A Multiplex Label-Free Approach to Avian Influenza Surveillance and Serology

Influenza serology has traditionally relied on techniques such as hemagglutination inhibition, microneutralization, and ELISA. These assays are complex, challenging to implement in a format allowing detection of several types of antibody-analyte interactions at once (multiplex), and troublesome to implement in the field. As an alternative, we have developed a hemagglutinin microarray on the Arrayed Imaging Reflectometry (AIR) platform. AIR provides sensitive, rapid, and label-free multiplex detection of targets in complex analyte samples such as serum. In preliminary work, we demonstrated the application of this array to the testing of human samples from a vaccine trial. Here, we report the application of an expanded label-free hemagglutinin microarray to the analysis of avian serum samples. Samples from influenza virus challenge experiments in mallards yielded strong, selective detection of antibodies to the challenge antigen in most cases. Samples acquired in the field from mallards were also analyzed, and compared with viral hemagglutinin inhibition and microneutralization assays. We find that the AIR hemagglutinin microarray can provide a simple and robust alternative to standard methods, offering substantially greater information density from a simple workflow.     

Immunostimulation of antibody-producing cells and humoral antibody to fish bacterins by a biological response modifier

Numbers of splenic antibody-producing cells and humoral antibody titres were elevated during immunization regimes in rainbow trout when the bacterins Yersinia ruckeri or Aeromonas salmonicida O-antigen preparations were mixed with the immunostimulator FK-565. Fish sampled 14 days after injection showed a marked increase in the immune response when doses of 5, 10 or 100 μg of antigen were used. The immunostimulator may aid initial antigen uptake and processing.     

Economic assessment of plant cell culture for the production of ajmalicine

Cost estimates have been prepared for commercial-scale production of ajmalicine-rich Catnaranthus roseus biomass using plant cell culture. At the current state of the technology the cost would be approximately $7.30/lb dry biomass ($3215/kg ajmalicine). Naturally-grown C. roseus roots have a 50% lower ajmalicine concentration but would cost only ca. $0.70/lb ($619/kg ajmalicine). The principal reason for the high cost of the plant cell culture route is not the slow specific growth rate (0.35 day(-1)), but rather the slow specific product accumulation rate (0.26 mg/g day). This rate will have to be increased by a factor of 40 to make the process competitive.     

Transient responses of hybridoma cells to nutrient additions in continuous culture: I. Glucose pulse and step changes

Glucose and glutamine are the main nutrients used by mammalian cells in culture. Each provides unique biosynthetic precursors but are complementary for production of other metabolites and energy. The transient and steady-state responses of hybridoma growth and metabolism to glucose pulse and step changes have been examined. Metabolic quotients are reported for oxygen, glucose, lactate, ammonia, glutamine, alanine, and other amino acids. The glucose consumption rate increased by 100-200% immediately after glucose was added to the reactor, and the increased glycolytic ATP production appears to be responsible for the concurrent rapid decrease in the oxygen consumption rate. The effects on glutamine consumption were delayed, probably due to buffering by the TCA cycle and interrelated pathways. A period of increased biosynthetic activity, as evidenced by an increase in the estimated specific ATP production rate and lower by-product yields from glutamine, preceded the increase in cell concentration after the glucose step change. The biosynthetic yield of cells from ATP was calculated, and it was estimated that maintenance accounted for about 60% of the energy used by the cells at a specific growth rate of 0.66 day(-1). The estimated 22% ATP production due to glycoysis was twice as great as that before the step change.     

Enzymatic production of L-tryptophan in a reverse micelle reactor

The enzymatic production of tryptophan from indole and serine was investigated in a micellar solution of the surfactant Brij 56 in cyclohexane. An anion exchanger was employed to facilitate the transfer of tryptophan and serine between the water pool of the reverse micelle and the bulk organic phase. The influence of potassium ion, water content, pH, and co-surfactant on enzyme activity is reported. Kinetic studies indicate that the enzyme is not inhibited by indole in the micellar system and that the enzyme is more stable in reverse micelles than in bulk water. The design of a continuous reverse micelle reactor, which accommodates both product recovery and enzyme reactivation, is discussed.     

Amino acid metabolism in several tissues of the obese Zucker rat as indicated by the tissue accumulation of α-amino[1-14C]isobutyrate

Measurements of the tissue accumulation of α-amino[1-¹⁴C]isobutyrate [1-¹⁴C]AIB) in lean (+/?) and obese (fa/fa) Zucker rats showed an augmented tissue/plasma ratio in the liver of the obese animals. In contrast, brown adipose tissue AIB accumulation was lower in the fa/fa animals. In response to a 24h starvation period AIB accumulation was significantly elevated in the liver and plasma of the lean animals and was unchanged in the liver of the fa/fa animals. The circulating concentration of alanine and branched-chain amino acids was elevated in the fa/fa animals as compared to their lean counterparts. These observations suggest that amino acid uptake is not involved in the impaired muscle development observed in the obese Zucker rat and that the ability of brown adipose tissue for amino acid utilization is decreased in the obese animals suggesting that this may partially explain the impaired thermoregulatory capacity observed in brown adipose tissue of obese Zucker rats.     

Effects of tumour necrosis factor-α (cachectin) on glucose metabolism in the rat

Intravenous administration of a single dose (20 g) of recombinant tumour necrosis factor- (TNF, cachectin) to rats decreased the rate of intestinal glucose absorption. In vivo, the oxidation of [U-¹⁴C]glucose to ¹⁴CO₂ was significantly increased by the cytokine. In addition, [¹⁴C]lipid accumulation from [U-¹⁴C]glucose was increased both in liver and brown adipose tissue of the TNF-injected animals. The decrease observed in intestinal glucose absorption was not associated with changes in intestinal metabolism. There was no difference in glucose metabolism by isolated enterocytes from either control or TNF-injected rats whether in the absence or presence of different concentrations of the cytokine in the incubation medium. In contrast, tumour necrosis factor altered the rate of gastric emptying as measured by the gastrointestinal distribution of [³H]inulin following an intragastric glucose load. These results suggest that the cytokine profoundly alters glucose metabolism by increasing its whole-body oxidation rate and delaying intestinal absorption through a reduced gastric emptying.     

Extensive dermatophytoses caused by Trichophyton mentagrophytes and Microsporum canis in a patient with AIDS

Retrospective studies have shown the occurrence of episodes of deep or superficial fungal infections in 58 to 81% of HIV/AIDS patients as a result of impairment of cell immunity. We describe a case of disseminated cutaneous dermatophytoses caused by Trichophyton mentagrophytes and Microsporum canis in a patients with AIDS. Diagnostic and therapeutic problems in relation to this unsual presentation are emphasized as well as the importance of an early mycologic diagnosis to prescribe antifungal therapy.     

Design and mathematical description of differential contactors used in extractive fermentations

The performance of differential contactors for use in extractive fermentation is complicated by the effects of product formation in the contactor. When product formation is significant, approximate analytical solutions are presented for the performance of the contactor for two limiting cases: high and low substrate concentrations. When products are formed at a constant rate, there is a minimum raffinate solute concentration that can be obtained, in contrast to the behavior of a column in the absence of product formation. General equations describing the behavior of the system for product formation with backmixing in both phases are presented. The case of a stripping factor not equal to unity is considered.