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Residual host cell proteins (HCPs) in the drug product can affect product quality, stability, and/or safety. In particular, highly active hydrolytic enzymes at sub-ppm levels can negatively impact the shelf life of drug products but are challenging to identify by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) due to their high dynamic range between HCPs and biotherapeutic proteins. We employed new strategies to address the challenge: (1) native digest at a high protein concentration; (2) sodium deoxycholate added during the reduction step to minimize the inadvertent omission of HCPs observed with native digestion; and (3) solid phase extraction with 50% MeCN elution prior to LC-MS/MS analysis to ensure effective mAb removal. A 50 cm long nanoflow charged surface hybrid column was also packed to allow for higher sample load for increased sensitivity. Our workflow has increased the sensitivity for HCP identification by 10- to 100-fold over previous reports and showed the robustness as low as 0.1 ppm for identifying HCPs (34.5 to 66.2 kDa MW). The method capability was further confirmed by consistently identifying85% of 48 UPS-1 proteins (0.10 to 1.34 ppm, 6.3 to 82.9 kDa MW) in a monoclonal antibody (mAb) and the largest number (746) of mouse proteins from NIST mAb reported to date by a single analysis. Our work has filled a significant gap in HCP analysis for detecting and demonstrating HCP clearance, in particular, extremely low-level hydrolases in drug process development.

Hydrolytic degradation of the polysorbate 20 (PS20) surfactant in protein-based liquid formulations releases free fatty acids (FFAs), which can accumulate to form particles in drug products during real-time (long-term) storage. To identify formulation conditions that mitigate the risk of particle formation, we conducted a longitudinal study using purified recombinant monoclonal antibody (mAb) formulated in 24 conditions. In this real-time stability study at 5 °C, three key formulation parameters—mAb concentration, initial PS20 concentration, and pH—were varied across representative ranges in a full-factorial design. A longitudinal regression analysis was used to evaluate the effects of these parameters and their interactions on PS20 degradation (via measurements of PS20, FFAs, and PS20 ester distribution) and on particle formation (via visible particle observations and subvisible particle counts). The time-dependent onset of visible particles trended with the rise in subvisible particle counts and FFA levels and fall in PS20 concentration. In the ranges studied here, lower mAb concentration and higher initial PS20 concentration delayed the onset of particles, whereas pH had a negligible effect. These observations were consistent with the general trends predicted by our previously published FFA solubility model. Taken together, these findings highlight the complex relationships between formulation parameters, PS20 degradation, and particle formation.

We present optical and ultraviolet photometry, as well as optical spectra, for the type II supernova (SN) 2015bf. Our observations cover the phases from ∼2 to ∼200 d after explosion. The first spectrum is characterized by a blue continuum with a blackbody temperature of ∼24 000 K and flash-ionized emission lines. After about 1 week, the spectra of SN 2015bf evolve like those of a regular SN II. From the luminosity of the narrow emission component of H α, we deduce that the mass-loss rate is larger than ${\sim}3.7\times 10^{-3}\, {\rm M_\odot \, yr^{-1}}$. The disappearance of the flash features in the first week after explosion indicates that the circumstellar material is confined within ∼6 × 1014 cm. Thus, we suggest that the progenitor of SN 2015bf experienced violent mass loss shortly before the supernova explosion. The multiband light curves show that SN 2015bf has a high peak luminosity with an absolute visual magnitude MV = −18.11 ± 0.08 mag and a fast post-peak decline with a V-band decay of 1.22 ± 0.09 mag within ∼50 d after maximum light. Moreover, the R-band tail luminosity of SN 2015bf is fainter than that of SNe II with similar peak by 1–2 mag, suggesting a small amount of 56Ni (${\sim}0.009\, {\rm M_\odot }$) synthesized during the explosion. Such a low nickel mass indicates that the progenitor of SN 2015bf could be a super-asymptotic-giant-branch star that collapsed owing to electron capture.

Hydrolytic degradation of polysorbate during 2–8°C storage of monoclonal antibody drug products has been attributed to residual enzymes (e.g., esterases) from bioprocessing steps. Robust detection of esterase activity using sensitive, non-polysorbate surrogate substrates can provide an alternate method to assess polysorbate degradation risk, if the correlation between the esterase activity and polysorbate degradation is established. A general esterase activity assay was developed as a monitoring and characterization tool during bioprocess development of monoclonal antibodies. We report a fluorescence plate-based assay for quantifying esterase activity, utilizing 4-methylumbelliferyl caprylate (MU-C8) as the esterase substrate. The assay was first assessed for substrate, inhibitor and pH specificity using both model enzymes and purified protein samples. The assay was then extensively tested to understand sample matrix effects on activity rates. The use of this high-throughput method will allow for rapid characterization of protein samples in under three hours. The esterase activity correlated directly with polysorbate degradation and can provide valuable information on polysorbate degradation risk throughout drug development.

Human skin perceives physical stimuli applied to the body and mitigates the risk of physical interaction through its soft and resilient mechanical properties. Social robots would benefit from whole-body robotic skin (or tactile sensors) resembling human skin in realizing a safe, intuitive, and contact-rich human-robot interaction. However, existing soft tactile sensors show several drawbacks (complex structure, poor scalability, and fragility), which limit their application in whole-body robotic skin. Here, we introduce biomimetic robotic skin based on hydrogel-elastomer hybrids and tomographic imaging. The developed skin consists of a tough hydrogel and a silicone elastomer forming a skin-inspired multilayer structure, achieving sufficient softness and resilience for protection. The sensor structure can also be easily repaired with adhesives even after severe damage (incision). For multimodal tactile sensation, electrodes and microphones are deployed in the sensor structure to measure local resistance changes and vibration due to touch. The ionic hydrogel layer is deformed owing to an external force, and the resulting local conductivity changes are measured via electrodes. The microphones also detect the vibration generated from touch to determine the location and type of dynamic tactile stimuli. The measurement data are then converted into multimodal tactile information through tomographic imaging and deep neural networks. We further implement a sensorized cosmetic prosthesis, demonstrating that our design could be used to implement deformable or complex-shaped robotic skin.

Chinese hamster ovary (CHO) cell engineering based on CRISPR/Cas9 knockout (KO) technology requires the delivery of guide RNA (gRNA) and Cas9 enzyme for efficient gene targeting. With an ever-increasing list of promising gene targets, developing and optimizing a multiplex gene KO protocol is crucial for rapid CHO cell engineering. Here, we describe a method that can support efficient targeting and KO of up to ten genes through sequential transfections. This method utilizes Cas9 protein to first screen multiple synthetic gRNAs per gene, followed by Sanger sequencing indel analysis, to identify effective gRNA sequences. Using sequential transfections of these potent gRNAs led to the isolation of single cell clones with the targeted deletion of all ten genes (as confirmed by Sanger sequencing at the DNA level and mass spectrometry at the protein level). Screening 704 single cell clones yielded 6 clones in which all 10 genes were deleted through sequential transfections, demonstrating the success of this decaplex gene editing strategy. This pragmatic approach substantially reduces the time and effort required to generate multiple gene knockouts in CHO cells. This article is protected by copyright. All rights reserved.

Degradation of polysorbate (PS) by hydrolytically active host cell proteins (HCPs) in drug products may impair the protein-stabilizing properties of PS and lead to the formation of particles due to the accumulation of poorly soluble free fatty acids upon long-term storage. The identification of the causative enzymes is challenging due to their low-abundance even when using state-of-the-art instrumentation and workflows. To overcome these challenges, we developed a rigorous enrichment strategy for HCPs, utilizing both Protein A and anti-HCP affinity chromatography, which facilitated the in-depth characterization of the HCP population in a monoclonal antibody formulation prone to PS hydrolysis. Based on the HCPs identified by liquid chromatography coupled to tandem mass spectrometry, a number of enzymes annotated as hydrolases were recombinantly expressed and characterized in terms of polysorbate degradation. Among the selected candidates, Lipoprotein Lipase, Lysosomal Acid Lipase (LIPA) and Palmitoyl-Protein Thioesterase 1 (PPT1) exhibited notable activity towards PS. To our knowledge, this is the first report to identify LIPA and PPT1 as residual HCPs that can contribute to PS degradation in a biological product.

We report velocity-delay maps for prominent broad emission lines, Ly_alpha, CIV, HeII and H_beta, in the spectrum of NGC5548. The emission-line responses inhabit the interior of a virial envelope. The velocity-delay maps reveal stratified ionization structure. The HeII response inside 5-10 light-days has a broad single-peaked velocity profile. The Ly_alpha, CIV, and H_beta responses peak inside 10 light-days, extend outside 20 light-days, and exhibit a velocity profile with two peaks separated by 5000 km/s in the 10 to 20 light-day delay range. The velocity-delay maps show that the M-shaped lag vs velocity structure found in previous cross-correlation analysis is the signature of a Keplerian disk with a well-defined outer edge at R=20 light-days. The outer wings of the M arise from the virial envelope, and the U-shaped interior of the M is the lower half of an ellipse in the velocity-delay plane. The far-side response is weaker than that from the near side, so that we see clearly the lower half, but only faintly the upper half, of the velocity--delay ellipse. The delay tau=(R/c)(1-sin(i))=5 light-days at line center is from the near edge of the inclined ring, giving the inclination i=45 deg. A black hole mass of M=7x10^7 Msun is consistent with the velocity-delay structure. A barber-pole pattern with stripes moving from red to blue across the CIV and possibly Ly_alpha line profiles suggests the presence of azimuthal structure rotating around the far side of the broad-line region and may be the signature of precession or orbital motion of structures in the inner disk. Further HST observations of NGC 5548 over a multi-year timespan but with a cadence of perhaps 10 days rather than 1 day could help to clarify the nature of this new AGN phenomenon., 19 pages, 9 figures, ApJ in press

Hydrolytic degradation of polysorbate during 2-8°C storage of monoclonal antibody drug products has been attributed to residual enzymes (e.g., esterases) from bioprocessing steps. Robust detection of esterase activity using sensitive, non-polysorbate surrogate substrates can provide an alternate method to assess polysorbate degradation risk, if the correlation between the esterase activity and polysorbate degradation is established.A general esterase activity assay was developed as a monitoring and characterization tool during bioprocess development of monoclonal antibodies.We report a fluorescence plate-based assay for quantifying esterase activity, utilizing 4-methylumbelliferyl caprylate (MU-C8) as the esterase substrate. The assay was first assessed for substrate, inhibitor and pH specificity using both model enzymes and purified protein samples. The assay was then extensively tested to understand sample matrix effects on activity rates.The use of this high-throughput method will allow for rapid characterization of protein samples in under three hours. The esterase activity correlated directly with polysorbate degradation and can provide valuable information on polysorbate degradation risk throughout drug development.

Cryopreservation provides the foundation for research, development, and manufacturing operations in the CHO-based biopharmaceutical industry. Despite its criticality, studies are lacking that explicitly demonstrate that the routine cell banking process and the potential stress and damage during cryopreservation and recovery from thaw have no lasting detrimental effects on CHO cells. Statistics are also scarce on the decline of cell-specific productivity (Qp ) over time for recombinant CHO cells developed using the glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection system. To address these gaps, we evaluated the impact of freeze-thaw on 24 recombinant CHO cell lines (generated by the GS/MSX selection system) using a series of production culture assays. Across the panel of cell lines expressing one of three monoclonal antibodies (mAbs), freeze-thaw did not result in any significant impact beyond the initial post-thaw passages. Production cultures sourced from cryopreserved cells and their non-cryopreserved counterparts yielded similar performance (growth, viability, and productivity), product quality (size, charge, and glycosylation distributions), and flow cytometric profiles (intracellular mAb expression). However, many production cultures yielded lower Qp at increased cell age: 17 of the 24 cell lines displayed ≥20% Qp decline after ∼2-3 months of passaging, irrespective of whether the cells were previously cryopreserved. The frequency of Qp decline underscores the continued need for understanding the underlying mechanisms and for careful clone selection. Because our experiments were designed to decouple the effects of cryopreservation from those of cell age, we could conclusively rule out freeze-thaw as a cause for Qp decline. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:463-477, 2018.

We present the results of an extensive observational campaign on the nearby Type Ibn SN 2015G, including data from radio through ultraviolet wavelengths. SN 2015G was asymmetric, showing late-time nebular lines redshifted by ~1000 km/s. It shared many features with the prototypical SN In 2006jc, including extremely strong He I emssion lines and a late-time blue pseudocontinuum. The young SN 2015G showed narrow P-Cygni profiles of He I, but never in its evolution did it show any signature of hydrogen - arguing for a dense, ionized, and hydrogen-free circumstellar medium moving outward with a velocity of ~1000 km/s and created by relatively recent mass loss from the progenitor star. Ultraviolet through infrared observations show that the fading SN 2015G (which was probably discovered some 20 days post-peak) had a spectral energy distribution that was well described by a simple, single-component blackbody. Archival HST images provide upper limits on the luminosity of SN 2015G's progenitor, while nondetections of any luminous radio afterglow and optical nondetections of outbursts over the past two decades provide constraints upon its mass-loss history., Comment: 19 pages, 13 figures. Published in MNRAS

We present BVRI and unfiltered light curves of 93 Type Ia supernovae (SNe Ia) from the Lick Observatory Supernova Search (LOSS) follow-up program conducted between 2005 and 2018. Our sample consists of 78 spectroscopically normal SNe Ia, with the remainder divided between distinct subclasses (three SN 1991bg-like, three SN 1991T-like, four SNe Iax, two peculiar, and three super-Chandrasekhar events), and has a median redshift of 0.0192. The SNe in our sample have a median coverage of 16 photometric epochs at a cadence of 5.4 days, and the median first observed epoch is ~4.6 days before maximum B-band light. We describe how the SNe in our sample are discovered, observed, and processed, and we compare the results from our newly developed automated photometry pipeline to those from the previous processing pipeline used by LOSS. After investigating potential biases, we derive a final systematic uncertainty of 0.03 mag in BVRI for our dataset. We perform an analysis of our light curves with particular focus on using template fitting to measure the parameters that are useful in standardising SNe Ia as distance indicators. All of the data are available to the community, and we encourage future studies to incorporate our light curves in their analyses., Comment: 29 pages, 13 figures, accepted for publication in MNRAS

The extent of afucosylation, which refers to the absence of core fucose on Fc glycans, can correlate positively with the antibody-dependent cellular cytotoxicity (ADCC) activity of a monoclonal antibody (mAb). Therefore, it is important to maintain consistent afucosylation during cell culture process scale-up in bioreactors for a mAb with ADCC activity. However, there is currently a lack of understanding about the impact of partial pressure of carbon dioxide (pCO2 )-a parameter that can vary with bioreactor scale-on afucosylation. Using a small-scale (3 L) bioreactor model that can modulate pCO 2 levels through modified configurations and gassing strategies, we identified three cell culture process parameters that influence afucosylation of a mAb produced by a recombinant Chinese Hamster Ovary (CHO) cell line: pCO 2 , media hold duration (at 37°C), and manganese. These three-independent parameters demonstrated a synergistic effect on mAb afucosylation; increase in pCO 2 , media hold duration, and manganese consistently increased afucosylation. Our investigations into the underlying mechanisms through proteomic analysis indicated that the synergistic interactions downregulated pathways related to guanosine diphosphate-fucose synthesis and fucosylation, and upregulated manganese transport into the CHO cells. These new findings highlight the importance of considering potential differences in culture environment and operations across bioreactor scales, and understanding the impact of their interactions on product quality.

Gamma-ray burst (GRB) 150910A was detected by {\it Swift}/BAT, and then rapidly observed by {\it Swift}/XRT, {\it Swift}/UVOT, and ground-based telescopes. We report Lick Observatory spectroscopic and photometric observations of GRB~150910A, and we investigate the physical origins of both the optical and X-ray afterglows, incorporating data obtained with BAT and XRT. The light curves show that the jet emission episode lasts $\sim 360$~s with a sharp pulse from BAT to XRT (Episode I). In Episode II, the optical emission has a smooth onset bump followed by a normal decay ($\alpha_{\rm R,2} \approx -1.36$), as predicted in the standard external shock model, while the X-ray emission exhibits a plateau ($\alpha_{\rm X,1} \approx -0.36$) followed by a steep decay ($\alpha_{\rm X,2} \approx -2.12$). The light curves show obvious chromatic behavior with an excess in the X-ray flux. Our results suggest that GRB 150910A is an unusual GRB driven by a newly-born magnetar with its extremely energetic magnetic dipole (MD) wind in Episode II, which overwhelmingly dominates the observed early X-ray plateau. The radiative efficiency of the jet prompt emission is $\eta_{\gamma} \approx 11\%$. The MD wind emission was detected in both the BAT and XRT bands, making it the brightest among the current sample of MD winds seen by XRT. We infer the initial spin period ($P_0$) and the surface polar cap magnetic field strength ($B_p$) of the magnetar as $1.02 \times 10^{15}~{\rm G} \leq B_{p} \leq 1.80 \times 10^{15}~{\rm G}$ and 1~ms $\leq P_{0}v\leq 1.77$~ms, and the radiative efficiency of the wind is $\eta_w \geq 32\%$., Comment: Accepted for publication in ApJ; 26 pages, 8 figures, 3 tables

Protein carbonylation is a posttranslational modification referring to the occurrence of aldehydes and ketones in proteins. The current understanding of how carbonylation, in particular, metal-catalyzed carbonylation, occurs in recombinant mAbs during production and storage is very limited. To facilitate investigations into mAb carbonylation, we developed a protein carbonylation assay with improved assay robustness and precision over the conventional assays. We applied this assay to investigate mAb carbonylation under production, storage, and stress conditions and showed that iron, hydrogen peroxide, and polysorbate 20 at pharmaceutically relevant levels critically influence the extent of mAb carbonylation. In addition, we found that while carbonylation correlates with mAb aggregation in several cases, carbonylation cannot be used as a general indicator for aggregation. Furthermore, we observed that mAb carbonylation level can decrease during storage, which indicates that carbonylation products may not be stable. Finally, we report for the first time a positive correlation between carbonylation and acidic charge heterogeneity of mAbs that underwent metal-catalyzed oxidation. This finding shows that the impact of protein carbonylation on product quality for mAbs is not limited to aggregation but also extends to charge heterogeneity.

We present the complete sample of stripped-envelope supernova (SN) spectra observed by the Lick Observatory Supernova Search (LOSS) collaboration over the last three decades: 888 spectra of 302 SNe, 652 published here for the first time, with 384 spectra (of 92 SNe) having photometrically-determined phases. After correcting for redshift and Milky Way dust reddening and reevaluating the spectroscopic classifications for each SN, we construct mean spectra of the three major spectral subtypes (Types IIb, Ib, and Ic) binned by phase. We compare measures of line strengths and widths made from this sample to the results of previous efforts, confirming that O I {\lambda}7774 absorption is stronger and found at higher velocity in Type Ic SNe than in Types Ib or IIb SNe in the first 30 days after peak brightness, though the widths of nebular emission lines are consistent across subtypes. We also highlight newly available observations for a few rare subpopulations of interest., Comment: 13 pages; 14 figures; 3 tables. Accepted for publication in MNRAS

To understand the diversity in the cell culture harvest (i.e., feedstock) provided for downstream processing, we compared host cell protein (HCP) profiles using three Chinese Hamster Ovary (CHO) cell lines in null runs which did not generate any recombinant product. Despite differences in CHO lineage, upstream process, and culture performance, the cell lines yielded similar cell-specific productivities for immunogenic HCPs. To compare the dynamics of HCP production, we searched for correlations between the time-course profiles of HCP (as measured by multi-analyte ELISA) and those of two intracellular HCP species, phospholipase B-like 2 (PLBL2) and lactate dehydrogenase (LDH). Across the cell lines, proteins in the day 14 supernatants analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) showed different spot patterns. However, subsequent analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) indicated otherwise: the total number of peptides and proteins identified were comparable, and 80% of the top 1,000 proteins identified were common to all three lines. Finally, to assess the impact of culture viability on extracellular HCP profiles, we analyzed supernatants from a cell line whose viability dropped after day 10. The amounts of HCP and PLBL2 (quantified by their respective ELISAs) as well as the numbers and major populations of HCPs (identified by LC-MS/MS) were similar across days 10, 14, and 17, during which viabilities declined from ∼80% to 107 cells/mL)—operated in fed-batch mode and exhibiting high viabilities (>70%) throughout the culture duration—can accumulate a considerable amount of immunogenic HCP (∼1–2 g/L) in the extracellular environment at the time of harvest (day 14). This work also demonstrates the potential of using LC-MS/MS to overcome the limitations associated with ELISA and 2D-PAGE for HCP analysis. Biotechnol. Bioeng. 2015;112: 2068–2083. © 2015 Wiley Periodicals, Inc.

The tsunami generated on 12 July 1993 by the Hokkaido–Nansei–Oki earthquake (Mw = 7.8) brought about a maximum wave run-up of 31.7 m, the highest recorded in Japan during the 20th century, near the Monai Valley on the west coast of Okushiri Island (Hokkaido Tsunami Survey Group, 1993). To reproduce the extreme run-up height, the three-dimensional non-hydrostatic model (Flow Science, 2012), referred to here as the NH-model, has been locally applied with open boundary conditions supplied in an offline manner by the three-dimensional hydrostatic model (Ribeiro et al., 2011), referred to here as the H-model. The area of the H-model is sufficiently large to cover the entire fault region with one-way nested multiple domains. For the initial water deformation, Okada's fault model (1985) using the sub-fault parameters is applied. Three NH-model experiments have been performed, namely without islands, with one island and with two islands. The experiments with one island and with two islands give rise to values close to the observation with maximum run-up heights of about 32.3 and 30.8 m, respectively, while the experiment without islands gives rise to about 25.2 m. The diffraction of the tsunami wave primarily by Muen Island, located in the south, and the southward topographic guiding of the tsunami run-up at the coast are, as in the laboratory simulation (Yoneyama et al., 2002), found to result in the extreme run-up height near Monai Valley. The presence of Hira Island enhances the diffraction of tsunami waves but its contribution to the extreme run-up height is marginal.

Copper concentration can impact lactate metabolism in Chinese Hamster ovary (CHO) cells. In our previous study, a 20-fold increase in initial copper concentration enabled CHO cultures to shift from net lactate production to net lactate consumption, and achieve higher cell growth and productivity. In this follow-up study, we used transcriptomics to investigate the mechanism of action (MOA) of copper that mediates this beneficial metabolism shift. From microarray profiling (days 0-7), the number of differentially expressed genes increased considerably after the lactate shift (>day 3). To uncouple the effects of copper at early time points (days 0-3) from that of lactate per se (>day 3), and to validate microarray hits, we analyzed samples before the lactate shift by RNA-Seq. Out of 6,398 overlapping genes analyzed by both transcriptomic methods, only the early growth response 1 gene-coding for a transcription factor that activates signaling pathways in response to environmental stimuli-satisfied the differential expression criteria (fold change ≥ 1.5; P < 0.05). Gene expression correlation and biological pathway analyses further confirmed that copper differences exerted minimal transcriptional impact on the CHO cultures before the lactate shift. By contrast, genes associated with hypoxia network and oxidative stress response were upregulated after the lactate shift. These upregulations should boost cell proliferation and survival, but do not account for the preceding shift in lactate metabolism. The findings here indicate that the primary MOA of copper that enabled the shift in lactate metabolism is not at the transcriptional level.

In recent years, several automated scale-down bioreactor systems have been developed to increase efficiency in cell culture process development. ambr™ is an automated workstation that provides individual monitoring and control of culture dissolved oxygen and pH in single-use, stirred-tank bioreactors at a working volume of 10-15 mL. To evaluate the ambr™ system, we compared the performance of four recombinant Chinese hamster ovary cell lines in a fed-batch process in parallel ambr™, 2-L bench-top bioreactors, and shake flasks. Cultures in ambr™ matched 2-L bioreactors in controlling the environment (temperature, dissolved oxygen, and pH) and in culture performance (growth, viability, glucose, lactate, Na(+), osmolality, titer, and product quality). However, cultures in shake flasks did not show comparable performance to the ambr™ and 2-L bioreactors.

During production of therapeutic monoclonal antibodies (mAb), it is highly desirable to remove and control antibody aggregates in the manufacturing process to minimize the potential risk of immunogenicity to patients. During process development for the production of a recombinant IgG in a CHO cell line, we observed atypical high variability from 1 to 20% mAb aggregates formed during cell culture that negatively impacted antibody purification. Analytical characterization revealed the IgG aggregates were mediated by hydrophobic interactions likely caused by misfolded antibody during intracellular processing. Strikingly, data analysis showed an inverse correlation of lower cell culture temperature producing higher aggregate levels. All cultures at 37°C exhibited ≤ 5% aggregates at harvest. Aggregate levels increased 4-12-fold in 33°C cultures when compared to 37°C, with a corresponding 2-4-fold increase in heavy chain (HC) and light chain (LC) mRNA. Additionally, 37°C cases showed a greater excess of LC to HC mRNA levels. Endoplasmic reticulum (ER) chaperone expression and ER size also increased 25-75% at 33°C versus 37°C but to a lesser extent than LC and HC mRNA, consistent with a potential limiting ER folding capacity at 33°C for this cell line. Finally, we identified a 2-5-fold increase in mAb aggregate formation at 33°C compared to 37°C cultures for three additional CHO cell lines. Taken together, our observations indicate that low culture temperature can increase antibody aggregate formation in CHO cells by increasing LC and HC transcripts coupled with limited ER machinery.

The biopharmaceutical industry is increasing its use of the WAVE Bioreactor for culturing cells. Although this disposable bioreactor can be equipped to provide real-time pH and dissolved oxygen (DO) monitoring and control, our goal was to develop a process for culturing CHO cells in this system without relying on pH and DO feedback controls. After identifying challenges in culturing cells without controlling for pH and DO in the WAVE Bioreactor, we characterized O(2) and CO(2) transfer in the system. From these cell-free studies, we identified rock rate and rock angle as key parameters affecting O(2) transfer. We also identified the concentration of CO(2) in the incoming gas and the rate of gas flow into the headspace as key parameters affecting CO(2) transfer--and therefore pH--in the disposable culture chamber. Using a full-factorial design to evaluate the rock rate, rock angle, and gas flow rate defined for this WAVE Bioreactor process, we found comparable cell growth and pH profiles in the ranges tested for these three parameters in two CHO cell lines. This process supported cell growth, and maintained pH and DO within our desired range--pH 6.8-7.2 and DO exceeding 20% of air saturation--for six CHO cell lines, and it also demonstrated comparable cell growth and viability with the stirred-tank bioreactor process with online pH and DO control. By eliminating the use of pH and DO probes, this process provides a simple and more cost-effective method for culturing cells in the WAVE Bioreactor.

Protein glycation is a non-enzymatic glycosylation that can occur to proteins in the human body, and it is implicated in the pathogenesis of multiple chronic diseases. Glycation can also occur to recombinant antibodies during cell culture, which generates structural heterogeneity in the product. In a previous study, we discovered unusually high levels of glycation (>50%) in a recombinant monoclonal antibody (rhuMAb) produced by CHO cells. Prior to that discovery, we had not encountered such high levels of glycation in other in-house therapeutic antibodies. Our goal here is to develop cell culture strategies to decrease rhuMAb glycation in a reliable, reproducible, and scalable manner. Because glycation is a post-translational chemical reaction between a reducing sugar and a protein amine group, we hypothesized that lowering the concentration of glucose--the only source of reducing sugar in our fed-batch cultures--would lower the extent of rhuMAb glycation. When we decreased the supply of glucose to bioreactors from bolus nutrient and glucose feeds, rhuMAb glycation decreased to below 20% at both 2-L and 400-L scales. When we maintained glucose concentrations at lower levels in bioreactors with continuous feeds, we could further decrease rhuMAb glycation levels to below 10%. These results show that we can control glycation of secreted proteins by controlling the glucose concentration in the cell culture. In addition, our data suggest that rhuMAb glycation occurring during the cell culture process may be approximated as a second-order chemical reaction that is first order with respect to both glucose and non-glycated rhuMAb. The basic principles of this glycation model should apply to other recombinant proteins secreted during cell culture.

Large-scale fed-batch cell culture processes of CHO cells are the standard platform for the clinical and commercial production of monoclonal antibodies. Lactate is one of the major by-products of CHO fed-batch culture. In pH-controlled bioreactors, accumulation of high levels of lactate is accompanied by high osmolality due to the addition of base to control pH of the cell culture medium, potentially leading to lower cell growth and lower therapeutic protein production during manufacturing. Lactate dehydrogenase (LDH) is an enzyme that catalyzes the conversion of the substrate, pyruvate, into lactate and many factors including pyruvate concentration modulate LDH activity. Alternately, pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenases (PDHs), to be metabolized in the TCA cycle. PDH activity is inhibited when phosphorylated by pyruvate dehydrogenase kinases (PDHKs). In this study, we knocked down the gene expression of lactate dehydrogenase A (LDHa) and PDHKs to investigate the effect on lactate metabolism and protein production. We found that LDHa and PDHKs can be successfully downregulated simultaneously using a single targeting vector carrying small inhibitory RNAs (siRNA) for LDHa and PDHKs. Moreover, our fed-batch shake flask evaluation data using siRNA-mediated LDHa/PDHKs knockdown clones showed that downregulating LDHa and PDHKs in CHO cells expressing a therapeutic monoclonal antibody reduced lactate production, increased specific productivity and volumetric antibody production by approximately 90%, 75% and 68%, respectively, without appreciable impact on cell growth. Similar trends of lower lactate level and higher antibody productivity on average in siRNA clones were also observed from evaluations performed in bioreactors.

Biotechnological companies and regulatory agencies are pursuing the complete characterization of protein therapeutics in every detail as a means to mitigate risks of product quality related safety issues. During the characterization of a recombinant humanized monoclonal antibody (referred to as rhuMAb), electrospray mass spectrometric analysis suggested that the light chain was highly glycated. The glycated and unglycated materials, separated using boronate affinity chromatography, were fully characterized using tryptic peptide mapping and tandem mass spectrometry. Using an automatic SEQUEST search of the single protein database for this antibody and extensive manual investigations of the mass spectra of the matched peptides, multiple tentative glycation sites in the light and heavy chains were observed in the highly glycated (>53%) samples. A predominant glycation site was identified and confirmed to be lysine 49 on the light chain, by performing extensive sequence analysis on an isolated glycated peptide utilizing Edman degradation analysis and MALDI-TOF/TOF mass spectrometry. Sequence alignments of rhuMAb with 12 other recombinant monoclonal antibodies and computer modeling of the Fab part of rhuMAb suggest that the unusually high level of glycation of lysine residue 49, which is located adjacent to the second complementarity-determining region (CDR2) in the light chain, is due to a spatial proximity effect in catalyzing the Amadori rearrangement by aspartic acid residue 31 in the CDR1 on the light chain.

Bordetella bronchiseptica is a Gram-negative bacterium equipped with several colonization factors that allow it to establish a persistent infection of the murine respiratory tract. Previous studies indicate that B. bronchiseptica adenylate cyclase toxin (ACT) and the type III secretion system (TTSS) synergize to drive dendritic cells into an altered phenotype to down-regulate the host immune response. In this study, we examined the effects of B. bronchiseptica ACT and TTSS on murine bone marrow-derived macrophages. We demonstrate that ACT and TTSS are required for the inhibition of Ag-driven CD4+ T cell proliferation by bacteria-infected macrophages. We identify PGE2 as the mediator of this inhibition, and we show that ACT and the TTSS synergize to increase macrophage production of PGE2. We further demonstrate that B. bronchiseptica can modulate normal macrophage function and drive the immune response toward a Th17 phenotype classified by the significant production of IL-17. In this study, we show that B. bronchiseptica-infected macrophages can induce IL-17 production from naive CD4+ splenocytes, and that lung tissues from B. bronchiseptica-infected mice exhibit a strong Th17 immune response. ACT inhibited surface expression of CD40 and CD86, suppressed TNF-α production, and up-regulated IL-6 production. TTSS also synergized with ACT to up-regulate IL-10 and PGE2 secretion. These findings indicate that persistent colonization by B. bronchiseptica may rely on the ability of the bacteria to differentially modulate both macrophage and dendritic cell function leading to an altered adaptive immune response and subsequent bacterial colonization.

We have previously reported that the respiratory pathogen Bordetella bronchiseptica can form biofilms in vitro. In this report, we demonstrate the disruption of B. bronchiseptica biofilms by rhamnolipids secreted from Pseudomonas aeruginosa. This suggests that biosurfactants such as rhamnolipids may be utilized as antimicrobial agents for removing Bordetella biofilms.

Bordetella bronchisepticautilizes a type III secretion system (TTSS) to establish a persistent infection of the murine respiratory tract. Previous studies have shown that theBordetellaTTSS mediated cytotoxicity in different cell types, inhibition of NF-κB in epithelial cells, and differentiation of dendritic cells into a semimature state. Here we demonstrate modulation of mitogen-activated protein kinase (MAPK) signaling pathways and altered cytokine production in macrophages and dendritic cells by theBordetellaTTSS. In macrophages, the MAPKs ERK and p38 were downregulated. This resulted in attenuated production of interleukin- (IL-)6 and IL-10. In contrast, the Th-1-polarizing cytokine IL-12 was produced at very low levels and remained unmodulated by theBordetellaTTSS. In dendritic cells, ERK was transiently activated, but this failed to alter cytokine profiles. These results suggest that theBordetellaTTSS modulates antigen-presenting cells in a cell type-specific manner and the secretion of high levels of IL-6 and IL-10 by macrophages might be important for pathogen clearance.

Expression of innate immune genes such as beta-defensins is induced in airway epithelium by bacterial components via activation of NF-kappaB. We show here that live Gram-negative bacteria can similarly stimulate this pathway, resulting in upregulation of the beta-defensin tracheal antimicrobial peptide (TAP) in primary cultures of bovine tracheal epithelial cells (TECs), by a Toll-like receptor 4 (TLR4)-mediated pathway. The Gram-negative airway pathogen Bordetella bronchiseptica possesses a type III secretion system previously suggested to inhibit the nuclear translocation of NF-kappaB in a cell line by immunohistochemistry. We therefore hypothesized that this pathogen might interfere in the innate immune response of the epithelium. Exposure of TECs to wild-type B. bronchiseptica suppressed the activation of NF-kappaB and the subsequent induction of TAP mRNA levels, whereas a type III secretion-defective strain did not. These results suggest a mechanism for bacterial evasion of the innate immune response in the airway, which could allow for the observed persistent colonization of this pathogen.

Bordetella species utilize the BvgAS ( Bordetella virulence gene) two-component signal transduction system to sense the environment and regulate gene expression among at least three phases: a virulent Bvg + phase, a nonvirulent Bvg − phase, and an intermediate Bvg i phase. Genes expressed in the Bvg + phase encode known virulence factors, including adhesins such as filamentous hemagglutinin (FHA) and fimbriae, as well as toxins such as the bifunctional adenylate cyclase/hemolysin (ACY). Previous studies showed that in the Bvg i phase, FHA and fimbriae continue to be expressed, but ACY expression is significantly downregulated. In this report, we determine that Bordetella bronchiseptica can form biofilms in vitro and that the generation of biofilm is maximal in the Bvg i phase. We show that FHA is required for maximal biofilm formation and that fimbriae may also contribute to this phenotype. However, expression of ACY inhibits biofilm formation, most likely via interactions with FHA. Therefore, the coordinated regulation of adhesins and ACY expression leads to maximal biofilm formation in the Bvg i phase in B. bronchiseptica .

Bordetella bronchiseptica establishes persistent infection of the murine respiratory tract. We hypothesize that long-term colonization is mediated in part by bacteria-driven modulation of dendritic cells (DCs) leading to altered adaptive immune responses. Bone marrow-derived DCs (BMDCs) from C57BL/6 mice infected with live B. bronchiseptica exhibited high surface expression of MHCII, CD86, and CD80. However, B. bronchiseptica-infected BMDCs did not exhibit significant increases in CD40 surface expression and IL-12 secretion compared with BMDCs treated with heat-killed B. bronchiseptica. The B. bronchiseptica type III secretion system (TTSS) mediated the increase in MHCII, CD86, and CD80 surface expression, while the inhibition of CD40 and IL-12 expression was mediated by adenylate cyclase toxin (ACT). IL-6 secretion was independent of the TTSS and ACT. These phenotypic changes may result from differential regulation of MAPK signaling in DCs. Wild-type B. bronchiseptica activated the ERK 1/2 signaling pathway in a TTSS-dependent manner. Additionally, ACT was found to inhibit p38 signaling. These data suggest that B. bronchiseptica drive DC into a semimature phenotype by altering MAPK signaling. These semimature DCs may induce tolerogenic immune responses that allow the persistent colonization of B. bronchiseptica in the host respiratory tract.

Summary The BvgAS virulence control system regulates the expression of type III secretion genes in Bordetella subspecies that infect humans and other mammals. We have identified five open reading frames, btrS, btrU, btrX, btrW and btrV, that are activated by BvgAS and encode regulatory factors that control type III secretion at the levels of transcription, protein expression and secretion. The btrS gene product bears sequence similarity to ECF (extracytoplasmic function) sigma factors and is required for transcription of the bsc locus. btrU, btrW and btrV encode proteins predicted to contain PP2C-like Ser phosphatase, HPK (His protein kinase)-like Ser kinase and STAS anti-sigma factor antagonist domains, respectively, which are characteristic of Gram-positive partner switching proteins in Bacillus subtilis. BtrU and BtrW are required for secretion of proteins that are exported by the bsc type III secretion system, whereas BtrV is specifically required for protein synthesis and/or stability. Bordetella species have thus evolved a unique cascade to differentially regulate type III secretion that combines a canonical phosphorelay system with an ECF sigma factor and a set of proteins with domain signatures that define partner switchers, which were traditionally thought to function only in Gram-positive bacteria. The presence of multiple layers and mechanisms of regulation most likely reflects the need to integrate multiple signals in controlling type III secretion. The bsc and btr loci are nearly identical between broad-host-range and human-specific Bordetella. Comparative analysis of Bordetella subspecies revealed that, whereas bsc and btr loci were transcribed in all subspecies, only broad-host-range strains expressed a functional type III secretion system in vitro. The block in type III secretion is post-transcriptional in human-adapted strains, and signal recognition appears to be a point of divergence between subspecies.

The genomes of three closely related bordetellae are currently being sequenced, thus providing an opportunity for comparative genomic approaches driven by an understanding of the comparative biology of these three bacteria. Although the other strains being sequenced are well studied, the strain ofBordetella parapertussischosen for sequencing is a recent human clinical isolate (strain 12822) that has yet to be characterized in detail. This investigation reports the first phenotypic characterization of this strain, which will likely become the prototype for this species in comparison with the prototype strains ofB. pertussis(Tohama I),B. bronchiseptica(RB50), and other isolates ofB. parapertussis. Multiple in vitro and in vivo assays distinguished each species.B. parapertussiswas more similar toB. bronchisepticathan toB. pertussisin many assays, including in BvgS signaling characteristics, presence of urease activity, regulation of urease expression by BvgAS, virulence in the respiratory tracts of immunocompromised mice, induction of anti-Bordetellaantibodies, and serum antimicrobial resistance. In other assays,B. parapertussiswas distinct from all other species (in pigment production) or more similar toB. pertussis(by lack of motility and cytotoxicity to a macrophage-like cell line). These results begin to provide phenotypes that can be related to genetic differences identified in the genomic sequences of bordetellae.

To understand the diversity in the cell culture harvest (i.e., feedstock) provided for downstream processing, we compared host cell protein (HCP) profiles using three Chinese Hamster Ovary (CHO) cell lines in null runs which did not generate any recombinant product. Despite differences in CHO lineage, upstream process, and culture performance, the cell lines yielded similar cell-specific productivities for immunogenic HCPs. To compare the dynamics of HCP production, we searched for correlations between the time-course profiles of HCP (as measured by multi-analyte ELISA) and those of two intracellular HCP species, phospholipase B-like 2 (PLBL2) and lactate dehydrogenase (LDH). Across the cell lines, proteins in the day 14 supernatants analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) showed different spot patterns. However, subsequent analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) indicated otherwise: the total number of peptides and proteins identified were comparable, and 80% of the top 1,000 proteins identified were common to all three lines. Finally, to assess the impact of culture viability on extracellular HCP profiles, we analyzed supernatants from a cell line whose viability dropped after day 10. The amounts of HCP and PLBL2 (quantified by their respective ELISAs) as well as the numbers and major populations of HCPs (identified by LC-MS/MS) were similar across days 10, 14, and 17, during which viabilities declined from ∼80% to20% and extracellular LDH levels increased several-fold. Our findings indicate that the CHO-derived HCPs in the feedstock for downstream processing may not be as diverse across cell lines and upstream processes, or change as dramatically upon viability decline as originally expected. In addition, our findings show that high density CHO cultures (10(7) cells/mL)-operated in fed-batch mode and exhibiting high viabilities (70%) throughout the culture duration-can accumulate a considerable amount of immunogenic HCP (∼1-2 g/L) in the extracellular environment at the time of harvest (day 14). This work also demonstrates the potential of using LC-MS/MS to overcome the limitations associated with ELISA and 2D-PAGE for HCP analysis.

The tsunami generated on 12 July 1993 by Hokkaido-Nansei-Oki earthquake (Mw = 7.8) has brought about the maximum wave run-up of 31.7 m, the highest record in Japan of 20th century, near the Monai Valley on the west coast of the Okushiri island (Hokkaido Tsunami Survey Group, 1993). To reproduce the extreme run-up height the three-dimensional non-hydrostatic model (Flow Science, 2012) denoted by NH-model has been locally applied with open boundary conditions supplied in an offline manner by the three-dimensional hydrostatic model (Ribeiro et al., 2011) denoted by H-model which is sufficiently large to cover the entire fault region with one-way nested multiple domains. For the initial water deformation Okada's fault model (1985) using the 3 sub-fault parameters is applied. Three non-hydrostatic model experiments have been performed, namely experiment without island, with one island and with two islands. The experiments with one island and with two islands give rise to values close to the observation with maximum run-up heights of about 32.3 and 30.8 m, respectively, while the experiment without islands gives rise to about 25.2 m. The diffraction of tsunami wave primarily by Muen Island located at the South and the southward topographic guiding of tsunami run-up at the coast are as in the laboratory simulation (Yoneyama et al., 2002) found to result in the extreme run-up height near the Monai Valley. The presence of Hira Island enhances the diffraction of tsunami waves but its contribution to the extreme run-up height is marginal.

Recent reports highlight the impact of copper on lactate metabolism: CHO cell cultures with higher initial copper levels shift to net lactate consumption and yield lower final lactate and higher titers. These studies investigated the effects of copper on metabolite and transcript profiles, but did not measure in detail the dependences of cell culture performance and product quality on copper concentrations. To more thoroughly map these dependences, we explored the effects of various copper treatments on four recombinant CHO cell lines. In the first cell line, when extracellular copper remained above the limit of detection (LOD), cultures shifted to net lactate consumption and yielded comparable performances irrespective of the differences in copper levels; when extracellular copper dropped below LOD (∼13 nM), cultures failed to shift to net lactate consumption, and yielded significantly lower product titers. Across the four cell lines, the ability to grow and consume lactate seemed to depend on the presence of a minimum level of copper, beyond which there were no further gains in culture performance. Although this minimum cellular copper requirement could not be directly quantified, we estimated its probable range for the first cell line by applying several assumptions. Even when different copper concentrations did not affect cell culture performance, they affected product quality profiles: higher initial copper concentrations increased the basic variants in the recombinant IgG1 products. Therefore, in optimizing chemically defined media, it is important to select a copper concentration that is adequate and achieves desired product quality attributes.

Adherence to ciliated respiratory epithelial cells is considered a critical early step in Bordetella pathogenesis. For Bordetella pertussis , the etiologic agent of whooping cough, several factors have been shown to mediate adherence to cells and cell lines in vitro. These putative adhesins include filamentous hemagglutinin (FHA), fimbriae, pertactin, and pertussis toxin. Determining the precise roles of each of these factors in vivo, however, has been difficult, due in part to the lack of natural-host animal models for use with B. pertussis . Using the closely related species Bordetella bronchiseptica , and by constructing both deletion mutation and ectopic expression mutants, we have shown that FHA is both necessary and sufficient for mediating adherence to a rat lung epithelial (L2) cell line. Using a rat model of respiratory infection, we have shown that FHA is absolutely required, but not sufficient, for tracheal colonization in healthy, unanesthetized animals. FHA was not required for initial tracheal colonization in anesthetized animals, however, suggesting that its role in establishment may be dedicated to overcoming the clearance action of the mucociliary escalator.

The DNA fingerprints of 170 human isolates and ten ovine isolates of Bordetella parapertussis were examined by arbitrarily-primed PCR/RAPD with 29 primers. Based on this technique, all the human isolates appear highly genetically homogeneous. The ovine isolates could be distinguished from human isolates and they showed diversity among themselves. Therefore, human isolates of B. parapertussis are a highly clonal group adapted to infect humans and they are distinct from polymorphic ovine isolates.

THIOMABs are recombinant antibodies with reactive cysteine residues used for forming THIOMAB-drug conjugates (TDCs). We recently reported a new impurity associated with THIOMABs: one of the engineered cysteines forms a disulfide bond with an extra light chain (LC) to generate a triple light chain antibody (3LC). In our previous investigations, increased LC expression increased 3LC levels, whereas increased glutathione (GSH) production decreased 3LC levels. In this work, on three stably transfected CHO cell lines, we investigated the effects of temperature, pH, dissolved oxygen (DO), and hydrolysate on 3LC formation during THIOMAB fed-batch cell culture production. Although pH between 6.8 and 7.0 had no significant impact on 3LC formation, temperature at 35°C instead of 33 or 31°C generated the lowest 3LC values for two cell lines. The decreased 3LC level correlated with increased GSH production. We implemented a 35°C temperature process for large-scale (2,000 L) production of a THIOMAB. This process reduced 3LC levels by ∼50% compared with a 33°C temperature process. By contrast, DO and hydrolysate had modest effect on 3LC levels for the model cell line studied. Overall, we did not find significant changes in LC expression under the conditions tested, whereas changes in GSH production were more evident. By investigating the impact of bioreactor process and medium conditions on 3LC levels, we identified strategies that reduced 3LC levels.

Pharmacokinetic parameters of third generation cephalosporins vary widely, requiring different dosage regimens and adjustment methods for each agent. Although their antibacterial spectrum favours their usage in infections caused by aerobic Gram-negative organisms, due to their limited post-antibiotic effect against these organisms, dosage regimens should ensure that free drug concentrations at the site of infection remain above the minimum inhibitory concentration for as much of the dosage interval as possible in patients with normal host defence mechanisms and for the entire dosage interval in immunocompromised patients. Altered protein binding encountered in various disease states can affect both microbiological and pharmacokinetic properties especially for drugs with high protein binding. Since the concentrations at the site of action are often different from those in serum, a higher or lower range of dosages needs to be selected depending on the target site. Decreased renal function affects the elimination of most third generation cephalosporins, whereas the presence of hepatic disease does not generally necessitate dosage adjustment. Because of the complex age-related physiological changes in paediatric and elderly patients, dosage should be adjusted on the basis of the reported pharmacokinetic data in these populations. The usual recommended dose may or may not be optimal in a given condition depending on the complex interactions between pharmacokinetic, microbiological and other host factors.

THIOMABs are recombinant antibodies engineered with reactive cysteines, which can be covalently conjugated to drugs of interest to generate targeted therapeutics. During the analysis of THIOMABs secreted by stably transfected Chinese Hamster Ovary (CHO) cells, we discovered the existence of a new species--Triple Light Chain Antibody (3LC). This 3LC species is the product of a disulfide bond formed between an extra light chain and one of the engineered cysteines on the THIOMAB. We characterized the 3LC by size exclusion chromatography, mass spectrometry, and microchip electrophoresis. We also investigated the potential causes of 3LC formation during cell culture, focusing on the effects of free light chain (LC) polypeptide concentration, THIOMAB amino acid sequence, and glutathione (GSH) production. In studies covering 12 THIOMABs produced by 66 stable cell lines, increased free LC polypeptide expression--evaluated as the ratio of mRNA encoding for LC to the mRNA encoding for heavy chain (HC)--correlated with increased 3LC levels. The amino acid sequence of the THIOMAB molecule also impacted its susceptibility to 3LC formation: hydrophilic LC polypeptides showed elevated 3LC levels. Finally, increased GSH production--evaluated as the ratio of the cell-specific production rate of GSH (q(GSH)) to the cell-specific production rate of THIOMAB (q(p))--corresponded to decreased 3LC levels. In time-lapse studies, changes in extracellular 3LC levels during cell culture corresponded to changes in mRNA LC/HC ratio and q(GSH)/q(p) ratio. In summary, we found that cell lines with low mRNA LC/HC ratio and high q(GSH)/q(p) ratio yielded the lowest levels of 3LC. These findings provide us with factors to consider in selecting a cell line to produce THIOMABs with minimal levels of the 3LC impurity.

The pharmacokinetics of prophylactic antibodies may differ in cardiac and aortic aneurysm surgery for at least two reasons: aortic aneurysm surgery generally entails a greater blood volume loss and replacement, and aortic aneurysm surgery usually does not require extracorporeal cardiopulmonary bypass. We prospectively studied two different cefamandole dosing regimens in patients undergoing aortic aneurysm surgery (phase 1, 1 gm intravenously at the induction of anesthesia; phase 2, 2 gm intravenously at the induction of anesthesia followed by 1 gm intravenously every 2 hours during surgery). In phase 1 and 2 plasma levels were measured at the time of skin incision, aortic cross-clamping, aortic unclamping, and skin closure. In phase 2 cefamandole elimination in urine and cell-saver effluent was also determined. An adequate plasma level of 10 μg/ml was maintained in only 4 of 14 patients in phase 1, but in 10 of 10 patients in phase 2. Cefamandole loss in cell-saver effluent was 136 ± 100 mg, which was 13% of the measured renally excreted amount. As has been previously shown in cardiac surgery, a cefamandole prophylactic antibiotic regimen of 2 gm intravenously at the induction of anesthesia followed by 1 gm every 2 hours during surgery provides a dependable and practical dosing regimen in patients undergoing aortic aneurysm surgery.

The absorption of ciprofloxacin was higher when administered through a nasoduodenal tube than through a nasogastric tube, indicating that even though acidic gastric pH is needed for rapid disintegration, dissolved ciprofloxacin might not be stable in the gastric environment. If the length of exposure or the different gastric environment in each individual affects the overall absorption of ciprofloxacin, this could explain the reported variability in ciprofloxacin absorption and suggests the need for the development of an enteric-coated tablet. Further studies are needed to fully characterize the absorption of ciprofloxacin in patients with different illnesses, gastric transit times, gastrointestinal environments and of different ages.

Bordetella bronchiseptica chronically infects a wide range of mammals, and resides primarily in the nasal cavity of the infected host. Multiple virulence factors of Bordetella species have been studied in the context of lower respiratory tract infections, but relatively less is known about the bacterial life cycle in the nasal cavity. Evidences were discovered for Bvg intermediate (Bvg(i)) phase expression in vivo and that the major adhesin filamentous hemagglutinin plays a major role in the colonization of B. bronchiseptica in the unciliated olfactory epithelia of the nasal cavity.

Chronic bacterial infection reflects a balance between the host immune response and bacterial factors that promote colonization and immune evasion. Bordetella bronchiseptica uses a type III secretion system (TTSS) to persist in the lower respiratory tract of mice. We hypothesize that colonization is facilitated by bacteria-driven modulation of dendritic cells (DCs), which leads to an immunosuppressive adaptive host response. Migration of DCs to the draining lymph nodes of the respiratory tract was significantly increased in mice infected with wild-type B. bronchiseptica compared with mice infected with TTSS mutant bacteria. Reduced colonization by TTSS-deficient bacteria was evident by 7 days after infection, whereas colonization by wild-type bacteria remained high. This decrease in colonization correlated with peak IFN-γ production by restimulated splenocytes from infected animals. Wild-type bacteria also elicited peak IFN-γ production on day 7, but the quantity was significantly lower than that elicited by TTSS mutant bacteria. Additionally, wild-type bacteria elicited higher levels of the immunosuppressive cytokine IL-10 compared with the TTSS mutant bacteria. B. bronchiseptica colonization in IL-10−/− mice was significantly reduced compared with infections in wild-type mice. These findings suggest that B. bronchiseptica use the TTSS to rapidly drive respiratory DCs to secondary lymphoid tissues where these APCs stimulate an immunosuppressive response characterized by increased IL-10 and decreased IFN-γ production that favors bacterial persistence.

Bordetella bronchiseptica utilizes a type III secretion system (TTSS) for induction of non-apoptotic cytotoxicity in host cells and modulation of host immunity. The identity of Bordetella TTSS effectors, however, has remained elusive. Here we report a genome-wide screen for TTSS effectors based on shared biophysical and functional characteristics of class I chaperones and their frequent colocalization with TTSS effectors. When applied to B. bronchiseptica, the screen identified the first TTSS chaperone-effector locus, btcA-bteA, and we experimentally confirmed its function. Expression of bteA is co-ordinated with expression of TTSS apparatus genes, BteA is secreted through the TTSS of B. bronchiseptica, it is required for cytotoxicity towards mammalian cells, and it is highly conserved in the human-adapted subspecies B. pertussis and B. parapertussis. Transfection of bteA into epithlieal cells results in rapid cell death, indicating that BteA alone is sufficient to induce potent cytotoxicity. Finally, an in vitro interaction between BteA and BtcA was demonstrated. The search for TTSS chaperones and effectors was then expanded to other bacterial genomes, including mammalian and insect pathogens, where we identified a large number of novel candidate chaperones and effectors. Although the majority of putative effectors are proteins of unknown function, several have similarities to eukaryotic protein domains or previously identified effectors from other species.