Elevated bioactivity of the tolerogenic cytokines, interleukin-10 and transforming growth factor-beta, in the blood of acutely malnourished weanling mice

The main objective of this investigation was to determine the influence of acute deficits of protein and energy on the blood levels of interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta), physiologically the main anti-inflammatory and tolerogenic cytokines. In four 14-day experiments, male and female C57BL/6J mice, initially 19 days old, consumed a complete purified diet either ad libitum or in restricted daily quantities, or had free access to an isocaloric purified low-protein diet. A zero-time control group (19 days old) was included. In the first two experiments, serum IL-10 levels were assessed by sandwich enzyme-linked immunosorbent assay (ELISA) and bioassay. The mean serum IL-10 bioactivities were higher (P < or = 0.05) in both malnourished groups (low-protein and restricted intake: 15.8 and 12.2 ng/ml, respectively) than in the zero-time and age-matched control groups (6.3 and 7.3 ng/ml, respectively), whereas serum IL-10 immunoactivity was high only in the restricted intake group (e.g., second experiment: 17.0 pg/ml vs. 5.4, 3.7, and 3.1 pg/ml in the zero-time control, age-matched control and low-protein group, respectively). The third and fourth experiments centered on plasma TGF-beta immunoactivity (sandwich ELISA) and bioactivity, respectively. The ELISA revealed a high mean plasma TGF-beta1 level (P < or = 0.05) in the low-protein group only, but TGF-beta bioactivity (beta1 isoform, although 15% beta2 in the restricted intake group) was high in both malnourished groups (8.7 and 9.3 ng/ml in the low-protein and restricted groups, respectively) relative to the age-matched control group (0.5 ng/ml). Thus, metabolically distinct weanling systems mimicking marasmus and incipient kwashiorkor both exhibit a blood cytokine profile that points to a tolerogenic microenvironment within immune response compartments. A model emerges in which malnutrition-associated immune competence, at least in advanced weight loss, centers on cytokine-mediated peripheral tolerance that reduces the risk of catabolically induced autoimmune disease, but this is at the cost of attenuated responsiveness to infectious agents.     

Identification and validation of Asteraceae miRNAs by the expressed sequence tag analysis

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a vital role in the regulation of gene expression. Despite their identification in hundreds of plant species, few miRNAs have been identified in the Asteraceae, a large family that comprises approximately one tenth of all flowering plants. In this study, we used the expressed sequence tag (EST) analysis to identify potential conserved miRNAs and their putative target genes in the Asteraceae. We applied quantitative Real-Time PCR (qRT-PCR) to confirm the expression of eight potential miRNAs in Carthamus tinctorius and Helianthus annuus. We also performed qRT-PCR analysis to investigate the differential expression pattern of five newly identified miRNAs during five different cotyledon growth stages in safflower. Using these methods, we successfully identified and characterized 151 potentially conserved miRNAs, belonging to 26 miRNA families, in 11 genus of Asteraceae. EST analysis predicted that the newly identified conserved Asteraceae miRNAs target 130 total protein-coding ESTs in sunflower and safflower, as well as 433 additional target genes in other plant species. We experimentally confirmed the existence of seven predicted miRNAs, (miR156, miR159, miR160, miR162, miR166, miR396, and miR398) in safflower and sunflower seedlings. We also observed that five out of eight miRNAs are differentially expressed during cotyledon development. Our results indicate that miRNAs may be involved in the regulation of gene expression during seed germination and the formation of the cotyledons in the Asteraceae. The findings of this study might ultimately help in the understanding of miRNA-mediated gene regulation in important crop species.     

Real-Time RT-PCR on SAG1 and BAG1 Gene Expression during Stage Conversion in Immunosuppressed Mice Infected with Toxoplasma gondii Tehran Strain

Toxoplasmic encephalitis is caused by reactivation of bradyzoites to rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immunocompromised hosts. Diagnosis of this life-threatening disease is problematic, because it is difficult to discriminate between these 2 stages. Toxoplasma PCR assays using gDNA as a template have been unable to discriminate between an increase or decrease in SAG1 and BAG1 expression between the active tachyzoite stage and the latent bradyzoite stage. In the present study, real-time RT-PCR assay was used to detect the expression of bradyzoite (BAG1)- and tachyzoite-specific genes (SAG1) during bradyzoite/tachyzoite stage conversion in mice infected with T. gondii Tehran strain after dexamethasone sodium phosphate (DXM) administration. The conversion reaction was observed in the lungs and brain tissues of experimental mice, indicated by SAG1 expression at day 6 after DXM administration, and continued until day 14. Bradyzoites were also detected in both organs throughout the study; however, it decreased at day 14 significantly. It is suggested that during the reactivation period, bradyzoites not only escape from the cysts and reinvade neighboring cells as tachyzoites, but also converted to new bradyzoites. In summary, the real-time RT-PCR assay provided a reliable, fast, and quantitative way of detecting T. gondii reactivation in an animal model. Thus, this method may be useful for diagnosing stage conversion in clinical specimens of immunocompromised patients (HIV or transplant patients) for early identification of tachyzoite-bradyzoite stage conversion.     

Production and Evaluation of Toxoplasma gondii Recombinant GRA7 for Serodiagnosis of Human Infections

The precise diagnosis of the acute toxoplasmosis in pregnant women and immunocompromsied patients has critical importance. Most of the commercially available assays use the whole Toxoplasma soluble extract as the antigen. However, the assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the lack of a purified standardized antigen. The aim of this study was production and evaluation of the usefulness of the recombinant Toxoplasma gondii GRA7 antigen for the serodiagnosis of Toxoplasma gondii IgM and IgG by ELISA. A total of 70 T. gondii IgM positive sera, 74 T. gondii IgG positive sera, and 60 sera from subjects who were not infected with T. gondii were examined. These sera were shown different absorbance values in ELISA test. To control the specificity of the rGRA7 other parasitic diseases, for example, echinococcosis, malaria, leishmaniasis, fascioliasis, and strongyloidiasis were tested of which none showed positive results. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (com ELISA) were 89% and 90%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 96% and 90%, respectively. The results obtained here show that this antigen is useful for diagnostic purposes.     

Isolation of alpha-lactalbumin,beta-lactoglobulin,and bovine serum albumin from cow's milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity

The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Globulins were then precipitated from whey by half-saturated ammonium sulfate and beta-lactoglobulin was purified further using Sephadex G-50 gel filtration. The proteins in the supernatant were also fractionated using diethylaminoethyl cellulose chromatography in which beta-lactoglobulin was separated from alpha-lactalbumin and bovine serum albumin. The latter two proteins that co-eluted in anion-exchange chromatography were then gently isolated from each other by Sephadex G-50 gel filtration. Pure beta-lactoglobulin was also obtained by anion-exchange chromatography of the ammonium sulfate-precipitated globulins. Using enzyme-linked immunosorbent assay (ELISA), Western blotting, and ELISA inhibition assay, antigenicity of the purified proteins was evaluated. Our results showed high purity and well-preserved antigenicity of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin thus purified.     

Blood concentrations of Th2-type immunoglobulins are selectively increased in weanling mice subjected to acute malnutrition

Male and female C57BL/6J mice, initially 19 days old, consumed a complete purified diet either ad libitum (age-matched control) or in restricted daily quantities (energy deficiency), or they consumed a purified isocaloric low-protein diet ad libitum (protein and energy deficit). In a 14-day experimental period, malnourished animals lost approximately 1.5% of their initial body weight daily. Zero-time controls, 19 days old, were also included in the study. Serum levels of Th2-type (IgG1 and IgE) and Th1-type (IgG2a and IgG3) immunoglobulins were quantified by enzyme-linked immunosorbent assay, and total IgG concentration was also assessed. Both malnourished groups exhibited high serum concentrations of IgG1 and IgE relative to the age-matched control group, whereas levels of the Th1-type immunoglobulins were unaffected. Total IgG concentration in the malnourished groups reflected the usual finding in humans (i.e., no effect or elevated). The results are consistent with the proposition of Th2-polarized immune competence in acute weanling deficiencies of energy, protein, or both.     

The Effect of Iron–Vitamin C Co-supplementation on Biomarkers of Oxidative Stress in Iron-Deficient Female Youth

There is no study that assessed the effect of co-supplementation of iron and vitamin C on biomarkers of oxidative stress in non-anemic iron-deficient females. We investigated the effects of iron vs. iron?+?vitamin C co-supplementation on biomarkers of oxidative stress in iron-deficient girls. In a double-blind randomized controlled clinical trial, performed among 60 non-anemic iron-deficient girls, participants were randomly assigned to receive either 50 mg/day elemental iron supplements or 50 mg/day elemental iron?+?500 mg/day ascorbic acid for 12 weeks. Fasting blood samples were taken at baseline, weeks 6 and 12 for assessment of biomarkers of oxidative stress. Compared with the baseline levels, both iron and iron?+?vitamin C supplementation resulted in a significant reduction in serum malondialdehyde (MDA) levels (P _time?<?0.001) and remarkable elevation in serum total antioxidant capacity (TAC; P _time?<?0.001) and vitamin C levels (P _time?=?0.001); however, comparing the two groups we failed to find an additional effect of iron?+?vitamin C supplementation to that of iron alone on serum TAC and MDA levels (P _group was not statistically significant). Iron?+?vitamin C supplementation influenced serum vitamin C levels much more than that by iron alone (P _group?<?0.01). We also found a significant interaction term between time and group about serum vitamin C levels while this interaction was not significant about serum TAC and MDA levels. In conclusion, we found that iron supplementation with/without vitamin C improve biomarkers of oxidative stress among non-anemic iron-deficient females and may strengthen the antioxidant defense system by decreasing reactive oxygen species. Co-supplementation of iron?+?vitamin C has no further effect on oxidative stress compared with iron alone.     

Comparison of the chaperon activity of glycerol and α-casein on amyloid formation of κ-casein in the presence of glycine and arginine

Amyloids are insoluble fibers which arise from inappropriately folded versions of proteins and have been associated with the pathology of many neurodegenerative diseases. α-Casein is one of the major components of the casein family which is known to show chaperone-like activity. Glycerol is a polyol compound which acts as a chemical chaperone to increase protein stability and inhibit protein aggregation. In this study, the effect of arginine and glycine on the chaperone ability of α-casein and glycerol against order aggregation of κ-casein was investigated and compared. We found that these additives reduced the chaperone ability of α-casein against the amyloid formation of κ-casein, especially in the presence of arginine. Importantly, our results show that the chaperone action of glycerol is enhanced in the presence of both arginine and glycine. Accordingly, our results suggest that these small molecules associated with glycerol, especially glycine, should be considered as a mechanism for the treatment of amyloid disease.