The immunodominant epitope of centromere-associated protein A displays homology with the transcription factor forkhead box E3 (FOXE3)

Some patients with systemic sclerosis express autoantibodies to centromere-associated protein A (CENP-A), but the exact CENP-A epitope is unknown and it is possible that another protein primes these antibodies. This study aimed to define the amino acids recognized by these antibodies and discover other proteins that may be targeted by or may prime them. Peptide Ap^17–30, the immunodominant epitope of CENP-A, was used to purify anti-CENP-A Ig from sera of 8 patients. Anti-Ap^17–30 Ig reacted dose-dependently with Ap^17–30. Panning a phage display peptide library with anti-Ap^17–30 IgG identified two overlapping motifs (m), pt1m and pt8m, permitting patients classification into subgroups based on their having antibodies for only pt1m, pt8m, or both. The pt1m matched residues 53–62 of forkhead box E3; this peptide (FOXE3p^53–62) behaved similarly in binding and inhibition assays with anti-Ap^17–30 IgG and elicited anti-Ap^17–30 antibodies in mice. This study sets the ground for future investigations on a possible relationship between antibody specificity and clinical manifestations of systemic sclerosis, as well as on a possible role of FOXE3 in priming these antibodies.     

Effect of active immunization with the C-terminal 67-94 (R-28) region of human seminal plasma inhibin on the fecundity of adult male rabbits

PROBLEM: To characterize the specificity of antibodies to the synthetic C-terminal 67-94 (R-28) peptide of human seminal plasma inhibin (hSPI), and to examine the effect of active immunization on the fecundity of adult male animals. METHOD OF STUDY: The specificity of R-28-DT antibodies was tested using enzyme-linked immunosorbent assay, immunohistochemical and immunofluorescence staining, and Western blotting. For fertility studies, adult male rats and rabbits were immunized and mated with females of the same strain. RESULTS: Rabbit antibodies to R-28-DT recognized native hSPI, as demonstrated by sperm agglutination in vivo and in vitro, on the sperm head by immunofluorescence staining, and in the columnar epithelial cells of the prostate by immunohistochemical staining. Immunization with the R-28-DT conjugate elicited a poor antibody response in male rats and their fecundity remained unaffected, while in male rabbits it elicited a good immune response with reduction in their fertility. CONCLUSION: R-28-DT antibodies recognized the native hSPI in the prostatic epithelium and agglutinated washed rat, rabbit, monkey, and human spermatozoa in vitro. Immunization of rabbits caused agglutination of spermatozoa resulting in a decrease in their fecundity. The conjugated R-28 peptide of hSPI offers promise as a male contraceptive.     

Effects of Wy-18, 251 (3-(P-Chlorophenyl) Thiazolo [3, 2-A] Benzimidazole-2-Acetic Acid), Levamisole and Indomethacin on the Generation of Murine T Suppressor Cells In Vitro

Abstract

In vitro culture of normal BALB/c spleen cells with staphylococcal enterotoxin B (SEB) activates antigen non-specific suppressor T cells (Ts) which can be assayed by their ability to suppress antibody production in a plaque assay. Addition of the experimental immunomodulatory drug Wy-18, 251 (10–100 μM) to cultures of spleen cells plus SEB significantly increased Ts activity relative to cultures without the drug. Similar results were obtained with levamisole, but, in contrast, indomethacin (0.1–10 μM) inhibited SEB-induced suppressor cell activity. The ability of Wy-18, 251 to augment Ts activity could be therapeutically useful in the treatment of those autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, in which hyperactive B cell function is a characteristic feature.     

Structures required for poly(A) tail-independent translation overlap with, but are distinct from, cap-independent translation and RNA replication signals at the 3' end of Tobacco necrosis virus RNA

Tobacco necrosis necrovirus (TNV) RNA lacks both a 5' cap and a poly(A) tail but is translated efficiently, owing in part to a Barley yellow dwarf virus (BYDV)-like cap-independent translation element (BTE) in its 3' untranslated region (UTR). Here, we identify sequence downstream of the BTE that is necessary for poly(A) tail-independent translation in vivo by using RNA encoding a luciferase reporter gene flanked by viral UTRs. Deletions and point mutations caused loss of translation that was restored by adding a poly(A) tail, and not by adding a 5' cap. The two 3'-proximal stem-loops in the viral genome contribute to poly(A) tail-independent translation, as well as RNA replication. For all necroviruses, we predict a conserved 3' UTR secondary structure that includes the BTE at one end of a long helical axis and the stem-loops required for poly(A) tail-independent translation and RNA replication at the other end. This work shows that a viral genome can harbor distinct cap- and poly(A) tail-mimic sequences in the 3' UTR.     

Monoclonal antibodies to B and T lymphocyte attenuator (BTLA) have no effect on in vitro B cell proliferation and act to inhibit in vitro T cell proliferation when presented in a cis, but not trans, format relative to the activating stimulus

B and T lymphocyte attenuator (BTLA) is an immunoglobulin superfamily member surface protein expressed on B and T cells. Its ligand, herpesvirus entry mediator (HVEM), is believed to act as a monomeric agonist that signals via the CRD1 of HVEM to inhibit lymphocyte activation: HVEM is also the receptor for lymphotoxin-α and LIGHT, which both bind in the CRD2 and CRD3 domains of the HVEM molecule, and for CD160 which competes with BTLA. We have shown that recombinant HVEM and a panel of different monoclonal antibodies specifically bind murine BTLA on both B and T cells and that some antibodies inhibit anti-CD3ε-induced T cell proliferation in vitro, but only when constrained appropriately with a putatively cross-linking reagent. The antibodies had no significant effect on in vitro T cell proliferation in a mixed lymphocyte reaction (MLR) assay nor on in vitro DO11.10 antigen-induced T cell proliferation. None of these antibodies, nor HVEM-Fc, had any significant effect on in vitro B cell proliferation induced by anti-immunoglobulin M antibodies (±anti-CD40) or lipopolysaccharide. We further elucidated the requirements for inhibition of in vitro T cell proliferation using a beads-based system to demonstrate that the antibodies that inhibited T cell proliferation in vitro were required to be presented to the T cell in a cis, and not trans, format relative to the anti-CD3ε stimulus. We also found that antibodies that inhibited T cell proliferation in vitro had no significant effect on the antibody captured interleukin-2 associated with the in vivo activation of DO11.10 T cells transferred to syngeneic recipient BALB/c mice. These data suggest that there may be specific structural requirements for the BTLA molecule to exert its effect on lymphocyte activation and proliferation.     

Brain γ‐aminobutyric acid (GABA) detection in vivo with the J‐editing 1H MRS technique: a comprehensive methodological evaluation of sensitivity enhancement,macromolecule contamination and test–retest reliability

Abnormalities in brain γ‐aminobutyric acid (GABA) have been implicated in various neuropsychiatric and neurological disorders. However, in vivo GABA detection by ¹H MRS presents significant challenges arising from the low brain concentration, overlap by much stronger resonances and contamination by mobile macromolecule (MM) signals. This study addresses these impediments to reliable brain GABA detection with the J‐editing difference technique on a 3‐T MR system in healthy human subjects by: (i) assessing the sensitivity gains attainable with an eight‐channel phased‐array head coil; (ii) determining the magnitude and anatomic variation of the contamination of GABA by MM; and (iii) estimating the test–retest reliability of the measurement of GABA with this method. Sensitivity gains and test–retest reliability were examined in the dorsolateral prefrontal cortex (DLPFC), whereas MM levels were compared across three cortical regions: DLPFC, the medial prefrontal cortex (MPFC) and the occipital cortex (OCC). A three‐fold higher GABA detection sensitivity was attained with the eight‐channel head coil compared with the standard single‐channel head coil in DLPFC. Despite significant anatomical variation in GABA + MM and MM across the three brain regions (p < 0.05), the contribution of MM to GABA + MM was relatively stable across the three voxels, ranging from 41% to 49%, a non‐significant regional variation (p = 0.58). The test–retest reliability of GABA measurement, expressed as either the ratio to voxel tissue water (W) or to total creatine, was found to be very high for both the single‐channel coil and the eight‐channel phased‐array coil. For the eight‐channel coil, for example, Pearson's correlation coefficient of test vs. retest for GABA/W was 0.98 (R² = 0.96, p = 0.0007), the percentage coefficient of variation (CV) was 1.25% and the intraclass correlation coefficient (ICC) was 0.98. Similar reliability was also found for the co‐edited resonance of combined glutamate and glutamine (Glx) for both coils. Copyright © 2016 John Wiley & Sons, Ltd.