Phenotyping of circulating CD8⁺ T cell subsets in human cutaneous leishmaniasis

Recovery from CL is usually accompanied with long-lasting protection and induction of strong immune response. The phenotypes, generation and maintenance of central (=T_CM) and effector (=T_EM) memory T cell subsets in human leishmaniasis are not well known. Profile of T cell subsets were analyzed on peripheral CD8⁺ T cells from volunteers with history of cutaneous leishmaniasis (HCL).In HCL and control groups, mean frequencies of CCR7⁺CD45RA⁺CD8⁺ naïve and CCR7^?CD45RA^?CD8⁺ T_EM cells were higher than other subsets before culture, but after stimulation with soluble Leishmania antigen, the frequency of naïve T cells was significantly decreased and the frequency of T_EM cells was significantly increased. T_EM phenotype composed the highest portion of proliferating Carboxy Fluorescein diacetate Succinimidyl Ester (CFSE)-dim population which was significantly higher in HCL volunteers than in control group. Stimulation of isolated CD8⁺ memory T cells, but not naïve T cells, from HCL volunteers induced a significantly higher IFN-γ production compared with that of healthy controls. Intracellular IFN-γ staining provided the same result.Memory population is shown to be responsible for Leishmania-induced IFN-γ production. Leishmania-reactive proliferating T_EM cells were identified as the most frequent subset which may play a role in recall immune response and protection against Leishmania infection.     

Altered serum pro-inflammatory cytokines in children with Down's syndrome

There are reports showing that pro-inflammatory cytokines are dysregulated in patients with Down's syndrome (DS). However, most of these reports concern adults. We analyzed cytokine levels in serum samples from children with DS, and compared them with samples from intellectually disabled (ID), and healthy, control children. Blood samples were collected from 24 DS, 24 age-/sex-matched ID, and 24 age-/sex-matched healthy, control children. Serum levels of the cytokines IL-5, IL-10, IL-13, IFN-γ, and TNF-α were measured using a sandwich ELISA method, . The age range of the children was 1-15 years, with a mean ± SD of 5.75 ± 4.36 years. TNF-α levels were significantly higher in the DS and ID groups compared with those found in healthy, control children (P<0.05). The DS and ID groups had significantly higher IFN-γ levels compared with healthy, control children (P = 0.0002 and P<0.01, respectively), with significant higher levels in the DS than the ID group (P<0.05). Serum from the ID group showed significantly higher IL-10 levels compared with those from the DS group (P<0.05), but not the healthy, control group. Significant correlations were found between the differences in TNF-α and IFN-γ levels, in both ID (rs = 0.558; P = 0.005) and DS children (rs = 0.405; P<0.05). There were no significant differences found in serum levels of IL-13 between the groups, and IL-5 was not detectable in any of the serum samples. Levels of TNF-α and IFN-γ were increased, and IL-10 decreased in serum from children with DS. It may be that these differences contribute to the clinical symptoms seen in DS: consequently, these pro-inflammatory cytokines might be useful as early biomarkers of the disorders associated with DS.     

Comparison of stool antigen immunoassay and serology for screening for Helicobacter pylori infection in intellectually disabled children

Diagnosis of active Helicobacter pylori infection in intellectually disabled (ID) children is problematic because they are unable to cooperate with performance of invasive tests. In this study, the non‐invasive methods of measuring serum IgG antibody concentrations and performing stool antigen tests were used to screen for H. pylori infection in ID children. Eighty‐seven children with intellectual disabilities were studied. The amount of serum IgG antibody against H. pylori was measured by the ELISA method. Stool samples were examined using an amplified IDEIA HpStAR kit. To assess categorical variables, X², Fisher's exact and Kappa tests were used. The stool antigen tests showed that 93.1% of the children had H. pylori antigen and the serology test that 85.1% of children were positive for H. pylori IgG antibodies. Agreement between results of H. pylori stool antigen (HpSA) testing and IgG antibody serology was 82.8%; however, according to the kappa measure of agreement this agreement is not statistically significant (value, 0.128; P = 0.19). Discordant results were observed for 15 children (17.2%): 11 (12.6%) who were positive on HpSA test but negative by serology and 4 (4.6%) who were IgG seropositive but had negative HpSA tests. This study showed a notably higher rate of H. pylori infection in ID children than has been reported by others for non‐ID children from the same geographical area. The HpSA test is a valid method for primary screening for H. pylori infection in ID children; it detects the specific antigens shed during active infections and has less cross‐reactivity than serological tests that detect antibodies. HpSA is a sensitive non‐invasive method for detecting infection in ID children and may serve as an accurate alternative to serology.     

YSL3-mediated copper distribution is required for fertility,seed size and protein accumulation in Brachypodium

Addressing the looming global food security crisis requires the development of high-yielding crops. In agricultural soils, deficiency in the micronutrient copper significantly decreases grain yield in wheat (Triticum aestivum), a globally important crop. In cereals, grain yield is determined by inflorescence architecture, flower fertility, grain size, and weight. Whether copper is involved in these processes, and how it is delivered to the reproductive organs is not well understood. We show that copper deficiency alters not only the grain set but also flower development in both wheat and its recognized model, Brachypodium distachyon. We then show that the Brachypodium yellow stripe-like 3 (YSL3) transporter localizes to the phloem, transports copper in frog (Xenopus laevis) oocytes, and facilitates copper delivery to reproductive organs and grains. Failure to deliver copper, but not iron, zinc, or manganese to these structures in the ysl3 CRISPR-Cas9 mutant results in delayed flowering, altered inflorescence architecture, reduced floret fertility, grain size, weight, and protein accumulation. These defects are rescued by copper supplementation and are complemented by YSL3 cDNA. This knowledge will help to devise sustainable approaches for improving grain yield in regions where soil quality is a major obstacle for crop production. Copper distribution by a phloem-localized transporter is essential for the transition to flowering, inflorescence architecture, floret fertility, size, weight, and protein accumulation in seeds.

Copper distribution by a phloem-localized transporter is essential for the transition to flowering, inflorescence architecture, floret fertility, size, weight and protein accumulation in seeds.     

Subsite mapping of purified glucoamylase I, II, III produced by Arthrobotrys amerospora ATCC 34468

Arthrobotrys amerospora ATCC 34468 produced glucoamylase in a medium containing maize starch as carbon source. On native PAGE, crude glucoamylase showed three isoenzymes which were designated as Glu I, Glu II, Glu III according to their electrophoretic mobility. These were purified by column chromatography techniques. The energy of binding for each glucoamylase was calculated using Hiromi's kinetic based calculation. At subsite 1, the binding energies for Glu I, II and III were found to be negative.     

Expression of nerve growth factor receptor during human peripheral nerve development

The expression of NGF receptors on human Schwann cells during development and myelination and in culture was analyzed using a murine monoclonal antibody to human NGF receptor. Nonmyelinated femoral nerves from 13- to 14-week fetuses stained strongly for NGF receptor, whereas tissues from later stages of development showed a decrease in the staining intensity. These changes correlated with the initiation of myelination (17-19 weeks), as observed by phase-contrast and electron microscopy, and the reactivity with monoclonal antibody 4C5, a marker of mature Schwann cells. In adult nerves, only the perineurium and few endoneurial cells were stained with anti-NGF receptor antibody. Cultured human fetal Schwann cells were positive for NGF receptor by immunofluorescence irregardless of donor age or length of time in culture. The decreased staining of NGF receptor with nerve maturation may reflect a dependence of antigen expression on Schwann cell differentiation and/or neuron-Schwann cell interaction.     

Peptide 53-78 of myelin P2 protein is a T cell epitope for the induction of experimental autoimmune neuritis.

We have recently described the clinical and pathological features of experimental autoimmune neuritis (EAN) in Lewis rats inoculated with varying doses of a synthetic peptide corresponding to the amino acid residues 53-78 of bovine P2 protein (SP-26). Immunization with this synthetic peptide was able to induce severe clinical and pathological characteristics of EAN. We are now reporting that, SP-26 T cell lines derived from spleen and lymph node cell populations of such immunized rats, upon being triggered by SP-26, can adoptively transfer severe clinical and histological signs of EAN to naive syngeneic recipients. The disease appears 7-8 days postinoculation of the cells and persist 5-10 days. The pathological features were indistinguishable from SP-26-induced active EAN which appears 12-15 days after sensitization. Examination of the surface phenotype of the cells that were used for the passive transfer of EAN by FACS analysis, showed majority of the cells to be CD4+, Ia+ cells.     

Arabidopsis Ca2+-ATPases 1, 2, and 7 in the endoplasmic reticulum contribute to growth and pollen fitness

Generating cellular Ca²⁺ signals requires coordinated transport activities from both Ca²⁺ influx and efflux pathways. In Arabidopsis (Arabidopsis thaliana), multiple efflux pathways exist, some of which involve Ca²⁺-pumps belonging to the Autoinhibited Ca²⁺-ATPase (ACA) family. Here, we show that ACA1, 2, and 7 localize to the endoplasmic reticulum (ER) and are important for plant growth and pollen fertility. While phenotypes for plants harboring single-gene knockouts (KOs) were weak or undetected, a triple KO of aca1/2/7 displayed a 2.6-fold decrease in pollen transmission efficiency, whereas inheritance through female gametes was normal. The triple KO also resulted in smaller rosettes showing a high frequency of lesions. Both vegetative and reproductive phenotypes were rescued by transgenes encoding either ACA1, 2, or 7, suggesting that all three isoforms are biochemically redundant. Lesions were suppressed by expression of a transgene encoding NahG, an enzyme that degrades salicylic acid (SA). Triple KO mutants showed elevated mRNA expression for two SA-inducible marker genes, Pathogenesis-related1 (PR1) and PR2. The aca1/2/7 lesion phenotype was similar but less severe than SA-dependent lesions associated with a double KO of vacuolar pumps aca4 and 11. Imaging of Ca²⁺ dynamics triggered by blue light or the pathogen elicitor flg22 revealed that aca1/2/7 mutants display Ca²⁺ transients with increased magnitudes and durations. Together, these results indicate that ER-localized ACAs play important roles in regulating Ca²⁺ signals, and that the loss of these pumps results in male fertility and vegetative growth deficiencies.

Autoinhibited Ca²⁺ pumps in the endoplasmic reticulum make important contributions to controlling the magnitude and duration of Ca²⁺ signals.