Double-pionic fusion of nuclear systems and the "ABC" effect: approaching a puzzle by exclusive and kinematically complete measurements.

The ABC effect-a puzzling low-mass enhancement in the pipi invariant mass spectrum, first observed by Abashian, Booth, and Crowe-is well known from inclusive measurements of two-pion production in nuclear fusion reactions. Here we report on the first exclusive and kinematically complete measurements of the most basic double-pionic fusion reaction pn-->dpi;{0}pi;{0} at beam energies of 1.03 and 1.35 GeV. The measurements, which have been carried out at CELSIUS-WASA, reveal the ABC effect to be a (pipi)_{I=L=0} channel phenomenon associated with both a resonancelike energy dependence in the integral cross section and the formation of a DeltaDelta system in the intermediate state. A corresponding simple s-channel resonance ansatz provides a surprisingly good description of the data.

Exclusive measurement of the pp --> pppi(+)pi(-) reaction near threshold.

The pp-->pp pi(+) pi(-) reaction has been measured exclusively near threshold at CELSIUS. The total cross sections are nearly an order of magnitude lower than expected from previous inclusive measurements. The differential cross sections reveal pp-->pp(*)(1440)-->pp sigma = pp(pi(+)pi(-))(I = l = 0) as the dominant process as well as significant contributions from p(*)-->Delta(++)pi(-)-->psigma. The observed anisotropy in the proton angular dependence is consistent with heavy-meson exchange. In the invariant mass spectra, no narrow structures of statistical relevance (3sigma) are found.

Mutational analysis of chicken interleukin 2.

Chicken interleukin 2 (chIL-2) has low, but significant, homology to both mammalian IL-2 and mammalian IL-15. In view of its unique phylogenetic position and potential use as a vaccine adjuvant, a detailed mutational analysis for critical functional sites was undertaken. It was found that Asp17 is a critical N terminal contact site for binding to the putative chIL-2 receptor, which is similar to results obtained for mammalian IL-2 and IL-15. Analysis of the C terminus did not reveal a single critical amino acid. However, deletion mutant studies demonstrated that removal of C terminal amino acids yielded proteins with decreased bioactivity and that this decrease was a function of the number and kind of amino acids removed. This study is the first non-mammalian IL-2 mutational analysis and proposes a model for the interaction between chIL-2 and its receptor.

Production and in vitro characterization of recombinant chicken interleukin-2.

Mammalian interleukin-2 (IL-2) is a well-characterized cytokine that plays key roles in T cell differentiation and activation, B cell development, and natural killer (NK) cell stimulation. Chicken IL-2, which is the first nonmammalian IL-2 cloned, differs substantially from mammalian IL-2 molecules. We undertook to study the functions of chicken IL-2 by producing recombinant molecules in prokaryotic and eukaryotic expression systems, determining the in vitro properties of these molecules, and examining the kinetics of endogenous IL-2 production in vitro. Recombinant chicken IL-2 (rChIL-2) produced in prokaryotic and eukaryotic expression systems induced proliferation of chicken splenocytes in vitro, demonstrating that glycosylation is not required for this activity. Polyclonal antibodies generated against prokaryotically produced rChIL-2 inhibited proliferation of splenocytes induced by eukaryotically and prokaryotically produced rChIL-2, as well as endogenously produced cIL-2 obtained from ConA-stimulated splenocytes. Human IL-2 or IL-15-induced CTLL proliferation was not blocked by rChIL-2 or polyclonal anti-rChIL-2 antibodies, indicating that chicken IL-2 cannot act as an antagonist of the mammalian IL-2 response. Endogenous chicken IL-2 appears to occur in vitro as a monomer of about 14.2 kDa and is secreted within 4 h after ConA stimulation. The production of rChIL-2 provides us with a useful tool for studying avian immunology as well as a potential vaccine-enhancing agent.

Interstrain variability of autoimmune encephalomyelitis in rats: multiple encephalitogenic myelin basic protein epitopes for DA rats.

We investigated T cell epitopes of guinea pig myelin basic protein (MBP) that induce experimental autoimmune encephalomyelitis (EAE) in DA rats, using synthetic peptides that correspond to regions of the guinea pig MBP molecule that are homologous to rat MBP. Four peptides were encephalitogenic when tested in DA rats. MBP63-81, which partially overlaps the dominant encephalitogenic MBP epitope for Lewis (LEW) rats, caused severe EAE in the DA strain but did not elicit EAE in LEW rats. MBP66-81 and MBP63-76 were also encephalitogenic for DA but not LEW rats. MBP79-99 also induced EAE in DA rats, although MBP87-99, the minor encephalitogenic LEW epitope, was inactive. This indicates that part of the 79-86 sequence is necessary for encephalitogenic activity in the DA strain. MBP101-120, and MBP142-167 were also encephalitogenic for DA rats. T cells from DA rats immunized with intact MBP proliferated in response to the whole protein and to MBP79-99, but were not stimulated to a significant extent by the other encephalitogenic peptides, suggesting that these may represent cryptic or subdominant epitopes. However, MBP63-81-specific T cell lines could be isolated by repeated restimulation with peptide, indicating that the peptide-specific T cells were present in DA rats at low frequency.

Regulation of cytokine gene expression in experimental autoimmune encephalomyelitis.

We previously reported that recovery of Lewis rats from experimental autoimmune encephalomyelitis (EAE) is associated with the appearance of suppressor T cells (Ts). These Ts secrete TGF-beta which down-regulates the production of inflammatory cytokines by the effector T cells that mediate this disease. In the present study, we immunized Lewis rats with myelin basic protein (MBP)+CFA, and evaluated purified T cells and MBP-activated spleen cells (SpC) during the paralytic phase (day 12) and after recovery (days 30-33) for TGF-beta and interferon (IFN)-gamma mRNA. We used reverse transcriptase-polymerase chain reaction (RT-PCR), quantitated on the basis of beta-actin mRNA. Abundant IFN-gamma mRNA was present in MBP-activated SpC obtained on day 12. In contrast, only trace IFN-gamma mRNA was detected in day 30 activated SpC, and no IFN-gamma mRNA was present in purified, nonactivated T cells obtained at either time. The level of IFN-gamma mRNA correlated with secretion of IFN-gamma as determined by ELISA on SpC culture supernatants, and with severity of adoptively transferred EAE by the activated SpC. Thus, it appears that IFN-gamma mRNA is both transcribed and translated in response to antigen activation, resulting in secretion of IFN-gamma by the disease-inducing Te. In contrast, when we used RT-PCR to investigate the expression of TGF-beta mRNA, we found the transcript present in isolated T cells and MBP-activated SpC obtained from rats at both days 12 and 30. The presence of TGF-beta mRNA at time points corresponding to both clinical EAE and recovery suggests post-transcriptional regulation of the production of this immunoregulatory cytokine.

Encephalitogenic T cells are present in Lewis rats protected from autoimmune encephalomyelitis by coimmunization with MBP73-84 and its analog.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) that is mediated by T helper 1 (Th1) CD4+ T cells. Lewis rats can be protected from actively induced EAE by coimmunization with the encephalitogenic myelin basic protein (MBP) epitope 73-84 and its single alanine-substituted analog 1028. Although analog 1028 cannot induce either active or passive EAE, it does elicit a Th1-like response that is cross-reactive with MBP73-84. Analog 1028 can effectively inhibit clinical EAE in a dose-dependent manner when rats are coimmunized with the encephalitogenic peptide MBP73-84 and 1028 in complete Freund adjuvant (CFA). Stimulation of cells from MBP73-84:1028-coimmunized protected rats proliferate and secrete interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in vitro in response to MBP73-84. Furthermore, coimmunized protected rats harbor a population of MBP73-84-reactive potentially encephalitogenic T cells, because splenocytes from these rats can be stimulated to transfer passive EAE to naive recipients. Thus, the protection of coimmunized rats by analog 1028 is not due to the inhibition of priming of MBP73-84-reactive T cells or alteration of the cytokine secretion profile of the MBP73-84-reactive cell population. Rather, MBP73-84-reactive potentially encephalitogenic T cells are primed in these protected animals.

A comparative study of experimental autoimmune encephalomyelitis in Lewis and DA rats.

We compared the T cell responses of Lewis (LEW) and DA rats to guinea pig myelin basic protein (MBP), the synthetic peptides corresponding to the epitopes that are encephalitogenic in the LEW strain (MBP73-86, MBP68-86, and MBP87-99), and bovine proteolipid protein (PLP). DA and LEW rats were susceptible to experimental autoimmune encephalomyelitis (EAE) induced with MBP or MBP68-86, but the peptide was less active in DA rats than in intact MBP molecule. MBP73-86 and MBP87-99 induced EAE in LEW rats but not in the DA strain. MBP89-169 was also encephalitogenic in DA rats. Encephalitogenic CDa+ T cell lines and clones derived from MBP-sensitized DA rats secreted IFN-gamma and TNF-alpha and proliferated to MBP and MBP89-169, but not to MBP68-88. However, T cells from MBP68-86-sensitized DA or LEW rats proliferated specifically in an I-A-restricted response to MBP68-86. T cells from MBP87-99-immunized LEW rats responded to MBP87-99 in the context of I-E, whereas the peptide-specific response of MBP87-99 immunized DA rats was I-A-restricted, although FACScan analysis indicated that DA rats express both I-A and I-E. DA rats were also highly susceptible to EAE induced with PLP; 0.6 nmol was Encephalitogenic for DA rats, but did not induce clinical EAE in LEW rats. Although both DA and LEW rats are highly susceptible to EAE, we demonstrate marked differences in the array of myelin epitopes capable of inducing the disease, as well as the MHC restriction of these epitopes, between the two rat strains.

Variable susceptibility of Lewis rats to experimental autoimmune encephalomyelitis.

This report is to alert other investigators that Lewis rats, which are widely employed in studies of autoimmune diseases, vary considerably with respect to susceptibility to experimental autoimmune encephalomyelitis (EAE), depending upon commercial source.