An HLA-binding-motif-aided peptide epitope library: A novel library design for the screening of HLA-DR4-restricted antigenic peptides recognized by CD4+T cells

Susceptibility to a series of autoimmune diseases is strongly associated with particular HLA class II alleles. Identification of T cell clones and antigenic epitopes bound by HLA class II molecules involved in autoimmune diseases is critical to understanding the etiology of these HLA class II-associated diseases. However, establishment of T cell clones in autoimmune diseases is difficult because the antigenic peptides are unknown. Peptide library methods which include all possible peptide sequences offer a potentially powerful tool for the detection of cross-reactive antigenic peptides recognized by T cells. Here, we reduced the number of peptides per mixture by utilizing the known binding motifs of peptides for the HLA-DRB1*0405 molecule and evaluated the effectiveness of this library design. Each library mixture evoked a strong proliferative response in the unprimed peripheral blood lymphocytes (PBL) from HLA-DRB1*0405-positive donors but little or no response in the PBL from HLA-DRB1*0405-negative donors. The library also detected antigenic peptides that activated three antigen-specific T cell lines restricted by HLA-DRB1*0405, with different specificities. The motif-based approach thus presents a powerful method for monitoring T cells in large, heterogeneous T cell populations and is useful for the identification of the mimic peptide epitopes of T cell lines and clones. Received: October 3, 1997 / Accepted: October 23, 1997     

LYMPHOMATOID GRANULOMATOSIS: Light Microscopic, Electron Microscopic and Immunohistochemical Study

A case of lymphomatoid granulomatosis (LYG) involving the lungs, skin, stomach, and possibly the left kidney in a 60-year-old man is presented. The infiltrates in the lungs, stomach, and skin showed a polymorphic appearance, and consisted predominantly of lymphocytes of mature and blastic form and of a few neutrophils, plasma cells, and histiocytes. Most lymphoid cells showed irregularly shaped nuclei and clustered dense bodies, characteristics indicative of T lymphocytes. An immunohistochemical study confirmed the T cell origin of the lymphocytes; i.e. they were positive for Leu-1, Leu-3a and la-like antigens but negative for Leu-2a antigen and the antibodies against light chains. The homogeniety of the major population of infiltrates in LYG indicates that at least some forms of LYG may be neoplastic or pre-neoplastic lymphocytic disorders which may ultimately progress to malignant lymphoma. ACTA PATHOL. JPN. 35 : 711–721, 1985.     

Plasma brain natriuretic peptide and the evaluation of volume overload in infants and children with congenital heart disease

This study was designed to explore whether it was possible to evaluate the severity of VSD, PDA, and ASD by measuring brain natriuretic peptide (BNP) levels. We also investigated normal BNP levels in children to provide a baseline for our study. We measured BNP levels in 253 normal children, including 11 normal neonates, and in 91 VSD patients, 29 PDA patients, and 34 ASD patients. BNP levels showed no age-related differences in normal children (the mean value: 5.3 +/- 3.8 pg/ml). In the healthy neonates, BNP levels rose from 10.4 +/- 11.9 pg/ml in cord blood to 118.8 +/- 83.2 pg/ml on day 0, then fell to 15.3 +/- 7.8 pg/ml by day 7. In VSD and PDA patients, BNP levels correlated significantly with Qp/Qs, LVEDV, and peak RVP/LVP. In ASD patients, BNP levels correlated with Qp/Qs and RVEDV. Especially, in VSD patients, as an index corresponding to 1.5-2.0 of the Qp/Qs ratio, BNP levels of 20-35 pg/ml were found to be best with regard to both sensitivity and specificity. In the healthy neonates, BNP levels changed rapidly after birth. In VSD, PDA, and ASD patients, BNP levels were well-correlated with the severity of the disease. Especially, in VSD patients, it that appears BNP levels may be useful in evaluating surgical indications, with 20-35 pg/ml levels being the appropriate cut-off value.     

Regulation of Th2 cell differentiation by mel-18, a mammalian polycomb group gene

Polycomb group (PcG) gene products regulate homeobox gene expression in Drosophila and vertebrates and also cell cycle progression of immature lymphocytes. In a gene-disrupted mouse for polycomb group gene mel-18, mature peripheral T cells exhibited normal anti-TCR-induced proliferation; however, the production of Th2 cytokines (IL-4, IL-5, and IL-13) was significantly reduced, whereas production of IFNgamma was modestly enhanced. Th2 cell differentiation was impaired, and the defect was associated with decreased levels in demethylation of the IL-4 gene. Significantly, reduced GATA3 induction was demonstrated. In vivo antigen-induced IgG1 production and Nippostrongylus brasiliensis-induced eosinophilia were significantly affected, reflecting the deficit in Th2 cell differentiation. Thus, the PcG gene products play a critical role in the control of Th2 cell differentiation and Th2-dependent immune responses.     

Enhancement of lymphocyte response to PHA by lysosomal enzymes from polymorphonuclear leukocytes of RA joint fluid

The effect of polymorphonuclear leukocyte (PMN) granule lysates obtained from joint fluid of RA an the in vitro DNA synthesis of PHA-stimulated autologous lymphocytes from joint fluid was studied. Lymphocytes were cultured for 3 days with or without PMN lysates in 2 ml of RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum (FCS). The lymphocytes were stimulated with phytohemagglutinin (PHA-M). The DNA synthesis was measured by counting the [³H]thymidine incorporation. Lymphocytes from RA joint fluid stimulated with PHA-M showed 19,466±987 cpm (mean±SE per 10⁶ cells in the absence of PMN lysates. Upon addition PMN lysates to the PHA-stimulated lymphocytes, the maximum in vitro DNA synthesis increased to 44,877±1338 cpm. The enhancing effect of PMN lysates was abolished by plasma inhibitors or by passage through a column of protease inhibitor (Trasylol). It was concluded, therefore, that the enhancing effect of PMN lysates on PHA-stimulated lymphocytes may be associated with lysosomal proteases. Based on experiments using separated T and B lymphocytes, the enhancing effect of PMN lysates was considered to result from the activation of T lymphocytes. The results obtained in the present study suggest an important role for lysosomal proteases in the perpetuation of rheumatoid synovitis.     

Effect of Pseudomonas aeruginosa exotoxin A on endotoxin-induced tumour necrosis factor production in murine lung

The ability of several Pseudomonas aeruginosa exo-enzymes, including exotoxin A (ETA), to induce inflammation and their influence on endotoxin-induced tumour necrosis factor (TNF) production in murine lung were evaluated. Intratracheal administration of lipopolysaccharide (LPS; 0.1-10 microg/mouse), 2(-1) LD50 of P. aeruginosa alkaline protease (7.5 microg/mouse) and elastase (1.2 microg/mouse) elevated total cell number and the percentage of neutrophils in broncho-alveolar lavage fluid (BALF), whereas ETA (0.1 microg/mouse) did not. LPS induced TNF production in BALF in a dose-dependent manner, whereas the P. aeruginosa exo-enzymes did not. When ETA was inoculated into the respiratory tract before LPS, production of TNF in BALF was significantly suppressed in a dose-dependent manner. ETA also suppressed TNF production by alveolar macrophages (AMs) stimulated with LPS in vitro. Flow cytometric analysis showed that ETA markedly reduced the expression of CD14 and CD11c/CD18 on the surface of AMs. ETA also depressed partially the expression of TNF-alpha mRNA in AMs. These findings suggest that ETA regulates TNF production in murine lung by suppressing LPS receptor expression, mRNA expression and protein synthesis and/or secretion of TNF.     

Purification of fully activated Clostridium botulinum serotype B toxin for treatment of patients with dystonia

Clostridium botulinum serotype B toxins 12S and 16S were separated by using a beta-lactose gel column at pH 6.0; toxin 12S passed through the column, whereas toxin 16S bound to the column and eluted with lactose. The fully activated neurotoxin was obtained by applying the trypsin-treated 16S toxin on the same column at pH 8.0; the neurotoxin passed through the column, whereas remaining nontoxic components bound to the column. The toxicity of this purified fully activated neurotoxin was retained for a long period by addition of albumin in the preparation.     

Study on CH50 levels in factor D-depleted serum

It is generally accepted that levels of serum whole complement activity (CH50) reflect the activities of complement (C) components of the classical C pathway (CP), since CH50 is assayed by use of sensitized sheep erythrocytes (EA). However, the alternative C pathway (AP) is considered to be also activated simultaneously in the process of activation of serum CP by EA. Thus, serum CH50 levels may possibly reflect not only CP but also AP activation in CH50 assay. We studied on the influence of AP activation during CH50 assay on CH50 levels, by comparison of CH50 levels in serum samples before and after treatment of factor D depletion. Polystyrene beads carrying polyanion, poly (2-acrylamide 2-methylpropane sulfonate) (PAMPS-beads), on the surface were prepared and used for preparation for factor D-depleted serum. After treatment of pooled normal human serum (NHS) with PAMPS-beads (2.5 mg/ml of serum), serum ACH50 level decreased to be undetectable, indicating that AP activation is prohibited in PAMPS-beads-treated serum. When isolated factor D was added to this PAMPS-beads-treated serum, ACH50 level recovered to that of before treatment. Immunoblot analysis revealed that factor D band observed in NHS disappeared completely after PAMPS-beads treatment. From these results, it is clear that factor-D deficient serum is prepared by PAMPS-beads treatment. Besides, since serum CH50 level was not decreased by PAMPS-beads treatment, it may be concluded that CH50 level is not affected by AP activation during CH50 assay.