Exhausted cytotoxic control of Epstein-Barr virus in human lupus

Systemic Lupus Erythematosus (SLE) pathology has long been associated with an increased Epstein-Barr Virus (EBV) seropositivity, viremia and cross-reactive serum antibodies specific for both virus and self. It has therefore been postulated that EBV triggers SLE immunopathology, although the mechanism remains elusive. Here, we investigate whether frequent peaks of EBV viral load in SLE patients are a consequence of dysfunctional anti-EBV CD8⁺ T cell responses. Both inactive and active SLE patients (n = 76 and 42, respectively), have significantly elevated EBV viral loads (P = 0.003 and 0.002, respectively) compared to age- and sex-matched healthy controls (n = 29). Interestingly, less EBV-specific CD8⁺ T cells are able to secrete multiple cytokines (IFN-γ, TNF-α, IL-2 and MIP-1β) in inactive and active SLE patients compared to controls (P = 0.0003 and 0.0084, respectively). Moreover, EBV-specific CD8⁺ T cells are also less cytotoxic in SLE patients than in controls (CD107a expression: P = 0.0009, Granzyme B release: P = 0.0001). Importantly, cytomegalovirus (CMV)-specific responses were not found significantly altered in SLE patients. Furthermore, we demonstrate that EBV-specific CD8⁺ T cell impairment is a consequence of their Programmed Death 1 (PD-1) receptor up-regulation, as blocking this pathway reverses the dysfunctional phenotype. Finally, prospective monitoring of lupus patients revealed that disease flares precede EBV reactivation. In conclusion, EBV-specific CD8⁺ T cell responses in SLE patients are functionally impaired, but EBV reactivation appears to be an aggravating consequence rather than a cause of SLE immunopathology. We therefore propose that autoimmune B cell activation during flares drives frequent EBV reactivation, which contributes in a vicious circle to the perpetuation of immune activation in SLE patients.     

Regulatory T Cell Responses to High-Dose Methylprednisolone in Active Systemic Lupus Erythematosus

Background/Purpose

A slight increase in the proportion of circulating regulatory T (Treg) cells has been reported in systemic lupus erythematosus (SLE) patients taking oral prednisone. The effects of intravenous (IV) high dose methylprednisolone (MP) on Tregs have not yet been described, especially in active SLE.

Methods

We prospectively analyzed the proportion of circulating CD4⁺ Treg cell subsets defined as follows: (1) naïve Treg (nTreg) FoxP3^lowCD45RA⁺ cells; (2) effector Treg (eTreg) FoxP3^highCD45RA⁻ cells; and (3) non-suppressive FoxP3^lowCD45RA⁻ cells (non-regulatory Foxp3^low T cells). Peripheral blood mononuclear cells of patients with active SLE were analyzed before the first infusion of IV high dose MP (day 0) and the following days (day 1, day 2, ±day 3 and ±day 8). The activity of SLE was assessed by the SLEDAI score.

Results

Seventeen patients were included. Following MP infusions, the median (range) percentage of eTregs significantly increased from 1.62% (0.53–8.43) at day 0 to 2.80% (0.83–14.60) at day 1 (p = 0.003 versus day 0), 4.64% (0.50–12.40) at day 2 (p = 0.06 versus day 1) and 7.50% (1.02–20.70) at day 3 (p = 0.008 versus day 2), and declined to baseline values at day 8. Expanding eTreg cells were actively proliferating, as they expressed Ki-67. The frequency of non-regulatory FoxP3^low T cells decreased from 6.39% (3.20–17.70) at day 0 to 4.74% (1.03–9.72) at day 2 (p = 0.005); nTreg frequency did not change. All patients clinically improved immediately after MP pulses. The absence of flare after one year of follow up was associated with a higher frequency of eTregs at day 2.

Conclusion

IV high dose MP induces a rapid, dramatic and transient increase in circulating regulatory T cells. This increase may participate in the preventive effect of MP on subsequent flares in SLE.     

Activity and specificity of TRV-mediated gene editing in plants

Plant trait engineering requires efficient targeted genome-editing technologies. Clustered regularly interspaced palindromic repeats (CRISPRs)/ CRISPR associated (Cas) type II system is used for targeted genome-editing applications across eukaryotic species including plants. Delivery of genome engineering reagents and recovery of mutants remain challenging tasks for in planta applications. Recently, we reported the development of Tobacco rattle virus (TRV)-mediated genome editing in Nicotiana benthamiana. TRV infects the growing points and possesses small genome size; which facilitate cloning, multiplexing, and agroinfections. Here, we report on the persistent activity and specificity of the TRV-mediated CRISPR/Cas9 system for targeted modification of the Nicotiana benthamiana genome. Our data reveal the persistence of the TRV- mediated Cas9 activity for up to 30 d post-agroinefection. Further, our data indicate that TRV-mediated genome editing exhibited no off-target activities at potential off-targets indicating the precision of the system for plant genome engineering. Taken together, our data establish the feasibility and exciting possibilities of using virus-mediated CRISPR/Cas9 for targeted engineering of plant genomes.     

Effectiveness of various Pseudomonas spp. and Burkholderia caryophylli containing ACC-deaminase for improving growth and yield of wheat (Triticum aestivum L.)

This study assessed the possible role of different traits in selected plant growth-promoting rhizobacteria (PGPR) for improving wheat growth and yield under natural conditions. Rhizobacteria exhibiting 1-aminocyclopropane-1-carboxylate (ACC)-deaminase activity were isolated and screened for their growth-promoting activity in wheat under axenic conditions. Five isolates belonging to Pseudomonas and one Burkholderia caryophylli isolate that showed promising performances under axenic conditions were selected and characterized for in vitro ACC-deaminase activity, chitinase activity, auxin production, P solubilization, and root colonization. These isolates were then used as inocula for wheat cultivated under natural conditions in pot and/or field trials. Significant increases in root elongation, root weight, tillers per pot, 1,000-grain weight, and grain and straw yields were observed in response to inoculation with PGPR in the pot trials. Inoculation with these PGPR was also effective under field conditions and increased the wheat growth and yield significantly. However, the efficacy of the strains was inconsistent under the axenic, pot, and field conditions. Pseudomonasfluorescens (ACC50), which exhibited a relatively high in vitro ACC-deaminase activity, chitinase activity, auxin production, and P solubilization and more intensive root colonization, was the most efficient isolate under the field conditions. Therefore, these results demonstrated that ACC-deaminase activity is an efficient parameter for the selection of promising PGPR under axenic conditions. However, additional traits of PGPR, including auxin production, chitinase activity, P solubilization, and root colonization, are also important for selecting PGPR as biofertilizers.     

Excretion of urinary cadmium, copper, and zinc in cadmium-exposed and nonexposed subjects, with special reference to urinary excretion of β2-microglobulin and metallothioneinand metallothionein

The objectives of this study were to examine the association between urinary excretion of cadmium (U-Cd), copper (U-Cu), and zinc (U-Zn) and the severity of two different indicators of renal toxicity (urinary excretion of beta2-microglobulin [U-beta2-MG] and metallothionein [U-MT]) in Cd-exposed subjects compared to controls, and to assess the physiologic mechanisms by which the exposure to environmental Cd affects U-Cd, U-Cu, and U-Zn. The target population included 3508 Cd-exposed and 294 nonexposed participants who received a health survey conducted among the population of the Kakehashi River basin. Increases of U-Cd, U-beta2-MG, and U-MT in the Cd-exposed population were observed relative to excretion of these substances in controls. Regression analysis using a general linear model revealed that the correlations between U-Cd or U-Cu, and U-beta2-MG and between U-Cd, U-Cu or U-Zn, and U-MT were statistically significant in both sexes, but the correlation between U-Zn and U-beta2-MG excretion was significant only in men. These results suggest U-Cd and U-Cu is affected by dysfunction in renal tubular absorption (indicated by U-beta2-MG), whereas not only U-Cd and U-Cu but also U-Zn appear to be a function of renal cellular desquamation (indicated by U-MT).     

Transgenic Tobacco Plants Expressing a Dimeric Single-chain Variable Fragment (scFv) Antibody Against <Emphasis Type="Italic">Salmonella enterica</Emphasis> Serotype Paratyphi B

Transgenic tobacco plants were produced that express an anti-Salmonella enterica single-chain variable fragment (scFv) antibody that binds to the lipopolysaccharide (LPS) of S. enterica Paratyphi B. The coding sequence of this scFv was optimized for expression in tobacco, synthesized and subsequently placed behind three different promoters: an enhanced tobacco constitutive ubiquitous promoter (EntCUP4), and single- and double-enhancer versions of the Cauliflower Mosaic Virus 35S promoter (CaMV 35S). These chimeric genes were introduced into Nicotiana tabacum cv. 81V9 by Agrobacterium-mediated transformation and 50 primary transgenic (T₀) plants per construct were produced. Among these plants, 23 were selected for the ability to express active scFv as determined by enzyme-linked immunosorbent assay (ELISA) using S. enterica LPS as antigen. Expanded bed adsorption-immobilized metal affinity chromatography (EBA-IMAC) was used to purify 41.7 μg of scFv/g from leaf tissue. Gel filtration and surface plasmon resonance (SPR) analyses demonstrated that the purified scFv was active as a dimer or higher-order multimer. In order to identify T₁ plants suitable for development of homozygous lines with heritable scFv expression, kanamycin-resistance segregation analyses were performed to determine the number of T-DNA loci in each T₀ plant, and quantitative ELISA and immunoblot analyses were used to compare expression of active and total anti-Salmonella scFv, respectively, in the T₁ generation. As S. enterica causes millions of enteric fevers and hundreds of thousands of deaths worldwide each year, large-scale production and purification of this scFv will have potential for uses in diagnosis and detection, as a therapeutic agent, and in applications such as water system purification.     

Judicious use of kinetin to improve growth and yield of rice in nickel contaminated soil

The present study was conducted to evaluate the effect of kinetin on growth and yield of rice in the presence and absence of nickel contamination. Rice seedlings were dipped in kinetin solution (10^?3, 10^?4 and 10 M^?5) for 2 hours and transplanted in pots having soil contaminated with nickel sulfate @ 130 mg kg^?1. Experiment was laid out according to completely randomized design with four replications. Results revealed that kinetin significantly improved growth and yield of rice grown in nickel contamination. Kinetin @ 10^?4 M showed maximum improvement in plant height, paddy yield, 1000 grain weight, number of tillers and panicles up to 9.76, 15.72, 11.77, 11.87, and 10.90%, respectively, as compared to plants grown in contaminated soil without kinetin. Kinetin also improved the uptake of nutrients (NPK) in straw and grain of plants grown in Ni contaminated soil. Plants treated with kinetin had more concentration of Ni in shoot but less in grain compared to plants grown in Ni contaminated soil without application of kinetin. The application of kinetin can reduce stress effect on plants through improvement in the biomass of plant. This strategy could be used to increase the phytoextraction of Ni from the contaminated soil.     

Drought stress amelioration in wheat through inoculation with Burkholderia phytofirmans strain PsJN

Plant growth promoting endophytic bacteria Burkholderia phytofirmans PsJN was used to investigate the potential to ameliorate the effects of drought stress on growth, physiology and yield of wheat (Triticum aestivum L.) under natural field conditions. Inoculated and uninoculated (control) seeds of wheat cultivar Sahar 2006 was sown in the field. The plants were exposed to drought stress at different stages of growth (tillering stage and flowering stage) by skipping the respective irrigation. The results showed that drought stress adversely affected the physiological, biochemical and growth parameters of wheat seedlings. It decreased the CO₂ assimilation, stomatal conductance, relative water content, transpiration rate and chlorophyll contents in wheat. Inoculation of wheat with PsJN significantly diluted the adverse effects of drought on relative water contents and CO₂ assimilation rate thus improving the photosynthetic rate, water use efficiency and chlorophyll content over the uninoculated control. Grain yield was also decreased when plants were exposed to drought stress at the tillering and flowering stage, but inoculation resulted in better grain yield (up to 21 and 18 % higher, respectively) than the respective uninoculated control. Similarly, inoculation improved the ionic balance, antioxidant levels, and also increased the nitrogen, phosphorus, potassium and protein concentration in the grains of wheat. The results suggested that B. phytofirmans strain PsJN could be effectively used to improve the growth, physiology and quality of wheat under drought conditions.     

The role of mycorrhizae and plant growth promoting rhizobacteria (PGPR) in improving crop productivity under stressful environments

Both biotic and abiotic stresses are major constrains to agricultural production. Under stress conditions, plant growth is affected by a number of factors such as hormonal and nutritional imbalance, ion toxicity, physiological disorders, susceptibility to diseases, etc. Plant growth under stress conditions may be enhanced by the application of microbial inoculation including plant growth promoting rhizobacteria (PGPR) and mycorrhizal fungi. These microbes can promote plant growth by regulating nutritional and hormonal balance, producing plant growth regulators, solubilizing nutrients and inducing resistance against plant pathogens. In addition to their interactions with plants, these microbes also show synergistic as well as antagonistic interactions with other microbes in the soil environment. These interactions may be vital for sustainable agriculture because they mainly depend on biological processes rather than on agrochemicals to maintain plant growth and development as well as proper soil health under stress conditions. A number of research articles can be deciphered from the literature, which shows the role of rhizobacteria and mycorrhizae alone and/or in combination in enhancing plant growth under stress conditions. However, in contrast, a few review papers are available which discuss the synergistic interactions between rhizobacteria and mycorrhizae for enhancing plant growth under normal (non-stress) or stressful environments. Biological interactions between PGPR and mycorrhizal fungi are believed to cause a cumulative effect on all rhizosphere components, and these interactions are also affected by environmental factors such as soil type, nutrition, moisture and temperature. The present review comprehensively discusses recent developments on the effectiveness of PGPR and mycorrhizal fungi for enhancing plant growth under stressful environments. The key mechanisms involved in plant stress tolerance and the effectiveness of microbial inoculation for enhancing plant growth under stress conditions have been discussed at length in this review. Growth promotion by single and dual inoculation of PGPR and mycorrhizal fungi under stress conditions have also been discussed and reviewed comprehensively.     

Spatial distribution of toxic metals in drinking water sources and their associated health risk in district buner,Northern Pakistan

Vigorous knowledge on the occurrence and distribution of toxic metals (TMs) in theenvironment is needed to assess their toxicological impacts on human health especially in developing countries like Pakistan. For the first time, the concentrations of TMs like Ni, Zn, Pb, Cr, and Cd in both ground and surface water and their potential health risk in the district Buner (Pakistan) were determined using Inductive Coupled Plasma Mass Spectrometry (ICP-MS, Agilent Technologies, 7500 CX, USA) and their results were compared with their safe limits defined by Pakistan Environmental Protection Agency (Pak-EPA) and World Health Organization WHO (2008 WHO (World Health Organization). 2008. Guidelines for drinking water quality, First Addendum to third ed. Recommendations, vol 1. Geneva, Switzerland [Google Scholar]). The concentrations of TMs, like Pb (43% and 86%), Ni (63% and 32%), Cr (41%), and Cd (1% and 20%) in drinking water samples exceeded their respective permissible limits set by Pak-EPA and WHO (2008), respectively. The mean concentrations of TMs were found in decreasing order Ni > Pb > Cr > Cd > Zn. The HRI values of TMs in drinking water samples for both adults and children were observed <1 indicating no health risk to the local population. Excessive marble industries are present in the study area; therefore, it is necessary to reduce metal contamination via proper disposal and treatment of marble wastewater, for which the government should take serious action in the study area.