Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells

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Protective and therapeutic efficacy study of divalent fusion protein rL7/L12-Omp25 against B. abortus 544 in presence of IFNγ

Brucella as intracellular pathogen requires a coordinate interaction between Th1 subset of gamma interferon-secreting CD4 T cells and CD8 T cells for optimal protective immunity. It was previously recognized that L7/L12 as T cell-reactive antigen from the pathogen. On other hand, Omp25 was found as another antigen to provide protection against the Brucella infection by eliciting both Th1 and Th2 type of immune responses in mice. Here, we analyzed the prophylactic and therapeutic efficacy of a divalent fusion protein (rL7/L12-Omp25) comprising these two promising immunogens of Brucella in the presence of murine IFN-gamma in mice against B. abortus 544 challenge. rIFN-gamma with rL7/L12-Omp25 resulted in superior immune response when compared to the animal vaccine strain B. abortus S19. The vaccine candidate caused dominance of IgG1 over IgG2a and upregulated cytokine secretion (IFN-gamma, TNF-α, and IL-10) among immunized mice. Moreover, the antigen in combination with murine IFN-gamma elicited stronger cell-mediated immune response among the immunized animals when compared to standard vaccine (S19). The registered log protection unit among challenged mice with B. abortus 544 pathogen was 2.16, p = 0.0001 when rL7/L12-Omp25 was administered alone and 2.4, p = 0.0001 when it was administered along with rIFN-gamma. However, the molecule upon administration with murine IFN-gamma imparted very minimal or no therapeutic effect against brucellosis. To conclude, our study demonstrates the potential of rL7/L12-Omp25 as an immunogen of prospective and efficient prophylaxis as it is capable of eliciting both cell-mediated and humoral immune responses against brucellosis.

     

Polysome shift assay for direct measurement of miRNA inhibition by anti-miRNA drugs

Anti-miRNA (anti-miR) oligonucleotide drugs are being developed to inhibit overactive miRNAs linked to disease. To help facilitate the transition from concept to clinic, new research tools are required. Here we report a novel method—miRNA Polysome Shift Assay (miPSA)—for direct measurement of miRNA engagement by anti-miR, which is more robust than conventional pharmacodynamics using downstream target gene derepression. The method takes advantage of size differences between active and inhibited miRNA complexes. Active miRNAs bind target mRNAs in high molecular weight polysome complexes, while inhibited miRNAs are sterically blocked by anti-miRs from forming this interaction. These two states can be assessed by fractionating tissue or cell lysates using differential ultracentrifugation through sucrose gradients. Accordingly, anti-miR treatment causes a specific shift of cognate miRNA from heavy to light density fractions. The magnitude of this shift is dose-responsive and maintains a linear relationship with downstream target gene derepression while providing a substantially higher dynamic window for aiding drug discovery. In contrast, we found that the commonly used ‘RT-interference’ approach, which assumes that inhibited miRNA is undetectable by RT-qPCR, can yield unreliable results that poorly reflect the binding stoichiometry of anti-miR to miRNA. We also demonstrate that the miPSA has additional utility in assessing anti-miR cross-reactivity with miRNAs sharing similar seed sequences.     

The effects of zinc and lanthanum on calcium uptake by mitochondria and fragmented sarcoplasmic reticulum of frog skeletal muscle

Calcium uptake by mitochondria and fragmented sarcoplasmic reticulum (FSR) isolated from frog skeletal muscle was studied. These fractions were characterized by electron microscopy, succinic dehydrogenase assay and by using mitochondrial inhibitors. With high (100 μM) Ca in the medium, the Ca accumulating capacity of the two fractions was similar. Zinc in concentrations of 5–10 μM in the medium had no effect on Ca uptake by either fraction whereas higher concentration of Zn (25 μM) reduced Ca uptake in both fractions. Five micromolar lanthanum lowered Ca uptake by 70% in mitochondria but had no effect on Ca uptake by FSR. With 10 and 25 μM La, Ca uptake by FSR decreased by 12 and 20% respectively. Addition of La (5 μM) to Ca-loaded mitochondria had no effect indicating that La could only interfere with the Ca binding step and was unable to release Ca that was already stored. In the medium that originally contained low (10 μM) Ca FSR was able to reduce the Ca concentration below 0.1 μM. In contrast mitochondria, although possessing an equal capacity for Ca uptake were unable to accumulate Ca from the medium when Ca was lowered to approximately 4 μM. Presence of 5–10 μM La in the low Ca medium had no effect on the total amount of Ca taken up by FSR in two minutes but reduced the rate of Ca uptake significantly. The relation of the effects of Zn and La on the isolated fractions to their reported effects on the contractile response of skeletal muscle is discussed.     

Luteinizing hormone in blood plasma of post-partum buffaloes (Bubalus bubalis)

The changes in luteinizing hormone concentration were measured by heterologous double-antibody radioimmunoassay in blood plasma of 28 Murrah buffaloes. The LH concentration fluctuated between 0.22 to 0.78 ng/ml during first 21 days post-partum. The level increased significantly (P < 0.001) at estrus. The basal LH concentration of second and third week post-partum was inversely related to the first post-partum ovulation interval.     

Effect of estrogen treatment on calcium uptake by the rat uterine smooth muscle

The effect of estrogenization on cellular 45Ca uptake by the isolated uterine strips was studied. Whereas estrogenization 1 h before sacrifice of ovariectomized rats caused no significant change in Ca uptake by the isolated uteri, exposure to estrogen 24 h before sacrifice resulted in a significant increase in uptake. Following continuous exposure for 72 h to estrogen, the uptake of Ca was increasd by more than two-fold. Increased Ca uptake following estrogenization may largely account for the increased contractile responses of the uterine smooth muscle particularly those induced by potassium depolarization.     

Milk progesterone levels to monitor reproductive status of Murrah buffalo

Concentration of progesterone in whole milk was used to diagnose pregnancy in 44 lactating buffaloes. Milk samples were taken from days 19 to 27 post breeding and analysed for progesterone by radio-immunoassay. A level exceeding 10 ng/ml of milk was taken as an indication of pregnancy. Using this criterion, the accuracy of the pregnancy test from a single milk sample on 19, 21, 23, 25 and 27 days after insemination ranged from 71.42 to 86.95% and 86 to 87.50% for pregnant and non-pregnant animals, respectively. Milk progesterone concentrations were significantly higher (P / 0.001) in pregnant compared with those in non-pregnant buffaloes on day 19, 21, 23, 25 and 27 post-insemination.     

Determination of Linkages in β-d-Glucans From Phaseolus aureus by exo-β-(1→3)-d-Glucanase

An alkali-insoluble glucan synthesized from UDP-d-glucose by the particulate enzyme system from Phaseolus aureus is hydrolyzed by a highly purified exo-beta-(1 --> 3)-d-glucanase to d-glucose, to the extent of 91% in 24 hr.The alkali-insoluble glucan from GDP-d-glucose formed by the particulate enzyme system from the same plant which is known to be (from chemical data) a beta-(1 --> 4)-d-glucan (cellulose) is not acted upon by this glucanase.     

The mycoflora of domesticated and wild bees (Apoidea)

Fungi including yeasts are common in the honey stomachs and provisions of diverse bees. They may be parasites, commensals or mutualistic. Yeasts, singly or in association with bacteria, are pioneer colonizers during a microbial succession in larval cells of many subterranean bees. They are followed by fungi such asAspergillus, Penicillium, Emericellopsis, Sartorya, Pseudoarachniotus, Gymnoascus, Carpenteles andFusarium. Aspergillus flavus andSaccharomyces spp. are pathogenic to many species of bees, and fungi are the main cause of declining alkali bee populations. There are 124 species of fungi, including 36 new records, associated with Apoidea; 49 species are associated with alkali bees.Cooperative investigations of the Plant Sciences Research (L. R. Batra) and Entomology (G. E. Bohart) Divisions, Agricultural Research Service, U.S. Department of Agriculture, and the Utah Agricultural Experiment Station, Logan, Utah.