms highly aligned nanofibers, displaying the migratory sequence of Tenascin-C glycoprotein as epitope. In this in vivo work, we have formed in situ a gel of aligned PA nanofibers presenting a migratory Tenascin-C signal sequence in the ventral horn of the rostral migratory stream, creating a track reaching the neocortex. Seven days posttransplant, doublecortin positive cells were observed migrating inside and alongside the injected biomaterial, reaching the cortex. We observed a 24-fold increase in number of redirected neuroblasts for the E2 Ten-C PA-injected animals compared to control. We also found injecting the E2 Ten-C PA to cause minimal neuroinflammatory response. Analysing GFAP+ astrocytes and Iba1+ microglia activation, the PA does not elicit a stronger neuroinflammatory response than would be expected from a small needle stab wound. Redirecting endogenous neuroblasts and increasing the number of cells reaching a site of injury using PAs may open up new avenues for utilizing the pool of neuroblasts and neural stem cells within the adult brain for regenerating damaged brain tissue and replacing neurons lost to injury.

Donor TSH level is associated with clinical pregnancy among oocyte donation cycles.

PURPOSE: The purpose of the study is to evaluate the association between donor TSH level (independent of recipient TSH level) and recipient pregnancy outcome among fresh donor oocyte IVF cycles. METHODS: This is a retrospective cohort study investigating 232 consecutive fresh donor-recipient cycles (200 total oocyte donors) at an academic medical center. Main outcome measures include clinical pregnancy and live birth. RESULTS: Cycles were categorized into two groups based on donor TSH level (< 2.5 and ≥ 2.5 mIU/L). After controlling for multiple donor and recipient characteristics, the probability of clinical pregnancy was significantly lower among donors with TSH levels ≥2.5 mIU/L compared to those with TSH values <2.5 mIU/L (43.1 %, 95 % CI 28.5-58.9, versus 66.7 %, 95 % CI 58.6-73.9, respectively, p = 0.01). The difference in live birth rates between the two groups did not achieve statistical significance (43.1 %, 95 % CI 28.8-58.6, versus 58.0 %, 95 % CI 50.0-65.6, respectively, p = 0.09). CONCLUSIONS: Donor TSH level, independent of recipient TSH level, is associated with recipient clinical pregnancy. These findings suggest that thyroid function may impact the likelihood of pregnancy at the level of the oocyte.

Long-term follow-up after preoperative trastuzumab and chemotherapy for HER2-overexpressing breast cancer.

BACKGROUND: Neoadjuvant chemotherapy and trastuzumab is an established treatment for locally advanced HER2-positive breast cancer, providing favorable rates of clinical response and pCR. Minimal data describe long-term outcomes after neoadjuvant HER2-directed therapy. This study aimed to explore long-term efficacy and toxicity after neoadjuvant trastuzumab and chemotherapy for HER2-positive breast cancer. PATIENTS AND METHODS: Eligible patients participated in 1 of 2 single-arm phase II neoadjuvant trials, receiving either paclitaxel/trastuzumab (TH) or vinorelbine/trastuzumab (NH) for stage II-III HER2-positive disease. Postoperative chemotherapy, with or without trastuzumab, was offered. Charts were reviewed to identify recurrence, death, and treatment-related toxicities. Association of long-term outcomes with baseline characteristics and pathological response to primary therapy was explored. RESULTS: Eighty patients were identified; 33 (41.3%) received TH and 47 (58.8%) received NH. Fourteen (17.5%) had pCR at surgery. Most (96.3%) received anthracycline-based adjuvant chemotherapy; 78.7% of NH patients also received adjuvant trastuzumab. At a median follow-up of 8.8 years, 23 (28.8%) patients have experienced recurrence, with 16 breast cancer-related deaths. Four-year RFS in patients with pCR was 92.9% (95% confidence interval [CI], 79.4%-100%) versus 72.4% without pCR (95% CI, 63.9%-82.1%). All initial symptomatic cardiotoxicity resolved during extended follow-up. New symptomatic cardiotoxicity in long-term follow-up was rare, primarily occurring in patients requiring retreatment with a cardiotoxic agent. CONCLUSION: Neoadjuvant chemotherapy and trastuzumab for HER2-positive breast cancer resulted in favorable long-term survival with minimal late toxicity. Trends in this data set suggest an association between pCR and improved long-term RFS. Retreatment with cardiotoxic agents might increase risk of late cardiotoxicity.

Reversible changes in cell morphology due to cytoskeletal rearrangements measured in real-time by QCM-D.

The mechanical properties and responses of cells to external stimuli (including drugs) are closely connected to important phenomena such as cell spreading, motility, activity, and potentially even differentiation. Here, reversible changes in the viscoelastic properties of surface-attached fibroblasts were induced by the cytoskeleton-perturbing agent cytochalasin D, and studied in real-time by the quartz crystal microbalance with dissipation (QCM-D) technique. QCM-D is a surface sensitive technique that measures changes in (dynamically coupled) mass and viscoelastic properties close to the sensor surface, within a distance into the cell that is usually only a fraction of its size. In this work, QCM-D was combined with light microscopy to study in situ cell attachment and spreading. Overtone-dependent changes of the QCM-D responses (frequency and dissipation shifts) were first recorded, as fibroblast cells attached to protein-coated sensors in a window equipped flow module. Then, as the cell layer had stabilised, morphological changes were induced in the cells by injecting cytochalasin D. This caused changes in the QCM-D signals that were reversible in the sense that they disappeared upon removal of cytochalasin D. These results are compared to other cell QCM-D studies. Our results stress the combination of QCM-D and light microscopy to help interpret QCM-D results obtained in cell assays and thus suggests a direction to develop the QCM-D technique as an even more useful tool for real-time cell studies.

A method to integrate patterned electrospun fibers with microfluidic systems to generate complex microenvironments for cell culture applications.

The properties of a cell's microenvironment are one of the main driving forces in cellular fate processes and phenotype expression invivo. The ability to create controlled cell microenvironments invitro becomes increasingly important for studying or controlling phenotype expression in tissue engineering and drug discovery applications. This includes the capability to modify material surface properties within well-defined liquid environments in cell culture systems. One successful approach to mimic extra cellular matrix is with porous electrospun polymer fiber scaffolds, while microfluidic networks have been shown to efficiently generate spatially and temporally defined liquid microenvironments. Here, a method to integrate electrospun fibers with microfluidic networks was developed in order to form complex cell microenvironments with the capability to vary relevant parameters. Spatially defined regions of electrospun fibers of both aligned and random orientation were patterned on glass substrates that were irreversibly bonded to microfluidic networks produced in poly-dimethyl-siloxane. Concentration gradients obtained in the fiber containing channels were characterized experimentally and compared with values obtained by computational fluid dynamic simulations. Velocity and shear stress profiles, as well as vortex formation, were calculated to evaluate the influence of fiber pads on fluidic properties. The suitability of the system to support cell attachment and growth was demonstrated with a fibroblast cell line. The potential of the platform was further verified by a functional investigation of neural stem cell alignment in response to orientation of electrospun fibers versus a microfluidic generated chemoattractant gradient of stromal cell-derived factor 1 alpha. The described method is a competitive strategy to create complex microenvironments invitro that allow detailed studies on the interplay of topography, substrate surface properties, and soluble microenvironment on cellular fate processes.

Issues of ligand accessibility and mobility in initial cell attachment.

The influence of lateral ligand mobility on cell attachment and receptor clustering has previously been explored for membrane-anchored molecules involved in cell-cell adhesion. In this study, we considered instead a cell binding motif from the extracellular matrix. Even though the lateral mobility of extracellular matrix ligands in membranes does not occur in vivo, we believe it is of interest for cell engineering in vitro. As is the case for cell-cell adhesion molecules, lateral mobility of extracellular matrix ligands could influence cell attachment and, subsequently, cell behavior in cell culture. In this paper, the accessibility and functionality of extracellular matrix ligands presented at surfaces were evaluated for the conditions of laterally mobile versus non-mobile ligands by studying ligand-antibody binding events and early cell attachment as a function of ligand concentration. We compare the initial attachment of rat-derived adult hippocampal progenitor (AHP) cells on laterally mobile, supported phospholipid bilayer membranes to non-mobile, poly-L-lysine-grafted-poly(ethylene glycol) (PLL-g-PEG) polymer films functionalized with a range of laminin-derived IKVAV-containing peptide densities. To this end, synthesis of a new PLL-g-PEG/PEG-IKVAV polymer is described. The characterization of available IKVAV peptides on both surface presentations schemes was explored by studying the mass uptake of anti-IKVAV antibodies using a combination of the surface-sensitive techniques quartz crystal microbalance with dissipation monitoring, surface plasmon resonance spectroscopy, and optical waveguide lightmode spectroscopy. IKVAV-containing peptides presented on laterally mobile, supported phospholipid bilayers and non-mobile PLL-g-PEG were recognized by the anti-IKVAV antibody in a dose-dependent manner, indicating that the amount of available IKVAV ligands increases proportionally with ligand density over the concentrations tested. Attachment of AHP cells to IKVAV-functionalized PLL-g-PEG and supported phospholipid bilayers followed a sigmoidal dependence on peptide concentration, with a critical concentration of approximately 3 pmol/cm2 IKVAV ligands required to support initial AHP cell attachment for both surface modifications. There appeared to be little influence of IKVAV peptide mobility on the initial attachment of AHP cells. Although the spread in the cell attachment data was larger for the PLL-g-PEG surface modification, this was reduced when observed after 24 h, indicating that the cells might need longer times to establish attachment strengths equivalent to those observed on peptide-functionalized supported lipid bilayers. The present study is a step toward understanding the influence of extracellular-matrix-derived ligand mobility on cell fate. Further analysis should focus on the systematic tuning of lateral ligand diffusion, as well as a comparison between the response of non-spreading cells (i.e., AHPs), versus spreading cells (i.e., fibroblasts).

DHA-induced changes of supported lipid membrane morphology.

Docosahexaenoic acid (DHA) is a polyunsaturated long fatty acid known to have fundamental effects on cell membrane function. Here, the effect of DHA on phosphocholine-supported lipid bilayers was measured using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique. Above a concentration of 60 muM (i.e., near the critical micelle concentration), DHA had drastic effects on the viscoelastic properties of the supported membranes, suggesting a more complex process and structure than simple insertion of molecules in the bilayer. Fluorescence microscopy revealed the spontaneous formation of elongated out-growths from the bilayers, which were remarkable for their length ( approximately 100 mum) and extensive coverage of the surface. These results demonstrate the applicability of QCM-D as a method to screen for conditions where membrane remodeling occurs but also that complementary techniques are required to describe in more detail the changes in viscoelastic properties of the membrane. These results are highly relevant for the present rapid development in the field of model lipid membranes aiming toward increased knowledge about processes occurring at biological surfaces.

Simulation of proliferation of neural stem cells on a surface with emphasis on spatial constraints on cell division.

We present Monte Carlo lattice simulations of proliferation of cells on a surface in the situation when the cell-cell adhesion is relatively strong and the cells may form islands and/or flattened hemispheres. The model parameters were chosen to mimic proliferation of adult rat neural stem cells (or, more specifically, adult hippocampal progenitor cells) deposited on polyornithine and laminin coated polystyrene. The results obtained show that the spatial constraints on cell division may result in slowdown of the exponential growth. Depending on the rules used for cell division, this effect may be either nearly negligible or appreciable. In the latter case, the scale of the deviations from the exponential growth is comparable with that observed in our experiments. In the simulations, the slowdown of the growth starts however somewhat earlier and occurs in a less abrupt manner. This seems to indicate that the spatial constraints on division of cells are not the main factor behind the experimentally observed termination of the growth.

Model porous surfaces for systematic studies of material-cell interactions.

A model system for studying cell-surface interactions, based on microfabricated cell culture substrates, has been developed and is described here. Porous surfaces consisting of interconnecting channels with openings of subcellular dimensions are generated on flat, single crystal, silicon substrates. Channel size (width, depth), distribution, and surface coating can be varied independently and used for systematic investigation of how topographical, chemical, and elastic surface properties influence cell or tissue biological responses. Model porous surfaces have been produced by using two different microfabrication methods. Submicron-sized channels with very high depth-to-width aspect ratios (up to 30) have been made by using electron beam lithography and anisotropic reactive ion etching into single-crystal silicon. Another method uses thick-resist photolithography, which can be used to produce channels wider than 1 microm and with depth-to-width aspect ratios below 20 in an epoxy polymer. Preliminary cell culture tests show that fibroblasts bridge 0.8- to 1.8-microm-wide channels with very few exceptions (i.e., a continuous space below the cell-surface interface is created). It has also been shown that variation of channel periodicity significantly affects fibroblast morphology and attachment density. With this model system, it is possible to load the channels with bioactive substances intended to interact with cells at or near the surface in a time-dependent manner.

An in vivo study of bone response to implants topographically modified by laser micromachining.

Dental implants topographically modified by laser ablation of periodic arrays of micron-sized craters, were studied in a two-part laboratory investigation. The patterned and control (turned) implants were inserted in rabbit femur and tibia. After 12 weeks the fixation in the bone was evaluated mechanically or by histomorphometry (all threads along the implant and the three best consecutive threads were analysed). In the pilot study no difference was found with respect to bone-to-implant contact and peak removal torque. Significantly more bone was found for the control implants when measuring the bone area inside the threads in the tibia. In the second part of the study, the pattern was improved and significantly more bone-to-implant contact was found for the laser-machined implants. The second part of the study also demonstrated significantly greater peak removal torque values in the tibia with the test implants than the control implants.

Influence of Polydispersity on Adsorption of Nanoparticles.

The adsorption of polydisperse, interacting nanoparticles is studied experimentally and discussed in terms of the random sequential adsorption model. Two kinds of polystyrene particles with different size variation (41+/-6 and 107+/-5 nm) were used in adsorption experiments at or close to saturation. The dried monolayer particle films were analyzed with scanning electron microscopy. Selective adsorption of smaller particles resulted in altered size distributions on the surface compared to that in solution. Varying the ionic strength was seen to influence the effective polydispersity of the particles. With increasing salt concentration there was a relative increase in the adsorption of smaller particles, resulting in a large shift toward smaller particle sizes in the size distribution on the surface. Polydispersity gave a slight increase in coverage at high salt concentrations and a decrease in the ordering of the particles on the surface. Copyright 2001 Academic Press.