Selective, efficient modulation of activated CD4+ αßT cells by the novel humanized antibody GZ-αßTCR targeting human αßTCR.

Allograft rejection and immunosuppression are two major issues in transplantation medicine. The specific targeting of alloreactive T cells, the initiators and promoters of allograft rejection, would be a promising strategy to reduce unwanted T-cell responses and side effects of lifelong immunosuppression. The novel humanized monoclonal antibody GZ-αßTCR, specific for the human αßT-cell receptor, was tested in vitro and in vivo for its specificity and efficacy to modulate the αßT-cell compartment. GZ-αßTCR moderately induced apoptosis in resting αßT cells in vitro, an effect considerably amplified in activated T cells. A single dose of GZ-αßTCR significantly reduced human CD45(+)CD3(+) T cells in vivo, with a preferential modulation of CD4(+) αßT cells. Importantly, naive T cells, the T-cell subset from which alloreactivity emanates, were significantly reduced. Simultaneously, a significant, compensatory increase of γδ T cells was observed in vitro and in vivo in both humanized mouse models examined. GZ-αßTCR did not induce cytokines and was well tolerated. Thus, specificity and high efficacy make GZ-αßTCR a powerful tool to selectively eliminate putatively detrimental T-cell subsets, a major goal in transplantation medicine. At the same time, GZ-αßTCR spares γδ and natural killer cells, thus leaving the recipient's immune system competent for cell-mediated immunoregulation and cell-mediated immunity.

Effects of diet type and enzyme addition on growth performance and gut health of broiler chickens during subclinical Clostridium perfringens challenge.

The effects of diet type (corn- vs. wheat-based) and multicarbohydrase addition on growth performance, digesta pH and viscosity, intestinal populations of Clostridium perfringens and lactic acid bacteria, and gut lesion score (from 0 to 4, where 0 = no gross lesions, 4 = severe extensive necrosis) of broiler chickens during oral challenge with C. perfringens (none or 10(8) cfu/bird on d 13) were studied in a 39-d experiment. A total of 1,216 male Ross-308 chickens was assigned to 8 dietary treatments in a randomized complete block design providing 8 replicate pens per treatment. Diets were formulated to meet the NRC protein requirement but were suboptimal in energy level. When compared with birds fed corn-based diets, chickens fed wheat-based diets had inferior (P < 0.01) final BW (2.49 vs. 2.59 kg) and feed conversion ratio (FCR; 1.83 vs. 1.78). Pathogen challenge significantly (P < 0.05) impaired growth performance and increased C. perfringens numbers and average lesion score. Increased (P < 0.01) C. perfringens counts (2.4 vs. 1.5 log(10) cfu/g of digesta) and intestinal lesion score (0.9 vs. 0.4) were observed for challenged birds fed wheat-based diets. No difference in digesta pH and lactic acid bacteria numbers were found among the treatments. Enzyme addition to both the corn- and wheat-based diets increased bird final BW (2.57 vs. 2.51 kg; P < 0.01), decreased overall FCR (1.78 vs. 1.83; P < 0.01), and, in those consuming wheat-based diets, reduced digesta viscosity (from 4.1 to 2.7 mPa.s; P < 0.01). Enzyme supplementation assisted the challenged birds in maintaining their optimal growth performance by improving (P < 0.05) average daily gain (59.5 vs. 56.9 g) in those consuming corn-based diets and FCR (1.83 vs. 1.90) in those consuming wheat-based diets to values similar to those observed in control birds (59.7 g/d and 1.84, respectively). In conclusion, enzyme addition improved growth performance and mitigated the negative effects of C. perfringens challenge.

Phytosterol effects on milk and yogurt microflora.

Phytosterols play a major role in functional foods. Their ability to reduce serum cholesterol in humans has been widely proven and they are now being added to various dairy based products. The present study investigated the potential antimicrobial activity of phytosterols in milk and their effect on yogurt starter cultures. A commercial phytosterol (0.26% to 1.8%, w/v) preparation (CPP) consisting of ss-sitosterol, campesterol, sitostanol, and campestanol had no effect on the standard plate count (SPC) and psychrotroph population in pasteurized milk stored at 4 degrees C. In addition, a challenge study employing Pseudomonas spp. in milk at 4 to 7 degrees C confirmed that the CPP was not antimicrobial. However, the addition of a dispersible CPP consisting of 0.72% phytosterol containing 0.02% to 0.03% sodium stearoyl lactylate (SSL) did appear to affect the SPC and psychrotrophic bacteria in refrigerated milk. The dispersible preparations did not, however, inhibit the growth of Pseudomonas. An investigation into the antimicrobial activity of SSL revealed that it alone had no effect on the SPC in milk. The CPP had no effect on growth and acid development by Lactobacillus bulgaricus and Streptococcus thermophilus during yogurt production at 33 degrees C and storage at 4 degrees C for 30 d. This is seen as a beneficial feature since growth and acid development by these organisms are crucial for yogurt quality. Saccaromyces cerevisiae and Aspergillus ochraceous added to yogurt as typical contaminants also were not inhibited. While the CCP was somewhat antimicrobial when formulated with dispersing agents, it otherwise had no antimicrobial activity.

Effect of low-protein amino acid-supplemented diets on the growth performance, gut morphology, organ weights and digesta characteristics of weaned pigs.

A 21-day study was conducted to determine whether isoleucine might limit the performance of piglets fed low-crude protein (CP), amino acid (AA)-supplemented diets and to investigate the potential benefits of low-CP diets on gastrointestinal health in weaned pigs. Ninety-six piglets (initial BW = 6.44 ± 0.14 kg), housed four per pen, were randomly assigned to one of four diets, resulting in six replicate pens per diet. Dietary treatments were as follows: (1) 210 g/kg CP diet, (2) 190 g/kg CP diet deficient in isoleucine, (3) 190 g/kg CP diet supplemented with crystalline isoleucine up to the level in the 210 g/kg CP diet and (4) 170 g/kg CP diet supplemented with isoleucine and valine on the ideal protein ratio basis (60% and 70% relative to lysine, respectively). Pigs were allowed to adapt to the new environment for 4 days before the experiment commenced. Overall, pigs fed the 210 g/kg CP diet had higher (P < 0.05) average daily gain and lower (P < 0.05) feed : gain ratio compared with those fed the other diets. The faecal consistency score of pigs fed the 210 g/kg CP diet was higher (P < 0.05) than those fed the other diets. Pigs fed the 170 g/kg diet had lower (P=0.02) small intestine weight than those fed the 210 g/kg CP diet. Pigs fed the 210 g/kg CP diet had deeper (P < 0.05) crypt in the duodenum and ileum and higher (P < 0.05) ammonia N concentration in caecal digesta than those fed the other diets. There were no effects of diet on microbial population and volatile fatty acid concentration in the caecal digesta except for propionic acid whose concentration was higher (P < 0.05) for pigs fed the 170 g/kg diet than those fed the 190+isoleucine and the 210 g/kg CP diets. The results indicate that the low-CP, AA-supplemented diet reduced crypt hypertrophy, ammonia N concentration in the caecal digesta, small intestine weight and the performance of piglets. Also, the results of the current study were inconclusive with respect to whether isoleucine may limit the performance of pigs fed a low-CP, AA-supplemented diet.

Effects of superheated steam on Geobacillus stearothermophilus spore viability.

AIMS: To examine the effect of processing with superheated steam (SS) on Geobacillus stearothermophilus ATCC 10149 spores. METHODS AND RESULTS: Two inoculum levels of spores of G. stearothermophilus were mixed with sterile sand and exposed to SS at 105-175 degrees C. The decimal reduction time (D-value) and the thermal resistance constant (z-value) were calculated. The effect of cooling of spores between periods of exposure to SS was also examined. A mean z-value of 25.4 degrees C was calculated for both inoculum levels for SS processing temperatures between 130 degrees C and 175 degrees C. CONCLUSIONS: Spore response to SS treatment depends on inoculum size. SS treatment may be effective for reduction in viability of thermally resistant bacterial spores provided treatments are separated by intermittent cooling periods. SIGNIFICANCE AND IMPACT OF THE STUDY: There is a need for technologies that require short thermal processing times to eliminate bacterial spores in foods. The SS processing technique has the potential to reduce microbial load and to modify food texture with less energy in comparison to commonly used hot air treatment. This work provides information on the effect of SS processing parameters on the viability of G. stearothermophilus spores.

Growth performance, gastrointestinal microbial activity, and nutrient digestibility in early-weaned pigs fed diets containing flaxseed and carbohydrase enzyme.

The effects of ground flaxseed (FS) and a multicarbohydrase enzyme (C) supplement on piglet performance, gastrointestinal microbial activity, and nutrient digestibility were investigated in a 28-d trial. The enzyme supplement provided 500 units of pectinase, 50 units of cellulase, 400 units of mannanase, 1,200 units of xylanase, 450 units of glucanase, and 45 units of galactanase per kilogram of diet. Ninety-six pigs were weaned at 17 d of age (BW, 6.1 +/- 0.4 kg, mean +/- SD) and assigned to treatments based on a 2 x 2 factorial arrangement in a completely randomized design, with 6 pens per diet (4 pigs per pen). The diets contained wheat, barley, peas, soybean meal, and canola meal with 0 or 12% FS, and were fed without or with C. Flaxseed was included by changing the levels of the other ingredients to balance the diets for DE and nutrients. Diets had similar nutrient contents and met the NRC (1998) nutrient specifications, with the exception of DE, CP, and AA, which were 95, 94, and 97% of the NRC requirements, respectively. Diets were fed in a 2-phase feeding program (2 wk/phase). Feed intake and BW were measured weekly, and 1 pig per pen with a BW nearest the pen average was bled weekly to evaluate plasma urea nitrogen. On d 28, fresh fecal samples were collected from each pen and 1 pig per pen with a BW nearest the pen average was killed to evaluate intestinal microbial activity and nutrient digestibility. A dietary effect on piglet performance was observed only in wk 3, when the FS diets decreased (P = 0.005) ADG and G:F, tended to decrease (P = 0.070) ADFI, and increased (P = 0.027) plasma urea nitrogen. An interaction between FS and C was observed for ileal digesta viscosity (P = 0.045), such that C increased viscosity in the FS diet but had no effect in the non-FS diet. Flaxseed and C interacted to affect ileal ammonia content (P = 0.049), such that in the absence of FS, pigs fed the diet with C had lower ammonia than those on the diet without C. Flaxseed and C affected other ileal parameters independently. Pigs fed the FS diets had decreased (P = 0.003 to 0.033) anaerobic spore counts, organic acid, DM, CP, and nonstarch polysaccharide (NSP) digestibility compared with pigs fed the non-FS diets, whereas pigs fed the C-supplemented diets had greater (P = 0.009 to 0.061) lactobacilli counts, lactate, DM, and NSP digestibility than pigs fed the unsupplemented diets. In conclusion, FS reduced ileal microbial activity, nutrient digestibilities, and piglet performance in wk 3. The multicarbohydrase supplement increased ileal DM and NSP digestibilities as well as lactobacilli counts and lactate.

Suitability of pea starch and calcium alginate as antimicrobial coatings on chicken skin.

The effect of incorporating trisodium phosphate (TSP) in pea starch (PS) and acidified sodium chlorite (ASC) in calcium alginate upon the antimicrobial activity of TSP and ASC was studied against a 3-strain cocktail of Salmonella inoculated on chicken skin. The influence of polymer coating concentration on skin pH, coating-skin adhesion, and coating absorption upon antimicrobial performance were investigated. Aqueous solutions of 0.5 to 4.8% (wt/vol) PS were prepared with 10% (wt/vol) TSP (PS + TSP coating), and alginate + ASC coatings contained 1% (wt/vol) calcium chloride in 1,200 ppm of ASC mixed with an aqueous solution of 0.5, 1.0, or 1.5% (wt/vol) sodium alginate. Coating drops (10 microL) were placed on chicken skin thighs, and the angle formed by the tangent of the liquid surface at the skin interface (contact angle) was measured using a digital camera to assess coating-skin adhesion. Excised skins were mounted in a ring holder, and 5 mL of the coatings was applied to the skin. Weight changes in the skins that were related to coating absorptiveness were recorded. The TSP dissolved in 3.5% PS and ASC in 1% alginate reduced Salmonella by 1.6 log cfu/g and 1.4 log cfu/g, respectively, within 24 h. These reductions were significantly greater than those caused by TSP or ASC alone in water for up to 120 h. In coatings, TSP and ASC caused significant elevation or reduction of skin surface pH for up to 120 h, respectively. The TSP destabilized PS with 88% of the coating having dripped from the skin 1 h later. Coatings with 0.5% PS were absorbed quickly by the skin and had high skin adhesion, whereas those with >3.5% PS had low skin adhesion and slow absorption. Alginate coatings with or without ASC were stable, and about 50% of the coating weight was retained at 120 h. The latter coatings appeared to have low absorptiveness because the skin gained approximately 1.0% of its weight within 60 min following application. These findings indicate that effects of the agents in coatings on skin pH, the extent of coating adhesion, and absorption may contribute to overall antimicrobial behaviors.

Performance responses and indicators of gastrointestinal health in early-weaned pigs fed low-protein amino acid-supplemented diets.

The effects of low-protein AA-supplemented diets on piglet performance, visceral organ mass, incidence of diarrhea, intestinal microbial population, and fermentation were studied in a 3-wk trial. After a 7-d adaptation period, 96 piglets (approximately 6.2 kg of initial BW) were assigned to 4 corn-wheat, soybean meal-based dietary treatments in a completely randomized design to give 6 replicate pens per treatment (n = 4 piglets per pen). The treatments were a control wheat-corn-soybean meal-based phase I diet containing 23% CP, or the same diet with CP reduced to 21%, 19%, or 17% and supplemented with crystalline AA to achieve equal standardized ileal digestible contents of Lys, Met plus Cys, Thr, and Trp in all diets. Diets were formulated to similar nutrient levels and provided ad libitum. Blood from all pigs was taken on d 0, 7, 14, and 21 for determining plasma urea N. Weekly feed intake, BW changes, and G:F were determined. On d 21, 2 pigs per pen were randomly selected and killed to determine small intestinal morphology, digesta pH and ammonia levels, and luminal microbial counts. Average daily feed intake, ADG, and G:F were not affected (P > 0.10) by reducing CP to 21%, but a reduction to 19% or 17% decreased ADFI (P < 0.001) and ADG (linear, P < 0.001; quadratic, P < 0.05) over the 3-wk study period. Reducing CP to 19% had no effect (P > 0.10) on G:F; however, this response criterion was decreased linearly (P < 0.001) over the 3-wk study period as dietary CP declined. Water usage was only numerically decreased (P > 0.10) with dietary CP reduction. Plasma urea N was decreased linearly (P < 0.01) with CP reduction. Reducing CP from 23 to 17% had a linear (P < 0.05) and cubic effect on stomach and liver weights, respectively. Although histological data showed some differences among diets, no distinct trend was evident. Ammonia N in ileal digesta was reduced linearly (P < 0.01) as dietary CP was decreased. With the exception of valeric acid, VFA levels in ileal digesta of piglets fed low-protein diets were generally lower (P < 0.05) compared with the control diet. Diet had no effect on intestinal microbial counts (P > 0.10). The results show that piglet performance may suffer when dietary CP is reduced by 4 or more percentage units from 23% and support the hypothesis that low-CP diets help maintain enteric health in pigs by lowering toxic microbial metabolites such as ammonia.

Survival of Escherichia coli O157:H7 in frozen foods: impact of the cold shock response.

The survival of Escherichia coli O157:H7 strains in both frozen foods and trypticase soy broth (TSB) was investigated following cold shocking at 10 degrees C for 1.5 h. Using both trypticase soy agar (TSA) and violet red bile agar (VRBA) as recovery media, it was demonstrated that survival levels between cold shocked (CS) and non-cold shocked (NS) E. coli in ground beef or pork were not significantly different (P < or = 0.05). In contrast, cold shocking E. coli in either milk, whole egg or sausage resulted in a significant(P < or = 0.05) enhancement in survival. For milk, survival levels of CS E. coli, by 28 days of frozen storage, were 1.89 and 1.66 log10 cfu/ml higher on TSA and VRBA, respectively, when compared to NS cells. In egg these values were 0.64 and 1.31, while in sausage, values of 0.74 and 1.19 were obtained. In TSB (pH 7) survival of CS E. coli for some strains was about 3 log10 cfu/ml higher when compared to NS cells. Acidification of TSB (pH 5), however, appeared to negate the protective effects of the cold shock treatment. In milk, increasing the differential between the growth and cold shock temperatures resulted in higher numbers of survivors. In this regard the growth-cold shock temperature protocol giving optimum protection was 37-10 degrees C. In contrast, growth of E. coli at 20 degrees C followed by cold shocking at 10 degrees C did not result in any significant freeze protection. In addition, increased protection due to cold shocking was correlated with the appearance of a novel protein appearing at pI 4.8 following isoelectric focusing analysis, thus demonstrating an alteration of protein synthesis.

Membrane ion-exchange chromatography for process-scale antibody purification.

The large-scale production of recombinant monoclonal antibodies demands economical purification processes with high throughputs. The potential for ion-exchange membrane adsorbers to replace traditional ion-exchange columns was evaluated. Breakthrough capacities of commercially available cation-exchange membranes were determined as a function of flow-rate and layer number. Due to economic and process restrictions, cation-exchange membranes may not currently be advantageous for process-scale antibody purification in a bind and elute mode. However, anion-exchange membranes in a flow-through mode may provide a reasonable alternative to columns for the removal of low levels of impurities such as DNA, host cell protein, and virus.

Expanded bed adsorption in the purification of monoclonal antibodies: a comparison of process alternatives.

Expanded bed adsorption (EBA) was examined as the initial capture/purification step in the purification of monoclonal antibodies from Chinese hamster ovary (CHO) cultures. Two process alternatives each using EBA were compared to a conventional Protein A process without EBA. One alternative used Protein A affinity EBA followed by packed-bed cation and anion-exchange steps. The other alternative used cation-exchange EBA as the capture step followed by packed-bed Protein A and anion-exchange steps. The process using Protein A EBA produced comparable purity (host cell protein, DNA, Protein A, antibody aggregate) to the conventional process. However, the Protein A EBA column showed a significant decrease in dynamic capacity with a limited number of cycles. The process using cation EBA achieved comparable levels of host cell proteins (HCP) and DNA but not antibody aggregate or leached Protein A compared to the conventional process.

Effects of exogenous serotonin on a motor behavior and shelter competition in juvenile lobsters (Homarus americanus).

Three experiments were conducted to determine (1) the pharmacodynamics of 5-hydroxytryptamine in juvenile lobsters; (2) the effects of 5-hydroxytryptamine, using a range of dosages, on a motor behavior used to escape an aversive situation; and (3) the effect of doses that did and did not inhibit this motor behavior on measures of dominance and shelter competition. The fate of 5-hydroxytryptamine in hemolymph over a 60-min post-injection period showed that the concentration fell rapidly to a low plateau that was maintained for at least 1 h. Low doses of 5-hydroxytryptamine did not affect locomotor behavior, but higher doses inhibited it. Dominance and subsequent possession of a shelter were unaffected by a low dose of 5-hydroxytryptamine but a higher dose that inhibited locomotion resulted in lobsters that lost fights and did not secure or retain possession of the shelter. In the context of dominance and shelter competition, we were unable to demonstrate any advantage of the low dose of exogenous 5-hydroxytryptamine and a severe disadvantage with the higher dose. Previous reports of transient increases in aggression in 5-hydroxytryptamine-treated subordinate lobsters did not take into account motor inhibition as a possible critical variable in aggression.

Expanded bed protein A affinity chromatography of a recombinant humanized monoclonal antibody: process development, operation, and comparison with a packed bed method.

We show that expanded bed protein A affinity chromatography using Streamline rProtein A media is an efficient method for purifying a recombinant humanized monoclonal antibody from unclarified Chinese hamster ovary cell culture fluid and that it provides purification performance comparable to using a packed bed. We determined that the dynamic capacity of the expanded bed media is related to flow rate (measured in column volumes per hour) by a power function, which allows a high capacity at a low flow rate. At 250 cm h-1 with a 25 cm bed height (10 column volumes h-1), the dynamic capacity is 30 g l-1. The yield and purity (measured by the amount of host cell proteins, DNA, SDS-PAGE, and turbidity) of the antibody purified by expanded bed is comparable to the yield and purity obtained on a standard packed bed method using Prosep A media.

Performance comparison of protein A affinity-chromatography sorbents for purifying recombinant monoclonal antibodies.

We describe the performance characteristics of five Protein A affinity-chromatography sorbents (Sepharose Fast Flow, Poros 50, Poros LP, Prosep and Streamline) for purifying a recombinant humanized monoclonal antibody from clarified Chinese hamster ovary cell culture fluid. We measured the dynamic capacity at varying flow rates, maximum capacity, pressure drop and production rate. For purified antibody, we measured yield and purity (by SDS/PAGE, the amount of DNA, the amount of host-cell proteins and the amount of Protein A). We found that, whereas all sorbents provided significant and essentially equivalent antibody purification, there were differences in capacity and pressure drop, which affected the production rate and had implications for process applications.

Real-time control of antibody loading during protein A affinity chromatography using an on-line assay.

We show that an on-line chromatographic assay can reliably control antibody loading in real-time during protein A affinity chromatography purification of a recombinant antibody from clarified Chinese hamster ovary cell culture fluid. The on-line assay directly sampled preparative column effluent and provided real-time measurement of antibody breakthrough during loading. The on-line assay used protein A immobilized on perfusion chromatography media, equilibrated with phosphate-buffered saline at pH 7.2 and eluted with phosphate-buffered saline at pH 2.2. The assay reliably ended loading at 1% breakthrough with minimal yield loss. Reproducible yield and purity were obtained over 23 consecutive cycles. Yield remained constant while breakthrough capacity and the antibody concentration in the load changed.

Real-time monitoring of recombinant antibody breakthrough during protein A affinity chromatography.

An on-line assay was developed to monitor antibody breakthrough in real time during Protein A affinity chromatography of recombinant antibodies. When loading cell culture fluid on to a Protein A affinity column, antibody breakthrough cannot be measured by UV absorbance because of the flow-through of UV-absorbing impurities. An assay using perfusion chromatography media with immobilized Protein A is a rapid, antibody-specific assay. It directly samples preparative column effluent, allowing real-time measurement of antibody breakthrough during loading of Protein A affinity chromatography. Breakthrough curves were generated for three column media at five flow rates, showing the effects of diffusion on the shape of the breakthrough curve. The breakthrough curves were used to measure dynamic capacity.

Non-flammable preparative reversed-phase liquid chromatography of recombinant human insulin-like growth factor-I.

Acetonitrile is used as an eluent for reversed-phase chromatography. However, because it is a flammable solvent, using acetonitrile on a large scale requires expensive equipment and facilities specially designed for flammable solvents. Using a non-flammable solvent as an eluent eliminates this expense. A method was developed to purify recombinant human insulin-like growth factor I by reversed-phase high-performance liquid chromatography using gradient elution with hexylene glycol, a non-flammable replacement for acetonitrile. The separation produced equivalent yield, purity and throughput as reversed-phase chromatography using elution with acetonitrile.

Recovery of foodborne microorganisms from potentially lethal radiation damage.

A two-stage recovery protocol was examined for microorganisms following gamma irradiation in phosphate buffer at 0 degrees C. In the first stage, survivors were recovered on basal yeast extract agar and held at various temperatures suboptimal for their growth for 20 h (resuscitation protocol). In the second stage the survivors were incubated for an additional 24 h, but in this case at their optimum temperature for growth. Controls consisted of survivors which were not subjected to the resuscitation protocol (direct incubation at their optimum growth temperature). The ratio of survivors enumerated with and without the resuscitation protocol (control) at each specified temperature was used to formulate a recovery factor(RF). An RF was determined for each treatment dose. Results of this study indicated that the number of Escherichia coli, Salmonella serotype typhimurium and Brochothrix thermosphacta survivors increased following a resuscitation protocol (RF > 2.0). Overall, optimum resuscitation temperatures ranged from 14 to 22 degrees C. The extent of recovery also appeared dose dependent, with larger treatment doses giving rise to a higher RF. S. serotype typhimurium irradiated at 1.5 kGy exhibited the highest RF, 161, when resuscitated at 22 degrees C. Listeria monocytogenes, Yersinia enterocolitica, Staphylococcus aureus, Aeromonas hydrophila and Saccharomyces cerevisiae exhibited an RF < 2.0 regardless of resuscitation temperature. Results of this study indicated that the use of suboptimal holding temperatures as part of a recovery protocol may have advantages, especially with respect to the enumeration of E. coli and salmonellae survivors in irradiated foods such as poultry.