Coupled Broad Ion Beam-Scanning Electron Microscopy (BIB-SEM) for polishing and three dimensional (3D) serial section tomography (SST).

Here we describe the first automated fully integrated in-microscope broad ion beam (BIB) system. Ar+-BIB has several advantages over Ga+ focused ion beam (FIB) and Xe+ plasma-FIB (PFIB) methods inducing less beam damage, especially for ion beam sensitive materials. It can mill areas several orders of magnitude larger (up to millimetre scale), and is not confined to the edge of the sample with associated curtaining issues. BIB is shown to have sputter rates up to five times higher than comparable FIB techniques. This new coupled BIB-SEM system (commercial name 'iPrep™II') enables in-microscope surface polishing to remove contaminants or damage for two dimensional (2D) imaging, as well as automated serial section tomography (SST) by milling and imaging hundreds of slices, cost and time efficiently. The milled slice thickness can be controlled from a few nanometers up to a micrometre. A novel sample transfer, handling and interlock system allows automated and sequential BIB polishing, scanning electron microscopy (SEM) and analysis by secondary electron (SE) imaging, electron back scatter diffraction (EBSD) and energy dispersive spectroscopy (EDS) for 3D microstructure analysis. Furthermore, insulating surfaces can be sputter coated after milling each slice to reduce charging during SEM analysis. The performance of the instrument is demonstrated through a series of case studies across the materials, earth and life sciences exploiting the imaging, crystallographic and chemical mapping capabilities. These include the study of butterfly defects in bearing steels, meta-stable intermetallic phases in bronze bearings, shale gas rock, aluminium plasma electrolytic oxide (PEO) coatings as well as liver and mouse brain tissues.

Improving product quality and productivity of bispecific molecules through the application of continuous perfusion principles.

Bispecific protein scaffolds can be more complex than traditional monoclonal antibodies (MAbs) because two different sites/domains for epitope binding are needed. Because of this increased molecular complexity, bispecific molecules are difficult to express and can be more prone to physical and chemical degradation compared to MAbs, leading to higher levels of protein aggregates, clipped species, or modified residues in cell culture. In this study, we investigated cell culture performance for the production of three types of bispecific molecules developed at Amgen. In particular, we cultured a total of six CHO cell lines in both an approximately 12-day fed-batch process and an approximately 40-day high-density perfusion process. Harvested cell culture fluid from each process was purified and analyzed for product quality attributes including aggregate levels, clipped species, charge variants, individual amino acid modifications and host cell protein (HCP) content. Our studies showed that in average, the intensified perfusion process increased 15-fold the integrated viable cell density and the total harvested product (and fivefold the daily volumetric productivity) compared to fed-batch. Furthermore, bispecific product quality improved in perfusion culture (as analyzed in affinity-capture pools) with reduction in levels of aggregates (up to 72% decrease), clipped species (up to 75% decrease), acidic variants (up to 76% decrease), deamidated/isomerized species in complementarity-determining regions, and HCP (up to 84% decrease). In summary, the intensified perfusion process exhibited better productivity and product quality, highlighting the potential to use it as part of a continuous manufacturing process for bispecific scaffolds.

Distinguishing deformation mechanisms in elastocapillary experiments.

Soft materials are known to deform due to a variety of mechanisms, including capillarity, buoyancy, and swelling. In this paper, we present experiments on polyvinylsiloxane gel threads partially-immersed in three liquids with different solubility, wettability, and swellability. Our results demonstrate that deformations due to capillarity, buoyancy, and swelling can be of similar magnitude as such threads come to static equilibrium. To account for all three effects being present in a single system, we derive a model capable of explaining the observed data and use it to determine the force law at the three-phase contact line. The results show that the measured forces are consistent with the expected Young-Dupré equation, and do not require the inclusion of a tangential contact line force.

Nonlocal rheology of dense granular flow in annular shear experiments.

The flow of dense granular materials at low inertial numbers cannot be fully characterized by local rheological models; several nonlocal rheologies have recently been developed to address these shortcomings. To test the efficacy of these models across different packing fractions and shear rates, we perform experiments in a quasi-2D annular shear cell with a fixed outer wall and a rotating inner wall, using photoelastic particles. The apparatus is designed to measure both the stress ratio µ (the ratio of shear to normal stress) and the inertial number I through the use of a torque sensor, laser-cut leaf springs, and particle-tracking. We obtain µ(I) curves for several different packing fractions and rotation rates, and successfully find that a single set of model parameters is able to capture the full range of data collected once we account for frictional drag with the bottom plate. Our measurements confirm the prediction that there is a growing lengthscale at a finite value µs, associated with a frictional yield criterion. Finally, we newly identify the physical mechanism behind this transition at µs by observing that it corresponds to a drop in the susceptibility to force chain fluctuations.

Expansion shock waves in regularized shallow-water theory.

We identify a new type of shock wave by constructing a stationary expansion shock solution of a class of regularized shallow-water equations that include the Benjamin-Bona-Mahony and Boussinesq equations. An expansion shock exhibits divergent characteristics, thereby contravening the classical Lax entropy condition. The persistence of the expansion shock in initial value problems is analysed and justified using matched asymptotic expansions and numerical simulations. The expansion shock's existence is traced to the presence of a non-local dispersive term in the governing equation. We establish the algebraic decay of the shock as it is gradually eroded by a simple wave on either side. More generally, we observe a robustness of the expansion shock in the presence of weak dissipation and in simulations of asymmetric initial conditions where a train of solitary waves is shed from one side of the shock.

Continuous bind-and-elute protein A capture chromatography: Optimization under process scale column constraints and comparison to batch operation.

Recently, continuous downstream processing has become a topic of discussion and analysis at conferences while no industrial applications of continuous downstream processing for biopharmaceutical manufacturing have been reported. There is significant potential to increase the productivity of a Protein A capture step by converting the operation to simulated moving bed (SMB) mode. In this mode, shorter columns are operated at higher process flow and corresponding short residence times. The ability to significantly shorten the product residence time during loading without appreciable capacity loss can dramatically increase productivity of the capture step and consequently reduce the amount of Protein A resin required in the process. Previous studies have not considered the physical limitations of how short columns can be packed and the flow rate limitations due to pressure drop of stacked columns. In this study, we are evaluating the process behavior of a continuous Protein A capture column cycling operation under the known pressure drop constraints of a compressible media. The results are compared to the same resin operated under traditional batch operating conditions. We analyze the optimum system design point for a range of feed concentrations, bed heights, and load residence times and determine achievable productivity for any feed concentration and any column bed height. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:938-948, 2016.

Elastocapillary deformations on partially-wetting substrates: rival contact-line models.

A partially-wetting liquid can deform the underlying elastic substrate upon which it rests. This situation requires the development of theoretical models to describe the wetting forces imparted by the drop onto the solid substrate, particularly those at the contact-line. We construct a general solution using a displacement potential function for the elastic deformations within a finite elastic substrate associated with these wetting forces, and compare the results for several different contact-line models. Our work incorporates internal contributions to the surface stress from both liquid/solid Σls and Σsg solid/gas solid surface tensions (surface stress), which results in a non-standard boundary-value problem that we solve using a dual integral equation. We compare our results to relevant experiments and conclude that the generalization of solid surface tension Σls ≠ Σsg is an essential feature in any model of partial-wetting. The comparisons also allow us to systematically eliminate some proposed contact-line models.

The role of gamma interferon in DNA vaccine-induced tumor immunity targeting simian virus 40 large tumor antigen.

The central role of CD4+ T lymphocytes in mediating DNA vaccine-induced tumor immunity against the viral oncoprotein simian virus 40 (SV40) large tumor antigen (Tag) has previously been described by our laboratory. In the present study, we extend our previous findings by examining the roles of IFN-γ and Th1-associated effector cells within the context of DNA immunization in a murine model of pulmonary metastasis. Immunization of BALB/c mice with plasmid DNA encoding SV40 Tag (pCMV-Tag) generated IFN-γ-secreting T lymphocytes that produced this cytokine upon in vitro stimulation with mKSA tumor cells. The role of IFN-γ as a mediator of protection against mKSA tumor development was assessed via in vivo IFN-γ neutralization, and these experiments demonstrated a requirement for this cytokine in the induction immune phase. Neutralization of IFN-γ was associated with a reduction in Th1 cytokine-producing CD4+ and CD8+ splenocytes, as assessed by flow cytometry analysis, and provided further evidence for the role of CD4+ T lymphocytes as drivers of the cellular immune response. Depletion of NK cells and CD8+ T lymphocytes demonstrated the expendability of these cell types individually, but showed a requirement for a resident cytotoxic cell population within the immune effector phase. Our findings demonstrate the importance of IFN-γ in the induction of protective immunity stimulated by pCMV-Tag DNA-based vaccine and help to clarify the general mechanisms by which DNA vaccines trigger immunity to tumor cells.

CD4+ T lymphocytes are critical mediators of tumor immunity to simian virus 40 large tumor antigen induced by vaccination with plasmid DNA.

A mechanistic analysis of tumor immunity directed toward the viral oncoprotein simian virus 40 (SV40) large tumor antigen (Tag) has previously been described by our laboratory for scenarios of recombinant Tag immunization in BALB/c mice. In the present study, we performed a preliminary characterization of the immune components necessary for systemic tumor immunity induced upon immunization with plasmid DNA encoding SV40 Tag as a transgene (pCMV-Tag). Antibody responses to SV40 Tag were observed via indirect enzyme-linked immunosorbent assay following three intramuscular (i.m.) injections of pCMV-Tag and were typified by a mixed Th1/Th2 response. Complete tumor immunity within a murine model of pulmonary metastasis was achieved upon two i.m. injections of pCMV-Tag, as assessed by examination of tumor foci in mouse lungs, without a detectable antibody response to SV40 Tag. Induction-phase and effector-phase depletions of T cell subsets were performed in vivo via administration of depleting rat monoclonal antibodies, and these experiments demonstrated that CD4(+) T lymphocytes are required in both phases of the adaptive immune response. Conversely, depletion of CD8(+) T lymphocytes did not impair tumor immunity in either immune phase and resulted in the premature production of antibodies to SV40 Tag. Our findings are unique in that a dominant role could be ascribed to CD4(+) T lymphocytes within a model of DNA vaccine-induced tumor immunity to Tag-expressing tumor cells. Additionally, our findings provide insight into the general mechanisms of vaccine-induced tumor immunity directed toward tumors bearing distinct tumor-associated antigens.

Vaccines and immunotherapeutics for the treatment of malignant disease.

The employment of the immune system to treat malignant disease represents an active area of biomedical research. The specificity of the immune response and potential for establishing long-term tumor immunity compels researchers to continue investigations into immunotherapeutic approaches for cancer. A number of immunotherapeutic strategies have arisen for the treatment of malignant disease, including various vaccination schemes, cytokine therapy, adoptive cellular therapy, and monoclonal antibody therapy. This paper describes each of these strategies and discusses some of the associated successes and limitations. Emphasis is placed on the integration of techniques to promote optimal scenarios for eliminating cancer.

Shear-driven size segregation of granular materials: modeling and experiment.

Granular materials segregate by size under shear, and the ability to quantitatively predict the time required to achieve complete segregation is a key test of our understanding of the segregation process. In this paper, we apply the Gray-Thornton model of segregation (developed for linear shear profiles) to a granular flow with an exponential shear profile, and evaluate its ability to describe the observed segregation dynamics. Our experiment is conducted in an annular Couette cell with a moving lower boundary. The granular material is initially prepared in an unstable configuration with a layer of small particles above a layer of large particles. Under shear, the sample mixes and then resegregates so that the large particles are located in the top half of the system in the final state. During this segregation process, we measure the velocity profile and use the resulting exponential fit as input parameters to the model. To make a direct comparison between the continuum model and the observed segregation dynamics, we map the local concentration (from the model) to changes in packing fraction; this provides a way to make a semiquantitative comparison with the measured global dilation. We observe that the resulting model successfully captures the presence of a fast mixing process and relatively slower resegregation process, but the model predicts a finite resegregation time, while in the experiment resegregation occurs only exponentially in time.

Role of the innate immune response and tumor immunity associated with simian virus 40 large tumor antigen.

We examined properties of the innate immune response against the tumor-specific antigen simian virus 40 (SV40) large tumor antigen (Tag) following experimental pulmonary metastasis in naive mice. Approximately 14 days after mKSA tumor cell challenge, expression of inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha), interleukin-2 (IL-2), and RANTES was upregulated in splenocytes harvested from mice, as assessed by flow cytometry and antibody array assays. This response was hypothesized to activate and induce tumor-directed NK cell lysis since IL-2-stimulated NK cells mediated tumor cell destruction in vitro. The necessary function of NK cells was further validated in vivo through selected antibody depletion of NK cells, which resulted in an overwhelming lung tumor burden relative to that in animals receiving a control rabbit IgG depletion regimen. Interestingly, mice achieved increased protection from experimental pulmonary metastasis when NK cells were further activated indirectly through in vivo administration of poly(I:C), a Toll-like receptor 3 (TLR3) agonist. In a separate study, mice receiving treatments of poly(I:C) and recombinant SV40 Tag protein immunization mounted effective tumor immunity in an established experimental pulmonary metastasis setting. Initiating broad-based immunity with poly(I:C) was observed to induce a Th1 bias in the SV40 Tag antibody response that led to successful antitumor responses not observed in animals treated only with poly(I:C) or SV40 Tag. These data have direct implications for immunotherapeutic strategies incorporating methods to elicit inflammatory reactions, particularly NK cell-driven lysis, against malignant cell types that express a tumor-specific antigen such as SV40 Tag.

Tumor immunity against a simian virus 40 oncoprotein requires CD8+ T lymphocytes in the effector immune phase.

The required activities of CD4(+) T cells and antibody against the virally encoded oncoprotein simian virus 40 (SV40) Tag have previously been demonstrated by our laboratory to be mediators in achieving antitumor responses and tumor protection through antibody-dependent cell-mediated cytotoxicity (ADCC). In this study, we further characterize the necessary immune cell components that lead to systemic tumor immunity within an experimental pulmonary metastatic model as the result of SV40 Tag immunization and antibody production. Immunized animals depleted of CD8(+) T cells at the onset of experimental tumor cell challenge developed lung tumor foci and had an overall decreased survival due to lung tumor burden, suggesting a role for CD8(+) T cells in the effector phase of the immune response. Lymphocytes and splenocytes harvested from SV40 Tag-immunized mice experimentally inoculated with tumor cells synthesized increased in vitro levels of the Th1 cytokine gamma interferon (IFN-gamma), as assessed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry assays. CD8(+) T-cell activity was also heightened in SV40 Tag-immunized and tumor cell-challenged mice, based upon intracellular production of perforin, confirming the cytolytic properties of CD8(+) T cells against tumor cell challenge. Altogether, these data point to the role of recombinant SV40 Tag protein immunization in initiating a cytotoxic T-lymphocyte (CTL) response during tumor cell dissemination and growth. The downstream activity of CD8(+) T cells within this model is likely initiated from SV40 Tag-specific antibody mediating ADCC tumor cell destruction.

INTS6/DICE1 inhibits growth of human androgen-independent prostate cancer cells by altering the cell cycle profile and Wnt signaling.

BACKGROUND: The gene encoding integrator complex subunit 6 (INTS6), previously known as deleted in cancer cells 1 (DICE1, OMIM 604331) was found to be frequently affected by allelic deletion and promoter hypermethylation in prostate cancer specimens and cell lines. A missense mutation has been detected in prostate cancer cell line LNCaP. Together, these results suggest INTS6/DICE1 as a putative tumor suppressor gene in prostate cancer. In this study, we examined the growth inhibitory effects of INTS6/DICE1 on prostate cancer cells. RESULTS: Markedly decreased INTS6/DICE1 mRNA levels were detected in prostate cancer cell lines LNCaP, DU145 and PC3 as well as CPTX1532 as compared to a cell line derived from normal prostate tissue, NPTX1532. Exogenous re-expression of INTS6/DICE1 cDNA in androgen-independent PC3 and DU145 cell lines substantially suppressed their ability to form colonies in vitro. This growth inhibition was not due to immediate induction of apoptosis. Rather, prostate cancer cells arrested in G1 phase of the cell cycle. Expression profiling of members of the Wnt signaling pathway revealed up-regulation of several genes including disheveled inhibitor CXXC finger 4 (CXXC4), frizzled homologue 7 (FZD7), transcription factor 7-like 1 (TCF7L1), and down-regulation of cyclin D1. CONCLUSION: These results show for the first time a link between INTS6/DICE1 function, cell cycle regulation and cell-cell communication involving members of the Wnt signaling pathway.

Evidence of simian virus 40 exposure in a colony of captive baboons.

Simian virus 40 (SV40) is a polyomavirus for which non-human primates are the permissive host. The baboon (Papio spp.) is an old world monkey that is used in a variety of research investigations; however, natural infection of SV40 among baboons has not been thoroughly examined or reported. Initially, we were interested in determining the prevalence of SV40 infection among a captive colony of baboons based on the presence of antibodies to SV40 large T-antigen (Tag). An overall seroprevalence rate of >50% was found after screening sera from 142 baboons in the colony based on ELISA. Endpoint titer values for serum antibody binding to SV40 Tag reached as high as 1280 for 5 out of 142 baboons. Peptide binding assays revealed that a range of SV40 Tag epitopes are immunogenic in the baboon, and that individual animals differ in their humoral immune responses to SV40 Tag based on epitope recognition. Specificity to SV40 Tag and not some other primate polyomavirus encoded large Tag was further examined by serologic reactivity to peptide epitopes unique to SV40 Tag. Additional serology was performed to assess SV40 Tag reactivity by Western blot and whether antibodies were capable of neutralizing SV40 infectivity in vitro. Although antibodies with high levels of SV40 neutralization were observed in a number of the baboons, there was a lack of correlation between viral neutralization and antibodies to SV40 Tag. Further examination using molecular-based diagnosis and SV40 Tag specific real-time quantitative PCR determined that some of the baboons appeared to be exposed to SV40. DNA sequence analysis of the PCR products confirmed that SV40 Tag specific sequences were detected in baboons.

Viral clearance using disposable systems in monoclonal antibody commercial downstream processing.

Once highly selective protein A affinity is chosen for robust mAb downstream processing, the major role of polishing steps is to remove product related impurities, trace amounts of host cell proteins, DNA/RNA, and potential viral contaminants. Disposable systems can act as powerful options either to replace or in addition to polishing column chromatography to ensure product purity and excellent viral clearance power for patients' safety. In this presentation, the implementation of three disposable systems such as depth filtration, membrane chromatography, and nanometer filtration technology in a commercial process are introduced. The data set of viral clearance with these systems is presented. Application advantages and disadvantages including cost analysis are further discussed.

A NOD/SCID tumor model for human ovarian cancer that allows tracking of tumor progression through the biomarker Sp17.

No experimental animal model employing a primary human ovarian carcinoma (OC) cell line is presently available that tracks the progression of this cell line with an identifiable marker. This hinders investigations related to developing new approaches for treating OC. Here, we describe the development of a tumor model in NOD/SCID mice for human OC that makes use of the endogenously expressed tumor specific sperm protein 17 (Sp17) cancer testis antigen. In this model, human SKOV-3 OC cell lines were intra-peritoneally seeded. Subsequently viable SKOV-3 cells were recovered from primary organ cell cultures from the liver ovaries, abdomen, and ascitic fluid, and their presence was confirmed by the detection of Sp17 mRNA by RT-PCR and Sp17 protein by immunocytochemistry and FACS analysis. When SKOV-3 tumor cells were administered intravenously the mice developed primarily lung tumor foci. This model makes it possible to evaluate new immunotherapeutic strategies for the treatment of human OC based on the biomarker Sp17.

Fc gamma receptors play a dominant role in protective tumor immunity against a virus-encoded tumor-specific antigen in a murine model of experimental pulmonary metastases.

Simian virus 40 (SV40) large tumor antigen (Tag) represents a virus-encoded tumor-specific antigen expressed in many types of human cancers and a potential immunologic target for antitumor responses. Fc receptors are important mediators in the regulation and execution of host effector mechanisms against conditions including infectious diseases, autoimmunity, and cancer. By examining tumor protection in SV40 Tag-immunized wild-type BALB/c mice using an experimental pulmonary metastasis model, we attempted to address whether engagement of the immunoglobulin G Fc receptors (FcgammaRs) on effector cells is necessary to mediate antitumor responses. All immunized BALB/c FcgammaR-/- knockout mice developed anti-SV40 Tag antibody responses prior to experimental challenge with a tumorigenic cell line expressing SV40 Tag. However, all mice deficient in the activating FcgammaRI (CD64) and FcgammaRIII (CD16) were unable to mount protective immunologic responses against tumor challenge and developed tumor lung foci. In contrast, mice lacking the inhibitory receptor FcgammaRII (CD32) demonstrated resistance to tumorigenesis. These results underscore the importance of effector cell populations expressing FcgammaRI/III within this murine tumor model system, and along with the production of a specific humoral immune response, antibody-dependent cell-mediated cytotoxicity (ADCC) may be a functioning mechanism of tumor clearance. Additionally, these data demonstrate the potential utility of ADCC as a viable approach for targeting vaccination strategies that promote FcgammaRI/III scavenging pathways against cancer.

Cytokine expression in peripheral blood lymphocytes before and after aspirin desensitization in aspirin-exacerbated respiratory disease.

Aspirin intolerance is the hallmark of aspirin-exacerbated respiratory disease (AERD). Overproduction of cysteinyl-leukotrienes (Cys-LTs) has been implicated as major mediators of AERD; however, the LT receptor antagonist montelukast is only partially effective in inhibiting aspirin responses. Several studies have documented the importance of cytokine production by T lymphocytes in asthma. Peripheral blood lymphocyte (PBL) cytokine expression and its relation to aspirin desensitization in aspirin-sensitive patients with asthma have not been studied. This study was performed to examine PBL cytokine expression in aspirin-sensitive patients who have asthma before and after aspirin desensitization. A 42-year-old white woman with a history of severe asthma, nasal polyps, aspirin sensitivity, and chronic sinusitis was treated with aspirin desensitization. Blood was taken before and after aspirin desensitization, and PBL cytokine expression was studied by flow cytometry. Aspirin desensitization differentially affects interferon (IFN) gamma expression. This effect results in an increase in IFN-gamma expression by CD4(+) lymphocytes and a decrease in IFN-gamma expression by CD8(+) lymphocytes. Aspirin desensitization in an aspirin-sensitive patient with asthma resulted in an increase in IFN-gamma expression by CD4(+) lymphocytes and a decrease in IFN-gamma expression by CD8(+) lymphocytes, the significance of which needs additional investigation.