Antigen recognition in autoimmune encephalomyelitis and the potential for peptide-mediated immunotherapy

Peptide binding and lymph node T cell activation studies have been used to characterize T cell recognition of an encephalitogenic T cell autoantigen from myelin basic protein in (PL/J x SJL)F1 mice. Amino acids that determine interactions with either the restriction element of the major histocompatibility complex (MHC) or the encephalitogenic T cell receptor are defined. This information enables the design of peptides that bind MHC yet do not cross-react with the autoantigen. A peptide analog of the encephalitogenic epitope is shown to be "heteroclitic" for MHC binding and activation of encephalitogenic T cells in vitro. This analog is not immunogenic for encephalitogenic T cells in vivo and is shown to inhibit disease that is induced by the autoantigen itself.     

Ocular vergence under natural conditions. II. Gaze shifts between real targets differing in distance and direction

Horizontal binocular eye movements of three subjects were recorded with the scleral sensor coil--revolving magnetic field technique during voluntary shifts of gaze between pairs of stationary, real, continuously visible targets. The target pairs were located either along the median plane (requiring symmetrical vergence), or on either side of the median plane (requiring asymmetrical vergence). Symmetrical vergence was primarily smooth, but it was often assisted by small, disjunctive saccades. Peak vergence speeds were very high; they increased from about 50 degrees s-1 for vergence changes of 5 degrees to between 150 and 200 degrees s-1 for vergence changes of 34 degrees. Differences between convergence and divergence were idiosyncratic. Asymmetrical vergence, requiring a vergence of 11 degrees combined with a version of 45 degrees, was largely saccadic. Unequal saccades mediated virtually all (95%) of the vergence required in the divergent direction, whereas 75% of the vergence required in the convergent direction was mediated by unequal saccades, with the remaining convergence mediated by smooth vergence, following completion of the saccades. Peak divergence speeds during these saccades were very high (180 degrees s-1 for a change of vergence of 11 degrees); much faster than the smooth, symmetrical vergence change of comparable size (14 degrees). Peak convergent saccadic speeds were about 20% lower. This difference in peak speed was caused by an initial, transient divergence, observed at the beginning of all horizontal saccades. The waveform of disjunctive saccades did not have the same shape as the waveform of conjugate saccades of similar size. The smaller saccade of the disjunctive pair was stretched out in time so as to have the same duration as its larger, companion saccade. These results permitted the conclusion that the subsystems controlling saccades and vergence are not independent. Vergence responses were relatively slow and incomplete with monocular viewing, which excluded disparity as a cue. Monocularly stimulated vergence decreased as a function of the increasing presbyopia of our three subjects. Subjects were able to generate some vergence in darkness towards previously seen and remembered targets. Such responses, however, were slow, irregular and evanescent. In conclusion, vergence shifts between targets, which provided all natural cues to distance, were fast and accurate; they appeared adequate to provide effective binocular vision under natural conditions. This result could not have been expected on the basis of previous observations, all of which had been made with severely reduced cues to depth.(ABSTRACT TRUNCATED AT 400 WORDS)     

Elevated levels of plasma lipofuscins in patients with chronic renal failure

The fluorescence excitation and emission spectra observed in plasma from patients with chronic renal failure were reproduced by the generation of soluble lipofuscins in normal plasma samples by incubation with mixtures of L-dopa, dopamine, L-norepinephrine, L-epinephrine, 3-hydroxy-DL-kynurenine and 3-hydroxy-anthranilic acid for 24 h at 37 degrees C. Relative fluorescence intensity measurements consistently showed elevated plasma levels of the soluble lipofuscins in chronic renal failure: the means (n = 27) were 73.9 +/- 33.4 (SD) and 71.1 +/- 14.8 at emissions 413 nm and 445 nm respectively, in contrast to those of normal plasma samples (n = 11), 18.2 +/- 5.3 and 23.1 +/- 5.6. The maximum or shoulder at approximately 413 nm represents soluble lipofuscin that can be generated from 3-hydroxyanthranilic acid and the maximum or shoulder at approximately 445 nm represents soluble lipofuscins derived from the precursors listed above and probably from other related precursors. Gravimetric measurements also showed elevated levels of melanins in the plasma samples of patients with chronic renal failure: 2.72 +/- 0.38 mg/ml (n = 16), as compared to normal values: 1.70 +/- 0.10 mg/ml (n = 6). In individual patients haemodialysis reduced the fluorescence intensities to a range of 65-99% and the melanin levels to a range of 86-99% of the pre-dialysis values.     

Bacteriocuprein superoxide dismutases in pseudomonads.

Two new instances of the rare bacteriocuprein form of superoxide dismutase have been discovered in Pseudomonas diminuta and P. maltophilia. Each species contains a manganese superoxide dismutase as well. Eight other strains of Pseudomonas and Xanthomonas spp. lacked bacteriocupreins and contained either a manganese or an iron superoxide dismutase. Native molecular weights and isoelectric points were determined for all these bacterial dismutases. A monospecific polyclonal antibody was prepared against the bacteriocuprein from Photobacterium leiognathi; it was not cross-reactive with the bacteriocuprein from either Pseudomonas strain. Bacteriocupreins have previously been identified in only two procaryotes, P. leiognathi and Caulobacter crescentus. The discovery of the Pseudomonas bacteriocupreins reveals a broader distribution, raising the possibility that bacteriocupreins are a continuous line of descent among procaryotes and not isolated evolutionary occurrences, as previous data suggested.     

Crystallographic characterization of a Cu,Zn superoxide dismutase from Photobacterium leiognathi

Crystals of a copper-zinc superoxide dismutase from Photobacterium leiognathi, a luminescent marine bacterium that is the species-specific symbiont of the ponyfish, have been obtained from 2-methyl-2,4-pentanediol solutions. The space group was determined using screenless small-angle precession photographs, and was confirmed by analyzing area detector diffraction data with the XENGEN programs for indexing and refinement. The crystals are monoclinic, space group C2 (a = 126.4 A, b = 87.0 A, c = 44.4 A, beta = 92.8 A), and have two 32,000 Mr dimers per asymmetric unit. The crystals diffract to at least 2.7 A resolution, are resistant to radiation damage, and are suitable for determination of the structure by X-ray diffraction.     

Multiple T and B cell epitopes in the S1 subunit ("A"-monomer) of the pertussis toxin molecule

The immunogenicity and reactogenicity of Bordetella pertussis vaccine are mediated in part by the S1 subunit of pertussis toxin (PT). To identify the immune epitopes in the S1 subunit of PT, synthetic peptides were prepared and tested for their capacity to induce antibodies in mice with different MHC genotypes. In BALB/c mice, peptides corresponding to sequences 1-17, 70-82 and 189-199 generate T cell proliferative responses, induce the production of antibodies capable of neutralization of the toxin in the Chinese hamster ovary-cell assay, and protect mice from a shock-like syndrome caused by alternate injections of BSA and PT. Protection and neutralization correlated with the ability of these peptides to elicit high anti-PT titers. Different B cell epitopes were detected in other inbred mouse strains. The antibody reactivity against synthetic peptides from two infants vaccinated with pertussis vaccine was tested. These infants had antibodies reactive to a variety of epitopes in the S1 subunit, including peptides 1-17, 70-82, 99-112, 135-145, and 189-199. Thus, it appears that there are multiple T and B cell epitopes in the S1 subunit of PT.     

The iron superoxide dismutase of Legionella pneumophila is essential for viability.

Legionella pneumophila, the causative agent of Legionnaires' disease, contains two superoxide dismutases (SODs), a cytoplasmic iron enzyme (FeSOD) and a periplasmic copper-zinc SOD. To study the role of the FeSOD in L. pneumophila, the cloned FeSOD gene (sodB) was inactivated with Tn903dIIlacZ, forming a sodB::lacZ gene fusion. By using this fusion, expression of sodB was shown to be unaffected by a variety of conditions, including several that influence sod expression in Escherichia coli: aeration, oxidants, the redox cycling compound paraquat, manipulation of iron levels in the medium, and the stage of growth. A reproducible twofold decrease in sodB expression was found during growth on agar medium containing charcoal, a potential scavenger of oxyradicals, in comparison with growth on the same medium without charcoal. No induction was seen during growth in human macrophages. Additional copies of sodB+ in trans increased resistance to paraquat. Construction of a sodB mutant was attempted by allelic exchange of the sodB::lacZ fusion with the chromosomal copy of sodB. The mutant could not be isolated, and the allelic exchange was possible only if wild-type sodB was present in trans. These results indicate that the periplasmic copper-zinc SOD cannot replace the FeSOD. The data strongly suggest that sodB is an essential gene and that FeSOD is required for the viability of L. pneumophila. In contrast, Sod- mutants of E. coli and Streptococcus mutans grow aerobically and SOD is not required for viability in these species.     

Dendritic and B-cell function during antibody responses in normal and immunodeficient (xid) mouse spleen cultures

Dendritic cells (DC) act as accessory cells for T-dependent antibody responses in two ways. One is to induce a class of stimulating factors (BSF) which allow B lymphocytes to respond to heterologous red cells as antigen. xid DC induce the production of these BSF, but xid B cells totally lack responsiveness. A second mechanism of DC function applies to red cell and haptenated-protein antigens. Here DC, helper T lymphocytes, and antigen-specific B cells interact in discrete clusters. Then the B cells become responsive to BSF. xid DC are fully active in this pathway, and xid B cells develop significant (10-20% of control) responses. This partial reduction in xid B-cell function could be due to the poor viability of xid lymphocytes in vitro. There is a comparable reduction in xid polyclonal responses to alloreactive helper T blasts. The other severe deficit in xid involves antibody formation to haptens on polysaccharide carriers. This response in normal mice is not influenced by DC or by BSF. The only similarity between DNP-Ficoll and RBC plus BSF responses is that both utilize B lymphocytes that do not associate with DC-T clusters, even though helper cells for DNP-Ficoll and for RBC are present in the culture. We conclude that DC function is not altered in xid. The main deficit seems to be in a B-cell activation pathway that is shared by polysaccharide carriers and some but not all BSF, and/or in a B-cell subpopulation that does not interact with carrier-specific helper cells. We speculate that this B-cell alteration primarily involves the Ig delta-poor marginal zone subpopulation of splenic B lymphocytes.     

Bioassays for the detection of a cytokine that enhances the development of cytolytic T lymphocytes

Interleukin 2 (IL-2) is an important growth factor for cytolytic T lymphocytes (CTL). Other factors here termed cytolytic differentiation factor(s) (CDF) may be required for CTL responses, but it has been difficult to identify suitable bioassays. We report a polyclonal assay in which lytic activity develops after 2 days of culture with lectin and lymphokines. CDF is required for the development of Thy-1+, CD8+, CD4- CTL in this assay. The responsive T cells are thymocytes from certain strains of mice (A, Swiss NCS, B6.H-2k) or peripheral T cells cultured in the presence of high doses of hydrocortisone acetate. If these precautions are taken, little or no lytic activity develops in the presence of rIL-2 alone. CDF is present in the media of mitogen-stimulated mouse spleen or human blood mononuclear cells, but not in the conditioned media of a number of mouse and human cell lines. It is immunologically distinct from IL-2, but only acts in concert with IL-2 to induce CTL. A large panel of available mouse and human cytokines do not synergize with Il-2 in the bioassay. These include interferons, colony-stimulating factors, lymphotoxin/tumor necrosis factor, and IL-1, -3, and -4. Therefore a distinct CDF(s) seems essential for the induction of at least some CTL. The assays described here should be useful in purifying the molecule responsible for this biological activity.