Development and validation of an ELISA for quantitation of bovine viral diarrhea virus antigen in the critical stages of vaccine production.

Bovine Viral Diarrhea Virus (BVDV) is the causative agent of a worldwide disease. The virus infects bovines of all ages, causing reproductive problems and contaminating biological products of high commercial value. The large-scale production of BVDV vaccines presents the challenge of processing antigenic proteins that are highly susceptible to the processing environment. Potency testing requires the immunization of cattle in order to determine the neutralizing antibodies titers induced by the vaccine. An alternative to the in vivo test is an in vitro measurement of key viral antigens. This paper describes the development and validation of a sandwich-type indirect ELISA that is able to detect and quantify BVDV E2 glycoprotein in live and inactivated BVDV. Validation parameters such as repeatability, intermediate precision, and reproducibility indicated that the developed ELISA constitutes an advanced tool for evaluating the BVDV antigen throughout manufacturing and vaccine release testing.

The effects of obesity and standing time on postural sway during prolonged quiet standing.

This study examined the effects of obesity level, standing time and their interaction on postural sway during a prolonged quiet upright standing task. Ten extremely obese (BMI > 40 kg/m(2)) and 10 non-obese (18.5 kg/m(2) < BMI < 24.9 kg/m(2)) participants performed quiet upright standing on a force plate for over 18 min. Eleven postural sway measures were computed for each 1-min time interval based on the centre-of-pressure data from the force plate. ANOVA and regression analyses showed that for all the 11 postural sway measures, the extremely obese group had higher postural sway than the non-obese at the beginning of the prolonged standing task and postural sway increased significantly faster for the extremely obese group than the non-obese over time. The results suggest that obesity may impair postural control and may be a risk factor of balance loss and falls, especially during prolonged physical work activities. The research findings are relevant to identifying and reducing risks of balance loss and falls in various workplace settings for a wide variety of workers.

Scale-up of Escherichia coli growth and recombinant protein expression conditions from microwell to laboratory and pilot scale based on matched k(L)a.

Fermentation optimization experiments are ideally performed at small scale to reduce time, cost and resource requirements. Currently microwell plates (MWPs) are under investigation for this purpose as the format is ideally suited to automated high-throughput experimentation. In order to translate an optimized small-scale fermentation process to laboratory and pilot scale stirred-tank reactors (STRs) it is necessary to characterize key engineering parameters at both scales given the differences in geometry and the mechanisms of aeration and agitation. In this study oxygen mass transfer coefficients are determined in three MWP formats and in 7.5 L and 75 L STRs. k(L)a values were determined in cell-free media using the dynamic gassing-out technique over a range of agitation conditions. Previously optimized culture conditions at the MWP scale were then scaled up to the larger STR scales on the basis of matched k(L)a values. The accurate reproduction of MWP (3 mL) E. coli BL21 (DE3) culture kinetics at the two larger scales was shown in terms of cell growth, protein expression, and substrate utilization for k(L)a values that provided effective mixing and gas-liquid distribution at each scale. This work suggests that k(L)a provides a useful initial scale-up criterion for MWP culture conditions which enabled a 15,000-fold scale translation in this particular case. This work complements our earlier studies on the application of DoE techniques to MWP fermentation optimization and in so doing provides a generic framework for the generation of large quantities of soluble protein in a rapid and cost-effective manner.

Framework for the rapid optimization of soluble protein expression in Escherichia coli combining microscale experiments and statistical experimental design.

A major bottleneck in drug discovery is the production of soluble human recombinant protein in sufficient quantities for analysis. This problem is compounded by the complex relationship between protein yield and the large number of variables which affect it. Here, we describe a generic framework for the rapid identification and optimization of factors affecting soluble protein yield in microwell plate fermentations as a prelude to the predictive and reliable scaleup of optimized culture conditions. Recombinant expression of firefly luciferase in Escherichia coli was used as a model system. Two rounds of statistical design of experiments (DoE) were employed to first screen (D-optimal design) and then optimize (central composite face design) the yield of soluble protein. Biological variables from the initial screening experiments included medium type and growth and induction conditions. To provide insight into the impact of the engineering environment on cell growth and expression, plate geometry, shaking speed, and liquid fill volume were included as factors since these strongly influence oxygen transfer into the wells. Compared to standard reference conditions, both the screening and optimization designs gave up to 3-fold increases in the soluble protein yield, i.e., a 9-fold increase overall. In general the highest protein yields were obtained when cells were induced at a relatively low biomass concentration and then allowed to grow slowly up to a high final biomass concentration, >8 g.L-1. Consideration and analysis of the model results showed 6 of the original 10 variables to be important at the screening stage and 3 after optimization. The latter included the microwell plate shaking speeds pre- and postinduction, indicating the importance of oxygen transfer into the microwells and identifying this as a critical parameter for subsequent scale translation studies. The optimization process, also known as response surface methodology (RSM), predicted there to be a distinct optimum set of conditions for protein expression which could be verified experimentally. This work provides a generic approach to protein expression optimization in which both biological and engineering variables are investigated from the initial screening stage. The application of DoE reduces the total number of experiments needed to be performed, while experimentation at the microwell scale increases experimental throughput and reduces cost.

Genetic selection for resistance or susceptibility to oral tolerance imparts correlation to both Immunoglobulin E level and mast cell number phenotypes with a profound impact on the atopic potential of the individual.

BACKGROUND: Immunological oral tolerance is being studied with great interest due to its therapeutic potential in allergy and autoimmunity processes, although the cellular and molecular mechanisms linking these different phenomena remain elusive. In the present study, two mouse lines with extreme phenotypes for susceptibility [TS Line] or resistance [TR Line] to oral tolerance and their [TS x TR]F2 segregants were used in order to evaluate the impact of these traits on the atopic potential of the individuals. OBJECTIVE: Demonstrate whether the tr and ts genes, cumulated during 18 generations of bidirectional genetic selection, influence expression of two important immunobiological traits (IgE and mast cell) critical to allergic response. METHODS: Mice with extreme phenotypes for oral tolerance to ovalbumin (OVA), produced by assortative mating (TS and TR Line), and their (TS x TR)F2 segregating were used. Serum IgE levels assayed by ELISA, and mastocytes counted with toluidine blue staining were evaluated in naïve mice. Anaphylaxis was induced by intravenous injection of OVA, intestinal inflammation by oral administration of OVA 7 days after immunization, and pulmonary inflammation by intranasal and nebulization OVA challenges. Specific IgE was dosed by passive cutaneous anaphylaxis. RESULTS: The naïve TS mice have a 20-fold lower serum IgE level and two- to threefold diminished mast cell numbers in mucosal sites, when compared with TR-mice, which were highly susceptible to allergic inflammation and anaphylactic shock. The associations of oral tolerance, serum IgE levels and mast cell numbers in naïve animals were confirmed analysing the simultaneous presence of these traits in individuals of a [TS x TR]F2 -segregating population. CONCLUSION: The results suggest that the complex of genes controlling TS and TR phenotypes play a main role in the regulation of the atopic potential of the individual. The studies of these traits in interline F2 segregants demonstrated a co-segregation of TS and TR phenotypes with IgE responsiveness and mast cell numbers. Thus, the opposite capacity of the genetically modified mice may be involved in co-adaptative mechanisms reflecting a dynamic relation between gene frequencies in a natural population. These correlations give circumstantial evidence to support clinical applications of oral tolerance in allergic and autoimmune diseases.

Subject variation in caprine anterior cruciate ligament reconstruction.

We studied the subject and treatment contributions to anterior cruciate ligament (ACL) reconstruction biomechanics by reexaming the results of two bilateral reconstruction studies. Bilateral reconstruction allows a comparison between treatments exposed to the same subject related healing factors. The studies examined the effects of gamma irradiation and the effects of initial graft size and initial graft laxity. In both studies different treatments were applied to contralateral limbs. We found that the subject was the best predictor of outcome, while the surgical treatments had little influence on outcome. There was a large variation between subjects despite similar treatments, and little difference between contralateral limbs despite different surgical treatments. At 26 weeks, the graft cross sectional area and modulus were most strongly influenced (p < 0.002) by the subject. We interpret this as a subject related factor is regulating the quantity and quality of the healing tissue. Potential sources of subject related factors include the subject's pre-operative condition, the activity during the post-operative period, and an intrinsic biologic response. By better understanding the source of subject variation, more successful and consistent ACL reconstructions might be achieved.

Purification of plasmid DNA by an integrated operation comprising tangential flow filtration and nitrocellulose adsorption.

There is an increasing interest in the development of scaleable and reproducible plasmid DNA purification protocols for vaccine and gene therapy. The use of an integrated unit operation, comprising tangential flow microfiltration coupled with the adsorption of contaminants onto nitrocellulose membranes as a single processing step was examined in this work. Experiments were performed using a custom-built tangential flow microfiltration rig (membrane area=12.5 cm(2)). Tangential flow filtration-adsorption of E. coli lysates containing a plasmid product removed most solids (>75%) and decreased chromosomal DNA contamination by 75% w/w. Total plasmid DNA concentration and supercoiled content of the permeate were virtually identical to those of the feed, indicating a recovery yield of 100% (transmission equal to 1). Results were similar for E. coli lysates containing either a 6.9 kb or a 20 kb plasmid. Significant reductions in RNA, endotoxin, and protein levels were also observed. The reproducibility and potential for scale up of this integrated filtration-adsorption operation makes it at attractive option for intermediate- to large-scale pharmaceutical-grade plasmid processing.

The effects of graft width and graft laxity on the outcome of caprine anterior cruciate ligament reconstruction.

We studied how initial graft size and initial graft laxity affected the biomechanics of anterior cruciate ligament (ACL) reconstruction at six months. Sixteen goats had bilateral reconstructions staged eight weeks apart. Autografts 4 and 7 mm wide were taken from the central patellar tendon (PT). Lax grafts were created by adding 4 mm slack to the graft before fixing. We reconstructed each joint using a combination of width and laxity treatments. Both factors were changed for the contralateral joint and all combinations appeared with equal frequency. At six months we measured the joint extension limit, anterior-posterior (AP) translation, and osteoarthritic changes. The grafts were then tested to failure to determine their mechanical properties. After six months the difference in initial treatments had disappeared: there was no difference in graft cross-section due to the different initial widths and there was no difference in joint AP translation due to the initial graft laxity. We did observe that wide grafts were associated with a block to extension, decreased joint AP translation, and increased articular cartilage damage and osteophyte formation. While AP translation was reduced, it was correlated with decreased extension, possibly indicating an increase in scar tissue formation rather than a more functional graft. Neither graft width nor graft laxity produced differences in any graft mechanical properties. This suggests that the use of larger grafts to prevent increased AP translation has undesirable complications. Ultimately, we conclude that neither of these surgical treatments strongly affects the biomechanical result of caprine ACL reconstruction.

Staged reduction of gastroschisis: a simple method.

Staged reduction of abdominal contents using a silastic sheet has become standard management in gastroschisis where primary closure is not possible. With the introduction of a pre-made Silastic silo coupled to a spring-loaded ring (Ben Tec, Sacramento, CA), the procedure can be done at the bedside. We present a simple technique utilizing a disposable umbilical-cord clamp that makes reduction a fast, one-physician procedure and present a preoperative step that facilitates tension-free closure of the abdominal fascia.

The formation of plasmid DNA loaded pharmaceutical powders using supercritical fluid technology.

The invention of novel drugs based on biological macromolecules requires the development of specialized formulation methods. Supercritical fluid technology offers the possibility to produce dry powder formulations suitable for inhalation or needle-free injection. In this article we describe the first application of a process involving supercritical carbon dioxide for the production of plasmid DNA-loaded particles. The technique of solution enhanced dispersion by supercritical fluids (SEDS) is used to coformulate the 6.9 kb plasmid pSV beta with mannitol as excipient. After initial experiments showed a high degradation of the plasmid during powder formation, a systematic investigation of the process revealed pH effects to be crucial for the recovery of intact DNA. The application of high-buffer concentration led to an increase of the recovered supercoiled proportion from 7% to 80%.

Two-bundle posterior cruciate ligament reconstruction. An in vitro analysis of graft placement and tension.

This study had two purposes: first, to determine how femoral attachment location affects the load sharing between the two bundles of a Y-type posterior cruciate ligament reconstruction, and second, to determine how the bundles, separately and in combination, control posterior tibial translation throughout the full range of knee flexion. One and two-bundle reconstructions were performed in 12 cadaveric knees. The one-bundle reconstructions were attached within the femoral posterior cruciate ligament footprint at one of three locations, high and shallow (S1), mid and shallow (S2), or mid and deep (D). The two-bundle reconstructions comprised an S1 bundle with either an S2 or a D bundle. Posterior translation and bundle tension were measured as the knee was flexed from full extension to 1,200 of flexion while a posterior force of either 50 or 100 N was applied to the proximal tibia. The shallow one-bundle reconstruction restored posterior translation to within 2 mm of that of the intact knee over the entire range of knee flexion. The deep reconstruction did not control abnormal posterior translation above 45 degrees. The tension in the shallow bundles increased with knee flexion, and the deep bundle tension remained nearly constant throughout knee flexion. Both two-bundle reconstructions controlled posterior translation, but with different load-sharing characteristics. The S1-S2 configuration resisted posterior tibial translation as both bundles became taut in flexion. In contrast, the S1-D configuration resisted posterior translation in a reciprocal fashion with the D bundle tension being the greatest in extension and the S1 bundle tension being the greatest tension in flexion.

Biochemical engineering approaches to the challenges of producing pure plasmid DNA.

Plasmid-based genes offer promise for a new generation of vaccines and for gene therapy, but the size and character of plasmids pose new challenges to biochemical engineers. By acknowledging these and using bioprocess-design information based on fundamental studies of the system's properties, it will be possible to create efficient and consistent processes for these materials. This review addresses the purity required, the key issue of the sensitivity of the chromosomal DNA contaminant and larger plasmids to hydrodynamic forces, and the impact of this and other characteristics of plasmids on the recovery and purification of DNA for pharmaceutical purposes.

Quantitation of supercoiled circular content in plasmid DNA solutions using a fluorescence-based method.

A method for quantifying the proportion of supercoiled circular (SC) forms in DNA solutions is described. The method (SCFluo) takes advantage of the reversible denaturation property of SC forms and the high specificity of the PicoGreen fluorochrome for double-stranded (ds)DNA. Fluorescence values of forms capable of reversible denaturation after a 5 min heating, 2 min cooling step are normalised to fluorescence values of total dsDNA present in the preparation. For samples with a SC content >20-30%, good regression fits were obtained when values derived from densitometric scanning of an agarose gel and those derived from the SCFluo method were compared. The method represents an attractive alternative to currently established methods because it is simple, rapid and quantitative. During large-scale processing and long-term storage, enzymatic, chemical and shear degradation may substantially decrease the SC content of plasmid DNA preparations. Regulations for pharmaceutical grade products for use in gene therapy and DNA vaccination may require >90% of the plasmid to be in the SC form. In the present study the SC content of 6.9, 13 and 20 kb plasmid preparations that had been subjected to chemical and shear degradation was successfully quantified using the new method.

A conceptualization of the repetition compulsion.

The concept of the repetition compulsion remains an enigma. Its etiology is not fully understood and the purpose it serves continues to be a mystery. Although it is often theorized that the compulsion to repeat may function to facilitate mastery of a past trauma, mastery is rarely achieved. In this article the concept of the repetition compulsion is reviewed and the unanswered questions that continue to exist about this phenomenon are summarized. A way to conceptualize the compulsion to repeat is then offered. The compulsion to repeat as it specifically relates to the attempt to master a previous trauma is reviewed, followed by a examination of the relationship between the compulsion to repeat and reenactments. Finally, how the compulsion to repeat can be viewed as a posttraumatic stress response and the implications of understanding it in this fashion is then examined.

Removal of contaminant nucleic acids by nitrocellulose filtration during pharmaceutical-grade plasmid DNA processing.

Pharmaceutical-grade plasmid DNA for use in vaccines and gene therapy requires the development of reproducible and scaleable downstream processes. Shearing of chromosomal DNA at the commencement of the purification results in fragments that are difficult to separate from supercoiled plasmid DNA. Regulatory standards will probably require that the level of chromosomal DNA contamination is kept below 0.01 mg mg(-1) plasmid DNA. This work reports the use of nitrocellulose membranes to decrease chromosomal DNA contamination in plasmid DNA preparations derived from a 450-l bioreactor. Clarified lysates, resuspended PEG precipitates and anion exchange chromatography elutes were filtered through nitrocellulose. In all the cases, chromosomal DNA was selectively retained by the membrane while most supercoiled plasmid DNA was recovered in the filtrate. Contamination levels dropped from over 27% to below 1% as measured by Southern analysis. Under ionic strength conditions equal to or above 1.5 M NaCl, a fraction of the contaminant RNA was also retained by the nitrocellulose membrane.

Rapid quantitation and monitoring of plasmid DNA using an ultrasensitive DNA-binding dye.

A sensitive fluorescence-based method for monitoring plasmid DNA during production was investigated. This simple method of assaying for plasmid DNA allows rapid monitoring of plasmid yields from a recombinant Escherichia coli fed-batch fermentation. The assay has several advantages over traditional methods of plasmid DNA measurement. The fluorescent dye is highly specific and can measure total plasmid DNA concentration in about 5 min. The assay is sensitive over a wide range of plasmid concentrations of between 15 and 280 ng/mL, even in the presence of impurities that occur within alkaline lysate preparations. The technique can also be applied to monitoring fermentation and downstream purification steps.

Evolution of the modified Rossetti fundoplication in children: surgical technique and results.

OBJECTIVE: To compare the modified Rossetti fundoplication with the classic Nissen. SUMMARY BACKGROUND DATA: The traditional surgical treatment of gastroesophageal reflux in children has been the classic Nissen fundoplication, defined by liver mobilization, crural repair, takedown of short gastric vessels, and floppy wrap. The authors have progressed in our technique of fundoplication and now perform a modified Rossetti fundoplication, defined by liver retraction without mobilization, no crural repair, short gastric vessels left intact, and 2-cm floppy wrap. METHODS: A retrospective chart review was performed on 407 pediatric patients who had open fundoplications (Jan. 13, 1993, to Feb. 25, 1998). Two groups were analyzed: the Nissen group (171 patients) and the Rossetti group (236 patients). Groups were compared for incidence of recurrent reflux, dysphagia, hiatal hernia, need for esophageal dilation, revision of fundoplication, time to discharge, and operative time. RESULTS: Incidence of dysphagia (3.7% vs. 3.3%), postoperative hiatal hernia (1.9% vs. 1.4%), need for esophageal dilation (1.2% vs. 0.5%), and need for fundoplication revision (2.5% vs. 2.3%) were similar between the groups. The mean operative time was significantly decreased in the Rossetti group (65 +/- 25 minutes) versus the Nissen group (73 +/- 33 minutes). Recurrent reflux occurred significantly more often in the Nissen group (11.2%) than in the Rossetti group (5.1 %). CONCLUSION: The modified Rossetti fundoplication has a low complication rate and is the authors' preferred method for the surgical treatment of gastroesophageal reflux in children.

Once daily, high dose versus divided, low dose gentamicin for open fractures.

A prospective, randomized study was performed on 75 Gustilo Grades II and III open fractures to determine the efficacy of once daily, high dose aminoglycoside therapy, compared with more conventional dosing, in reducing the infection rate when used in conjunction with an aggressive operative treatment protocol. All patients enrolled in the study were treated with immediate irrigation, debridement, operative stabilization of the fracture, and 1 g of cefazolin every 8 hours. At the time of admission patients were randomized to two groups. Patients in Group I received gentamicin 5 mg/kg divided into twice daily doses, and patients in Group II received gentamicin 6 mg/kg given once daily. All patients were monitored for renal toxicity and observed for radiographic and clinical signs of infection until fracture union. The results of the study revealed no statistically significant difference between once daily, high dose versus divided, low dose gentamicin in infection rates. Thus, daily dosing of gentamicin was found to be safe, effective, and cost efficient in the treatment of open fractures when combined with a cephalosporin and aggressive operative debridement and stabilization.