Czeslawa Kowal

Molecular mimicry between bacterial and self antigen in a patient with systemic lupus erythematosus.

The importance of microbial infection as a trigger for the induction of systemic lupus erythematosus is frequently debated. Clinical observations indicate that anti‐viral and anti‐bacterial responses are often accompanied by self reactivity, and anti‐pneumococcal antibodies elicited in non‐autoimmune individuals by pneumococcal vaccine express lupus‐associated anti‐DNA idiotypes. To explore the relationship between protective and pathogenic antibodies in humans, we have used the phage display immunoglobulin expression system to generate a combinatorial library from spleen cells of a lupus patient immunized with a polyvalent pneumococcal polysaccharide vaccine prior to splenectomy. From this library, monovalent antigen‐binding fragments expressing the 3I Vκ1‐associated idiotype were isolated. This idiotype is expressed on up to 90 % of anti‐DNA antibodies in the serum of lupus patients and on anti‐pneumococcal antibodies in the serum of non‐autoimmune individuals. Eight 3I+ monovalent antigen‐binding fragments reacting with pneumococcal polysaccharide, DNA or both were analyzed. Four of these fragments were cross‐reactive with both foreign and self antigen, demonstrating that a high percentage of anti‐bacterial antibodies produced in a patient with lupus bind double‐stranded DNA. These studies provide support at the molecular level for a potential role of molecular mimicry in the generation of anti‐DNA antibodies. In addition, this is, to our knowledge, the first panel of fully sequenced human anti‐pneumococcal antibodies.

Naked DNA vaccination differentially modulates autoimmune responses in experimental autoimmune encephalomyelitis

Vaccination with naked DNA represents a therapeutic strategy currently under consideration in multiple sclerosis (MS). In this study, we tested the potential therapeutic effect of vaccination with a naked DNA construct encoding proteolipid protein (pRc/CMV-PLP) upon the outcome of subsequent sensitization for experimental autoimmune encephalomyelitis (EAE) actively-induced in SJL mice with PLP139-151 peptide in adjuvant. Intramuscular vaccination with the naked DNA pRc/CMV-PLP construct led to PLP expression in local muscle tissue that persisted for about 8 weeks. Early sensitization for EAE (4 weeks after DNA vaccination) caused recipient mice to develop a severe, exacerbated form of disease (in comparison to control mice), while late sensitization (>10 weeks) resulted in a milder, ameliorated form. In the groups sensitized <10 weeks post-DNA vaccination with pRc/CMV-PLP induction of a Th1-type cytokine response was noted. In contrast, sensitization >10 weeks post-DNA vaccination led to peripheral tolerance as evidenced by a decrease in T cell proliferation and cytotoxic T cell response, no Th2 response, and no increase in apoptosis. These data are novel in that they demonstrate a differential effect of DNA vaccination and have important implications for its use as a mechanism to enhance or modulate immune reactivity.

Protective effect of naked DNA immunization in experimental autoimmune encephalitis

581 Reactive Changes of Thymocytes and Retieuloepithelium during Autotransplantation of the Thymus A.A. Stadnikov, O.Y. Nickolaeva. StateMedJcalAcademy, Drenbur~ Russia 584 Systemic lntedeukin-12 Displays Antitumor Activity in the Mouse Central Nervous System K. Shim i ~ , H. Kishim a, Osaka UniversityMedicalSchool, Japan, Y. Miyao, Suita Municipal Hospital, Japan, K. Tamurn, Osaka University Medical School, Japan, M. Tamura, Minoh MunicipaI Hospital, Japan, M. Sasaki, Osaka University Medical School, Japan

Molecular mapping of the 8.12 SLE-associated idiotype specificity at the single amino acid level.

The 8.12 idiotype is expressed in elevated titer in the serum of patients with systemic lupus erythematosus and is a marker for a subpopulation of anti-DNA antibodies that possess a V(lambda)II encoded light chain. This study utilized a eukaryotic expression system to identify the structural basis for expression of this idiotype. Reversion of the 8.12+ DSC light chain to the hslv215.23/DPL11 germline gene reveals that the 8.12 idiotype is encoded in the germline. The 8.12+ DSC and the 8.12 AS17 light chains, both belonging to the V(lambda)II family, were subjected to site directed mutagenesis, to localize amino acids important for expression of the 8.12 idiotype. Point mutations were performed in CDR1, CDR2, FR3 and CDR3, in positions where the 8.12+ DSC differs from the 8.12-AS17. Amino acids in CDR1 and the CDR2 proximal region of FR3, but not the J proximal region of CDR3, play a crucial role in 8.12 reactivity. The 3-D structure of Mcg, a human IgG1, with which DSC shares a sequence homology of 92.3% has been examined to visualize the effect of each of the mutations and to identify the surface on DSC that comprises the idiotype.

An EαEβ heterodimer dramatically alters selection of functional T-cell subsets in A.CA transgenic mice

The A.CA strain (H-2f) lacks expression of the class II E molecule because of a stop codon in the leader sequence of the Ea gene and defective transcription of the Eb gene (Vu et al. 1989; Tacchini-Cottier et al. 1988). We previously reported that A.CA mice possessing an Ea d transgene express the Eo~ d polypeptide chain on the surface of lymphoid cells, presumably in association with AI3 chains. (Ishikawa et al. 1991). We have established transgenic mice expressing both the o~ and ~3 chain of the E d molecule to investigate the role in thymic selection of Eo~E~3 isotype-matched heterodimers in comparison with Ec~A[3 mixed-isotype heterodimers. A 17 kilobase (kb) Kpn I fragment (Fig. 1 A) containing the Eb gene of the macrophage cell line, BAC1.2 (H-2d#) was isolated from a genomic library generated in Charon 40. The gene spanning from -5.7 kb to 11 kb was microinjected directly into A.CA fertilized eggs. The founder shows a characteristic 2 kb band present in Eco Rl-digested genomic DNA (Fig. 1 B). Northern blot analysis shows that transcripts of the transgene were detected in spleen, thymus, lung, and kidney, but not in heart or liver (Fig. 1 C). The Tg(o,Eb) line of the Eb transgenic mice was established and crossed to two A.CA strains transgenic for the Ec~a, Tg(a, Ea), and Tg(b, Ea) to obtain Tg(a/o, Ea/Eb) and Tg(b/o, Ea/Eb) double transgenic mice, respectively. Fluorescence-activated cell sorter (FACS) analysis of lymph node cells stained with the 14.4.4S anti-E~ antibody showed that Tg(b/o, Ea/Eb) double transgenic mice express a higher density of E d molecules than Tg(a/o, Ea/Eb) double transgenic mice, even higher than the density of E d seen in BALB/c mice (Fig. 2A). In this mouse, the expression of E d is elevated on B220 + cells (data not shown). Single transgenic mice with the

Autoantibodies and cognitive impairment in systemic lupus erythematosus

Many patients with systemic lupus erythematosus experience cognitive decline as their disease progresses. The pathogenic mechanisms include thrombosis, vasculitis, and drug toxicity. We have demonstrated that a subset of anti-DNA antibodies cross-reacts with N-methyl-D-aspartate NMDA receptors and can cause excitotoxic cell death. In mice with high serum titers of these antibodies, there is no evidence of brain damage until there is a breach in the blood–brain barrier. With a break in the blood–brain barrier induced by administration of lipopolysaccharide, the antibodies bind preferentially to hippocampal neurons that express N-methyl-D-aspartate receptors at high density. The antibodies mediate a noninflammatory apoptotic cell death. By 1 week following lipopolysaccharide administration, there is a 30% loss of hippocampal neurons. At 1 month there is no further loss of neurons, suggesting that the blood–brain barrier closes and the antibodies have no further access to brain tissue. Mice experiencing hippocampal damage from antibody show decreased N-acetyaspartate in the hippocampus by magnetic resonance spectroscopy. An abnormal metabolism is detectable only in the hippocampus, confirming the selectivity of the damage. These mice also display impaired performance on tasks of memory function that are dependent on the integrity of the hippocampus. Thus, these studies provide a new model for cognitive impairment in systemic lupus and suggest that antibodies may mediate, through noninflammatory mechanisms, changes in cognitive function.