The potency and durability of DNA- and protein-based vaccines against Leishmania major evaluated using low-dose, intradermal challenge

DNA- and protein- based vaccines against cutaneous leishmaniasis due to Leishmania major were evaluated using a challenge model that more closely reproduces the pathology and immunity associated with sand fly-transmitted infection. C57BL/6 mice were vaccinated s.c. with a mixture of plasmid DNAs encoding the Leishmania Ags LACK, LmSTI1, and TSA (AgDNA), or with autoclaved L. major promastigotes (ALM) plus rIL-12, and the mice were challenged by inoculation of 100 metacyclic promastigotes in the ear dermis. When challenged at 2 wk postvaccination, mice receiving AgDNA or ALM/rIL-12 were completely protected against the development of dermal lesions, and both groups had a 100-fold reduction in peak dermal parasite loads compared with controls. When challenged at 12 wk, mice vaccinated with ALM/rIL-12 maintained partial protection against dermal lesions and their parasite loads were no longer significantly reduced, whereas the mice vaccinated with AgDNA remained completely protected and had a 1000-fold reduction in dermal parasite loads. Mice vaccinated with AgDNA also harbored few, if any, parasites in the skin during the chronic phase, and their ability to transmit L. major to vector sand flies was completely abrogated. The durable protection in mice vaccinated with AgDNA was associated with the recruitment of both CD8(+) and CD4(+) T cells to the site of intradermal challenge and with IFN-gamma production by CD8(+) T cells in lymph nodes draining the challenge site. These data suggest that under conditions of natural challenge, DNA vaccination has the capacity to confer complete protection against cutaneous leishmaniasis and to prevent the establishment of infection reservoirs.     

Inhibition of IgG-triggered human eosinophil function by IL-4

Triggering of eosinophil secretory and cytotoxic functions by stimulation of the IgG and IgE FcR is thought to have major importance in the pathophysiology of tissue eosinophilia. We studied the ability of human rIL-4 to regulate this triggering event in human eosinophils. At doses ranging from 0.1 to 10 pg/ml, IL-4 suppressed eosinophil secretion of beta-glucuronidase and arylsulfatase by up to 65% after stimulation with IgG-coated Sepharose beads. This effect required prolonged preincubation (16 h) of eosinophils with IL-4; no effect was detected after 1 h preincubation. Enzyme secretion stimulated by IgE-coated beads was not affected. Further, IL-4 (after 16 h preincubation), suppressed eosinophil antibody-dependent killing of schistosomula (Schistosoma mansoni) targets by 24 to 39% in four experiments (p less than 0.05). Flow microfluorimetry analysis showed that IL-4 reduced the expression of IgG FcR, but not IgE FcR, suggesting that this mechanism underlies the suppression of IgG-mediated secretion. Taken collectively, these results demonstrate a mechanism for T lymphocyte suppression of IgG-stimulated eosinophil functions via IL-4.     

Overcoming challenges in WAVE Bioreactors without feedback controls for pH and dissolved oxygen

The biopharmaceutical industry is increasing its use of the WAVE Bioreactor for culturing cells. Although this disposable bioreactor can be equipped to provide real-time pH and dissolved oxygen (DO) monitoring and control, our goal was to develop a process for culturing CHO cells in this system without relying on pH and DO feedback controls. After identifying challenges in culturing cells without controlling for pH and DO in the WAVE Bioreactor, we characterized O(2) and CO(2) transfer in the system. From these cell-free studies, we identified rock rate and rock angle as key parameters affecting O(2) transfer. We also identified the concentration of CO(2) in the incoming gas and the rate of gas flow into the headspace as key parameters affecting CO(2) transfer--and therefore pH--in the disposable culture chamber. Using a full-factorial design to evaluate the rock rate, rock angle, and gas flow rate defined for this WAVE Bioreactor process, we found comparable cell growth and pH profiles in the ranges tested for these three parameters in two CHO cell lines. This process supported cell growth, and maintained pH and DO within our desired range--pH 6.8-7.2 and DO exceeding 20% of air saturation--for six CHO cell lines, and it also demonstrated comparable cell growth and viability with the stirred-tank bioreactor process with online pH and DO control. By eliminating the use of pH and DO probes, this process provides a simple and more cost-effective method for culturing cells in the WAVE Bioreactor.     

Bio-efficacy of different oil carriers in ultra low volume sprayer for management of pod borer,Helicoverpa armigera (Hübner) in pigeonpea ecosystem

A study was conducted to evaluate the bioefficacy of different oil carriers (sunflower oil, seamum oil, neem oil, mineral oil), sugar solution and water formulation to be used in ultra low volume sprayer (ULV) along with rynaxypyr 18.5 SC (Coragen) for the management of pod borer at the Agricultural Research Station, Gulbarga, Karnataka, India during the kharif season of 2011–2012. The results revealed that the sesamum oil carrier fits well with rynaxypyr 18.5 SC and recorded the lowest mean pod borer larval population after last spray (1.0 per five plants) the lowest pod damage (23.20%), grain damage (18.30%) and higher grain yield (9.43?q/ha) with a high B:C ratio of 3.64 in the plot sprayed with the sesamum oil carrier. The findings suggested that the sesamum oil carrier in ULV sprayer is suitable and practicable in pigeonpea ecosystem for effective management of pod borer with no risk of phytotoxicity, spray drift and operational difficulties. The foregoing studies indicated that the performance of sesamum oil carriers was superior to other carriers in terms of suppression of larval population, reduction in pod damage and harnessing higher yield.     

Mineralization of pristine chitosan film through biomimetic process

The biomineralization of pristine chitosan film without any prior surface treatment was evaluated by immersing the film in simulated body fluid (SBF) at 37 °C for 3 weeks. The film was prepared by solvent casting method using chitosan of known degree of deacetylation (DD). The formation of the hydroxyapatite (HA) phase on the film surface after immersion was studied periodically by X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), energy dispersive X-ray analysis (EDX) and scanning electron microscopy (SEM) methods. The electron micrographs showed the morphology of the deposited apatite as small globules appearing uniformly throughout the films surfaces. The Ca/P ratio of the apatite was found to increase with increase in immersion time and approaching towards the stoichiometric value of the HA phase. The mineralized chitosan film could be of promising support to hard tissue regeneration.     

Inhibition of telomerase activity and human telomerase reverse transcriptase gene expression by histone deacetylase inhibitor in human brain cancer cells

The aim of the present study is to investigate the effect of histone deacetylase inhibitor, trichostatin A (TSA) on the cell growth, apoptosis, genomic DNA damage and the expression of telomerase and associated factors in human normal and brain cancer cells. Here, human normal un-transformed fibroblasts (MRC-5), human normal hTERT-immortalised fibroblasts (hTERT-BJ1) and human brain cancer cell lines (glioblastoma cell line, A-172 and medulloblastoma cell line, ONS-76) were treated with 0.5–3.0 μM TSA for 24 h. Exposure to TSA resulted in apoptosis in a dose-dependent manner in the brain cancer cells. Glioblastoma cell line (A-172) displayed higher sensitivity to TSA-induced cell killing effect and apoptosis than the medulloblastoma cell line (ONS-76). The brain cancer cell lines and hTERT-BJ1 cell line displayed significant inhibition in telomerase activity and hTERT mRNA level after 2 μM TSA treatment. Elevated expressions of p53 and p21 with a decrease in cyclin-D level supported the observation on cell cycle arrest following TSA treatment. Upregulation of Bax and cytochrome c correlated with the apoptotic events in TSA-treated cells. This study suggests that telomerase and hTERT might be the primary targets of TSA which may have the potential to be used as a telomerase inhibitor in cancer therapy.     

A minimal element in 5'UTR of insulin mRNA mediates its translational regulation by glucose

Glucose induced translation of insulin in pancreatic beta cells is mediated by the 5'UTR of insulin mRNA. We determined the minimal sequence/structure in the 5'UTR of rat insulin gene1 for this regulation. We show that specific factors in the pancreatic islets bind to the 5'UTR of the insulin mRNA upon glucose stimulation. We identified a minimal 29-nucleotide element in the 5'UTR that is sufficient to form the complex, and confer glucose mediated translation activation. Conserved residues in the predicted stem loop region of the un-translated region (UTR) seem to be important for the complex formation and the translation regulation.     

Analysis of Maize (Zea mays L.) Seedling Roots with the High-Throughput Image Analysis Tool ARIA (Automatic Root Image Analysis)

The maize root system is crucial for plant establishment as well as water and nutrient uptake. There is substantial genetic and phenotypic variation for root architecture, which gives opportunity for selection. Root traits, however, have not been used as selection criterion mainly due to the difficulty in measuring them, as well as their quantitative mode of inheritance. Seedling root traits offer an opportunity to study multiple individuals and to enable repeated measurements per year as compared to adult root phenotyping. We developed a new software framework to capture various traits from a single image of seedling roots. This framework is based on the mathematical notion of converting images of roots into an equivalent graph. This allows automated querying of multiple traits simply as graph operations. This framework is furthermore extendable to 3D tomography image data. In order to evaluate this tool, a subset of the 384 inbred lines from the Ames panel, for which extensive genotype by sequencing data are available, was investigated. A genome wide association study was applied to this panel for two traits, Total Root Length and Total Surface Area, captured from seedling root images from WinRhizo Pro 9.0 and the current framework (called ARIA) for comparison using 135,311 single nucleotide polymorphism markers. The trait Total Root Length was found to have significant SNPs in similar regions of the genome when analyzed by both programs. This high-throughput trait capture software system allows for large phenotyping experiments and can help to establish relationships between developmental stages between seedling and adult traits in the future.