Recognition of a novel naturally processed, A2 restricted, HCV-NS4 epitope triggers IFN-gamma release in absence of detectable cytopathicity

Using short term CTL lines derived from HLA A2/K^b transgenic mice and IFN-gamma release assays we demonstrate that the NS4.1769 epitope, is generated from natural processing of the NS4 antigen, and presented in the context of the A2/K^b molecules. Interestingly, T cell recognition of the naturally processed form of the NS4.1769 epitope was associated with significant IFN-gamma release, but no direct cytolytic activity. Epitopes of this phenotype might be of interest, in terms of therapy of chronic HCV infection by associating the benefit of localized lymphokine release with low or absent direct cytopathicity.     

Ultrasonic nebulization of hypertonic solution: a new method for obtaining specimens from nasal mucosa for morphologic and biochemical analysis in allergic rhinitis

Various techniques are used to collect specimens from the nasal mucosa for morphologic and biochemical analysis. The purpose of this study was to devise a method that overcomes some of the disadvantages (e.g., invasive procedure, samples not suitable for cytologic and biochemical analysis, lack of standardization, and poor reproducibility) of these techniques. The new method requires subjects, with neck extended, to inhale an ultrasonic nebulization of a hypertonic (3% NaCl) solution (UNHS) for 5 min. They then blow their nose into a Petri dish, one nostril at a time with the other one blocked. The secretions are dispersed with 0.1% dithiothreitol in phosphate buffer solution for 20 min. Total cell count (TCC) is evaluated, and the cellular suspension is divided into two aliquots: one is centrifuged and the supernatants are collected for eosinophil cationic protein (ECP) measurements; the other is cytocentrifuged and the slides, stained with Diff-Quik, are used for differential cell count. The results obtained with the UNHS and nasal lavage (NL) methods were compared. Eleven nonatopic healthy subjects and 19 allergic rhinitic patients were studied. Total cell count (×10⁵) was significantly higher with UNHS than with NL (13.0±12.3 vs 1.911.6; P<0.0]) The differential cell count was similar with the two procedures. ECP levels (μg/l) were higher with UNHS than with NL (39.1+38.2 V.S 16.7±41.2; P<0.01). For evaluation of reproducibility, four healthy and six rhinitic subjects underwent UNHS on two occasions within 5 days, and the results of two samples (sample 1 vs sample 2) were analyzed. Reproducibility was good as to TCC, differential cell count, and ECP     

Variable regions of antibodies to synthetic polypeptides--I. Characterization of an idiotope expressed on antibodies and T-cell factors

Spleen cells from a Lewis rat immunized with affinity-purified B10 anti-(T,G)-A-L antibody were fused with the non-secreting murine hybridoma SP2/0. Cell lines secreting monoclonal antibodies specific for mu- and kappa-chains, as well as an idiotope on anti-(T,G)-A-L antibodies, were isolated and characterized. The anti-mu and -kappa antibodies, are true anti-isotypes, reacting with sera from all strains of mice tested. The anti-idiotope antibodies recognize a determinant on antibodies binding a GT-containing epitope. The proportion of anti-GAT antibody bearing the idiotope varies markedly in different murine strains. A 1000-fold higher level of antibody from Igha mice than from Ighb and Ighe mice is required to give an equivalent inhibition of the idiotope-anti-idiotope reaction. Analysis of monoclonal antibodies expressing the idiotope indicates that the affinity of binding between idiotope and anti-idiotope can vary by as much as two orders of magnitude. Immunoadsorbants prepared with anti-idiotope antibody bind suppressor factor secreted by a GAT-specific T-cell hybridoma.     

Purification of bovine conglutinin using pepsin digestion

This paper describes a new method for the purification of bovine conglutinin based on the relative resistance of this protein to pepsin digestion. First, conglutinin is purified by absorption on yeast, then the preparation is treated with 2% pepsin (w/w) at 4°C for 18 hr, and finally gel filtrated on agarose A5m. The yield is 60–75% and conglutinin thus prepared appears physically, immunochemically and functionally intact. This procedure allows for a rapid production of sufficient amounts of conglutinin for immune complex detection or purification methods.     

A simple technique for evaluation of methods of cell separation

A simple method is described for labelling cells with fluorescein and using them in artificial mixtures to assess cell separation procedures. The method facilitates the examination of the variables in a separation procedure. It is thus possible to tailor a separation procedure (for example panning with monoclonal antibody) to suit the specific requirements of the experiment.     

Identification of parasite proteins in a membrane preparation enriched for the surface membrane of erythrocytes infected with Plasmodium knowlesi

A subcellular fraction enriched in erythrocyte membranes has been isolated from rhesus monkey erythrocytes infected with Plasmodium knowlesi. Infected cells were lysed by centrifugation through a zone of hypotonic buffer and membranes isolated by equilibrium density gradient centrifugation in the same tube. The purified membrane fraction was shown to include the erythrocyte surface membrane by several methods: electron microscopy, identification of Coomassie Blue stained erythrocyte membrane proteins, identification of band 3 with a monoclonal antibody, and identification of radioiodinated cell surface proteins. The resulting ghosts were shown to be specifically reactive with monkey sera against the variant surface antigens of P. knowlesi by indirect immunofluorescence and membrane agglutination. No reactivity was seen with a monoclonal antibody (13C11) against the intracellular schizont surface. A number of metabolically labelled parasite proteins were enriched in this membrane function, including peptides of 277, 208, 173, 153, 134, 109, 80, 60 and 48 kDa and the variant surface antigens of variable molecular mass (180-207 kDa). These proteins were distinct from the major parasite proteins of total infected erythrocytes and isolated merozoites. The major glucosamine labelled glycoprotein of the internal schizont (230 kDa) was not found in this fraction. Moreover, no fragment of this parasite glycoprotein was found in this membrane fraction, indicating that no part of this molecule is transported to the erythrocyte surface. In contrast, the variant antigen of P. knowlesi, known to be on the erythrocyte surface, could be readily identified as peptides unique to specific cloned parasite lines. We propose that the other nine parasite proteins found within this membrane fraction represent a starting point for the identification of other parasite proteins transported to the surface membrane of the infected erythrocyte.     

Purification of biologically active rubella virus antigens by immunoaffinity chromatography

A general procedure for isolating biologically active rubella virus antigens (VPI, Mr = 61,000; VP2, Mr = 45,000; VP3, Mr = 36,000) by monoclonal antibody affinity chromatography is described. Complexes formed between monoclonal antibodies and rubella virus antigens were found to be stable either at low pH or in Tris buffer containing detergent and high salt, but were efficiently dissociated by 5% diethanolamine, pH 11.5, or 50 mM lithium diiodosalicylate buffer, pH 8.0. Chromatographically purified rubella viral antigens retained their antigenicity as determined by enzyme-linked immunosorbent assays. Biological studies showed that rubella structural proteins VP2 and VP3 had no hemagglutinin function while the mixture of VP1 and VP2 and VP3 directly demonstrated hemagglutination activity. These results indicate that VP1 is at least in part responsible for the hemagglutinin function of rubella virus.     

Induction of immunoglobulin-producing human peripheral blood lymphocytes in serum-free medium

Induction of immunoglobulin-secreting cells from human peripheral blood lymphocytes in a serum-free culture medium was studied. Albumin, transferrin, insulin and fibronectin can replace serum entirely for support of pokeweed mitogen (PWM)-stimulated B lymphocytes, measured by a reverse hemolytic plaque assay using protein A-coated red cells. In this serum-free system, growth and maturation to IgM and IgG secretion occur at the same or higher efficiency as in conventional serum-containing medium, with maximum numbers of plaque-forming cells on day 6 at optimal dose of PWM, 0.5 ～ 5 μg/ml. This system can be used to avoid the interference from undefined serum components.     

A monoclonal antibody (RH1-38) which inhibits multiple systems of cell-mediated cytotoxicity--I. Identification of a novel epitope on a killer cell surface bimolecular complex important in the cytotoxic response

This paper presents the initial characterization of a mouse monoclonal antibody (RH1-38) which blocks, in the absence of complement, three different systems of cell-mediated cytotoxicity. This monoclonal antibody markedly inhibits cytotoxicity mediated by human natural killer cells, a monocyte-like cell [phorbol myristate acetate (PMA) stimulated HL-60], and cytotoxic T-lymphocytes generated in a mixed leukocyte reaction. RH1-38 is not nonspecifically toxic to cells since antibody-dependent cellular cytotoxicity was not inhibited and viability as assessed by trypan blue exclusion was not affected. Inhibition is specific since control hybridoma culture supernatants, parent (NS-1) ascites supernatant, monoclonal anti-HLA and normal mouse IgG were not significantly inhibitory. In the NK system, the inhibitory effect appears to be due to binding of monoclonal antibody to effector cell surface since exposure of targets to antibody followed by washing yielded no inhibition of killing. Inhibition requires the antigen-binding portion of the antibody molecule and thus appears to be related to steric hindrance of an effector cell surface molecule which is important in the expression of cell-mediated cytotoxicity. Immunoprecipitation of surface-radioiodinated membranes from PMA-stimulated HL-60 cells and analysis on sodium dodecyl sulfate-polyacrylamide gels revealed a bimolecular complex (195,000 and 125,000 daltons) without significant change under reducing conditions. Control immunoprecipitates yielded no peaks of activity. This monoclonal antibody should serve as a useful probe of the function and biochemistry of a killer cell surface antigen important in the expression of cell-mediated cytotoxicity. Since RH1-38 inhibits cytotoxicity mediated by at least three apparently unrelated effector cells, the relevant antigen may be part of a common mechanistic step. As the companion paper demonstrates, this monoclonal antibody does not affect the conjugation step, but appears to block a late step in the NK cytolytic mechanism. Thus, RH1-38 recognizes either an epitope district from previously-described anti-LFA-1 antibodies or alternatively recognizes a distinct functional killer cell surface molecule.     

Immunocytochemical localization of a granuliberin-like peptide in Rana pipiens brain

The histamine-releasing peptide “granuliberin”, originally isolated from the skin of Rana rugosa, was localized by immunofluorescence within nerve cell bodies and fibers in the brain of Rana pipiens. The granuliberin-positive neurons were characterized ultrastructurally by electron microscopic observation of ultrathin sections stained either with immunoperoxidase or with conventional stains. Granuliberin-positive nerve cell bodies were seen throughout the hypothalamus, from the suprachiasmatic area rostrally to the full length of the periinfundibular grey matter caudally. Similarly positive nerve fibers were localized in the hypothalamus radiating upwards to the optic vesicles in the midbrain, extending through the preoptic area into the subpallium in the forebrain, and throughout the white matter surrounding the floor and lateral walls of the fourth ventricle in the brainstem. The granuliberin-positive nerve cells showed the presence of variable numbers of small cytoplasmic neurosecretory granules. Possessing an electron dense elongated core, and measuring 250–350 nm in their largest diameter. The functional significance of a granuliberin-like peptide in neurons of the frog brain is not known.