Total Titers Research Articles

Engineered production of fungal anticancer cyclooligomer depsipeptides in Saccharomyces cerevisiae

Two fungal cyclooligomer depsipeptide synthetases (CODSs), BbBEAS (352kDa) and BbBSLS (348kDa) from Beauveria bassiana ATCC 7159, were reconstituted in Saccharomyces cerevisiae BJ5464-NpgA, leading to the production of the corresponding anticancer natural products, beauvericins and bassianolide, respectively. The titers of beauvericins (33.8±1.4mg/l) and bassianolide (21.7±0.1mg/l) in the engineered S. cerevisiae BJ5464-NpgA strains were comparable to those in the native producer B. bassiana. Feeding d-hydroxyisovaleric acid (d-Hiv) and the corresponding l-amino acid precursors improved the production of beauvericins and bassianolide. However, the high price of d-Hiv limits its application in large-scale production of these cyclooligomer depsipeptides. Alternatively, we engineered another enzyme, ketoisovalerate reductase (KIVR) from B. bassiana, into S. cerevisiae BJ5464-NpgA for enhanced in situ synthesis of this expensive substrate. Co-expression of BbBEAS and KIVR in the yeast led to significant improvement of the production of beauvericins. The total titer of beauvericin and its congeners (beauvericins A–C) was increased to 61.7±3.0mg/l and reached 2.6-fold of that in the native producer B. bassiana ATCC 7159. Supplement of l-Val at 10mM improved the supply of ketoisovalerate, the substrate of KIVR, which consequently further increased the total titer of beauvericins to 105.8±2.1mg/l. Using this yeast system, we functionally characterized an unknown CODS from Fusarium venenatum NRRL 26139 as a beauvericin synthetase, which was named as FvBEAS. Our work thus provides a useful approach for functional reconstitution and engineering of fungal CODSs for efficient production of this family of anticancer molecules.

Saccharification of recalcitrant biomass and integration options for lignocellulosic sugars from Catchlight Energy’s sugar process (CLE Sugar)

BackgroundWoody biomass is one of the most abundant biomass feedstocks, besides agriculture residuals in the United States. The sustainable harvest residuals and thinnings alone are estimated at about 75 million tons/year. These forest residuals and thinnings could produce the equivalent of 5 billion gallons of lignocellulosic ethanol annually. Softwood biomass is the most recalcitrant biomass in pretreatment before an enzymatic hydrolysis. To utilize the most recalcitrant lignocellulosic materials, an efficient, industrially scalable and cost effective pretreatment method is needed.ResultsObtaining a high yield of sugar from recalcitrant biomass generally requires a high severity of pretreatment with aggressive chemistry, followed by extensive conditioning, and large doses of enzymes. Catchlight Energy’s Sugar process, CLE Sugar, uses a low intensity, high throughput variation of bisulfite pulping to pretreat recalcitrant biomass, such as softwood forest residuals. By leveraging well-proven bisulfite technology and the rapid progress of enzyme suppliers, CLE Sugar can achieve a high yield of total biomass carbohydrate conversion to monomeric lignocellulosic sugars. For example, 85.8% of biomass carbohydrates are saccharified for un-debarked Loblolly pine chips (softwood), and 94.0% for debarked maple chips (hardwood). Furan compound formation was 1.29% of biomass feedstock for Loblolly pine and 1.10% for maple. At 17% solids hydrolysis of pretreated softwood, an enzyme dose of 0.075 g Sigma enzyme mixture/g dry pretreated (unwashed) biomass was needed to achieve 8.1% total sugar titer in the hydrolysate and an overall prehydrolysate liquor plus enzymatic hydrolysis conversion yield of 76.6%. At a much lower enzyme dosage of 0.044 g CTec2 enzyme product/g dry (unwashed) pretreated softwood, hydrolysis at 17% solids achieved 9.2% total sugar titer in the hydrolysate with an overall sugar yield of 85.0% in the combined prehydrolysate liquor and enzymatic hydrolysate. CLE Sugar has been demonstrated to be effective on hardwood and herbaceous biomass, making it truly feedstock flexible.ConclusionsDifferent options exist for integrating lignocellulosic sugar into sugar-using operations. A sugar conversion plant may be adjacent to a CLE Sugar plant, and the CLE Sugar can be concentrated from the initial 10% sugar as needed. Concentrated sugars, however, can be shipped to remote sites such as ethanol plants or other sugar users. In such cases, options for shipping a dense form of sugars include (1) pretreated biomass with enzyme addition, (2) lignocellulosic sugar syrup, and (3) lignocellulosic sugar solid. These could provide the advantage of maximizing the use of existing assets.

Functionality of cross-reactive antibodies against Moraxella catarrhalis

Event Abstract Back to Event Functionality of cross-reactive antibodies against Moraxella catarrhalis Daria Augustyniak1*, Monika Piekut1 and Grażyna Majkowska-Skrobek1 1 University of Wroclaw, Department of Pathogen Biology and Immunology, Institute of Genetics and Microbiology, Poland Introduction: Antibody-dependent immune response against human respiratory tract pathogen Moraxella catarrhalis is critical for its effective elimination [1-5]. Outer membrane proteins (OMPs) and lipooligosaccharide are the major surface-exposed immune targets in this bacterium [1-3,6].The goal of the study was to characterize the broad-spectrum effectiveness of cross-reacting antibodies produced in response to whole cells (Mc) or purified outer membrane proteins (OMPs) of this pathogen. Methods: Antisera were obtained from mice immunized with whole bacteria (anti-Mc sera) or Zwittergent-isolated OMPs (anti-OMPMc sera) [3]. The titers of cross-reactive antibodies were measured by whole-cell ELISA whereas their avidities by ELISA elution assay with sodium thiocyanate. The following functional methods were used: (1) the antibody-dependent bactericidal assay, (2) the opsonophagocytic assay with human THP-1 cell line, and (3) the blocking adhesion assay with human A549 epithelial cell line. Results: There were high titers of cross-reactive antibodies both in anti-Mc and anti-OMPMc sera. The titers of anti-OMPMc sera were higher than that of anti-Mc sera. The cross-reactive IgG produced exclusively to OMPs had stronger avidity comparing to those elicited against whole M. catarrhalis cells. Additionally, these antibodies played a pivotal role in complement-mediated killing of heterologous M. catarrhalis isolates. The positive relationship between total titer of complement-fixing subclasses IgG2a and IgG2b and bactericidal titer was found for both anti-Mc and anti-OMPMc sera. The opsonophagocytic potency as well as reduction of bacterial attachment to human epithelium was much stronger for cross-reactive anti-OMPMc than cross-reactive anti-Mc antibodies. Conclusion: The presence of cross-reactive antibodies with bactericidal, opsonophagocytic and bacterial adhesion blocking potency may be additional source to control host-Moraxella catarrhalis interaction especially when the host immune status is prone to a new infection.

Continuous butanol fermentation from inexpensive sugar-based feedstocks by Clostridium saccharobutylicum DSM 13864

Corn stover hydrolysate (CSH) and cane molasses were studied for butanol fermentation by Clostridium saccharobutylicum DSM 13864 in continuous fermentation. Using cane molasses as substrate, solvent of 13.75g/L (butanol 8.65g/L) and productivity of 0.439g/L/h were achieved in a four-stage continuous fermentation at a gradient dilution mode of 0.15–0.15–0.125–0.1h−1. In continuous fermentation using CSH as substrate, total solvent titer of 11.43g/L (butanol 7.81g/L) and productivity of 0.429g/L/h were reached at a dilution rate of 0.15h−1, and the steady process was continuously operated for 220h without compromise in solvent titer.

Introducing a single secondary alcohol dehydrogenase into butanol-tolerant Clostridium acetobutylicum Rh8 switches ABE fermentation to high level IBE fermentation

BackgroundPreviously we have developed a butanol tolerant mutant of Clostridium acetobutylicum Rh8, from the wild type strain DSM 1731. Strain Rh8 can tolerate up to 19 g/L butanol, with solvent titer improved accordingly, thus exhibiting industrial application potential. To test if strain Rh8 can be used for production of high level mixed alcohols, a single secondary alcohol dehydrogenase from Clostridium beijerinckii NRRL B593 was overexpressed in strain Rh8 under the control of thl promoter.ResultsThe heterogenous gene sADH was functionally expressed in C. acetobutylicum Rh8. This simple, one-step engineering approach switched the traditional ABE (acetone-butanol-ethanol) fermentation to IBE (isopropanol-butanol-ethanol) fermentation. The total alcohol titer reached 23.88 g/l (7.6 g/l isopropanol, 15 g/l butanol, and 1.28 g/l ethanol) with a yield to glucose of 31.42%. The acid (butyrate and acetate) assimilation rate in isopropanol producing strain Rh8(psADH) was increased.ConclusionsThe improved butanol tolerance and the enhanced solvent biosynthesis machinery in strain Rh8 is beneficial for production of high concentration of mixed alcohols. Strain Rh8 can thus be considered as a good host for further engineering of solvent/alcohol production.

Quantification of AAV Particle Titers by Infrared Fluorescence Scanning of Coomassie-Stained Sodium Dodecyl Sulfate–Polyacrylamide Gels

Adeno-associated virus (AAV)-based vectors have gained increasing attention as gene delivery vehicles in basic and preclinical studies as well as in human gene therapy trials. Especially for the latter two-for both safety and therapeutic efficacy reasons-a detailed characterization of all relevant parameters of the vector preparation is essential. Two important parameters that are routinely used to analyze recombinant AAV vectors are (1) the titer of viral particles containing a (recombinant) viral genome and (2) the purity of the vector preparation, most commonly assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. An important, third parameter, the titer of total viral particles, that is, the combined titer of both genome-containing and empty viral capsids, is rarely determined. Here, we describe a simple and inexpensive method that allows the simultaneous assessment of both vector purity and the determination of the total viral particle titer. This method, which was validated by comparison with established methods to determine viral particle titers, is based on the fact that Coomassie Brilliant Blue, when bound to proteins, fluoresces in the infrared spectrum. Viral samples are separated by SDS-PAGE followed by Coomassie Brilliant Blue staining and gel analysis with an infrared laser-scanning device. In combination with a protein standard, our method allows the rapid and accurate determination of viral particle titers simultaneously with the assessment of vector purity.

Immunogenicity of commercial, formaldehyde and binary ethylenimine inactivated Newcastle disease virus vaccines in specific pathogen free chickens.

Newcastle disease (ND) is one of the most important diseases that affect birds; the epizootic nature of the disease has caused severe economic losses in the poultry industry worldwide. In this experiment ND virus (NDV) was inactivated by two different chemicals binary ethylenimine (BEI) and formaldehyde. Formaldehyde was used at 0.1%, while BEI was used at concentrations of 1 to 4 mM. NDV inactivation with BEI was done in various incubation temperatures and periods and the best result (30 °C, 4 mM BEI and 21 hrs treatment) used as an experimental vaccine. Prepared inactivated NDV vaccines and a commercial vaccine were tested for their efficiency in generating humoral immune response in different groups of specific pathogen free (SPF) chicks. Test groups received 0.2 ml formaldehyde inactivated NDV (NDVF), BEI inactivated NDV (NDVEI) and Razi institute produced NDV vaccine (NDVR) subcutaneously respectively. HI Log 2 total mean titer of NDVEI group (8.42 ± 0.12) were significantly higher than NDVF (7.64 ± 0.16) and NDVR (7.86 ± 0.11) groups (p

High HBV-DNA Titer in Surrounding Liver Rather Than in Hepatocellular Carcinoma Tissue Predisposes to Recurrence After Curative Surgical Resection

In this study the authors intended to investigate the relationship between intrahepatic hepatitis B virus (HBV)-DNA concentrations and posthepatectomy recurrence of HBV-associated hepatocellular carcinoma (HCC). High HBV-DNA level is strongly associated with HCC development in chronic HBV infection and considered to be a risk factor of HCC recurrence. A total of 109 patients with HBV-associated HCC who underwent curative surgical resection were followed up every 3 to 6 months for a median of 82 months. Intrahepatic total HBV-DNA titer was measured in HCC and surrounding liver tissues using a TaqMan probe-based real-time polymerase chain reaction method. HBV-DNA titers in HCC and surrounding liver were compared in accordance with patients' clinical, radiologic, and histopathological characteristics. The relationships between HBV-DNA titers in HCC or surrounding liver tissues and cumulative HCC recurrence rates were determined. Of the 109 patients, 67 (62%) showed posthepatectomy recurrence of HCC. In all patients, total HBV-DNA titers were significantly higher in HCCs than in surrounding liver tissues (P=0.019). HCC recurred more frequently in patients with higher than those with lower HBV-DNA titers in surrounding liver tissues (P=0.009). In contrast, the HCC recurrence rates were similar in patients with higher and those with lower HBV-DNA titers in HCC specimens (P=0.301). Multivariate analysis showed that tumor size >5 cm (P=0.008), the presence of portal vein thrombus (P=0.001), and high HBV-DNA titer in surrounding liver tissues (P=0.002) were independent risk factors for posthepatectomy HCC recurrence in patients with HBV-associated HCC. In patients with HBV-associated HCC, high HBV-DNA titer in surrounding liver rather than in the HCC itself is associated with posthepatectomy HCC recurrence after curative surgical resection.

Mini‐scale bioprocessing systems for highly parallel animal cell cultures

Animal cells have been used extensively in therapeutic protein production. The growth of animal cells and the expression of therapeutic proteins are highly dependent on the culturing environments. A large number of experimental permutations need to be explored to identify the optimal culturing conditions. Miniaturized bioreactors are well suited for such tasks as they offer high-throughput parallel operation and reduce cost of reagents. They can also be automated and be coupled to downstream analytical units for online measurements of culture products. This review summarizes the current status of miniaturized bioreactors for animal cell cultivation based on the design categories: microtiter plates, flasks, stirred tank reactors, novel designs with active mixing, and microfluidic cell culture devices. We compare cell density and product titer, for batch or fed-batch modes for each system. Monitoring/controlling devices for engineering parameters such as pH, dissolved oxygen, and dissolved carbon dioxide, which could be applied to such systems, are summarized. Finally, mini-scale tools for process performance evaluation for animal cell cultures are discussed: total cell density, cell viability, product titer and quality, substrates, and metabolites profiles.

Cationic liposomes as adjuvants for influenza hemagglutinin: More than charge alone

Cationic liposomes are known as potent adjuvants for subunit vaccines. The purpose of this work was to study whether the content and the physicochemical properties of the positively charged compound affect the adjuvanticity of cationic liposomes. Cationic liposomes containing a cationic compound (DDA, DPTAP, DC-Chol, or eDPPC) and a neutral phospholipid (DPPC) were prepared by the film hydration-extrusion method and loaded with influenza hemagglutinin (HA) by adsorption. The liposomes were characterized (hydrodynamic diameter, zeta potential, membrane fluidity, HA loading) and their adjuvanticity was tested in mice. The formulations were administered twice subcutaneously and mouse sera were analyzed for HA-specific antibodies by ELISA and for HA-neutralizing antibodies by hemagglutination inhibition (HI) assay.First, the influence of cationic lipid concentration in the DC-Chol/DPPC liposomes (10 vs. 50mol%) was investigated. The DC-Chol/DPPC (50:50) liposomes showed a higher zeta potential and HA loading, resulting in stronger immunogenicity of the HA/DC-Chol/DPPC (50:50) liposomes compared to the corresponding (10:90) liposomes.Next, we used liposomes composed of 50mol% cationic lipids to investigate the influence of the nature of the cationic compound on the adjuvant effect. Liposomes made of the four cationic compounds showed similar hydrodynamic diameters (between 100 and 170nm), zeta potentials (between +40 and +50mV), HA loading (between 55% and 76%) and melting temperatures (between 40 and 55°C), except for the DC-Chol liposomes, which did not show any phase transition. HA adjuvanted with the DC-Chol/DPPC (50:50) liposomes elicited significantly higher total IgG1 and IgG2a titers compared to the other liposomal HA formulations and non-adjuvanted HA. A similar trend was observed for the HI titers. These results show that the adjuvanticity of cationic liposomes depends on both the content and the physicochemical properties of the charged compound.

Reevaluation of Methionine Requirement Based on Performance and Immune Responses in Broiler Breeder Hens

This experiment was conducted to study methionine requirements in broiler breeder hens aged from 26-35 weeks. The treatments were consisted of six levels of methionine (0.2, 0.25, 0.3, 0.35, 0.4 and 0.45%), with five replicates of eight birds (Seven hens and one rooster). The results showed that different levels of methionine had no significant (p>0.05) effects on egg weight, unsettable (double yolk, small size) and settable eggs. The methionine levels significantly (p 0.05), but total titer against sheep red blood cell (SRBC) and IgG responses were influenced (p<0.05). Using of two-slope quadratic broken-line analysis indicated that methionine requirements for egg mass (g/h/d), feed conversion ratio, settable eggs, SRBC and IgG were 446, 450, 494, 580 and 492 (mg/d), respectively. In conclusion, methionine requirement for settable eggs was similar to those needed for the optimum immune responses. Since, settable eggs are an important factor in broiler breeder industries, so the authors recommend 494 mg/d methionine for broiler breeder hens. This amount is higher than those needed for egg mass and feed conversion ratio.

Metabolic Engineering of Clostridium acetobutylicum ATCC 824 for Isopropanol-Butanol-Ethanol Fermentation

Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineered C. acetobutylicum can be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B-593 (i.e., adh(B-593)) in C. acetobutylicum ATCC 824. To increase the total alcohol titer, a synthetic acetone operon (act operon; adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in the buk gene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged by in situ removal of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.

Effects of clonal variation on growth, metabolism, and productivity in response to trophic factor stimulation: a study of Chinese hamster ovary cells producing a recombinant monoclonal antibody.

The growth, metabolism, and productivity of five Chinese hamster ovary (CHO) clones were explored in response to stimulation with insulin (5mg/L) and LONG(®)R(3)IGF-I (20μg/L or 100μg/L). All five clones were derived from the same parental CHO cell line (DG44) and produced the same recombinant monoclonal antibody, with varying specific productivities. There was no uniform response among the clones to stimulation with the different trophic factors. One of the high productivity clones (clone D) exhibited significantly better growth in response to LONG(®)R(3)IGF-I; whereas the other clones showed equivalent or slightly better growth in the presence of insulin. Three out of the five clones had higher specific productivities in the presence of insulin (although not statistically significant); one was invariant, and the final clone exhibited slightly higher specific productivity in the presence of LONG(®)R(3)IGF-I. Total product titers exhibited moderate variation between culture conditions, again with neither trophic factor being clearly superior. Overall product titers were affected by variations in both integrated viable cell density and specific productivity. Nutrient uptake and metabolite generation patterns varied strongly between clones and much less with culture conditions. These results point to the need for careful clonal analysis when selecting clones, particularly for platform processes where media and culture conditions are predetermined.

Modification of the trypsin cleavage site of rotavirus VP4 to a furin-sensitive form does not enhance replication efficiency

The infectivity of rotavirus (RV) is dependent on an activation process triggered by the proteolytic cleavage of its spike protein VP4. This activation cleavage is performed by exogenous trypsin in the lumen of the intestines in vivo. Here, we report the generation and characterization of a recombinant RV expressing cDNA-derived VP4 with a modified cleavage site (arginine at position 247) recognized by endogenous furin as well as exogenous trypsin. Unexpectedly, the mutant virus (KU//rVP4-R247Furin) was incapable of plaque formation without an exogenous protease, although the mutant VP4s on virions were efficiently cleaved by endogenous furin. Furthermore, KU//rVP4-R247Furin showed impaired infectivity in MA104 and CV-1 cells even in the presence of trypsin compared with the parental virus carrying authentic VP4 (KU//rVP4). Although the total titre of KU//rVP4-R247Furin was comparable to that of KU//rVP4, the extracellular titre of KU//rVP4-R247Furin was markedly lower than its cell-associated titre in comparison with that of KU//rVP4. In contrast, the two viruses showed similar growth in a furin-defective LoVo cell line. These results suggest that intracellular cleavage of VP4 by furin may be disadvantageous for RV infectivity, possibly due to an inefficient virus release process.

Study on some predelivery immunoparameters in pregnant women in Al-Ramadi city

Serum Protein plays major role to maintain balance of blood, so total protein may be elevated or decreased due to some pathological & physiological changes in human body during pregnancy period. On the other hand the serological diagnosis of antibodies level during pregnancy is usually based on the detection of IgG and IgM antibodies as markers for diagnosis. The aim of this study was to investigate some immunoparameters including IgM, IgG, and assay of TSP, TSA & TSG in pregnant women attending Maternity & Child Hospital in Ramadi City, West of Iraq. Sera of 50 pregnant women were collected & quantitative assays by Single Radial Immunodiffusion test (Mancini test) for detection of (IgM & IgG) concentration & spectrophotometric method for Total serum protein, Total serum Albumin & Total serum Globulin reader were achieved against these serum samples.Serum immunoglobulin (IgM & IgG) were measured in 50 pregnant women. (22) with natural delivery & (28) Cesarean delivery pregnant women. The mean IgG level was increased in pregnant women aged under 25 years, while IgM levels were markedly increased at (25-39) years. A total mean titer for IgG & IgM were found to be 1006.2071± 408.12282 & 48.8571±28.81962 respectively.Total serum protein reports separate values for total protein , albumin & Globulin. Values of serum protein reflect high globulin concentration, while albumin concentration in pregnant women recorded significant differences It is mean values for age group (15-24) & (25- 39) years are 3.4479 ± .72373 & 3.0044±.33212 respectively. There was significant differences in albumin concentration through age group in pregnant women. Most values of serum routine liver function tests during normal pregnancy remain below the upper normal limits in no pregnant women. When liver disease is suspected & considered pathologic further should prompt evaluation. Furthermore, IgG concentration was predominantly increase in their level during pregnancy in association with transported IgG through placenta to the fetus during late pregnancy period

Natural antibodies in bovine milk and blood plasma: Variability among cows, repeatability within cows, and relation between milk and plasma titers

Innate immunity plays an important role in preventing (barrier function) or combating infection (effector function). An important humoral component of innate immunity is formed by natural antibodies (NAb). The objectives of this study were to determine presence, variation among cows and repeatability within cows over time of total NAb titers directed to the pathogen-associated molecular patterns lipopolysaccharide, lipoteichoic acid (LTA) and peptidoglycan, and titers of NAb directed to the glycoprotein keyhole limpet hemocyanin in milk and plasma of individual cows. Furthermore in milk the antibody isotypes IgG1, IgG2, IgM and IgA binding LTA were analyzed. Ten milk and blood samples were obtained from each of 20 clinically healthy dairy cows from first to seventh parity during a period of 3 weeks. Total NAb binding lipopolysaccharide, LTA, peptidoglycan, and keyhole limpet hemocyanin were detected in milk and plasma, with titers considerably higher in plasma than in milk. Total NAb titers showed significant variation among cows, and repeatability within cows over time (ranging from 0.60 to 0.93). Titers of NAb in milk and plasma were positively correlated (correlation ranging from 0.69 to 0.91). Natural antibodies in milk binding LTA were of all 4 isotypes tested, although IgG2 was on average only present at low titers. All 4 isotypes in milk binding LTA also showed variation among cows, and repeatability within cows over time (ranging from 0.84 to 0.92). We conclude that NAb can be measured in a consistent and repeatable manner in bovine milk and blood plasma.

Antisense inhibition of a spermidine synthase gene highlights the role of polyamines for stress alleviation in pear shoots subjected to salinity and cadmium

Three transgenic European pear ( Pyrus communis L.) lines with reduced spermidine synthase ( SPDS) expression and spermidine (Spd) titers were developed using a construct containing an apple SPDS gene ( MdSPDS1) in antisense orientation. After exposure to either salt or cadmium stress, growth inhibition was more severe in the antisense lines than in the wild-type (WT). The antioxidant system, as shown by glutathione (GSH) content, activity of glutathione reductase (GR) and superoxide dismutase (SOD), and proline accumulation, was not effectively induced under stress in the antisense lines as compared with the WT. The reduction in antioxidant system function in the antisense lines was accompanied by a greater accumulation of malondialdehyde (MDA), an indicator of lipid peroxidation. Growth inhibition, Spd level, and parameters indicative of the antioxidant system were significantly ameliorated by exogenous Spd application. Under either salt or cadmium stress, GSH content, GR and SOD activity, and proline accumulation were positively correlated with Spd, putrescine (Put), and total polyamine titers. Conversely, MDA level showed a significantly negative correlation with these polyamines under both stress conditions. Thus, the responses to stress treatments were first identified in the SPDS antisense European pears, and the results provide further evidence for the important role of polyamines in both salt and cadmium stress tolerance, in which the polyamines act, at least in part, by influencing the antioxidant system.

Maternal Intake of Fish Oil but not of Linseed Oil Reduces the Antibody Response in Neonatal Mice

Dietary levels of n-3 PUFA are believed to influence the immune system. The importance of the source of n-3 PUFA is debated. This study addressed how the content and source of n-3 PUFA in the maternal diet influenced tissue FA composition and the immune response to ovalbumin (OVA) in mice pups. From the day of conception and throughout lactation, dams were fed diets containing 4% fat from linseed oil (LSO), fish oil (FO) or a n-3 PUFA-deficient diet (DEF). Pups were injected with OVA within 24h of birth and sacrificed at weaning (day 21). Overall, the content of n-3 PUFA in milk, liver and spleen reflected the source and only minor differences were observed in brain phospholipid 22:6n-3. The source had only limited influence on the n-3 PUFA accretion in peripheral tissue, with most pronounced differences in the spleen. The marine PUFA-group had reduced levels of total OVA-specific antibodies and OVA-IgG1 titers in the pup blood, while the response in the LSO-group did not differ from that in the DEF-group. There were no statistical differences in the cytokine responses to OVA-stimulated splenocytes, but the decrease in IgG1 was paralleled by an increase in IFNγ-production and a decrease in IL-6-production. Our results indicate that maternal intake of FO, but not of LSO, changes the offspring's antigen-specific response and potentially increases Th1-polarization.

A Short Burst of Oral Corticosteroid for Children with Acute Asthma: Is There an Impact on Immunity?

Background: We wished to examine the impact of a short course of oral corticosteroids on the immune function of children with acute asthma and explore the mechanism of immune suppression, if any. Methods: We conducted a prospective cohort study of children aged 3–17 years presenting with acute asthma to the emergency department. The exposure of interest was a short course of systemic corticosteroids. In emergency department, corticosteroid-treated and nonexposed children were immunized with 3 antigens, namely, a neoantigen (Bacteriophage φX 174) and 2 recall antigens (diphtheria and tetanus). After 5 ± 1 weeks, children were reimmunized with Bacteriophage φX 174. Antibody titers were measured 1, 2 (peak), and 5 weeks after the primary immunization, and 1 week (peak) after the secondary one. Results: We enrolled 41 children, 27 treated with and 14 unexposed to oral corticosteroids. Corticosteroid-treated children displayed an 85% lower adjusted total (IgG and IgM) peak titers to the primary Bacteriophage immunization compared to controls [ratio of geometric means (95% confidence interval): 15% (5% to 43%)]. There were no group differences in the rise of diphtheria or tetanus antibodies. After the secondary Bacteriophage immunization, corticosteroid-treated patients showed no significant group difference in peak total titers [ratio of geometric means (95% confidence interval): 1.84% (0.66%, 5.10%)], amplification and absolute, and percent IgG titers as compared to unexposed children. Conclusions: In children with acute asthma, a short course of oral corticosteroids may be associated with depressed immune response to a new antigen, with recovery within 6 weeks and no apparent impact on response to known antigens.

Experimental infection with different bacterial strains in larvae and juvenile Litopenaeus vannamei reared in Santa Catarina State, Brazil

This study evaluated the pathogenic characteristics of bacteria isolated from Litopenaeus vannamei during an outbreak at the Laboratory of Marine Shrimp, UFSC, Santa Catarina State, Brazil. Their virulence potential in larvae and juvenile shrimp and the effects on the total haemocyte count, phenoloxidase activity and serum agglutinate titre were examined after experimental infection. Bacterial strains were isolated from larvae and adult shrimps, identified by the AP120E biochemical system as: two strains of Vibrio alginolyticus, three of Aeromonas salmonicida and one of Pasteurella multocida sp. and Pasteurella sp. All the bacterial strains isolated in this study caused mortality in shrimp. One strain of V. alginolyticus was responsible for 97.3 and 88.7% mortality in larvae and juvenil shrimps, respectively. The shrimp immunological system was influenced by experimental infection with V. alginolyticus. Decrease in the total haemocyte count and increase in the phenoloxidase activity and the serum agglutinate titre (p < 0.05) were observed. The results showed the high pathogenicity of V. alginolyticus isolated from larvae and juvenile reared marine shrimp.