An association between a lymphocyte antigen in sheep and the response to vaccination against the parasite Trichostrongylus colubriformis

Lymphocyte antigens were tested in sheep which had been selected for responsiveness to vaccination against the intestinal nematode Trichostrongylus colubriformis. These sheep had been bred in an assortative mating programme which produced offspring designated as either “high responders” or “low responders”, with highly heritable resistance or susceptibility.Ovine lymphocyte antigen (OLA) typing antisera were obtained from parous ewes in the course of matings which produced the high and low responder flocks. A particular antigen (SY1) was found to be present in high frequency on the lymphocytes of high responder (72·2%) and in lower frequency (21·9%) on the lymphocytes of low responder rams. In ewes, the frequency for high responders was 65·7% and for low responders it was 33·5%. A similar association between the SY1 antigen and low faecal egg count was found in random-bred sheep which had been vaccinated with irradiated larvae and challenged with normal larvae. The conclusion was drawn that this lymphocyte antigen was likely to be part of the sheep major histocompatibility complex which influenced the immune response of sheep to vaccination against the parasite.     

Electron-microscopic and immunocytochemical analyses of Weibel-Palade bodies in the human umbilical vein during pregnancy

Summary The present study was done to elucidate the biological significance of the Weibel-Palade body of human umbilical vein endothelial cells. Quantitative determinations of these endothelial-specific granules throughout pregnancy revealed that their numbers and size per cell profile were maintained at low levels from 12 to 19 weeks of gestation; then both rapidly increased from 33 weeks to full term. This increase coincided with the development of the rough endoplasmic reticulum and an increase in the number of endothelial cell pinocytotic vesicles. Light-microscopic peroxidase anti-peroxidase and electron-microscopic protein A-gold techniques provided evidence that factor VIII-related antigen was localized in the Weibel-Palade bodies. Furthermore, in vitro treatment of incubated umbilical vein tissue with compound 48/80, a histamine releaser, induced degranulation of Weibel-Palade bodies from the endothelium. The present study indicates that Weibel-Palade bodies are storage sites of both histamine and factor VIII-related antigen and have an important role in the obliteration of this vessel.     

Dynamics of the Coreceptor-LCK Interactions during T Cell Development Shape the Self-Reactivity of Peripheral CD4 and CD8 T Cells

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Characterization of messenger RNA from epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi

The cell-free translation products of polyribosomal and post-polyribosomal mRNAs from the non-infective epimastigotes and the infective metacyclic trypomastigotes of the parasitic protozoan Trypanosoma cruzi were compared by two-dimensional polyacrylamide gel electrophoresis. The result show that although many polypeptides are conserved, quantitative and qualitative differences are observed between both differentiation stages. The results also indicate the existence of post-polyribosomal mRNAs in equilibrium with polyribosomal counterparts. The immunoprecipitation of the in vitro synthesized polypeptides with chagasic human serum and the serum raised against an 85-kDa glycoprotein (P2-WGA), potentially involved in the process of T. cruzi penetration into mammalian cells, shows that while the chagasic serum recognizes the same 72-kDa, 68-kDa and 46-kDa polypeptides in both differentiation stages, the anti-P2-WGA serum immunoprecipitates a single 48-kDa polypeptide from in vitro translation products of metacyclic trypomastigotes.     

Probing binding mechanism of interleukin-6 and olokizumab: in silico design of potential lead antibodies for autoimmune and inflammatory diseases

Computer-aided antibody engineering has been successful in the design of new biologics for disease diagnosis and therapeutic interventions. Interleukin-6 (IL-6), a well-recognized drug target for various autoimmune and inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, and psoriasis, was investigated in silico to design potential lead antibodies. Here, crystal structure of IL-6 along with monoclonal antibody olokizumab was explored to predict antigen–antibody (Ag???Ab)-interacting residues using DiscoTope, Paratome, and PyMOL. Tyr56, Tyr103 in heavy chain and Gly30, Ile31 in light chain of olokizumab were mutated with residues Ser, Thr, Tyr, Trp, and Phe. A set of 899 mutant macromolecules were designed, and binding affinity of these macromolecules to IL-6 was evaluated through Ag???Ab docking (ZDOCK, ClusPro, and Rosetta server), binding free-energy calculations using Molecular Mechanics/Poisson Boltzman Surface Area (MM/PBSA) method, and interaction energy estimation. In comparison to olokizumab, eight newly designed theoretical antibodies demonstrated better result in all assessments. Therefore, these newly designed macromolecules were proposed as potential lead antibodies to serve as a therapeutics option for IL-6-mediated diseases.     

Binding of B‐cell maturation antigen to B‐cell activating factor induces survival of multiple myeloma cells by activating Akt and JNK signaling pathways

B‐cell maturation antigen (BCMA) is expressed on normal and malignant plasma cells and represents a potential target for therapeutic intervention. In this study, we characterized the mechanism underlying the protein kinase B (Akt) and c‐Jun N‐terminal kinase (JNK) pathways and BCMA interactions in regulating multiple myeloma (MM) cell survival. It was found that the expression levels of B cell‐activating factor (BAFF) and BCMA were increased in MM cells as compared with those in normal controls. The proliferation of U266 cells was induced by recombinant human BAFF (rhBAFF) and could also be decreased by BCMA siRNA. The expression of Bcl‐2 protein was up‐regulated, and Bax protein was down‐regulated after rhBAFF treatment, which could be reversed by BCMA siRNA. Similarly, the protein p‐JNK and p‐Akt were activated by rhBAFF and could be changed by BCMA siRNA. In addition, the BCMA mRNA and protein expression levels were decreased after treatment with Akt and JNK pathway inhibitors. These results suggest that Akt and JNK pathways are involved in the regulation of BCMA. A novel BAFF/BCMA signalling pathway in MM may be a new therapeutic target for MM. Copyright © 2016 John Wiley & Sons, Ltd.     

Comparison of the relative sensitivity of human lymphocytes and mouse splenocytes to two spindle poisons

Aneugenic compounds act on non-DNA targets to exert genotoxicity via an indirect mechanism. In contrast to DNA-binding agents, these compounds are expected to possess threshold levels of activity. Therefore, the risk for adverse effects following human exposure to an aneugen could be minimal, if the threshold of activity has been clearly determined in vivo and in vitro and providing the human exposure level is below this threshold. Thus, the development of a single-cell model to allow comparisons between in vitro and in vivo threshold values for aneugenic compounds is of importance.The in vivo micronucleus test is one of the main assays used in genetic toxicology, and is often performed in the mouse. Thus, an extensive database is available in the literature. However, there are only few data concerning the in vitro micronucleus assay using mouse cells, as the majority of in vitro micronucleus assays have been performed using human lymphocytes. In addition, there is a lack of data concerning thresholds for any compound using this model.First, we evaluated whether the use of mouse splenocytes would be an acceptable alternative to that of human lymphocytes to identify aneugens. To allow valid comparisons, the two protocols were first harmonized. Thus, phytohemagglutinin (PHA) and concanavalin A were used as specific mitogens for human lymphocytes and mouse splenocytes, respectively, in order to achieve similar cell-proliferation rates. To achieve similar and sufficient numbers of binucleated cells, cytochalasin B was added 44 and 56 h after culture initiation of the human and mouse cells, respectively.Second, we compared the sensitivity of the mouse protocol with that of the human protocol by exposing the cells to the aneugens nocodazole and paclitaxel.There was good reproducibility of the cytotoxic/genotoxic responses of the two cell models following exposure to the aneugens. The sensitivity of the mouse splenocytes to paclitaxel was higher than that of the human lymphocytes. The two cell types were equally sensitive to nocodazole.     

Identification of Tumor Antigens in the HLA Peptidome of Patient-derived Xenograft Tumors in Mouse

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Highlights

• •Sufficient tumor tissues are often unavailable large HLA peptidome discovery.
• •Using patient derived xenograft (PDX) tumors can overcome this limitation.
• •The large PDX HLA peptidomes expand significantly those of the original biopsies.
• •The HLA peptidomes of the PDX tumors included many tumor antigens.

     

Human lymphocyte tubulin: Purification and characterization in normal and leukemic cells

Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119 000. The proteins was shown to consist of two subunits, with molecular weights of 61 000 and 58 000 comparable to the α and β polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 ± 0.86 · 10⁶M^?1 and a level in normal lymphocytes of 1.21 · 10^?2 ± 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and K_a for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocyte tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity or function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.     

Determination of human factor VIII (AHG-associated protein)-antigen in endothelial cells from Xenopus laevis (XTH cells)

Summary AHG-associated protein (AHG-a.p.), the antigen of the blood-clotting factor VIII complex, is a specific endothelial cell marker. Primary (p-XTH) and established (XTH-2) endothelial cells from the hearts of Xenopus laevis tadpoles were assayed for the presence of this marker by means of immunological cross-reaction (recognition of common antigenic sites) with antiserum against human AHG-a.p. Radial imtnunodiffusion and rocket immunoelectrophoresis proved to be insufficiently sensitive, whereas immunofluorescence and a newly evaluated ELISA technique gave positive results. The very high sensitivity of the ELISA (less than 1/240000 of the AHG-a.p. in 0.1 ml human standard plasma can be detected) and the removal of interfering proteins by gel filtration also revealed the presence of AHG-a.p. in the fetal calf serum used in the culture medium; earlier investigations into this subject by a one-step radioimmunoassay had reported negative results. Specially adapted XTH-2 cells were grown in a proteinand serum-free hydrolysate medium in order to demonstrate the presence of a Xenopus-derived antigen that was immunoreactive with the anti-human AHG-a.p.