The complete genomic DNA sequence of the highly attenuated vaccinia strain modified vaccinia Ankara (MVA) was determined. The genome of MVA is 178 kb in length, significantly smaller than that of the vaccinia Copenhagen genome, which is 192 kb. The 193 open reading frames (ORFs) mapped in the MVA genome probably correspond to 177 genes, 25 of which are split and/or have suffered mutations resulting in truncated proteins. The left terminal genomic region of MVA contains four large deletions and one large insertion relative to the Copenhagen strain. In addition, many ORFs in this region are fragmented, leaving only eight genes structurally intact and therefore presumably functional. The inserted DNA codes for a cluster of genes that is also found in the vaccinia WR strain and in cowpox virus and includes a highly fragmented gene homologous to the cowpox virus host range gene, providing further evidence that a cowpox-like virus was the ancestor of vaccinia. Surprisingly, the central conserved region of the genome also contains some fragmented genes, including ORF F5L, encoding a major membrane protein, and ORFs F11L and O1L, encoding proteins of 39.7 and 77.6 kDa, respectively. The right terminal genomic region carries three large deletions all classical poxviral immune evasion genes and all ankyrin-like genes located in this region are fragmented except for those encoding the interleukin-1 beta receptor and the 68-kDa ankyrin-like protein B18R. Thus, the attenuated phenotype of MVA is the result of numerous mutations, particularly affecting the host interactive proteins, including the ankyrin-like genes, but also involving some structural proteins.

Acquired thrombotic thrombocytopenic purpura (TTP) has been linked to severe deficiency of ADAMTS-13 activity caused by autoantibodies inhibitory to ADAMTS-13. We report data on a patient with confirmed TTP who had severely reduced ADAMTS-13 activity but showed no ADAMTS-13 inhibition in a widely used fluid phase activity assay. With a newly developed enzyme-linked immunosorbent assay, using immobilized recombinant ADAMTS-13, we found high titers of IgM and IgG antibodies that bound to ADAMTS-13, but did not neutralize protease activity. These autoantibodies probably influenced the half-life of ADAMTS-13 or its binding to the endothelial cell surface, thereby compromising ADAMTS-13 activity in vivo. Given that ADAMTS-13 may interact physiologically with various receptors or ligands, the occurrence, distribution, and the epitope mapping of nonneutralizing antibodies will be an important area for future research. (Blood. 2003;102:3241-3243)

Memory B cells are responsible for the rapidly emerging antibody response after antigen reexposure. The signals required for the restimulation of memory B cells have not been fully explained. We used a murine model of anti-factor VIII (FVIII) antibody responses in hemophilia A to study the requirements for the restimula-tion of FVIII-specific memory B cells and their differentiation into anti-FVIII anti-body-producing cells. We were particularly interested in the significance of activated T cells and costimulatory interactions. Our results indicate that the re-stimulation of FVIII-specific memory B cells is strictly dependent on interactions with activated T cells. These activated T cells can be specific for either FVIII or third-party antigens. Restimulation by T cells specific for third-party antigens requires the presence of FVIII, indicating that signals induced by B-cell receptor (BCR) triggering and by interactions with activated T cells are important. The blockade of B7-1 or B7-2 as well as the blockade of CD40L inhibits the restimulation and differentiation of FVIII-specific memory B cells in vitro and in vivo. The interference with inducible costimulator-inducible costimulator ligand (ICOS-ICOSL) interactions, however, does not cause any modulation. As expected, the production of anti-FVIII antibodies by plasma cells is not dependent on any of the costimulatory interactions tested.

Severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 can lead to thrombotic thrombocytopenic purpura (TTP), a disease associated with the widespread formation of platelet-rich thrombi in many organs. Autoantibodies that inactivate ADAMTS13 are the most frequent cause of acquired TTP. Little is known about epitope specificity and reactivity of anti-ADAMTS13 antibodies. In this study, a series of ADAMTS13 domains were expressed in Escherichia coli, and the reactivity of purified recombinant fragments with anti-ADAMTS13 autoantibodies from 25 patients with severe ADAMTS13 deficiency was evaluated in vitro. All TTP plasmas contained antibodies directed against the cysteine-rich spacer (cys-rich/spacer) domain of ADAMTS13. In the plasma of 3 patients, antibodies were detected that reacted exclusively with the cys-rich/spacer domain, underscoring the importance of this region for functional activity of ADAMTS13. In 64% of the plasmas, antibodies reacted with the 2 CUB domains, and in 56% they reacted with the isolated first thrombospondin type 1 (TSP-1) repeat and with the compound fragment consisting of the catalytic, the disintegrin-like, and the TSP1-1 domain. Less frequently, in 28% of the plasmas, antibodies reacted with the TSP1 repeats 2 to 8. Unexpectedly, antibodies reacted with the propeptide region in 20% of the plasmas. In conclusion, this study shows that even though anti-ADAMTS13 autoantibodies react with multiple domains of the protease, the cys-rich/spacer domain is consistently involved in antibody reactivity. (Blood. 2004;103:4514-4519)

The ospC gene was amplified by the polymerase chain reaction from each of 76 Lyme disease Borrelia strains. Restriction fragment length polymorphism (RFLP) analysis demonstrated 33 distinct RFLP types; two additional RFLP types were identified from published ospC sequences. For each RFLP type, at least one ospC gene was sequenced and the degree of sequence relatedness examined by construction of an ospC gene tree. The genes were extremely diverse, with sequence identity ranging from 74.4% to 99.0%; the majority of changes are localized within the central portion of the molecule. A comparison of ospC sequences suggests that recombination occurs frequently between ospC alleles; this genetic exchange is proposed to be mediated by lateral transfer of ospC sequences. Evidence indicates that recombination occurs between ospC genes from the same Borrelia species (i.e. B. afzelii and B. garinii) as well as between different Borrelia species (i.e. B. afzelii and B. garinii, B. burgdorferi and genogroup DN127).

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SummaryThe analysis of anti-factor VIII (FVIII) antibody-secreting cells (ASC) at different anatomic sites provides valuable information about the nature of the anti-FVIII immune response in hemophilic mice after treatment with human FVIII. An Elispot system is described that is suitable for analyzing frequencies and IgG subclasses of anti-FVIII ASC at the single-cell level. Hemophilic mice were treated with four doses of FVIII. Anti-FVIII antibodies in blood as well as anti-FVIII ASC in spleen and bone marrow were analyzed after each dose of FVIII and subsequently up to 22 weeks after termination of the FVIII treatment. Anti-FVIII ASC first appeared in the spleen where they were detectable after two intravenous doses of FVIII. Their appearance correlated with that of anti-FVIII antibodies in blood plasma. Anti- FVIII ASC in bone marrow were detectable after three doses of FVIII and were probably cells that initially formed in the spleen and subsequently migrated to the bone marrow. Whereas the frequency of anti- FVIII ASC in the spleen increased up to the fourth dose of FVIII and declined thereafter, in the bone marrow it remained constant for up to at least 22 weeks after the termination of the FVIII treatment. Titers of anti-FVIII antibodies in blood plasma increased up to the fourth dose of FVIII, then remained high constantly for 14 weeks and decreased but the antibodies were still detectable for up to at least 22 weeks after the fourth dose of FVIII. The IgG-subclass distribution of anti-FVIII ASC was similar in spleen and bone marrow and matched the subclasses of anti-FVIII antibodies in blood plasma indicating that both organs contribute to circulating antibodies in the blood.

Summary. We investigated the effect of proteases derived from Ficus carica (common fig) on human blood coagulation. The milky sap (latex) of several Ficus (F.) species contain ficin, which is a mixture of proteases. Ficin derived from Ficus carica shortened the activated partial thromboplastin time and the prothrombin time of normal plasmas and plasmas deficient in coagulation factors, except plasma deficient in factor X (FX) and generated activated FX (FXa) in defibrinated plasma. Chromatographic separation of ficin from Ficus carica yielded six proteolytic fractions with a different specificity towards FX. We isolated two factor X activators with molecular masses of 23AE2 and 23AE5 kDa, and studied their action on purified human FX. . These data suggest the haemostatic potency of Ficus proteases is based on activation of human coagulation factor X.